Gene Therapy & Molecular Biology Volume 5 Issue A by Niko Boulikas

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Gene Therapy & Molecular Biology FROM BASIC MECHANISMS TO CLINICAL APPLICATIONS

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Gene Therapy and Molecular Biology Vol 5

Table of contents Gene Therapy and Molecular Biology Vol 5, December 2003

Pages

Type of Article

Article title

Authors (corresponding author is in boldface)

1-8

Research Article

A novel expression vector induced by heat, !-radiation and chemotherapy

Farha H. Vasanawala, Tom Tsang, Abdul Fellah, Peter Yorgin, and David T. Harris

9-30

Review Article

Misregulation of pre-mRNA splicing that causes human diseases. Concepts and therapeutic strategies

Oliver Stoss, Peter Stoilov, Rosette Daoud, Annette M. Hartmann, Manuela Olbrich, and Stefan Stamm

31-37

Research Article

Th2-type immune response induced by Yasuhiro Kajihara, Shuhei Hashiguchi, Yuji Ito, and Kazuhisa Sugimura a phage clone displaying a CTLA4binding domain mimic-motif

39-46

Review Article

Segregation of partly melted molecules and its application to the isolation of methylated CpG islands in human cancer cells

Masahiko Shiraishi, Leonard S. Lerman, Adam J. Oates, Xu Li,,Ying H. Chuu, Azumi Sekiguchi, and Takao Sekiya

47-53

Review Article

PNA (peptide nucleic acid) antigene/antisense can access intact viable cells and downregulate target genes

Lidia C Boffa, Elisabetta M.Carpaneto, Benedetta Granelli, Maria R. Mariani

55-61

Review Article

Delivery of plasmid DNA by in vivo electroporation

Loree Heller and M. Lee Lucas

63-79

Review Article

Potential roles of p53 in recombination

Nuray Akyüz, Gisa S. Boehden, Christine Janz, Silke Süße, and Lisa Wiesmüller

81-86

Research Article

Characterisation of the p53 gene in the Sacha B Geutskens, Diana JM van den Wollenberg, Marjolijin M van der Eb,, rat CC531 colon carcinoma

Hans van Ormondt, Aart G Jochemsen, Rob C Hoeben

87-100

Review Article

Recombinant adenoviruses as expression vectors and as probes for DNA repair in human cells

Andrew J. Rainbow, Bruce C. McKay, and Murray A. Francis

101-110

Review Article

Chromatin remodeling and developmental gene regulation by thyroid hormone receptor

Laurent M. Sachs, Peter L. Jones, Victor Shaochung Hsia, and Yun-Bo Shi

Gene Therapy and Molecular Biology Vol 5

111-120

Review Article

Signal transduction pathways in cancer cells; novel targets for therapeutic intervention

Christos A. Tsatsanis and Demetrios A. Spandidos

121-130

Review Article

Control of pre-mRNA processing by extracellular signals: emerging molecular mechanisms

Rossette Daoud, Peter Stoilov, Oliver Stoss, Mark Hübener, Maria da Penha Berzaghi, Annette M. Hartmann, Manuela Olbrich, and Stefan Stamm

131-145

Review Article

Herpes Simplex Virus vector-based gene therapy for malignant glioma

Edward A. Burton and Joseph C. Glorioso

147-156

Research Article

Viral vectors carrying a markersuicide fusion gene (TK-GFP) as tools for TK/GCV –mediated cancer gene therapy

Sami Loimas, Tiina Pasanen, Andreia Gomes, Susana Bizarro, Richard A. Morgan, Juhani Jänne and Jarmo Wahlfors

157-162

Research Article

Aberrant DNA methylation of p16 onco-suppressor gene in human cervical carcinoma

Luciano Mariani, Giuseppe Zardo, Cesare Rapone, Anna Reale, Giuseppe Netri, Serena Buontempo, Adriana de Capoa and, Paola Caiafa

Gene Therapy and Molecular Biology Vol 5, page 1

Gene Ther Mol Biol Vol 5, 1-8, 2000

A novel expression vector induced by heat, !radiation and chemotherapy Research Article

Farha H. Vasanawala1, Tom Tsang1, Abdul Fellah2, Peter Yorgin2 and David T. Harris1* 1

Department of Microbiology & Immunology and 2Department of Pediatrics, University of Arizona, Tucson, AZ. USA.

_________________________________________________________________________________________________ * Correspondence: David Harris, Ph.D, Dept. Microbiology and Immunology, Bldg # 90, Main Campus, University of Arizona, Tucson, AZ 85721 USA. Tel: (520) 621-5127; Fax: (520) 621-6703; E-mail: davidh@u.arizona.edu Key words: expression vector, inducible promoter, heat, radiation, chemotherapy Received: 19 April 2000; accepted: 29 April 2000

Summary In many gene therapy applications and molecular biology manipulations it is desirable to be able to control the expression of the therapeutic gene. In this study a new expression vector, pHOT-MCS, was constructed using a 451 bp fragment from the human heat shock protein 70B (HSP 70) promoter. The vector has a large multiple cloning site and the neomycin selectable marker, making it more user-friendly. Using the human Interleukin-2 (IL-2) gene as a marker, it was demonstrated that heat can induce the pHOT-IL-2 plasmid to express 2 fold more IL-2 than levels obtained with the human cytomegalovirus (CMV) promoter. Using the Enhanced Green Fluorescence Protein (EGFP) gene as a reporter gene, a human breast carcinoma cell line (MCF-7) was transfected with pHOT-EGFP and stably transfected cells selected with neomycin. The stable transfectants were subjected to three different experimental conditions; heat treatment at 42Â°C for one hour, treatment with geldanamycin at 1Âľg/ml (an anti-leukemic drug) or 3000 rads of !-radiation. EGFP expression was measured for up to 72 hours by flow cytometry. The non-treated cells expressed a basal level of EGFP that increased 387% above background at 24 hours after heat-shock. Cells treated with Geldanamycin had a 208% increase in EGFP intensity at 24 hours which was maintained up to 72 hours as compared to the non-treated cells. Exposure of cells to 3000 rads of !-radiation had a 150% increase in EGFP expression at 48 hours post-treatment as compared to the non-treated cells. Induction of heat shock proteins by heat, radiation and geldanamycin was confirmed by Western blot analysis. This inducible gene expression system may be applicable to clinical use in synergy with other types of standard therapy (e.g, hyperthermia, radiation and chemotherapy).

cytomegalovirus and Rous sarcoma virus). An alternative method is the use of expression vectors that can be induced to express therapeutic genes by one or more of the aforementioned conventional therapies. Thus, vectors with inducible gene expression could be advantageous in some gene therapy protocols. The human HSP70B promoter is a well characterized promoter (Schiller et al, 1988) known to be induced by a family of heat shock factors that respond to diverse forms of physiological and environmental stress including high temperatures, heavy metals, oxidative stress and antiinflammatory drugs (Morimoto et al, 1997). The HSP70B

I. Introduction The major therapies currently used to treat cancer include radiotherapy, chemotherapy, hyperthermia and immunotherapy. Gene therapy is emerging as a new treatment for cancer, with encouraging clinical results. With the emerging view of combinatorial therapy as an approach to cancer treatment (Feyerabend et al, 1997; Otte, 1988), there is a need to integrate gene therapy with conventional therapies. Most gene therapy approaches have utilized constitutive expression of therapeutic genes (e.g. co-receptors such as B7.1 and B7.2, cytokines such as IL-2 and GM-CSF) by viral promoters (e.g,

Vasanawala et al: A novel, inducible expression vector

promoter contains heat shock regulatory sequences that bind to the heat shock transcription factor, thus "turning on" any gene downstream of the promoter. The HSP70B promoter has previously been incorporated in heterologous systems to express foreign genes in an inducible manner (Dreano et al, 1986). These vectors are difficult to use due to their large size (>10kb), small multiple cloning sites and lack of a selectable marker, such as neomycin. In the present study a new user-friendly expression vector was constructed using a fragment of the HSP70B promoter and was tested for inducible gene expression. This promoter was compared to the CMV promoter, which has been shown in several studies to be one of the strongest promoters available (Boshart et al, 1985). Results showed that when heat-induced, the heat shock promoter fragment was twice as strong as the CMV promoter. Our results also demonstrated that this expression vector was inducible by !-radiation and the anti-leukemic drug, geldanamycin (a benzoquinoid ansamycin antibiotic and a potent inhibitor of a protein kinase (Uehora et al, 1986) known to induce heat shock proteins (Conde eet al, 1997)). The enhanced green florescence protein (EGFP) was used as a reporter gene to detect induction of the expression vector by the different treatments.

Figure 1: Map of the HOT-MCS expression plasmid. The plasmid was generated by replacing the CMV promoter of the pcDNA3 expression plasmid with a 400 bp BamH1/Hind III fragment of the human HSP70B promoter derived from the p173OR plasmid.

II. Results A. Construction of vectors The HOT-MCS vector (Figure 1) was generated by replacing the cytomegalovirus (CMV) promoter of pcDNA3 (Invitrogen, San Diego, CA) by a BamHI- Hind III fragment of the human HSP70B promoter from the p173OR plasmid (StressGen, Victoria, BC). The fulllength HSP70B promoter is 2.3 kilo base pairs (kbp) in size, while the BamHI-HindIII fragment of the promoter is 451 bp in size. This adaptation resulted in a smaller vector, which was advantageous for further cloning. The pHOTMCS vector also has a neomycin selectable marker that can be useful for in vitro research work. The pHOT-MCS vector has a large multiple cloning site which facilitates the cloning of genes into the vector. Thus, this newly constructed pHOT-MCS vector has many advantages over the p173OR plasmid.

Percent "-gal positive cells at: PLASMID p173OR pHOT-"-gal

37째C 0% 0%

42째C 27% 23%

Table 1: Inducible gene expression by a fragment of the human HSP promoter. SW480 human colon carcinoma cells were transfected with either the HOT-"-gal or the p173OR expression plasmid using the lipid DMRIE/DOPE as described in Materials and Methods. Transiently transfected cells were exposed for 1 hour to either 37째C or 42째C as described. Fortyeight hours after transfection the cells were harvested and stained for "-gal using standard staining procedures (Invitrogen, San Diego, CA). Data are expressed as the percentage of cells staining positive for each treatment.

B. Characterization and comparison of the inducible promoter expression vector The pHOT-MCS plasmid containing a betagalactosidase reporter gene ("-gal) was evaluated for its ability to be induced by heat shock treatment. SW480 cells were transfected with the plasmid by the lipofection technique as described in Materials and Methods.

Gene Therapy and Molecular Biology Vol 5, page 3

Figure 2: Promoter strength comparison by IL-2 production. MCF-7 cells were plated overnight at a density of 0.5x106 cells per well in a six well plate. The cells were transfected with the HOT-IL-2 plasmid using the lipid Novafector. The cells were heated to 42°C for 60 min and returned to 37°C. Twenty-four hours later the supernatants were harvested and analyzed by ELISA for IL-2 levels. Results are the mean of triplicate experiments.

Figure 3: Increase in EGFP expression. Mean fluorescence channel (MFC) of EGFP expression following treatment with heat, radiation or chemotherapy. MCF-7 cells were treated as described in Materials and Methods, harvested at the indicated time points and analyzed by flow cytometry for EGFP intensity.

C. Induction of the transgene by heat shock, !-radiation and geldanamycin

Twenty-four hours after transfection the cells were heated to 42°C for one hour. Twenty-four hours after heat shock treatment the cells were stained for "-gal gene expression. The p173OR plasmid expressing "-gal under the control of the full-length 2.3kb HSP promoter was used as a positive control. The analyses (Table 1) revealed that "gal gene expression was not induced at 37°C, whereas the pHOT- "-gal plasmid transfected cells stained positive 24 hours after heat treatment. Results were comparable with both vectors. These results confirmed that the 451 bp BamHI /Hind III fragment of HSP70B promoter maintained it's ability to be induced by heat shock. The promoter strength of the inducible expression vector was also tested against the CMV promoter. The IL2 gene was cloned into the EcoRI site of the multiple cloning site of the pHOT-MCS vector. The HOT-IL-2 plasmid and the pcDNA3-IL-2 plasmid were transfected into MCF-7 cells with the lipid Novafector. Twenty-four hours after transfection the cells were heated and 24 hours after heat treatment supernatants were collected and assayed for IL-2. Results (Figure 2) showed that the pHOT-MCS vector promoter was twice as strong as the CMV promoter when induced by heat treatment (67 units v/s 31 units of IL-2).

Due to the difficulty in quantitating and comparing "-gal assays and the expense of IL-2 assays, the enhanced green fluorescent protein (EGFP) reporter gene was cloned into the HOT-MCS plasmid yielding the HOTEGFP plasmid. This plasmid can easily be detected by flow cytometry and was used for further experiments. MCF-7 cells were transfected with HOT-EGFP by the calcium phosphate method and stable transfectants were selected and maintained with the antibiotic G418. Stable, as compared to transient, transfections were chosen in order to eliminate differences in transfection efficiencies between experiments thereby allowing direct comparisons of increases in gene expression. Stably transfected MCF-7 cells were plated overnight into 35mm, 6 well tissue culture plates at a density of 1x106 cells/plate. The next day adherent cells were treated with either heat (42°C, 1hour), !-radiation (3000 rads, a previously determined optimal level) or geldanamycin (1µg/ml). Synergy of gene induction was also analyzed for by using !-radiation and geldanamycin treatments together. Four, 24, 48, and 72 hours after treatment the cells were harvested and assayed for EGFP expression by flow cytometry. All transfected cells displayed a basal level of EGFP expression. Thus, results from the cell treatments are shown as the mean florescence channel (Figure 3) and as the percent increase in EGFP intensity

Vasanawala et al: A novel, inducible expression vector

(Table 2) over the basal level. Flow cytometric analyses indicated that heat shock treatment induced the highest level of gene expression. As early as 4 hours posttreatment there was a 243% increase in EGFP expression above the basal level. Maximal EGFP expression was observed at 24 hours after heat treatment (387% increase above background) after which it started to decline. Geldanamycin was also observed to induce the HSP promoter, with EGFP intensity increasing to 208% above background at 24 hours post-treatment. !-radiation also induced the HSP promoter, although to a weaker extent than geldanamycin treatment. The highest level of gene expression was seen at 48 hours post-radiation treatment, with an increase of 150% over background levels. Finally, a combination of geldanamycin and radiation treatments together was not observed to increase gene expression (i.e. act synergistically) above that seen with either treatment alone (data not shown). These results were significant in that it demonstrated that the HOT-EGFP vector could be induced not only by heat shock, but also by !-radiation and chemotherapy.

HSP72/73 protein expression at 4 hours post-treatment, but the levels were not significant at the later timepoints.

III. Discussion We have constructed a novel, user-friendly inducible expression vector, that possesses a large multiple cloning site and the neomycin gene as a selectable marker. This vector has the useful property of being inducible by heat, chemotherapy, and radiation. Using pcDNA3 as the backbone plasmid, pHOT-MCS was derived by replacing the CMV promoter with a heat inducible promoter, the human HSP70B promoter. As the size of a plasmid may affect transfection efficiency, such that smaller plasmids transfect with a higher efficiency than larger ones, size was an important factor in our plasmid design. Thus, a 451 bp fragment of the human HSP70B promoter was used rather than the entire 2.3kbp promoter. From previously published work (Schiller et al, 1988), the 451 bp fragment was expected to be as heat inducible as the parental promoter since it contains the heat shock element (HSE) sequences and the TATA box. Results using "-gal as the reporter gene indicated that the 451bp fragment of the human HSP70B promoter was indeed sufficient for heat inducible gene expression. Currently, the most commonly used promoter is the CMV promoter and it was imperative to compare the pHOTMCS plasmid with this promoter. The results showed that the 451 bp fragment of the human HSP70B promoter upon treatment with heat was twice as strong as the CMV promoter. Thus, promoter strength was not compromised in the construction of the HOT-MCS plasmid. In further experiments, EGFP was used as the reporter gene rather than "-gal due to the ease of assaying EGFP gene expression by flow cytometry. Thus, the EGFP gene was cloned into the HOT-MCS plasmid, yielding the HOT-EGFP plasmid. The reporter gene was easily detected by flow cytometry (Figure 3).

D. Analysis of heat shock proteins by western blots As the time course for induction of EGFP expression by both geldanamycin and !-radiation treatments were different from that of heat-shock treatment, Western blots were performed on the treated cells at these time points to quantitate HSP72/73 production. As each of the treatments theoretically induced EGFP expression via the HSP promoter, the treatments should have induced an increase in cellular heat shock proteins. Western blot analyses (Figure 4) for HSP72/73 expression indicated that all three treatments (heat, radiation and geldanamycin) induced the HSP 72/73 proteins. The highest level of HSP 72/73 production was induced by heat at 4 hours posttreatment. However, at 24 hours post-treatment and thereafter, treatment with geldanamycin induced higher levels of HSP72/73 expression. !-radiation also induced

4 hours

24 hours

48 hours

72 hours

Heat

243

387

376

327

Geldanamycin

120

208

201

Radiation

113

138

150

154

Table 2: Percent Increase in EGFP Intensity. Mean Fluorescence Channel units from Figure 3 were converted into percent intensity increases above background. An increase in 75 units of MFC = a doubling in intensity. The above formula was used to calculate the percent increase in EGFP intensity over background, which was considered to be 100%.

Gene Therapy and Molecular Biology Vol 5, page 5

Figure 4. HSP72/72 protein expression after treatment with either heat, radiation or chemotherapy. MCF-7 cells were treated as described in Figure 2 and heat shock protein expression quantitated by western blotting and densitometry at the indicated time points. Data are presented optical density for each measurement.

and !-radiation. As these various treatments turned on the reporter gene, each of the treatments must have induced heat shock proteins in the cell since the heat shock promoter was activated. Thus, western blots detecting heat shock proteins were performed to possibly elaborate the mechanisms of gene induction. The western blot experiments indicated that heat shock increased HSP72/73 production at 4 hours and a similar effect was also seen with geldanamycin treatment. This increase in HSP72/73 levels was however, not reflected in an increase in EGFP intensity with geldanamycin treatment. Thus, it can be concluded that different mechanisms were affecting gene expression, which needs to be further characterized. In conclusion, we have constructed a vector that opens up the possibility of a combinatorial type of therapy using gene therapy and chemotherapy, hyperthermia or radiation. Synergistic effects between these therapies may be more beneficial than any one therapy alone. Further, it may be possible to utilize lower doses of chemotherapy or radiation in combination with the above gene therapy vector expressing a biologically active gene (e.g, HSV-tk or cytokines) to achieve less toxic clinical results.

MCF-7 cells stably transfected with HOT-EGFP were used instead of transient transfections to eliminate differences in transfection efficiencies between experiments. Stably transfected cells allowed for direct comparisons in increased gene expression by the different experimental treatments. Since stable transfectants were used, EGFP gene expression was also observed in nontreated (but transfected) cells. This background expression was considered to be the background level and increases in gene expression were calculated as the fold-increase over the basal level. It was a novel finding that the HSP70B promoter fragment was not only inducible by heat but also by !radiation and geldanamycin. These treatments represent radiation therapy and chemotherapy, and thus present an opportunity to combine gene therapy with existing cancer therapies such as hyperthermia therapy, chemotherapy and radiation therapy. Although, the fluorescence intensities with geldanamycin and !-radiation werenâ&#x20AC;&#x2122;t equivalent to that observed with heat shock, the levels were still significantly increased above basal levels. There was a difference in the induction of gene expression between the three treatments. Heat shock induced gene expression rapidly by 4 hours, while maximal gene induction was seen at 24 hours with geldanamycin and !-radiation treatments. Thus, different mechanisms of heat induction occurred with geldanamycin

IV. Materials and Methods A. Vector construction Three reporter constructs were made from pHOT-MCS (see results for further details). One construct contained beta-

Vasanawala et al: A novel, inducible expression vector

levels using MEDGENIX IL-2 EASIA Kit (Biosource Europa, Belgium).

galactosidase ("-gal) as the reporter gene (from the pCMVB plasmid, Clontech, Palo Alto, CA) cloned into the Not I site of pHOT-MCS. For the IL-2 construct, the human IL-2 gene (a gift from Dr. Evan Hersh, University of Arizona) was first adapted for EcoR1 site. Briefly, a 0.5kb BamHI-PstI DNA fragment containing the IL-2 gene was inserted into the Sac-KiSS-# (Tsang et al, 1996) following a complete digestion with BamHI and a partial digestion with PstI to create the plasmid pSac-KiSSIL-2. The IL-2 gene was then excised from the pSac-KiSS-IL-2 as an EcoRI fragment and inserted into the EcoRI site of pHOTMCS to generate pHOT-IL-2. The third reporter construct contained EGFP (Clontech, Palo Alto, CA) as a reporter gene, which was inserted into the Kpn I – Not I multiple cloning site of pHOT-MCS.

E. Flow cytometry Flow cytometric analysis was performed using a FACStar flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA). Data was acquired utilizing a COHERENT (Palo Alto, CA) 90-5 5W argon ion water-cooled laser tuned to 488 nm at 100mW power for excitation. Emitted fluorescence was collected with a standard 530/30 band pass filter. A minimum of 10,000 events were collected in a 'live' gate. Data was acquired and analyzed on an HP340 computer with Lysys II (Becton Dickinson Immunocytometry Systems Mountain View, CA) software. Data was collected as mean florescence channel of EGFP. An increase in 75 units of mean florescence channel indicates a doubling in fluorescence intensity (as per personal communications from Becton Dickinson, according to the formula: PLUS

B. Cell lines and transfections MCF-7, a human breast carcinoma cell line, was transfected with the pHOT-EGFP vector by standard calcium phosphate methodologies (Sambrook et al, 1989). Stable transfectants were obtained by selection with G418 (400 µg/ml) and were maintained in complete RPMI medium, (GIBCO, Gaithersburg, MD) supplemented with 10% fetal bovine serum (JRH Biosciences, Lenaxa, KS). The cells were maintained at 37°C in 5% CO2. For IL-2 studies, MCF-7 cells were seeded in 35mm plates (Falcon, Franklin Lakes, NJ) at 0.5x106 cells/plate. Cells were transfected with the plasmids using the lipid Novafector (VennNova, Pompano Beach, FL) at a ratio of 1µg DNA to 4 µl of lipid for 6 hours. SW480, a human colon carcinoma cell line, was transfected using DMRIE-DOPE (Vical Inc, San Diego, CA) (Parker et al, 1996).

channel value x = 10 channel per decade where x= log channel Data is represented as the percent increase in EGFP intensity over background intensity.

F. Western blots Western immunoblots were performed to estimate production of heat shock proteins. Cells to be assayed were washed with phosphate buffered saline and resuspended in cell lysis buffer. Total protein was estimated by the BCA protein assay (Pierce, Rockford, IL). Protein samples (30mg each) were fractionated for Western blot analysis by separation on denaturing SDS-PAGE and transferred onto nitrocellulose filters. Filters were blocked by soaking the membrane in buffer containing 3% milk in TTBS (Tris-buffered saline containing 1% Tween-20) to minimize non-specific binding. After three washes in TTBS the membranes were incubated with anti-HSP 72/73 antibody (StressGen, Victoria, BC). Goat anti-mouse IgG-horse radish peroxidase (Pierce, Rockford, IL) was used as a secondary/developing antibody. Both incubations were performed at room temperature for 1 hour. The membrane was washed three times with TTBS and incubated in substrate reagent containing peroxide for 5 min. Heat shock proteins were detected by exposure to ELC hyperfilm and developed for autoradiogram. Heat shock protein expression was quantitated by scanning densitometry.

For "-gal studies, SW480 cells were plated out at 4x105 cells/well in a six well tissue culture plate (Falcon, Franklin Lakes, NJ). A lipid to DNA ratio of 4:1 was used for transfections. Transfections were performed in reduced serum media OPTI-MEM (GIBCO, Gaithersburg, MD). Four hours after transfections, 0.5 ml of 30% FBS in OPTI-MEM was added to each well. The next day an additional 1ml of 10% FBS in OPTI-MEM was added. Forty-eight hours after transfection the cells were trypsinized and stained for "-gal using the Invitrogen "-gal staining kit (Invitrogen, Carlsbad, CA).

C. Cell treatments Transfected cells were plated overnight in a 35mm2 tissue culture dish (Falcon, Franklin Lakes, NJ) at a density of 1x106 cells in 5ml of RPMI medium. The following day the cells were treated as follows. Heat shock treatment was performed by sealing the plate with parafilm and immersing it in a 42°C water bath for 60 min. Geldanamycin (Sigma, St. Louis, MO) was added to the cell cultures at a concentration of 1µg/ml. !radiation treatment was performed using a 60Co !- irradiation unit. The cells were exposed to a total of 3000 rads (225 rads/min) of radiation in a single dose. All treated cells were trypsinized and assayed by flow cytometry for EGFP expression at 4, 24, 48 and 72 hours post-treatment.

Acknowledgements The authors would like to thank Dr. Ashok Gupta for his technical assistance. The authors would also like to thank Barb Carolus for all her help with flow cytometry. For plasmid requests please contact Dr. David Harris at davidh@u.arizona.edu or write to Dr. David T. Harris. Dept. of Microbiology and Immunology, Building # 90, University of Arizona, Tucson, AZ 85721.

D. IL-2 assay Cell supernatants were harvested 24 hours after heat treatments. The supernatants were assayed by ELISA for IL-2

Gene Therapy and Molecular Biology Vol 5, page 7

benzoquinonoid ansamycins accompanies inactivation of p60src in rat kidney cells infected with Rous sarcoma virus. Mol Cell Biol 6, 2198-2205.

References: Boshart M, Weber F, Jahn G, Dorsch-Hasler K, Fleckenstein B. and Schaffner W. (1985) A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell 41, 521-530. Conde AG, Lau SS, Dillman WH, Mestril R, (1997) Induction of heat shock proteins by tyrosine kinase inhibitors in rat cardiomyocytes and myogenic cells confers protection against simulated ischemia. J Mol Cell Cardiol 26, 19271935. Dreano M, Brochot J, Myers A, Cheng-Meyer C, Rungger D, Voellmy R, and Broomley P, (1986) High-level, heatregulated synthesis of proteins in eukaryotic cells. Gene 49, 1-20. Feyerabend T, Steeves, R, Wiedmann G. J, Richter, E. and Robins, H. I. (1997) Rationale and clinical status of local hyperthermia, radiation, and chemotheapy in locally advanced malignancies. Anticancer Res 17, 2895-2900. Morimoto R. I, Kline, M. P, Bimston, D. N, Cotto, J. J. (1997) The heat-shock response: regulation and function of heatshock proteins and molecular chaperones. Essays Biochem. 32, 17-30. Otte J, (1988) Hyperthermia in cancer therpay. Eur J Pediatr 147, 560-565. Parker SE, Khatibi S, Margalith M, Anderson D, Yankauckas M. Gromkowski SH, Latimer T, Lew D, Marquet M, Manthorpe M, Hobart P, Hersh E, Stopeck AT. and Norman J, (1996) Plasmid DNA gene therapy: studies with the human interleukin-2 gene in tumor cells in vitro and in the murine B16 melanoma model in vivo. Cancer Gene Ther. 3, 175185. Sambrook J, Fritsch, EF, and Maniatis T, (1989) Molecular Cloning, A laboratory manual Volume 3, 16.32. Schiller PJ. Amin J. Ananthan ME. Brown WA. Scott and R Voellmy, (1988) Cis-acting elements involved in the regulated expression of a human HSP70 gene. J Mol Biol 203, 97. Tsang TC, Harris DT, Akporiaye ET, Schluter SF, Bowden GT, and Hersh EM, (1996) Biotechniques 20:51-53. Uehora Y, Hori M, Takeuchi T, and Umezawa H, (1986) Phenotypic change from transformed to normal induced by

Vasanawala et al: A novel, inducible expression vector

(From left): Jean Boyer, David Harris, Tom Tsang and Farha Vasanawala (Crete 1998)

Gene Therapy and Molecular Biology Vol 5, page 9

Gene Ther Mol Biol Vol 5, 9-30, 2000

Misregulation of pre-mRNA splicing that causes human diseases. Concepts and therapeutic strategies Review Article

Oliver Stoss1, Peter Stoilov1, Rosette Daoud, Annette M. Hartmann, Manuela Olbrich, and Stefan Stamm* Max-Planck Institute of Neurobiology, Am Klopferspitz 18a, D-82152 Martinsried, Germany

__________________________________________________________________________________ *Correspondence: Stefan Stamm, Ph.D., Phone: +49 89 8578 3625, Fax: +49 89 8578 3749, E-mail: stefan@stamms-lab.net Key words: alternative splicing, kinases, tau, FTDP-17, signal transduction 1 authors contributed equally to the work Received: 27 August 2000; accepted: 4 October 2000

Summary About one third of all human genes are subject to alternative splicing. The molecular mechanisms that regulate alternative splice site usage are beginning to emerge and show that transcription and pre-mRNA processing are integrated processes that can be modified by cellular signals. Several diseases are caused by mutations in sequences that regulate pre-mRNA processing. Their molecular characterization indicates that contributions of pre-mRNA splicing defects to human diseases have been underestimated and could account for pleiotropic phenotypes. The understanding o f the molecular mechanisms allows the development of therapeutic strategies. PKG-I: protein kinase G-I; p o l I I : polymerase II; PTP-1B: protein tyrosine phosphatase 1B; S A M 6 8 : src associated in mitosis; SELEX: systematic evolution of ligands by exponential enrichment; snRNP: small nuclear riboprotein; SR: protein: protein with serine-arginine-rich domain; SRPK: SR protein kinase; STAR: signal transduction and activation of RNA; TCR : T cell receptor; TNF : tumor necrosis factor; U2AF: U2 auxiliary factor; UTR : untranslated region.

A b b r e v i a t i o n s : ACTH : adrenocorticotrophic hormone; CFTR: cystic fibrosis transmembrane conductance regulator; CKI : casein kinase I; C l k : Cdc2-like kinase; ConA: Concanavalin A; EGF: epidermal growth factor; ESE : exonic splicing enhancer; ESS: exonic splicing silencer; FGF: fibroblast growth factor; hnRNP: heterogeneous nuclear protein; IFN: interferon; IL: interleukin; ISE: intronic splicing enhancer; ISS: intronic splicing silencer; MKK: mitogen-activated protein kinase kinase; NGF: nerve growth factor; PDGF: platelet-derived growth factor; PI3-K: Phosphatidylinositide-3-kinase; PKC: protein kinase C;

splicing (Mironov et al, 1999; Brett et al, 2000). Alternative splicing is a mechanism where parts of the premRNA are either excluded from, or included in the mature mRNA. This process can be regulated in a cell-type or developmental-specific way (Stamm et al, 1994, 2000), that can be used to regulate gene expression at the level of pre-mRNA processing. Furthermore, it allows different protein isoforms to be created from a single gene. In many cases, stop codons are introduced by alternative splicing (Stamm et al, 1994, 2000), which usually changes the carboxy terminus of proteins. This can affect the physiological function of a protein, as shown by several examples:

I. Introduction A. Splicing machinery

and

basal

splicing

In eukaryotes, the primary transcript generated by polymerase II undergoes an extensive maturation process that involves capping, polyadenylation, editing, and premRNA splicing. Pre-mRNA splicing removes intervening sequences (introns) and joins the remaining sequences (exons) to form mature mRNA that is finally exported into the cytosol. With a few exceptions, all human polII transcripts are spliced, and it is estimated that about 30% of all pre-mRNA transcripts are subject to alternative

Stoss et al: RNA splicing and disease

splice sites adhere better to a consensus than others (Breitbart et al, 1987; Stamm et al, 1994; Stamm et al, 2000). Interestingly, a new class of exons has been discovered that uses AT and AC instead of the GT/AG flanking nucleotides (Tarn and Steitz, 1997). Due to the degenerate nature of splice sites, it is difficult to predict exons in genomic sequences, and current computer programs cannot accurately predict exons from genomic DNA (Thanaraj, 2000). This contrasts the high accuracy and fidelity characteristic for splice-site selection in vivo. One reason for the specificity observed in vivo are additional regulatory elements known as exonic or intronic enhancers or silencers (Figure 1 , Table 1). These elements are again characterized by loose consensus sequences. The enhancers can be subdivided into purinerich (GAR-type), pyrimidine-rich and AC-rich (ACE) enhancers (Cooper and Mattox, 1997). Enhancers bind to proteins that are able to recruit components of the basal splicing machinery, which results in recognition of splicesites located near an enhancer (Hertel et al, 1997). The degeneracy of splicing enhancers is most likely necessary to allow for the amino acid usage needed. The importance of splice-site enhancers becomes apparent when they are changed by mutation, which can alter their interaction with trans-acting factors. Interestingly, some of these mutations are silent, e.g. they do not change the amino acid usage, but generate an aberrant gene product by causing an abnormal splicing product. It remains to be determined whether all missense mutations cause a pathological state by an amino acid exchange or are actually unrecognized splicing mutations. Such an analysis could be made by analyzing the premRNA processing of the mutation by RT-PCR or RNAse protection. An overview of diseases caused by splicing enhancers/silencers is shown in Table 2. Proteins binding to enhancer or silencer sequences can be subdivided into two major groups: members of the SR family of proteins (Manley and Tacke, 1996) and hnRNPs (Weighardt et al, 1996). Binding of individual proteins to enhancer sequences is intrinsically weak and not highly specific. However, multiple proteins bind to all known exon enhancers forming a complicated RNA:protein complex. This binding involves protein:protein as well as protein:RNA interactions, and results in the specific recognition of an exon (Figure 1). As a result, proper splice-site recognition is governed by the ratio of various proteins involved, as well as the enhancer and silencer sequences.

(i ) creation of soluble instead of membrane-bound receptors (Baumbach et al, 1989; Eipper et al, 1992; Toksoz et al, 1992; Zhang et al, 1994; Hughes and Crispe, 1995; Tabiti et al, 1996); (i i ) altered ligand affinity (Sugimoto et al, 1993; Xing et al, 1994; Suzuki et al, 1995); (i i i ) protein truncations producing inactive variants (Swaroop et al, 1992; van der Logt et al, 1992; Duncan et al, 1995; Sharma et al, 1995; Eissa et al, 1996); and (i v ) changes of endocytotic pathways (Wang and Ross, 1995). In addition, inclusion or skipping of alternative exons can (v) add or delete protein modules that change the affinity towards ligands (Danoff et al, 1991; Giros et al, 1991; Guiramand et al, 1995; Strohmaier et al, 1996); (v i ) modulate enzymatic activity (O'Malley et al, 1995); (v i i ) create different hormones (Amara et al, 1982; Courty et al, 1995); and (v i i i ) change properties of ion channels (Sommer et al, 1990; Kuhse et al, 1991; Xie and McCobb, 1998). Finally, (i x ) numerous transcription factors are subject to alternative splicing, which contributes to control of gene expression (reviewed in Lopez, 1995). A recent compilation and statistical analysis of alternative exons (Stamm et al, 2000) is available on the world wide web under www.stamms-lab.net. Proper splicing regulation is important for an organism, since it has been estimated that up to 15% of genetic defects caused by point mutations in humans manifest themselves as pre-mRNA splicing defects caused by changing splice site sequences (Krawczak et al, 1992; Nakai and Sakamoto, 1994). These mutations can be viewed as new sources of variation in human evolution that was probably accelerated by alternative splicing mechanisms, allowing the combination of different RNA processing events to generate appropriate mRNAs as a result of changing cellular needs (Herbert and Rich, 1999). Significant progress has been made in understanding the mechanism of constitutive splicing. Three major ciselements on the RNA define an exon, the 5' and 3' splice sites and the branch point (Berget, 1995). These elements are recognized by the spliceosome, a 60S complex containing small nuclear RNAs (U1, U2, U4, U5, U6) and over 50 different proteins (Neubauer et al, 1998). In the spliceosome, U1 snRNP, U2AF, SF1, U6 snRNP, and U2 snRNP are the trans-acting factors that ultimately recognize the 5', 3' splice sites and the branch point (reviewed in Green, 1991; Kr채mer, 1996; Elliot, 2000; Moore, 2000). Sequence compilations of the 5', 3' splice sites and the branch point revealed that they follow only a loose consensus sequence (Breitbart et al, 1987). Only the GT and AG nucleotides directly flanking the exon, together with the branch point adenosine (Figure 1A), are always conserved, whereas in all other positions nucleotides can deviate from the consensus. However, some positions in

B. Splice-site recognition is influenced by the relative concentration of proteins that form a complex to recognize exon-intron borders In contrast to constitutive splicing, the mechanisms regulating alternative exon usage are less well understood. It is clear that the relative concentration of splicingassociated proteins is responsible for alternative splice-site

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Figure 1: cis and trans factors involved in pre-mRNA splicing. A) E l e m e n t s i n v o l v e d i n a l t e r n a t i v e s p l i c i n g o f p r e - m R N A . Exons are indicated as boxes, introns as thin lines. Splicing regulator elements (enhancers or silencers) are shown as gray boxes in exons or as thin boxes in introns. The 5' splicesite (CAGguaagu) and 3' splice-site (y)10 ncagG, as well as the branch point (ynyyray), are indicated (y=c or u, n=a, g, c or u). Upper-case letters refer to nucleotides that remain in the mature mRNA. Two major groups of proteins, hnRNPs (yellow) and SR or SR related proteins (orange), bind to splicing regulator elements; the protein:RNA interaction is shown in green. This protein complex assembling around an exon enhancer stabilizes binding of the U1 snRNP close to the 5' splice-site, due to protein:protein interaction between an SR protein and the RS domain of U170K (shown in red). This allows hybridization (thick red line with stripes) of the U1 snRNA (red) with the 5' splice-site. The formation of the multi-protein:RNA complex allows discrimination between proper splice-site (bold letters) and cryptic splice-sites (small gt ag) that are frequent in pre-mRNA sequences. Factors at the 3' splice-site include U2AF which recognizes pyrimidine rich regions of the 3' splice-sites, and is antagonized by binding of several hnRNPs (e.g hnRNP I) to elements of the 3' splice-site. orange: SR and SR related proteins; yellow: hnRNPs; green: protein:RNA interaction; red: protein:protein interaction; thick red line with stripes: RNA:RNA interaction B) The RNA factory. RNA is generated after genes are recognized by transcription factors (TF) by RNA polymerase II (polII) (dark blue). Exons present on the RNA are recognized by SR proteins and hnRNPs that interact with exonic or intronic sequence elements. SR proteins interact with factors assembled around the promoter and can form protein networks across exons. SR proteins directly interact with the carboxy terminal domain of RNA polII (polII-CTD), which assembles proteins near active sites of transcription. Among polII interacting proteins is scaffold attachment factor B (SAF-B) that can couple SR proteins and RNA polII to chromatin organizing elements (S/MAR, thick green line). The processed RNA is coated with hnRNPs and transported into the cytoplasm, where it is translated into protein. SR proteins and hnRNPs are recruited from storage sites (speckles) through phosphorylation. Some SR proteins and hnRNPs shuttle between nucleus and cytoplasm. Protein shuttling can be regulated by phosphorylation or arginine methylation.

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Table 1. Compilation of RNA elements that influence splice-site selection (previous two pages) The first column shows the gene and exon that contains the RNA element, which is characterized in the next columns according to its type (ESE: exonic sequence element; ISE: intronic sequence element) and sequence. Trans-acting factors are indicated in bold under the sequence if they were identified experimentally. Most RNA elements will work in combination with additional RNA regulatory sequences that are not shown. Meth. indicates the experimental method used: 1: deletion analysis; 2: in vivo splicing assay; 3: in vitro splicing assay; 4: gel mobility shifts; 5: mutagenisis; 6: in vitro binding; 7: UV-crosslink; 8: competition experiments; 9: SELEX; 10: immunoprecipitation; 11: spliceosomal complex formation; 12: nuclease protection R= G or A; W=A or U.

Stoss et al: RNA splicing and disease

in pre-mRNA processing (Du and Warren, 1996; Kim et al, 1996; Yuryev et al, 1996; Bourquin et al, 1997; Corden and Patturajan, 1997). Furthermore, these complexes are most likely associated with S/MAR elements (Bode et al, 2000) via SAF-B, an hnRNP-like protein that can link factors involved in pre-mRNA processing, and the CTD to chromatin-organizing elements (Nayler et al, 1998c). Finally, it was shown directly that RNA polymerase II can stimulate splicing reaction in vivo (Hirose et al, 1999) and targets splicing factors to sites of active transcription (Misteli and Spector, 1999). These close interactions most likely influence splice-site usage and it has been shown that the particular promoter usage influences splice-site selection (Cramer et al, 1999). Together, these data indicate that pre-mRNA is generated and processed by a large complex that was termed 'RNA factory' (McCracken et al, 1997). It is most likely that 5' capping (Cho et al, 1997), polyadenylation (McCracken et al, 1997), and editing (Higuchi et al, 2000) activities are also part of this complex. When cells are stained with antibodies against splicing factors, the proteins are concentrated in 20-40 large nuclear structures that are called speckles. Using imaging techniques, it has been shown that splicing and transcription take place concomitantly in the vicinity of speckles (Jiménez-Garcia and Spector, 1993; Huang and Spector, 1996). Within speckles, no transcriptional activity could be detected (Fay et al, 1997), indicating that these structures serve as storage particles. Speckles are dynamic (Misteli et al, 1997) structures that can release splicing components when these are phosphorylated (Nayler et al, 1998b). A new subnuclear structure, the YT bodies, was discovered that forms around speckles and often partially overlaps with them (Nayler et al, 2000). YT bodies contain YT521-B, a protein that binds to factors implicated in pre-mRNA processing and is subject to tyrosine phosphorylation through src kinases (Hartmann et al, 1999). YT bodies change in response to the tyrosine phosphorylation status of the cell (Nayler et al, 2000 and our unpublished results) and harbor sites of transcription (Nayler et al, 2000), suggesting that the RNA factory can be modulated by tyrosine phosphorylation in YT bodies. Finally, proteins implicated in splicing shuttle between the nucleus and the cytosol. After a stress-induced change of the cellular phosphorylation status they accumulate in the cytosol (van Oordt et al, 2000), which affects premRNA processing patterns in the nucleus, because the nuclear concentration of the splicing factors is changed. Together, these data suggest that the concentration of factors involved in splice-site selection, which dictates exon usage, can be controlled by several ways: (i) a specific amount of the factor expressed in a tissue, (ii) release from storage sites by phosphorylation, (iii) export from the nucleus, (iv) sequestration by protein-binding partners that, e.g., assemble at an active promoter, and (v) local concentration at different sites of the nucleus. Together, these mechanisms allow the cell to process a given pre-mRNA with a specific set of splicing regulatory proteins, such as SR proteins and hnRNPs.

selection (Black, 1995; Grabowski, 1998; Varani and Nagai, 1998) . It has been shown experimentally, that the relative concentration of SR proteins and hnRNPs can dictate splice- site selection, both in vivo and in vitro (Mayeda and Krainer, 1992; Cáceres et al, 1994; Screaton et al, 1995; Wang and Manley, 1995). The expression levels of various SR proteins (Ayane et al, 1991; Mayeda and Krainer, 1992; Zahler et al, 1993; Screaton et al, 1995) and hnRNPs (Kamma et al, 1995) vary amongst tissues and could therefore account for differences in splicesite selection. Several examples of antagonistic splicing factors have been described (Cáceres et al, 1994; Mayeda et al, 1993; Gallego et al, 1997; Jumaa and Nielsen, 1997; Polydorides et al, 2000). Here, one factor promotes inclusion of an exon and the other factor promotes its skipping. In most of these cases, it remains to be determined whether this antagonistic effect is achieved by (i ) an actual competition of the factors for an RNA binding site, (i i ) through sequestration of the factors by protein:protein interaction and, (i i i ) by changes in the composition of protein complexes recognizing the splicing enhancer.In addition, cell-type specific splicing factors have been detected. In Drosophila, for example, the expression of the SR protein transformer is female-specific (Boggs et al, 1987) and determines the sex by directing alternative splicing decisions. Other tissue-specific factors include the male germline specific transformer-2 variant in D. melanogaster (Mattox et al, 1990) and D. virilis (Chandler et al, 1997), an isoform of its mammalian homologue htra2-beta3 that is expressed only in some tissues (Nayler et al, 1998a), the muscle specific protein Nop30 (Stoss et al, 1999a) , the neuron-specific factor NOVA-1 (Jensen et al, 2000) as well as testis and brain enriched factor rSLM-2 (Stoss et al, submitted) and NSSR (Komatsu et al, 1999). For most of these factors, the tissue-specific target genes remain to be determined. However, a combination of knockout experiments and biochemical analysis allowed the identification of doublesex, fruitless, and transformer-2 as a target of the transformer-2/transformer complex in Drosophila ( Hoshijima et al, 1991; Mattox and Baker, 1991; Heinrichs et al, 1998) and glycine receptor alpha2 and GABA(A) pre-mRNA as a target for NOVA-1 (Jensen et al, 2000). Although this analysis is currently limited, it is likely that a given splicing factor will influence several pre-mRNAs.

C. Coupling transcription

splicing

and

Transcription and splicing can be separated by biochemical means. Especially when RNA splicing is studied in vitro, the two processes are uncoupled, since the RNA is made synthetically. However, when transcription and splicing factors were analyzed by microscopy methods (Misteli, 2000) and in yeast two-hybrid screens, an intimate association became apparent. A number of studies used the carboxyl terminal domain of the RNA polII and found several interacting proteins, some of which, e.g. snRNPs and SR like proteins, were most likely involved

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Table 2: Mutation in RNA regulatory elements that cause disease Mutations that disrupt RNA regulatory elements and cause a disease are listed. The name of the gene is under the disease and is underlined. Point mutations that change splice-sites (Krawczak et al, 1992; Nakai and Sakamoto, 1994) are not added to the table, if they are not part of exonic elements. ESE: exonic sequence element; ISE: intronic sequence element, IVS: intron.

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itself can be regulated by a membrane bound guanylyl cyclase receptor that is activated by natriuretic peptides or by a cytoplasmic guanylyl cyclase which is activated by nitric oxide (NO). Phosphorylation of SF1 on Ser20 inhibits the SF1-U2AF65 interaction, leading to a block of pre-spliceosome assembly.

II. Change of splice-site selection in response to an external stimulus A. Overview Alternative splicing pathways are not static, since the use of alternative exons can change during development (for a summary see: Stamm et al, 1994, 2000), or in response to outside stimuli. For example, insulin administration influences the incorporation of the alternative exon 11 of the insulin receptor (Sell et al, 1994) and activates exon ßII inclusion in the PKC gene (Chalfant et al, 1998); serum deprivation alters usage of the serine/arginine-rich protein 20 (SRp20) exon 4 (Jumaa and Nielsen, 1997); and neuronal activity changes the alternative splicing pattern of clathrin light chain B, the NMDAR1 receptor, and c-fos (Daoud et al, 1999). In the brain, stress changes splicing patterns of potassium channels (Xie and McCobb, 1998) and of acetylcholin esterase (Kaufer et al, 1998). ConA has been shown to change splicing patterns of the class 1b major histocompatibility complex molecule Qa-2 (Tabaczewski et al, 1994) and the splicing patterns of tumor necrosis factor ß are regulated by src kinases (Gondran and Dautry, 1999; Neel et al, 1995). Finally, programmed cell death is concomitant with a change in the alternative splicing patterns of several cell death regulatory proteins (reviewed in Jiang and Wu, 1999). As can be seen in Table 3, numerous extracellular stimuli, such as growth factors, cytokines, calcium concentration, and extracellular pH can change alternative exon usage. Since these alternative exons are in mRNAs of diverse biological functions, it is likely that the change of alternative splicing in response to an extracellular signal is a general regulatory mechanism in higher eukaryotes. Although for some cases the signal transduction pathways have been established, the molecular mechanism that transduces the signal to the spliceosome remains largely obscure. For several systems, it was demonstrated that changes in alternative splicing do not require de novo protein biosynthesis. Examples include the splicing of exon v5 of the CD44 gene in response to TPA (König et al, 1998) or the differential splicing of the Ca-ATPase transcript upon a rise in intracellular calcium (Zacharias and Strehler, 1996). It is likely that these changes in splicing patterns are the result of posttranscriptional modifications of regulatory proteins, e.g. phosphorylation, methylation, and glycosylation. However, it is largely unknown which factors are affected. In the following, we summarize several protein groups that are likely endpoints of signal transduction pathways in the spliceosome.

C. hnRNPA1 hnRNP A1 has also been implicated as a mediator of signal transduction. This protein antagonizes the action of SR proteins that promote distal 5' splice-site usage in E1A and ß-globin pre-mRNAs bearing thalassemia mutations (Cáceres et al, 1994; Mayeda and Krainer, 1992). In addition, hnRNPA1 controls inclusion of exon 7b of its own transcript (Blanchette and Chabot, 1999) and of exon 2 of the HIV Tat-pre-mRNA (Caputi et al, 1999). Two signal transduction pathways have been described to change the RNA binding properties and the intracellular localization of hnRNPA1. Stimulation of the PDGF receptor causes phosphorylation of hnRNP A1 by PKCzeta (Municio et al, 1995). This phosphorylation impairs the RNA binding and strand annealing activity of hnRNPA1. Furthermore, hnRNP A1 is phosphorylated by the MKK3/6-p38 signaling cascade after cellular stress induced by osmotic stress or UV irradiation, but the direct kinase remains to be determined (van Oordt et al, 2000). Stress induced phosphorylation leads to the cytoplasmic accumulation of hnRNP A1 and results in a change of the alternative splicing pattern of the adenovirus E1A premRNA splicing reporter.

D. STAR proteins Other likely candidates for proteins that can transduce a signal to the spliceosome are STAR proteins. STAR is an abbreviation for signal transduction and activation of RNA (Jones and Schedl, 1995; Vernet and Artzt, 1997). This protein family is also called GSG for GRP33, Sam68, GLD-1 (Jones and Schedl, 1995). Its members belong to an expanding group of proteins that share an amino-terminal maxi-KH-RNA binding domain as well as proline and tyrosine-rich regions present in many adapter proteins involved in signal transduction (Richard et al, 1995). The binding properties of STAR proteins suggest their involvement in splice-site selection. For example, Sam68 has been found to crosslink to a splicing regulator region on the rat tropomyosin pre-mRNA (Grossman et al, 1998), and also binds to FBP21, a protein implicated in splicing (Bredford et al, 2000). A protein related to SAM68 is SLM-2, for Sam68 like molecule (Di Fruscio et al, 1999). In humans, the protein is called T-STAR and was shown to interact with RBM, an hnRNP G like protein previously implicated in splice-site regulation (Venables et al, 1999, 2000). We identified the rat homolog and demonstrated that it regulates alternative splicing of CD44, htra2-beta, and tau pre-mRNAs by

B. SF1 Some signal transduction pathways to spliceosomal components have been investigated in detail. One paradigm is SF1 (Berglund et al, 1998; Rain et al, 1998), a factor that recognizes the branch point and is therefore important for the formation of the spliceosomal A complex. SF1 has recently been identified as a target of PKG-I (Wang et al, 1999). This kinase is activated by cGMP. The cGMP level

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Table 3: Stimuli that change alternative splicing patterns Signals known to influence alternative splicing patterns are shown in the first column. Known proteins involved in signal propagation to the splicing machinery are indicated in the second column and the gene which changes its alternative splicing pattern is shown in the fourth column. In some cases, it has also been determined whether de novo protein biosynthesis is necessary for the observed change of a given splicing pattern, which is shown in the fourth column.

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binding to purine-rich enhancer sequences (Stoss et al, submitted). The physiological importance of STAR proteins becomes apparent in mutations of the quaking locus. The molecular defect is a mutation in the STAR family member QKI, which results in severe defects in myelination in the nervous system (Ebersole et al, 1996). SAM68 is phosphorylated by the tyrosine kinases Src or Fyn during mitosis (Fumagalli et al, 1994). SAM68 tyrosine phosphorylation is inducible by insulin in fibroblasts, or after TCR stimulation (Fusaki et al, 1997; Lang et al, 1997; Sanchez-Margalet and Najib, 1999). The tyrosine phosphorylation results in a decrease of the RNA binding affinity and leads to the dissociation of Sam68 multimers which could have a direct influence on the regulation of alternative splicing (Chen et al, 1997; Wang et al, 1995). We were able to show that rSam68, rSLM-1, and rSLM-2 bind to the scaffold attachment factor B (SAFB), a component that binds to DNA-nuclear matrix attachment regions as well as to RNA polymerase II and various SR proteins (Stoss et al, submitted). This association again emphasizes the intimate connection between pre-mRNA processing, transcription, and chromatin structure. The exact change of protein:protein interaction caused by tyrosine phosphorylation of complex components and its influence on alternative splicing remain to be determined, but will most likely influence splice-site selection.

In addition, there are three other kinases that are able to phosphorylate SR proteins: CKIa, the Lamin B receptor and topoisomerase I (Gross et al, 1999; Nikolakaki et al, 1996; Rossi et al, 1996). In contrast to the Cdc2-like kinases, the substrates of CKIa must already be phosphorylated, suggesting a phosphorylation hierarchy in the regulation of SR proteins. The lamin B receptor is part of a nuclear envelope complex that also phosphorylates lamin A and B. This kinase phosphorylates RS domains and seems to have substrate specificities identical to SRPK1 (Papoutsopoulou et al, 1999b). Phosphorylation of SR proteins has numerous effects on protein:RNA and protein:protein interactions. For example, phosphorylation of SRp40 enhances its RNA binding affinity (Tacke et al, 1997) and phosphorylation of ASF/SF2 stimulates its binding to U170K, a component of the U1snRNP (Xiao and Manley, 1998). This type of covalent modification of SR proteins has a direct effect on their activity in the splicing reaction (Prasad et al, 1999; Tazi et al, 1993). An important consequence of SR protein phosphorylation is the release of these proteins from their storage compartments, the speckles (Colwill et al, 1996b; Gui et al, 1993; Koizumi et al, 1999), and their recruitment to sites of transcription (Misteli, 2000; Misteli et al, 1998). In addition, phosphorylation influences the ability of some SR proteins to shuttle between the nucleus and the cytoplasm (Cรกceres et al, 1998; Yeakley et al, 1999). In a manner that is similar to hnRNP A1 (van Oordt et al, 2000), this could result in a rapid change of the nuclear concentration of SR proteins, which changes alternative splice-site selection. Together, these data show that pre-mRNA splicing can be regulated by external stimuli. Some of the signal transduction pathways to the spliceosome are beginning to emerge and result in regulated serine and tyrosine phosphorylation of splicing proteins.

E. SR proteins and their kinases Finally, in humans, SR proteins are phosphorylated by four different Cdc2-like kinases of the LAMMER family (Clks) and two structurally related kinases called SRPK1 and SRPK2 (Gui et al, 1993; Koizumi et al, 1999; Nayler et al, 1997; Prasad et al, 1999; Stojdl and Bell, 1999; Wang et al, 1998). These kinases all contain an RS domain; they interact with SR proteins and their overexpression leads to the disassembly of speckles (Nayler et al, 1998b). A direct involvement of Cdc2-like kinases in the regulation of alternative splicing has recently been shown for the Clk1-, E1A- and SRp20-, and tau pre-mRNAs (Duncan et al, 1997; Hartmann et al, 2000; Stoss et al, 1999b). However, there are important differences among these kinases. First, the Cdc2-like kinases are ubiquitously expressed, whereas the SRPKs show a more differentiated expression pattern with SRPK1 predominantly expressed in testis and SRPK2 in brain (Papoutsopoulou et al, 1999a; Wang et al, 1998). In addition, SRPK1 shows higher specificity towards ASF/SF2 in comparison with Clk/STY, since Clk/STY phosphorylates Ser-Arg, Ser-Lys, or Ser-Pro sites, wheras SRPK1 shows a strong preference for Ser-Arg sites (Colwill et al, 1996a). There is also evidence that these kinases phosphorylate SR proteins at different sites. Finally, there are differences in the intracellular localization, since the Clks are predominantly nuclear and colocalize with speckles, whereas SRPK1 was primarily found in the cytoplasm and is believed to phosphorylate the cytosolic SR protein fraction.

III. Diseases caused by splicing defects A. Overview Most of the diseases associated with defects in premRNA processing result from a loss of function due to mutations in regulatory elements of a single gene. These mutations have previously been compiled (Krawczak et al, 1992; Nakai and Sakamoto, 1994) and are available on the web (cookie.imcb.osaka-u.ac.jp/nakai/asdb.html). A few diseases have been attributed to a change in trans-acting factors. Knockout experiments of essential splicing factors have proven lethal (Hirsch et al, 2000; Wang et al, 1996). However, the knockout of an enzyme involved in premRNA editing (Higuchi et al, 2000) and overexpression of mutated SR proteins in Drosophila (Kraus and Lis, 1994) causes phenotypes that cannot be linked to a single mRNA, which shows that defects in pre-mRNA processing factors can result in a complex pathological state. The combinatorial nature of trans-acting factors raises the interesting possibility that pleiotropic diseases with a variable phenotype might be caused by alterations

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of trans-acting factors. Using minigenes of CFTR mutations, it has been shown that the splicing patterns of mutated alleles strongly depend on the cell type (NissimRafinia et al, 2000), indicating that variations in transacting factors could be the reason for a variable penetrance of mutations among individuals with different ethnic backgrounds (McInnes et al, 1992; Rave-Harel et al, 1997). Similarly, a contribution of pre-mRNA processing could explain why natural mutations in human genes frequently have tissue- or cell-type specific effects. In these cases, the mutated gene is similarly expressed in all cells, but is processed in a tissue specific manner, since the relative concentrations of splicing factors vary among tissues (Hanamura et al, 1998). Since diseases caused by splicing defects have been recently reviewed (Philips and Cooper, 2000), we will concentrate on three diseases, FTDP-17, spinal muscular atrophy, and !-thalassemia to illustrate the complex relationships between trans-acting factors and their corresponding cis-elements, and to outline possible therapeutic approaches.

10 revealed a complicated set of cis-acting elements that is disrupted by natural mutations (D'Souza et al, 1999). Some of these mutations, such as L284L, are silent, but lead to disease by interrupting an exonic element that causes missplicing. Since alternative splice-site usage can be regulated by the relative concentration of SR proteins or hnRNPs, several such trans-acting factors were tested in vivo, and it was found that some SR or SR related proteins (SF2/ASF, SRp75 and U2AF65) stimulate exon 10 skipping (Gao et al, 2000). It was shown that SR proteins are released from their storage compartments, the speckles, by Cdc2-like kinases (clk1-4). Those kinases were found to strongly inhibit missplicing of exon 10, even in several mutations that activate exon 10 usage (Hartmann et al, submitted). These examples show that missplicing can be reversed in vivo by activating regulatory proteins through their kinases. It is possible that lower molecular weight substances can be isolated that cause the release of specific regulatory factors, by either activating the appropriate kinase or blocking the corresponding phosphatase. In addition, recent results show that drugs such as aminoglycoside antibiotics which can directly interact with regulatory RNA structures, may also have a therapeutic potential (Varani et al, 2000).

B. FTDP-17: frontotemporal dementia with parkinsonism linked to chromosome 17 Frontotemporal dementias (Figure 2A) represent a rare form of presenile dementias that are clinically defined by behavioral and personality changes, psychomotor stereotypes, as well as loss of judgment and insight. The neuropathological findings include an asymmetric frontotemporal atrophy and the presence of filamentous tau deposits. The disease was mapped to the tau locus on chromosome 17. Tau is a microtubule-associated protein. Knockout experiments revealed that tau is not essential for brain formation (Harada et al, 1994), although it is involved in the pathology of several neurodegenerative diseases (Spillantini and Goedert, 1998). Tau transcripts undergo complex regulated splicing in the mammalian nervous system. The alternative splicing of one of its exons, exon 10, is species-specific. This exon is alternatively spliced in adult humans, but is constitutively used in the adult rodent brain. In addition, the usage of exon 10 is regulated during development and increases when neuronal development proceeds. In the protein, it encodes one of the four microtubuli binding sites of tau. Some of these tau mutations that activate exon 10 usages were shown to cause an accelerated aggregation of tau into filaments (Nacharaju et al, 1999), which is a hallmark of several neurodegenerative diseases, e.g. Alzheimer's disease. A disruption of the proper balance of tau isoforms with three and four microtuble binding sites is observed in the pathology of several tauopathies, including FTDP, Picks disease, corticobasal degeneration, Guam amytrophic lateral sclerosis/parkinsonism dementia complex. Secondary structure predictions suggest a stem loop structure at the 5' splice-site of exon 10 that contributes to its regulation (Grover et al, 1999; Jiang et al, 2000); however the in vivo relevance of this structure remains to be proven. In addition, mapping of elements in tau exon

C. Spinal muscular atrophy (SMA) Proximal spinal muscular atrophy (SMA, Figure 2B) is a neurodegenerative disorder with progressive paralysis caused by the loss of alpha-motor neurons in the spinal cord. With an incidence of 1 in 10,000 live births and a carrier frequency of 1 in 50, SMA is the second-most common autosomal recessive disorder and the most frequent genetic cause of infantile death (Pearn, 1980). The gene responsible for the disease was identified as SMN1 (survival of motor neurons, Lefebver, 1995) and the disease is caused by loss of (96.4%) or mutations in (3.6%) the SMN1 gene (Wirth, 2000). A nearly identical copy of the SMN1 gene exists but cannot compensate for the absence of SMN1, because it is processed differently. Due to a single nucleotide difference in exon 7, this exon is skipped in SMN2. Therefore the proteins generated by both genes differ in their carboxy terminus, which is most likely crucial for the function. The protein generated by SMN2 encodes a truncated, less stable protein with reduced self-oligomerization activity ( Lefebvre et al, 1995, 1997; Coovert et al, 1997). The exon enhancer containing the single nucleotide difference has been characterized (Lorson et al, 1999) and was found to be of the GAR type. A systematic search for trans-acting factors identified human transformer2-beta, a member of the SR related family of proteins (Hofmann et al, 2000). An increase of the concentration of htra2-beta1 results in stimulation of exon 7 increase. A mRNA generated by this pathway would encode for a protein that can complement for the loss of SMN1. This example demonstrates that pre-mRNA processing can be maniplulated in vivo to complement the loss of a gene product.

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Figure 2: Examples of diseases that are caused by errors in pre-mRNA splicing A) Frontotemporal dementia w i t h P a r k i n s o n i s m (FTDP-17) a s an e x a m p l e o f mutation i n s p l i c i n g e n h a n c e r s a n d s i l e n c e r s . FTDP-17 is caused by a misregulation of tau exon 10 usage. Exon 10 splicing is tightly regulated by exonic enhancer and silencer sequences in exon 10. It is unclear whether a secondary structure that masks the 5' splice-site is important in vivo. Mutations close to exon 10, which are observed in FTDP-17 patients (shown in red), favor exon 10 inclusion. The Cdc2-like kinases clk1-4 are able to revert the increase in exon 10 inclusion. This opens new therapeutic possibilities to treat FTDP-17 and related diseases by screening for factors or substances that selectively affect the regulation of alternative splicing. B ) S p i n a l m u s c u l a r a t r o p h y ( S M A ) : c o m p e n s a t i o n o f a n o n - f u n c t i o n a l g e n e . Positional cloning strategies led to the identification of the survival of motorneuron (SMN) genes as one of the genes affected in SMA. Two non-equal copies of SMN exist on chromosome 5q13. Full-length SMN can only be generated from the telomeric copy (SMN-1). A silent C/T transition of the sixth nucleotide of the centromeric copy (SMN-2) (in red) disturbs splicing of exon 7, leading to the generation of a truncated SMN protein. SMA is caused by failure to express the telomeric SMN-1 gene. One possibility to compensate for the loss of SMN-1 expression is overexpression of htra2-beta1. This SR like protein can switch the splicing pattern of the SMN-2 pre-mRNA towards the inclusion of exon 7, leading to a functional SMN-2 copy. C) !- t h a l a s s e m i a : A p o i n t m u t a t i o n a c t i v a t e s a c r y p t i c s p l i c e - s i t e . The second intron of the thalassemic !-globin gene harbors a C to T mutation at nucleotide 654. This creates an additional 5' splice-site that activates a cryptic splice-site at nucleotide 579 of the !-globin pre-mRNA, leading to the retention of an intronic region. Antisense oligonucleotides can be used to mask the aberrant splice-sites, resulting in the formation of the desired gene product.

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the underling molecular pathology. Furthermore, such a tool might be used to detect the effect of trans-acting factors in vivo and could ultimately be used to unveil misregulation of trans-acting factors in complex diseases.

D. !-thalassemia !-thalassemias are autosomal recessive diseases (Figure 2C), in which the amount of !-globin is reduced. Individuals carrying a single mutated gene are less prone to malaria infection. About 3% of the world population, mostly people living in regions endemic with malaria, or their descendants, are carriers. !-thalassemia causes a hypochromic, microcytic and hemolytic anemia. The imbalance of globin synthesis causes the "-chains to precipitate and damages the red blood cells. More than 100 mutations of the !-globin gene leading to thalassemia have been described (Wetherall and Clegg, 1981), among them are at least 51 point mutations, which mostly affect premRNA processing (Kazzazin and Boehm, 1988; Krawczak et al, 1992). These mutations either destroy the 5' or 3' splice-sites or generate cryptic splice-sites that are usually located in the introns (Figure 2C). As a result, no functional !-globin protein is produced. Since point mutations often manifest themselves as defects in premRNA splicing, missplicing of !-thalassemias is a model for a large number of mutations (Krawczak et al, 1992; Nakai and Sakamoto, 1994). Cryptic splice-sites of thalassemic !-globin can be changed in vivo by overexpression of the SR protein SF2/ASF (Cáceres et al, 1994). Furthermore, the mutated cryptic sites can be blocked by a complementary oligonucleotide (Schmajuk et al, 1999; Sierakowska et al, 2000), that enhances the formation of the desired !-globin product in cell-culture systems. This example illustrates that cryptic splice-sites can be masked in vivo, which promotes the formation of the desired gene product.

B. Suppression of point mutations by oligonucleotides The majority of known pathological states associated with splicing are generated by point mutations that either destroy splice-sites or generate new, cryptic sites in the vicinity of normally used exons (Krawczak et al, 1992). It has been demonstrated that antisense nucleic acids binding to the aberrant splice-sites can inhibit the usage of the wrong sites and promote the formation of the normal gene product. Among nucleic acids, modified RNA oligonucleotides have been used (Sierakowska et al, 2000). Blocking the aberrant splice-sites forces the splicing machinery to reselect the original splice-site and can restore the correct gene product. Currently, 2'-O-methyl oligoribonucleoside phosphoro-thioates are the most widely used nucleic acids, since they do not induce RNase H mediated cleavage of targeted RNA and seem to have only minor effects on cell viability, morphology, and growth rates. Diseases targeted include ß-thalassemias (Schmajuk et al, 1999), cystic fibrosis (Friedman et al, 1999), muscular dystrophy mRNA (Wilton et al, 1999), and eosinophilic diseases (Karras et al, 2000). Furthermore, apoptosis can be influenced by oligonucleotides directed against Bcl-x splice variants (Taylor et al, 1999). The oligonucleotide approach offers a high specificity to target a mutated gene. Most studies have been performed in cell culture systems, where the oligonucleotide approach works in multiple cellular contexts, which argues for its broad applicability to suppress an aberrant splice-site selection. It remains to be seen whether this approach can also be used to modify exon enhancer usage.

IV. Detection and treatment of splicing defects A. Alternative splicing as an indicator of disease Since pre-RNA pathways can adapt according to environmental signals, the splicing pattern of pre-mRNAs is most likely a reflection of the cellular state. There are numerous examples in which a change of exon usage is associated with a pathological state. The most prominent example is cancer. Some of the changes found in splicesite selection that are associated with cancer are shown in Table 4. One of the best described genes that shows the importance of pre-RNA processing for tumor progression and metastasis is CD44. In this gene, at least 12 exons are alternatively spliced (Screaton et al, 1992) and their usage relates to metastatic potential. With the completion of the human gene project and the progress in array techniques, it will be possible to detect differences in splicing patterns between a normal and pathological state. Arrays with exon-specific oligonucleotides will make it possible to discover changes in alternative splicing patterns. Given the numerous examples in which changes in exon usage are associated with disease, the development of such a DNA exon chip might help in the diagnosis of cancer and the elucidation of

C. Modification of trans-acting factor action Since the selection of splice-sites is dependent on the relative concentration of regulatory proteins, a change of the concentration of a protein could possibly correct a pathological ratio of exon inclusion to exon skipping. For example, overexpression of SR protein and their kinases clk1-4 can revert missplicing of tau exon 10 (Gao et al, 2000; Hartmann et al, submitted); overexpression of htra2beta1 can change the splicing pattern of SMN2 to complement loss of SMN1 in spinal muscular atrophy (Hofmann et al, 2000); and the levels of hnRNPA1 and SF2/ASF regulate alternative splicing of mutated alleles of the cystic fibrosis transmembrane conductance regulator (Nissim-Rafinia et al, 2000) and mutated ß-globin genes (Cáceres et al, 1994). Since most splicing factors are released from nuclear storage compartments, a promising strategy might be the identification of specific antagonist/agonists for splicing factor kinases from a chemical library.

Stoss et al: RNA splicing and disease

Table 4: Examples of genes that change their splicing pattern during cancer formation and progression. Only a few examples are given

References

Furthermore, it is likely that release of splicing factors can occur in response to a stimulation of a receptor and specific agonists could be found to arrest this process. Such molecules will be easier to deliver pharmacologically because of their small size; however such agonists remain to be identified, and their specificity proven.

Amara, S.G., V. Jonas, M.G. Rosenfeld, E.S. Ong, and R.M. Evans. 1 9 8 2 . Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature. 298, 240-244. Ayane, M., U. Preuss, G. Kรถhler, and P.J. Nielsen. 1 9 9 1 . A differentially expressed murine RNA encoding a protein with similarities to two types of nucleic acid binding motifs. N u c l . A c i d s R e s . 19, 1273-1278.

V. Conclusions The basic mechanisms regulating alternative splicesite selection have been deciphered in recent years. The abundant usage of alternative pre-mRNA processing has most likely generated an evolutionary advantage for vertebrates, since the formation of protein isoforms required for specialized functions was greatly accelerated. Disadvantages of this mechanism is the misregulation of pre-mRNA processing that is apparent in several human diseases. We hypothesize that the role of pre-mRNA defects in human disease is just beginning to emerge and is currently largely underestimated. Since splice-site selection is regulated by extracellular signals, the analysis of these signal transduction pathways might provide new insights and novel chances for therapy. The connection of the molecular mechanisms governing splice-site selection with the detailed analysis of human diseases will provide opportunities for diagnosis and treatment.

Baumbach, W.R., D.L. Horner, and J.S. Logan. 1 9 8 9 . The growth hormone-binding protein in rat serum is an alternatively spliced form of the rat growth hormone receptor. G e n e s D e v . 3, 1199-1205. Berget, S.M. 1 9 9 5 . Exon Recognition in splicing. J . B i o l . C h e m . 270, 2411-2414.

vertebrate

Berglund, J.A., M.L. Fleming, and M. Rosbash. 1 9 9 8 . The KH domain of the branchpoint sequence binding protein determines specificity for the pre-mRNA branchpoint sequence. RNA. 4, 998-1006. Black, D.L. 1 9 9 5 . Finding splice sites within a wilderness of RNA. RNA. 1, 763-771. Blanchette, M., and B. Chabot. 1 9 9 9 . Modulation of exon skipping by high-affinity hnRNP A1-binding sites and by intron elements that repress splice site utilization. EMBO J. 18, 1939-1952.

Acknowledgments Work in the laboratory is supported by the MaxPlanck Society, the DFG (Sta 399 3-1), the HSFP (RG 562/96) and the EU (Bio4-98-0259).

Bode, J., C. Benham, K. A., and C. Mielke. 2 0 0 0 . Transcriptional augmentation, modulation of gene

Gene Therapy and Molecular Biology Vol 5, page 23

expression by scaffold/matrix-attached regions (S/MAR elements). Crit. Rev. Eukar. Gene Exp. 10, 73-90.

carboxy-terminal domain. G e n e s a n d D e v . 15, 33193326.

Boggs, R.T., P. Gregor, S. Idriss, J.M. Belote, and M. McKeown. 1 9 8 7 . Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene. C e l l . 50, 739-747.

Colwill, K., L.L. Feng, G.D. Gish, J.F. C_ceres, T. Pawson, and X.D. Fu. 1 9 9 6 a . SRKP1 and Clk/Sty protein kinases show distinct substrate specificities for serine/argininerich splicing factors. J . B i o l . C h e m . 271, 2456924575.

Bourquin, J.-P., I. Stagljar, P. Meier, P. Moosmann, J. Silke, T. Baechi, O. Georgiev, and W. Schaffner. 1 9 9 7 . A serine/arginine-rich nuclear matrix cyclophilin interacts with the c-terminal domain of RNA polymerase II. N u c l . A c i d s R e s . 25, 2055-2061.

Colwill, K., T. Pawson, B. Andrews, J. Prasad, J.L. Manley, J.C. Bell, and P.I. Duncan. 1 9 9 6 b . The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution. EMBO J. 15, 265-275.

Bredford, M.T., A. Frankel, M.B. Yaffe, S. Clarke, P. Leder, and S. Richard. 2 0 0 0 . Arginine methylation inhibits the binding of proline-rich ligands to SH3, but not WW domains. J . B i o l . C h e m . 275, 16030-16036.

Cooper, T.A., and W. Mattox. 1 9 9 7 . The regulation of splice-site selection, and its role in human disease. A m . J. Hum. Genet. 61, 259-266. Coovert, D.D., T.T. Le, P.E. McAndrew, J. Strasswimmer, T.O. Crawford, J.R. Mendell, S.E. Coulson, E.J. Androphy, T.W. Prior, and A.H. Burghes. 1 9 9 7 . The survival motor neuron protein in spinal muscular atrophy. H u m . M o l . G e n e t . 6, 1205-1214.

Breitbart, R.E., A. Andreadis, and B. Nadal-Ginard. 1 9 8 7 . Alternative splicing, a ubiquitous mechanism for the generation of multiple protein isoforms from single genes. A n n u . R e v . B i o c h e m . 56, 467-495. Brett, D., J. Hanke, G. Lehmann, S. Haase, S. Delbrück, S. Krueger, J. Reich, and P. Bork. 2 0 0 0 . EST comparison indicates 38% of human mRNAs contain possible alternative splice forms. F E B S l e t t . 474, 83-86.

Corden, J.L., and M. Patturajan. 1 9 9 7 . A CTD function linking transcription to splicing. Trends B i o c h e m . S c i . , 413-419. Courty, Y., C.I. Rosinski, and F. Rougeon. 1 9 9 5 . Various transcripts are generated from the VCSA1 gene by alternative splicing and poly(A) processing in the rat submandibular gland. Gene. 162, 291-296.

Cáceres, J., S. Stamm, D.M. Helfman, and A.R. Krainer. 1 9 9 4 . Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors. S c i e n c e . 265, 1706-1709.

Cramer, P., J.F. Caceres, D. Cazalla, S. Kadener, A.F. Muro, F.E. Baralle, and A.R. Kornblihtt. 1 9 9 9 . Coupling of transcription with alternative splicing, RNA polII promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer. M o l . C e l l . 4, 251-258.

Cáceres, J.F., G.R. Screaton, and A.R. Krainer. 1 9 9 8 . A specific subset of SR proteins shuttles continously between the nucleus and the caytoplasm. G e n e s D e v . 12, 55-66. Caputi, M., A. Mayeda, A.R. Krainer, and A.M. Zahler. 1 9 9 9 . hnRNP A/B proteins are required for inhibition of HIV-1 pre-mRNA splicing. EMBO J. 18, 4060-4067.

D'Souza, I., P. Poorkai, M. Hong, D. Nochlin, V.M.-Y. Lee, T.D. Bird, and G.D. Schellenberg. 1 9 9 9 . Missense and silent tau gene mutations cause frontotemporal dementia with parkinsonism-chromosome 17 type, by affecting multiple alternative RNA splicing regulatory elements. P r o c . N a t l . A c a d . S c i . U S A. 96, 5598-5603.

Chalfant, C.E., J.E. Watson, L.D. Bisnauth, J.B. Kang, N. Patel, L.M. Obeid, D.C. Eichler, and D.R. Cooper. 1 9 9 8 . Insulin regulates protein kinase CbetaII expression through enhanced exon inclusion in L6 skeletal muscle cells. A novel mechanism of insulin- and insulin-like growth factor-i-induced 5' splice site selection. J . B i o l . C h e m . 273, 910-916.

Danoff, S.K., C.D. Ferris, C. Donath, G.A. Fischer, S. Munemitsu, A. Ullrich, S.H. Snyder, and C.A. Ross. 1 9 9 1 . Inositol 1,4,5,-trisphosphate receptors, Distinct neuronal and nonneuronal forms derived by alternative splicing differ in phosphorylation. P r o c . N a t l . A c a d . S c i . U S A . 88, 2951-2955.

Chandler, D., M.E. McGuffin, J. Piskur, J. Yao, B. Baker, and W. Mattox. 1 9 9 7 . Evolutionary conservation of regulatory strategies for the sex determination factor transformer-2. M o l . C e l l . B i o l . , 2908-2919.

Daoud, R., M. Berzaghi, F. Siedler, M. Hübener, and S. Stamm. 1 9 9 9 . Activity dependent regulation of alternative splicing patterns in the rat brain. Eur. J . N e u r o s c i . 11, 788-802.

Chen, T., B.B. Damaj, C. Herrera, P. Lasko, and S. Richard. 1 9 9 7 . Self-association of the single-KH-domain family members Sam68, GRP33, and Qk1, role of the KH domain. M o l . C e l l . B i o l . 17, 5707-5718.

Cho, E.J., T. Takagi, C.R. Moore, and S. Buratowski. 1 9 9 7 . mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II

Fruscio, M., T. Chen, and S. Richard. 1 9 9 9 . Characterization of Sam68-like mammalian proteins SLM-1 and SLM-2, SLM-1 is a Src substrate during mitosis. P r o c . N a t l . Acad. S c i . USA. 96, 27102715.

Stoss et al: RNA splicing and disease

10, whose missplicing causes frontotemporal dementia. J. Neurochem. 74, 490-500.

Du, L., and L. Warren. 1 9 9 6 . A functional interaction between the Carboxy-terminal domain of RNA polymerase II and pre-mRNA splicing. J . C e l l B i o l . 136, 5-18.

Giros, B., M.-P. Martres, C. Pilon, P. Sokoloff, and J.-C. Schwartz. 1 9 9 1 . Shorter variants of the D3 dopamine receptor produced through various patterns of alternative splicing. B i o c h e m . B i o p h y s . R e s . C o m . 176, 1584-1592.

Duncan, P.I., B.W. Howell, R.M. Marius, S. Drmanic, E.M. Douville, and J.C. Bell. 1 9 9 5 . Alternative splicing of STY, a nuclear dual specificity kinase. J . B i o l . C h e m . 270, 21524-21531.

Gondran, P., and F. Dautry. 1 9 9 9 . Regulation of mRNA splicing and transport by the tyrosine kinase activity of src. O n c o g e n e . 18, 2547-2555.

Duncan, P.I., D.F. Stojdl, R.M. Marius, and J.C. Bell. 1 9 9 7 . In vivo regulation of alternative pre-mRNA splicing by the Clk1 protein kinase. M o l . C e l l . B i o l . 17, 59966001.

Grabowski, P. 1 9 9 8 . Splicing regulation in neurons, tinkering with cell-specific control. C e l l . 92, 709-712.

Ebersole, T.A., Q. Chen, M.J. Justice, and K. Artzt. 1 9 9 6 . The quaking gene product necessary in embryogenesis and myelination combines features of RNA binding and signal transduction proteins. Nature genetics . 12, 260-265.

Green, M.J. 1 9 9 1 . Biochemical mechanism of constitutive and regulated pre-mRNA splicing. Annu. R e v . C e l l B i o l . 7, 559-599.

Eipper, B.A., C.B. Green, T.A. Campbell, D.A. Stoffers, H.T. Keutmann, R.E. Mains, and L. Ouafik. 1 9 9 2 . Alternative splicing and endoproteolytic processing generate tissuespecific forms of pituitary peptidylglycine alphaamidating monooxygenase (PAM). J . B i o l . C h e m . 267, 4008-4015.

Gross, S.D., J.C. Loijens, and R.A. Anderson. 1 9 9 9 . The casein kinaseI alpha isoform is both physically positioned and functionally competent to regulate multiple events of mRNA metabolism. J . C e l l S c i . 112, 2647-2656. Grossman, J.S., M.I. Meyer, Y.C. Wang, G.J. Mulligan, R. Kobayashi, and D.M. Helfman. 1 9 9 8 . The use of antibodies to the polypyrimidine tract binding protein (PTB) to analyze the protein components that assemble on alternatively spliced pre-mRNAs that use distant branch points. RNA. 4, 613-625.

Eissa, N.T., A.J. Strauss, C.M. Haggerty, E.K. Choo, S.C. Chu, and J. Moss. 1 9 9 6 . Alternative splicing of human inducible nitric-oxide synthase messenger-RNA - tissuespecific regulation and induction by cytokines. J . B i o l . C h e m . 271, 27184-27187. Elliot, D.J. 2 0 0 0 . Splicing and the single cell. H i s t o l . H i s t o p a t h o l . 15, 239-249.

Grover, A., H. Houlden, M. Baker, J. Adamson, J. Lewis, G. Prihar, S. Pickering-Brown, K. Duff, and M. Hutton. 1 9 9 9 . 5' splice site mutation in tau associated with the inherited dementia FTDP-17 affect a stem-loop structure that regulates alternative splicing of exon 10. J . B i o l . C h e m . 274, 15134-11543.

Fay, F.S., K.L. Taneja, S. Shenoy, L. Lifshitz, and R.H. Singer. 1 9 9 7 . Quantitative digital analysis of diffuse and concentrated nuclear distributions of nascent transcripts, SC35 and poly(A). E x p . C e l l R e s . 231, 27-37.

Gui, J.-F., W.S. Lane, and X.-D. Fu. 1 9 9 3 . A serine kinase regulates intracellular localization of splicing factors in the cell cycle. Nature. 369, 678-682.

Friedman, K.J., J. Kole, J.A. Cohn, M.R. Knowles, L.M. Silverman, and R. Kole. 1 9 9 9 . Correction of aberrant splicing of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by antisense oligonucleotides. J . B i o l . C h e m . 274, 36193-36199.

Guiramand, J., J.P. Montmayeur, J. Ceraline, M. Bhatia, and E. Borrelli. 1 9 9 5 . Alternative splicing of the dopamine D2 receptor directs specificity of coupling to G-proteins. J . B i o l . C h e m . 270, 7354-7358.

Fumagalli, S., N. Totty, J. Hsuan, and S.A. Courtneidge. 1 9 9 4 . A target for Src in mitosis. Nature. 368, 871874.

Hanamura, A., J.F. Caceres, A. Mayeda, B.R. Franza, and A.R. Krainer. 1 9 9 8 . Regulated tissue-specific expression of antagonistic pre-mRNA splicing factors. R N A . 4, 430444.

Fusaki, N., A. Iwamatsu, M. Iwashima, and J. Fujisawa. 1 9 9 7 . Interaction between Sam68 and Src family tyrosine kinaes, Fyn and Lck, in T Cell receptor signaling. J . B i o l . C h e m . 272, 6214-6219.

Harada, A., K. Oguchi, S. Okabe, J. Kuno, S. Terada, T. Ohshima, R. Sato-Yoshitake, Y. Takei, T. Noda, and N. Hirokawa. 1 9 9 4 . Altered microtubule organization in small-calibre axons of mice lacking tau protein. Nature. 369, 488-491.

Gallego, M.E., R. Gattoni, J. Stevenin, J. Marie, and A. Expert-Bezancon. 1 9 9 7 . The SR splicing factors ASF/SF2 and SC35 have antagonistic effects on intronic enhancer-dependent splicing of the beta-tropomyosin alternative exon 6A. EMBO J. 16, 1772-1784.

Hartmann, A.M., E. Mandelkow, M. Goedert, D. Rujescu, A. Andreadis, and S. Stamm. Regulation of alternative splicing of human tau exon 10 by phosphorylation of the cdc2 like kinases clk1-4. , submitted.

Gao, Q.S., J. Memmott, R. Lafyatis, S. Stamm, G. Screaton, and A. Andreadis. 2 0 0 0 . Complex regulation of tau exon

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Hartmann, A.M., O. Nayler, F.W. Schwaiger, A. Obermeier, and S. Stamm. 1 9 9 9 . The interaction and colocalisation of SAM68 with the splicing associated factor YT521-B in nuclear dots is regulated by the Src family kinase p59 fyn .

Jiang, Z.-H., and J.Y. Wu. 1 9 9 9 . Alternative splicing and programmed cell death. P r o c . S o c . E x p . B i o l . M e d . 220, 64-72. Jiménez-Garcia, L.F., and D. Spector. 1 9 9 3 . In vivo evidence that transcription and splicing are coordinated by a recruiting mechanism. C e l l . 73, 47-60.

M o l . B i o l . C e l l , 3909-3926. Heinrichs, V., L.C. Ryner, and B. Baker. 1 9 9 8 . Regulation of sex-specific selection of fruitless 5' splice sites by transformer and transformer-2. M o l . C e l l . B i o l . 18, 450-458. Herbert, A., and A. Rich. 1 9 9 9 . RNA processing and the evolution of eukaryotes. N a t . G e n e t . 21, 265-269.

Jones, A.R., and T. Schedl. 1 9 9 5 . Mutations in gld-1, a female germ-cell-specific tumor suppressor gene in Caenorhabditis elegans, affect a conserved domain also found in Src-associated protein Sam68. G e n e s D e v . 9 , 1491-1504.

Hertel, K.J., K.W. Lynch, and T. Maniatis. 1 9 9 7 . Common themes in the function of transcription and splicing enhancers. C u r r . O p i n . C e l l B i o l . 9, 350-357.

Jumaa, H., and P.J. Nielsen. 1 9 9 7 . The splicing factor SRp20 modifies splicing of its own mRNA and ASF/SF2 antagonizes this regulation. EMBO J. 16, 5077-5085.

Higuchi, M., S. Maas, F.N. Single, J. Hartner, A. Rozov, N. Burnashev, D. Feldmeyer, R. Sprengel, and P.H. Seeburg. 2 0 0 0 . Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature. 406, 78-81.

Kamma, H., D.S. Portman, and G. Dreyfuss. 1 9 9 5 . Cell type specific expression of hnRNP proteins. E x p . C e l l R e s . 221, 187-196. Karras, J.G., R.A. McKay, N.M. Dean, and B.P. Monia. 2 0 0 0 . Deletion of individual exons and induction of soluble murine interleukin-5 receptor-alpha chain expression through antisense oligonucleotide-mediated redirection of pre-mRNA splicing. M o l . Pharamcol. 58, 380-387.

Hirose, Y., R. Tacke, and J.L. Manley. 1 9 9 9 . Phosphorylated RNA polymerase II stimulates pre-mRNA splicing. Genes and Dev. 15, 1234-1239. Hirsch, E., U. Fischer, T. Oohashi, C. Suchanek, B. Richter, J. Kellermann, S. Stamm, and R. Fässler. 2 0 0 0 . PeriImplantation lethality in mice lacking the Sm motifcontaining protein Lsm4. M o l . C e l l . B i o l . 20, 10551062.

Kaufer, D., A. Friedman, S. Seidman, and H. Soreq. 1 9 9 8 . Acute stress facilitates long-lasting changes in choinergic gene expression. Nature. 393, 373-377. Kazzazin, H.H., and C.D. Boehm. 1 9 8 8 . Molecular basis and prenatal diagnosis of beta-thalassemia. B l o o d . 72, 1107-1116.

Hofmann, Y., C.L. Lorson, S. Stamm, E.J. Androphy, and B. Wirth. 2 0 0 0 . Htra2-beta1 stimulates an exonic splicing enhancer an can restore full-length SMN expression to survival of motor neuron 2 (SMN2). P r o c . N a t l . A c a d . S c i . 97, 9618-9623.

Kim, E., L. Du, D.B. Bregman, and S.L. Warren. 1 9 9 6 . Splicing factors associate with hyperphosphorylated RNA polymerase II in the absence of pre-mRNA. J . C e l l B i o l . 136, 19-28.

Hoshijima, K., K. Inoue, I. Higuchi, H. Sakamoto, and Y. Shimura. 1 9 9 1 . Control of doublesex alternative splicing by transformer and transformer-2 in Drosophila. S c i e n c e . 1991, 833-836.

Koizumi, J., Y. Okamoto, H. Onogi, A. Mayeda, A.R. Krainer, and M. Hagiwara. 1 9 9 9 . The subcellular localization of SF2/ASF is regulated by direct interaction with SR protein kinases (SRPKs). J . B i o l . C h e m . 274, 11125-11131.

Huang, S., and D.L. Spector. 1 9 9 6 . Intron-dependent recruitment of pre-mRNA splicing factors to sites of transcription. J . C e l l B i o l . 133, 719-732.

Komatsu, M., E. Kominami, K. Arahata, and T. Tsukahara. 1 9 9 9 . Cloning and characterization of two neural-salient serine/arginine-rich (NSSR) proteins involved in the regulation of alternative splicing in neurons. G e n e s t o C e l l s . 4, 593-606.

Hughes, D.P., and I.N. Crispe. 1 9 9 5 . A naturally occurring soluble isoform of murine fas generated by alternative splicing. J . E x p . M e d . 182, 1395-1401.

König, H., H. Ponta, and P. Herrlich. 1 9 9 8 . Coupling of signal transduction to alternative pre-mRNA splicing by a composite splice regulator. EMBO J. 10, 2904-2913.

Jensen, K.B., B.K. Drege, G. Stefani, R. Zhong, R.J. Buchanovich, H.J. Okano, Y.Y. Yang, and R.B. Darnell. 2 0 0 0 . Nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability. Neuron. 25, 359-371.

Krämer, A. 1 9 9 6 . The structure and function of proteins involved in mammalian pre-mRNA splicing. Annu. R e v . B i o c h e m . 65, 367-409.

Jiang, Z., J. Cote, J.M. Kwon, A.M. Goate, and J.Y. Wu. 2 0 0 0 . Aberrant splicing of the tau pre-mRNA caused by intronic mutations associated with the inherited dementia frontotemporal dementia with parkinsonism linked to chromosome 17. M o l . C e l l . B i o l . 20, 4036-4048.

Kraus, M.E., and J.T. Lis. 1 9 9 4 . The concentration of B52, an essential splicing factor and regulator of splice site choice in vitro, is critical for Drosophila development. M o l . C e l l . B i o l . 14, 5360-5370.

Stoss et al: RNA splicing and disease

dramatically different phenotypes in patients from different racial backgrounds. J . C l i n . I n v e s t . 90, 306314.

Krawczak, M., J. Reiss, and D.N. Cooper. 1 9 9 2 . The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes, causes and consequences. Hum. Genet. 90, 41-54.

Mironov, A.A., J.W. Fickett, and M.S. Gelfand. 1 9 9 9 . Frequent alternative splicing of human genes. G e n o m e R e s . 9, 1288-1293.

Kuhse, J., A. Kuryatov, Y. Maulet, M.L. Malosio, V. Schmieden, and H. Betz. 1 9 9 1 . Alternative splicing generates two isoforms of the a2 subunit of the inhibitory glycine receptor. F E B S l e t t . 283, 73-77.

Misteli, T. 2 0 0 0 . Cell biology of transcription and premRNA splicing, nuclear architecture meets nuclear function. J . C e l l S c i . 113, 1841-1849.

Lang, V., D. Mege, M. Semichon, H. Gary-Gouy, and G. Bismuth. 1 9 9 7 . A dual participation of ZAP-70 and src protein tyrosine kinases is required for TCR-induced tyrosine phosphorylation of Sam68 in Jurkat T cells. Eur. J. Immunol. 27, 3360-3367.

Misteli, T., J.F. Caceres, J.Q. Clement, A.R. Krainer, M.F. Wilkinson, and D.L. Spector. 1998. Serine phosphorylation of SR proteins is required for their recruitment to sites of transcription in vivo. J . C e l l . B i o l . 143, 297-307.

Lefebvre, S., L. Burglen, S. Reboullet, O. Clermont, P. Burlet, L. Viollet, B. Benichou, C. Cruaud, P. Millasseau, M. Zeviani, and et al. 1 9 9 5 . Identification and characterization of a spinal muscular atrophy-determining gene. C e l l . 80, 155-165.

Misteli, T., J.F. C_ceres, and D.L. Spector. 1 9 9 7 . The dynamics of a pre-mRNA splicing factor in living cells. Nature. 387, 523-527. Misteli, T., and D.L. Spector. 1 9 9 9 . RNA polymerase II targets pre-mRNA splicing factors to transcription sites in vivo. M o l . C e l l . 3, 697-705.

Lefebvre, S., P. Burlet, Q. Liu, S. Bertrandy, O. Clermont, A. Munnich, G. Dreyfuss, and J. Melki. 1 9 9 7 . Correlation between severity and SMN protein level in spinal muscular atrophy. N a t . G e n e t . 16, 265-269.

Moore, M.J. 2 0 0 0 . Intron recognition comes of AGe. N a t . S t r u c t . B i o l . 7, 14-16.

Lorson, C.L., E. Hahnen, E.J. Androphy, and B. Wirth. 1 9 9 9 . A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. P r o c . N a t l . A c a d . S c i . U S A. 96, 6307-6311.

Municio, M.M., J. Lozano, P. Sanchez, J. Moscat, and M.T. Diaz-Meco. 1 9 9 5 . Identification of heterogeneous ribonucleoprotein A1 as a novel substrate for protein kinase C zeta. H . B i o l . C h e m . 270, 15884-15891.

Manley, J.L., and R. Tacke. 1 9 9 6 . SR proteins and splicing control. Genes and Dev. 10, 1569-1579.

Nacharaju, P., J. Lewis, C. Easson, S. Yen, J. Hackett, M. Hutton, and S.H. Yen. 1 9 9 9 . Accelerated filament formation from tau protein with specific FTDP-17 missense mutations. F E B S l e t t . 447, 195-197.

Mattox, W., and B.S. Baker. 1 9 9 1 . Autoregulation of the splicing of transcripts from the transformer-2 gene of Drosophila. Genes and Dev. 5, 786-796.

Nakai, K., and H. Sakamoto. 1 9 9 4 . Construction of a novel database containing aberrant splicing mutations of mammalian genes. Gene. 14, 171-177.

Mattox, W., M.J. Palmer, and B.S. Baker. 1 9 9 0 . Alternative splicing of the sex determination gene transformer-2 is sex-specific in the germ line but not in the soma. G e n e s and Dev. 4, 789-805.

Nayler, O., C. Cap, and S. Stamm. 1 9 9 8 a . Human transformer-2-beta gene, Complete nucleotide sequence, chromosomal localisation and generation of a tissue specific isoform. G e n o m i c s . 53, 191-202.

Mayeda, A., D.M. Helfman, and A.R. Krainer. 1 9 9 3 . Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and premRNA splicing factor SF2/ASF [published erratum appears in Mol Cell Biol 1993 Jul;13(7), 4458]. M o l . C e l l . B i o l . 13, 2993-3001.

Nayler, O., A.M. Hartmann, and S. Stamm. 2 0 0 0 . The ERrepeat protein YT521-B localizes to a novel subnuclear compartment. J . C e l l B i o l . , in press. Nayler, O., F. Schnorrer, S. Stamm, and A. Ullrich. 1 9 9 8 b . The cellular localization of the murine serine/argininerich protein kinase CLK2 is regulated by serine 141 autophosphorylation. J . B i o l . C h e m . 273, 3434134348.

Mayeda, A., and A.R. Krainer. 1 9 9 2 . Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2. C e l l . 68, 365-375. McCracken, S., N. Fong, K. Yankulov, S. Ballantyne, G. Pan, J. Greenblatt, S.D. Patterson, M. Wickens, and D.L. Bentley. 1 9 9 7 . The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature. 385, 357-361.

Nayler, O., S. Stamm, and A. Ullrich. 1 9 9 7 . Characterisation and comparison of four SR protein kinases. B i o c h e m . J . 326, 693-700. Nayler, O., W. Str채tling, J.-P. Bourquin, I. Stagljar, L. Lindemann, H. Jasper, A.M. Hartmann, F.O. Fackelmeyer, A. Ullrich, and S. Stamm. 1 9 9 8 c . SAF-B couples

McInnes, B., M. Potier, N. Wakamatsu, S.B. Melancon, M.H. Klavins, S. Tsuji, and D.J. Mahuran. 1 9 9 2 . An unusual splicing mutation in the HEXB gene is associated with

Gene Therapy and Molecular Biology Vol 5, page 27

transcription and pre-mRNA splicing to SAR/MAR elements. N u c l e i c A c i d s R e s . 26, 3542-3549.

and Saccharomyces cerevisiae pre-mRNA splicing factor SF1. RNA. 4, 551-565.

Neel, H., P. Gondran, D. Weil, and F. Dautry. 1 9 9 5 . Regulation of pre-mRNA processing by src. C u r r . B i o l . 5, 413-422.

Rave-Harel, N., E. Kerem, M. Nissim-Rafinia, I. Madjar, R. Goshen, A. Augarten, A. Rahat, A. Hurwitz, A. Darvasi, and B. Kerem. 1 9 9 7 . The molecular basis of partial penetrance of splicing mutations in cystic fibrosis. A m . J. Hum. Genet. 60, 87-94.

Neubauer, G., A. King, J. Rappsilber, C. Calvio, M. Watson, P. Ajuh, J. Sleeman, A. Lamond, and M. Mann. 1 9 9 8 . Mass spectometry and EST-database searching allows characterization of the multiprotein spliceosome complex. N a t . G e n e t . 20, 46-50.

Richard, S., D. Yu, K.J. Blumer, D. Hausladen, M.W. Olszowy, P.A. Connelly, and A.S. Shaw. 1 9 9 5 . Association of p62, a multifunctional SH2- and SH3domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1. M o l . C e l l . B i o l . 15, 186-197.

Nikolakaki, E., G. Simos, S.D. Georgatos, and T. Giannakouros. 1 9 9 6 . A nuclear envelope-associated kinase phosphorylates arginine-serine motifs and modulates interactions between the lamin B receptor and other nuclear proteins. J . B i o l . C h e m . 271, 83658372.

Rossi, F., E. Labourier, T. Forne, G. Divita, J. Derancourt, J.F. Riou, E. Antione, G. Cathela, C. Brunel, and J. Tazi. 1 9 9 6 . Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I. Nature. 381, 80-82.

Nissim-Rafinia, M., O. Chiba-Falek, G. Sharon, A. Boss, and B. Kerem. 2 0 0 0 . Cellular and viral splicing factors can modify the splicing pattern of CFTR transcripts carrying splicing mutations. H u m . M o l . G e n e t . 2000, 17711778.

Sanchez-Margalet, V., and S. Najib. 1 9 9 9 . p68 Sam is a substrate of the insulin receptor and associates with the SH2 domains of p85 PI3K. FEBS Lett. 455, 307-310. Schmajuk, G., H. Sierakowska, and R. Kole. 1 9 9 9 . Antisense oligonucleotides with different backbones. Modification of splicing pathways and efficacy of uptake. J . B i o l . C h e m . 274, 21783-21789.

O'Malley, K.L., S. Harmon, M. Moffat, A. Uhland-Smith, and S. Wong. 1 9 9 5 . The human aromatic L-amino acid decarboxylase gene can be alternatively spliced to generate unique protein isoforms. J . N e u r o c h e m . 65, 2409-2416.

Screaton, G.R., M.V. Bell, D.G. Jackson, F.B. Cornelis, U. Gerth, and J.I. Bell. 1 9 9 2 . Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. P r o c . N a t l . A c a d . S c i . 89, 12160-12164.

Papoutsopoulou, S., E. Nikolakaki, G. Chalepakis, V. Kruft, P. Chevaillier, and T. Giannakouros. 1 9 9 9 a . SR proteinspecific kinase 1 is highly expresed in testis and phosphorylates protamine 1. N u c l e i c A c i d s R e s . 27, 2972-2980.

Screaton, G.R., J.F. Caceres, A. Mayeda, M.V. Bell, M. Plebanski, D.G. Jackson, J.I. Bell, and A.R. Krainer. 1 9 9 5 . Identification and characterization of three members of the human SR family of pre-mRNA splicing factors. EMBO J. 14, 4336-4349.

Papoutsopoulou, S., E. Nikolakaki, and T. Giannakouros. 1 9 9 9 b . SRPK1 and LBR protein kinases show identical substrate specificities. B i o c h e m . B i o p h y s . R e s . Commun. 255, 602-607.

Sell, S.M., D. Reese, and V.M. Ossowski. 1 9 9 4 . Insulininducible changes in insulin receptor mRNA splice variants. J . B i o l . C h e m . 269, 30769-30772.

Pearn, J. 1 9 8 0 . Classification of spinal muscular atrophies. Lancet. 8174, 919-922. Philips, A.V., and T.A. Cooper. 2 0 0 0 . RNA processing and human disease. C e l l M o l . L i f e S c i . 57, 235-249.

Sharma, E., F. Zhao, A. Bult, and P.J. Lombroso. 1 9 9 5 . Identification of two alternatively spliced transcripts of STEP, a subfamily of brain-enriched protein tyrosine phosphatases. M o l . B r a i n R e s . 32, 87-93.

Polydorides, A.D., H.J. Okano, Y.Y.L. Yang, G. Stefani, and R.B. Darnell. 2 0 0 0 . A brain-enriched polypyrimidine tract-binding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing. P r o c . N a t l . A c a d . S c i . U S A. 97, 6350-6355.

Sierakowska, H., L. Gorman, S.H. Kang, and R. Kole. 2 0 0 0 . Antisense oligonucleotides and RNAs as modulators of pre-mRNA splicing. M e t h o d s E n z y m o l . 313, 506521.

Prasad, J., K. Colwill, T. Pawson, and J. Manley. 1 9 9 9 . The protein kinase Clk/Sty directly modulates SR protein activity, both hyper- and hypophosphorylation inhibit splicing. M o l . C e l l . B i o l . 19, 6991-7000.

Sommer, B., K. Keinänen, T.A. Verdoorn, W. Wisden, N. Burnashev, A. Herb, M. Köhler, T. Takagi, B. Sakman, and P.H. Seeburg. 1 9 9 0 . Flip and Flop, A cell-specific functional switch in glutamate-operated channels of the CNS. S c i e n c e . 249, 1580-1585.

Rain, J.-C., Z. Rafi, Z. Rhani, P. Legrain, and A. Krämer. 1 9 9 8 . Conservation of functional domains involved in RNA binding and protein-protein interactions in human

Stoss et al: RNA splicing and disease

splicing enhancer. P r o c . N a t l . A c a d . S c i . U S A . 94, 1148-1153.

Spillantini, M.G., and M. Goedert. 1 9 9 8 . Tau protein pathology in neurodegenerative diseases. TINS. 21, 428433.

Tarn, W.-Y., and J.A. Steitz. 1 9 9 7 . Pre-mRNA splicing, the discovery of a new spliceosome doubles the challenge. TIBS, 132-137.

Stamm, S., M.Q. Zhang, T.G. Marr, and D.M. Helfman. 1 9 9 4 . A sequence compilation and comparison of exons that are alternatively spliced in neurons. N u c l e i c A c i d s R e s . 22, 1515-1526.

Taylor, J.K., Q.Q. Zhang, J.R. Wyatt, and N.M. Dean. 1 9 9 9 . Induction of endogenous Bcl-xS through the control of Bcl-x pre-mRNA splicing by antisense oligonucleotides. N a t . B i o t e c h n o l . 17, 1097-1100.

Stamm, S., J. Zhu, K. Nakai, P. Stoilov, O. Stoss, and M.Q. Zhang. 2 0 0 0 . An alternative exon database (AEDB) and its statistical analysis. i n p r e s s .

Tazi, J., U. Kornst채dt, F. Rossi, P. Jeanteur, G. Cathala, C. Brunel, and R. L체hrmann. 1 9 9 3 . Thiophosphorylation of U1-70K protein inhibits pre-mRNA splicing. Nature. 363, 283.

Stojdl, D.F., and J.C. Bell. 1 9 9 9 . SR protein kinases, the splice of life. B i o c h e m . C e l l B i o l . 77, 293-298. Stoss, O., F.W. Schwaiger, T.A. Cooper, and S. Stamm. 1 9 9 9 a . Alternative splicing determines the intracellular localization of the novel muscle-specific protein Nop30 and its interaction with the splicing factor SRp30c. J . B i o l . C h e m . , 10951-10962.

Thanaraj, T.A. 2 0 0 0 . Positional characterisation of false positives from computational prediction of human splice sites. N u c l . A c i d s R e s . 28, 744-754. Toksoz, D., K.M. Zsebo, K.A. Smith, S. Hu, D. Brankow, S.V. Suggs, F.H. Martin, and D.A. Williams. 1 9 9 2 . Support of human hematopoiesis in long-term bone marrow cultures by murine stromal cells selectively expressing the membrane-bound and secreted forms of the human homolog of the steel gene product, stem cell factor. P r o c . N a t l . A c a d . S c i . U S A . 89, 73507354.

Stoss, O., P. Stoilov, A.M. Hartmann, O. Nayler, and S. Stamm. 1 9 9 9 b . The in vivo minigene approach to analyze tissue-specific splicing. B r a i n R e s . P r o t . 4 , 383-394. Strohmaier, C., B.D. Carter, R. Urfer, Y.-A. Barde, and G. Dechant. 1 9 9 6 . A splice variant of the neurotrophin receptor trkB with increased specificity for brain-derived neurotrophic factor. EMBO J. 15, 3332-3337.

van der Logt, C.P., P.H. Reitsma, and R.M. Bertina. 1 9 9 2 . Alternative splicing is responsible for the presence of two tissue factor mRNA species in LPS stimulated human monocytes. Thromb. Haemost. 67, 272-276.

Sugimoto, Y., M. Negishi, Y. Hayashi, T. Namba, A. Honda, A. Watabe, M. Hirata, S. Narumiya, and A. Ichikawa. 1 9 9 3 . Two isoforms of the EP3 receptor with different carboxyl-terminal domains. Identical ligand binding properties and different coupling properties with Gi proteins. J . B i o l . C h e m . 268, 2712-2718.

van Oordt, W., M.T. Diaz-Meco, J. Lozano, A.R. Krainer, J. Moscat, and J.F. Caceres. 2 0 0 0 . The MKK 3/6-p38signaling cascade alters the subcellular distribution of hnRNPA1 and modulates alternative splicing regulation. J . C e l l B i o l . 149, 307-316.

Suzuki, A., M. Yoshida, and E. Ozawa. 1 9 9 5 . Mammalian alpha1- and beta1-syntrophin bind to the alternative splice prone region of dystrophin COOH terminus. J . C e l l . B i o l . 128, 373-381.

Varani, G., and K. Nagai. 1 9 9 8 . RNA recognition by RNP proteins during RNA processing. Annu. R e v . B i o p h y s . B i o m o l . S t r u c t . 27, 407-445.

Swaroop, M., K. Bradley, T. Ohura, T. Tahara, M.D. Roper, L.E. Rosenberg, and J.P. Kraus. 1 9 9 2 . Rat cystathionine beta-synthase. Gene organization and alternative splicing. J . B i o l . C h e m . 267, 11455-11461.

Venables, J.P., D.J. Elliot, O.V. Makarova, E.M. Makarova, H.J. Cooke, and I.C. Eperon. 2 0 0 0 . RBMY, a probable human spermatogenisis factor, and other hnRNP G proteins interact with tra2beta and affect splicing. Hum. M o l . G e n e t . 9, 685-694.

Tabaczewski, P., H. Shirwan, K. Lewis, and I. Stroynowski. 1 9 9 4 . Alternative splicing of class Ib major histocompatibility complex transcripts in vivo leads to the expression of soluble Qa-2 molecules in murine blood. P r o c . N a t l . A c a d . S c i . U S A . 91, 1883-1887.

Venables, J.P., C. Vernet, S.L. Chew, D.J. Elliott, R.B. Cowmaedow, J. Wu, H.J. Cooke, K. Artzt, and I.C. Eperon. 1 9 9 9 . T-STAR/ETOILE, a novel relative of SAM68 that interacts with an RNA-binding protein implicated in spermatogenesis. H u m . M o l . G e n e t . 8 , 959-969.

Tabiti, K., L. Cui, V.J.S. Chhatwal, S. Moochhala, S.S. Ngoi, and C.J. Pallen. 1 9 9 6 . Novel alternative splicing predicts a secreted extracellular isoform of the human receptor-like protein-tyrosine-phosphatase lar. Gene. 175, 7-13.

Vernet, C., and K. Artzt. 1 9 9 7 . STAR, a gene family involved in signal transduction and activation of RNA. Trends Genet. 13, 479-484.

Tacke, R., Y. Chen, and J.L. Manley. 1 9 9 7 . Sequencespecific RNA binding by an SR protein requires RS domian phosphorylation, creation of an SRp40-specific

Wang, H.-Y., W. Lin, J.A. Dyck, J.M. Yeakley, Z. Songyang, L. Cantley, and X.-D. Fu. 1 9 9 8 . SRPK2, A differentially

Gene Therapy and Molecular Biology Vol 5, page 29

expressed SR protein-specific kinase involved in mediating the interaction and localization of pre-mRNA splicing factors in mammalina cells. J . C e l l B i o l . 140, 737-750.

Yuryev, A., M. Patturajann, Y. Litingtung, R.V. Joshi, C. Gentile, M. Gebara, and J.L. Corden. 1 9 9 6 . The Cterminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins. P r o c . N a t l . A c a d . S c i . U S A . 93, 69756980.

Wang, J., and J.L. Manley. 1 9 9 5 . Overexpression of the SR proteins ASF/SF2 and SC35 influences alternative splicing in vivo in diverse ways. RNA. 1, 335-346.

Zacharias, D.A., and E.E. Strehler. 1 9 9 6 . Change in plasma membrane Ca2+-ATPase splice-variant expression in response to a rise in intracellular Ca2+. C u r r . B i o l . 6, 1642-1652.

Wang, J., and E.M. Ross. 1 9 9 5 . The carboxyl-terminal anchorage domain of the turkey beta 1-adrenergic receptor is encoded by an alternatively spliced exon. J . B i o l . C h e m . 270, 6488-6495.

Zahler, A.M., K.M. Neugebauer, W.S. Lane, and M.B. Roth. 1 9 9 3 . Distinct functions of SR proteins in alternative pre-mRNA splicing. S c i e n c e . 260, 219-222.

Wang, J., Y. Takagaki, and J.L. Manley. 1 9 9 6 . Targeted disruption of an essential vertebrate gene, ASF/SF2 is required for cell viability. G e n e s D e v . 15, 2588-2599.

Zhang, K., E.E. Max, H.-K. Cheah, and A. Saxon. 1 9 9 4 . Complex alternative RNA splicing of epsilonimmunoglobulin transcripts produces mRNAs encoding four potential seccreted protein isoforms. J . B i o l . C h e m . 269, 456-462.

Wang, L.L., S. Richard, and A.S. Shaw. 1 9 9 5 . p62 association with RNA is regulated by tyrosine phosphorylation. J . B i o l . C h e m . 270, 2010-2013. Wang, X., S. Bruderer, Z. Rafi, J. Xue, P.J. Milburn, A. Kr채mer, and P.J. Robinson. 1 9 9 9 . Phosphorylation of splicing factor SF1 on Ser20 by cGMP-dependent protein kinase regulates spliceosome assembly. E M B O J . 18, 4549-4559. Weighardt, F., G. Biamonti, and S. Riva. 1 9 9 6 . The role of heterogeneous nuclear ribonucleoproteins (hnRNP) in RNA metabolism. B i o E s s a y s . 18, 747-756. Wetherall, D.J., and F.H.C. Clegg. 1981. The thalassemia syndromes. Blackwell Scientific Publications, Oxford. Wilton, S.D., F. Lioyd, K. Carville, S. Fletcher, K. Honeyman, S. Agrawal, and R. Kole. 1 9 9 9 . Specific removal of the nonsense mutation from the mdx dystrophin mRNA using antisense oligonucleotides. Neuromuscul. Disord. 9, 330-338. Wirth, B. 2 0 0 0 . An update of the mutation spectrum of the survival motor neuron gene (SMN1) in autosomal recessive spinal muscular atrophy (SMA). Hum. Mutat. 15, 228-237. Xiao, S.-H., and J.L. Manley. 1 9 9 8 . Phosphorylationdephosphorylation differentially affects activities of splicing factor ASF/SF2. EMBO J. 17, 6359-6367. Xie, J., and D.P. McCobb. 1 9 9 8 . Control of alternative splicing of potassium channels by stress hormones. S c i e n c e . 280, 443-446. Xing, S., Q. Tong, T. Suzuki, and S.M. Jhiang. 1 9 9 4 . Alternative splicing of the ret proto-oncogene at intron 4. B i o c h e m . B i o p h y s . R e s . Commun. 205, 15261532. Yeakley, J.M., H. Tronchere, J. Olesen, J.A. Dyck, H.Y. Wang, and X.D. Fu. 1 9 9 9 . Phosphorylation regulates in vivo interaction and molecular targeting of serine/arginine-rich pre-mRNA splicing factors. J . C e l l . B i o l . 145, 447-455.

Stefan Stamm

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Gene Ther Mol Biol Vol 5, 31-37, 2000

Th2-type immune response induced by a phage clone displaying a CTLA4-binding domain mimicmotif Research Article

Yasuhiro Kajihara, Shuhei Hashiguchi , Yuji Ito, and Kazuhisa Sugimura* Department of Bioengineering, Faculty of Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan

_____________________________________________________________________________________ *Correspondence: Kazuhisa Sugimura, Department of Bioengineering, Faculty of Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan, Phone: +81-99-285-8345; Fax: +81-99-258-4706, E-mail: kazu@be.kagoshima-u.ac.jp Key words: CTLA-4, Th2, immune deviation, peptide mimic, phage library, molecular design, vaccine Received: 4 May 2000; accepted: 21 September 2000

Summary We have recently isolated a phage clone, F2, which displays the CTLA4-binding domain mimic from a phage display library. To investigate the in vivo effects of an F2 motif on the regulation of immune responses, we immunized Balb/c mice intraperitoneally with varying doses o f an F 2 phage i n a phosphate buffered saline and followed the resulting antibody and cytokine responses. It was shown that the F2 phage enhanced the IgG antibody response to phage particles in comparison to control phages that were randomly selected from the library. When the antigen specificity o f the induced antibody response was examined, the production of an anti-g3p antibody was preferentially increased while an anti-g8p antibody was slightly down-regulated by the immunization of F2 in comparison to control phage clones. The increase of an anti-g3p antibody response was found in the isotype of IgG1 but not in the IgM or IgG2a. When the cytokine production was examined by culturing spleen cells f r o m t h e s e m i c e u n d e r s t i m u l a t i o n w i t h a n t i - C D 3 m A b , IL-4 production was approximately twice higher in the F2-primed cells than in the L4-primed cells while IFN-! production was higher in L4p r i m e d c e l l s t h a n i n t h e F 2 - p r i m e d c e l l s . Thus, these results suggested that the F 2 phage clone bearing g3p with the CTLA4-binding domain mimic-motif induced the Th2-type response when compared to control phage clones. than had initially been thought (Krummel and Allison, 1995). In principle, the interactions of co-stimulatory molecules were possible with four kinds of combinations: CD28-CD80, CD28-CD86, CTLA4-CD80, and CTLA4CD86. However, the functional difference between CD80 and CD86 is still obscure although considerable interest has focused on the possible role of the CD80 and CD86 co-stimulatory molecules expressed on antigen-presenting cells (APC) in skewing CD4+ T cells to either the Th1 or Th2 phenotype (Hathcock et al, 1994; Schweitzer et al, 1997; Manickasingham et al, 1998). Recently, we selected phage clones from a phage display library by employing a CTLA4-conformation recognizing a monoclonal antibody (mAb) (Fukumoto et al, 1998). A phage clone, F2, is specifically recognized with the anti-CTLA4 mAb and able to bind to CD80. The F2 motif consists of the unique 15-amino-acid sequence

I. Introduction T-cell co-stimulatory receptors CD28 and CTLA4 deliver opposite signals on T-cell activation, mediating augmentation and inhibition of T-cell responses, respectively (Linsley, 1995; Thompson, 1995; Bluestone, 1997; Thompson and Allison, 1997). These two receptors use the same ligands, CD80 (B7-1) and CD86 (B7-2), expressed on the antigen-presenting cells (Azuma et al, 1993; Freeman et al, 1993). The kinetic study on the expression of these molecules suggested that CD28 may be responsible for CD86, and CTLA4 is responsible for CD80, respectively, since CTLA4 and CD80 expression reaches maximal levels 2-3 days after antigenic stimulation (Hathcock et al, 1994; Schweitzer et al, 1997). However, CTLA4 may have more of a role in regulating T-cell responses at earlier stages in the process

Kajihara et al: Th2-type immune response

with an internal disulfide bond and is inserted in gene 3 proteins (g3p), which display only three to five copies in contrast to approximately 2700 copies of gene 8 proteins (g8p) per fd phage. The HPLC-purified g3p (F2-g3p) is also recognized with anti-CTLA4 mAb but not anti-CD28 mAb and binds to CD80 but not to CD86. When hen egg lysozyme (HEL)-primed lymph node cells were stimulated with HEL in the presence of the F2-g3p in vitro, cell proliferation was highly potentiated (Fukumoto et al, 1998). In the absence of antigenic stimulation, the F2-g3p induced no T-cell proliferation, indicating the costimulatory nature of the F2-g3p. Thus, the F2 motif represents a peptide mimic of the CTLA4-binding domain. In this study, we examined the effect of F2 phage as an immunogen on the anti-phage immune response in vivo. When the phages were administered intraperitoneally (i.p.) in mice in the form of a phosphate-buffered saline (PBS) solution, F2 phage induced an anti-phage antibody response two to three times higher than that of the control phage. In this augmented response, the anti-g3p antibody production was predominantly increased, whereas the antig8p antibody production was rather decreased. The isotype of the increased antibody was IgG1 but not IgM or IgG2a. When the spleen cells were stimulated with the immobilized anti-CD3 mAb in vitro, cells derived from F2-immunized mice showed the augmented response in IL-

4 production while a weaker response was shown in IFN-! production in comparison to those of the control phageprimed mice. Thus, an F2 motif that interferes with the interaction of CTLA4 with CD80 but not CD86 preferentially generated the Th2-type immune response in vivo, suggesting that the predominant interaction of CD86 with CD28 in the absence of CTLA4/CD80 signaling may skew the immune response to the Th2-type in vivo.

II. Results A. In vivo antibody response of Balb/c mice immunized with fd phage clones Balb/c mice were administered i.p. with a PBS solution containing varying doses of wild type (wt), F2 or a control phage (L4), which was randomly selected from the library. The anti-phage IgG antibody responses were followed by using control phage (K7)-coated plastic plates (K7-ELISA). As shown in Figure 1, 50 µg of F2 phage induced a marked anti-phage antibody response when compared to those of L4 and wt clones. In the case of an L4 or wt clone, 50 and 5 µg of phage induced almost comparable IgG antibody responses, while 0.5 µg barely induced a response. In the next experiments, mice were

F i g u r e 1 . Antibody responses induced with intraperitoneal administration of PBS solution containing varying doses of phage clones: Balb/c mice were administered i.p. with 0.5, 5, and 50 µg of either F2, L4, or a wild-type (wt) phage clone, in PBS. An antiphage IgG antibody was measured by a control phage (K7)-coated ELISA every week after the immunization. K7 was randomly selected from the phage library. Each line indicates a response pattern of a mouse. The serum was tested at a dilution of 1: 1080.

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administered 5 µg of a wt, L4, or F2 phage clone, and the antibody production was estimated by either the wt or K7 phage-coated ELISA plate. The wt-ELISA estimated the amount of anti-phage antibodies except for the anti-g3p antibody because the wt lacks g3p due to a frame shift, while K7-ELISA measured the amount of whole antiphage antibodies including the anti-g3p antibody. When the antisera were assayed with K7-ELISA, F2 induced an IgG response that was approximately four times higher in comparison to those of the L4 or wt (Figure 2A). In contrast, when the same antisera were estimated by wtELISA, the magnitudes of antibody responses were almost at the same levels among these groups (Figure 2B). These results suggested that the amplified IgG response by F2-immunization might be directed to the specificity against g3p but not to the other constituents of phage in comparison to the wt or L4-immunization.

F i g u r e 3 . Preferential enhancement of an anti-g3p antibody response by the introduction of the F2 motif: Balb/c mice immunized with 50 µg of various phage clones. Sera were collected four weeks after immunization. The amount of antiphage IgG antibodies was measured by phage (K7 " or wt )coated ELISA (panel A: serum dilution:1/1080) or purified g3p-( ) or g8p ( ! )-coated ELISA (panel B: serum dilution:1/500) plates. Each value represents the mean of three mice per group ± S.E.

B. F2 motif enhanced the antibody response to g3p but not to g8p molecules In order to determine the antigen specificity of antibodies amplified by the F2 phage clone, the antisera were assayed by using both phage clone- and HPLCpurified g3p/g8p-coated plastic plates. The sera from the L4-immunized mice showed a value by K7-ELISA that was as high as the value of the wt-ELISA (Figure 3A).

In contrast, the sera of the F2-immunized mice showed a value with the K7-ELISA that was approximately twice as high as that shown by the wtELISA. These sera were assayed by g3p or g8p-coated plates (Figure 2B). The sera from the L4-immunized mice reacted to both g3p and g8p with a slightly higher value of the anti-g8p antibody, while the sera of the F2immunized mice exhibited a value to g3p that was approximately twice as high as that to g8p. The sera of the wt-immunized mice predominantly reacted to g8p but not to g3p (data not shown). Thus, the F2 motif influenced the immune response to its carrier protein molecule but not to other phage proteins associating with g3p.

C. IgG1 production was preferentially augmented by the F2 motif

Figure 2. F2 induced the augmented anti-phage IgG antibody response relative to the control phage clones: Balb/c mice were immunized i.p. with 5 µg of various phage clones (F2 !, L4 ! ,WT ") in PBS. Antibodies were measured by a control phage (K7)-coated ELISA (panel A) and wt phage-coated ELISA (panel B). The serum was tested at the dilution of 1: 1080.

Next, we examined the immunoglobulin isotype on the amplified anti-g3p antibody response induced by the introduction of the F2 motif. As shown in Figure 4, L4 and F2 induced almost the same kinetic patterns of the IgM antibody responses. However, the IgG1 isotype was preferentially produced in the augmented anti-g3p antibody response by the immunization of F2. The IgG2a isotype was barely detected by the immunization of F2 or L4. These results suggested that the F2 motif might skew the immune response to the Th2-type. The F2 motif does not appear to accelerate the time course of the IgG antibody response because two weeks after the F2-immunization, only the IgM and not the IgG1 was significantly produced.

Kajihara et al: Th2-type immune response

D. Cytokine production affected by the F2 motif in g3p

proliferation in vitro was much more remarkable as described in previous studies (Fukumoto et al, 1998a, 1998b). The augmented response was shown in the production of the anti-g3p but not in the anti-g8p antibody (F i g u r e 3 ). The anti-g8p antibody response was rather decreased by the introduction of the F2 motif in the g3p molecules. We have demonstrated in in vitro studies that the addition of F2-g3p augments the proliferation of the hen egg lysozyme (HEL)-primed lymphocytes when cells are stimulated with HEL, indicating that the F2 motif is not necessarilly fused with the antigen (Fukumoto et al, 1998). The F2-g3p inhibited the interaction of CD80/CTLA4 and did not inhibit the interaction of CD86/CTLA4 or CD86/CD28 (Fukumoto et al, 1998). It is, therefore, conceivable that the fusion of the F2 motif with the antigen, g3p, results in their efficient binding to the g3p-specific T lymphocytes in vivo . The enhancement of this response was markedly demonstrated by the amount of IgG1 but not IgM or IgG2a isotypes (Figure 4). A characteristic cytokine profile of the F2-primed spleen cells was detected by stimulating the cells with an anti-CD3 mAb, but it was not with the F2 or L4 phage. These results may be attributed to the very small size of the F2-responding populations that were generated. The IL-4 produced from these F2-responding T cells may stimulate the generation of the bystander Th2type T-cell populations. This amplifying process might enable us to detect IL-4 or IFN-! in our cell culture system when cells were stimulated with anti-CD3 mAb. Thus, these results suggested that the inhibition of the CTLA4/CD80 interaction with a peptide mimic of the CTLA4-binding domain may have skewed the response to the Th2 pathway, implying that the predominant interaction of CD86 with CD28, in the absence of CTLA4/CD80 signaling, preferentially induces the Th2 immune response in vivo.

In order to examine the effect of the F2 motif on the T-cell activation, spleen cells derived from L4- or F2immunized mice were stimulated with anti-CD3 mAb in vitro, and IL-4 and IFN-! that were produced in supernatants were measured by ELISA. As shown in Figure 5, the control phage of L4-primed cells showed a significant amount of IFN-! production and a very weak IL-4 production. However, in the case of the F2-primed cells, the IFN-! production was rather decreased, and in contrast, the IL-4 production was augmented in comparison to the L4-primed cells. These results suggested that the anti-g3p antibody response might be skewed toward Th2-type immune responses by the insertion of the F2 motif. We carried out the same kind of experiments using L4 or F2 phage as an in vitro-stimulant instead of anti-CD3 mAb. In these cases, we failed to detect a significant production of IFN-! or IL-4 in culture supernatants (data not shown).

III. Discussion A phage clone, F2, was isolated from a phage display library by using a CTLA4-conformation recognizing a monoclonal antibody (Fukumoto et al, 1998). The HPLCpurified F2-g3p exhibited the ability to bind to CD80 and inhibited the interaction of CTLA4 and CD80. These characteristics of F2-g3p appeared to result in the marked augmentation of the antigen-stimulated T-cell proliferation in vitro (Fukumoto et al, 1998). In this study, we characterized the immune response to fd phage by simply administering the F2 phage intraperitoneally without an adjuvant to Balb/c mice. We demonstrated here that the F2 motif augmented the antiphage antibody responses approximately two to three times higher than those of the control phage-primed mice, although the F2-mediated augmentation on T-cell

F i g u r e 4 . The F2 motif augmented the anti g3p IgG1 antibody response: Balb/c mice immunized with 50 Âľg of F2 or L4 phage clones. Anti-g3p (upper panel) or -g8p antibodies (lower panel) were measured on the immunoglobulin subclasses, which were detected by AP-anti-mouse IgM (panel A), IgG1 (panel B), and IgG2a antibody (panel C). Sera were tested at a dilution of 1:500. Each value represents the mean of five mice per group Âą S.E.

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F i g u r e 5 . IL-4 production was augmented in F2-primed spleen cells relative to L4-primed spleen cells in vitro: Spleen cells were obtained from Balb/c mice, which had been immunized i.p. with 50 µg F2 or L4 phage in PBS seven weeks before. The cells were cultured in the anti-CD3 Ab-immobilized culture plates (10 µg/ well) for 48 hr (IL-4) or 72 hr (IFN-!). Murine IFN-! or IL-4 in supernatants was measured using sandwich ELISA. Each value represents the mean of five mice per group ± S.E.

Regarding these results, studies by Freeman and coworkers indicated that CD86, but not CD80, costimulation during the anti-CD3 Ab-mediated CD4+ T-cell activation skewed the cells to produce IL-4, suggesting that CD80 and CD86 may provide distinct signals during the development of CD4 + T-cell responses (Freeman et al, 1995). Consistent with this, studies by Kuchroo and coworkers indicated that the administration of anti-CD86 Abs to mice during priming with proteolipid protein for induction of experimental autoimmune encephalomyelitis (EAE) skewed CD4+ T-cell development to the Th1 phenotype and exacerbated disease, whereas the administration of anti-CD80 Ab skewed CD4+ T-cell development to the Th2 phenotype and the induction of disease was blocked or decreased (Kuchroo et al, 1995). These results have suggested that CD4+ T-cell engagement of CD80 may direct development to the Th1 phenotype and engagement of CD86 may direct development to the Th2 phenotype. A similar finding was reported by Khoury and Gallon et al. using a derivative of CTLA4, CTLA4IgY100F (Khoury et al, 1996). This molecule binds to CD80 but not to CD86, which is the same characteristic as F2-g3p. These reagents are able to avoid the potential of signaling, which is induced by the addition of anti-CTLA4 antibodies. Using the CTLA4IgY100F in the induction of experimental autoimmune encephalomyelitis (EAE), they showed that CD28-CD80 interaction may lead the response

to the Th1 pathway. However, other studies have reported that the qualitative differences were not detected in the capacities of murine CD80 and CD86 to induce IL-4 production (Natesan et al, 1996). In the case of Schweizer et al, they showed that CD86 has a more important role than CD80 in initiating antibody responses in the absence of an adjuvant and that CD86, and to a lesser extent CD80, makes significant contributions to the production of both IL-4 and IFN-! . CD80 and CD86 contribute to the magnitude of Tcell activation, but they do not appear to selectively regulate Th1 versus Th2 differentiation (Schweitzer et al, 1997; Schweitzer and Sharpe, 1998). A recent study by Anderson et al. shows that a CTLA4 blockade can enhance or inhibit the clonal expansion of different T cells that respond to the same antigen, depending on both the T-cell activation state and the strength of the T-cell receptor signal delivered during T-cell stimulation (Anderson et al, 2000). Thus, it is still unclear if there is any difference in the role of CD80 as opposed to CD86 on the interaction with CTLA4. In our case, we have shown here that the selective inhibitor of CD80-binding, F2-g3p induced the skewing toward a Th2-type response when administered in mice in vivo. The influences of the F2-motif on the generation of cytotoxic T cells or delayed-type hypersensitivity-responsible T cells remains to be investigated.

Kajihara et al: Th2-type immune response

10 5 /0.2ml/well) in flat-bottomed 96-well plates (Iwaki Glass, Tokyo) for three days and pulsing cells with 0.5 µCi 3 Hthymidine (Amersham, St. Louis, MO) for the final 18 hr.

As functional motifs of co-stimulatory molecules appear to manipulate the immune responses by introducing it into the target molecule, this strategy may lead us to novel approaches to gene therapy and DNA vaccine developments.

Acknowledgments This work was partly supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, and Culture.

IV. Materials and Methods A. Mice and antibodies Balb/c mice (female) were purchased from Nihon SLC Co. (Fukuoka). Alkaline phosphatase (AP)-conjugated antimouse IgG, !1, !2a, and antibody were obtained from ZYMED (San Francisco, CA). AP-conjugated anti-µ antibody was purchased from Southern Biotechnology Associates, Inc. (Birmingham, AL). Anti-CD3 mAb (cat. # 01081D) was purchased from PharMingen (San Diego, CA).

Reference Anderson DE, Bieganowska KD, Bar-Or A, Oliveira EM, Carreno B, Collins M, and Hafler DA, (2 0 0 0 ) Paradoxical inhibition of T-cell function in response to CTLA-4 blockade; heterogeneity within the human T-cell population. Nat Med 6, 211-4. Azuma M, Ito D, Yagita H, Okumura K, Phillips JH, Lanier LL, and Somoza C, (1 9 9 3 ) B70 antigen is a second ligand for CTLA-4 and CD28. Nature 366, 76-9. Bluestone JA, (1 9 9 7 ) Is CTLA-4 a master switch for peripheral T cell tolerance? J Immunol 158, 1989-93. Freeman GJ, Boussiotis VA, Anumanthan A, Bernstein GM, Ke XY, Rennert PD, Gray GS, Gribben JG, and Nadler LM, (1 9 9 5 ) B7-1 and B7-2 do not deliver identical costimulatory signals, since B7- 2 but not B7-1 preferentially costimulates the initial production of IL- 4. Immunity 2, 523-32. Freeman GJ, Gribben JG, Boussiotis VA, Ng JW, Restivo VA, Jr, Lombard LA, Gray GS, and Nadler LM, (1 9 9 3 ) Cloning of B7-2: a CTLA-4 counter-receptor that costimulates human T cell proliferation [see comments]. S c i e n c e 262, 909-11. Fukumoto, T, Torigoe, N, Kawabata, S, Murakami, M, Uede, T, Nishi, T, Ito, Y, and Sugimura, K, (1 9 9 8 a ) Peptide mimics of the CTLA4-binding domain stimulate T-cell proliferation [see comments]. Nat B i o t e c h n o l 16, 267-70. Fukumoto, T, Torigoe, N, Ito, Y, Kajiwara, Y, and Sugimura, K, (1 9 9 8 b ) T cell proliferation-augmenting activities of the gene 3 protein derived from a phage library clone with CD80-binding activity. J Immunol 161, 6622-8. Hathcock KS, Laszlo G, Pucillo C, Linsley P, and Hodes RJ, (1 9 9 4 ) Comparative analysis of B7-1 and B7-2 costimulatory ligands: expression and function. J Exp Med 180, 631-40. Khoury SJ, Gallon L, Verburg RR, Chandraker A, Peach R, Linsley PS, Turka LA, Hancock WW, and Sayegh MH, (1996) Ex vivo treatment of antigen-presenting cells with CTLA4Ig and encephalitogenic peptide prevents experimental autoimmune encephalomyelitis in the Lewis rat. J Immunol 157, 3700-5. Krummel MF, and Allison JP, (1 9 9 5 ) CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation [see comments]. J Exp Med 182, 459-65. Kuchroo VK, Das MP, Brown JA, Ranger AM, Zamvil SS, Sobel RA, Weiner HL, Nabavi N, and Glimcher LH, (1 9 9 5 ) B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy. C e l l 80, 707-18. Linsley PS, (1 9 9 5 ) Distinct roles for CD28 and cytotoxic T lymphocyte-associated molecule- 4 receptors during T cell activation? [comment]. J Exp Med 182, 289-92. Manickasingham SP, Anderton SM, Burkhart C, and Wraith DC (1 9 9 8 ) Qualitative and quantitative effects of

B. Phage proteins The fd (fUSE5) phage clones were isolated and the phage proteins were purified as described previously (Fukumoto et al, 1998; Nishi et al, 1996). Briefly, the fd phages (7mg/ml) were incubated with 1% sodium dodecyl sulfate (SDS) at 37˚C for 20 min. The g3p and g8p were purified by size-exclusion chromatography on a HiLoad superdex 200 (26/60) column (Pharmacia, Uppsala, Sweden) as described. The F2 phage displays the motif of GFVCSGIFAVGVGGRC at the fifth position of the N-terminal of g3p molecule (Fukumoto et al, 1998).

C. ELISA ELISA was performed as described previously (Fukumoto et al, 1998; Fukumoto et al, 1998). Plastic plates (Nunc) were coated with phages (4 x 109 transducing unit [TU]/ 40 µl/ well) or phage proteins (30 ng / 40 µl/well) in 50 mM Tris HCl, pH 7.5 and 150 mM NaCl (TBS) containing 0.02% NaN3 . Blocking was done by using 350 µl of 1% bovine serum albumin (BSA, Sigma, St. Louis, MO). The plates were washed five times with TBS containing 0.05% Tween 20 (TBS/Tween) and once with TBS. After the incubation with varying concentrations of mouse antiserum for 1 hr at 4˚C, APconjugated anti mouse IgG, !1, !2a, or µ antibody was added at a dilution of 1:250. The substrate (85 µl) consisted of 1 mg / ml p-nitrophenolphosphate (Wako Co., Osaka), and 10% diethanolamine (Wako Co., Osaka) in TBS. Absorbance was read at 405 nm by a microplate photometer (InterMed NJ2300, Tokyo). All assays were carried out after the dilution rates of sera were determined on their linearity for ELISA. The cytokine ELISA was performed using the IL-4 plate (cat. # M4000) and IFN-!-plate (cat. # MIF00) of R&D systems Co. (Minneapolis, MN), according to the manufacturer's description. Statistical analysis was carried out by a Student's t-test.

D. Cytokine production assay The cell culture was carried out as described (Fukumoto et al, 1998; Fukumoto et al, 1998). Briefly, cells (2 x 10 6 /1ml/well of 24 well-plate) were stimulated with varying doses of phage clones, anti-CD3 mAb (10 µg/ml). The supernatants were harvested 72 hr later for the assay on the cytokine production. In parallel with these cultures, T-cell proliferation was monitored by culturing cells (1.5 x

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CD28/B7-mediated costimulation on naive T cells in vitro. J Immunol 161, 3827-35. Natesan M, Razi-Wolf Z, and Reiser H (1 9 9 6 ) Costimulation of IL-4 production by murine B7-1 and B7-2 molecules. J Immunol 156, 2783-91. Nishi, T, Budde, R. J, McMurray, J. S, Obeyesekere, N. U, Safdar, N, Levin, V. A, and Saya, H, (1 9 9 6 ) Tightbinding inhibitory sequences against pp60 (c-src) identified using a random 15-amino-acid peptide library. FEBS Lett 399, 237-40.

lacking expression of CD80 or CD86. J Immunol 158, 2713-22. Schweitzer AN, and Sharpe AH (1 9 9 8 ) Studies using antigenpresenting cells lacking expression of both B7-1 (CD80) and B7-2 (CD86) show distinct requirements for B7 molecules during priming versus restimulation of Th2 but not Th1 cytokine production. J I m m u n o l 161, 276271. Thompson CB, (1 9 9 5 ) Distinct roles for the costimulatory ligands B7-1 and B7-2 in T helper cell differentiation? C e l l 81, 979-82. Thompson CB, and Allison JP, (1 9 9 7 ) The emerging role of CTLA-4 as an immune attenuator. Immunity 7, 445-50.

Schweitzer, A. N, Borriello, F, Wong, R. C, Abbas, A. K, and Sharpe, A. H, (1 9 9 7 ) Role of costimulators in T cell differentiation: studies using antigen- presenting cells

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Gene Therapy and Molecular Biology Vol 5, page 39

Gene Ther Mol Biol Vol 5, 39-46, 2000

Segregation of partly melted molecules and its application to the isolation of methylated CpG islands in human cancer cells Review Article

Masahiko Shiraishi 1*, Leonard S. Lerman2, Adam J. Oates1, Xu Li1,3, Ying H. Chuu1, Azumi Sekiguchi1, and Takao Sekiya1 1

DNA Methylation and Genome Function Project, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku 104-0045, Tokyo, Japan, and 2Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. 3 Present address: Department of Biochemistry, Nantong Medical College, Jiangsu 226001, P. R. China

_____________________________________________________________________________________ * Correspondence: Masahiko Shiraishi, DNA Methylation and Genome Function Project, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku 104-0045, Tokyo, Japan; Tel: +81-3-3542-2511 (ext. 4809); Fax: +81-3-5565-9535; E-mail: mshirais@ncc.go.jp Key words: denaturing gradient gel electrophoresis, DNA methylation, methylated DNA binding column, adenocarcinomas of the lung, epigenetics Received: 12 Jume 2000; accepted: 26 Jume 2000

Summary Segregation of partly melted molecules is a convenient and efficient method for isolation of DNA fragments associated with CpG islands. DNA fragments digested with restriction endonucleases are subjected to denaturing gradient gel electrophoresis. DNA fragments derived from CpG islands are preferentially retained in the gel after prolonged field exposure because of their lower rate of strand dissociation. An independent technique, methylated-DNA-binding column chromatography, permits separation of DNA fragments on the basis of the number of methyl-CpG sequences in the fragment, and it enables separation of methylated CpG islands from those that are not methylated. Segregation of partly melted molecules and methylated-DNA-binding column chromatography were successfully combined to isolate CpG islands methylated in human adenocarcinomas of the lung. The methylated CpG island library will be valuable in order to elucidate epigenetic process in carcinogenesis. promoter elements. Although methylation often results in gene silencing, CpG islands are not methylated in all somatic tissues, even where associated genes are not expressed. A small fraction of CpG islands are methylated in normal somatic cell DNA. So far as is known, CpG islands on inactive X chromosome and those associated with imprinted genes are methylated in normal DNA, genes associated with these CpG islands are transcriptionally silenced. Some tumor suppressor genes are known to be inactivated by CpG island methylation (for a recent review, see Baylin et al, 1998). Denaturing gradient gel electrophoresis (DGGE) takes advantage of the change in electrophoretic mobility of DNA fragments accompanying partial melting, and permits sequence-determined separation of DNA fragments (Fischer and Lerman, 1979). The electrophoretic mobility of partly melted DNA fragments, consisting of both

I. Introduction In the human genome, G+C content of which is 40%, the appearance of CpG dinucleotides would be expected one every 25 base pair (bp), if all four bases are evenly distributed throughout the genome, but the actual frequency is about one every 150 bp. This indicates that distribution of four bases is not uniform and that a CpG dinucleotide appeared less frequently than the simple assumption of random distribution predicts. Furthermore, the 5-position of cytosine of most CpG’s is methylated in the genome, but methylation is rare in certain regions. Mosaic methylation is a characteristic feature of vertebrate genomes. There are regions in normal somatic cell DNA where non-methylated CpGs are significantly clustered, called CpG islands (Bird, 1986; Cross and Bird, 1995). A CpG island is about 1–2 kilobase (kb) in size, and frequently associated with 5’ region of many genes including

Shiraishi et al: Segregation in human cancer cells

helical and dissociated portions, in polyacrylamide gel is much lower than that of fully helical or fully dissociated molecules, and the transition in each fragment to low residual mobility results in stable band pattern. The position corresponding to this transition is called the retardation level. In some cases, two DNA fragments which differ by only one base pair have different melting temperature, and retard in a gradient gel at different levels. DGGE is useful for detection of point mutations (Fischer and Lerman, 1983). DNA methylation affects Tm, and correspondingly the retardation level, when it occurs in the domain having the lowest Tm. Difference in methylation at one base, that is, non-methylation, hemimethylation, or symmetrical methylation, can be resolved by DGGE (Collins and Myers, 1987). We have found that DGGE can be applied to the study of DNA methylation associated with CpG islands (Shiraishi et al, 1995), although DGGE itself does not here depend on methylation. In this article, we describe how DNA fragments associated with CpG islands are isolated on the basis of reduced rate of strand dissociation and its application to the isolation of methylated CpG islands, with emphasis on methodology.

level at which the mobility decreases abruptly in the gel, while the residual regions determine the stability of the partly melted structure. The separation depends on the markedly reduced electrophoretic mobility, which occurs when a part of DNA fragment melts, resulting in a structure that is partly helical and partly random chain. On prolonged field exposure, the retarded fragments will fade away through strand dissociation. If the stability of the helical part is appreciably higher than that of the melted part, the dissociation rate will be low and the retarded partly melted molecule remains in the gel for some hours. It would be reasonable to speculate that DNA fragments derived from the edges of CpG islands consist of at least two different melting domains, because the G+C-rich nature of CpG island sequence results in high Tm, while flanking non-island sequences are not G+C-rich and would be lower melting. Thus, preferential retention of DNA fragments derived from the edges of CpG islands after prolonged field exposure is strongly expected (F i g u r e 1 ). That DGGE can be used for the isolation of DNA fragments associated with CpG islands was implicit when Fischer and Lerman described detection of point mutations by DGGE showing that it depends on partial melting (Fischer and Lerman, 1983). Later on, Myers et al proposed and showed that addition of 300-bp G+C-rich sequence (GC-clamp) to DNA fragments lacking a high Tm domain would ensure partial melting (Myers et al, 1985a, b). Sheffield et al also showed that addition of short (40â&#x20AC;&#x201C;45 bp) GC-clamp was sufficient to protect the DNA fragment from strand dissociation and permit retention in a gel (Sheffield et al, 1989). It can be expected that CpG island sequences would serve as â&#x20AC;&#x153;naturalâ&#x20AC;? GC-clamps, as demonstrated by attachment of one to the mouse !major globin promoter (Myers et al, 1985a, b).

II. Segregation of partly melted molecules A. Background DGGE is a technique to separate DNA fragments on the basis of local variation in base composition within the DNA fragments (Lerman et al, 1984). It is effective only when the molecule is comprised of domains having different melting temperatures. The Tm of the lowest melting domain of sufficient length determines retardation

F i g u r e 1 . Schematic model of separation. The polyacrylamide gel contains a linear gradient of chemical denaturant (urea and formamide), low at the top and high at the bottom. Red and blue bars indicate DNA fragments having a G+C-rich region and those without a G+C-rich region, respectively. Once part of a DNA molecule melts, pronounced drop in electrophoretic mobility occurs, and low residual mobility restricts migration into more strongly denaturing regions. If the helical portion is stable enough, that fragment persists in the gel, while all others become dissociated and run out of the gel.

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F i g u r e 3 . A retained fragment, R16-2, contained the sequence identical to the 5’-end of the prostacyclin synthase cDNA and a putative promoter region (Shiraishi and Sekiya, 1996). It is well known that the isolation of 5’ upstream region of a gene is notoriously difficult by the conventional cDNA synthesis approach. Therefore, SPM is also useful for isolation of promoter regions. The melting map of the fragment, R16-2, is shown in F i g u r e 3 . A region of high Tm is identified in about position 400 to the end. It is expected that this G+C-rich region is thermally stable and serves to tether the partly melted molecule. This region is also inferred to be the 5’ edge of the island, since there is a clear boundary of CpG distribution at about position 350.

B. Preferential isolation of DNA fragments associated with CpG islands Long cloned DNA fragments have to be fragmented appropriately in order for island fragments to be enriched by DGGE. It was shown that digestion of DNA fragments with four restriction endonucleases, Tsp509 I (AATT), Mse I (TTAA), Nla III (CATG), and Bfa I (CTAG), yields DNA fragments of appropriate size for DGGE (Shiraishi et al, 1995). Since occurrence of these sites are rare in CpG islands but abundant in the remaining bulk genomic DNA, this treatment keeps the integrity of CpG island relatively intact, while the rest generally undergoes severe fragmentation. When cloned DNA molecules containing entire region of some known genes with CpG island were digested with four restriction endonucleases and subjected to DGGE, DNA fragments associated with CpG islands formed stable bands which persisted through continued application of the field, but others did not (Shiraishi et al, 1995). DNA fragments that persisted in the gel were recovered from all CpG islands that were analyzed. Cosmid clones randomly selected from a human genomic library were analyzed similarly (F i g u r e 2 ). Digestion yielded hundreds of fragments. When the digests are subjected to DGGE, only a few selected fragments, three per cosmid clone on the average, were retained in the gel after 11 hours run. Nucleotide sequence analysis revealed that about half of the retained fragments were considered to be derived from CpG islands (Shiraishi et al, 1995). Thus, the method, named segregation of partly melted molecules (SPM), provides a useful means to isolate DNA fragments associated with CpG islands (Shiraishi et al, 1995). A representative result is shown in

F i g u r e 2 . SPM analysis of cosmid clones. Cosmid clones were serially digested with four restriction endonucleases, Tsp 509 I, Mse I, Nla III, and Bfa I. Conventional polyacrylamide gel electrophoresis shows that numerous fragments were yielded after digestion. After DGGE, only limited number of fragments was retained in the gel (Shiraishi et al, 1995). R16-2 is a fragment whose profiles are shown in Figure 3 (Shiraishi and Sekiya, 1996).

Shiraishi et al: Segregation in human cancer cells

The melting map of the fragment, R16-2, is shown in F i g u r e 3 . A region of high Tm is identified in about position 400 to the end. It is expected that this G+C-rich region is thermally stable and serves to tether the partly melted molecule. This region is also inferred to be the 5â&#x20AC;&#x2122; edge of the island, since there is a clear boundary of CpG distribution at about position 350.

C. Application to gene hunting Since 56% of human genes are reported to be associated with CpG islands (Larsen et al, 1992), detection of CpG islands in unsequenced DNA fragments can provide markers for unidentified genes. When P1 artificial chromosome clones covering a 400-kb region of human chromosomal region 11q13, a well known region enriched with CpG islands (Craig and Bickmore, 1994), were subjected to SPM analysis (F i g u r e 4 ), the expected numbers of CpG islands were isolated (Shiraishi et al, 1998). This result suggested that SPM is an efficient method for isolation of bits of gene sequences from long unsequenced DNA fragments. The isolation of DNA fragments associated with CpG islands by means of DGGE stands on the different basis from that of current practice that makes use of clustering or presence of restriction sites characteristic for CpG islands, some of which are BssH II (GCGCGC), Eag I (CGGCCG), and Sac II (CCGCGG) (Lindsay and Bird, 1987; Bickmore and Bird, 1992; Valdes et al, 1994). The SPM method would be advantageous when unbiased isolation of DNA fragments associated with CpG islands is attempted since these restriction sites are not always present in all CpG islands.

F i g u r e 3 . The melting map of fragment R16-2 and corresponding gene structure. The contour shows the midpoint of the melting equilibrium at each base pair (Lerman and Silverstein, 1987). Arrows down and up indicate CpG dinucleotides and GC-box sequences (GGGCGG or CCGCCC), respectively.

D. Rationalization The system depends on both extensive digestion of DNA fragments with restriction endonucleases and relative rate of dissociation of partly melted molecules, which are locally G+C-rich during DGGE. However, theory for dissociation rates at temperature lower than that sufficient for full dissociation is not established. Instead, a heuristic approach was adopted to explain the molecular basis for retention and examine the generality of the principle. RHST is an index, which attempts to relate the rate of dissociation to the length and sequence of the unmelted portion of the molecule, where the index is the reciprocal of the estimated relative rate (Shiraishi et al, 1995, 1998). RHST would be large for retained fragments and small for disappearing fragments.

F i g u r e 5 . Separation of methylated and non-methylated CpG islands in genomic DNA. DNAs from male and female tissues were digested with Tsp509 I, subjected to an MBD column, and eluted with a stepwise gradient of salt concentration as described (Shiraishi et al, 1999a). DNA from each fraction was subjected to PCR-based detection of fragments containing the CpG island of the human HPRT gene.

fragment; q is an arbitrary constant that is uniform for all fragments. The summation extends only over pairs calculated by MELT (Lerman and Silverstein, 1987) to be helical at the retardation level. It was shown that RHST values could discriminate retention and non-retention when the followings were assumed; Tm taken to be equivalent (bath temperature + denaturant) gel temperature at which melted domain is approximately 75% helical, the mobility in the gel is reduced to about 28% of initial velocity, equivalent to 95-bp melting, and q value of 1.546 (Shiraishi et al, 1998). All retained fragments showed RHST values not less than 3.56x103, while non-retained ones showed RHST values not greater than 3.19x103. Fragments more or less uniformly dense in G+C are not retained in the gel since they are already at the edge of dissociation as partial melting begins. Their RHST values

RHST = # {exp([Tm(i) " Tret] / q) "1} where Tm is the calculated temperature for each pair at which the unimolecular probability of helicity of each pair in the stable segment falls to a chosen threshold. Tret represents the equivalent temperature at the gradient level of the retarded

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are small. Both experimental results and calculation showed that not the entire island but rather the edge of the island can be generally isolated by the SPM method. High RHST values are not only found for CpG island fragments; some DNA fragments containing repetitive sequences also have high RHST values. Alu repetitive sequences are CpG-rich and contain poly(A) stretches (for a recent review, see Schmid, 1996), and can have high RHST values. When P1 clones from human chromosomal region 11q13 was analyzed by SPM method, about 40% of retained fragments contained Alu sequences, and RHST values of these fragments were high (Shiraishi et al, 1998). These results reflects the observation that both CpG islands and Alu sequences are enriched in R band regions of chromosomes (Korenberg and Rykaowski, 1988; Craig and Bickmore, 1994), and strongly suggest that RHST is a good indicator of retention.

and that non-methylation is negatively displayed as the absence of a signal.

2. Modification by chemical reagents Although some chemical reactions permit discrimination of cytosine and methylcytosine (for recent reviews, see Rein et al, 1998; Thomassin et al, 1999; Oakeley 1999), their application to the analysis of genomic methylation status was very limited due to complexity of mammalian genomes. The pyrimidine ring of cytosine is cleaved by hydrazine treatment, but when 5-position of cytosine is methylated, the ring is not cleaved. These properties were applied to determine methylation status of the mouse IgM heavy chain constant region gene CÂľ in combination with Southern hybridization (Church and Gilbert, 1984). However, in addition to technical difficulties, indication of the presence of methylcytosine as the absence of signal makes interpretation difficult. A major breakthrough was brought to analysis of DNA methylation when chemical modification of cytosine by sodium bisulfite, conversion of cytosine to uracil (Hayatsu et al, 1970; Shapiro et al, 1970), was combined with PCR to permit positive display of 5-methylcytosine in individual DNA strand (Frommer et al, 1992). Treatment of single-stranded DNA with sodium bisulfite, followed by hydrolysis, converts nonmethylated cytosine into uracil, while methylated cytosine remains intact. Since adenine makes a base pair with uracil, subsequent PCR converts original nonmethylated cytosines to thymine. Consequently a non-methylated C-G pair becomes converted to a T-A pair, while a methylated C-G pair remains as a C-G pair. This reaction can be applied to the analysis of any CpG sequence, and permits positive display of methylcytosine. Bisulfite modification method has now become popular, and unraveled previously unknown features of sequence. One of the unexpected findings revealed by bisulfite modification method is that methylated CpG islands, such as those on inactive X chromosomes, are not uniformly or densely methylated, contrary to previous thought. Hornstra and Yang reported that the CpG island associated with the human HPRT gene on an inactive X chromosome is not uniformly methylated (Hornstra and Yang, 1994). CpG sites in the GC-box region were methylation-free even in the CpG island on inactive X chromosome. Although the interpretation of this finding awaits further experiments, we are now closer to the precise nature of methylation.

III. Isolation of methylated CpG islands in human cancer cells A. Methods methylation

for

analysis

DNA

It is well known that cancer is caused by accumulation of genetic and epigenetic aberrations. Epigenetic aberrations are those that can not be explained by alteration of nucleotide sequence, and CpG island methylation is representative. Epigenetic process in carcinogenesis is less well understood compared with genetic process, partly due to limitation of available methods. Before describing application of SPM to the isolation of methylated CpG islands, we briefly summarize methods for analysis of DNA methylation in order to know their features.

1. Sensitivity to digestion restriction endonucleases

with

some

Until the appearance of bisulfite modification method described in the next section, sensitivity to digestion with some restriction endonucleases, first described in 1978 (Bird and Southern, 1978), was practically the only method to analyze methylation status of a complex genome. The most frequently used combination of restriction endonucleases is that of Msp I and Hpa II. Both endonucleases recognize CCGG sequence and cleave DNA at that site. However, when the internal C is methylated, the modified sequence becomes resistant to cleavage by Hpa II, but not by Msp I. Subsequent Southern hybridization or PCR permits discrimination of methylation and non-methylation as presence or absence of appropriate fragments. Restriction landmark genomic scanning (RLGS) is a method involving two-dimensional gel electrophoresis that permits genomic scan on the basis of methylation status of Not I sites (Hatada et al, 1991). The results of methods using restriction endonucleases are strongly affected by incomplete digestion, which often results in overestimation of methylation, especially in PCR-based experiments. Moreover, the analysis is restricted to methylation status of specific restriction sites, which are not always a representative of the target region,

3. Methylated chromatography

DNA

binding

column

An approach that stands on a different principle in terms of discrimination of methylation and nonmethylation was developed recently. Rat nuclear protein MeCP2 (Lewis et al, 1992) binds DNA at a mCpG site, but not at a CpG site (Meehan et al, 1992) The DNA binding domain of this protein is comprised of 85 amino acids (Nan et al, 1993). A methylated DNA binding column (MBD column) is an affinity matrix that contains

Shiraishi et al: Segregation in human cancer cells

a polypeptide derived from the methyl-CpG binding domain (Cross et al, 1994). Since the stoichiometry of binding is one protein to one mCpG (Nan et al, 1993), it is expected that highly methylated DNA fragments bind to the column tightly, while poorly methylated DNA fragments bind only weakly. By MBD column chromatography, DNA fragments with the same nucleotide sequence but different methylation status can be separated (Cross et al, 1994; Shiraishi et al, 1999a). A representative result of MBD column chromatography experiment is shown in F i g u r e 5 . The human HPRT gene is an X-liked gene having a CpG island, and one allele is inactivated in female. There is only one non-methylated HPRT-CpG island in male DNA, while there are two HPRT-CpG islands in female DNA, one is methylated and the other is not methylated. Most part of the island is contained in a 0.9-kb Tsp509 I fragment and there are 86 CpG residues in it (data not shown). When Tsp509 I digests of male DNA was analyzed by MBD column chromatography, DNA fragments containing the CpG island were detected only in low salt fraction (fractions around number 16). In contrast, corresponding fragment of female DNA were detected both in lower (fractions around number 16) and higher (fractions around 36) salt fractions. These results show that DNA fragments from methylated CpG islands and those from non-methylated CpG islands can be separated by MBD column chromatography. Not only number of mCpGs but also mCpG density seems to be a factor that affects affinity (Shiraishi et al, 1999b; Brock et al, 1999), although possibility of sequence-specific preferential binding can not be excluded. Clearly this method is insensitive to discriminate heterogeneity in methylation within the same DNA fragment and small change in total number of mCpGs. The nature of this method, which is not destructive and not influenced by methylation status of specific restriction sites, is advantageous for comprehensive isolation of methylated CpG islands.

associated with CpG islands methylated in cancer were isolated (Shiraishi et al, 1999a). Many CpG islands thus obtained from cancer were also methylated in noncancerous portion of the lung, possibly only in one allele. These results suggest that number of CpG islands specifically methylated in cancer is lower than that of normally differentially methylated ones.

IV Perspective In this article, we introduced approaches to study CpG island and DNA methylation standing on novel principles; these may play an important role in cancer research (Terada, 1999). There are many issues yet to be clarified in the field of DNA methylation, such as molecular mechanism of gene silencing by methylation and comprehensive identification of genes that are inactivated by methylation and involved in carcinogenesis. Development of new experimental techniques and their application will be a key to solve these problems.

Acknowledgments We thank the Ministry of Education, Science, Sports, and Culture and the Ministry of Health and Welfare of Japan for research support.

References Baylin S, Herman J, Graff J, Vertino P, and Issa J-P (1 9 9 8 ) Alteration in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res 72, 141–196. Bickmore W and Bird A (1 9 9 2 ) Use of restriction endonucleases to detect and isolate genes from mammalian cells. M e t h o d s E n z y m o l 216, 224–244. Bird A and Southern E (1 9 7 8 ) Use of restriction enzymes to study eukaryotic DNA methylation: I. The methylation pattern in ribosomal DNA from Xenopus laevis. J M o l B i o l 118, 27-47. Bird A (1 9 8 6 ) CpG-rich islands and the function of DNA methylation. Nature 321, 209–213. Church G and Gilbert W (1 9 8 4 ) Genomic sequencing. Proc Natl Acad Sci USA 81, 1991–1995. Collins M and Myers R (1 9 8 7 ) Alterations in DNA helix stability due to base modofication can be evaluated using denaturing gradient gel electrophoresis. J M o l B i o l 198, 737–744. Costello J, Fruhwald M, Smiraglia D, Rush L, Robertson G, Gao X, Wright F, Feramisco J, Peltomaki P, Lang J, Schuller D, Yu L, Bloomfield C, Caligiuri M, Yates A, Nishikawa R, Su Huang H, Petrelli N, Zhang X, O'Dorisio M, Held W, Cavenee W, and Plass C (2 0 0 0 ) Aberrant CpG-island methylation has non-random and tumortype–specific patterns. Nature Genet 25, 132–138. Craig J and Bickmore W (1 9 9 4 ) The distribution of CpG islands in mammalian chromosomes. Nature Genet 7 , 376–382. Cross S, Charlton J, Nan X, and Bird A (1 9 9 4 ) Purification of CpG islands using a methylated DNA binding column. Nature Genet 6, 236–244. Cross S and Bird A (1 9 9 5 ) CpG islands and Genes. Curr Opin Genet Dev 5, 309–314.

B. Isolation of methylated CpG islands Isolation of CpG methylated islands in cancer cells has been drawing growing interest since it provides valuable information on cancer epigenetics, which is very limited now. Several methods for this purpose have been reported; arbitrary primed PCR method (Gonzalgo et al, 1997; Huang et al, 1997; Gonzalgo and Jones, 1998), subtraction (Ushijima et al, 1997; Huang et al, 1999, Toyota et al, 1999), and RLGS (Costello et al, 2000) are those primarily dependent on the methylation status of specific restriction sites. In contrast, MBD column chromatography permits separation of methylated DNA fragment independent of methylation status of any internal restriction sites, and seems to be excellent for unbiased, comprehensive isolation of methylated CpG islands. Using MBD column chromatography, highly methylated DNA fragments in human adenocarcinomas of the lung was enriched and then cloned. By SPM analysis of the clones, DNA fragments

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Fischer S and Lerman L (1 9 7 9 ) Length-independent separation of DNA restriction fragments in twodimensional gel electrophoresis. C e l l 16, 191–200. Fischer S and Lerman L (1 9 8 3 ) DNA fragments differing by single base-pair substitutions are separated by denaturing gradient gel: correspondence with melting theory. Proc Natl Acad Sci USA 80, 1579–1583. Frommer M, McDonald L, Millar D, Collis C, Watt F, Grigg G, Molloy, and Paul C (1 9 9 2 ) A genomic sequencing protocol that yields a positive display of 5methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA 89, 1872–1831. Gonzalgo M, Liang G, Spruck III C, Zingg J-M, Rideout III W, and Jones P (1 9 9 7 ) Identification and characterization of differentially methylated regions of genomic DNA by methylation-sensitive arbitary primed PCR. Cancer Res 57, 594–599. Graham G, Charlton J, and Bird A (1 9 9 9 ) Densely methylated sequences that are preferentially localized at telomereproximal regions of human chromosomes. Gene 240, 269–277. Hatada I, Hayashizaki Y, Hirotsune S, Komatsubara H, and Mukai T (1 9 9 1 ) A genomic scanning method for higher organisms using restriction sites as landmarks. Proc Natl Acad Sci USA 88, 9523–9527. Hayatsu H, Wataya Y, Kai K, and Iida S (1 9 7 0 ) Reaction of sodium bisulfite with uracil, cytosine, and their derivatives. B i o c h e m i s t r y 9, 2858–2865. Huang T, Laux D, Hamlin B, Tran P, Tran H, and Lubahn D (1 9 9 7 ) Identification of DNA methylation markers for human breast carcinomas using the methylation-sensitive restriction fingerprinting technique. Cancer R e s 57, 1030–1034. Huang T, Perry M, and Laux D (1 9 9 9 ) Methylation profiling of CpG islands in human breast cancer cells. H u m M o l Genet 8, 459–470. Hornstra I and Yang T (1 9 9 4 ) High-resolution methylation analysis of the human hypoxanthine phosphoribosyltransferase gene 5’ region on the active and inactive X chromosomes: Correlation with binding sites for transcription factors. M o l C e l l B i o l 14, 1419–1430. John R and Cross S (1 9 9 8 ) Gene detection by the identification of CpG islands. G e n o m e A n a l y s i s : A Laboratory Manual, Vol. 2, eds. Birren B, Hieter P, and Myers R (Cold Spring Harbor Lab. Press, Plainview, NY), 217–285. Korenberg J and Rykaowski M (1 9 8 8 ) Human genome organization: Alu, lines, and the molecular structure of metaphase chromosoma bands. C e l l 53, 391–400. Larsen F, Gundersen G, Lopez R, and Prydz H (1 9 9 2 ) CpG islands as gene markers in the human genome. G e n o m i c s 13, 1095–1107. Lerman L, Fischer S, Hurley I, Silverstein K, and Lumelsky N (1 9 8 4 ) Sequence-determined DNA separations A n n R e v B i o p h y s B i o e n g 13, 399–423. Lerman L and Silverstein K (1 9 8 7 ) Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. M e t h o d s Enzymol 155, 482–501. Lewis J, Meehan R, Henzel W, Maurer-Fogy I, Jeppesen P, Klein P, and Bird A (1 9 9 2 ) Purification, sequence, and cellular localization of a novel chromatin protein that binds to methylated DNA. C e l l 69, 905–914. Liang G, Salem, C, Yu M, Nguyen H, Gonzales F, Nguyen T, Nichols P, and Jones P (1 9 9 8 ) DNA methylation

differences associated with tumor tissues identified by genome scanning analysis. G e n o m i c s 53, 260–268. Lindsay S and Bird A (1 9 8 7 ) Use of restriction enzymes to detect potential gene sequences in mammalian DNA. Nature 327, 336–338. Meehan R, Lewis J, and Bird A (1 9 9 2 ) Characterization of MeCP2, a vertebrate DNA binding protein with affinity for methylated DNA. N u c l e i c Acids R e s 20, 5085–5092. Myers R, Fischer S, Maniatis T, and Lerman L (1 9 8 5 a ) Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis. N u c l e i c A c i d s R e s 13, 3111–3129. Myers R, Fischer S, Lerman L, and Maniatis T (1 9 8 5 b ) Nearly all single base substitution in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. N u c l e i c A c i d s R e s 13, 3131–3145. Nan X, Meehan R, and Bird A (1 9 9 3 ) Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2. N u c l e i c A c i d s R e s 21, 4886–4892. Oakeley E (1 9 9 9 ) DNA methylation analysis: a review of current methodologies. Pharmacol Ther 84, 389–400. Rein T, DePamphilis M, and Zorbas H (1 9 9 8 ) Identifying 5methylcytosine and related modifications in DNA genomes. N u c l e i c A c i d s R e s 26, 2255–2264. Sheffield V, Cox D, Lerman L, and Myers R (1 9 8 9 ) Attachment of a 40-base-pair G+C-rich sequence (GCclamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of singlebase changes. Proc Natl Acad Sci USA 86, 232–236. Schmid C (1 9 9 6 ) Alu: structure, origin, evolution, significance and function of one-tenth of human DNA. P r o g N u c l A c i d R e s M o l B i o l 53, 283–319. Shapiro R, Servis R, and Welcher M (1 9 7 0 ) Reactions of uracil and cytosine derivatives with sodium bisulfite. A specific deamination method. J Am Chem S o c 92, 422–424. Shiraishi M, Lerman L, and Sekiya T (1 9 9 5 ) Preferential isolation of DNA fragments associated with CpG islands. Proc Natl Acad Sci USA 92, 4229–4233. Shiraishi M and Sekiya T (1 9 9 6 ) Isolation of CpG island fragments: a putative promoter region of the human prostacyclin synthase gene. Proc Jpn Acad 72B, 101–103. Shiraishi M, Oates A, Xu L, Hosoda F, Ohki M, Alitalo T, Lerman L, and Sekiya T (1 9 9 8 ) The isolation of CpG islands from human chromosomal regions 11q13 and Xp22 by segregation of partly melted molecules. N u c l e i c A c i d s R e s 26, 5544–5550. Shiraishi M, Chuu Y, and Sekiya T (1 9 9 9 a ) Isolation of DNA fragments associated with methylated CpG islands in human adenocarcinomas of the lung using a methylated DNA binding column and denaturing gradient gel electrophoresis. Proc Natl Acad S c i USA 96, 2913–2918. Shiraishi M, Sekiguchi A, Chuu Y, and Sekiya T (1 9 9 9 b ) Tight interaction between densely methylated DNA fragments and the methyl-CpG binding domain of the rat MeCP2 protein attached to a solid support. B i o l C h e m 380, 1127–1131. Terada M (1 9 9 9 ) Segregation of partly melted molecules: A step toward elucidation of epigenetic aberration in cancer. Jpn J Cancer Res 90, 1397–1398.

Shiraishi et al: Segregation in human cancer cells

Thomassin H, Oakeley E, and Grange T (1 9 9 9 ) Identification of 5-methylcytosine in complex genomes. M e t h o d s 19, 465–475. Toyota M, Ho C, Ahuja N, Jair K-W, Ohe-Toyota M, Baylin S, and Issa J-P (1 9 9 9 ) Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification. Cancer Res 59, 2307–2312. Ushijima T, Morimura K, Hosoya Y, Okonogi H, Tatematsu M, Sugimura T, and Nagao M (1 9 9 7 ) Establishment of methylation-sensitive-representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors. Proc Natl Acad Sci USA 94, 2284–2289. Rein T, DePamphilis M, and Zorbas H (1 9 9 8 ) Identifying 5methylcytosine and related modifications in DNA genomes. Nuc l e ic A c id s R e s 26, 2255–2264. Valdes J, Tagle D, and Collins F (1 9 9 4 ) Island rescue PCR: a rapid and efficient method for isolating transcribed sequences from yeast artificial chromosomes and cosmids. Proc Natl Acad Sci USA 91, 5377–5381.

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Gene Ther Mol Biol Vol 5, 47-53, 2000

PNA (peptide nucleic acid) anti-gene/antisense can access intact viable cells and downregulate target genes Review Article

Lidia C Boffa*, Elisabetta M.Carpaneto, Benedetta Granelli, Maria R. Mariani National Cancer Institute, IST, L.go R. Benzi 10, 16132 Genoa, Italy

_____________________________________________________________________________________ * Correspondence: Lidia Boffa, National Cancer Institute, IST, L.go R. Benzi 10, 16132 Genoa, Italy, Tel: -39-010-5600214; Fax: -39-010-5600217; E-mail: boffa@hp380.ist.unige.it Key words: Peptide Nucleic Acid (PNA), anti-gene, antisense, cell /nuclear localization vectors, gene downregulation Received: 27 June 2000; accepted: July 2000

Summary In this paper we summarize our recent data on the anti-gene properties of PNA constructs both with a classical Nuclear Localization Signal peptide, that appears to be effective in all the cell lines tested, and with other vectors designed t o be specific for c e l l s carrying their receptors on the nuclear membrane. We discuss the cellular localization pattern of PNA constructs, the consequent regulatory effects on the target gene and their influence in the cellular metabolism. The data are discussed from the perspective of the very recent literature on the access of PNAs antisense/anti-gene in intact viable cells and their consequent regulatory effect in the form of: (i) PNA linked to cellular/nuclear peptidic localization vectors; (ii) PNA linked to non peptidic vectors; (iii) unmodified PNA. RNA target. Structural information on the four possible PNA complexes has been obtained by NMR spectroscopy for PNA/RNA (Brown et al, 1994) and for PNA/DNA duplexes (Eriksson and Nielsen, 1996) and by X-ray crystallography for PNA2/DNA triplex (Betts et al, 1995) and for PNA/PNA duplex (Rasmussen et al, 1997). The overall conclusion from these studies is that the rather flexible PNA oligomer is able to a large extent to adapt its conformation to its rigid complementary oligonucleotide. In terms of sugar conformation, in PNA/RNA duplexes the RNA strand is basically A-form and in PNA/DNA duplexes the DNA strand is close to Bform. The advantages of using PNA oligomers over the conventional antisense oligonucleotides, that have been used for some time to try to downregulate target gene expression, are numerous partially due to the high flexibility and absence of charge of the artificial backbone: they are resistant to nucleases and proteases (Demidov et al, 1994) and consequently have a longer life span in the cellular environment than any other oligonucleotide; they can invade duplex DNA and hybridize with complementary sequences with such a superior thermal stability (Giesen et al, 1998), resulting from the decrease in electrostatic repulsion, so that they can successfully compete and eventually displace the natural complementary strand; they

I. Introduction Peptide Nucleic Acids (PNA) are a recent development in the field of oligonucleotides analogues. PNAs were originally conceived as oligonucleotide homologues that could be used in the sequence-specific targeting of double-stranded DNA (Nielsen et al, 1991; Egholm et al, 1992). They are constructed in such a way to have a neutral charge, achiral, pseudo peptide backbone of N-(2-aminoethyl) glycine polymer. Each unit is linked to a purine or pyrimidine base to create the specific sequence required for hybridization to the targeted polynucleotide. Therefore PNA is chemically more closely related to peptides and proteins than to nucleic acids. In vitro PNA/DNA and PNA/RNA duplexes are, in general, thermally more stable than the corresponding DNA/DNA or RNA/RNA duplexes and PNA2/DNA triplexes formed between homopyrimidine PNAs and sequence complementary homopurine DNA show even higher stability (Egholm et al, 1993; Nielsen et al, 1994). Although PNAs may bind complementary oligonucleotide or PNA targets in both orientations (parallel and antiparallel) with significant efficiency, the most stable duplexes are formed with an antiparallel Watsonâ&#x20AC;&#x201C;Crick orientation (the PNA N-terminus facing the 3' end of the oligonucleotide) and, in case of triplex helix formation, the Hoogsteen PNA strand should be parallel to the DNA or

Boffa et al: A new and innovative approach to the in “vivo” antisense and anti-gene therapy

have a higher mismatch discrimination therefore forming strong, selective duplexes upon binding to complementary DNA or RNA sequences (Almarsson et al, 1993; Egholm et al, 1993). In cell chromatin the PNA/DNA hydrogenbonded double is further stabilized by three factors: in the cellular environment PNA/DNA hybrids are more stable than their homologues DNA/DNA since they are ionic strength independent (Jensen et al, 1997); PNA/DNA binding is more tight if DNA is supercoiled than if it is linear; PNA/DNA hybridization is favored in transcriptionally active, open chromatin (Bentin and Nielsen, 1996; Boffa et al, 1997). The greater stability of PNA hybrids with the complementary DNA of transcriptionally active chromatin was used as a tool for selection and characterization of active chromatin fragment as large as 23 kb (Boffa et al, 1995). Experiments with permeabilized cells and isolated nuclei (Boffa et al, 1996; Boffa et al, 1997) have shown that complex sequence PNAs are highly effective in blocking transcription of the targeted gene without inhibiting RNA synthesis in unrelated genes. Unfortunately at 37ºC PNAs easily enter the cells by endocytosis but are readily sequestered by cytoplasmic vesicles (endosomes and lysosomes) before they can enter the nucleus (Bonham et al, 1995).

upon exposure to the anti-gene PNA-NLS than to the unmodified matching PNA (Figure 1). In particular, all cells were subjected to run on transcription assay in the presence of [!-32P]UTP. Total RNA was purified and the newly synthesized, radioactively labeled mRNA was analyzed by hybridization for its content in specific sequences, located not only at the PNA/DNA binding site but also upstream and downstream from it, that were previously blotted on an appropriate membrane. We showed that the PNA binding to its target sequence in the c-myc gene strongly inhibited the sense transcription of 4 sequences downstream from the PNA/DNA hybridization site and that the extent of this inhibition depended on the distance of the sequences from the PNA block. Recently (Cutrona et al, 2000) we have described the anti-gene effect of the above described construct in live cultured cells. When Burkitt’s lymphoma derived cell lines (BL) were exposed to the c-myc anti-gene PNA-NLS this molecule was localized predominantly in the cell nuclei. The PNA nuclear localization was not only due to the basic nature of the peptide, but also to the specific amino acids sequence. In particular previous studies have determined that the nuclear localization function of the peptide was strictly dependent on the presence of lysine as the third amino acid (termed Lys128, as from the original sequence of SV40 NLS) (Colledge et al, 1986). In order to demonstrate that only the correct original NLS sequence can specifically confer a cellular/nuclear localization to the bound PNA, we designed a scrambled NLS (KKVKPKR) mutated at the third amino acid to be used as a negative control (NLSscr). BL cells were exposed in culture to PNAmyc +/-NLS or NLSscr tagged with a fluorophore Rhodamine (Rho) ( Rho-PNA-myc, Rho-PNA-myc-NLS or Rho-PNA-myc-NLSscr) and analyzed by confocal microscopy. Maximum cellular fluorescence intensity was obtained at 24 h (Figure 2). Rho-PNA-myc (a) and RhoPNA-myc-NLSscr (b) were localized in the cytoplasm, while Rho-PNA-myc-NLS (c) was clearly detectable in the cell nuclei. The PNA myc-NLS access to the cell nuclei caused a rapid consequent downregulation of c-myc transcription as from the time course of MYC expression determined by Western blot and Northern blot analysis of c-myc mRNA (Figure 3).

II. Review

A. PNA linked to cellular/nuclear peptidic localization vectors Cellular and nuclear localization signal peptides have successfully been used for some time to carry bulky uncharged molecules into live cells. Several recent reports have demonstrated that PNAs conjugated to such vectors are quite efficiently taken up by some eukaryotic cells.

1. SV 40 Nuclear Localization Signal peptide (NLS) PKKKRKV is a basic Nuclear Localization Signal peptide (NLS) that was shown first to mediate the transfer of SV40 large T antigen across the nuclear membrane (Kalderon et al, 1984) and later to facilitate the nuclear delivery of large proteins (Gorlich and Mattaj, 1996). Our Laboratory first described the construct of this NLS with a PNA (Boffa et al, 1997) and observed that it was capable of remarkably decreasing the time of access of PNA to nuclei of permeabilized cells in vitro without altering their anti-gene effects. Permeabilized cells were exposed for increasing time to a 17mer anti-gene PNA (+/NLS) complementary to a unique sequence at the beginning of the second exon of c-myc oncogene. In human adenocarcinoma derived cell line (COLO320-DM) at short time of exposure, we described a selective c-myc transcriptional inhibition that was significantly higher

Figure 1. c-myc and its specific anti-gene PNA

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Figure 2 Delivery of PNA-NLS to intact nuclei of BL cells. BL cells were exposed to Rho- PNA-myc (a), to Rho- PNA-myc-NLS (b ) or to Rho- PNA-myc-NLSscr (c ) and analyzed by confocal microscopy. The pictures show the section crossing the middle of the nuclei with the phase contrast and fluorescence images superimposed.

Figure 2. Inhibition of c-myc expression by PNA-myc-NLS. a. MYC expression determined by Western blot following exposure of BL cells to the indicated PNAs for 18h. b . Northern blot analysis of c-myc mRNA expression in BL cells exposed to the indicated PNAs for 18h.

PNA-myc-NLS-treated BL cells displayed a largely impaired growth capacity and a decrease in 3H-thymidine incorporation compared to those treated with control PNAmyc (not shown). Concomitantly, there was a substantial reduction in the proportion of cells in the S or G2M phases of the cell cycle as determined after 36 h following PNA treatment (Figure 4a). The presence of PNA-myc-NLS caused a decrease in viability of the cells in culture that became particularly evident by 72 h. Cell death, however, was unrelated to apoptosis that was not above the control values, as measured by Annexin-V staining (Figure 4b). A PNA-NLS construct was also shown to increase the transfection efficacy of plasmids containing the PNA complementary sequences (Branden et al, 1999).

to be able to translocate bulky or uncharged molecules through biological membranes and such internalization does not depend on classical endocytosis (Derossi et al, 1996). This peptide coupled to PNA (Simmons et al, 1997) was shown to confer permeability through cellular membranes. In particular the intracellular delivery property of this peptide was verified when conjugate with few different PNAs: i) antisense PNAs targeting the galanin receptor were shown to downregulate its expression by receptor activity assays and by Western blot (Pooga et al, 1998) in Bowers cells. Furthermore intrathecal injection of such a PNA conjugate into the brain of living rats reduced receptor activity in the brain, implicating in vivo antisense effects. Also, the behavioral response of the rats was compatible with a decreased galanin receptor level;

2. Antennapedia peptide A 16-amino acid peptide corresponding to the third helix of DNA binding domain of Antennapedia was shown

Boffa et al: A new and innovative approach to the in “vivo” antisense and anti-gene therapy

Figure 5 Chemical structure of PNA-dihydrotestoterone construct. Dihydrotestoterone was bound through a flexible linker to the N terminal position of PNA in order to confer the construct a better accessibility to nucleic acid in chromatin.

iii) two antisense PNAs to telomerase mRNA were selectively effective in downregulating the telomerase activity in human melanoma cells (Villa et al, 2000). In all cases the antisense was not only efficiently delivered to the cells but eventually migrates into the nuclei.

3. D-peptide analog of insulin growth factor (IGF1) IGF1 PNA conjugates displayed a much higher cellular uptake than unmodified PNAs but the uptake was in correlation with the level of expression of the IGF1 gene in the cells. In fact, in Jurkat cells that do not express the gene there is no PNA uptake, while in p6 cells, where the gene expression is high, a relevant uptake is detectable (Basu and Wickstrom, 1997) suggesting for the first time a possible cell-specific, tissue specific application of PNAs as gene-regulatory agents in vivo.

4. Hydrophobic tetrapeptide (FLFL) FLFL linked to PNA caused not only PNA internalization, but also remarkable stability of the complex in the cellular environment in Namalwa cells (Scarfì et al, 1997). This peptide could also internalize inducible Nitric Oxide synthase (iNOs) cDNA complementary DNA and significantly reduce the level of the enzyme in Macrophages in culture (Scarfì et al, 1999).

Figure 4 . PNA-myc-NLS effects on completion of a productive cell cycle and apoptosis a Flow-cytometric analysis of the cell cycle upon 36-h exposure to the indicated PNAs. b . BL cells were incubated with the indicated PNAs for different times. Apoptotic cells and dead cells were detected by Annexin-V binding and PI incorporation.

B. PNA linked to non peptidic vectors 1. Dihydrotestosterone (T)

T covalently linked to PNA acts as a vector (Figure 5) for targeting a c-myc anti-gene PNA selectively to cell nuclei of prostatic cancer LNCaP cells, which express Androgen Receptor (AR) gene, but not to DU145 cells, in which the AR gene is silent (Boffa et al, 2000). T vector was covalently linked to the N-terminal position of a PNA complementary to a unique sequence of c-myc oncogene (PNAmyc-T). A fluorophore (Rho) was also attached at the C-terminal position to localize the vector-free PNA and the PNA myc-T conjugate (PNAmycRho, PNAmyc-T-Rho) within the cells. The cellular uptake was

ii) antisense PNA to the prepro-oxytocin mRNA selectively and significantly decreased mRNA and protein product in neuronal cells in culture (Herrada et al, 1998);

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F i g u r e 6 . Effect of the different specific PNA constructs on c-myc expression. LNCaP and DU145 cells were treated for 24h with PNA-myc, PNA-myc-T, and PNA-myc-NLS. In this picture is shown a Western blot analysis of MYC .These findings suggest a strategy for targeting of cell-specific anti-gene therapy in prostatic carcinoma.

4. Adamantyl group

monitored by confocal fluorescence microscopy. PNAmycRho was detected in the cytoplasm of both prostatic cell lines, whereas PNAmyc-T-Rho was present only in nuclei of LNCaP (AR+) cells. The effects of the complete set of PNAs on expression of the c-myc gene in LNCaP and DU145 cells was monitored by Western blots of the MYC protein content of cell lysates: The results of a series of these analyses proved that in LNCaP cells only PNAmycT induced a significant and persistent decrease of MYC expression (Figure 6).

This group is probably the most innovative PNA vector. Adamantyl is a lipophilic group that when covalently attached to PNA (Ardhammar et al, 1999) shows at least a 3-fold improvement in PNA cellular uptake in a variety of cell lines in culture (Ljungstrom et al, 1999).

C. Unmodified PNA PNAs have been shown to enter to a small extent in cultured cells, for example neurons, probably through a mechanism of endocytosis (Aldrian-Herrada et al, 1998). Antisense PNAs uptake by cultured human myoblasts was shown to cause a specific inhibition of replication of mutant mitochondrial DNA (Taylor et al, 1997). In the literature there are recent reports of antisense effects of unmodified PNAs uptake in live mice brain. The first study (Tyler et al, 1998) was on a 14-mer PNA directed against the neurotensin, NTR1, (position +103) and mu opioid (position -70) receptors mRNAs. PNAs were injected into the periaqueductal gray (PAG) of rats. Neurotensin as well as opioids are well known to exert an antinociceptive effect. In addition, neurotensin induces hypothermia. Behavioral studies of anti-NTR1 or antiopioid mu receptors PNA treated animals showed dramatically reduced responses to neurotensin and morphine, respectively. Furthermore, hypothermia induced

2. OX26 murine monoclonal antibody to the rat transferrin receptor OX26 linked to the antisense PNA for the rev gene mRNA of the human HIV1 virus, not only was able to cross the blood-brain barrier (BBB) but also retained the capacity to bind the target mRNA, if injected in rats (Pardridge et al, 1995). This model system has recently been proposed also in the treatment of Alzheimerâ&#x20AC;&#x2122;s disease (Pardridge et al, 1998).

3. Spermine Covalent conjugation of Spermine to PNAs was shown to increase PNA solubility with consequent increase in cellular accessibility and a 2-fold acceleration of the rate of molecular association with complementary DNA (Gangamani et al, 1997).

Boffa et al: A new and innovative approach to the in “vivo” antisense and anti-gene therapy

invasion by a peptide nucleic acid. P r o c N a t l A c a d S c i USA. 92, 1901-1905. Boffa L.C, Carpaneto E.M, Mariani M.R, Louissaint M ,and Allfrey V.G (1 9 9 7 ) Contrasting effects of PNA invasion of the chimeric DMMYC gene on transcription of its myc and PVT domains. O n c o l o g y R e s . 9, 41-51 Boffa L.C, Morris P.L, Carpaneto E.M, Louissaint M ,and Allfrey V.G. (1 9 9 6 ) Invasion of the CAG triplet repeats by a complementary peptide nucleic acid inhibits transcription of the androgen receptor and TATA-binding protein genes and correlates with refolding of an active nucleosome containing a unique AR gene sequence. J . B i o l . C h e m 271, 13228-13233, Boffa L.C, Scarfi’ S, Mariani M.R, Damonte G,. Allfrey V.G, Benatti U ,and. Morris P.L. (2 0 0 0 ) Dihydrotestosterone as a Selective Cellular/Nuclear Localization Vector for Anti-Gene Peptide Nucleic Acid in Prostatic Carcinoma Cells. Cancer Res 60, 2258-2262. Bonham M.A, Brown S, Boyd A.L, Brown P.H, Bruckenstein D.A, Hanvey J.C, Thomson S.A, Pipe A, Hassman F, Bisi J.E, Froehler B.C, Matteucci M.D, Wagner R.W, Noble S.A ,and Babiss L.E. (1 9 9 5 ) An assessment of the antisense properties of RNase H-competent and stericblocking oligomers. N u c l e i c Acids R e s 23, 11971203. Branden L.J., Mohamed A.J ,and Edward Smith C.I (1 9 9 9 )A peptide nucleic acid nuclear localization signal fusion that mediates nuclear transport of DNA. Nat B i o t e c h n o l . 17, 784-787. Brown S.C, Thomson S.A, Veal J.M ,and Davis D.G (1 9 9 4 ) NMR solution structure of a peptide nucleic acid complexed with RNA. S c i e n c e 265, 777–780 Colledge W.H, Richardson W.D, Edge M.D ,and Smith A.E. (1 9 8 6 ) Extensive mutagenesis of the nuclear localization signal of simian virus 40 large T antigen. M o l C e l l B i o l . 6, 4136-4138 Cutrona G., Carpaneto E.M, Ulivi M, Roncella S, Landt O, Ferrarini M ,and Boffa L.C.(2 0 0 0 ) Effects in live cells of a c-myc anti-gene PNA liked to a nuclear localization signal. N a t B i o t e c h n o l 18, 300-303. Demidov V.V, Potaman V., Frank-Kamenetskii M.D., Buchardt O, Egholm M.E.,and Nielsen P.E (1 9 9 4 ) Stability of Peptide Nucleic Acids in human serum and cellular extracts. B ioc he m Phar mac ol 48, 1310-1313. Derossi D, Calvet S, Trembleau A, Brunissen A, Chassaing G ,and Prochiantz A (1 9 9 6 ) Cell internalization of the third helix of Antennapedia homeodomain is receptor indipendent J B i o l C h e m 271, 18188-18193. Egholm M, Buchardt O, Christensen L, Behrens C, Freier S, Driver D.A, Berg R.H, Kim S.K, Norden B ,and Nielsen P.E. (1 9 9 3 ) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogenbonding rules. Nature 365, 566-568. Egholm M, Buchardt O, Christensen L, Behrens C, Freier S.M, Driver D.A, Berg R.H, Kim S.K, Nordén B, and Nielsen P.E (1 9 9 3 ) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen bonding rules. Nature 36, 556–568. Egholm M, Buchardt O, Nielsen P.E ,and Berg R.H (1 9 9 2 ) Peptide Nucleic Acids (PNA). Oligonucleotide analogues with an achiral peptide backbone. J Am Chem S o c 114, 1895-1897. Eriksson M, and Nielsen P.E (1 9 9 6 ) Solution structure of a peptide nucleic acid-DNA duplex. Struct B i o l 3, 410–413. Fraser G.L, Holmgren J, Clarke P.B ,.and Wahlestedt C. (2 0 0 0 ) Antisense inhibition of delta–oppioid receptor

by neurotensin was substantially reduced. These effects where reversible and specific. A similar pattern of results was obtained in a subsequent study (Tyler et al, 1999) with NTR1 antisense (injected intraperitoneally) and sense (injected directly into the PAG of rats) PNA. The PNA uptake into the brain was detect by a gel shift assay. A 50% decrease of NTR1 mRNA level in brain induced only by the specific sense PNA, determined by quantitative PCR, suggested an anti-gene mechanism. Moreover a recent study described the non toxicity of an antisense PNA targeted toward mRNA of the opioid receptor gene injected repeatedly in mice (Fraser et al, 2000).

III. Conclusion We hope to have been able to clearly illustrate the recent body of evidence in support of the novel anti-gene/antisense PNAs-cellular/nuclear vector constructs capacity to access intact viable cells and consequently to downregulate target genes. These new modified PNAs could really provide an innovative and effective approach to the in “vivo” antisense and anti-gene therapy.

Acknowledgments We acknowledge the support of the Italian Association for Cancer research (AIRC) and National Research Council (CNR “Biotechnologies”).

References Aldrian-Herrada G, Desarmenien M.G, Orcel H, BoissinAgasse L, Mery J, Brugidou J ,and Rabie A. (1 9 9 8 ) .A peptide nucleic acid (PNA) is more rapidly internalized in cultured neurons when coupled to retro-inverso delivery peptide. The antisense activity depresses the target mRNA and protein in magnocellular oxytocin neurons. N u c l e i c A c i d s R e s . 26,4910-4916. Almarsson O, Bruice T.C, Kerr J .and Zuckermann R.N (1 9 9 3 ) Molecular mechanics calculations of the structures of polyamide nucleic acid DNA duplexes and triple helical hybrids. Proc Natl Acad Sci USA. 90 ,7518-7522 Ardhammar M, Norden B, Nielsen P.E, Malmstrom B.G ,and Wittung-Stafshede P. (1 9 9 9 ) In vitro membrane penetration of modified peptide nucleic acid (PNA). J B i o m o l S t r u c t D y n 17,33-40 Basu S ,and Wickstrom E (1 9 9 7 ) Synthesis and characterization of a peptide nucleic acid conjugated to a dpeptide anslog of insulin-like growth factor 1 for increased cellular uptake. B i o c o n j u g Chem 8, 481488. Bentin T ,and Nielsen P.E (1 9 9 6 ) Enhanced peptide nucleic acid binding to supercoiled DNA: possible implications for DNA “breathing” dynamics. B i o c h e m i s t r y 35, 8863-8869. Betts L, Josey J.A, Veal J.M ,and Jordan S.R(1 9 9 5 ) A nucleic acid triple helix formed by a peptide nucleic acidDNA complex. S c i e n c e , 270, 1838–1841. Boado R.J, Tsukamoto H ,and Pardridge W. (1 9 9 8 ) Drug delivery of antisense molecules to the brain for treatment of Alzheimer’s disease. J Pharm Sci 87,1308-1315 Boffa L.C, Carpaneto E.M ,and Allfrey V.G (1 9 9 5 ) Isolation of active genes containing CAG repeats by DNA strand

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gene function in vivo by peptide nucleic acids. M o l Pharm 57,725-731.. Gangamani B.P, Kumar V.A ,and Ganesh N.K. (1 9 9 7 ) Spermine conjugated peptide nucleic acids (spPNA): UV and fluorescence studies of PNA-DNA hybrids with improved stability. Biochem Biophys Res Commun 240, 778-782. Giesen U, Kleider W, Berding C, Geiger A, _rum H ,and Nielsen PE (1 9 9 8 ) A formula for thermal stability (Tm) prediction of PNA/DNA duplexes. N u c l e i c A c i d s R e s 26, 5004–5006. Gorlich D ,and Mattaj I.W (1 9 9 6 ). Nucleocytoplasmic transport. S c i e n c e 271, 1513-1518. Jensen K.K, Orum H, Nielsen P.E ,and Norden B. (1 9 9 7 ) Kinetics for hybridization of peptide nucleic acids (PNA) with DNA and RNA studied with the BIAcore technique B i o c h e m i s t r y 36, 5072–5077 Kalderon D, Roberts B.L Richardson W.D ,and Smith E.A (1 9 8 4 ) Short amino acid sequence able to specify nuclear localization. C e l l 39, 499-509. Ljungstrom T, Knudsen H ,and Nielsen P.E (1 9 9 9 )Cellular uptake of adamantyl conjugated peptide nucleic acids. B i o c o n j u g C h e m 10,965-972. Nielsen P.E, Egholm M ,and Buchardt O (1 9 9 4 ) Evidence for (PNA)2/DNA triplex structure upon binding of PNA to dsDNA by strand displacement. J M o l R e c o g n i t 7, 165–170. Nielsen PE, Egholm M, Berg R.H,and Buchardt O (1 9 9 1 ) Sequence selective recognition of DNA by strand displacement with a thymine-substituted polyamide. S c i e n c e 254, 1497–1500. Pardridge W, Boado RJ ,and Kang Y.S (1 9 9 5 ) Vectormediated delivery of polyamine (“peptide”) nucleic acid analogue through the blood barrier in vivo. P r o c N a t l Acad Sci USA 92,5592-5596 Pooga H, Soomets U, Hällbrink M, Valkna A, Saar K, Rezaei K, Kahl U, Hao J.X, Xu X.J, Wiesenfeld-Hallin Z, Hokfelt T, Bartfai T ,and Langel U (1 9 9 8 ) Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo N a t B i o t e c h n o l 16, 857861 Rasmussen H, Kastrup J.S, Nielsen J.N, Nielsen J.M ,and Nielsen P.E (1 9 9 7 ) Crystal structure of a peptide nucleic acid (PNA) duplex at 1.7 Å resolution. N a t S t r u c t B i o l 4, 98–101. Scarfi’ S, Gaspsarini A, Damonte G ,and Benatti U.(1 9 9 7) Synthesis,uptake and intracellular metabolism of a hydrophobic tetra-peptide nucleic acid(PNA)-biotin molecule. B i o c h e m B i o p h y s Res Commun 236,323-326 Scarfi’ S, Giovine M, Gaspsarini A, Damonte G, Millo E, Pozzolino M ,and Benatti U.(1 9 9 9 ) Modified peptide nucleic acids are internalized in mouse macrophages RAW 264.7 and inhibit inducible nitric oxide synthase. FEBS l e t t e r s 451,264-268. Simmons C.G, Pitts A.E, Mayfield L.D, Shay J.W ,and Corey D.R (1 9 9 7 ) Synthesis and membrane permeability of PNA-peptide conjugates B i o o r g M e d C h e m Lett 7, 3001–3006. Taylor R.W, Chinnery P.F, Turnbull D.M ,and Lightowlers R.N (1 9 9 7 ) Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids. Nat Genet 15, 212–215. Tyler B.M, Jansen K, McCormick D.J, Douglas C.L, Boules M, Stewart J.A, Zhao L, Lacy B, Cusak B, Fauq A ,and Richelson E. (1 9 9 9 ). Peptide nucleic acids targeted to the neurotensin receptor and administered i.p. cross the blood

barrier and specifically reduce gene expression. Proc Natl Acad Sci USA 96, 7053-7058 Tyler B.M, McCormick D.J, Hoshall C.V, Douglas C.L, Jansen K, Lacy B.W, Cusack B ,and Richelson E (1 9 9 8 ) Specific gene blockade shows that peptide nucleic acids readily enter neuronal cells in vivo.FEBS Lett 421, 280–284 Villa R, FoliniM, Lualdi S, Veronese S, Daidone M.R ,and Zaffaroni N. (2 0 0 0 ) Inhibition of telomerase activity by a cell-penetrating peptide nucleic acid construct in human melanoma cells. FEBS Letters 473, 241-248.

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Gene Ther Mol Biol Vol 5, 55-61, 2000

Delivery of plasmid DNA by in vivo electroporation Review Article

Loree Heller1* and M. Lee Lucas2 1

Center for Molecular Delivery, University of South Florida, Tampa, FL 33612, 2Department of Medical Microbiology, University of South Florida, Tampa, FL 33612

__________________________________________________________________________________ * Correspondence: Loree Heller, University of South Florida, Center for Molecular Delivery, MDC16, 12901 Bruce B. Downs Blvd., Tampa, FL 33612; Telephone: 813-974-8184; Fax: 813-974-2669; Email: lheller@com1.med.usf.edu Key words: gene therapy, electroporation Received: 13 June 2000; accepted: 13 July 2000

Summary For gene therapy, i n v i v o delivery of plasmid DNA offers an alternative to viral delivery methods. Since the efficiency of plasmid delivery to tissues is generally lower than viral delivery, several methods have been introduced t o augment i n v i v o plasmid delivery including liposome conjugation, particle mediated delivery, and electroporation. I n v i v o electroporation has reached clinical trials for the delivery of chemotherapeutic agents to cancers, and a number of preclinical studies have been performed demonstrating that this technique also enhances plasmid delivery and expression of both reporter and therapeutic genes or cDNAs. This delivery has been performed to a n u m b e r o f t i s s u e s i n c l u d i n g s k i n , m u s c l e , l i v e r , t e s t e s a n d t u m o r s e m p l o y i n g a w i d e range of electrical conditions and electrodes. While this preclinical research i s promising, further optimization of electrical conditions and electrodes may be necessary for clinical use. With the availability of a variety of therapeutic gene delivery techniques, it will be possible to tailor gene therapies to individual diseases. liposomes, microparticle bombardment and electroporation. During in vivo electroporation, electric pulses are applied directly to the tissue to enhance uptake of extracellular molecules (reviewed in Jaroszeski et al, 2000). The delivery of chemotherapeutic agents to tumors by this method was first demonstrated in subcutaneously injected hepatocellular carcinomas (Okino et al, 1987). This technique, termed electrochemotherapy, results in substantial, but localized tumor necrosis. Electrochemotherapy is a highly effective anti-tumor regimen and has been demonstrated preclinically in variety of cutaneous and internal tumors, including rat (Jaroszeski et al, 1997a) and rabbit (Ramirez et al, 1998) hepatocellular carcinomas, Lewis lung carcinoma (Kanesada, 1990), sarcomas and melanomas (Mir et al, 1991), mammary tumors (Belehradek et al, 1991), gliomas (Salford et al, 1993), fibrosarcomas (Sersa et al, 1995), and melanomas (Heller et al, 1995). This work was extended to clinical trials in head and neck squamous cell carcinoma, melanoma, basal cell carcinoma, Kaposiâ&#x20AC;&#x2122;s sarcoma, and adenocarcinoma (Jaroszeski et al, 1997b; Heller et al, 1999). In these clinical trials, objective responses varied from 72 to 100%.

I. Introduction Gene therapy has the potential to play a role in the effective treatment of a variety of diseases. Biological gene therapy employs genetically engineered viruses to deliver the desired gene or cDNA. Several types of viral vectors including recombinant retroviruses, adenoviruses, and adeno-associated viruses have been described or employed in gene delivery (Kay, 1997). While gene therapy with these vectors may result in high protein levels and long term expression, short term, lower levels of expression of molecules such as immune modifiers may also be desirable. Ideally, multiple gene delivery techniques may be necessary to fit multiple treatment regimens. The delivery of plasmid DNA encoding the gene or cDNA of interest may also be used for gene therapy. Plasmid DNA is neither replicated nor integrated into the host cell genome, but remains in its episomal form (Nichols et al, 1995), and expression is generally short term in tissues other than muscle. DNA injection does not result in the production of anti-DNA antibodies (Jiao et al, 1992; Robertson, 1994), which allows for multiple treatments. Since the efficiency of plasmid delivery is lower than that of viral delivery, several methods have been introduced to increase delivery in vivo, including

Heller and Lucas: Delivery of plasmid DNA by in vivo electroporation

Table 1. Luciferase expression after muscle electroporation of plasmid DNA Mir et al, 1999 Pulse description Electrode µg p l a s m i d Muscle treated Fold increase in expression Peak measured expression Observed length of expression

Vicat et al, 2000

Square LVLP Caliper 15

Mathiesen et al, 1999 Bipolar trains Wire pair 25

mouse tibial cranial (transcutaneous)

rat soleus (surgically exposed)

mouse tibialis anterior (exposed)

100

500

mouse gastrocnemius (transcutaneous) 1000

Day 7

Day 3

Day 1

Day 2

9 months

3 days

6 months

7 days

Square HVSP Caliper 10

Lucas et al, submitted EEP Needle 100

LVLP, low voltage long pulses; HVSP, high voltage short pulses; EEP, exponentially enhanced pulses

II. Reporter gene delivery with in vivo electroporation

approximately 30% of cells in the electroporated area expressed !-galactosidase and this expression was still detectable 21 days later. The expression was dose dependent, with peak expression occurring with delivery of 25 µg plasmid. GFP expression in normal liver from delivery of plasmid DNA was examined using pulses that were less intense but 500 fold longer (eight 50 ms at a variety of field strengths) and a disk electrode (Suzuki et al, 1998). The highest expression was noted at 250 V/cm, while the extreme damage noted at 500 V/cm probably contributed to the lack of expression. This expression was 3.5 fold higher than with 50 ms pulses than with 25 or 99 ms pulses at this field strength. These investigators found a DNA dose dependent expression to 80 µg plasmid DNA, which may be due to the different electroporation parameters used. Clearly, different pulsing parameters may be used to effectively enhance plasmid DNA deliver to the same tissue. Delivery by lipid conjugation, microparticle bombardment and electroporation using equal quantities of plasmid DNA was directly compared in chicken embryos (Muramatsu et al, 1997a). For liposome-mediated delivery, DNA was complexed per manufacturer’s instructions (Lipofectamine, Gibco BRL, USA) and injected. In microparticle bombardment, DNA was bound to 0.9 mg tungsten beads and delivered by nitrogen gas at 20 kfg/cm2 at a distance of 6 cm. For electroporation delivery, tissue received three 50 ms pulses at 31.25 V/cm with a plate electrode after plasmid injection. No significant difference was noted in embryo viability or number of embryos positive for !-galactosidase staining 48 hours after delivery, although electroporation appeared to transfect a larger area with a greater intensity of expression. Using CAT as a reporter, this group also compared several electroporation conditions for optimization of DNA delivery in surgically exposed mouse testes (Muramatsu et

Delivery of DNA to cells by the application of electric pulses was first reported in 1982 (Wong and Neumann, 1982). In vivo electroporation to enhance plasmid delivery was later demonstrated in the skin cells of newborn mice using exponential pulses with a clip electrode (Titomirov et al, 1991). Optimal transformation of skin cells was noted after two pulses in opposing polarities of a field strength of 300-400 V/cm and a pulse length of 0.1-0.3 ms. In experiments using three much longer, higher amplitude exponential pulses and the addition of pressure with a caliper electrode (1200V/cm, 10-20 ms), !-galactosidase expression was detected to a depth of 370 µm in the skin of hairless mice after topical administration of plasmid DNA (Zhang et al, 1996). More recently, delivery to skin with square wave pulses after intradermal injection has been demonstrated (Heller et al, submitted). While plasmid delivery with caliper electrodes and eight short (0.1 ms), high field strength (1500 V/cm) pulses increased luciferase reporter expression10 fold in the treated skin 24 hours after treatment, no increase was seen using eight long (20 ms) lower field strength (100 V/cm) pulses. Skin delivery of a plasmid encoding IL12 induced a systemic response as well, in the form of increased serum levels of interferon " . In these experiments, plasmid delivery with a custom designed electrode induced higher expression than delivery with simple calipers. These results emphasize the importance of optimizing both pulsing protocols and electrode configurations with respect to tissue type to avoid damage and maximize protein expression. Electroporation enhances plasmid expression in other tissues. Delivery to normal rat liver was first characterized using six 0.1 ms pulses at a variety of field strengths using a 6 needle array (Heller et al, 1996). Maximum luciferase expression occurred at 1000 to 1500 V/cm, electrical conditions very similar to those used for drug delivery in clinical trials. 48 hours after treatment,

Gene Therapy and Molecular Biology Vol 5, page 57

Table 2. Serum protein expression after muscle electroporation of plasmid DNA

Pulse description Electrode Muscle treated Fold increase in expression Peak measured expression Observed length of expression Reporter

Aihara et al, 1998 Square LVLP

Rizzuto et al, 1999

Maruyama et al, 2000 Square LVLP

Widera et al, 2000 Square LVLP

Needle pair Mouse tibialis anterior (transcutaneous) 119

Parallel wires Mouse quadriceps (exposed)

Needle pair Rat thigh (exposed)

>100

Needle pair Mouse tibialis anterior (transcutaneous) 400

Day 5-7

Day 7

Day 5

< 42 days

84 days

>21 days

20 days

Interleukin-5

Erythropoietin

HBsAg

Bipolar trains

LVLP, low voltage long pulses; mice received 50 µg plasmid; rats received 400 µg plasmid

al, 1997b). In each case, eight low voltage, long pulses were delivered. Tissue damage due to heat generation was observed after severe conditions such as 100V for 50 ms, so only conditions that caused minimal damage were presented. The optimal conditions elucidated in this experiment were 50 V for 10ms or 25 V for 50 ms. Furthermore, CAT activity was examined after liposome, microparticle bombardment, and electroporation deliveries to mouse testes (Muramatsu et al, 1998). Microparticle bombardment and electroporation both significantly increased expression, while liposome delivery had no significant effect on expression. Much of the focus of in vivo electroporation has been on muscle delivery. Mouse skeletal muscle injected with plasmid DNA alone expressed reporter genes in both dividing and non-dividing cells (Wolff et al, 1990). This expression can be significantly augmented by the addition of electric pulses (Aihara et al, 1998). In addition, individual variability in transgene expression is reduced after electroporation (Mir et al, 1999). The data from several groups that have examined luciferase reporter expression after muscle electroporation is summarized in T a b l e 1. Mir et al (1999) found maximum luciferase expression after delivery of eight 20 ms pulses at 200 V/cm with a caliper electrode in mouse tibial cranial muscles. This expression lasted 9 months. Electroporation also enhanced luciferase plasmid DNA expression in various mouse, rat, and rabbit muscles, and expression of FGF1 was detected in muscle after plasmid electroporation of mouse and monkey muscle. Similar long, low voltage pulses (ten 40 ms) were compared to trains of bipolar pulses (ten 1000 Hz trains of on thousand 0.4 ms pulses each), both at a field strength of 100-180 V/cm (Mathiesen, 1999). The pulsing protocols, which resulted in the same total pulse time, induced similar levels of expression. Although more muscle damage was observed after the long pulses, the muscle cells had regenerated by 2 weeks after treatment.

While one group found a 50 fold decrease in luciferase expression after plasmid delivery with eight 0.1 ms 1200 V/cm pulses (Mir et al, 1999), another found increasing expression with increasing field strength with 0.1 ms pulses to an expression maximum at 1800 V/cm (Vicat et al, 2000). This expression increased linearly with DNA concentration up to 50 µg. This group also reported more stable expression (>6 months) with shorter pulses. This difference may be due to the more intense immune infiltrate observed after longer pulses. A 20 fold increase in expression after short, high voltage electroporation delivery was confirmed using 0.1 ms pulses at 1500 V/cm (Lucas et al, submitted), although in these experiments, long, low voltage pulses or exponentially enhanced pulses induced an even larger (1000 fold) increase in luciferase expression. Since these groups compared similar pulsing parameters, other variables such as the electrode used may be affecting the level of delivery. Clearly, though, plasmid delivery to muscle is enhanced by several different pulse types. In vivo muscle electroporation also enhances systemic expression of plasmid DNA. A comparison of electroporation enhanced serum protein expression after in vivo electroporation is summarized in Table 2. Since several different serum reporter plasmids were used, the increase in expression noted may not be directly comparable. Electroporation significantly increased serum expression of interleukin-5 after muscle injection (Aihara et al, 1998). In this case, three 50 ms 100 V/cm square wave pulses were delivered with a needle pair. Serum expression of mouse erythropoietin was demonstrated after electroporation of as little as 1 µg plasmid DNA into muscle with ten 1 second pulse trains of on thousand square bipolar pulses (0.2 ms each, 90 V/cm, Rizzuto et al, 1999). Erythropoietin levels peaked at day 7 and were elevated for at least 84 days. No muscle damage from electroporation was detected histologically 24 hours after treatment. At one week, areas of muscle fibers with central nuclei and small necrotic areas with lympocyte

Heller and Lucas: Delivery of plasmid DNA by in vivo electroporation

infiltrations were observed, constituting up to 10-20% of the total electroporated muscle. At one month after treatment, muscles appeared normal. A plasmid encoding rat erythropoietin was also delivered to rat muscle at 4 sites with 100 µg plasmid each with eight 50 ms 200 V/cm pulses delivered with a needle pair (Maruyama et al, 2000). In this case, serum erythropoietin expression peaked at day 7 and continued for 32 days, with a corresponding increase in hematocrit continuing at least to day 32. Systemic expression of a hepatitis B surface antigen was also observed after in vivo electroporation of 50 µg plasmid (Widera et al, 2000). After delivery of six 50 ms 100 V/cm pulses with a needle pair, antigen expression peaked 5 days after treatment and continued at least 20 days post injection in nude mice, while this expression began to wane at day 13 in immunocompetent mice, possibly due to a cellular immune response against transfected muscle cells or clearance of antigen in antigenantibody complexes. Electrically enhanced gene transfer to muscle, which can result in both local and systemic transgene expression, may prove an effective tool for treating a variety of diseases. A wide range of electrical conditions has also been used to enhance plasmid delivery directly to several tumor types. Increased plasmid encoded !-galactosidase expression was first demonstrated after intra-arterial injection of plasmid DNA, followed by eight 0.099 ms 600 V/cm electric pulses applied directly to rat brain tumors (Nishi et al, 1996). Plasmid delivery by hemagglutinating virus of Japan (HSV) liposomes, microparticle bombardment and electroporation was compared in rat bladder cancers (Harimoto et al, 1998). In this experiment, eight 50 ms pulses at a field strength of 143-1000 V/cm were applied with a needle type electrode. These researchers found that all three delivery methods may be suitable for therapy of localized bladder tumors. Expression of !-galactosidase was also detected in B16 mouse melanomas after intratumor injection of 12 µg plasmid and application of ten 5 ms pulses at 800-900 V/cm with caliper electrodes (Rols et al, 1999). No gene transfer was detected in these experiments with short (µs) pulses. In contrast, 15% of B16 melanoma cells were positive for !-galactosidase expression after intratumor delivery of 100 µg plasmid with fourteen 0.1 ms pulses at a field strength of 1500 V/cm (Niu et al, 1999). In these experiments, pulses were delivered segmentally so that each tissue area received only two pulses. The significant difference between these two results may be due to different electrode configurations or to the use of different reporter plasmids. Using 0.1 ms pulses, a plasmid encoding luciferase was delivered to rat liver tumors (Heller et al, 2000). Expression at 48 hours was highest at 2000 V/cm, although this extreme field strength burned the tissue immediately around the electrodes. Therefore, 1500 V/cm, which caused no evidence of tissue damage, was used for subsequent experiments. The level of luciferase expression in response to electroporation increased as the amount of plasmid increased up to 2 µg/mm 3 initial tumor volume. Histochemical staining for !-galactosidase expression was also enhanced by electroporation (Figure 1). In an

interesting set of experiments in mouse breast tumors, in vivo electroporation of lipid complexed and naked DNA was compared (Wells et al, 2000). Using caliper electrodes and six 1 ms pulses, luciferase expression was augmented 2 orders of magnitude maximally 48 hours after delivery at 1100 V/cm. No statistical difference in expression was noted between electroporation of naked plasmid or lipoplexes.

III. Preclinical gene therapy with in vivo electroporation Electroporation enhanced plasmid delivery to muscle may induce a clinically relevant response. After delivery of a plasmid encoding erythropoietin, the responding hematocrit is significantly increased in mice up to 6 months (Rizzuto et al, 1999) after delivery of as little as 1 µg plasmid or in rats up to 32 days after delivery of 400 µg plasmid (Maruyama et al, 2000). An immune response to viral proteins can also be induced after muscle electroporation of plasmid DNA. In mice, strong antibody responses to HbsAg and to HIV gag protein were detected two weeks after delivery of 3 µg or 10 µg plasmid respectively (Widera et al, 2000). In addition, four weeks after plasmid delivery, an anti-gag T cell response was observed after challenge with a vaccinia virus expressing HIV gag. Although there is no consensus as to the “best” muscle electroporation conditions, the experiments described here demonstrate that electroporation of plasmid DNA into muscle has potential as a gene therapy in a clinical setting, enhancing both intramuscular and systemic transgene expression. Electroporation enhanced plasmid delivery directly to tumors may also induce a clinically relevant response. After delivery of a plasmid encoding human monocyte chemoattractant protein-1 into rat brain tumors, large numbers of macrophages and lymphocytes were observed in the tumor tissues (Nishi et al, 1996). Electroporation enhanced delivery of a plasmid encoding a dominant negative Stat3 variant into B16 mouse melanomas inhibited tumor growth and caused tumor regression mediated by apoptosis (Niu et al, 1999). The combination of electrochemotherapy and cytokine plasmid delivery by electroporation into B16 melanomas prevents tumor recurrence and induces long term antitumor immunity in mice (Heller et al, submitted). After electroporation of plasmids encoding diphtheria toxin or herpes simplex thymidine kinase followed by gancyclovir administration, the growth of subcutaneously inoculated colon adenocarcinomas was significantly inhibited (Goto et al, 2000).

IV. Conclusions and clinical considerations While electroporation is highly effective at enhancing tissue expression of transgenes, it will be necessary to optimize two variables for plasmid DNA delivery, quite possibly for each tissue or tumor type: the pulsing conditions and the electrodes used for pulse delivery.

Gene Therapy and Molecular Biology Vol 5, page 59

Figure 1. Histochemical staining for !-galactosidase expression in rat hepatocellular carcinomas (Heller et al, 2000). 15 µm frozen sections were stained for expression 48 hours after delivery. (a) Injection only of 50 µg pSV-!gal (Promega, Madison, WI, USA); (b) Injection of 50 µg pSV-!gal followed by two 100 µs pulses at 1250 V/cm per tissue area.

Several pulse shapes will enhance DNA delivery in vivo, including exponential, square wave, and bipolar trains. In each case, the total number, length, and field strength of each pulse can be varied considerably. Effective pulse lengths range from 0.1 to 50 ms, while effective field strengths range from 200 to 1800 V/cm. The potential for damage resulting from delivery, which may result from irreversible electroporation or from heat damage after long or high intensity pulses, must be balanced against the increased DNA expression. This damage may be much more important in plasmid delivery to healthy tissues such as muscle than delivery directly to tumor tissue. The potential damage from electric pulses to rabbit liver, pancreas, kidney or spleen after delivery of eight 0.1 ms pulses at a field strength of 850 V/cm was assessed histologically (Ramirez et al, 1998). Immediately after pulse application, the primary effect was edema formation. At days 2 and 7 after pulse delivery, localized necrosis and fibrosis was limited to the electrode contact sites. In depth studies of the effect of six 0.1 ms 1300V/cm pulses on skin, muscle, nerves, and blood vessels in the hind limbs of rats were also performed (Richard Heller, personal communication). Histological analysis of the tissues after one to three treatments showed short term (3 days), localized necrosis to skin and muscle that started to resolve within 14 days and completely resolved by 56 days after treatment. In addition, all animals regained full limb function within 6 minutes after the therapy was administered. For clinical consideration, detailed experiments elucidating damage from long, low voltage pulses will also be necessary. Electrode design must also be optimized (Gilbert et al, 1997). Caliper or plate electrodes are easily available and simple to use, although the treated tissue must be accessible and in each case the voltage must be calculated based on the thickness of tissue “gripped”. In this case, electric pulses may be delivered over a large surface area,

but this delivery also requires higher voltages to maintain the field strength. Needle electrodes increase the depth of delivery and allow treatment of tissue without altering tissue shape. The treated area can be better defined as well, and the specific voltage used can be standardized from sample to sample. Particularly for clinical use, it will be necessary to design pulsing protocols and electrodes specifically for each application of gene therapy.

References Aihara H, Miyazaki JI. (1 9 9 8 ) Gene transfer into muscle by electroporation in vivo. N a t B i o t e c h 6, 867-870. Belehradek Jr. J, Orlowski S, Poddevin B, Paoletti C, and Mir LM. (1 9 9 1 ) Electrochemotherapy of spontaneous mammory tumors in mice. Eur J Cancer 27, 73-76. Gilbert R, Jaroszeski MJ, and Heller R. (1 9 9 7 ) Novel electrode designs for electrochemotherapy. B i o c h i m B i o p h y s A c t a 1334, 9-14. Goto T, Nishi T, Tamura T, Dev SB, Takeshima H, Kochi M, Yoshizato K, Kuratsu J-I, Sakata T, Hofmann GA, and Ushio Y. (2 0 0 0 ) Highly efficient electro-gene therapy of solid tumor by using an expression plasmid for the herpes simplex virus thymidine kinase gene. P r o c N a t l A c a d Sci USA 97, 354-359. Harimoto K, Sugimura K, Lee CR, Kuratsukuri K, and Kishimoto T. (1 9 9 8 ) In vivo gene transfer methods in the bladder without viral vectors. B r J U r o l 81, 870874. Heller L, Jaroszeski MJ, Coppola D, Pottinger C, Gilbert R, and Heller R. (2 0 0 0 ) Electrically mediated plasmid DNA delivery to hepatocellular carcinomas in vivo. Gene Ther 7, 826-829. Heller L, Pottinger C, Jaroszeski MJ, Gilbert R, and Heller R. In vivo electroporation of plasmids encoding GM-CSF or interleukin-2 into existing B16 melanomas combined with electrochemotherapy induces long term antitumor immunity, submitted for publication. Heller R, Gilbert R, and Jaroszeski M. (1 9 9 9 ) Clinical applications of electrochemotherapy. Adv Drug D e l R e v 35, 119-129.

Heller and Lucas: Delivery of plasmid DNA by in vivo electroporation

Heller R, Jaroszeski M, Atkin A, Moradpour D, Gilbert R, Wands J, and Nicolau C. (1 9 9 6 ) In vivo electroinjection and expression in rat liver. FEBS Letters 389,225-228. Heller R, Jaroszeski M, Leo-Messina J, Perrot R, Van Voorhis N, Reintgen D, Gilbert R. (1 9 9 5 ) Treatment of B16 mouse melanoma with the combination of electropermeabilization chemotherapy. B i o e l e c t r o c h e m B i o e n e r g 36, 83-87. Heller R, Schultz J, Lucas ML, Jaroszeski MJ, Heller LC, Gilbert RA, Moelling K, and Nicolau C. Intradermal delivery of IL-12 plasmid DNA by in vivo electroporation, submitted for publication. Jaroszeski MJ, Gilbert R, and Heller R. (1 9 9 7 a ) In vivo antitumor effects of electrochemotherapy in a hepatoma model. B i o c h i m i c B i o p h y s A c t a 1334, 15-18. Jaroszeski MJ, Gilbert R, and Heller R. (1 9 9 7 b ) Electrochemotherapy: An emerging drug delivery method for the treatment of cancer. Adv Drug D e l R e v 26, 185-197. Jaroszeski MJ, Heller R, and Gilbert R. (2 0 0 0 ) Electrochemotherapy, electrogenetherapy, and transdermal drug delivery. Humana Press, Totowa, NJ. Jiao S, Williams P, Berg RK, Hodgeman BA, Liu L, Repetto G, and Wolff JA. (1 9 9 2 ) Direct gene transfer into nonhuman primate myofibers in vivo. Hum Gene Ther 3, 21-33. Kanesada K. (1 9 9 0 ) Anticancer effect of high voltage pulses combined with concentration dependent anticancer drugs on Lewis lung carcinoma. J J p n S o c C a n c e r Ther 25, 2640-2648. Kay MA, Liu D, Hoogerbrugge PM. (1 9 9 1 ) Gene therapy. Proc Natl Acad Sci USA 94, 12744-12746. Lucas ML, Jaroszeski MJ, Gilbert R, and Heller R. In vivo delivery of plasmid DNA using an exponentially enhanced pulse (EEP), a new wave form for electroporation, submitted for publication. Maruyama H, Sugawa M, Moriguchi Y, Imazeki I, Ishikawa Y, Ataka K, Hasegawa S, Ito Y, Higuchi N, Kazama JJ, Gejyo F, and Miyazaki, J-I. (2 0 0 0 ) Continuous erythropoietin delivery by muscle-targeted gene transfer using in vivo electroporation. Hum Gene Ther 11,429-437. Mathiesen, I. ( 1 9 9 9 ) Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Ther 6,508-514. Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D, Caillaud J-M, Delaere P, Branellec D, Schwartz B, and Scherman D. (1 9 9 9 ) High-efficiency gene transfer into skeletal muscle mediated by electric pulses. P r o c N a t l A c a d S c i U S A 96, 4262-4267. Mir LM, Orlowski S, Belehradek Jr J, and Paoletti C. (1 9 9 1 ) Electrochemotherapy potentiation of antitumor effect of bleomycin by local electric pulses. Eur J Cancer 27, 68-72. Muramatsu T, Mizutani Y, Ohmori Y, and Okumura J-i. ( 1 9 9 7 a ) Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo. B i o c h e m B i o p h y s R e s Com 230,376-380. Muramatsu T, Nakamura A, and Park H-M. (1 9 9 8 ) In vivo electroporation: A powerful and convenient means of nonviral gene transfer to tissues of living animals. I n t J Mol Med 1,55-62. Muramatsu T, Shibata O, Ryoki S, Ohmori Y, and Okumura, Ji. (1 9 9 7 b ) Foreign gene expression in the mouse testis by localized in vivo gene transfer. B i o c h e m B i o p h y s Res Com 233,45-49.

Nichols WW, Ledwith BJ, Manam SV, and Troilo PJ. (1 9 9 5 ) Potential DNA vaccine integration into host cell genome. Ann New York Acad Sci 772, 30-39. Nishi T, Yoshizato K, Yamashiro S, Takeshima H, Sato K, Hamada K, Kitamura I, Yoshimura T, Saya H, Kuratsu J, and Ushio Y. ( 1 9 9 6 ) High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation. Cancer R e s 56, 1050-1055. Niu G, Heller R, Catlett-Falcone R, Coppola D, Jaroszeski M, Dalton W, Jove R, and Yu H. (1 9 9 9 ) Gene therapy with dominant-negative Stat3 suppresses growth of the murine melanoma B16 tumor in vivo. C a n c e r R e s 59, 50595063. Okino M, Marumoto M, Kanesada H, Kuga K, and Mohri H. (1 9 8 7 ) Electrical impulse chemotherapy for rat solid tumors. Proc Jpn Cancer Congress 46, 420. Ramirez LH, Orlowski S, An D, Bindoula G, Dzodic R, Ardouin P, Bognel C, Belehradek Jr. J., Munck J-N, and Mir LM. (1 9 9 8 ) Electrochemotherapy on liver tumours in rabbits. Br J Cancer 77, 2104-2111. Rizzuto G, Cappelletti M, Maione D, Savino R, Lazzaro D, Costa P, Mathiesen I, Cortese R, Ciliberto G, Laufer R, La Monica N and Fattori E. (1 9 9 9 ) Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. P r o c N a t l A c a d S c i U S A 96, 6417-22. Robertson JS. (1 9 9 4 ) Safety considerations for nucleic acid vaccines. V a c c i n e 12, 1526-1528. Rols M-P, Delteil C, Golzio M, Dumond P, Cros S, and Teissie J. (1 9 9 8 ) In vivo electrically mediated protein and gene transfer in murine melanoma. Nat B i o t e c h 16, 168171. Salford LG, Persoon BRR, Brun A, Ceberg CP, Kongstad PC, and Mir LM. (1 9 9 3 ) A new brain tumor therapy combining bleomycin with in vivo electropermeabilization. B i o c h e m B i o p h y s R e s Com 194, 938-943. Sersa G, Cemazar M, and Miklavcic D. (1 9 9 5 ) Antitumor effectiveness of electrochemotherapy with cisdiamminedichloroplatinum (II) in mice. Cancer Res 55, 3450-3455. Suzuki T, Shin B-C, Fujikura K., Matsuzaki T, and Takata K. (1 9 9 8 ) Direct gene transfer into rat liver cells by in vivo electroporation. FEBS Letters 425,436-440. Titomirov AV, Sukharev S, and Kistanova E. (1 9 9 1 ) In vivo electroporation and stable transformation of newborn mice by plasmid DNA. B i o c h i m B i o p h y s A c t a 1088, 131-134. Vicat JM, Boisseau S, Jourdes P, Laine M, Wion D, BoualiBenazzouz R, Benabid AL, and Berger F. (2 0 0 0 ) Muscle transfection by electroporation with high-voltage and short-pulse currents provides high-level and long-lasting gene expression. Hum Gene Ther 11,909-916. Wells JM, Li LH, Sen A, Jahreis GP, and Hiu SW. (20 0 0 ) Electroporation-enhanced gene delivery in mammary tumors. Gene Ther 7, 541-547. Widera G, Austin M, Rabussay D, Goldbeck C, Barnett SW, Chen M, Leung L, Otten GR, Thudium K, Selby MJ, and Ulmer JB. (2 0 0 0 ) Increased DNA vaccine delivery and immunogenicity by electroporation in vivo. J I m m u n o 164,4635-4640. Wolff .A, Malone RA, Williams P, Chong W, Acsadi G, Jani A, and Felgner PL. (1 9 9 0 ) Direct gene transfer into mouse muscle in vivo. S c i e n c e 247, 1465-1468.

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Wong T-K and Neumann, E. (1982) Electric field mediated gene transfer. B i o c h e m B i o p h y s R e s Com 107, 584-587. Zhang L, Lingna L, Hoffman GA, and Hoffman R. (1 9 9 6 ) Depth-targeted efficient gene delivery and expression in the skin by pulsed electric fields: An approach to gene therapy of skin aging and other diseases. B i o c h e m B i o p h y s R e s C o m 220,633-636.

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Gene Therapy and Molecular Biology Vol 5, page 63

Gene Ther Mol Biol Vol 5, 63-79, 2000

Potential roles of p53 in recombination Review Article

Nuray Akyüz, Gisa S. Boehden, Christine Janz, Silke Süße, and Lisa Wiesmüller* Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg _________________________________________________________________________________________________ *Correspondence: Lisa Wiesmüller, Ph.D., Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistrasse 52, 20251 Hamburg, Germany; Tel.: +49-40-48051-235; Fax: +49-40-48051-117; Email: wiesmuel@uke.unihamburg.de Key Words: double strand breaks / homologous recombination / nonhomologous end-joining / strand exchange / tumor suppressor

Received: 27 August 2000; accepted: 7 October 2000

Summary Functional inactivation of p53 by mutation or by interactions with viral tumor antigens is associated with a deficit to maintain the genomic stability. Until recently, p53 was believed to exclusively avoid the manifestation of DNA damage by transcriptionally signaling transient cell cycle arrest or apoptotic cell death in response to cellular stress situations. New ideas on a direct involvement of p53 in DNA repair originated from the discoveries of interactions with replication and repair proteins and of repair-related enzymatic activities, such as the 3!-5! exonuclease activity. Moreover, physical and genetic links were established between p53 and factors involved in homologous DNA recombination processes, namely the initial strand transferase RAD51 and the RAD51 complex partner BRCA1 and BRCA2. Importantly, several groups reported that p53 suppresses spontaneous as well as radiation-induced inter- and intrachromosomal homologous recombination events by at least one to two orders of magnitude. The identification of cancer-related separation of function mutations demonstrated that p53 regulates homologous recombination processes independently of its activities in transcription and growth control, and suggested that functions in recombinative repair contribute to tumor suppression. Mechanistic studies indicated that the p53dependent regulation of DNA-exchange events is tied to the generation of DNA strand exchange intermediates by RAD51. The recognition of certain mismatched bases within these heteroduplex joints by p53 has lead to a model suggesting that p53 monitors the fidelity of homologous recombination events in a manner complementary to the mismatch repair factor MSH2. The possibility of an involvement of p53 in replication-associated recombination processes is discussed.

I. Introduction Clues to a role of p53 in DNA repair Soon after having established p53 as the most frequently altered gene in human tumors in the 1980s (Caron de Fromentel and Soussi, 1992; Hollstein et al, 1994; 1996), p53 was understood as a major component of the DNA damage response pathway (Lane, 1992; Ko and Prives, 1996; Levine, 1997). After the introduction of DNA injuries the level of posttranslationally modified p53 protein rises, which in turn induces a transient cell cycle arrest or apoptotic cell death. The p53 response is triggered most rapidly by DNA strand breaks, which can be introduced either directly, e.g. via ionizing irradiation,

or indirectly after the conversion of DNA adducts or single-stranded breaks by DNA repair or replication (Nelson and Kastan, 1994). DNA damage activates p53 through posttranslational modifications by specific kinases, such as the strand break sensor ATM, by acetylases, and by the poly(ADP-ribose)polymerase, which prevent proteolysis via the ARF-MDM2 pathway or enhance binding of p53 to consensus sequences within the genome (Lane, 1998; Wang et al, 1998; Lakin and Jackson, 1999). After the generation of mice nullizygous for p53, it became clear that p53 not only responds to DNA damage, but in fact represents a central player in genome stabilization, thereby counteracting the multistep process

Akyüz et al: p53 in recombination

of tumorigenesis (Livingstone et al, 1992; Harvey et al, 1993; Ishizaki et al, 1994; Cleaver et al, 1999; Schwartz and Russell, 1999). Loss of p53 function was shown to be associated with genetic instabilities,which become manifested as aneuploidies, allelic losses, as well as increases in sister chromatid exchanges and in gene amplification rates. Until the 1990s genome stabilizing functions of p53 were explained by its ability to transcriptionally signal cell cycle arrest in the G1 phase, thereby giving time for DNA repair prior to the entry into S-phase (Lane, 1992). Among the products of the p53 target genes, the cyclin-dependent kinase inhibitor p21WAF1/CIP1 is essential for the execution of the cell cycle arrest at the G1/S transition and to sustain a G2 arrest under certain circumstances (Levine, 1997; Bunz et al, 1998). However, different from p53 knockout mice, which have a predisposition to form various tumors within months (Donehower et al, 1992), mice nullizygous for p21WAF1/CIP1 did not display increased cancersusceptibilities (Deng et al, 1995). As an alternative pathway to avoid the manifestation of DNA damage, p53 induces apoptotic cell death in certain cell types and in response to irrepairable DNA damage (Gottlieb and Oren, 1996; Levine, 1997). Apoptotic signaling involves transcription-independent pathways and the activation of target genes, such as BAX and IGF-BP3, encoding antagonists of the survival factor BCL-2 and of the insulin-like growth factor-1, respectively. However, even the concept that p53-mediated apoptosis in addition to the transcriptional upregulation of cell-cycle regulatory genes prevents spontaneous tumorigenesis turned out to be incomplete. This was indicated by the absence of tumor formation after treatment of mice with "-ray at doses of 68 Gy and a compound, which inactivates transcriptional and apoptotic functions of p53 (Komarov et al, 1999). Due to this observation it is also unlikely that the p53reponsive gene GADD45 plays a central role in p53dependent tumor suppression. GADD45 was proposed to promote nucleotide excision repair (NER) downstream of p53 (Smith et al, 1994). However, according to more recent reports alternative functions of GADD45 in chromatin remodelling and of GADD45-p21WAF1/CIP1 complexes in cell cycle regulation seem to be more likely (Kazantsev et al, 1995; Kearsey et al, 1995a, b; Carrier et al, 1999). Investigations on a direct participation of p53 in DNA repair were stimulated by a number of biochemical observations, which revealed activities of p53 in the recognition of DNA damage, DNA reannealing, strand transfer, and exonucleolytic DNA degradation (Albrechtsen et al, 1999). The C-terminal 30 amino acids of p53 (see Figure 1) recognize several DNA damagerelated structures, such as dsDNA and ssDNA ends, DNA gaps, and insertion/deletion mismatches (Bakalkin et al, 1994; Jayaraman and Prives, 1995; Reed et al, 1995; Lee et al, 1997; Protopova et al, 2000). They are also required for the RNA and DNA reannealing and strand transfer

activities (Oberosler et al, 1993; Brain and Jenkins, 1994; Wu et al, 1995). Noticeably, the same central region encompassing amino acids 83 to 323 within human p53, where tumorigenic mutations are clustered (Hollstein et al, 1991), recognizes DNA sequence-specifically and is necessary and sufficient for the 3´-5´ exonuclease activity on DNA (Mummenbrauer et al, 1996; Janus et al, 1999). Since its discovery the exonucleolytic activity intrinsic to p53 has been confirmed by several groups (Jean et al, 1997; Huang, 1998; Shakked, 2000). This finding raised the question, whether p53 is involved in fidelity control of DNA replication, in the removal of unpaired regions during the course of the post-replicative mismatch repair, or in DNA end processing for conservative or nonconservative pathways of double-strand break (DSB) repair. In agreement with a possible role of p53 in DNA replication, p53 was found in replicative foci of virusinfected cells (Wilcock and Lane, 1991; Fortunato and Spector, 1998). Furthermore, p53 physically interacts with the single-stranded DNA binding protein RPA and with polymerase # (Wold, 1997; Kühn et al, 1999). In agreement with a possible role of p53 in DNA repair, p53 binds to a plethora of repair-related proteins, namely via residues 300 to 393 to the two helicase components, XPB and XPD, of the dual transcription initiation/repair factor TFIIH, to CSB, a helicase involved in coupling transcription to NER, to topoisomerase I (TOPO I) and II (TOPO II), involved in transcription, replication, repair, and recombination, and to the Werner's Syndrome Protein (WRN), a helicase and exonuclease related to genomic stabilization and with putative functions in replication initiation (Xiao et al, 1994; Wang et al, 1994; Wang et al, 1995; Leveillard et al, 1996; Albor et al, 1998; Blander et al, 1999; Gobert et al, 1999; Cowell et al, 2000). The meaning of most of these interactions is not yet clear. Thus, uncertainties exist on a direct participation of p53 in NER by modulating TFIIH activities: Some groups reported on defective NER in cells with reduced levels of wild-type p53 (Smith et al, 1995; Wang et al, 1995; Mirzayans et al, 1996; Ford and Hanawalt, 1997). However, others saw an increase in sister chromatid exchanges (SCEs) after UV irradiation in p53-deficient cells rather than differences in repair (Ishizaki et al, 1994; Cleaver et al, 1999).

II. Links to homologous recombination A close functional relationship between p53 and the human RecA counterpart, HsRAD51, must be deduced from accumulating biochemical and genetic data (Lim and Hasty, 1996; Stürzbecher et al, 1996; Tsuzuki et al, 1996; Buchhop et al, 1997; Süße et al, 2000). In E. coli RecA is sufficient to execute ATP-dependent homologous pairing and strand exchange over a distance of 6 kb (Roca and Cox, 1990; Kowalczykowski, 1991; Radding, 1991; West, 1992). Like RecA also the human RAD51 protein (HsRAD51) binds to single- and double-stranded DNAs

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(ssDNA and dsDNA), assembles cooperatively into helical nucleoprotein filaments, synapses ssDNA with homologous dsDNA, and catalyzes strand exchange (Sung and Robberson, 1995; Baumann et al, 1996; Gupta et al, 1997, 1999; Baumann and West, 1999). However, HsRAD51 hydrolyzes ATP and promotes strand exchange at least 10 fold less efficiently than RecA, so that the length of heteroduplex DNA formed is limited to 1.3 kb. Therefore, homologous pairing and strand exchange by RAD51 in vivo are facilitated by the DNA-end binding protein RAD52, the DNA-dependent ATPase RAD54, and the single-stranded DNA binding protein RPA (reviewed in Baumann and West, 1998). Direct physical interactions between wild-type p53 and HsRAD51 (St端rzbecher et al, 1996) were indicated from the results of immunoprecipitation experiments with mammalian cell extracts and Ni2+-NTA- or GST-pull-down assays with recombinant proteins. The p53 point mutants p53(135tyr),

p53(249ser), and p53(273his) showed weaker interactions (Buchhop et al, 1997). According to the mapping analysis by the same authors, two segments of the central domain of p53, namely between amino acids 94 and 160 and between 264 and 315, are important for HsRAD51 binding. The highly conserved amino acids 125 to 220 of HsRAD51, comprising the nucleotide binding motif and, in analogy to RecA, the putative homooligomerization domain, were implicated in binding to p53. p53 has also been shown to interact with the products of the two major hereditary breast cancer susceptibility genes, BRCA1 and BRCA2, which form stable complexes with RAD51 in the nuclei of mitotic and meiotic cells (Scully et al, 1997; Chen et al, 1998; Marmorstein et al, 1998; Ouchi et al, 1998; Zhang et al, 1998). Functions in DNA repair, in histone acetylation, and in checkpoint signaling at the G2/M transition have been ascribed to BRCA2 (Patel et al,

Figure 1. The tumor suppressor p53 displays molecular interactions and biochemical activities related to transcription and to DNA repair. The amino acids, which are most frequently mutated during cancerogenesis, are indicated by vertical bars at the relative positions within the p53 molecule. The height of each bar reflects the occurrence in cancer patients. Well-established phosphorylation (P) and acetylation sites (A) and the modifying enzymes are indicated. The regions of p53, to which certain biochemical functions were ascribed, are indicated according to the mapping studies cited in the text. The domains interacting with transcription or repair factors, with the large tumor antigen of the Simian virus 40 (SV40Tag), and with the tyrosine kinase c-ABL are marked below the p53 scheme.

Akyüz et al: p53 in recombination

1998; Siddique et al, 1998; Chen et al, 1999a). BRCA1 was linked to transcriptional regulation, and transcriptional transactivation of target genes, such as p21WAF1/CIP1 and GADD45, seems to underly cell cycle control and the induction of apoptosis (Chen et al, 1999b; Venkitaram, 1999). Furthermore, a role in recombination was recently demonstrated, since BRCA1 directs DSB repair from nonhomologous end-joining (NHEJ) into the nonmutagenic pathway of homologous recombination (Moynahan et al, 1999). Consistently, after irradiation BRCA1 displays a mutually exclusive association with either RAD51, the initial strand transferase of homologous recombination, or with RAD50-hMRE11-p95 complexes, which participate in DSB repair via NHEJ and via homologous recombination pathways (Scully et al, 1997; Haber, 1998; Zhong et al, 1999). Indicating functions upstream of HsRAD51, BRCA1 and BRCA2 are required for the assembly of ionizing-radiation-induced RAD51 complexes (Yuan et al, 1999; Bhattacharyya et al, 2000). Indicating functions downstream of RAD51, the disruption of BRCA2-RAD51 complexes leads to a loss of the G2/M checkpoint control (Chen et al, 1999a). The importance of these recombination proteins during proliferation was convincingly demonstrated by the embryonic lethal phenotypes of knockout mice with deficiencies in either RAD50, RAD51, BRCA1, or BRCA2 (Lim and Hasty, 1996; Hakem et al, 1997; Ludwig et al, 1997; Luo et al, 1999). Strikingly, the concomitant knockout of p53 partially suppressed the arrest of embryo development in mice nullizygous for RAD51, BRCA1, and BRCA2. Inactivation of the p53 target gene p21WAF1/CIP1 had the same effect in BRCA1 and BRCA2 knockouts. The p53-dependent G1/S checkpoint response is significantly reduced in cell lines derived from Nijmegen breakage syndrome patients, which are devoid of p95, the subunit of the RAD50-MRE11-p95 complex which is required for the DNA damage induced phosphorylation of MRE11 (Jongmans et al, 1997; Carney et al, 1998; Dong et al, 1999). Moreover, adding to the complexity of this intricate regulatory network, the DSB sensing kinase ATM directly regulates the activities of p95, BRCA1, and p53 and indirectly promotes the assembly of recombinative repair complexes via activation of c-ABL-mediated RAD51 phosphorylation (Banin et al, 1998; Canman et al, 1998; Cortez et al, 1999; Chen et al, 1999c; Venkitaram, 1999; Lim et al, 2000; Morrison et al, 2000). Mice deficient of ATM are viable, but display a defect early in prophase I during male gametogenesis, resulting in apoptotis, whereas ATM/p53 or ATM/p21WAF1/CIP1 double mutant mice proceed to pachytene (Barlow et al, 1997). p53 is highly expressed during meiosis in spermatogenesis from preleptotene to early pachytene (Rotter et al, 1993; Sjöblom and Lähdetie, 1996). This raises the question, whether interactions of p53 with the recombination machinery, modulate the transmission of signals by p53. Indeed, an early hypothesis on the possible function of BRCA1 was to play

an auxiliary role in p53-dependent transcriptional transactivation (Ouchi et al, 1998), although later studies demonstrated additional p53-independent functions of BRCA1 in transcriptionally upregulating GADD45, in controlling the checkpoint during the G2/M-phase, and in inducing apoptosis (Venkitaram, 1999). Therefore, BRCA1 may link DSB repair and DNA damage signaling via p53-dependent and -independent pathways.

III. p53 Inhibits homologous recombination independent of its functions in transcriptional transactivation and in cell cycle control DSBs arise spontaneously due to errors in replication, recombination, or mitosis and can be induced experimentally by ionizing radiation. DSBs trigger both repair-associated and targeted recombination processes, which in turn made the DSB responsive molecule p53 a good candidate for being a regulatory factor of DNA exchange processes. In 1994 Xia and colleagues (Xia et al, 1994) demonstrated that after X-ray treatment loss of heterozygosity (LOH), due to inter-allelic homologous recombination or gene conversion, was observed more frequently in human lymphoblastoid cell lines with the mutant p53(237ile), as compared to isogenic cells with wild-type p53 (Figure 2). Unrestrained LOH in the same mutant p53 cells was later also observed after treatment with one of the chemical mutagens EMS, MMS, or mitomycin C (Honma et al, 1997). With respect to spontaneous recombination rates, several groups observed 5 to >100 fold rate elevations, when wild-type p53 was inactive (Meyn et al, 1994; Wiesmüller et al, 1996; Bertrand et al, 1997; Mekeel et al, 1997). In these studies, systems for probing recombination were either based on repeat sequences integrated into the cellular chromosomes or on mutated variants of the Simian virus 40 (SV40) genome, thereby taking advantage of the small, chromatin packaged viral genome, which is amplified episomally. Isogenic cell types of human and rodent origin, either differing in the endogenous p53 status or ectopically expressing wild-type p53 in a p53 null background or the dominant negative mutants p53 (143ala) and p53 (175his) in a wild-type p53 background, equally supported the notion of an inhibitory role of p53 in homologous recombination processes. Furthermore, a correlation could be drawn between p53 neutralization by viral proteins and the elevation of recombination rates. Overexpression of the human papilloma virus 16 (HPV16) E6 protein, which promotes p53 degradation by the ubiquitin-pathway, reproducibly caused an increase in DNA exchange frequencies by one to two orders of magnitude (Havre et al, 1995; Mekeel et al, 1997). A possible influence of the large tumorantigen of SV40 (Tag) in this process was investigated by designing viral test genomes with a mutation, leading to the single amino

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Figure 2. Separation of p53 functions in transcriptional transactivation, cell cycle control, and the inhibition of homologous recombination. Several groups have provided evidence for a role of wild-type p53 in suppressing homologous recombination (HR) independently of its transcriptional and checkpoint functions. In these studies transcriptional transactivation (TA) with respect to specific target genes, G1 arrest induction after p53 upregulation, and the exchange between homologous sequences on viral or cellular chromosomes (inhibition of HR) were monitored with respect to the endogenous p53 status, after expression of a mutated p53 transgene, or after expression of a cellular (HDM2) or a viral (SV40Tag, HPV-E6) protein antagonizing wild-type (wt) p53 functions. p53 was originating from mouse (mu), monkey (mo), or man (hu). Beyond the transforming p53 variants with wild-type p53 conformation [p53(248gln), p53(273his)] or with mutant p53 conformation [p53(237ile), p53$237-239, p53(175his), p53(273pro)], the temperature sensitive mutants p53(135val) and p53(143ala), the alternatively spliced (AS) form from mice, and variants with shortened C-terminus were analysed [p53(1-363), p53 (1-333)]. The references were: a, Meyn et al, 1994; b, Xia et al, 1994; c, Wiesmüller et al, 1996; d, Bertrand et al, 1997; e, Mekeel et al, 1997; f, Dudenhöffer et al, 1998; g, Saintigny et al, 1999; h, Dudenhöffer et al, 1999; i, Willers et al, 2000.

acid exchange 402asp->his in Tag, thereby specifically blocking p53-Tag interactions (Wiesmüller et al, 1996). The corresponding analysis revealed that the suppression of homologous recombination events by wild-type p53 can be alleviated by complex formation with SV40Tag. Tag was known to represent the causative agent of SV40 for the elevation of recombination frequencies with cellular and viral DNAs as well as for the stimulation of the closely related gene amplification events (Perry et al, 1992; Ishizaka et al, 1995; Cheng et al, 1997). Now, p53 appeared to be the missing link between the viral protein and the genomic instabilities conferred by SV40, and

possibly by other tumor viruses. Moreover, connections between p53 and RAD51 became apparent, when cell immortalization after stable transformation of human fibroblasts with Tag was demonstrated to increase chromosomal recombination in a RAD51-dependent manner (Xia et al, 1997). Thus, protecting RAD51mediated recombination from the interference by p53 via Tag complex formation allows unrestrained strand exchange by RAD51. Recently, Schiestl and coworkers (Aubrecht et al, 1999) provided in vivo evidence for an antagonistic role of p53 in homologous recombination processes by use of the pink eyed unstable (pun) mouse

Akyüz et al: p53 in recombination

model. Intrachromosomal homologous recombination, resulting in deletions at the pun locus, was scored by black spots on the gray fur of the offspring. Increased frequencies were noticed with p53+/- and p53-/- mice as compared to p53+/+ mice. This was true for spontaneous events as well as for the exchange frequencies enhanced by the administration of benzo[a]pyrene during embryogenesis, whereas different results were obtained after the corresponding treatment with X-rays (see chapter V). Even further, chromosomal instabilities were noticed in Ataxia telangiectasia (AT) patients, and ATM-/- mice displayed significantly elevated spontaneous recombination frequencies, suggesting that deregulated DNA exchange events are due to the reduced p53 response to DSBs in the absence of the upstream kinase ATM (Bishop et al, 2000). At this point the critical question was, whether the regulation of spontaneous homologous recombination processes is dependent on p53´s transcriptional activities or on its growth regulatory functions, i.e. whether DNA exchange processes are modulated indirectly by the products of target genes or by secondary effects of cell cycle control like the prevention of DNA synthesis. However, already in 1995 p53-dependent G1 arrest signaling via Rb was dissociated from functions in repair, when it was observed that E6 from different HPVs, but not HPV-E7 caused an increase in spontaneous mutagenesis (Havre et al, 1995). Later on, by use of the SV40-based recombination test system, it was shown that, after the inactivation of p53, rate changes of viral DNA synthesis did not correlate with the corresponding changes of recombination frequencies (Wiesmüller et al, 1996). Finally, p53 mutations were identified, which served to demonstrate that p53 regulates homologous recombination processes independently of its activities in transcription and growth control (Dudenhöffer et al, 1999; Saintigny et al, 1999; Willers et al, 2000). Striking examples were cancer-related p53 mutants with an alteration at amino acid position 273, which unveiled defects in the inhibition of homologous recombination processes, while retaining the ability to induce a G1 arrest (Figure 2). This finding suggested that functions in recombinative repair contribute to tumor suppression. Vice versa, a defectiveness in transcriptional and cell cycle control functions without the concomitant loss of the capacity to inhibit genetic exchanges was noticed with the temperature sensitive mutant p53(135val) and with wild-type p53 expressed together with the p53-antagonist HDM2. Furthermore, from the quantitative analysis of cell clones inducibly expressing different amounts of exogenous wild-type p53, it was noticed that low protein levels are sufficient for the inhibition of recombination processes, whereas growthrelated functions are exerted in a dose-dependent manner. It can be envisioned that p53 guarantees the maintenance of genomic stability in mitotically growing cells, because 1000 to 10000 p53 molecules are permanently available in order to monitor DNA exchange processes. During

cellular stress situations modified p53 accumulates and gains functions directed towards the regulation of growth. Therefore, p53 might play a dual role as a tumor suppressor in its latent and in its activated state, a model which is compatible with the opposite regulation of the exonuclease activity versus sequence-specific DNA binding after phosphorylation (Janus et al, 1999).

IV. Possible mechanisms underlying the regulation of homologous recombination by p53 Given that wild-type p53 controls DNA rearrangements independently of its transcriptional functions, further investigations were aiming at clues to the mechanism underlying the inhibition of recombination. To identify candidate pathways of homologous recombination, which are affected by p53, Lopez and colleagues designed recombination substrates with inverted or direct repeat sequences (Saintigny et al, 1999). Inverted repeat recombination substrates allowed to focus on mechanisms initiated by strand invasion, namely gene conversion or crossing-over events, the latter of which can lead to intrachromatid or unequal sister chromatid exchanges. In yeast these pathways require the RAD52 epistasis group members RAD51, RAD52, RAD54, and the RAD51 homologues RAD55, and RAD57, corresponding to HsRAD51, HsRAD52, HsRAD54, and some of the HsRAD51 homologues XRCC2, XRCC3, RAD51B, RAD51H3, RAD51C, and RAD51D in human cells (Lambert et al, 1999). In comparison, direct repeat sequences allow nonconservative mechanisms of singlestranded annealing (SSA) and replication slippage in addition to strand invasion pathways. SSA in yeast requires the NER endonuclease RAD1/RAD10, the mismatch repair factors MSH2 and MSH3, and the helicase SRS2, corresponding to XP-F/ERCC1, HsMSH2, HsMSH3, and possibly an unknown SRS2 homologue in human cells. Interestingly, wild-type p53 was shown to affect interand intramolecular homologous recombination processes with both types of substrates without altering the ratio of gene conversion versus crossing-over events (80 % versus 20 %). This indicated an interference of p53 with recombination involving RAD51-depedent strand invasion (Table 1). Further clues to the involvement of p53 in homologous recombination came from investigations on its DNA binding activites. Applying gel retardation assays and electron microscopy, wild-type p53 was demonstrated to interact with Holliday junctions and with 3-stranded recombination intermediates independent of the DNA sequence content (Lee et al, 1997; Dudenhöffer et al, 1998). Interactions of p53 homotetramers with recombination intermediates were characterized by a high specificity and binding affinity (KD = 0.1 nM), which was 100 fold higher than for the binding of sequence-specific

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Reference

Method

Result

Lim and Hasty, 1996

genetic crossing

p53 knockout alleviates the embryonic lethal phenotype of RAD51 mutant mice.

Stürzbecher et al, 1996

immunoprecipitation, pull down

physical interactions in vivo and in vitro

Wiesmüller et al 1996 Xia et al 1997

SV40 chromosome and plasmid recombination

Complex formation between p53 and SV40Tag alleviates the p53dependent suppression of homologous recombination. Tag stimulates recombination in a RAD51-dependent manner.

Saintigny et al, 1999

intrachromosomal recombination p53 regulates spontaneous and irradiation-induced recombination processes involving strand invasion. between repeat sequences

Süße et al, 2000

gel retardation, strand exchange, formation of ternary RAD51-p53-DNA complexes on strand exchange intermediates; stimulation of the exonuclease activity by and exonuclease assay RAD51-mediated generation of heteroduplex joints

Table 1. Links between RAD51 and p53.

transcriptional response elements (Dudenhöffer et al, 1998; Süße et al, 2000). Compatible with this finding, it was noticed that supercoiled DNA efficiently competes with p53-consensus-DNA in DNA binding assays with p53 (Palecek et al, 1997). Others observed that even sitespecific recognition of p53-response elements requires DNA bending or a conformation distinct from B-DNA (Kim et al, 1997; Nagaich et al, 1997). Furthermore, strong binding of p53 to DNA junctions, which are generated during homologous DNA exchange processes, was in agreement with the observation that low p53 protein levels, such as in the absence of a DNA damage stimulus, are sufficient for the suppression of homologous recombination processes (see chapter III). Therefore, strong junction DNA binding would provide the basis for a specific and immediate response to RAD51-dependent DNA exchange processes despite the multifunctionality of p53. The idea of a direct interaction between p53 and the recombination intermediate as a prerequisite for the suppression of homologous recombination was supported by DNA binding studies with differently mutated and truncated forms of p53. For each p53 mutant analysed, a perfect correlation was observed between the intensity of binding to 3-stranded junction DNAs and the ability to suppress homologous recombination (Dudenhöffer et al, 1999; Süße et al, 2000). These DNA binding studies were also compatible with the notion that the integrity of p53 within its central DNA binding and 3´-5´ exonuclease domain and within its C-terminally neighbouring oligomerization domain are essential for recombination inhibition. Substantiating the hypothesis of a direct interaction with newly generated recombination intermediates further, DNA-complex formation by p53 was found to be synergistically stimulated, when RAD51-

nucleoproteins were allowed to assemble on 3-stranded DNA junctions before the association of p53 (Süße et al, 2000). According to a novel model for the mechanism of recombination control, it was suggested that p53 monitors the fidelity of strand exchange events in close contact to the recombinase RAD51 (Dudenhöffer et al, 1998). This hypothesis was based on the discovery that high-affinity binding of heteroduplex joints was enhanced 10 fold, when certain types of mismatches were located within the heteroduplex part. Strikingly, when SV40-virus based recombination assays were designed to provoke the generation of mispairings within the heteroduplex after strand transfer, the same mismatches identified by in vitro binding assays caused maximal inhibition of homologous DNA exchange. The analysis of truncated p53 molecules showed that the extreme C-terminus, which is responsible for unspecific DNA binding, reannealing, and strand transfer (Oberosler et al, 1993; Bakalkin et al, 1994; Brain and Jenkins, 1994), is dispensable for recombination suppression and 3-stranded junction DNA binding by p53, but mediates the mismatch-dependent stimulation of junction DNA binding by p53 (Dudenhöffer et al, 1999). Therefore, this region negatively controls not only sequence-specific DNA binding (Hupp and Lane, 1994) and the exonuclease activity of the core (Janus et al, 1999), but also the inhibition of recombination. DNA damage binding, truncation, the interaction with a cofactor, acetylation by p300/CBP, and phosphorylation by the kinases CKII and PKC can neutralize the negative effect on sequence-specific DNA binding, and, by analogy, might also modulate regulatory activities in recombination (Bayle et al, 1995; Jayaraman and Prives, 1995; Lee et al, 1995; Reed et al, 1995; Selivanova et al,

Akyüz et al: p53 in recombination

1996; Steegenga et al, 1996; Gu and Roeder, 1997; Meek et al, 1997). Uncontrolled homologous recombination can be a source of detrimental genome rearrangements, when imperfectly homologous regions are involved. Erroneous exchange events give rise to deletions, duplications, contractions, and expansions of tandem repeat sequences (reviewed in Belmaaza and Chartrand, 1994), which accelerate the multistep process of tumor progression. The activity of the mismatch repair system has been shown to inhibit recombination between diverged sequences (Kolodner, 1995; Modrich and Lahue, 1996). In MSH2-/mice, deficient of the mammalian counterpart of MutS, genomic instabilities are associated with mismatch repair deficiencies and a hyperrecombinative phenotype (De Wind et al, 1995). Parallels of p53 to the functions of this mismatch repair factor in recombination immediately arise, as MSH2 is well-known to strongly (KD = 0.5 nM) bind Holliday junction DNAs (Alani et al, 1997; Marsischky et al, 1999). Strikingly, p53 shows maximal binding affinities for 3-stranded recombination intermediates with A-G and C-T mispairings, whereas MutS homodimers and MSH2/MSH6 heterodimers recognize G-T and A-C mismatches best (De Wind et al, 1995; Dudenhöffer et al, 1998). This observation suggested that p53 monitors the fidelity of strand exchange events in a manner similar to the mismatch repair factor MSH2. Indeed, synergistically increased cancer susceptibilities of the double knockouts indicate complementary anti-carcinogenic activities for these multi-functional proteins (Cranston et al, 1997). The question that remains is, if and how the exchange process is attenuated when p53 encounters mismatches in the strands aligned within heteroduplexes. MutS binds Holliday junctions and inhibits RecA-exerted strand exchange and branch migration upon encountering mismatches in the heteroduplex (Worth et al, 1994). In analogy, it is conceivable that p53´s interaction with RAD51 serves to inhibit or interrupt strand exchange by disturbing ATP-hydrolysis of RAD51 or the oligomerization of monomers (Stürzbecher et al, 1996). The association of p53 might be triggered or enhanced by the recognition of certain heterologies within nascent recombination intermediates (see Figure 3). Alternatively, p53 could be envisioned to actively dissolve intermediates comprising mispairings. Indeed, strand transfer and exonuclease experiments indicated that p53 protein performs exonuclease activity in a 3´-5´ orientation on the double-stranded termini of 3-stranded junction DNAs (Süße et al, 2000). As was predicted from the DNA binding affinities of p53, degradation was faster, when p53 was encountering an A-G mismatch within these specifically recognized structures. Interestingly, for DSB repair in yeast, Haber and coworkers (Paques and Haber, 1997; Sugawara et al, 1997) have shown that after strand invasion extended nonhomologous 3´ ends are removed by the RAD1/RAD10 endonuclease at the junction of the

duplex DNA. This NER endonuclease is recruited by the mismatch repair proteins MSH2 and MSH3, which recognize the branched DNA structure. Therefore, both p53 and MSH2 have the potential to directly or indirectly remove heterologies within recombination intermediates. In summary, p53 and hRAD51 seem to execute their recombinational repair functions in close neighbourhood to each other, and interact during or shortly after strand transfer possibly to facilitate the recognition of heteroduplexes by p53. According to a new model for the mechanism leading to fidelity control of recombination processes by p53, recognition of recombinative DNA structures by p53 would be followed by the nucleolytic destruction of heteroduplexes, when encompassing mispairings (Süße et al, 2000).

V. Possible roles in DNA end-joining, meiosis, and replication-associated recombination Recombination represents the final and irreplaceable repair mechanism under circumstances when DNA double-strand breaks or gaps appear. Different from yeast, where homologous recombination is the predominating pathway of DSB repair, the relative contribution of homology-directed repair of chromosomal DSBs in mammalian cells seems to account for up to 50 %, as studied by use of the rare-cutting, site-specific I-SceI nuclease (Sargent et al, 1997; Taghian and Nickoloff, 1997; Liang et al, 1998; Lambert et al, 1999; Lin et al, 1999; Moynahan et al, 1999; Dronkert et al, 2000). To check possible effects of p53 on the second major DSB repair pathway, NHEJ, Yang and colleagues examined a thyroid cell line with the temperature sensitive (ts) mutant p53(138val) in transfection assays with a linearized luciferase plasmid (Yang et al, 1997). At the permissive temperature p53 was shown to enhance DNA end-joining 3-4 fold, and at the nonpermissive temperature 2-3 fold, indicating a stimulatory influence of tsp53 in the wild-type and in the mutant conformation. Interestingly, the corresponding murine tsp53(135val) was shown to inhibit homologous recombination processes again at both temperatures (Willers et al, 2000). Therefore, one possible explanation might be that the upregulation of NHEJ simply reflects redirected repair after the downregulation of homologous recombination. However, the stimulatory effect on NHEJ was postulated to be dependent on the Cterminal reannealing activity of p53, and this was interpreted such that p53 joins broken DNA ends via short homologies (Tang et al, 1999). Compatible with an active participation of p53 in NHEJ, Schiestl and coworkers (Aubrecht et al, 1999) concluded from their experiments with p53+/+, p53+/-, and p53-/- mice that p53 is required for efficient recombination after X-ray treatment. Therefore, after irradiation RAD51-independent DSB repair pathways, such as SSA and NHEJ, could be enhanced by p53, whereas gene conversion and crossing

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Figure 3. Model for p53-dependent fidelity control of homologous recombination. During the generation of nascent heteroduplexes by RAD51, RPA, and auxiliary factors from the RAD52 epistasis group, mismatches can arise as a result of DNA exchange between imperfectly homologous sequences. MSH2, within heterodimers with MSH6 or MSH3, and p53 homotetramers independently surveil the fidelity of the initial strand exchange by transient interactions with the heteroduplex joint. Depending on the type of mismatch created, either the interactions by MSH2 or by p53 (shown here) are stabilized, thereby abrogating further strand exchange by RAD51 and/or allowing nucleolytic correction of the misaligned region.

over events would be suppressed in parallel. However, this interpretation might hold true only for certain recombination substrates or cell types, since other groups observed totally different results, which indicated that p53 suppresses NHEJ after irradiation (Mallya and Sikpi, 1999), and reduces the integration of linearized plasmid into the host chromosome (Lee et al, 1999). Furthermore, when monitored by the neutral Comet assay, DSB rejoining during and after the exposure to ionizing radiation was shown to increase with mutant versus wildtype p53 (Bristow et al, 1998). Therefore, the role of p53 in NHEJ must be considered far from being understood. There is some evidence for an indirect involvement of p53 in V(D)J joining. In pre-B cells, the accumulation

of wild-type p53 induces cell differentiation, which is manifested by immunoglobulin % light-chain gene expression after successful V(D)J recombination (AloniGrinstein et al, 1995; Bogue et al, 1996). However, the upregulation of p53 might simply be explained by V(D)J recombination triggering a p53-dependent DNA damage checkpoint (Guidos et al, 1996). In scid mice the knockout of p53 can rescue rearrangement at multiple TCR loci, which most likely involves a homology-dependent bypass pathway (Guidos et al, 1996). Thus, the proposed indirect involvement of p53 in V(D)J recombination can be explained by its regulatory role in homologous recombination processes.

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Homologous recombination processes are important for eukaryotic organisms not only during the mitotic life cycle in order to repair DNA damage, but also to create diversity and to ensure proper segregation of chromosomes during meiosis (Kucherlapati and Smith, 1988). From the fact that p53 expression in testes is stronger than in other tissues, p53 was proposed to be connected to meiotic recombination (Rotter et al, 1993; Sjöblom and Lähdetie, 1996). More specifically, its expression in mice and rats is most prominent in zygotene - early pachytene spermatocytes, at the meiotic stage when homologous chromosomes synapse for genetic exchange. Still, p53 expression can further be increased by radiation treatment. However, Gersten and Kemp (1997) did neither observe elevated rates for the targeted types of DNA exchange during meiosis nor during antigen receptor rearrangements in p53 knockout mice. Therefore, it is conceivable that during meiosis p53 only serves to eliminate defective meiotic spermatocytes by irradiationinduced apoptosis (Odorisio et al, 1998). Homologous DNA exchange processes in mitotically growing cells are suppressed by a factor of 1000 as compared to rates during meiotic recombination, so that complex control mechanisms must be involved to allow recombination between homologous chromosomes during meiosis and to avoid detrimental genome rearrangements during growth (Haber, 1997). Considering the experimental data describing the regulatory role of wild-type p53 in RAD51dependent DNA exchange processes (Table 1), p53mediated surveillance of homologous recombination seems to contribute to the control mechanisms, which suppress spontaneous DNA exchange events specifically in mitotically growing cells. In mitotically growing cells recombination processes are frequently associated with DNA replication in order to allow the bypass of unrepaired lesions, and to repair DSBs generated at replication forks passing a single-strand break (Cox, 1997; Haber, 1999; Cox et al, 2000; Flores-Rozas and Kolodner, 2000). Due to this vital function of recombination processes during the normal life cycle of cells, the lack of the central enzyme function causes high mortality rates in E. coli devoid of RecA (Roca and Cox, 1990), an early embryonic lethal phenotype of mice nullizygous for RAD51 (Lim and Hasty, 1996; Tsuzuki et al, 1996), and results in an extreme sensitivity towards ionizing radiation and methyl methanesulfonate (MMS) of S. cerevisiae cells carrying mutations in RAD51 (Game and Mortimer, 1974; Shinohara et al, 1992). Consistent with the sister chromatid being the preferred homologous template for the repair of damaged DNA, homologous recombination represents the predominant DSB repair pathway in replicating cells, as opposed to nonhomologous end-joining (reviewed in Haber, 1999; Lambert et al, 1999; Cox et al, 2000; Flores-Rozas and Kolodner, 2000). RAD51 levels rise at the beginning of Sphase and cause the formation of S-phase specific nuclear foci (Flygare et al, 1996; Tashiro et al, 1996; Chen et al,

1997; Scully et al, 1997). Most recently, Tashiro and colleagues (Tashiro et al, 2000) concluded from their combined pulse labeling and microirradiation studies that even within 15 min RAD51 is recruited to sites of DNA damage in regions of replicative DNA synthesis. Interestingly, p53 binds RPA, which participates in replication and recombination, and forms functional and extremely stable complexes with DNA polymerase # (Wold, 1997; Kühn et al, 1999), the DNA polymerase for lagging-strand synthesis, which in the yeast system has been shown to be involved in DSB repair (Holmes and Haber, 1999). p53 was also found to excise mispaired nucleotides from DNA in a polymerase # based in vitro replication assay (Huang, 1998). Therefore, p53 might function on heteroduplexes after strand invasion or/and during strand synthesis, namely by controlling the replicative extension of the 3´ invading end, especially during RAD51-mediated replication-associated recombination processes.

VI. Conclusions DNA in eukaryotic cells undergoes continuous damage and resynthesis. Therefore, multiple repair, fidelity control, and cell cycle checkpoint systems exist to avoid the accumulation of mutations, and to create barriers against the multistep process of cancerogenesis (Loeb and Loeb, 2000). During the last years numerous genetic studies have revealed overlapping functions in DNAmetabolizing processes and checkpoint control for several proteins, which are as divergent as 3´-5´ exonucleases (Lydall and Weinert, 1996; Onel et al, 1996), DNA polymerase & (Navas et al, 1995), and the mismatch repair factors hMSH2 and hMLH1 (reviewed in Kunkel, 1995). Until the 1990s, tumor suppression by p53 was solely explained by its cell cycle control functions in response to DNA damage (Lane, 1992). As soon as DNA repairrelated activities of p53 were discovered, new models of a dual role of p53 in checkpoint control and in repair were arising (Albrechtsen et al, 1999). So far, evidence for a role of p53 as a proofreader of DNA polymerases was mainly based on in vitro experiments (Mummenbrauer et al, 1996; Jean et al, 1997; Huang, 1998; Janus et al, 1999; Kühn et al, 1999; Shakked et al, 2000), although recently, p53 was found to reduce the frequency of chemically induced point mutations in vivo (Courtemanche and Anderson, 1999). More convincingly, in vitro and in vivo data from several groups substantiated that p53 suppresses spontaneous inter- and intrachromosomal homologous recombination by at least one to two orders of magnitude (Meyn et al, 1994; Xia et al, 1994; Wiesmüller et al, 1996; Bertrand et al, 1997; Mekeel et al, 1997; Dudenhöffer et al, 1999; Saintigny et al, 1999; Willers et al, 2000; Süße et al, 2000). Accurate recombination is especially important in actively dividing cells, when DSBs arise spontaneously during the process of DNA replication (Haber, 1999; Cox et al, 2000).

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Therefore, a hierarchy of regulatory factors coordinates recombinative repair. Thus, BRCA1 is engaged in channeling DSB repair from DNA end-joining into the precise pathway of homologous recombination in mammalian cells (Moynahan et al, 1999). In S. cerevisiae, with predominantly coding sequences and hardly any repeats, DNA end-joining is rare, and this might explain why a BRCA1 homologue and possibly other fidelity systems, such as p53, did not develop. The recognition of mismatches seems to enable MSH2 to prevent strand exchange (Alani et al, 1997), thereby creating a barrier to the recombination between repeated sequences that are diverged (Kolodner, 1995; Modrich and Lahue, 1996). Similar to the proposed role of MSH2, p53 might monitor the correct alignment of homologous sequences during recombinational repair. As would be expected for the unrestrained exchange of mismatched sequences, spontaneous and UV-induced sister chromatid exchanges rise in p53-deficient cells (Ishizaki et al, 1994; Cleaver et al, 1999; Schwartz and Russell, 1999). Consistently, a role of p53 in creating a threshold for recombination between short versus long homologies was proposed from studies on the stability of repetitive sequences (Gebow et al, 2000). Moreover, a role in surveillance of the fidelity of RAD51-dependent strand transfer processes by exonucleolytic correction of errors was proposed for p53 (Dudenhöffer et al, 1998; Süße et al, 2000). In agreement with this hypothesis, a correlation was recently reported to exist between p53 mutations in non-small cell lung cancer and the appearance of microsatellite instabilities at certain tetranucleotide repeats (Ahrendt et al, 2000). Importantly, some cancer-related p53 mutants are defective in modulating recombination, but still arrest cells in G1. This suggests that, after mutation of p53, deregulated recombination contributes to accelerated tumor formation. Therefore, characterizing both functions for different p53 mutants seems critical to the understanding of resistance phenotypes during cancer therapeutic treatments. Indeed, p53 has profound effects on the responses to genotoxic treatments, and these effects vary dramatically depending on the treatment (McGilland and Fisher, 1999) and depending on the p53 mutation (Benchimol, 1999). Thus, p53-deficient colon carcinoma cells are resistant towards an inhibitor of DNA synthesis, 5-Fluorouracil, whereas they respond extremely sensitive towards adriamycin or irradiation. Taken together, it seems that as a consequence of the individual p53 mutation and of the specific anti-cancer treatment applied either the impairment of DNA repair or the inability to execute apoptosis produce different cellular responses. The final goal will be to raise the curing rate by individual cancer therapies, developed according to the functional status of p53 in the respective tumor.

Acknowledgements Our work was supported by the Deutsche Forschungsgemeinschaft, grants Wi 1376/1-4 and -5 and grant 10-1417-De4 by the Deutsche Krebshilfe, the Dr. Mildred Scheel Stiftung. S.S. was supported by the FAZIT-Stiftung, Frankfurt a.M. The Heinrich-PetteInstitut is supported by the Freie und Hansestadt Hamburg and by the Bundesministerium für Gesundheit.

References Ahrendt, S.A., Decker, P.A., Doffek, K., Wang, B., Xu, L., Demeure, M.J., Jen, J., and Sidransky, D. (2000). Microsatellite instability at selected tertanucleotide repeats is associated with p53 mutations in non-small cell lung cancer. Cancer Res. 60, 2488-2491. Alani, E., Lee, S., Kane, M.F., Griffith, J., and Kolodner, R.D. (1997). Saccharomyces cerevisiae MSH2, a mispaired base recognition protein, also recognizes Holliday junctions in DNA. J. Mol. Biol. 265, 289-301. Albor, A., Kaku, S., and Kulesz-Martin, M. (1998). Wild-type and mutant forms of p53 activate human topoisomerase I: a possible mechanism for gain of function in mutants. Cancer Res. 5, 2091-2094. Albrechtsen, N., Dornreiter, I., Grosse, F., Kim, E., Wiesmüller, L., and Deppert, W. (1999). Maintenance of genomic integrity by p53: Complementary roles for activated and non-activated p53. Oncogene 8, 7706-7717. Aloni-Grinstein, R., Schwartz, D., and Rotter, V. (1995). Accumulation of wild-type p53 protein upon "-irradiation induces a G2 arrest-dependent immunoglobulin ' light chain gene expression. EMBO J. 14, 1392-1401 Aubrecht, J., Secretan, M.B., Bishop, A.J.R., and Schiestl, R.H. (1999). Involvement of p53 in X-ray induced intrachromosomal recombination in mice. Carcinogenesis 20, 2229-2236. Bakalkin, G., Yakovleva, T., Selivanova, G., Magnusson, K. P., Szekely, L., Kiseleva, E., Klein, G., Terenius, L., and Wiman, K. G. (1994). p53 binds single-stranded DNA ends and catalyzes DNA renaturation and strand transfer. Proc. Natl. Acad. Sci. USA 91,413-417. Banin, S., Moyal, L., Shieh, S., Taya, Y., Anderson, C.W., Chessa, L., Smorodinsky, N.I., Prives, C., Reiss, Y., Shiloh, Y., and Ziv, Y. (1998). Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281, 1674-1677. Barlow, C., Liyanage, M., Moens, P.B., Deng, C.-X., Ried, T., and Wynshaw-Boris, A. (1997). Partial rescue of the prophase I defects of Atm-deficient mice by p53 and p21 null alleles. Nature Genet. 17, 462-466. Baumann, P., Benson, F.E., and West, S.C. (1996). Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro. Cell 87, 757-766. Baumann, P., and West, S.C. (1998). Role of the human Rad51 protein in homologous recombination and double-strandedbreak repair. Trends Biochem. Sci. 23, 247-251. Baumann, P., and West, S.C. (1999). Heteroduplex formation by human Rad51 protein: Effects of DNA end-structure, hRP-A and hRad52. J. Mol. Biol. 29, 363-374.

Akyüz et al: p53 in recombination

Bayle, J.H., Elenbaas, B., and Levine, A.J. (1995). The carboxylterminal domain of the p53 protein regulates sequencespecific DNA binding through its nonspecific nucleic acidbinding activity. Proc. Natl. Acad. Sci. USA 92, 5729-5733. Belmaaza, A., and Chartrand, P. (1994). One-sided invasion events in homologous recombination at double-stranded breaks. Mutat. Res. 314, 199-208. Benchimol, S. (1999). Expression of different mutant p53 transgenes in neuroblastoma cells leads to different cellular responses to genotoxic agents. In book of abstracts Workshop p53: Twenty Years On, p19. Bertrand, P., Rouillard, D., Boulet, A., Levalois, C., Soussi, T., and Lopez, B.S. (1997). Increase of spontaneous intrachromosomal homologous recombination in mammalian cells expressing a mutant p53 protein. Oncogene 14, 11171122. Bhattacharyya, A., Ear, U.S., Koller, B.H., Weichselbaum, R.R., and Bishop, D.K. (2000). The breast cancer-susceptibility gene BRCA1 is required for subnuclear assembly of Rad51 and survival following treatment with the DNA crosslinking agent cisplatin. J. Biol. Chem. 275, 23899-23903. Bishop, A.J., Barlow, C., Wynshaw-Boris, A.J., and Schiestl, R.H. ( 2000). Atm deficiency causes an increased frequency of intrachromsomal homologous recombination in mice. Cancer Res. 60, 395-399. Blander, G., Kipnis, J., Leal, J.F.M., Yu, C.-E., Schellenberg, G.D., and Oren, M. (1999). Physical and functional interaction between p53 and the Werner_s syndrome protein. J. Biol. Chem. 274, 29463-29469. Bogue, M.A., Zhu, C., Aguilar-Cordova, E., Donehower, L.A., and Roth, D.B. (1996). p53 is required for both radiationinduced differentiation and rescue of V(D)J rearrangement in scid mouse thymocytes. Genes Dev. 10, 553-565. Brain, R., and Jenkins, J. R. (1994). Human p53 directs DNA strand reassociation and is photolabelled by 8-azido ATP. Oncogene 9,1775-1780. Bristow, R.G., Hu, Q., Jang, A., Chung, S., Peacock, J., Benchimol, S., and Hill, R. (1998). Radioresistant MTp53expressing rat embryo transformants exhibit increased DNAdsb rejoining during exposure to ionizing radiation. Oncogene 16, 1789-1802. Buchhop, S., Gibson, M.K., Wang, X.W., Wagner, P., Stürzbecher, H.-W., Harris, C.C. (1997). Interaction of p53 with the human Rad51 protein. Nucl. Acids Res. 25, 38683874. Bunz, F., Dutriaux, A., Lengauer, C., Waldman, T., Zhou, S., Brown, J.P., Sedivy, J.M., Kinzler, K.W., and Vogelstein B. (1998). Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282, 1497-1501. Canman, C.E., Lim, D.S., Cimprich, K.A., Taya, Y., Tamai, K., Sakaguchi, K., Appella, E., Kastan, M.B., and Siliciano, J.D. (1998). Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281, 1677-1679. Carney, J.P., Maser, R.S., Olivares, H., Davis, E.M., Le Beau, M., Yates III, J., Hays, L., Morgan, W.F., and Petrini, J.H.J. (1998). The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93, 477-486.

Caron de Fromentel, C., and Soussi, T. (1992). TP53 tumor suppressor gene: a model for investigating human mutagenesis. Genes Chromosomes Cancer 4, 1-15. Carrier, F., Georgel, P.T., Pourquier, P., Blake, M., Kontny, H.U., Antinore, M.J., Gariboldi, M., Myers, T.G., Weinstein, J.N., Pommier, Y., and Fornace Jr., A.J. (1999). Gadd45, a p53-responsive stress protein, modifies DNA accessibility on damaged chromatin. Mol. Cell. Biol. 19, 1673-1685. Chen, F., Nastasi, A., Shen, Z., Brenneman, M., Crissman, H., and Chen, D.J. (1997). Cell cycle-dependent protein expression of mammalian homologs of yeast DNA doublestrand break repair genes Rad51 and Rad52. Mutat. Res. 384, 2025-211. Chen, P.-L., Chen, C.-F., Chen, Y., Xiao, J., Sharp, Z.D., and Lee, W.-H. (1998). The BRC repeats in BRCA2 are critical for RAD51 binding and resistance to methyl methanesulfonate treatment. Proc. Natl. Acad. Sci. USA 95, 5287-5292. Chen, C.F., Chen, P.L., Zhong, Q., Sharp, Z.D., and Lee, W.H. (1999a). Expression of BRC repeats in breast cancer cells disrupts the BRCA2-Rad51 complex and leads to radiation hypersensitivity and loss of G(2)/M checkpoint control. J. Biol. Chem. 274, 32931-32935. Chen, Y., Lee, W.H., and Chew, H.K. (1999b). Emerging roles of BRCA1 in transcriptional regulation and DNA repair. J. Cell Physiol. 181, 385-392. Chen, G., Yuan, S.-S.F., Liu, W., Xu, Y., Trujillo, K., Song, B., Cong, F., Goff, S.P., Wu, Y., Arlinghaus, R., Baltimore, D., Gasser, P.J., Park, M.S., Sung, P., and Lee, E.Y.-H.P. (1999c). Radiation-induced assembly of Rad51 and Rad52 recombination complex requires ATM and c-Abl. J. Biol. Chem. 274, 12748-12752. Cheng, R.Z., Shammas, M.A., Li, J., and Reis, R.J.S. (1997). Expression of SV40 large T antigen stimulates reversion of a chromosmal gene duplication in human cells. Exp. Cell Res. 234, 300-312. Cleaver, E., Afzal, V., Feeney, L., McDowell, M., Sadinski, W., Volpe, J.P., Busch, D.B., Coleman, D.M., Ziffer, D.W., Yu, Y., Nagasawa, H., and Little, J.B. (1999). Increased ultraviolet sensitivity and chromosomal instability related to p53 function in the xeroderma pigmentosum variant. Cancer Res. 59, 1102-1108. Cortez, D., Wang, Y., Qin, J., and Elledge, S.J. (1999). Requirement of ATM-dependent phosphorylation of Brca1 in the DNA damage response to double-strand breaks. Science 286, 1162-1166. Courtemanche, C., and Anderson, A. (1999). The p53 tumor suppressor protein reduces point mutation frequency of a shuttle vector modified by the chemical mutagens (±)7,8dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, aflatoxin B1 and meta-choroperoxybenzoic acid. Oncogene 18, 4672-4680. Cowell, I.G., Okorokov, A.L., Cutts, S.A., Padget, K., Bell, M., Milner, J., and Austin, C.A. (2000). Human topoisomerase II# and IIß interact with the C-terminal region of p53. Exp. Cell. Res. 255, 86-94. Cox, M.M. (1997). Recombinational crossroads: Eukaryotic enzymes and the limits of bacterial precedents. Proc. Natl. Acad. Sci. USA 94, 11764-11766.

Gene Therapy and Molecular Biology Vol 5, page 75

Cox, M.M., Goodman, M.F., Kreuzer, K.N., Sherratt, D.J., Sandler, S.J., and Marians, K.J. (2000). The importance of repairing stalled replication forks. Nature 404, 37-40. Cranston, A., Bocker, T., Reitmair, A., Palazzo, J., Wilson, T., Mak, T., and Fishel, R. (1997). Female embryonic lethality in mice nullizygous for both Msh2 and p53. Nature Genet. 17, 114-118. De Wind, N., Dekker, M., Berns, A., Radman, M., and te Riele, H. (1995). Inactivation of the mouse msh2 gene results in mismatch repair deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer. Cell 82, 321-330. Deng, C., Zhang, P., Harper, J.W., Elledge, S.J., and Leder, P. (1995). Mice lacking p21 cip1/waf1 undergo normal development, but are defective in G1 checkpoint control. Cell 82,675-684. Donehower, L.A., Harvey, M., Slagle, B.L., MacArthur, M.J., Montgomery, C.A., Butel, J.S., and Bradley, A. (1992). Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356, 215-221. Dong, Z., Zhong, Q., and Chen, P.-L. (1999). The Nijmegen breakage syndrome protein is essential for Mre11 phosphorylation upon DNA damage. J. Biol. Chem. 274, 19513-19516. Dronkert, M.L.G., Beverloo, H.B., Johnson, R.D., Hoeijmakers, J.H.J., Jasin, M., and Kanaar, R. (2000). Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange. Mol. Cell. Biol. 20, 3147-3156. Dudenhöffer, C., Rohaly, G., Will, K., Deppert, W., and Wiesmüller, L. (1998). Specific mismatch recognition in heteroduplex intermediates by p53 suggests a role in fidelity control of homologous recombination. Mol. Cell. Biol. 18, 5332-5342. Dudenhöffer, C., Kurth, M., Janus, F., Deppert, W., and Wiesmüller, L. (1999). Dissociation of the recombination control and the sequence-specific transactivation function of p53. Oncogene 18, 5773-5784. Flores-Rozas, H., and Kolodner, R.D. (2000). Links between replication, recombination and genome instability. Trends Biochem. Sci. 25, 196-200. Flygare, J., Benson, F., and Hellgren, D. (1996). Expression of the human RAD51 gene during the cell cycle in primary human peripheral blood lymphocytes. Biochim. Biophys. Acta 1312, 231-236. Ford, J.M., and Hanawalt, P.C. (1997). Expression of wild-type p53 is required for efficient global genomic nucleotide excision repair in UV-irradiated human fibroblasts. J. Biol. Chem. 272, 28073-28080. Fortunato, E.A., and Spector, D.H. (1998). p53 and RPA are sequestered in viral replication centers in the nuclei of cells infected with human cytomegalovirus. J. Virol. 72, 20332039. Game, J.C., and Mortimer, R.K. (1974). A genetic study of Xray sensitive mutants in yeast. Mutat. Res. 24, 281-292. Gebow, D., Miselis, N. and Liber, H.L. (2000). Homologous and nonhomologous recombination resulting in deletion: effects of p53 status, microhomology, and repetitive DNA length and orientation. Mol. Cell. Biol. 20, 4028-4035.

Gersten, K.M., and Kemp, C.J. (1997). Normal meiotic recombination in p53-deficient mice. Nature Genet. 17, 378-179. Giovanella, B.C., Tainsky, M.A, Bradley, A., and Donehower, L.A. (1993). In vitro growth characteristics of embryo fibroblasts isolated from p53-deficient mice. Oncogene 8, 2457-2467. Gobert, C., Skladanowski, A., and Larsen, A.K. (1999). The interaction between p53 and DNA topoisomerase I is regulated differently in cells with wild-type and mutant p53. Proc. Natl. Acad. Sci. USA 96, 10355-10360. Gottlieb, T.M., and Oren, M. (1996). p53 in growth control and neoplasia. Biochim. Biophys. Acta 1287, 77-102. Gu, W., and Roeder, R.G. (1997). Activation of p53 sequencespecific DNA binding by acetylation of the p53 C-terminal domain. Cell 90, 595-606. Guidos, C.J., Williams, C.J., Grandal, I., Knowles, G., Huang, M.T.F., and Danska, J.S. (1996). V(D)J recombination activates a p53-dependent DNA damage checkpoint in scid lymphocyte precursors. Genes Dev. 10, 2038-2054. Gupta, R.C., Bazemore, L.R., Golub, E.I., and Radding, C.M. (1997). Activities of human recombination protein Rad51. Proc.Natl. Acad. Sci. USA 94, 463-468. Gupta, R.C., Folta-Stogniew, E., and Radding, C.M. (1999). Human Rad51 protein can form homologous joints in the absence of net strand exchange. J. Biol. Chem. 274, 12481256. Haber, J.E. (1997). A super new twist on the initiation of meiotic recombination Cell 89, 163-166. Haber, J. (1998). The many interfaces of Mre11. Cell 95, 583586. Haber, J. (1999). DNA recombination: the replication connection. Trends Biol. Sci. 24, 271-275. Hakem, R., de la Pompa, J.L., Elia, A., Potter, J., and Mak, T.W. (1997). Partial rescue of BRCA15-6 early embryonic lethality by p53 or p21 null mutation. Nature Genet. 16, 298-302. Harvey, M., Sands, A.T., Weiss, R.S., Hegi, M.E., Wiseman, R.W., Pantazis, P., Giovanella, B.C., Tainsky, M.A., Bradley, A. and Donehower, L.A. (1993). In vitro growth characteristics of embryo fibroblasts isolated from p53deficient mice. Oncogene 8, 2457-2467. Havre, P.A., Yuan, J., Hedrick, L., Cho, K.R., and Glazer, P.M. (1995). p53 inactivation by HPV16 E6 results in increased mutagenesis in human cells. Cancer Res. 55, 4420-4424. Hollstein, M., Sidransky, D., Vogelstein, B., and Harris, C.C. (1991). p53 mutations in human cancers. Science 253,49-53. Hollstein, M., Rice, K., Greenblatt, M.S., Soussi, T., Fuchs, R., Sorlie, T., Hovig, E., Smithsorensen, B., Montesano, R., and Harris, C.C. (1994). Database of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Res. 22, 3551-3555. Hollstein, M., Shomer, B., Greenblatt, M., Soussi, T., Hovig, E., Montesano, R., and Harris, C.C. (1996). Somatic point mutations in the p53 gene of human tumors and cell lines: updated compilation. Nucleic Acids Res. 24, 141-146. Holmes, A.M., and Haber, J.E. (1999). Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases. Cell 96, 415-424.

Aky端z et al: p53 in recombination

Honma, M., Hayashi, M., and Sofuni, T. (1997). Cytotoxic and mutagenic responses to X-rays and chemical mutagens in normal and p53-mutated lymphoblastoid cells. Mutat. Res. 374, 89-98. Huang, P. (1998). Excision of mismatched nucleotides from DNA: a potential mechanism for enhancing DNA replication fidelity by the wild-type p53 protein. Oncogene 17, 261-270. Hupp, T.R., and Lane, D.P. (1994). Allosteric activation of latent p53 tetramers. Curr. Biol. 4, 865-875. Ishizaka, Y., Chernov, M.V., Burns, C.M., and Stark, G.R. (1995). p53-dependent growth arrest of REF52 cells containing newly amplified DNA. Proc. Natl. Acad. Sci. USA 92, 3224-3228. Ishizaki, K., Ejima, Y., Matsunaga, T., Hara, R., Sakamoto, A., Ikenaga, M., Ikawa, Y., and Aizawa, S. (1994). Increased UV-induced SCEs but normal repair of DNA damage in p53deficient mouse cells. Int. J. Cancer 58, 254-257. Janus, F., Albrechtsen, N., Knippschild, U., Wiesm端ller, L., Grosse, F., and Deppert, W. (1999). Different regulation of the core domain activities 3_ to 5_exonuclease and sequence-specific DNA binding. Mol. Cell. Biol. 19, 21552168. Jayaraman, L., and Prives, C. (1995). Activation of p53 sequence-specific DNA binding by short single strands of DNA requires the p53 C-terminus. Cell 81,1021-1029. Jean, D., Gendron, D., Delbecchi, L., and Bourgaux, P. (1997). p53-mediated DNA renaturation can mimic strand exchange. Nucl. Acids Res. 25, 4004-4012. Jongmans, W., Vuillaume, M., Chrzanowska, K., Smeets, D., Sperling, K., and Hall, J. (1997). Nijmegen breakage syndrome cells fail to induce the p53-mediated DNA damage response following exposure to ionizing radiation. Mol. Cell. Biol. 17, 5016-5021. Kazantsev, A., and Sancar, A. (1995). Does the p53 up-regulated Gadd45 protein have a role in excision repair? Science 270, 1003-1004. Kearsey, J.M., Shivji, M.K.K., Hall, P.A., and Wood, R.D. (1995a). Does the p53 up-regulated Gadd45 protein have a role in excision repair? Science 270, 1004-1005. Kearsey, J.M., Coates, P.J., Prescott, A.R., and Hall, P.A. (1995b). Gadd45 is a nuclear cell cycle regulated protein which interacts with p21Cip1. Oncogene 11, 1675-1683. Kim, E., Albrechtsen, N., and Deppert, W. (1997). DNAconformation is an important determinant of sequencespecific DNA binding by tumor suppressor p53. Oncogene 15, 857-869. Ko, L.J., and Prives, C. (1996). p53: puzzle and paradigm. Genes and Dev. 10, 1054-1072. Kolodner, R.D. (1995). Mismatch repair: mechanisms and relationship to cancer susceptibility. Trends Biochem. Sci. 20, 397-401. Komarov, P.G., Komarova, E.A., Kondratov, R.V., ChristovTselkov, K., Coon, J.S., Chernov, M.V., and Gudkov, A.V. (1999). A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science 85, 1733-1737. Kowalczykowski, S.C. (1991). Biochemistry of genetic recombination: energetics and mechanism of DNA strand exchange. Annu. Rev. Biophys. Chem. 20, 539-575.

Kucherlapati, R., and Smith, G.R. (1988). Genetic Recombination. American Society for Microbiology, Washington, D.C. K端hn, C., M端ller, F., Melle, C., Nasheuer, H.P., Janus, F., Deppert, W., and Grosse, F. (1999). Surface plasmon resonance measurements reveal stable complex formation between p53 and DNA-polymerase #. Oncogene 18, 769774. Kunkel, T.A. (1995). The intricacies of eukaryotic spellchecking. Curr. Biol. 5, 1091-1094. Lakin, N.D., and Jackson, S.P. (1999). Regulation of p53 in response to DNA damage. Oncogene 18, 7644-7655. Lambert, S., Saintigny, Y., Delacote, F., Amiot, F., Chaput, B., Lecomte, M., Huck, S., Bertrand, P., and Lopez, B.S. (1999). Analysis of intrachromosomal homologous recombination in mammalian cells, using tandem repeat sequences. Mutat. Res. 433, 159-168. Lane, D.P. (1992). p53, guardian of the genome. Nature 358, 15-16. Lane, D. (1998). Awakening angels. Nature 394, 616-617. Lee, J.M., Abrahamson, J.L.A., Kandel, R., Donehower, L.A., and Bernstein, A. (1994). Susceptibility to radiationcarcinogenesis and accumulation of chromosomal breakage in p53 deficient mice. Oncogene 9, 3731-3736. Lee, S., Elenbaas, B., Levine, A., and Griffith, J. (1995). p53 and its 14 kDa C-terminal domain recognize primary DNA damage in the form of insertion/deletion mismatches. Cell 81, 1013-1020. Lee, S.M., Cavallo, L., and Griffith, J. (1997). Human p53 binds Holliday junctions strongly and facilitates their cleavage. J. Biol. Chem. 272, 7532-7539. Lee, H., Sun, D., Larner, J.M., and Wu, F.S. (1999). The tumor suppressor p53 can reduce stable transfection in the presence of irradiation. J. Biomed. Sci. 6, 285-292. Leveillard, T., Andera, L., Bisonnette, N., Schaeffer, L., Bracco, L., Egly, J.M., and Wasylyk, B. (1996). Functional interactions between p53 and the TFIIH complex are affected by tumour-associated mutations. EMBO J. 15, 1615-1624. Levine, A. (1997). p53, the cellular gatekeeper for growth and division. Cell 88, 323-331. Liang, F., Han, M., Romanienko, P.J., and Jasin, M. (1998). Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Proc. Natl. Acad. Sci. USA 95, 5172-5177. Lim, D.-S., and Hasty, P. (1996). A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a p53 mutation. Mol. Cell. Biol. 16, 7133-7143. Lim, D.-S., Kim, S.-T., Xu, B., Maser, R.S., Lin, J., Petrini, J.H.J., and Kastan, M.B. (2000). ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway. Nature 404, 613-617. Lin, Y., Lukacsovich, T., and Waldman, A.S. (1999). Multiple pathways for repair of DNA double-strand breaks in mammalian chromosomes. Mol. Cell. Biol. 19, 8353-8360. Livingstone, L.R., White, A., Sprouse, J., Livanos, E., Jacks, T., and Tlsty, T.D. (1992). Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell 70, 923-935.

Gene Therapy and Molecular Biology Vol 5, page 77

Loeb, K.R., and Loeb, L.A. (2000). Significance of multiple mutations in cancer. Carcinogenesis 21, 379-385. Ludwig, T., Chapman, D.L., Papaioannou, V.E., and Efstratiadis, A. (1997). Targeted mutations of breast cancer susceptibility gene homologs in mice: lethal phenotypes of Brca1, Brca2, Brca1/Brca2, Brca1/p53, Brca2/p53 nullizygous embryos. Genes & Dev. 11, 1226-1241. Luo, G., Yao, M.S., Bender, C.F., Mills, M., Bladl, A.R., Bradley, A., and Petrini, J.H.J. (1999). Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation. Proc. Natl. Acad. Sci. USA 6, 7376-7381. Lydall, D., and Weinert, T. (1996). From DNA damage to cell cycle arrest and suicide: a budding yeast perspective. Curr. Opin.Genet.Dev. 6, 4-11. Mallya, S.M., and Sikpi, M.O. (1999). Requirement for p53 in ionizing-radiation-inhibition of double-strand-break rejoining by human lymphoblasts. Mutat. Res. 23, 119-132. Marmorstein, L.Y., Ouchi, T., and Aaronson, S.A. (1998). The BRCA2 gene product functionally interacts with p53 and RAD51. Proc. Natl. Acad. Sci. USA 95, 13869-13874. Marsischky, G.T., Lee, S., Griffith, J., and Kolodner, R.D. (1999). Saccharomyces cerevisiae MSH2/6 complex interacts with Holliday junctions and facilitates their cleavage by phage resolution enzymes. J. Biol. Chem. 274, 7200-7206. McGilland, G., and Fisher, D.E. (1999). p53 and cancer therapy: a double-edged sword. J. Clin Invest. 104, 223-225. Meek, D.W., Campbell, L.E., Jardine, L.J., Knippschild, U., McKendrick, L., and Milne, D.M. (1997). Multi-site phosphorylation of p53 by protein kinases inducible by p53 and DNA damage. Biochem. Soc. Trans. 25, 416-419. Mekeel, K.L., Tang, W., Kachnic, L.A., Luo, C.-M., DeFrank, J.S., and Powell, S.N. (1997). Inactivation of p53 results in high rates of homologous recombination. Oncogene 14, 1847-1857. Meyn, M.S., Strasfeld, L., and Allen, C. (1994). Testing the role of p53 in the expression of genetic instability and apoptosis in ataxia-telangiectasia. Int. J. Radiat. Biol. 66, S141-S149. Mirzayans, R., Enns, L., Dietrich, K., Barley, R.D.C., and Paterson, M.C. (1996). Faulty DNA polymerase delta/epsilon-mediated excision repair in response to gamma radiation or ultraviolet light in p53-deficient fibroblast strains from affected members of a cancer-prone family with Li-Fraumeni syndrome. Carcinogenesis 17, 691-698. Modrich, P., and Lahue, R. (1996). Mismatch repair in replication fidelity, genetic recombination, and cancer biology. Annu. Rev. Biochem. 65, 101-133. Morrison, C., Sonoda, E., Takao, N., Shinohara, A., Yamamoto, K., and Takeda, S. (2000). The controlling role of ATM in homologous recombinational repair of DNA damage. EMBO J. 19, 463-471. Moynahan, M.E., Chiu, J.W., Koller, B.H., and Jasin, M. (1999). Brca1 controls homology-directed DNA repair. Mol. Cell 4, 511-518. Mummenbrauer, T., Janus, F., Müller, B., Wiesmüller, L., Deppert, W., and Große, F. (1996). p53 protein exhibits 3´to -5´ exonuclease activity. Cell 85, 1089-1099.

Nagaich, A.K., Appella, E., and Harrington, R.E. (1997). DNA bending is essential for the site-specific recognition of DNA response elements by the DNA binding domain of the tumor suppressor protein p53. J. Biol. Chem. 272, 14842-14849. Navas, T.A., Zhou, Z., and Elledge, S.J. (1995). DNA polymerase & links the DNA replication machinery to the S phase checkpoint. Cell 80, 29-39. Nelson, W.G., and Kastan, M.B. (1994). DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways. Mol. Cell. Biol. 14, 1815-1823. Oberosler, P., Hloch, P., Ramsperger, U., and Stahl, H. (1993). p53-catalyzed annealing of complementary single-stranded nucleic acids. EMBO J. 12, 2389-2396. Odorisio, T., Rodriguez, T.A., Evans, E.P., Clarke, A.R., and Burgoyne, P.S. (1998). The meiotic checkpoint monitoring synapsis eliminates spermatocytes via p53-independent apoptosis. Nat. Genet. 18, 257-261. Onel, K., Koff, A., Bennett, R.L., Unrau, P., and Holloman, W.K. (1996). The REC1 gene of Ustilago maydis, which encodes a 3_( 5_exonuclease, couples repair and completion of DNA synthesis to a mitotic checkpoint. Genetics 143, 165-174. Ouchi, T., Monteiro, A.N.A., August, A., Aaronson, S.A., and Hanafusa, H. (1998). BRCA1 regulates p53-dependent gene expression. Proc. Natl. Acad. Sci. USA 95, 2302-2306. Palecek, E., Vlk, D., Stankova, V., Brazda, V., Vojtesek, B., Hupp, T.R., Schaper, A., and Jovin, T.M. (1997). Tumor suppressor protein p53 binds preferentially to supercoiled DNA. Oncogene 15, 2201-2209. Paques, F., and Haber, J.E. (1997). Two pathways for removal of nonhomologous DNA ends during double-strand break repair in Saccharomyces cerevisiae. Mol. Cell. Biol. 17, 6765-6771. Patel, K.J., Yu, V.P.C.C., Lee, H., Corcoran, A., Thistlethwaite, F.C., Evans, M.J., Colledge, W.H., Friedman, L.S., Ponder, B.A.J., and Venkitaraman, A.R. (1998). Involvement of Brca2 in DNA repair. Mol. Cell 1, 347-357. Perry, M.E., Commane, M., and Stark, G.R. (1992). Simian virus 40 large tumor antigen alone or two cooperating oncogenes convert REF52 cells to a state permissive for gene amplification. Proc. Natl. Acad. Sci. USA 89, 8112-8116. Protopova, M., Zotchev, S.B., and Selivanova, G. (2000). Recognition of DNA lesions by p53: the role of the Cterminal and core domains. Journal of Biomolecular Structure and Dynamics, Mendel Brno 2000 abstract. Radding, C. (1991). Helical interactions in homologous pairing and strand exchange driven by RecA protein. J. Biol. Chem. 266, 5355-5358. Reed, M., Woelker, B., Wang, P., Wang, Y., Anderson, M. E., and Tegtmeyer, P. (1995). The C-terminal domain of p53 recognizes DNA damaged by ionizing radiation. Proc. Natl. Acad. Sci. USA 92, 9455-9459. Roca, A.I., and Cox, M.M. (1990). The RecA protein: structure and function. Crit. Rev. Biochem. Mol. Biol. 25, 415-456. Rotter, V., Schwartz, D., Alman, E., Goldfinger, N., Kapon, A., Meshorer, A., Donehower, L.A., and Levine, A.J. (1993). Mice with reduced levels of p53 protein exhibit the testicular giant-cell degenerative syndrome. Proc. Natl. Acad. Sci. USA 90, 9075-9079.

Akyüz et al: p53 in recombination

Saintigny, Y., Rouillard, D., Chaput, B., Soussi, T., and Lopez, B.S. (1999). Mutant p53 proteins stimulate spontaneous and radiation-induced intrachromosomal homologous recombination independently of the alteration of the transactivation activity and of the G1 checkpoint. Oncogene 18, 3553-3565. Sakaguchi, K., Herrera, J.E., Saito, S., Miki, T., Bustin, M., Vassilev, A., Anderson, C.W., and Appella, E. (1998). DNA damage activates p53 through a phosphorylation-acetylation cascade. Genes Dev. 12, 2831-2841. Sargent, R.G., Brenneman, M.A., and Wilson, J.H. (1997). Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination. Mol. Cell. Biol. 17, 267-277. Schwartz, J.L., and Russell, K.J. (1999). The effect of functional inactivation of TP53 by HPV-E6 transformation on the induction of chromosome aberrations by gamma rays in human tumor cells. Radiat. Res. 151, 385-390. Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T., and Livingston, D.M. (1997). Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell 88, 265-275. Selivanova, G., Iotsova, V., Kiseleva, E., Stroem, M., Bakalkin, G., Grafstroem, R.C., and Wiman, K.G. (1996). The singlestranded DNA end binding site of p53 coincides with the Cterminal regulatory region. Nucl. Acids Res. 24, 3560-3567. Siddique, H., Zou, J.-P., Rao, V.N., and Reddy, E.S. (1998). The BRCA2 is a histone acetyltransferase. Oncogene 16, 22832285. Shakked, Z., Yavnilovitch, M., Gilboa, A.J., Rotter, V., and Haran, T.E. (2000). DNA binding and exonuclease activity in the mourine alternatively-spliced p53 protein: an alternative structure and function? Journal of Biomolecular Structure and Dynamics, Mendel Brno 2000 abstract. Shinohara, A., Ogawa, H., and Ogawa, T. (1992). Rad51 protein involved in repair and recombination in Saccharomyces cerevisiae is a RecA-like protein. Cell 69, 457-470. Sjöblom, T., and Lähdetie, J. (1996). Expression of p53 in normal and "-irradiated rat testis suggests a role for p53 in meiotic recombination and repair. Oncogene 12, 2499-2505. Smith, M.L., Chen, I.-T., Zhan, Q., Bae, I., Chen, C.-Y., Gilmer, T.M., Kastan, M.B., O_Connor, P.M., and Fornace Jr., A.J. (1994). Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science 266, 1376-1380. Smith, M.L., Chen, I.-T., Zhan, Q., O_Connor, P.M., and Fornace Jr., A.J. (1995). Involvement of the p53 tumor suppressor in repair of u.v.-type of DNA damage. Oncogene 10, 1053-1059. Steegenga, W.T., van der Eb, A.J., and Jochemsen, A.G. (1996). How phosphorylation regulates the activity of p53. J. Mol. Biol. 263, 103-113. Stürzbecher, H.-W., Donzelmann, B., Henning, W., Knippschild, U., and Buchhop, S. (1996). p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction. EMBO J. 15, 1992-2002. Süße, S., Janz, C., Janus, F., Deppert, W., and Wiesmüller, L. (2000). Role of heteroduplex joints in the functional interactions between human Rad51 and wild-type p53. Oncogene, in press.

Sugawara, N., Paques, F., Colaiacovo, M., and Haber, J.E. (1997). Role of Msh2 and Msh3 repair proteins in doublestrand break-induced recombination. Proc. Natl. Acad. Sci. USA 94, 9214-9219. Sung, P., and Robberson, D.L. (1995). DNA strand exchange mediated by a RAD51-ssDNA nucleoprotein filament with polarity opposite to that of RecA. Cell 82, 453-461. Taghian, D.G., and Nickoloff, J.A. (1997). Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells. Mol. Cell. Biol. 17, 63866393. Tang, W., Willers, H., and Powell, S.N. (1999). p53 directly enhances rejoining of DNA double-strand breaks with cohesive ends in "-irradiated mouse fibroblasts. Cancer Res. 59, 2562-2565. Tashiro, S., Kotomura, N., Shinohara, A., Tanaka, K., Ueda, K., and Kamada, N. (1996). S phase specific formation of the human Rad51 protein nuclear foci in lymphocytes. Oncogene 12, 2165-2170. Tashiro, S., Walter, J., Shinohara, A., Kamada, N., and Cremer, T. (2000). Rad51 accumulation at sites of DNA damage and in postreplicative chromatin. J. Cell. Biol., in press. Tsuzuki, T., Fujii, Y., Sakumi, K., Tominaga, Y., Nakao, K., Sekiguchi, M., Matsushiro, A., Yoshimura, Y., and Morita, T. (1996). Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc. Natl. Acad. Sci. USA 93, 6236-6240. Venkitaraman, A.R. (1999). Breast cancer genes and DNA repair. Science 286, 1100-1102. Wang, Y., Reed, M., Wang, P., Mayr, G., Stenger, J.E., Anderson, M.E., Schwedes, J.F., and Tegtmeyer, P. (1994). p53 domains: structure, oligomerization, and transformation. Mol. Cell. Biol. 14, 5182-5191. Wang, X.W., Yeh, H., Schaeffer, L., Roy, R., Moncollin, V., Egly, J.-M., Wang, Z., Friedberg, E.C., Evans, M.K., Taffe, B.G., Bohr, V.A., Weeda, G., Hoeijmakers, J.H.J., Forrester, K., and Harris, C.C. (1995). p53 modulation of TFIIHassociated nucleotide excision repair activity. Nature Genet. 10, 188-195. Wang, X., Ohnishi, K., Takahashi, A., and Ohnishi, T. (1998). Poly(ADP-ribosyl)ation is required for p53-dependent signal transduction induced by radiation. Oncogene 17, 2819-2825. West, S. (1992). Enzymes and molecular mechanisms of homologous recombination. Annu. Rev. Biochem. 61, 603640. Wiesmüller, L., Cammenga, J., and Deppert, W.W. (1996). In vivo assay of p53 function in homologous recombination between Simian Virus 40 chromosomes. J. Virol. 70, 737744. Wilcock, D., and Lane, D.P. (1991). Localization of p53, retinoblastoma and host replication proteins at sites of viral replication in herpes-infected cells. Nature 349, 429-431. Willers, H., McCarthy, E.E., Wu, B., Wunsch, H., Tang, W., Taghian, D.G., Xia, F., and Powell, S.N. (2000). Dissociation of p53-mediated suppression of homologous recombination from G1/S cell cycle checkpoint control. Oncogene 19, 632-639.

Gene Therapy and Molecular Biology Vol 5, page 79

Wold, M.S. (1997). Replication protein A: a hetrotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu. Rev. Biochem. 66, 61-92. Worth, L., Clark, S., Radman, M., and Modrich, P. (1994). Mismatch repair proteins MutS and MutL inhibit RecAcatalyzed strand transfer between diverged DNAs. Proc. Natl. Acad. Sci. USA 91, 3238-3241. Wu, L., Bayle, J.H., Elenbaas, B., Pavletich, N.P., and Levine, A.J. (1995). Alternatively spliced forms in the carboxyterminal domain of the p53 protein regulate its ability to promote annealing of complementary single strands of nucleic acids. Mol. Cell. Biol. 15, 497-504. Xia, F., Amundson, S.A., Nickoloff, J.A., and Liber, H.L. (1994). Different capacities for recombination in closely related human lymphoblastoid cell lines with different mutational responses to X-irradiation. Mol. Cell. Biol. 14, 5850-5857. Xia, S.J., Shammas, M.A., and Shmookler Reis, R.J. (1997). Elevated recombination in immortal human cells is mediated by HsRAD51 recombinase. Mol. Cell. Biol. 17, 7151-7158. Xiao, H., Pearson, A., Coulombe, B., Truant, R., Zhang, S., Regier, J.L., Trizenberg, S.J., Reinberg, D., Flores, O., and Ingles, C.J. (1994). Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53. Mol. Cell. Biol. 14, 7013-7024. Yang, T.-t., Namba, H., Hara, T., Takmura, N., Nagayama, Y., Fukata, S., Ishikawa, N., Kuma, K., Ito, K., and Yamashita, S. (1997). p53 induced by ionizing radiation mediates DNA end-joining activity, but not apoptosis of thyroid cells. Oncogene 14, 1511-1519. Yuan, S.-S., Lee, S.-Y., Chen, G., Song, M., Tomlinson, G.E., and Lee, E.Y.-H.P. (1999). BRCA2 is required for ionizing radiation-induced assembly of Rad51 complex in vivo. Cancer Res. 59, 3547-3551. Zhang, H., Somasundram, K., Peng, Y., Tian, H., Zhang, H., Bi, D., Weber, B.L., and El-Deiry, W.S. (1998). BRCA1 physically associates with p53 and stimulates its transcriptional activity. Oncogene 16, 1713-1721. Zhong, Q., Chen, C.-F., Li, S., Chen, Y., Wang, C.-C., Xiao, J., Chen, P.-L., Sharp, Z.D., and Lee, W.-H. (1999). Association of BRCA1 with the hRad50-hMre11-p95 complex and the DNA damage response. Science 285, 747750.

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Gene Ther Mol Biol Vol 5, 81-86, 2000

Characterisation of the p53 gene in the rat CC531 colon carcinoma Research Article

Sacha B Geutskens1, Diana JM van den Wollenberg1, Marjolijin M van der Eb1,2, Hans van Ormondt1, Aart G Jochemsen1, Rob C Hoeben1* Departments of 1Molecular Cell Biology and 2Surgery, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands _________________________________________________________________________________________________ * Correspondence: Rob C Hoeben, PhD, Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands; Phone: 00 31 71 5276119; Fax: 00 31 71 5276284; E-mail: r.c.hoeben@lumc.nl Key words: colorectal cancer, mutated p53 Received: 6 November 2000; accepted: 7 November 2000

Summary The p53 gene was characterised in CC531 colon carcinoma of the Wag/Rij rat, a model frequently used for the evaluation of anti-cancer treatments. The gene incurred a large in-frame deletion, with junctions in exons 4 and 8, and encodes a protein of approximately 32 kDa, lacking the entire DNA-binding domain. No wild-type p53 allele is retained. Functional analysis shows that the mutated protein can repress the function of wtp53 protein.

and frequently used for studying effects of various anticancer treatments (Marinelli et al, 1991; Oldenburg et al, 1994; Veenhuizen et al, 1996; Griffini et al, 1997). Two-thirds of all colorectal tumours contain mutations in the p53 gene, illustrating the essential role of p53 in the aetiology of colorectal cancer (Soussi et al, 1996). Loss of p53 function, as frequently occurs in cancer cells, causes loss of growth control and abrogation of programmed cell death (Bellamy, 1997). Reintroduction of wild-type p53 (wtp53) arrests cell growth and may induce apoptosis, as has been described for various tumour cell lines (Xu et al, 1997; Anderson et al, 1998). A relationship has also been established between the presence of a mutation in the p53 gene and the clinical prognosis (Hamelin et al, 1994). In view of the prominent role of the p53 gene for colorectal cancer, we assessed the integrity of the p53 gene in the CC531 cancer cell line, and discovered a homozygous in-frame deletion within the coding region of the p53 mRNA, resulting in removal of the entire DNAbinding domain. The mutated protein is capable of inhibiting transcription activation by wtp53. Our data not only reveal the p53 status of this model, but also provide a genetic marker that allows development of a sensitive

I. Introduction Colorectal cancer is one of the most common malignant tumours and a major cause of cancer death in developed countries. Over 700, 000 men and women are found to have colorectal cancer globally each year (Soussi et al, 1996). The liver is the first vascular bed in which disseminating colorectal cancer cells are trapped. Liver metastases are detected in 20% of all patients undergoing resection of their primary colorectal tumour (Wanebo et al, 1978). Ultimately, about 75% of all colorectal cancer patients develop liver metastases. At present, the only chance of long-term survival is complete resection of these metastases, an operation that is exclusively performed on patients with no signs of irresectable extra-hepatic disease. Animal models are indispensable in the search of new approaches for the treatment of colorectal tumour metastases. One of the few well-characterised animal models for hepatic colorectal cancer makes use of the rat CC531 cell line. The CC531 colon carcinoma cell line is derived from a 1,2-dimethyl-hydrazine-induced tumour and syngeneic with the Wag/Rij rat (Thomas et al, 1993). It is a representative model for secondary liver metastases

Geutskens et al: Characterisation of p53 in the rat CC531 cell line

PCR-based assay for the detection of cancer cells amid normal cells.

600 bp was amplified, under various conditions (Figure 1A). Moreover, when internal primers (annealing to exons 4 and 8) were used no amplified product was detected (Figure 1A), suggesting a deletion within the coding region. Indeed, sequence analysis and comparison with the published sequence of the rat p53 gene (Hulla and Schneider, 1993), revealed that a large part of the coding region was absent. This in-frame deletion encompasses part of exon 4, exons 5-7 and part of exon 8 and removes amino acids 105 to 326.

II. Results A. PCR amplification and sequence analysis of the p53 mRNA and chromosomal DNA in CC531 cells To study whether the p53 gene of the CC531 cell line contains a mutation, RT-PCR was performed to amplify the entire coding region of the p53 gene. Instead of the expected 1200-bp fragment, only a fragment of ca.

Figure 1. A: Lane 1 shows the 600-bp p53-cDNA fragment obtained by PCR-amplification on RNA isolated from CC531 cells. In lanes 2, 3 and 4, a PCR fragment is absent if primer combinations are used spanning exon 1/exon 4, exon 8/exon 10 and exon 4/exon 8 respectively. Fragment sizes have been estimated by comparison to a 100 bp DNA marker (first lane). B: Schematic representation the genomic configuration of the coding region of p53 gene in CC531 cells. The exons are indicated. Below the graph the sequence of the fused exons 4 and 8 is given. The filled triangle, below the sequence marks the junction. C: PCR amplification of a 200-bp DNA fragment encoding the fused exons 4 and 8 (primers F4/R8) of the p53 gene from CC531derived chromosomal DNA (lane 2). No fragment is detected if primers F7/R8 are used (lane 1). A 300-bp fragment containing the 5â&#x20AC;&#x2122; exon 7/ 5â&#x20AC;&#x2122; exon 8 (F7/R8 primers) region is amplified if chromosomal DNA from Wag/Rij rat hepatocytes is used (lane 3). No fragment is detected if this DNA is amplified with F4/R8 primers (lane 4). Lane 5 and 6 depict H2O controls for primer pairs F7/R8 and F4/R8 respectively. Fragment sizes have been estimated by comparison to a 100-bp DNA marker (first lane).

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Thus, the resulting p53 protein lacks the entire specific DNA-binding domain, but still contains the domain responsible for tetramerization with other p53 proteins (Figure 1B). To verify that the deletion is not the result of aberrant RNA splicing, chromosomal DNA from CC531 cells was analysed. Therefore, forward primers were developed binding in the remaining part of exon 4 (F4) and in exon 7 (F7) and one reverse primer downstream of the junction in exon 8 (R8). If a wild-type p53 allele were present, it would be expected to yield a PCR-product of about 300-bp when F7 is combined with R8. If, on the other hand, the deletion occurs on the chromosomal DNA, a !200-bp product is expected if F4 is combined with R8. If the mutated allele is the only one present, no product should be seen with F7 and R8. Amplification of chromosomal DNA derived from CC531 cells yielded a 200-bp product with the F4/R8 primers and no product with the F7/R8 primers. In contrast, PCR amplification of chromosomal DNA isolated from Wag/Rij rat hepatocytes yielded no product with F4/R8 primers, but a fragment of ca.300-bp when F7/R8 primers were used (Figure 1C). These data demonstrate that the deletion is present on the chromosomal DNA level. Moreover, the CC531 cell line is homozygous for the mutated p53 allele.

rat CC531 and BxC22 cells, were analysed for presence of human and rat p53. Indeed, a smaller p53-protein of ca. 32 kDa was detected in CC531 cells, which co-migrates with a band seen in the Hep3B and HepG2 cells transfected with pCMVCC53 (Figure 2). Since the CC531-derived p53 protein lacks the DNA-binding domain, but still contains the tetramerization part, it was hypothesized that

Figure 2 Immunoblot analysis on cell lysates of BxC22 (lane A; rat wtp53), CC531 (lane B, rat mutp53), HepG2 (lane C, human wtp53) and Hep3B (lane F, human, no p53). HepG2 and Hep3B cells transfected with 10 µg (lane D and G) or 20 µg (lane E and H) of pCMVCC53. Blots have been incubated with PAb122, recognising the C terminus of rat and human wtp53 (blot A) or with DO-1, recognising human wtp53 (blot B). Both BxC22 and HepG2 express normal-size (ca. 53 kDa) wtp53. CC531-derived cell lysates and those transfected with the CC53-construct express a shorter p53 protein of ca. 32 kDa. Protein sizes have been estimated by comparison to a broad-range protein marker.

B. Analysis and function of the mutated p53 protein Because the deletion does retain the p53 open reading frame, a p53 protein lacking the central domain be synthesised. To investigate this possibility, the CC531 p53encoding cDNA was cloned in an expression vector and transfected into the human Hep3B (no p53) and HepG2 (wtp53) cell lines. After 48 h, these cells and, as controls,

Figure 3 Luciferase activity in HepG2 cell lysates. Lysates were made 48 h after transfection of HepG2 cells with the various constructs. Cells (1x105/well) were transfected with the calcium-phosphate precipitation method, with 1.5 µg pXAluc or pOLXALuc, 20 ng of either pCMVneo, pCMVwtp53 or pCMVCC53, and 20 ng pCMVlacZ as indicated. Precipitates were made in duplicate and the experiment was performed three times. Luciferase activities depicted are corrected for differences in transfection efficiencies determined with the ß-galactosidase assay.

Geutskens et al: Characterisation of p53 in the rat CC531 cell line

Eb et al, manuscript in preparation). Combining chemotherapeutics with the transfer of wtp53 might give a higher efficacy of anti-tumour treatment than with the antineoplastic agent alone. In addition, the characterisation of the junction of this deletion has allowed the differentiation of CC531 tumour and non-tumour cells via PCR. This technique permits detection and quantification of minute amounts of tumour cells in extra-hepatic tissues, such as lymph nodes and lungs and it will make the CC531 model even more valuable for exploration of new avenues for treatment of colorectal cancer.

the protein might act as a dominant-negative mutant. To investigate whether the 32-kDa p53 protein can affect transcription activation of wtp53, the pCMVCC53 vector was transfected into HepG2 cells, together with a p53responsive luciferase-reporter plasmid. Transfection of pOLXALuc, which contains a p53-responsive element (Steegenga et al, 1995) yields a higher luciferase activity than transfection with pXALuc, which lacks a p53reponsive element (Peltenburg and Schrier, 1994) (Figure 3). Co-transfection of a plasmid encoding human wtp53 further increased expression of pOLXALuc, but not that of pXALuc, indicating the validity of the approach. The activity of pOLXALuc was clearly reduced upon cotransfection with pCMVCC53, while pXALuc expression was not affected. This suggests that the 32-kDa p53 protein acts as a dominant inhibitor of wtp53 function. Expression of a non-p53-regulated reporter, lacZ, which is driven by the CMV promoter, did not show manifest differences between various co-transfections.

IV. Materials and Methods A. Tissue culture and cell lines The CC531 cell line is a moderately differentiated adenocarcinoma of the colon, syngeneic with Wag/Rij rats (Thomas et al, 1993). BxC22 is a wtp53-expressing Ad5 E1transformed Wag/Rij baby rat kidney cell line. HepG2 and Hep3B are human hepatoma cell lines expressing wtp53 (Hosono et al, 1991) or lacking p53 expression (Farshid and Tabor, 1992) respectively. All cell lines were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal-calf serum (GIBCO Laboratories, Grand Island, NY, USA), at 37 °C/5% CO2.

III. Discussion Genetic alteration of the tumour-suppressor gene p53 is frequently found in cancer, especially in the DNAbinding domain that spans exons 5 through 8 (Tullo et al, 1999; Veldhoen et al, 1999). In many studies, only this region is screened for the presence of mutations in clinical samples. The rat colon carcinoma studied here, showed a large deletion, removing amino acids 105 to 326. A smaller p53-protein of ca.32 kDa is translated which is recognized by a human and rat-p53 specific antibody, PAb 122, that is known to bind to the C-terminus of the wild-type protein. Although the DNA-binding domain is deleted, the 32-kDa p53 protein can impair transcriptional activation by regulation of wtp53. Such a dominant-negative effect has been described earlier (Chen, 1998; Roemer, 1999) and probably results from disruption of DNA binding of wtp53 by forming hetero-tetramers with the mutant p53 (Deb et al, 1999). This type of mutants is also thought to exhibit a gain of function by generating genomic instability, increasing oncogenic transformation (Gualberto et al, 1998). CC531 is a valuable model for secondary liver metastases in the rat and often used for studying the therapeutic effect of various anti-neoplastic agents (Marinelli et al, 1991; Oldenburg et al, 1994; Veenhuizen et al, 1996). The p53 protein is essential in the induction of apoptosis by several anti-cancer therapeutics (Tishler and Lamppu, 1996; Hagopian et al, 1997; Anderson et al, 1998). Mutations in the p53 gene are associated with drug resistance, so the p53 status of this model is highly important. The p53 deletion characterised here might explain the resistance of CC531 to cisplatinum treatment (Gheuens et al, 1993) that activates p53-dependent apoptotic pathways. Despite the dominant negative activity of the CC531 p53 protein, transfer of wtp53 rendered the CC531 colon carcinoma susceptible for apoptosis (Van der

B. Polymerase chain reaction (PCR) For reverse transcription-PCR (RT-PCR), RNA was isolated directly from tissue culture with RNAzolB according to the manufacturers protocol (Campro Scientific, Veenendaal, The Netherlands). RNA was treated with DNase I (Roche Diagnostics, Almere, The Netherlands) to degrade contaminating DNA. First-strand-complementary DNA (cDNA) of 1 µg of RNA was synthesized with SuperscriptII RNase H** reverse transcriptase (Gibco/Life Technologies, Breda, The Netherlands) and an Oligo-dT primer. The cDNA (1 µg) was amplified with primers complementary to the 5’ and 3’ ends of the coding region of wild-type rat p53 (forward primer: 5’ GTG GAT CCT GAA GAC TGG ATA ACT GTC 3’; reverse primer: 5’ AGT CGA CAG GAT GCA GAG GCT G 3’) (Van den Heuvel et al, 1990), with Pfu polymerase (Stratagene, Amsterdam, The Netherlands) in buffer supplied by the manufacturer. Primers complementary to internal sequences of the p53 coding region (forward primer: 5’ TAC CAC TAT CCA CTA CAA GTA CAT G 3’; reverse primer: 5’ TTT CTT CCT CTG TCC GAC GGT CTC 3’) were used as a control. The amplified 600-bp fragment was sequenced by BaseClear (Leiden, The Netherlands). Chromosomal DNA was isolated with the NP40 protocol (Maniatis et al, 1989). DNase was heat-inactivated and RNase (Merck, Darmstadt, Germany) was added to degrade RNA. One microgram of DNA was amplified with primers annealing to exon 8 (R8): 5’ AAT CCA ATA ATA ACC TTG GTA CCT T 3’, exon 7 (F7): 5’ TGT GCC TCC TCT TGT CCC 3’ or exon 4 (F4): 5’ CGA CAG GGT CAC CTA ATT CC 3’ of wild-type rat p53 chromosomal DNA with Taq polymerase (Roche Diagnostics, Almere, The Netherlands).

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Chen P (1998) In vitro analysis of the dominant negative effect of p53 mutants. J Mol Biol 281, 205-209 Deb D, Chakraborti AS, Lanyi A, Troyer DA, Deb S (1999) Disruption of functions of wild-type p53 by heterooligomerization. Int J Oncol 15, 413-422 Farshid M, Tabor E (1992) Expression of oncogenes and tumor suppressor genes in human hepatocellular carcinoma and hepatoblastoma cell lines. J Med Virol 38, 235-239 Gheuens E, Van der Heyden S, Elst H, Eggermont A, Van Oosterom A, De Bruijn E (1993) Multidrug resistance in rat colon carcinoma cell lines CC531, CC531mdr+ and CC531rev. Jpn J Cancer Res 84, 1201-1208 Griffini P, Smorenburg SM, Verbeek FJ, Van Noorden CJ (1997) Three dimensional reconstruction of colon carcinoma metastases in liver. J Microsc 187, 12-21 Gualberto A, Aldape K, Kozakiewicz K, Tlsty TD (1998) An oncogenic form of p53 confers a dominant, gain-of-function phenotype that disrupts spindle checkpoint control. Proc Natl Acad Sci USA 95, 5166-5171 Hagopian GS, Mills GB, Khokhar AR, Bast RC jr, Siddik ZH (1999) Expression of p53 in cisplatin-resistant ovarian cancer cell lines: modulation with the novel platinum analogue (1R,2R-diaminocyclohecane)(trans-diacetato)(dichloro)platinum. Clin Cancer Res 5, 655-666 Hamelin R, Laurent-Puig P, Olschwang S, Jego N, Asselain B, Remvikos Y, Girodet J, Salmon RJ, Thomas G (1994) Association of p53 mutations with short survival in colorectal cancer. Gastroenterology 106, 42-48 Hosono S, Lee CS, Chou MJ, Yang CS, Shih CH (1991) Molecular analysis of the p53 alleles in primary hepatocellular carcinomas and cell lines. Oncogene 6: 237243 Hulla JE and Schneider RP Sr. (1993) Structure of the rat p53 tumor suppressor gene. Nucleic Acids Res 21, 713-717 Maniatis T, Sambrook J, Fritch EF (1989) Molecular Cloning: a Laboratory Manual, 2nd edition. CSHL Press, Cold Spring Harbor, NY. Marinelli A, Van Dierendonck JH, Van Brakel GM, Irth H, Kuppen PJ, Tjaden UR, Van de Velde CJ (1991) Increasing the effective concentration of melphalan in experimental rat liver tumours: comparison of isolated liver perfusion and hepatic artery infusion. Br J Cancer 64, 1069-1075 Oldenburg J, Begg AC, Van Vugt MJ, Reuvekamp M, Schornagel JH, Pinedo HM, Los G (1994) Characterization of resistance mechanisms to cisdiamminedichloroplatinum (II) in three sublines of the CC531 colon adenocarcinoma cell line in vitro. Cancer Res 54, 487-493 Peltenburg LTC, Schrier PI (1994) Transcriptional suppression of HLA-B expression by c-myc is mediated through the core promotor elements. Immunogenetics 40: 54-61 Roemer K (1999) Mutant p53: gain-of-function oncoproteins and wild-type p53 inactivators. Biol Chem 380, 879-887 Schmieg FI, Simmons DT (1984) Intracellular location and kinetics of complex formation between simian virus 40 T antigen and cellular protein p53. J Virol 52, 350-355 Soussi T (1996) The p53 tumour suppressor gene: a model for molecular epidemiology of human cancer. Mol Med Today 2, 32-37 Steegenga WT, Van Laar T, Terleth C, Van der Eb AJ, Jochemsen AG (1995) Distinct modulation of p53 activity in transcription and cell-cycle regulation by the large (54 kDA) and small (21 kDA) adenovirus E1B proteins. Virology 212, 543-554

C. Western immunoblotting 6

Cells (total of 3x10 ) were lysed in 750 µl NP40/SDS (2%/0.2%) buffer (25 mM Tris pH 7.4, 50 mM NaCl, 0.5% deoxycholate). Protein lysates (40 µl) were size-fractionated by gel electrophoresis in 10% SDS-polyacrylamide. Proteins were transferred to Immobilon-P (0.45 µm, Millipore Corporation, Bedford, USA) and incubated with an antibody specifically recognizing human wtp53, DO-1, or with PAb 122, recognizing both human and rat p53 (Schmieg and Simmons,1984). As a second antibody horse-radish-peroxidase (HRP)-conjugated antibody, G"M-IgG (Jackson Immunoresearch Laboratories, Westgrave, USA) was used. The blots were visualized by exposion to Kodak XAR-films. The broad-range protein marker was used as a standard (BioRad laboratories, Veenendaal, The Netherlands).

D. Luciferase reporter assay For the expression of the mutant p53 cDNA, a vector containing the p53-coding region of CC531 under the regulation of the CMV promoter (pCMVCC53) was made by digesting the purified RT-PCR fragment with BamHI and SalI and subsequent ligation into pcDNA3.1+ (Invitrogen, Leek, The Netherlands) digested with BamHI and XhoI. Plasmid constructs expressing the neomycin resistance gene (pCMVneo), the E.coli ßgalactosidase gene (pCMVlacZ), or the human wtp53 cDNA (pCMVwtp53) were used and have been described earlier (Steegenga et al, 1995). The luciferase reporter constructs, pXALuc and pOLXALuc (containing the p53-consensus binding sequence) have also been described before (Peltenburg and Schrier, 1994; Steegenga et al, 1995). Cells (1x105/well) were transfected with the calciumphosphate precipitation method (van der Eb and Graham,1980), with 1.5 µg pXAluc or pOLXALuc, 20 ng of either pCMVneo, pCMVwtp53 or pCMVCC53, and 20 ng pCMVlacZ. Precipitates were made in duplicate and the experiment was performed three times. After 48 h, cells were lysed in 250 µl of cell-culture lysis reagent (Promega, Madison, WI, USA) and luciferase activity of 100 µl of lysate was determined as described before (Steegenga et al, 1995). The #-galactosidase activity resulting from cotransfection of the control plasmid pCMVlacZ was determined in a ß-galactosidase assay (Maniatis et al, 1989) and served as an internal control to correct for variations in transfection efficiency. In none of the experiments, ß-gal activity varied more than 20%.

Acknowledgements The sequence of the CC531 p53 cDNA is deposited in Genbank (accession number AY009504).

References Anderson SC, Johnson DE, Harris MP, Engler H, Hancock W, Huang W, Wills KN, Gregory RJ, Sutjipto S, Fen Wen S, Lofgren S, Shepard HM, Maneval DC (1998) p53 Gene therapy in a rat model of hepatocellular carcinoma: Intraarterial delivery of a recombinant adnovirus. Clin Cancer Res 4, 1649-1659 Bellamy CO (1997) p53 and apoptosis. Br Med Bul 53, 522-523

Geutskens et al: Characterisation of p53 in the rat CC531 cell line

Thomas C, Nijenhuis AM, Timens W, Kuppen PJ, Daemen T, Scherphof GL (1993) Liver metastasis model of colon cancer in the rat: immunohistochemical characterization. Invas Metas 13, 102-112 Tishler RB, Lamppu DM (1996) The interaction of taxol and vinblastine with radiation induction of p53 and p21 WAF1/CIP. Br J Cancer 27, S82-85 Tullo A, D'Erchia AM, Honda K, Mitry RR, Kelly MD, Habib NA, Saccone C, Sbisa E (1999) Characterization of p53 mutations in colorectal liver metastases and correlation with clinical parameters. Clin Cancer Res 5, 3523-3528 Van den Heuvel SJ, Van Laar T, Kast WM, Melief CJ, Zantema A, Van der Eb AJ (1990) Association between the cellular p53 and the adenovirus 5 E1B-55kd proteins reduces the oncogenicity of Ad-transformed cells. EMBO J 9, 26212629

Van der Eb AJ, Graham FL (1980) Assay of transforming activity of tumor virus DNA. Methods Enzymol 65, 826839 Veenhuizen RB, Marijnissen JP, Kenemans P, ReuvekampHelmers MC, â&#x20AC;&#x2DC;t Mannetje LW, Helmerhorst TJ, Stewart FA (1996) Intraperitoneal photodynamic therapy of the rat CC531 adenocarcinoma. Br J Cancer 73, 1387-1392 Xu M, Kumar D, Srinivas S, Detolla LJ, Yu SF, Stass SA, Mixson AJ (1997) Parental gene therapy with p53 inhibits human breast tumors in vivo through a bystander mechanism without evidence of toxicity. Hum Gene Ther 8, 177-185 .

Gene Therapy and Molecular Biology Vol 5, page 87

Gene Ther Mol Biol Vol 5, 87-100, 2000

Recombinant adenoviruses as expression vectors and as probes for DNA repair in human cells Review Article 1

Andrew J. Rainbow , Bruce C. McKay and Murray A. Francis 1

Department of Biology, McMaster University, Hamilton, Ontario L8S 4K1, Canada Canada Centre for Cancer Therapeutics, Ottawa Regional Cancer Centre, 501 Smyth Rd, Ottawa, Ontatio K1H 8L6, Canada. _________________________________________________________________________________________________ 2

*Correspondence: Dr. Andrew J. Rainbow, Department of Biology, McMaster University, Hamilton, Ontario L8S 4K1, Tel: (905) 5259140 Ext. 23544 Fax: (905)-522-6066; E-mail: rainbow@mcmaster.ca Key Words: recombinant adenovirus, host cell reactivation, nucleotide excision repair, enhanced reporter gene expression, inducible DNA repair, p53 tumour suppressor, ultraviolet light

Received: 25 August 2000; accepted: 10 September 2000

Summary There is widespread interest in the use of recombinant adenovirus (Ad) vectors for gene therapy of cancer and as tools in molecular biology research. There are also potential benefits to be gained by combining strategies for Adbased gene therapy of cancer with radiotherapy and chemotherapy. However, there is limited information available on the effects of cytotoxic agents on transgene expression which would allow a rational approach to combining these modalities. We have used recombinant nonrepilcating Ad expressing the lacZ gene under the control of the cytomegalovirus (CMV) immediate early promoter to assess the effects of cytotoxic agents on the expression of a reporter gene in human cells. Using this approach we are able to examine both constitutive and inducible expression of the reporter gene. Pretreatment of normal human cells with low UV fluence results in an enhanced expression of the reporter gene. The enhanced expression occurs at lower doses of the DNA damaging agent in cells deficient in the transcription coupled repair (TCR) pathway of nucleotide excision repair (NER) suggesting that the enhancement in human cells is triggered by persistent damage in actively transcribed genes. The enhancement is also reduced or absent in SV4O-transformed cells and cells expressing the human papilloma virus ('WV) E7 gene, but not in Li-Fraumeni syndrome (LFS) cells or cells expressing the 'WV E6 gene. Since SV4O-transformation and HPV E7 expression both abrogate the retinoblastoma (pRb) family of proteins, whereas 'WV E6 abrogates p53 and LFS cells express mutant p53, these results indicate that the enhanced expression depends on one or more of the pRb family of proteins, but not on p53. We have also used recombinant Ad expressing a lacZ reporter gene as a probe for DNA repair in human cells. Using this approach we have examined constitutive as well as inducible DNA repair of a UV-damaged reporter gene in human cells. We detected enhanced host cell reactivation (HCR) of a UVdamaged reporter gene in pre-heat-shock treated or pre-UV treated TCR proficient but not in pretreated TCR deficient human fibroblasts or LFS cells. These results suggest the existence of an inducible repair response for UVdamaged DNA in human cells which is dependent on the TCR pathway of NER and the wild type p53 tumour suppressor. These results have important implications for the use of recombinant Ad-based expression vectors under the control of the CMV promoter in gene therapy for cancer when used in combination with DNA damaging agents.

I. Introduction Adenovirus (Ad) vectors are a very efficient method for delivering foreign genes into mammalian cells both in vitro and in vivo (Hitt et al, 1997) and show great promise

for gene therapy of cancer (Boulikas, 1998; Stewart et al, 1999). Ad infects both dividing and non-dividing cells in a wide variety of tissues and cell types of many different species.

Rainbow et al: Recombinant adenoviruses as expression vectors

Figure 1. Enhanced expression of a recombinant adenovirus based reporter gene following pretreatment of human cells with UV. Cells were seeded in 96 well microtitre plates at a density of 2 x 104 cells/well, 18-24 h to treatment. For UV treatment of cells, the growth medium was aspirated and replaced with 40 ml phosphate buffered saline (PBS) and the cell monolayers were either left untreated or UV-irradiated with a range (A to D) of fluences. UV irradiation of cells was performed using a germicidal lamp (General Electric model G8T5) emitting predominantly 254 nm at a fluence rate of I J/m2/s. After UV treatment, cells were either infected or at mock infected for 90 mm at 37 ยบC with untreated Ad5HCMVSPlIaCZ in a total volume of 40 ml and the infected cells were incubated for a period of time (usually 12 - 48 h) before harvesting and scoring for !galactosidase activity as reported previously (Francis and Rainbow 2000). Lysates from mock infected wells served as a measure of background levels for !-galactosidase activity.

The Ad genome is relatively easy to manipulate using standard molecular biology techniques such that both replication proficient and replication deficient vectors can be easily produced and putified on a large scale (Graham and Prevec 1991; Hitt et al, 1995). Replication proficient Ad vectors with only the early region 3 (E3) deleted can accommodate up to about 4 kb of foreign DNA, whereas replication deficient Ad vectors deleted in both E3 and El can accommodate up to about 8 kb. Several reports have proposed the use of Ad transgene delivery in combination with radiation therapy and chemotherapy (for a recent review see Stackhouse and Buchsbaum 2000). To combine gene therapy and radiation therapy or chemotherapy into an effective combination of modalities for the treatment of cancer it is essential to understand the effects of radiation treatment and chemotherapy treatment of cells and transgenes on transgene expression. We have used a recombinant nonreplicating human Ad, either Ad5HCMVsp1lacZ (Morsy et al, 1993) or AdCA35 (Addison et al, 1997), expressing the lacZ gene under the control of the cytomegalovirus (CMV) immediate early promoter to assess the effects of cytotoxic agents on the expression of a reporter gene in mammalian cells. Using this approach we are able to examine both constitutive and inducible expression of the reporter gene in cells treated with DNA damaging agents (Figure 1 and 2). In addition we have used the same non-replicating Ad expressing the lacZ reporter gene as a probe for DNA

repair in mammalian cells. Using this approach were are able to examine both constitutive and inducible DNA repair of a UV-damaged reporter gene in several different mammalian cell types including normal human fibroblasts, repair deficient human fibroblasts and several different human tumour cells (Figures 3 and 5).

II. Recombinant adenovirus expression vectors for gene transfer into mammalian cells A. Adenovirus based transgene expression levels in mammalian cells The level of expression in cells infected by Ad vectors is greatly influenced by the promoter controlling expression of the transgene. Xu et al, (1995) have demonstrated that the human CMV immediate early promoter directs the highest level of expression in the widest variety of mammalian cell types in vitro when compared to that directed by the human b-actin, Ad major late, and SV4O early and late promoters. In addition, Addison et al, 1997 showed that the murine CMV immediate early promoter (Dorsch-Hasler et al, 1985) is also an extremely strong promoter and rivals the human CMV promoter for in vitro expression in both murine and human cells. In vivo, the highest level of expression reported to date occurred following intravenous delivery

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to the mouse of an Ad vector under the control of the human CMV promoter (Kolls et al, 1994).

B. Enhanced expression of a lacZ reporter gene driven by the CMV immediate early promoter following pretreatment of human cells with DNA damaging agents The CMV immediate early (IE) promoter is one of the most commonly used promoters in eukaryotic expression vectors, due primarily to its ability to yield high expression levels in many different mammalian cell types. Both stress-activated MAP protein kinases (Bruening et al, 1998) and ionising radiation (Tang et al, 1997) can each up regulate expression of transgenes driven by the CMV promoter. Our protocol for assessing transgene expression in cells treated with DNA damaging agents is outlined in Figure 1. We report that up regulation of transgene expression from the CMV promoter in human cells also results from pretreatment of cells with UV light (Francis and Rainbow 1997, 2000, Figure 2) or cisplatin (Francis 2000). UV radiation results in damage to DNA and the activation of cell surface receptors and their downstream signalling pathways. Although UV-induced DNA damage acts to directly block transcription, and cellular RNA levels immediately decrease following UV exposure (Mayne and Lehmann 1982), the expression of several cellular and viral genes are enhanced following UV exposure (Herrlich et al, 1994; Bender et al, 1997). These UV-inducible genes can be divided into those which respond immediately after UV exposure and those which have a response which is delayed and not observed until several hours after UV exposure (Herrlich et al, 1994; Bender et al, 1997). While the extent of the immediate response appears to depend on the magnitude of the initial UV insult, the strength and duration of the delayed response appears to be affected by the cells ability to repair UVinduced DNA damage, particularly to its DNA (reviewed in Bender et al, 1997). Evidence for this comes from studies using cell lines which are deficient in repair of UV-damaged DNA (Miskin and Ben-Ishai 1981; Blattner et al, 1998). UV-induced up regulation of the CMV driven transgene in human cells appears to be a delayed response and expression from CMV-driven constructs is enhanced following lower UV exposures to TCR deficient compared to TCR proficient human fibroblasts (Francis and Rainbow 1997, 2000; Figure 2). In addition, pre-infection of human fibroblasts with a UV-damaged Ad construct containing an actively transcribing gene can induce expression from a CMV driven transgene, while preinfection with a UVdamaged Ad control vector which does not contain an actively transcribing gene (which presumably has minimal transcription activity) does not (Francis and Rainbow 2000). Taken together, these data strongly suggest that persistent and unrepaired damage in active genes plays a direct role in eliciting enhanced

expression from CMV promoters. It is possible that it is the persistent stalling of RNA pol II at sites of unrepaired damage which acts as a trigger for this response following UV exposure as has been suggested for other UV-induced cellular responses (Yamaizumi and Sugano 1994; Ljungman and Zhang 1996; Ljungman et al, 1999). The p53 protein has been implicated in numerous cellular responses to UV, including DNA repair, and can regulate the expression of a large number of cellular genes (reviewed in McKay et al, 1999, Lakin and Jackson 1999, elDeiry 1998). We have examined UV-induced expression of a CMV-driven reporter construct in HeLa cells, 5V40transformed fibroblasts, Li-Fraumeni syndrome (LFS) fibroblasts, and spontaneously immortalised LFS sublines (Francis and Rainbow 2000). These cells have impaired p53 function as a result of human papilloma virus (HPV) E6-expression (Seedoif 1987; Scheffner et al 1990; Mietz et al, 1992), SV4O large

Figure 2. Enhanced expression of tbe CMV-driven bgalactosidase transgene in Ad5HCMVSp1lacZ following pretreatment of human fibroblasts with UV light. !-galactosidase reporter activity was quantitated 24 h following UV irradiation and subsequent infection at 10-20 plaque forming units per cell of normal (GM 38 (!)), XP-A (GM XP12BE (")), and CS-B (CS lAN (#)) fibroblasts with a highly purilied preparation of AdlHCMVsp1IacZ. Values were normalised to unirradiated controls. Each point is the average of 2 independent experiments (+1- SEM), each performed in 6 replicates. Adapted from Rainbow and Francis 2000

Rainbow et al: Recombinant adenoviruses as expression vectors

T antigen (SV40LT) expression (Segawa et al, 1993), geamime transmission of a mutant p53 allele (Malkin et al, 1990), and spontaneous loss of the wild type p53 allele from LFS fibroblasts (Yin et al, 1992), respectively. Although no UV induced expression of the CMV-driven lacZ gene from Ad5HCMVsp1IacZ was observed in any SV404ransformed line examined, a significant UVenhancement of reporter expression was observed in both ReLa and all LFS cell strains (Francis and Rainbow, 2000). These data suggest that p53 does not play an essential role in the UV-induced expression from CMV promoters, whereas some protein or pathway altered by SV4O-transformation does play an essential role in this response. Candidate proteins altered by SV40transfonnation include members of the retinoblastoma (Rb) family, which are known to play important roles in stress signalling, repair, and transcription (along with several other pathways). Since the pRb family of proteins are also altered in HeLa cells due to expression of the HPV E7 gene, the results of UV-enhanced expression of the reporter in HeLa cells n'ight suggest that pRb does not play an essential role. However, it has been reported that, although HPV E7 binds pRb and its family members p107 and p130, only pRb is targeted for degradation, while the levels of the two other proteins are not significantly altered by E7 expression (Berezutskaya et al, 1997) Furthermore, even pRb remains at significant levels and accumulates still higher in ReLa cells following UV exposure (Pedley et al, 1996). Thus it is possible that sufficient levels of one or more of the pRb family of proteins remain in HeLa cells to induce expression of reporter activity. In addition, more recent experiments using pRb-null or p53-null mouse embryo fibroblasts (Francis and Rainbow 1999a; Francis 2000) and human fibroblasts transformed with the HPV E7 or HPV E6 gene (Francis and Rainbow, unpublished data) support our earlier data indicating that p53 does not play an essential role and suggest a role for the pRb protein(s) in UVenhanced expression from a CMV-driven reporter. Since the pRb proteins and other pathways involving stressactivated MAP protein kinases (Bruening et al, 1998) are frequenfly altered in human tumour cells, the outcome of protocols combining gene therapy and radiotherapy or chemotherapy using CMV-driven transgenes may be tumour cell4ype specific. Other reports have suggested the use of ionizing radiation-activated gene therapy vectors for combined gene therapy and radiotherapy (Joki et al, 1995; Seung et al, 1995; Mauceri et al, 1996; Takahashi et al, 1997; Tang et al, 1997; Marples et al, 2000; Scott et al, 2000; Stackhouse and Buchsbaum 2000). Gamma-ray enhanced expression from a reporter gene is both cell-type specific and promoter specific (Tang et al, 1997; Marples et al, 2000) and gamma-ray enhanced expression from the CMV promoter was only detected when cells were irradiated and the transgenes were not. No amplification of the transgene was detected when both host cells and transgene were

subjected to irradiation (Tang et al, 1997). In contrast, gamma-ray induced expression of a plasmid born reporter gene under the control of a synthetic radio-responsive transcriptional enhancer could be repeated by additional radiation treatments in human tumour cells (Marples et al, 2000). These results suggest that depending on the promoter of the transgene, the timing sequence of genetherapy and radiotherapy or chemotherapy may be an important determinant of clinical outcome. It thus appears likely that with additional information on the various parameters controlling the up regulation of transgene expression, adenovirus-mediated gene therapy and radiotherapy or chemotherapy can potentially be formulated into synergistic protocols for the treatment of cancer.

III. Recombinant adenovirus as a probe for DNA repair in mammalian cells A. Nucleotide excision repair of damaged DNA The integrity of the human genome is constantly being compromised by alterations induced by a wide variety of exogenous physical and chemical agents, as well as by products of cellular metabolism. Several highly conserved repair pathways have evolved to remove damage from cellular DNA and disiuption of each of these DNA repair pathways is associated with carcinogenesis (as reviewed in Friedberg et al, 1995). The nucleotide excision repair (NBR) pathway repairs a wide range of bulky DNA adducts induced by numerous carcinogenic and antineoplastic compounds, including ultraviolet (UV) irradiation from the sun. NER can be divided into two interrelated subpathways: (1) transcription coupled repair (TCR) which preferentially removes DNA damage at a faster rate from the transcribed strand of actively transcribed genes, and (2) global genomic repair (GGR) which removes damage from throughout the entire genome and from the non-transcribed strand of active genes (Mellon et al, 1986, 1987). Individuals with the genetic diseases xeroderma pigmentosum (XP) have some deficiency in NER and show an increased incidence of a variety of skin cancers (Wei et al, 1993, Kraemer et al, 1994). Several additional links between NER and carcinogenesis have been reported. Mutations in the p53 tumour suppressor gene, the most commonly altered gene in malignancy (Hollstein et al, 1991), have also been shown to result in reduced NER (Ford and Hanawalt 1995; Smith et al, 1995; Wang et al, 1995; McKay et al, 1997; McKay et al, 1999; Therrien et al, 1999). Mismatch repair proteins have also been implicated in NER (Mellon et al, 1996) and mutations within genes encoding these proteins are associated with hereditary non-polyposis colorectal cancer (Fishel et al, 1993). Decreased NER has also been observed in a variety of tumour and transformed cell lines (Squires et al, 1982; Rainbow 1989). These reports suggest that DNA repair mechanisms are disrupted

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Figure 3. Host cell reactivation of a UVdamaged recombinant adenovirus based reporter gene in human cells. Cells were seeded in 96 well microtitre plates at a density of 2 x 104 cells/well and 18-24 h later infected for 90 mm at 37 째C with unirradiated or UV-irradjated AdS HCMVSp1lacZ in a total volume of 40 ml. AdSHCMVsp1lacZ was UV-irradiated with range of Iluences (A to D) using a germicidal lamp (General Electric model G8T5) emitting predominantly at 254 nm at an incident fluence rate of 2 J/m2/s. 1248 h later, infected cells were harvested and scored for !-galactosidase activity as reported previously (Francis and Rainbow 1999). Lysates from wells infected with heavily irradiated AdSHCMVspIIacZ (10,000 J/m2), X, served as a measure of background levels for !-galactosidsse activity .

in tumour cells and that DNA repair contributes to resistance to neoplasia. XP is composed of a minimum of seven complementation groups (XP-A-G), each displaying a general deficiency in NER which compromises at least the GR subpathway and usually TCR as well. The exception is XP-C which retains viable TCR in spite of a severe deficiency in GGR (Venema et al, 1990; Venema et al, 1991). The genetic disease Cockayne syndrome (CS) is also associated with a deficiency in NER, although unlike XP patients, CS patients do not display an increased risk of skin cancer. Two complementation groups of CS (CS-A and CS-B) have been identified, each of which exhibits a deficiency in TCR, while the GGR pathway appears to function normally (Venema et al, 1990a; van Hoffen et al, 1993). The XPB and XPD proteins are components of the transcription factor THIH (Schaeffer et al, 1993, Schaeffer et al, 1994) which plays a role in both NER and transcription by RNA polymerase II (RNAPII) (Drapkin et al, 1994). The CSA and CSB proteins coimmunoprecipitate (Henning et al, 1995) and are required for TCR. CSA interacts with THIH directly (Henning et al, 1995) whereas CSB does so via XPG (Iyer et al, 1996). TFIIH, XPG and the RPAIXPA/XPFIERCC1 complex (Park and Sancar 1994; Matsuda 1995) are required for both subpathways of NER, whereas the XPC/HHR23B complex (Masutani et al, 1994) appears to be required only for GGR (Venema et al, 1990a; Venema et al, 1991; Evans et al, 1993).

B. Host cell reactivation of DNA-damaged reporter genes in mammalian cells Host cell reactivation (HCR) of reporter gene activity has been assessed in mammalian cells using either recombinant Ad or plasmid constructs by a number of different laboratories. For plasmid constructs, this approach has typically involved the transfection of a DNA-damaged plasmid carrying a reporter gene into the cells of interest (Protic et al, 1988; Ganesan and Hanawalt 1994; Smith et al, 1995, Stevsner et al, 1995; Ganesan et al, 1999). Since primary human fibroblasts take up exogenous DNA 10-100 times less efficiently than either rodent or human cell lines derived from tumour tissue or transformed by viral antigens (Murname et al, 1985; Canaani et al, 1986; Hoejmakers et al, 1987), most experiments examining the reactivation of plasmid born UV-damaged reporters has been peiformed in tumour and transformed cell lines rather than primary human fibroblasts. However, many tumour and transformed cells have been reported to have a reduced DNA repair capacity for UV-damaged DNA (Squires et al, 1982; Rainbow 1989; McKay and Rainbow 1996; McKay 1997). Furthermore, the cellular response to DNA damage is stimulated by at least some transfection procedures (Renzing and Lane 1995; Seiget et al, 1995), leading to cell cycle arrest (Renzing and Lane 1995), suggesting that the transfection procedure itself may affect the outcome of DNA repair experiments. In contrast, recombinant nonreplicating Ad reporter constructs have the ability to infect and express high levels of recombinant gene products in most cell types including primary human fibroblasts. Furthermore, non-replicating Ad reporter constructs do not appear to elicit the DNA damage response, or inhibit host

Rainbow et al: Recombinant adenoviruses as expression vectors

DNA synthesis following infection (Blagoskionnay and el-Deity 1996). Recombinant non-replicating Ad constructs have been used to introduce UV-damaged (Valerie and Singhal 1995; McKay and Rainbow 1996; McKay et al, 1997; Francis and Rainbow 1999) or cisplatin damaged (Moorehead et al, 1996) reporter genes into non-treated human and rodent cells in order to assess the repair of damaged DNA in the absence of cellular stress using bost cell reactivation (HCR) of reporter gene activity as an endpoint. UVinduced lesions in the template strand of active genes inhibit progression of RNA polymerase II (Donahue et al, 1994) and a single UVinduced cyclobutane pyrimidine dimer (CPD) is thought to be sufficient to inhibit reporter gene expression (Protic-Sabaji and Kraemer 1985; Francis and Rainbow 1999). UVinduced DNA lesions are removed from plasmid born (Ganesan and Hanawalt 1994, Ganesan et al, 1999) and recombinant adenovirus born (Boszko 2000, Boszko and Rainbow 2000) reporter genes when introduced into repair proficient human cells and the removal is reduced when the same reporter genes are introduced into NER deficient

Figure 4. Nucleotide excision repair deficent cell strains show reduced HCR of UV-irradiated reporter activity. Lines represent repair proficient normal GM 3440 ("); and repair deficient XP-C (XP3BE (!)), XP-G (XP2BJ ($)) and CS-B (CS lAN (")) primary human fibroblasts. Each point is the average of 4 replicates, error bars represent one standard error. Untreated cells were infected with unirradiated or UV-irradiated virus at 10-20 plaque forming units per cell and scored for !-galactosidase activity 40-44 h later. Adapted from Francis and Rainbow 1999.

ceUs. Thus HCR of reporter gene activity is thought to require the repair of transcription blocking DNA lesions and reflectrepair of DNA lesions in the transcribed strand. A typical protocol to examine HCR of reporter gene activity for UV-irradiated recombinant Ad expressing the lacZ gene is shown in Figure 3. Using this approach we have shown that HCR of reporter gene activity for UV damaged DNA is reduced in several different nucleotide excision repair (NER) deficient cells of both human and rodent origin, including skin fibroblasts from patients with xeroderma pigmentosum from complementation group C (XPC) which showed HCR levels ranging from 25-75% that obtained in NER proficient normal primary human fibroblasts (Figure 4, McKay and Rainbow 1996; Francis and Rainbow 1999). The result for XP-C is surprising since XP-C cells are reported to be proficient in transcription coupled repair (TCR) and thus would be expected to reactivate UV induced lesions in the transcribed strand of the reporter gene. Blockage of RNA polymerase II by UV induced DNA lesions does not appear to be sufficient to promote the preferential repair of these transcription blocking lesions in non-UV-treated XP-C cells (McKay and Rainbow 1996; Francis and Rainbow 1999). Also of interest was the finding that CS fibroblast strains retained a considerable ability to repair the UV-damaged reporter gene in non-treated cells (5790% of normal levels) in spite of their being characterized as deficient in repair of the transcribed strand of active genes following UV irradiation of the cell (Francis and Rainbow 1999). It is therefore apparent that in the absence of UV exposure to the cell, damage in the transcribed strand of the recombinant Ad-based reporter gene is repaired to a large extent by the global genomic repair (GGR) pathway of NER in primary human fibroblasts, although significant TCR must also occur since HCR is also reduced in the CS strains. Since the Ad genome is approximately 5 orders of magnitude smaller than the human genome, the number of lesions introduced into cells with a UV-irradiated virus in our experiments is minimal compared to the number introduced into the host cell genome following UV treatments used to examine repair in cellular DNA. Several studies using plasmid born reporter genes have reported decreased HCR in NER deficient XP cells (Lehmann and Ooman 1985; Protic-Sabljic and Kraemer 1985; Barrett et al, 1991) and CS cells (Barrett et al 1991, Klocker et al, 1985) compared to repair proficient human cells. These studies have generally used 5V40-transformed repair deficient XP and CS cell lines and repair proficient "normal" cell lines derived from human tumours rather than primary fibroblast strains. SV4O transformed cells and many human tumours have alterations in the p53 tumour suppressor, the pRb tumour suppressor and other stress activated pathways which have been shown to affect the GGR and/or TCR pathway of NER (Ford and Hanawalt 1995, 1997; ; McKay et al, 1997; 1999; Ford et al, 1998; Therrien et al; 1999).

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Fignre 5. UV-enhanced host cell reactivation of a UV-damaged recombinant adenovirus based reporter gene in hmnan cells. Cells were seeded in 96 well microtitre plates at a density of 2 x 104 cells/well, 18-24 h prior to UV-treatrnent of cells. The growth medium was then aspirated, replaced with 40 ml phosphate buffered saline (PBS) and sets of cell monolayers were either UV-irradiated with a fluence F or were mockirradiated and received no UV. After treatment, both UV-irradiated and non4rradiated sets of cells were infected for 90 min at 37 ยบC in a total volume of 40 ml with unirradiated Ad5HCMVsp1lacZ or AdiHCMVsp1lacZ which had been UV-irradiated with arange of fluences (A to D). Infected cells were incubated for a period of time (usually 12 - 48 h) before harvesting and scoring for b-galactosidase activity as reported previously (Francis and Rainbow 1999). Lysates from wells infected with heavily irradiated Ad5HCMVspllacZ (10,000 J/m2), X, served as a measure of background levels for !-galactosidase activity. UV irradiation of cells and virus was as for Figures 1 and 3 and as described previously (Francis and Rainbow 1999). Enhanced HCR in UV-treated compared to non-treated cells suggests inducible repair of the UV-damaged reporter gene .

Therefore, a direct comparison of the relative contribution of TCR and GGR to repair in the transcribed strand of plasmid born reponter genes using 5V40-transformed cells and tumour cells with that obtained using an Ad based reponter in primary human fibroblasts may not be appropnate.

B. Enhanced host cell reactivation of a UV-damaged reporter gene following pretreatment of mammalian cells with DNA damaging agents 1. Evidence for inducible DNA repair. Examination of DNA repair in mammalian cells generally requires that the cells are treated with a DNA damaging agent in some manner, which makes it difficult to determine if the repair pathways are constitutively active or induced by the DNA damaging agent. In contrast, the use of viral probes for DNA repair allows the virus and cell to he treated with a DNA damaging agent independently and thus allows an examination of both constitutive and inducible pathways affecting survival of the virus or expression of the reponter gene. There are manyreponts showing that pretreatment of a variety of different mammalian cells with chemical or physical DNA damaging agents results in an increased survival (or enhanced reactivation) for several nuclear replicating

double stranded DNA viruses damaged by UV or ionising radiation (for a review see Rainbow 1981, Defais et al, 1983). It has been suggested that the enhanced reactivation of DNA-damaged viruses results, in part at least, from an induced DNA repair pathway (Jeeves and Rainbow 1983, 1983a, 1983h; Bennett and Rainbow 1988; Brown and Cenrutti 1989). We and others have examined HCR of a UV-damaged reporter gene in pre-treated compared to non-treated cells (McKay et al, 1997; Li and Ho 1998; Francis and Rainbow 1999; Boszko and Rainbow 2000). A typical protocol for these enhanced HCR experiments is shown in Figure 5 Using this approach we show that pre-treatment of normal human fibroblasts with low UV fluences (McKay et al, 1997; Francis and Rainbow 1999) as well as heat shock (McKay and Rainbow 1996; McKay et al, 1997) results in enhanced HCR of the UV-damaged reporter suggesting the presence of inducible DNA repair in human cells. Prior exposure of cells to low UV fluences or heat shock resulted in enhanced HCR for expression of the UV-damaged reporter gene in normal and XP-C fibroblast strains, but not in TCR deficient XP and CS strains (Figure 6). These results suggest that UV or heat shock treatment results in an induced repair of UVdamaged DNA in the transcribed strand of the reporter gene in

Rainbow et al: Recombinant adenoviruses as expression vectors

Figure 6. Pre-UV irradiation of cells results in enhanced HCR of UV-irradiated reporter activity in normal and XP-Cbut not in other TCR deficient cells. Results of typicalexperiments representing unirradiated (") and UV-irradiated (#) primary human fibroblasts. UV exposures to cells are indicated on the figure and cell strains presented are GM 3440 (normal), XP3BE (XP-C) CS1BE (CS-B), and XP2BI (XP-G). Immediately following UV exposure cells were infected with unirradiated orUV-irradiated virus at 10-20 plaque forming units per cell and scored for !-galactosidase activity 40-44 h later. Each point is the average of 4-6 replicates, error bars represent one standard error. Adapted ftom Francis and Rainbow 1999.

Figure 7. Pre-UV irradiation of cells results in enhanced HCR of UV-irradiated reporter activity in normal but not in Li-Fraumeni syndrome cells. Results of typical experiments representing unirradiated (") and UV-irradiated with 15 J/m2 (#) normal human fibroblasts and Li- Fraumeni syndrome (LFS) cells. Cell strains and cell lines presented are GM 9503 (normal), LFS: 087 mutlwt (heterozygous for a mutation in p53), LFS 087 mut (expressing only mutant p53). Immediately following UV exposure cells were infected with unirradiated or UV-irradiated virus at 10-20 plaque forming units per cell and scored for !-galactosidase activity 40-44 h later. Each point is the average of 3-6 replicates, error bars represent one standard error. Adapted from McKay et al, 1997 and Francis 2000.

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normal and XP-C cells through an enhancement of TCR or through a mechanism which involves the TCR pathway. More recently we have used a novel quantitative polymerase chain reaction (PCR) technique to examine direct removal of UV-induced photoproducts from lacZ reporter gene in AdHCMVsp1lacZ following infection of human fibroblasts. Using this technique we show a significant removal of UV photoproducts after infection of normal human fibroblasts, hut a reduced removal of lesions, compared to normal fibroblasts, after infection of NER deficient XP and CS fibroblasts. In addition, we show that pre-UY exposure of normal human fibroblasts results in an enhanced rate of removal of photoproducts from the reporter gene, giving evidence that UV-enhanced HCR for expression of the UVdamaged reporter gene results from enhanced removal of UV-induced lesions from DNA (Boszko 2000, Boszko and Rainbow 2000). Other evidence for damage-induced DNA repair pathways in mammalian cells comes from a number of studies including the enhanced DNA repair capacity of mammalian cells following carcinogen treatment (Protic et al, 1988), the p53-mediated enhancement of NER by the DNA damaged induced GADD45 gene (Smith et al, 1996, Smith et al, 1994) and the identification of a novel DNA repair response which is induced by irradiation of cells at the GuS border (Leadon et al, 1996). Pretreatment of normal human lung fibroblasts with the drug emodin enhances NER of UV and cisplatin damaged DNA (Chang et al, 1999) and pretreatment of cells with dinucleotides prior to UV irradiation increased the repair of UV-induced DNA damage as assessed by unscheduled DNA synthesis (Eller et al, 1997). Pre-treatment of human cells with quinacrine mustard resulted in an enhanced removal of UV-induced CPD from both the transcribed and the nontranscribed strand of the p53 gene (Ye et al, 1999), also giving evidence for an inducible NER response in human cells. Most of these studies provide evidence for an induction of the GGR rather than the TCR pathway of NER (as reviewed in McKay et al, 1999). Some mammalian cells exhibit a hypersensitivity to low doses of x-rays or cisplatin, but increased resistance following higher doses of these agents (Skov et al, 1994, Joiner et al, 1996, Caney et al, 2000). The increased radioresistance at higher x-ray doses is absent in some DNA repair deficient cell lines (Skov et al, 1994), and hypersensitivity at low doses of x-rays or cisplatin can he removed by pretreatment of cells with "priming doses" of a DNA damaging agent (Joiner et al, 1996, Caney et al, 2000), suggesting an inducible DNA repair response in mammalian cells. Pre- exposure of human cells with low "priming" doses of ionising radiation leads also to an enhanced removal of thymine glycols after higher doses (Le et al, 1998) providing evidence for an inducible repair of base damage in human cells.

2. Evidence for the involvement of p53 in NER Over the past few years it has become clear that p53 and/or p53 regulated gene products contribute to NER of UV-induced DNA damage in mammalian cells (Smith et al, 1994, 1995, 1996; Ford and Hanawalt 1995, 1997; Wang et al, 1995; McKay et al, 1997, 1997a; Ford et al, 1998; Li and Ho 1998). We have reported that UV and heat shock enhanced HCR for expression of the UVdamaged reporter gene was absent in Li-Fraumeni cells expressing mutant p53 (Figure 7, McKay et al, 1997, 1999) indicating a role for p53 in the induced DNA repair response. A similar p53 dependent enhancement in HCR for a CMV driven plasmid based and UV-damaged reporter gene has been reported in UVB-treated murine fibroblasts (Li and Ho 1998). In addition, thymine dinucleotides have been shown to induce the reactivation of a UV-damaged reporter gene under control of the SV4O early promoter, by a process which may also involve p53 (Eller et al, 1997). Furthermore, Huang et al 1998 report that transcription from a p53 driven promoter in the presence of wild-type p53 results in up regulation of both transcription and repair of a UV-damaged reporter gene, and that the enhanced DNA repair of the reporter gene is a separate and distinct activity of p53, but is dependent on p53 driven transcription. As discussed above, UV and heatshock enhanced HCR of the recombinant Ad-based and UV-damaged reporter gene are thought to reflect an induction of TCR or a repair process dependent on TCR. The absence of UV and heat-shock enhanced HCR of the UV-damaged reporter in LFS cells suggests further that either TCR or a repair process dependent on TCR requires functional p53. Hwang et al. 1999 have reported that transcription from the p48 gene, which is mutated in GGR-deficient, damage-specific DNA binding (DDB) protein deficient, XP-E cells (Hwang et al, 1998), is up regulated (in a p53dependent manner) in response to UV treatment in human cells. This provides a model for a UV-inducible GGR response in human cells which is dependent on p&8 transcription. A UV-induced increase in p48transcription would require removal of UV-induced lesions from the p48 gene and therefore be dependent on TCR as has been reported for other p53 responsive genes (McKay et al, 1999, McKay and Ljungman, 1999). Thus the UV-induced up regulation of p48 leading to enhanced GGR would be expected to be dependent on both wild-type p53 and TCR. It is thus possible that the p53 and TCR dependent UVenhanced repair of the UV-damaged reponter gene results from an up regulation of GGR in the transcribed strand of the reporter gene mediated by a UVinduced up regulation of the p48 gene product. Previous reports have also suggested that the DDB protein is responsible for the enhanced repair of UV-damaged expression vectors (Protic et al, 1989). However, some recent reports suggest that TCR also may be up regulated by a p53 dependent mechanism. Pre-treatment of human cells with low doses

Rainbow et al: Recombinant adenoviruses as expression vectors

of quinacrine mustard resulted in an enhanced rate of removal of CPD by NER (Ye et al, 1999). Although the enhanced rate of removal was greater for non-transcribed strand, an enhanced rate of removal also occurred for the transcribed strand of an exon 9 portion of the p53 gene, such that both GGR and TCR may be up regulated by pretreatment. In addition, Therrien et al, 1999 showed that the rate of repair of UV-induced CPD was reduced along both the transcribed and the non4ranscribed strands of the p53 and/or c-jun loci in Li-Fraumeni syndrome (LFS) cells expressing mutant p53 and human fibroblasts expressing the human papilloma virus (HPV) E6 oncoprotein that functionally inactivates p53. The reduction in the rate of CPD repair for the LFS cells compared to normal cells was considerably greater in the transcribed (6 fold) compared to the non-transcribed strand (3 fold) providing evidence that both TCR and GGR are dependent on wildtype p53 in UV-irradiated human cells. Our results for UV-enhanced HCR of a UV-damaged reporter gene are therefore also consistent with a model in which pretreatment of cells with UV results in an up regulation of TCR through a p53 dependent mechanism. It is possible that a p53 dependent up regulation of both GGR and TCR can contribute to UV-enhanced HCR of a UV-damaged reporter gene.

3. Gene therapy using Ad vectors expressing p53 and p53 responsive genes The p53 tumor supressor and several p53 responsive genes also play a role in arresting the cell cycle at the GI checkpoint in response to DNA damage and in inducing apoptosis in cells that have received extensive radiation damage (for a review see Hartwell and Kastan, 1994; Hinds and Weinberg, 1994). The p53 gene and other tumor suppressor genes have been found to be mutated in a variety of tumours and many of these mutations are thought to be responsible for the proliferative capacity and resistance of these cells to radiotherapy and chemotherapy. On this basis, both p53 and the p53 responsive gene p21waf1 have been proposed as gene therapy vectors to prevent replication of tumor cells. p21waf1 is a member of the family of cyclin-dependent kinase (CDK) inhibitors and plays a role in the maintenance of the cell cycle checkpoints and cell progression (Harper et al, 1993). Following infection of cells with Ad expressing a p53 transgene in vitro, the biological effects of p53 are readily detected, including the upregulation of p21wafl, an overall growth suppression, and an increased number of cells undergoing apoptosis for a variety of tumour cell lines carrying p53 mutations (Bacerietti and Graham, 1993; Liu et al, 1994; Yang et al, 1995). Furthermore, administration of p53 expressing Ad vectors has been found to be efficacious in several tumor models (Fujiwara et al, 1994; Lui et al, 1994; Yang et al, 1995). In vitro infection of a variety of tumour cells with p21waf1 recombinant Ad vectors induces a growth arrest at

the G 0/G1 checkpoint without inducing apoptosis (Eastam et al, 1995; Katayose et al, 1995; Yang et al 1995) and p21waf1 expressing Ad vectors have been reported to suppress tumour growth in vivo (Eastam et al, 1995; Yang et al, 1995). Ad constructs expressing p53 have been suggested as a means of sensitizing tumor cells to conventional radiotherapy and chemotherapy (Fugiwara et al, 1994). However, this approach may be detrimental in some situations. Down regulation of the p53 responsive GADD4S gene decreased DNA repair and sensitized cells to UVirradiation and cisplatin (Smith et al, 1996) whereas upregulation of the p53 responsive p21waf1 gene by Admediated transgene expression results in an increased resistance of cells to UV and cisplatin (McKay et al, 1998, Bulmer and Rainbow, unpublished data). Recently it has been reported that p53 expression protects against or confers sensitivity to UV-induced apoptosis depending on the timing of p53 expression relative to the genotoxic stress (McKay et al, 2000). Thus it is possible that upregulation of p53 and p53 responsive genes such as p21waf1 and GADD45 through the use of gene therapy vectors may result in the upregulation of p53 protective functions, including DNA repair, resulting in an enhanced resistance of tumor cells to radiation and chemotherapy. Furthermore, we have found that expression of p53 regulated gene products is both positively and negatively regulated by DNA damage depending on the cell type and the extent of such damage (McKay et al, 1998). Therefore, it may be difficult to predict the net effect of protective and cytotoxic functions of p53 in combined therapies.

Acknowledgements We thank Todd Bulmer, Cathy Hill, Jim Stavropoulos, Katharine Sodek, Ihor Boszko and Colleen Caney for their contributions to this work. This work was supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society.

References Addison CL, Fie MM, Kurisken D and Graham FL (1997) Corparison of the human versus murine cytomegalovirus immediate early promoters for transgene expression by admoviral vectors J Gen Virol 78,1653-1661. Bacerietti S and Graham FL (1993) Inhibition of cell proliferation by an adenovirus vector expressing the human wild type p53 protein. Int J Oncol 3, 781-788. Baaett, SF, Robbins JH, Tarone RE and Kreemer KH (1991) Evidence for defective repair of cyclobutane pyrimidine dimers with normal repair of other DNA photoproducts in a transcriptionally active gene transfected into Cockayne syndrorne cells. Mutat Res 255, 281-91. Bender K, Blattner C, Knebel A, Iordanov M, Herrrlich P and Rahmsdorf HJ (1997) UV-induced signal transduction. J Photochem Photobiol B:Biology 37, 1-17. Bennett CB and Rainbow AJ. (1998) Enhanced reactivation and nutagenesis of UV-irradiated adenovirus in normal human

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fibroblasts. Mutagenesis 3,157-164. Berezutskaya EB, Yu A, Morozov, P Raychaudhuri and Bagchi S (1997) Diferential regulation of the pocket domains of the retinoblastoma family proteins by the HPV16 E7 oncoprotein. Cell Growth Differ 8, 1277-1286. Blagosklonny MV and el-Diery WS (1996) In vitro evaluation of a p53-expressing adenovirus as an anticancer drug. Int J Cancer 67, 386-392. Blanner C, Bender K, Herrlich P and Rahmsdorf HJ (1998) Photoproducts in transcriptionally active DNA induce signal transduction to the delayed UV-responsive genes for collagenase and metallothionein. Oncogene 16,2827-2834. Boszko IP (2000) An examination of nucleotide excision repair in human cells by a novel quantitative polyinease chain reaction. M.Sc. Thesis, Department of Biology, McMaster University, Hamilton, Ontario, Canada Boszko IP and Rainbow AJ (2000) Enhanced repair of UVinduced DNA damage from a reporter gene following pretreatment of human cells with 1o,v dose UV light Radiation Research 2000: Proceedings of the Annual Meeting of the Association for Radiation Research, Bristol, April 2000, Poster Presentation Pl-16. Boulikas T (1998) Status of gene therapy in 1997: molecular mechanisms, disease targets, and clinical applications. Gene Ther Mol Biol 1, 1-172 Brown TC and Cerutti PA (1989) UV-enhanced reactivation of UV-damaged SV40 is dose to restoration of viral early gene functions. Mutat Res 2l8, 211-217. Bruening W, Giasson B, Mushynski W and Durham HD (1998) Activation of stress-activated MAP Protein kinases upregulates expression of transgenes driven by the cytomegalovirus immediate/early promoter. Nucleic Acids Res 26,486489. Cansani D, Nalman T, Teitz T and Berg P (1986) Immortalization of xeroderma pigmentosum cells by simian virus 40 DNA having a defective origin of replication. Somatic Cell Mol Genet 12,13-20. Carey C, Bulmer IT, Singh G, Lukka H and Rainbow AJ (1999) Pre-exposure of human squamous carcinoma cells to low doses of gamma-rays leads to an increased resistance to subsequent low-dose cisplatin treatment. Int J Radiat Biol 75, 963-972. Clang LC, Sheu HM, Huang YS, Tsai TR and Kun KW (1999) A novel function of emodin: enhancement of the nucleotide excision repair of UV- and cispiatin induced DNA damage in human cells. Biochemical Pharmacology 58,49-57. Defais MJ, Hanawalt IC and Sarasin A (1985) Viral probes for DNA repair. Adv Radiat Biol 268,1-37. Donahue BA, Yin S, Taylor JS, Ranes D and Hanawalt PC (1994) Transcript cleavage by RNA polymerase II arrested by a cyclobutane pyrimidine dimer in the DNA template. Proc Natl Acad Sci USA 91, 8502-6. Dorrsch-Hasler K, Keil GM, Weber F, Jasin M, Schaffner W and Koszinowski UH (1985) A long and complex enhancer activates transcription of the gene coding for the highly abundant immediate early mRNA in murine cytomegalovirus Proc Natl Acad Sci USA 82,8325-8329. Drapkin R, Reardon IT, Ansari A, Huang JC, Zawel L, Ann K, Sancar A and Reinberg D (1994) Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II. Nature 368,769-72. Eastham JA, Hall SL, Sehgal I, Wang J, Timme TLh, Yang G, ConnellCrowley L, Elledge SJ, Zhang W-W, Harper JW and Thompson TC (1995) In vivo gene therapy with p53 or p21

adenovirus prostate cancer. Cancer Res 55, 5151-5155. el-Deiry WS (1998) Regulation of p53 downstream genes. Semin Cancer Biol 8, 345-357. Eller MS, Maeda T, Magnoni C, Atwal D and Gilchrest BA (1997) Enhancement of DNA repair in human skin cells by thymidine dinucleotides: evidence for a p53-mediated mammalian SOS response. Proc Natl Acad Sci USA 94, 12627-32. Evans MK, Robbins JH, Ganges M., Tarone RE, Naim RS and Bohr VA (1993) Gene-specific DNA in xeroderma pigmentosum complementation groups A, C, D, and F. Relation to cellular survival and clinical features. J Biol Chem 268, 483947. Fishel R, Lescoe MK, Ran MRS, Copeland NG, Jenkins NA, Garber J, Kane M and Kolodner R (1993) The human mutator gene homologuMSH2 and its association with hereditary nonpolyposis colon cancer. Cell 75,1027-1038. Ford JM and Hanawalt PC (1995) Li-Fraurneni syndrome fibroblasts homozygous for p53 mutation are deficient in global DNA repair but exhibit normal transcription coupled repair and enhanced UV resistance, Proc Natl Acad Sci USA 92, 8876-8880. Ford JM and Hanawalt PC (1997) Expression of wild-type p53 is required ior efficient global nucleotide excision repair in UV-irradiated human fibroblasts. J Biol Chem 272, 2807328080. Ford JM, Baron EL and Hanawalt PC (1998) Human fibroblasts expressing the human papillomavirus E6 gene are deficient in global genomic nucleotide excision repair and sensitive to ultraviolet irradiation. Cancer Res 58, 599~O3 Francis M (2000) Characterisation of DNA damage inducible issponses and repair in human cells using recombinant adenovirus vectors. Ph.D. Thesis, Department of Biology, McMaster University, Hamilton, Ontario, Canada Francis MA and Rainbow AJ (1997) UV-enhanced expression of a reporter gene is induced at lower UV fluences in transcription-coupled repair deficient compared with normal human cells and is absent in SV4-transformed human cells. Photochem Photobiol 655 (Abstr. ThAM-E5), 1028. Francis MA and Rainbow AJ (1999) UV-enhanced reactivation of a UV-damaged reporter gene suggesting transcriptioncoupled repair is UV-inducible in human cells. Carcinogenesis 20, 19-26. Francis MA and Rainbow AJ (1999a) Role of the retinoblastoma (pRb) and p53 tumour suppressor proteins in UV-induced expression of a reporter gene from the human cytomegalovirus imnediate early promoter. Proceedings of the 11th International Congress of Radiation Research, Dublin, Ireland. Abst GE/14 pp.116 Francis MA and Rainbow AJ (2000) UV-enhanced expression of a reporter gene is induced at lower UV fluences in transcription-coupled repair deficient compared to normal human fibroblasts, and is absent in SV40-transformed counter parts. Photochem Photobiol 72, 554-561 Friedberg EC, Waler GC and Siede W (1995) DNA repair and mutagenesis, ASM Press, Washington, D.C. Fugiwara T, Grimm FA, Mukhopadhyay T, Zang W-W, OwenSchaub IB and Roth JA (1994) Induction of chemosensitivity in human lung cancer cells in vivo by adenovirus-mediated transfer of the wild-type p53 gene. Cancer Res 54, 22872291. Ganesan AK and Hanawalt PC (1994) Removal of cyclobutane pyrimidine dime from a UV-irradiated shuttle vector introduced into human cells. Somatic Cell Mol Genet 20,

Rainbow et al: Recombinant adenoviruses as expression vectors

233-42. Ganesan AK, Hunt J and Hanawalt PC (1999) Expression and nucleotide excision repair of a UV-irradiated reporter gene in unirradiated human cells. Mutat Res 433, 117-26. Graham FL and Prevec L (1991) Manipulation of AdenoviruS Vectors. In Murray, EJ. (ed.), Methods in Molecular Biology: Gene Transfer and Expression Protocols. The Humana Psess Inc., Clifton, NJ, Vol 7, pp. 109-128. Haper JW, Adarni GR, Wei N, Keyomarsi K and Elledge SJ (1993) The p2l cdk-interacting Protein Cip 1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75,805-816. Hartwell LH and Kastan MB (1994) Cell cycle control and cancer. Science 266, 1821-1828. Henning KA, Li L, Iyer N, McDaniel LD, Reagan MS, Legerski R, Schultz RA, Stefanini M, Lechmann AR, Mayne LV and Friedberg EC (1995) The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH. Cell 82, 555-64. Herrlich P, Sachsenmaier C, Radler-PohI A, Gebel S, Blattrer C and Rahmsdorf HJ (1994) The mammalian UV response: mechanism of DNA damage induced gene expression. Adv Enzyme Regul 34, 381-395. Hinds PW and Weinberg RA (1994) Tumour suppressor genes. Curr Opin Genet Dev 4, 135-141. Hit M, Bet Al, Addison C, Prevec L and Graham FL (1995) Techniques for human adenovirus vector construcion and characterization, in K Adolph (Ed), Meth Mol Genet 7, 1330. Hit MM, Addison CL and Graharn FL (1997) Human adenovirus vectors for gene transfer into mammalian cells. Adv Pharnac 40, 137-206. Hoeijmakers JH, Odijk H and Westerveld A (1987) Differences between rodent and human cell lines in the amount of intregrated DNA after transfection. Exp Cell Res 169, 111119. Hollstein M, Sidranski D, Vogelstein B and Harris CC (1991) ps3 mutations in human cancers. Science 253, 49-53. Huang J, Logsdon N, Schmieg H and Simmons DT (1998) p53mediated transcription induces resistance of DNA to UV inactivation. Oncogene 17, 401-411 Hwang BJ, Ford JM, Hanawalt PC and Chu G (1999) Expression of the p48 xeroderma pigmentosurn gene is p53-dependent and involved in global genomic repair. Proc Natl Acad Sci USA 96, 424-428. Hwang BL Toering S, FrancIse U and Chu G (1998) p48activates a UV-damaged-DNA binding factor and is defective in xeroderma pigmentosurn group E cells that lack binding activity. Mol Cell Biol 18,4391-4399. Iyer N, Reagan MS, Wu KJ, Canagarajah B and Friedherg BC (1996) Interactions involving the human RNA polymerase II transcription/nucleotide excision repair complex TFIIH, the nucleotide excision repair protein XPG, and Cockayne syndrome group B (CSB) protein. Biochemistry 35, 215767. Jeeves WP ad Rainbow AJ (1983) Gamma-ray-enhanced reactivation irradiated adenovirus xeroderma pigmentosurn and Cockayne syndrome fibroblasts. Radiat Res 94, 480498 Jeeves WP and Rainbow AJ (1983a) Uvenhanced reactivation of UV and gamma-irradiated adenovirus in normal human fibroblasts. Int J Radiat Biol 43, 599-623. Jeeves WP and Rainbow AJ (1983b) UV-enhanced reactivation of UVand gamma-irradiated adenovirus in Cockayne syndrome and xeroderma pigmentosurn fibroblasts. Int J Radiat Biol 73, 625-47.

Joiner MC, Lambin P, Malaise EP, Rohson T, Arrand JE, Skov K and Marples B. (1996) Hypersensitivity to very-low single radiation doses: its relationship to the adaptive response and induced radioresistance. Mutat Res 358, 171-83. Joki T, Nakamura M and Ohno T (1995) Activation of the EGR1 promoter induces expression of the herpes simplex virus thymidine kinase gene and sensitivity of human glioma cells to ganciclovir. Hum Gene Ther 6, 1507-1513. Kataynse D, Wersto R, Cowan KH and Seth P (1995) Bifiants of a recomlisnant adenovirus expressing WAF1/Cip1 on cell growth cell cycle, and apoptosis. Cell Growth Differ 6, 1207-1212. Kolls J, Beppel K, Silva M and Beutler B (1994) Prolonged and effective blockage of tumor necrosis activity through adenovirus mediated gene transfer. Proc Natl Acad Sci USA 91, 215-219. Klocker H, Schneider R, Burtscher AJ, Auer B, HirschKauffmann M and Schweiger M (1985) Transient expression of a plasmid gene a tool to study DNA repair in human cells: defect of DNA repair in Cockayne Syndrome; one thymine cyclobutane dimer is sufficient to block transcription. EurJ Cell Biol 39, 346-351. Kraemer KH, Levy DD, Parris CN, Gozokara EM, Moriwaki S, Adelberg S and Seidman MM (1994) xeroderma pigmentosurn and related disorders examining the linkage between defective DNA repair and cancer. J Inv Dermatol 103, 965-lOIS. Lakin ND and Jackson SP (1999) Regulation of p53 in response to DNA damage. Oncogene 18, 7644-7655. Le XC, Xing JZ, Lee J, Leadon SA and Weinfeld M (1998) lnductible repair of thymine glycols by an ultrasensitive assay for DNA damage. Science 280, 1066-1069. Leadon SA, Dunn AB and Ross CE (1996) A novel DNA repair response is induced in human cells exposed to ionizing radiation at the G1/S-phase border. Radait Res 146, 123-30. Lehmann AR and Oomen A (1985) Effect of DNA damage on the expression of the chloramphenirol acetyltransferase gene after transduction into diploid human fibroblasts. Nucleic Acids Res 13, 2087-95. Li G and Ho VC (1998) p53-dependent DNA repair and apoptosis respond differently to high and low dose ultraviolet radiation. Br J Dermatol 139, 3-10. Liu T-J, Zhang W-W, Taylor DL, Roth JA, Goepfert H and Clayman GL (1994) Growth suppression of human bead and neck cancer by introduction of a wild-type p53 gene via a recombinant adenovirus. Cancer Res 54, 3662-3667. Ljungman M and Zhang F (1996) Blockage of RNA polymerase as a possible trigger for UV. light- induced apoptosis. Oncogene 13,823-31. Ljungman M, Zhang F, Chen F, Rainbow AJ and McKay BC (1999) Inhibition of RNA polymerase II as a trigger for the p53 response. Oncogene 18, 583-592. Malkin D, Li FP, Strong LC, Fraumeni Jr. JF, Nelson CE, Kim DH, Kassel J, Gryka MA, Bischoff FZ, and Tainsky MA (1990) Germ line p53 mutations in a familiar syndrone of breast cancer, sarcomas, and other neoplasms. Science 250, 1233-1238. Marples B, Scott SD, Hendry JH, Embleton MI, Lashford LS and Margison GP (2000) Development of synthetic promoters for radiation-mediated gene therapy. Gene Ther 7, 511-517. Masutani C, Sugasawa K, Yanagisawa J, Sonoyama T, Ui M, Enonoto T, Takio K, Tanaka K, van der Spek PJ, Bootsma D, Hoeijmakers JHJ and Hanaoka F (1994) Purification and

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cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23. Embo J I3, 1831-43. Massuda T, Saijo M, Kuraoka I, Kobayashi T, Nakatsu Y, Nagai A, Enjoji T, Masutani C, Sugasawa K Hanaoka F, Yasui A and Tanaka K (1995) DNA repair protein XPA binds replication protein A (RPA). J Biol Chem, 270, 4152-7. Mauceri HJ, Nader N, Wayne JD, Hallahan DE, Hellway S and Weichselbaum RR (1996) Tumor necrosis factor alpha (TNF-#) gene therapy targeted by ionizing radiation selectively damages tumor vasculature. Cancer Res 56, 43114314. Mayne LV and Lehmann AR (1982) Failure of RNA synthesis to recover after UV irradiation. an early defect in cells from individuals with Cockayne's syndrome and xeroderma pigmentosum. Cancer Res 42, 1473-8 McKay BC (1997) The relationship between repair of ultraviolet light induced DNA damage in human cells and the p53 tumour suppresor. PhD. Thesis Department of Biology, McMaster University, Hamilton, Ontario, Canada McKay BC and Rainbow AJ (1996) Heat-shock enhanced reactivation of an UV-damaged reporter gene in human cells involves the transcription coupled DNA repair pathway. Mutat Res 363, 125-35. McKay BC and Ljungman M (1999) Role for p53 in the recovery of transcription arid protection against apoptosis induced by ultraviolet light. Neoplasia 1, 276-284. McKay BC, Francis MA and Rainbow AJ (1997) Wildtype p53 is tequired for heat shock and ultaviolet light enhanced repair of a UV-damaged reporter gene. Carcinogenesis 18, 245-9. McKay BC, Winrow C and Rainbow AJ (1997a) Capacity of UV irradiated human fibroblasts to support adenovirus DNA synthesis correlates with transcription coupled repair and is reduced in SV40 transformed cells and cells expressing mutant p53. Photochem Photobiol 66, 659- 664. McKay BC, Ljungman M and Rainbow AJ (1998) Persistent DNA damage induced by ultraviolet light inhibits p21wafl and bax expression. implications for DNA repair, UV sensitivity and the induction of apoptosis. Oncogene 17, 545-555. McKay BC, Ljungman M and Rainbow AJ (1999) Potential roles for p53 in nucleotide excision repair. Carcinogenesis 20, 1389-1396. McKay BC. Chen F, Perumalswami CR, Zhang F and Ljungman M (2000) The tumor suppressor p53 can both stimulate and inhibit UV-light induced apoptosis. Mol Biol Cell 11, inpress. Mellon I, Bohr VA, Smith CA and Hanawalt PC (1986) Preferential DNA repair of an active gene in human cells. Proc Natl Acad Sci USA 83, 8878-8882. Mellon I, Spivak G and Hanawalt PC (1987) Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell 51, 241-249. Mellon I, Rajpal DK, Koi M, Boland CR and Champe GN (1996) Transcription-coupled repair deficiency and mutations in human mismatch repair gene. Science 272, 557560 Mietz JA, Unger T, Huibregtse JM and Howley PM (1992) The transcriptional transactivation function of wild-type p43 is inhibited by SV40 large T-antigen and by HFV-16 E6 oncoprotein. EMBO J 11, 5013-5020. Miskin R and Ben-Ishai R (1981) Induction of plasminogen activator by UV light in normal and xeroderma pigmentosum fibroblasts. Proc Natl Acad Sci USA 78, 6236-6240.

Moorehead RA, Armstrong SA, Rainbow AJ and Singh G (1996) Nucleotide excision repair in the human ovarian carcinoma cell line (2008) and its cisplatin resistant variant (CI 3) Cancer Chemo Pharm 38, 245-253. Morsy MA, Alford EL, Bett A, Graham FL and Caskey CT (1993) Efficient adenoviral-mediated ornithine transcarbamylase expression in deficient mouse and human hepatocytes. J Clin Invst 92, 1580-6. Mumane W, Fuller LF and Painter RB (1985) Establishment and characterization of a permanent SV ori-transformed ataxia telangiectsia cell line. Exp Cell Res l58, 119-126. Prak CH and Sancar A (1994) Formation of a ternary complex by human XPA, ERCC1, and ERCC4(XPF) excision repair proteins. Proc Natl Acad Sci USA 91, 5017-21. Pedley J, Ablett EM, Pettit A, Meyer L Dunn IS, Sturm RA and Parsons PG (1996) Inhibition of retinoblastoma protein translation by UVB in human melanocytic cells and reduced cell cycle arrest following repeated irradiation. Oncogene 13,1335-1342. Protic-Sabljic M and Kraemer KH (1985) One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells. Proc Natl Acad Sci USA 82, 6622-6. Protic M, Roilides E, Levine AS and Dixon K (1988) Enhancment of DNA repair capacity of mammalian cells by carcinogen treatment. Somat Cell Mol Genet 14, 351-7. Protic M, Hirschfeld S, Tsang AP, Wagner M, Dixon K and Levine AS (1989) Induction of a novel damage-specific DNA binding protein correlates with enhances DNA rapair in primate cells. Mol Toxic 2, 255-270. Rainbow AJ (1981) Reactivation of viruses Short Term Tests for Chemical Carcinogens. HF. Stich and RHC San (Eds) SpringerVerlag, New York, pp 20-35 Rainbow AJ (1989) Defective repair of UV-damaged DNA in human tumor and SV40-transformed human cells but not in adenovirus-transformed human cells. Carcinogenesis 10, 1073-1077. Renzing J and Lane DP (1995) p53dependent growth arrest following calcium phosphate-mediated transfection of murine fibroblasts. Oncogene 10, 1865-1868. Schaeffer L, Roy R, Humbert S, Moncollin V, Venmeulen W, Hoeijmakers JH, Chambon P and Egly JM (1993) DNA repair helicase: a component of BTF2 (THIIH) basic transcription factor. Science 260, 58-63. Schaeffer L, Moncollin V, Roy R, Staub A, Mezzina M, Sarasin A, Weeda G, Hoeijmakers JH and Egly JM (1994) The ERCC2-DNA rapair protein is associated with the class II BTF2/THIIH transcription factor EMBO J 13, 2388-92. Schaeffner M, Werness BA, Huibregtse JM, Levine AJ and Howley PM (1990) The E6 oncoprotein encoded by human paplllomavirus types 16 and 18 promotes the degradation of p53. Cell 63, 1129-1136. Scott SD, Marples B, Hendry JH, Lashford 15, Embleton Mt, Hunter RD, Howell A and Marison GP (2000) A radiation controlled molecular switch for use in gene therapy of cancer. Gene Therapy 7, 1121-1125. Seedorf K, Oltersdorf T, Krammer G and Rowekamp W (1987) Identification of early proteins of the human papilloma vinuses type 16 (HPV 16) and type 18 (HPV 18) in cervical carcinoma cells. EMBO J 6, 139-144 Segawa K, Minowa A, Sugasawa K, Takano T and Hanaoka F (1993) Abrogation of p53-mediatrd transactivation by SV40 large T antigen. Oncogene 8, 543-548. Seung LP est al (1995) Genetic radiotherapy overcomes tumor resistance to cytotoxic agents. Cancer Res 55, 5561-5565.

Rainbow et al: Recombinant adenoviruses as expression vectors

Siegel J, Fritshe M, Mai S, Brandner G and Hess RD (1995) Enhanced p53 activity and accumulation in response to DNA damage upon transfection. Ongogene 11, 1362-1370. Skov K, Marples B, Matthews JB, Joiner M. and Thou H (1994) A preliminary investigation into the extent of increased radioresistance or hyper-radiosensitivity in cells of hamster cell lines known to he deficient DNA repair. Radiat Res 138, S1269. Smith M., Chen IT, Zhan Q, Bae I, Chen CY, Gilmer TM, Kastan MB, O'Connor PM and Formace Jr AJ (1994) Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science 266, 137680. Smith ML, Chen IT, Zhan Q, O'Cornor PM and Formace Jr AJ (1995) Involvement of the p53 tumor suppressor in repair of UV-type DNA damage, Oncogene 10, 1053-9. Smith ML, Kontny HU, Zhan Q, Sreenath A, O'Connor PM and Formace Jr AJ (1999) Antisense Gadd45 expression results in decreased DNA repair and sensitizes cells to UVirradiation or cisplatin. Oncogene 1, 2255-2263 Squires S, Johnson RT and Collins AR (1982) Initial rates of DNA incision in UV-irradiated human cells: differences between normal, xeroderma pigmentosum and tumour cells Mutat Res 95, 389404 Stackhouse MA and Buschsbaum DJ (2000) Radiation to control gene expression. Gene Ther 7, 1085-1086. Stevsner T, Fnandsen H and Autrup H (1995) Repair of DNA lesions induced by ultraviolet irradiation and aromatic amines in normal and pair-deficient lymphoblastoid cell lines. Carcinogenesis 16, 2855-2858 Stewan AK, Lassam NJ, Quirt IC, Balley DJ, Rotsein LE, Krajden M, Dessureault S, Gallinger S, Cappe D, Wan Y, Addison CL, Moen RC, Gauldie J and Graham FL (1999) Adenovector-mediated gene delivery of interleukin-2 in metastatic breast cancer and melanoma: results of a phase I clinical trial. Gene Ther 6, 350-363 Takahashi et al (1997) Induction of the suiside HSV TK gene by activation of the Egr 1 promoter with radioisotopes. Hum Gene Ther 8, 827-833. Tang DC, Jennelle RS, Sni Z, Garver Jr RI, Carbone DP, Loya F, Chang CH and Curiel DT (1997) Overexpression of adenovirus-encoded transgenes from the cytomegalovirus immediate early promoter in irradiated tumor cells. Hum Gene Ther 2l, 17-2124. Therrien JP, Drouin R, Baril C and Drobetsky EA (1999) Human cells compromised for p53 function exhibit defective global and transcription-coupled nucleotide excision repair, whereas cells compromised for Rb function are defective only in global repair. Proc Natl Acad Sci USA 96, 1503815043. Valerie K and Singhal A (1995) Host cell reactivation of reporter genes introduced into cells by adenovirus as a convenient way to measure cellular DNA repair. Mutat Res 336, 91100. van Hoffen A, Naarajan AT, Mayne LV, van Zeeland AA, Mullenders LH and Venema J (1993) Deficient repair of the transcribed strand of active genes m Cockayne's syndrome cells. Nucleic Acids Res 21, 5890-5895. Venema J, van Hoflen A, Naarajan AT, van Zeeland AA and Mullenders LH (1990) The residual repair capacity of in xeroderma pigmentosum complementation group C fibrobasts is highly specific for transcriptionally active DNA. Nucleic Acids Res 18,443448. Venema J, van Hoflen A, V Karcagi V, Naarajan AT, van Zeeland AA and Mullenders LH (1991) Xeroderma

pigmentosum complementation group C cells remove Pyrimidine dimers selectively from the transcribed strand of active genes. Mol Cell Biol 11,4128 4134. Venema J, Mullenders LH, Naarajan AT, van Zeeland AA and Mayne LV (1990a) The genetic defect in Cockayne syndrome is associated with a defect in repair of UV-induced DNA damage in transcriptionally active DNA. Proc Natl Acad Sci USA 87,47074711. Wang XW, Yeh H, Schaeffer L, Roy R, Moncollin V, Egly IM, Wang Z Freidberg BC, Evans MK, Taffe HG, Bohr VA, Weeda G, Hoeijmakers MU, Forrester K and Harris CC (1995) p53 modulation of TFIIH-associated nucleotide excision repair activity. Nature Genet 10, 188-95. Wei Q, Matanoski GM, Farner ER, Hedayati MA and Grossman L (1993) DNA repair and aging in basal cell carcinoma: a molecular epidemiology study. Proc Natl Acad Sci USA 90, l6l~l6l8. Wood CM, Timme TL, Hurt MM, Brinkley BR, Ledbetter DH and Moses ER (1987) Transformation of DNA-repair deficient human diploid fibroblasts with a simian virus 40 plasmid. Exp Cell Res 169,543-553 Xu ZZ, Krougliac V, Ptevec J, Graham FL and Both GW (1995) Investigations of promotet function in human and animal cells with human recombinant adenovirus expressing rotavirus antigen VP7s. J Gen Virol 76, 1971-1980 Yamaizumi M and Sugano T (1994) UV-induced nuclear accumulation of p53 is evoked through DNA damage of actively transcribed genes independent of the cell cycle. Oncogene 9, 2775-84 Yang C, Cinell C, Capogossi MC and Passaniti A (1995) Menovirus-mediated wild type p53 expression induced apoptosis and supresses tumorigenesis of prostate tumor cells. Cancer Res 55,42104213 Ye N, Bianchi MS, Bianchi NO and Hoiroquist GP (1999) Adaptive enhancement and kinetics of nucleotide excision repair in humans. Mutat Res 435, 43-61. Yin Y, Tainslty MA, Bischoff FZ, Strong LC and Wahl GM (1992) Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutantp 53 alleles. Cell 70, 937-948.

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Gene Ther Mol Biol Vol 5, 101-110, 2000

Chromatin remodeling and developmental gene regulation by thyroid hormone receptor Review Article

Laurent M. Sachs1, Peter L. Jones2, Victor Shaochung Hsia2, and Yun-Bo Shi2,3 1

Laboratoire de Physiologie, MNHN, UMR CNRS 8572, PARIS cedex 05, FRANCE, and Unit on Molecular Morphogenesis, Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, NIH, Bethesda, MD USA _________________________________________________________________________________________________ 2

* Correspondence: Yun-Bo Shi, Building 18T, Rm. 106, NICHD, NIH, Bethesda MD, 20892; Tel: (301)-402-1004; Fax: (301)-4021323; E-mail: Shi@helix.nih.gov Key Words: Xenopus laevis, Amphibian metamorphosis, histone acetylation, chromatin remodeling, thyroid hormone receptor

Received: 15 November 2000; accepted: 21 November 2000

Summary Thyroid horruone (TH) receptors (TRs) are dual function transcription factors. They activate or repress transcription in the presence or absence of TH, respectively. Using the Xenopus laevis oocyte as an in vivo system to assemble TH target promoters into chromatin under conditions mimicking somatic cells, we have shown that transcriptional repression by unilganded TR involves histone deacetylase while transcriptional activation by THbound TR leads to chromatin disruption. Using Xenopus laevis development as a developmental model, we have demonstrated that TR is constitutively bound to its target genes in chromatin. Transcriptional activation induced by TH is accompanied by the release of at least one histone deacetylase and increase in local histone acetylation. These studies together with the developmental expression profiles of TR genes suggest that TH-induced changes in chromatin remodeling play an important role in the dual functions of TR in frog development: gene repression in premetamorphic tadpoles when TH is absent and gene activadon during metamorphosis, a process induced by the endogenously synthesized TH.

I. Introduction Thyroid hormone (TH) plays important roles during development (Shi, 1999). In humans, TH tectable in the embryonic plasma by 6 months rises to high levels around birth (Tata, 1997). is postembryonic period, extensive tissue and organogenesis take place. TH deficiency during human development leads to developmental, such as mental retardation, short stature, and in the e form, cretinism (Hetzel, 1989; Shi, l999).Likewise, TH is critical for amphibian development. In fact, anurans depend upon TH to develop into adult, frogs (Dodd and Dodd, 1976; Shi, 1999). endogenous synthesis of TH leads to the f giant tadpoles that cannot metamorphose ion of exogenous TH to premetamorphic tadpoles causes precocious metamorphosis. Furthermore, most, if not all, organs are genetically predetermined to undergo specific changes and these changes are organ autonomous. Such properties have

made anuran metamorphosis one of the best-studied postemhryonic developmental process at morphological, cellular, and biochemical levels and paved way for current molecular investigations of the underlying mechanisms. Here we summarize some recent advances from studies in Xenopus laevis.

II. Chromatin remodeling by TRs The biological effects of TH are mostly, if not entirely, mediated by thyroid hormone receptors (TRs). TRs belong to the superfamily of nuclear hormone receptors, with two subfamilies of TRs in vertebrates, TR! and TR". TR can be divided roughly into 5 domains, A/B, C, D, E, and F, respectively, from the amino- to carboxyl-terminus (Krust et al, 1986). The DNA binding domain (domain C) is located in the amino half of the

Sachs et al: Gene repression and activation by TRs

protein and is the most highly conserved domain among different receptors of the supeifamily. The large ligand binding domain (domain F) is in the carboxyl half of the protein and is conserved among TRs in different species. The other domains vary in sizes and sequences among different nuclear receptors. The N-terminal A/B domain is highly variable in sequence and length, the shortest being the TRs in Xenopus laevis (Yaoita et al, 1990). At least in some TRs, this domain contains a transactivation function (AF), although its role in amphibian TRs is unclear. Another transactivation function domain is the AF-2 domain, which is located at the very C-terminus (F domain and part of the E domain). TH can both up- and down-regulate gene expression in target tissues or cells. The vast majority of the known TH response genes are up-regulated by the hormone and most studies of receptor function have been on these upregulated genes. The discussions here focus only on the mechanisms for this class of genes. Transcriptional activation by TH requires the binding of TRs, most likely as heterodimers with RXRs (9-cis retinoic acid receptors), to TREs (TII response elements) present in the regulatory regions of the TH-response genes. The binding of TREs by TR/RXR heterodimers is, however, independent of TH both in solution and in chromatin (Perlman et al, 1982; Wong et al, 1995). In the absence of TH, TR/RXR represses transcription of target promoters, while in the presence of TH, TR/RXR enhances transcription from these same promoters (Fondell et al, 1993; Tsai and O'Malley, 1994; Wong et al, 1995).

chromatin conditions. Xenopus oncytes have little endogenous TR to affect the transcription of a TREcontaining promoter (Wong and Shi, 1995). However, when exogenous Xenopus TRs and RXRs are cointroduced into the oocytes by injecting their mRNA into the cytoplasm, they can repress the transcription from both single-stranded and double-stranded DNA containing a TRE (Figure 1) (Wong and Shi, 1995; Wong et al, 1995; Hsia et al, 2000). On the other hand, maximal regulation by TH occurs when the single-stranded DNA is used. This is mainly due to more effective repression of the promoter by unliganded TR/RXR during replication-coupled chromatin assembly process (Wong et al, 1995). We have used two independent assays to investigate the effects of TR/RXR on chromatin structure (Wong et al, 1997a). These are the plasmid DNA supercoiling assay for measuring nucleosomal density andlor DNA wrapping conformation in the plasmid minichromosome, and the micrococcal nuclease digestion assay for determining the nucleosomal array stmcture of the plasmid minichromosome. Both assays have shown that the binding of TR/RXR alone deacetyla has little effect on the gross chromatin structure. On the by unlig other hand, the addition of TH to TR/RXR-containing (Figure 1), templates causes the disruption of the ordered chromatin. Furthermore, this chromatin disruption occurs even when transcription elongation is blocked. Thus, TH-bound leads TR/RXR heterodimers can disrupt chromatin structure through an active process, although the nature of the disruption is yet unclear.

A. Chromatin disruption by liganded TR/RXR Most of the functional studies of hormone receptors have been carried out in vitro or by transient transfection experiments in tissue culture cells. However, genomic DNA in eukaiyotic cells is associated with histones and other nuclear proteins and assembled into chromatin. Thus, to understand the mechanism of TR action, it is important to use properly chromatinized templates. We have made use of the ability of Xenopus oocyte to assemble exogenous DNA into chromatin (Almouzni et al, 1990) to investigate the mechanism of TR action. When single-stranded plasmid DNA is injected into a frog oocyte nucleus, it is quickly replicated (1-2 hr) and assembled into chromatin in a replication-coupled chromatin assembly pathway, mimicking the chromatin assembly process in somatic cells. The resulting template often produces low level of transcriptional activity. In contrast, when double-stranded promoter-containing plasmid DNA is injected into an oocyte nucleus, it is chromatinized more slowly (5-6 hr) with less well defined nucleosome arrays such that the transcnption from the promoter is often at high levels, Thus, by using different forms of promoter-containing plasmid DNA, it is possible to study the transcriptional regulation under different

Figure 1. Histone deacetylases is involved in transcnptional repression by TR. A double-stranded plasmid (pHL10) containing HIV- 1 promoter, which is regulated by TH (Hsia et al, 2000), was microinjected into frog oocytes with or without prior injection of TR"/RXR! mRNAs. The injected oncytes were treated with or without 5 ng/ml TSA or 50 nM T3 as indicated and the promoter activity was analyzed by primer extension. Note that the addition of TSA activated the promoter slightly. The presence of unliganded TR/RXR repressed the promoter activity. The addition of either T3 or TSA reversed the inhibition and further activated the promoter, supporting a role of histone deacetylase in the repression by unliganded TR/RXR. The plasmid pCMV-CAT containing a cytomegatovirus promoter driving the expression of CAT reporter gene was used as an internal control (Kass et al, 1997).

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B. Regulation of histone acetylation levels through histone acetyltransferases and deacetylases Both transcriptional repression by unliganded TRs and activation by TH-bound TRs involve TR-interacting cofactors (Chen and Li, 1998; McKenna et al, 1999; Xu et al, 1999; Rachez and Freedman, 2000). Many such factors have been isolated based on their ability to interact c with TRs in the presence or absence of T3 or under both conditions. The corepressors bind preferentially or exclusively to unliganded TR while the coactivators generally require TH for binding to TR. Interestingly, the corepressors appear to form multimeric complexes containing histone deacetylases while many coactivators themselves are histone acetyltransferases or acetylases (McKenna et al, 1999; Xu et al, 1999; Burke and Baniabmad, 2000; Hu and Lazar, 2000; Urnov et al, 2000). Our studies have suggested the existence of multiple corepressor complexes, both with and without histone deacetylase activity, in the frog oocyte (Jones et al, unpublished data). This raises the possibility that histone acetylation status may play a role ii transcriptional regulation by TR/RXR. Histone acetylation has long been implicated influence gene expression (Allfrey et al, 1964; Wolffe 1986; Struhl, 1998). Histone acetylation occurs at lysine residues on the amino-terminal tails of the histor leading to the neutralization of the positive charges histone tails and reduced affinity toward DNA (Hon al., 1993). Although we have failed to detect any gross changes in chromatin structure under conditions exp to alter histone acetylation levels of plasmid minichromosome (Wong et al, 1998), alteratic histone acetylation levels will likely chang nucleosomal conformation and chromatin access thus influencing transcription. Indeed, our studies in the oocyte have provided evidence for a role of histone acetylation in promoter activation (Figure 1) (Wong et al, 1998; Hsia et al, 2000) First, addition of a specific inhibitor of deacetylase, TSA (trichostatin A), can reverse the repression by unliganded TR/RXR, mimicking the addition of TH (Figure 1), indicating the involvement of histone deacetylase in the repression by TR/RXR. Conversely, overexpression of the catalytic subunit of a frog histone deacetylase complex (Rpd3) leads to transcriptional repression of a THinducible promoter. This deacetylase-induced repression can be reversed by either TR/RXR in the presence of TH or TSA (Wong et al, 1998).

C. A model for gene regulation by TR/RXR Although the studies so far are supportive of an important role for histone acetylation in transcriptional activation, other pathways are likely involved. First, we have shown that transcriptional activation by liganded TR/RXR leads to chromatin disruption but over-

expression or blocking the function of histone deacetylases has no such effect despite dramatic influences on transcription. In addition, many cofactors can interact with the transcriptional machinery directly (Burke and Baniahmad, 2000; Hu and Lazar, 2000; Rachez and Freedman, 2000). Finally, at least one coactivator complex, the DRIP/TRAP complex, has no histone acetyltransferase activity but can activate transcription from chromatin templates (Rachez and Freedman, 2000). Thus, transcriptional regulation by TR/RXR is likely to involve a complex, multi-step, multicomponent process. A potential model for TR/RXR function is outlined in Figure 2. Tn the absence of TH, TR/RXR recruits a corepressor and its associated deacetylase complex to the promoter, leading to histone deacetylation and transcriptional repression. Upon TH binding, the corepressor complex is dissociated and one or more coactivator complexes are recruited to the promoter. This recruitment may lead to increased histone acetylation (Utley et al, 1998; Sachs and Shi, 2000), chromatin disruption, and transcriptional activation.

III. Dual function of TRS in frog development Four TR genes, two TR! and two TR" genes, are present in Xenopus laevis (Figure 3) (Yaoita et al, 1990). The total dependence of anuran metamorphosis on TR offers an opportunity to study TR/RXR function during development. Expectedly, both TR! and TR" genes are highly expressed luring metamorphosis in Xenopus (Yaoita and Brown, 1990; Shi, 1999). In addition, RXR genes are also expressed during metamorphosis (Wong and Shi, 1995). More importantly, the expression of both TR and RXR genes correlates temporally w metamorphosis of individual organs. Thus, high levels of both TR and RXR mRNAs are present in the limb during early sages of metamorphosis (Stage 54-58) when limb morphogenesis takes place. Subsequently as limb undergoes growth with little morphological changes, both TR and RXR genes are down regulated. On the other hand, both TR and RXR genes are upregulated toward the end of metamorphosis (after stage 60), which corresponds to the period of tail resorption. Such correlation argues that TR/RXR heterodimers are indeed the mediators of the controlling effects of TII on metamorphosis in all organs (Shi et al, 1996). Interestingly, TR! and TR" genes are differentially regulated dunng development (Figure 3) (Yaoita and Brown, 1990). The TR! genes have little expression prior to metamorphosis and are themselves direct TH-response genes (Ranjan et al, 1994; Machuca et al, 1995). Their expression is upregulated by the rising concentration of endogenous TH during metamorphosis (Figure 3). In contrast, the TR! genes are activated shortly after the completion of embryogenesis and their mRNAs reach high levels by stage 45 when tadpole feeding begins (Figure 3)

Sachs et al: Gene repression and activation by TRs

Figure 2. A model for transcriptional regulation by TRs. TR functions as a heterodimer with RXR. In the absence of TH, the heterodimer represses gene transcription through the recruitment of a corepressor complex containing the corepressor such as N-CoR, Sin3A and histone deacetylase such as Rpd3. This leads to histone deacetylation and transcriptional repression. When TH is present, the corepressor complex is released and a coactivator complex containing coactivators such as SRC-l, CBP/p300, and P/CAF, and/or the DRIP/TRAP coactivator complex is recruited. The DRIP/TRAP complex may contact RNA polymerase directly to activate gene transcription. On the other hand, the SRC-l, CBP/p300, and P/CAF complexes may function through chromatin modification as they possess histone acetylase activity. In addition, transcriptional activation is associated with chromatin disruption, which may be due to the recruitment of chromatin remodeling machinery by TR/RXR.

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Figure 3. Developmental expression of TR genes suggests dual functions for TR in frog development. The TH-inducible gene stromelysin-3 (ST3) is expressed during late embryogenesis when little TR mRNA is present. As the TRa genes are activated, ST3 is repressed. When endogenous TH levels rise after stage 54 both ST3 and TR" genes are activated. The TR and RXR mRNA levels are based on (Yaoita and Brown, 1990; Wong and Shi, 1995). The ST3 mRNA levels are based on (Patterton et al, 1995). Thyroid hormone T4 levels are from (Leloup and Buscaglia, 1977).

The expression profiles together with the ability of TR to both repress and activate TH-inducible genes in the absence and presence of TH, respectively, suggest dual functionsfor TRs during development. In premetamorphic tadpoles, TRs, mainly TR!, act to repress TH-response important also for the gene regulation by TR. Thus, TR/RXR heterodimers function as transcriptional repressors of TH-inducible genes in premetamorphic tadpoles when TR is absent, and as transcriptional activators during metamorphosis when TH is available.

IV. Constitutive DNA-binding and involvement of histone acetylation in developmental gene regulation by TRS The studies in the frog oocyte and other model systems have provided strong evidence that TR/RXR may regulate gene transcription at least in part by recruiting histone deacetylase or acetylase (acetyltransferase) complexes, depending upon the absence or presence of TH, respectively. To investigate the possible involvement of histone acetylation in gene regulation by TR in vivo, we have treated tadpoles with TH or TSA, a specific drug for blocking histone deacetylases, and analyzed the effect on the expression of TH response genes (Sachs and Shi, 2000). Surprisingly, no detectable upregulation of TH response genes by TSA can be detected in whole animals, although T 3 induces the expression of TH response genes as expected (Figure 4B) Since TR-treatment leads to a

large array of very different changes in the premetamorphic tadpoles, it is possible that the regulation of TH response genes may be tissue/organ-specific, depending upon the changes in the tissues/organs. Thus, we have chosen the intestine and the tail to investigate the role of histone acetylation further. These two organs are among the few well-characterized organs that undergo extensive remodeling and are known to have the most dramatic upregulation of TH-response genes during metamorphosis. Premetamorphic tadpole intestine consists predominantly of a single tissue, the larval epithelium, which undergoes apoptosis and is replaced by the adult epithelium (Yoshizato, 1989; Shi, 1996). The tail, on the other hand, completely absorbs through an apoptotic pathway (Dodd and Dodd, 1976; Yoshizato, 1989; Shi, 1999). Thus, these two organs offer relatively homogeneous tissues for study tissue specific changes in gene expression and chromatin remodeling. Indeed, our studies on these two organs indicate that TSA induces precocious expression of most TH response genes analyzed, including the only two genes that have been shown to contain TREs (Ranjan et al, 1994; Machuca et al, 1995; Furlow and Brown, 1999), the TRb and TH/bZIP genes Figure. 4A) (Sachs and Shi, 2000). On the other hand, TSA had little effect on the expression of TR! genes, which are not direct TH response genes. Thus, these data support the involvement of histone deacetylase in the repression of TH response genes by unliganded TR/RXR.

Sachs et al: Gene repression and activation by TRs

It has long been known that TR is chromatinassociated in somatic cells (Penman et al, 1982). Furthermore, in the frog oocyte system, we have shown that TR/RXR can bind to TRE both prior to and subsequent of replication-coupled chromatin assembly (Wong and Shi, 1995; Wong et al, 1997a). However, a direct demonstration of TR/RXR binding to the TREs of its target genes is lacking in any developmental system. If TR/RXR indeed functions to repress TH response genes in premetamorphic tadpoles as suggested above, we would expect that they are bound to TREs of endogenous TH response genes independent of TH. To test this possibility, we have made use of the sensitive chromatin irnmunoprecipitation (ChIP) assay using antibodies against TR or RXR (Sachs and Shi, 2000). PCR analysis of the immunoprecipitates for the binding of TR or RXR to the TRE regions of the Xenopus TRb and TH/bZip genes, have demonstrated clearly that both TR and RXR are bound to the TREs in the intestine and tail (Figure 4C) (Sachs and Shi, 2000). Furthermore, tbe binding is independent of TH or TSA treatment, in agreement with studies in vitro and in the frog oocyte. The ChIP assay also offers an opportunity to study whether local histone acetylation levels change in response to TH binding to TR/RXR. This has been done by using an antibody against acetylated histone H4 on the two TH response genes (TRb and THIbZip) in Xenopus laevis intestine and tail. The results have shown that TH treatment of premetamorphic tadpoles leads to an increase of histone acetylation specifically at the TRE regions of TH response genes (Figure 5A) (Sachs and Shi, 2000) without affecting global histone acetylation or the acetylation of chrornatin far away from the TRE (Figure 5B). On the other hand, TSA treatment of premetamorphic tadpoles elevates global histone acetylation levels, including the TRE regions of TH response genes. Similarly, ChIP assay using an antibody against the histone deacetylase Rpd3, the only characterized deacetylase in Xenopts laevis, demonstrates that Rpd3 is present at the TRE regions of TH response genes and its binding is reduced upon TH treatment of premetamorpbic tadpoles (analyzed in whole animals as Rpd3 was not detectable in prernetamorphic intestine, Sachs and Shi, 2000). Thus, these data together suggest that TR/RXR is bound to TREs assembled into chromatin in vivo. In the absence of TH, TR/RXR recruits histone deacetylase complexes to silence transcnption, at least in the intestine and tail. In the presence of TH, histone deacetylase complexes are released and histone acetylase complexes are likely recruited by TR/RXR, resulting in increased histone acetylation and gene activation.

V. Conclusion TH regulates a wide range of biological processes across most animal species by influencing gene transcription through TR. The roles of TR! and TR" in regulating anuran metamorphosis are supported by their temporal and spatial expression profiles during development. Furthermore, these receptors appear to have dual functions depending upon the cell types and developmental stages when they expressed. In premetamorphic tadpoles, they are likely to function as unliganded transcriptional repressors to block the expression of TH response genes that are involved in metamorphosis, thus ensuring a proper period of tadpole growth. When TM becomes available during metamorphosis, it binds to the receptors and converts them into activators to upregulate the TH-inducible genes, thus initiating metamorphosis. Our studies involving over-expression of TRJRXR in embryos have provided some in vivo evidence that supports the involvement of TR/RXR heterodimers in repressing TM-inducible genes in the absence of TM and in activating them when TM is present. ChIP assays have directly shown that TRs are bound to TREs assembled into chromatin. Furthermore, our data support the model that in the absence of TM, they recruit histone deacetylase complexes to silence transcription at least in some organs/tissues. The binding of TM to chromatin-bound TR leads to local histone acetylation likely due to the release of deacetylase complexes and possible recruitment of acetylase complexes. These findings are also consistent with those from in vitro studies and from analyses in the frog oncyte system, where it has been shown that histone acetylation plays an important role in gene regulation by TR and that transcriptional activation by TM leads to additional chromatin remodeling. Thus a model for TR action based on a TM-dependent switch between transcriptional repression and activation involving chromatin remodeling provides one possible molecular mechanism for the dual functions of TRs in development.

Acknowledgements We would like to thank Ms. K. Pham for preparing the manuscript.

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Figure 4. TH and TSA induce transcription of TH response genes in premetasnorphic tadpole intestine. Stage 55 tadpoles were treated for two days with T3 (10 nM) or TSA (100 nM). A) T 3 arid TSA treatments increase TH response gene expression. The intestine was isolated for total RNA extraction. The RNA was used for analysis of TR !, TR" and TH/bZip mRNA expression by PCR. The expression of ribosomal protein gene rp18 was used as an internal control (Shi and Liang., 1994). Note that TR" is not a directly TH response gene and is not induced by TH or TSA during the treatment period. B) T 3 and TSA treatments do not alter overall mRNA levels of TH response genes in whole ariimals. Total RNA was extracted from whole ariimals arid used for PCR analysis of TR!, TR" and TH/bZip expression. C) TR/RXR binds to TREs in chromatin constitutively. Chromatin from T3- or TSA-treated stage 55 tadpole intestine was immunoprecipitated with antibodies against TR or RXR arid arialyzed by PCR for the presence of the TRE regions of the two TH response genes in the TR/RXR-bound chromatin fraction. Aliquots of the chromatin prior to immunoprecipitation were used directly in PCR as a DNA control (Input).

Figure 5. TH treatment increases histone H4 acetylation specifically at the TRE regions of TH response genes in premetamorphic tadpole intestine. Stage 55 tadpoles were treated for two days with T3 (10 nM) or TSA (100 nM). The intestine was isolated for extraction of the nuclei used for ChIP assay with an antibody against acetylated histone H4. Aliquots of the chromatin prior to immunoprecipitation were used directly in PCR as a DNA control (Input). T3 and TSA treatment leads to increases in histone H4 acetylation at TH response gene promoters (TRE regions) of both TR" arid TH/bZIP (A) but not in the transcribed region far from the promoter of TR" (B).

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References Allfrey V, Faulkner RM and Mirsky AE (1964) Acetylation and methylation of histines and ther possible role in the regulation of RNA synthesis. Proc Natl Acad Sci USA 51, 786-794 Almouzni G, Clark DL, M. M, and Wolffe AP. (1990). Chromatin assembly on replicating DNA in vitro. Nucl. Acids Res. 18, 5767-5774. Burke U, and Baniahmed A. (2000). Co-repressors 2000 [In Process Citation]. Faseb J 14, 1876-1888. Chen JD, and Li H. ( 1998). Coactivation and corepresion in transcriptional regulation by steroid/nuclear hormone receptors. Critical Reviews in Eukaryotic Gene Express. 8, 169-190. Dodd MHI, and Dodd JM. ( 1976). The biology of metamorphosis. In physiology of the amphibia (ed. B. Lofts). Acadmic Press, New York, 467-599. Fondell JD, Roy AL, and Roeder RG. ( 1993). Unliganded thyroid hormone receptor inhibits formation of a functional preinitiation complex: implications for active repression. Genes Dev 7, 1400-1410. Furlow JD, and Brown DD. (1999). In vitro and in vivo analysis of the regulation of a transcription factor gene by thyroid hormone during Xenopus laevis metamorphosis. Mol Endocrinol 13, 2076-2089. Hetzel BS. (1989). The story of iodine deficiency: An international challenge in nutrition. Oxford University Press, Oxford. Hong L, Schroth GP, Matthews HR, Yau P, and Bradbury EM. (1993). Studies of the DNA binding properties of histone H4 amino terminus. Thermal denaturation studies reveal that acetylation markedly reduces the binding constant of the H4 "tail" to DNA. J Biol Chem 268, 305-314. Hsia S-CY, Wang H, and Shi Y-B. (2000). Involvement of Chromatin and Histone Acetylation in the Regulation of HIVLTR by Thyroid Hormone Receptor. Cell Research, in press. Hu X, and Lazar MA. (2000). Transcriptional Repression by Nuclear Hormone Receptors. TEM 11:1, 6-10. Kass SU, Landsberger N, and Woiffe AP (1997) DNA methylation directs a time dependent repression of transcription initiation. Curr Biol. 7, 157-165. Krust A, Green S, Argos P, Kumar, Walter, Bornert J-M, and Chambon P. (1986). The chicken oestrogen receptor sequence: homology with v-erbA and the human oestrogen and glucocorticoid receptors. EMBO J 5, 89~-897. Leloup J, and Buscaglia M. (1977). La trijodothyronine: hormone de la metamorphose des amphibiens. C.R. Acad. Sci. 284, 2261-2263. Machuca I, Esslemont 6, Fairdough L, and Tata JR. (1995). Analysis of structure and expression of the Xenopus thyroid hormone receptor b gene to explain its autoregulation. Mol. Endocrinol 9, 96-107. McKenna NJ, Lanz RB, and O'Malley BW. (1999). Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 20, 321-344. Patterton D, Hayes WP, and Shi YB. (1995). Transcriptional activation of the matrix metalloproteinase gene stromelysin3 coincides with thyroid hormone-induced cell death during frog metamorphosis. Dev Biol 167, 252-262. Perlman Al, Stanley F, and Samuels HH. (1982). Thyroid

hormone nuclear receptor. Evidence for multimeric organization in chromafin. J Biol Chem 257, 930-938. Puzianowsak-Kuznicka M, Damjanovski 5, and Shi Y-B. ( 1997). Both thyroid Hormone and 9-cis Retinoic Acid receptors are Required to Efficien~y mediate the Effects of Thyroid Hormone on Embryonic Development and Specific Gene Regulation in Xenopus laevis. Mol. and Cell Biol. 17, 47384749. Rachez C, and Freedman LP. (2000). Mechanisms of gene regulation by vitamin D(3) receptor: a network of coactivator interactions. Gene 246 , 9-21. Ranjan M, Wong j, and Shi YB. (1994). Transcriptional repression of Xenopus TR beta gene is mediated by a thyroid hormone response element located near the start site. I Biol Chem 269 , 24699-24705 Sachs LM, and Shi Y-B. (2000). Targeted chromatin binding and histone acetylation in vivo by thyroid hormone receptor during amphibian development. PNAS in press. Shi Y-B. (1996). Thyroid hormone-regulated early and late genes during amphibian metamorphosis. In Metamorphosis:Post embryonic reprogramming of gene expression in amphibian and insect cells. (Eds. L. I. Gilbert, I. R. Tata and B. G. Atkinson). Academic Press, New york., 505-538 Shi Y-B. (1999). Amphibian Metamorphosis: From morphology to molecular biology. John Wiley & Sons, Inc., New York , 28 8pp Shi Y-B, and Liang. VC-T. (1994). Clonng and characterization of the ribosomal protein L8 gene from Xenopus laevis. Biochimica et Biophysica Acta.1217, 227-228. Shi Y-B, Wong I, puzianowska~Kuznicka M, and Stolow MA (1996) Tadpole competence and tissue-specific temporal regulation of amphibian metamorphosis: Roles of thyroid hormone and its receptors. BioEssays. 12, 391-399. Struhl K. (1998). Histone acetyation and transcriptional regulatory mechanisms. Genes & Develop 12, 599-606. Tata JR. (1997). How hormones regulate programmed cell death during amphibian metamorphosis. In programmed Cell Death (Eds., Shi, Y.G., Shi, y., Xu, y. and Scott, D.W.) pp Plenum Press, New York. Tsai Mi, and Oâ&#x20AC;&#x2122;Malley BW. (1994). Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Ann Rev Biochem 63, 451-486. Urnov FD, Yee I, Collingwood TN, Bauer A, Beug H, Shi Y-B, and Wolffe AP. (2000). Targeting of N-CoR-HDAC3 by the oncoprotein v-ErbA yieds a chromatin infrastructuredependent transcriptional repression pathway. EMBO I 19, 40744090. Utley RT, Ikeda K, Grant PA, Cote I, Steger Di, Eberharter A, John S, and Workman IL. (1998). Transcriptional activators direct histone acetyltransferase complexes to nucleosomes. Nature 394, 498-502. Woffe AP. (1986). Histone deacetylase: a regulator of transcription. Science 272, 371-372. Wong J, Patterton D, Imhof D, Guschin D, Shi Y-B, and Woiffe AP. (1998). Distinct requirements for chromatin assembly in transcriptional repression by thyroid hormone receptor and histone deacetylase. EMBO J. 17, 520-534. Wong J, and Shi Y-B. (1995). Coordinated regulation of and transcriptional activation by Xenopus thyroid hormone and retinoid X receptors. J Biol Chem 270, 18479-18483. Wong J, Shi Y-B, and Wolffe AP. (1997a). Determinants of chromatin disruption and transcriptional regulation instigated

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by the thyroid hormone receptor: hormone-regulated chromatin :1isruption is not sufficient for transcriptiinal activation. EMBO J. 16, 3158-3171. Wong J, Shi YB, and Wolffe AP. (1995). A role for nucleosome assembly in both silencing and activation of the Xenopus TR beta gene by the thyroid hormone receptor. Genes Dev 9,2696-2711. Xu L, Glass CK, and Rosenfeld MG. (1999). Coactivator and orepressor complexes in nuclear receptor function. Current Opinion in Genetics & Development 9,40-147. Yaoita Y, and Brown DD. (1990). A correlation of thyroid hormone eceptor gene expression with amphibian metamorphosis. Genes Dev 4, 1917-1924. Yaoita Y, Shi Y-B, and Brown DD. (1990). Xenopus laevis and 13 thyroid hormone receptors. PNAS 87, 7090-7094. Yoshizato K. (1989). Biochemistry and cell biology of amphibian metamorphosis with a special emphasis on the mechanism of removal of larval organs. Int. Rev. Cytol. 119, 97-149.

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Gene Ther Mol Biol Vol 5, 111-120, 2000

Signal transduction pathways in cancer cells; novel targets for therapeutic intervention Review Article

Christos A. Tsatsanis* and Demetrios A. Spandidos Medical School, University of Crete, Heraklion 71409, Crete, Greece _________________________________________________________________________________________________ * Correspondence: Christos Tsatsanis, email: tsatsani@med.uoc.gr Key Words: Human neoplasms, signal transduction, oncogenes, transcription factors, kinases Received: 26 August, 2000; Accepted: 26 August, 2000

Summary Oncogenic proteins participate in signal transduction cascades that induce cell transformation. Understanding the molecular events that take place during oncogenesis is necessary to find novel, more effective therapeutic interventions. Signal transduction in cancer cells involves signaling from the extracellular environment, through the membrane, into the cytoplasm and towards the nucleus where transcription is initiated to generate proteins that will eventually contribute to the oncogenic phenotype. Alterations in such signaling cascades via mutations, gene amplifications or deletions frequently occur in human neoplasms. The result of such alterations has an impact in the cell cycle control, the cell morphology and the regulation of apoptosis. An overview on the signaling molecules altered in human tumors and their potential role as therapeutic targets is presented.

I. Introduction Cancer originates in the genetic material of the tumor cell. Alterations that occur in the genetic material deregulate the cellular functions and lead to uncontrolled proliferation and alterations in the cell morphology. To find effective therapeutic interventions for cancer we need to understand the events that take place during cell transformation. The first step will be the identification of the genes that are altered in the tumor cell. Such genes are defined as oncogenes, and are usually either overexpressed or mutated in a way that they cannot be regulated as they used to leading to the oncogenic phenotype. A second category are the onco-suppressor genes, genes that normally function as brakes in the cell cycle or repair damaged DNA and when their function is lost the cell loses control of its division rate or acquires mutations that lead to faster proliferation (Fearon, 1997; Hanahan and Weinberg, 2000). Following the identification of these genes we need to elucidate the role of the proteins encoded by these genes in the cellular environment. In other words we need to understand the function of these proteins in the

normal cell and in the tumor cell. By understanding the mechanism through which these proteins induce the tumor we can interfere with therapeutic agents that will be able either to specifically inhibit the function of these genes and therefore eliminate these cells or perturb their proliferation and lead them to extinction (Denhardt, 1996). Oncogenic proteins participate in signal transduction pathways that play central role in the transmission of a signal from the extracellular environment, through the cell membrane, into the cytoplasm and to the nucleus where transcription is initiated to generate proteins that will eventually contribute to the oncogenic phenotype. The function of these proteins is vital for cell and tissue homeostasis and they control processes such as cell division, differentiation and apoptosis. All these molecules are potential targets for anti cancer drug design since inhibition or activation of their function will lead to elimination of the tumor cells.

Tsatsanis and Spandidos: Signal transduction pathways in cancer cells

Figure 1. Growth factors and other extracellular signals initiate cascades that lead to activation of transcription factors and gene expression

II. Growth factors and transmembrane receptors Growth factors normally play a role in controlling the proliferation and metabolic activation of certain cells. They act by binding on specific receptors of the cell membrane, which, in turn, transmit the signal into the cytoplasm. They are frequently found overexpressed in a variety of tumors. The result is that the respective receptors are stimulated at a higher rate and, therefore, the signal that is transmitted is constant. Often tumors are found to secrete growth factors such as epidermal growth factor (EGF), colony stimulating growth factor 1 (CSF1), insulin growth factor I (IGF-I) and platelet-derived growth factor (PDGF) (Kolibaba and Druker, 1997). These factors bind to their receptors and initiate growth and proliferative signals. This mechanism establishes an autocrine loop that leads to tumor growth. Alternatively, receptors can be mutated in a way that they transmit the signal without ligand binding. For instance tyrosine kinase receptors dimerize or oligomerize following ligand binding. The dimerization and the conformational changes that are induced by ligand binding

bring the cytoplasmic tails in such proximity to trigger autophosphorylation. Autophosphorylation in most cases activates a cascade of phosphorylation events that include phosphorylation of intracellular signaling molecules and recruitment of SH2 (src homology 2) domain-containing proteins that bind to specific tyrosine phosphorylated residues (Cooper and Howell, 1993; Rodrigues and Park, 1994). In various tumors tyrosine kinase receptors can be constitutively activated by mutations that render them active independent of ligand binding. Such mutations were found on NEU/c-erbB-2 (Bargmann and Weinberg, 1988; Weiner et al, 1989). Mutation of the transmembrane domain was also found in other viral oncogenes such as vROS, which obtains very broad substrate specificity (Zong et al, 1993). Alternatively, tyrosine kinases can become oncogenic by mutations that make them active independent of ligand binding or dimerization. Non-receptor tyrosine kinases are also activated by mutations that affect their negative regulation such as the mutation on tyrosine 527 of Src that leads to deregulation of its activation (Sawyers and Denny, 1994).

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There are several tyrosine kinases that are activated in tumors via mutations. Most of these mutations result from chromosomal translocations that give rise to hybrid gene products. A major example is the BCR-ABL that is a mutant protein caused by the reciprocal translocation between chromosomes 9 and 22, the Philadelphia chromosome, that juxtaposes sequences of the breakpoint cluster region BCR on chromosome 22 with the c-ABL kinase on chromosome 9 (Groffen et al, 1984; Heisterkamp et al, 1985). This translocation is present on 95% of chronic myelogenous leukemias, which account for 20% of the adult leukemias. The BCR-ABL fusion gene in CMLs produces a protein in which the first exon of c-ABL has been replaced by BCR sequences encoding 927 or 902 aminoacids (Shtivelman et al, 1985; BenNeriah et al, 1986). In other cases 185 kd BCR portion is fused with exons 2-11 of the c-ABL protein (Hermans et al, 1987). The BCR-ABL chimeric protein exhibits tyrosine kinase activity several fold higher than that of the c-ABL. This kinase can transform fibroblasts and is considered highly oncogenic (Daley et al, 1987; Lugo et al, 1990). The pathways that this protein uses to cause transformation are not clearly defined. It is known that it binds and activates GRB-2 (Pendergast et al, 1993) which, in turn, activates the Ras pathway, a key pathway for triggering MAPK activation and cell proliferation. Other cases of fusion proteins is this of TEL-ABL, present in acute lymphoblastic leukemia (ALL), in acute lymphoblastic leukemia (AML) and in chronic myeloblastic leukemia (CML) with a reciprocal t (9; 12) translocation which links the Ets-like transcription factor TEL with the ABL tyrosine kinase (Golub et al, 1996; Golub et al, 1996; Papadopoulos et al, 1995). TEL has also been found fused to the PDGF receptor (TELPDGFR) in chronic myelomonocytic leukemias (CMML) through an acquired translocation in hematopoietic cells, t(5;12)(q33;p13) (Berkowicz et al, 1991; Lerza et al, 1992; Golub et al, 1994). Receptors molecules such as the cytokine receptors can contribute to the oncogenic phenotype by transducing signals from cytokines often expressed by tumor cells, such as the TGF! in breast tumors (Chakravarthy et al, 1999). Antigen receptors also play a significant role in the tumor formation either by giving the tumor cell the ability to escape the immune system surveillance or by rendering hematopoietic cells sensitive to proliferation signals. Interfering with the mutated receptor kinases may contribute to inhibition of the signal they transmit and subsequent elimination of the tumor cell. Chemical inhibitors are currently in trial such as tyrosine kinase inhibitors and growth factors are being used in therapeutic strategies in order to induce tumor cells to differentiate into a non-proliferating type of cell or cause apoptosis. Gene therapy strategies can, therefore, be used to produce such substances locally and thus eliminating side effects.

III. Cytoplasmic molecules The signal initiated by the growth factors at the cell surface is then transmitted into the cytoplasm and transduced by a cascade of events that includes phosphorylation, farnesylation, ubiquitination and other changes that alter molecules in order to promote or inhibit their activity or interaction with other molecules. Kinases and phosphatases play an important role in the transduction of oncogenic signals. The MAPKinase and the PI3Kinase cascades play a central role during cell activation and proliferation. Several oncogenes are known to act on these pathways and several molecules that participate on these cascades when deregulated they become oncogenic. Ras, a wellstudied family of oncogenes, structurally altered in about 25% of all human tumors, functions on activating the MAPK cascade (Kinzler and Vogelstein, 1996; Spandidos and Anderson, 1990; Zachos and Spandidos, 1997). Raf1, a serine threonine kinase that is activated by Ras, is also activated in some myeloid leukemias (Okuda et al, 1994; Schmidt et al, 1994). Serine threonine kinases are another important group of oncogenes. This family of oncogenes includes the Akt family (Akt1, Akt2, Akt3). Akt2 was activated in pancreatic adenocarcinomas, small cell lung cancer, and ovarian cancers (Cheng et al, 1992; Bellacosa et al, 1995; Ruggeri et al, 1998). Akt3 has also been found activated in estrogen receptor deficient breast cancers and androgen independent prostate cancers (Nakatani et al, 1999). The Tpl-2/Cot oncogene is activated in breast (Sourvinos et al, 1999), thyroid and colon tumors (Ohara et al, 1995). The Tpl-2 oncogene activates the MAPKinase (Mitogen Activated Protein Kinase) and the SAPKinase (Stress Activated Protein Kinase) pathways (Patriotis et al, 1994; Salmeron et al, 1996). Activation of these two pathways leads to the activation of transcription factors such as AP1 and NFAT (Ballester et al, 1997; Tsatsanis et al, 1998). Tpl-2 also activates the transcription factor NFkB, a major transcription factor, by activating the kinase that phosphorylates and induces degradation of the NFkB inhibitor IkB" (Tsatsanis et al, 1998; Belich et al, 1999; Lin et al, 1999). Activation of these factors induces transcription of several genes that contribute to the tumor phenotype. The Akt proto-oncogene (Bellacosa et al, 1991) is activated by PDGF receptor via activation of the PI3Kinase, a kinase that phosphorylates lipids (Franke et al, 1995; Chan et al, 1999). Activation of Akt inhibits apoptosis by inhibiting BAD, a pro-apoptotic, Bcl-2binding protein (Franke et al, 1997; Khwaja, 1999; Wang et al, 1999). Akt is also involved in inducing cell cycle progression possibly by activating transcription factors such as NFkB (Ozes et al, 1999; Romashkova and Makarov, 1999). Akt kinase is known to induce phosphorylation of IkB" via NIKinase and IKK" (Ozes et al, 1999). It is also a transducer of growth factor signals such as PDGF, G-CSF, IL-2, hepatocyte growth factor,

Tsatsanis and Spandidos: Signal transduction pathways in cancer cells

IGF and other mitogenic signals. Most of these signals lead to phosphorylation of Akt which results in signals that lead to inhibition of apoptosis (Ahmed et al, 1997; Kennedy et al, 1997; Chan et al, 1999). When a combination of oncogenes is activated a particular phenotype is favored. For example, in breast tumor cells Akt phosphorylates Raf at a highly conserved serine residue in its regulatory domain in vivo. This phosphorylation of Raf by Akt inhibited activation of the Raf-MEK-ERK signaling pathway and shifted the cellular response from cell cycle arrest to proliferation (Zimmermann and Moelling, 1999). Such interactions can occur and determine the levels of crosstalk and fine regulation of different signaling pathways, the MAPK and the PI-3Kinase. Raf, Akt and Tpl-2 are kinases that contribute to the oncogenic phenotype through divergent mechanisms. On the one hand they can induce transcription of genes that are normally not expressed in these cells and on the other hand they can directly interfere with cell cycle machinery and promote progression through the cell cycle. Alternatively, they can inhibit programmed cell death and, therefore, allow the survival of a cell that carries other defects and would otherwise apoptose. Interference with the function of these kinases via chemotherapeutic agents requires caution since the effect could be the opposite from the expected. On the contrary such molecules can be used in gene therapy approaches by introducing mutated forms that will favor a particular function.

IV. Transcription factors Transmission of the signals from the cytoplasm will lead to the activation of transcription factors. Transcription factors are activated by several mechanisms during tumorigenesis and contribute to tumor formation. Phosphorylation and ubiquitination are mechanisms that control and regulate the activation of transcription factors. This is the case for NFkB where a sequence of phosphorylation events leads to degradation of its inhibitory molecule, IkB", and its subsequent translocation into the nucleus. IkB" is ubiquitinated and recognized by the proteasome complex where its degradation takes place. In the case of NFAT, dephosphorylation by calcineurin in the cytoplasm leads to its nuclear translocation and a nuclear kinase, GSK3 phosphorylates NFAT and pushes it to translocate into the cytoplasm (Beals et al, 1997). In tumor cells a transcription factor can be mutated and activated independent of extracellular or cytoplasmic signals. Expression of the transcription factors Ets-1 and Ets-2 is induced during cell proliferation but it has also been directly linked to a complex chromosomal translocation, t (6;18;21), in acute non lymphoblastic leukemias. Ets-2 is overexpressed during hepatic regeneration and hepatocellular carcinomas (Dittmer and

Nordheim, 1998). NFkB is a transcription factor that regulates expression of several genes and was activated in a series of tumors such as breast tumors, pancreatic adenocarcinomas, lung cancers and acute T cell leukemias (Bukowski et al, 1998; Sovak et al, 1997; Wang et al, 1999). In this case we do not know whether it is the immediate effect or the consequence of other oncogenes that have been activated and lead to NFkB induction. Regardless, inhibition of its activity in tumors may be beneficial for the elimination of the neoplastic cells. C-myc is a transcription factor implicated in a variety of human tumors. When overexpressed it dimerizes with Max, a complex that elicits growth signals, while the Mad-Max complex promotes differentiation signals (Bouchard et al, 1998; Brandt-Rauf and Pincus, 1998; Schmidt, 1999). Overexpression of c-myc has been involved in a series of human tumors including colon, stomach, cervix, breast and hematological neoplasms (Spandidos et al, 1991; Agnantis et al, 1992; Porter et al, 1994; Nesbit et al, 1999).

V. Cell cycle control proteins Deregulation of the cell cycle control is crucial for the development of a cancer cell since it has to proliferate at a faster than the normal rate. This effect can be direct, involving mutations of the cell cycle control proteins or indirect when an oncogenic protein targets the cell cycle regulators. Oncogenic processes exert their greatest effect by targeting particular regulators of the G1 to S phase progression. Control of the G1 to S progression is a crucial checkpoint for the cell fate. Deregulation of the checkpoint proteins can contribute to uncontrolled proliferation (Sherr, 1996). Progression from the G1 to S phase occurs when cyclins respond to growth factor signals. Thus, such signals can be initiated by different growth stimuli that transmit the signal to the cytoplasm where cyclins are bound to cyclin dependent kinases and control the restriction point. Release of the cyclin dependent kinases from the complex pinpoints the passage from G1 to S phase. Cyclins D1, D2 and D3 control that stage. They are bound to the cyclin dependent kinases CDK4 and CDK6 which, when released, phosphorylate the retinoblastoma protein Rb (Morgan, 1995). Phosphorylation of Rb is a critical point in the cell cycle progression since it appears to be necessary for the transcriptional initiation of several genes. Hyperphosphorylated form of Rb is present past the G1 to S restriction point and all through the cell cycle until cell division (Zhu et al, 1996). The cyclin/CDK complex is inhibited by a family of proteins that include p15, p16, p18 and p19, frequently mutated in human melanomas, gliomas and leukemias, that specifically interact with CDK4 and CDK6 and therefore block the function of D type cyclins (Nobori et al, 1994; Zhang et al, 1994). On

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Figure 2. Progression of a cell through the cell cycle is tightly controlled by inhibitors of the CDKs or inhibitors of the cyclins

the other hand the p21, p27, and p57 family of cyclin inhibitors are capable of interacting with cyclins type D, E and A exhibiting a broader spectrum of inhibition (elDeiry et al, 1993; Toyoshima and Hunter, 1994; Lee et al, 1995). Several types of tumors carry mutations on genes that control the cell cycle. Inactivation of the Rb gene is a primary event in retinoblastomas (Knudson, 1971), but overall the gene is targeted more often in adult cancers, particularly small-cell carcinomas of the lung (Sumitomo et al, 1999). Similarly, inherited loss of INK4a gene that encodes p16 confers susceptibility to melanoma (Palmero and Peters, 1996). Cyclin D1 is also overexpressed in many human cancers as a result of gene amplification or translocations targeting the D1 locus on human chromosome 11q13 (Masciullo et al, 1997). The gene encoding its catalytic partner CDK4, located on chromosome 12q13 is also amplified in sarcomas and gliomas (Nobori et al, 1994) although several other potential oncogenes including MDM2, the p53 antagonist, map on the same region (Hall and Peters, 1996).

Although cell cycle transition depends on the underlying CDK cycle, superimposed checkpoint controls help ensure that certain processes are completed before others begin. The role of such mechanisms is to act as a brake on the cell cycle in the face of stress and damage and allowing repair to take place. The best-studied checkpoint regulator is the p53 gene and is most frequently mutated in human cancer (Baker et al, 1989; Nigro et al, 1989). Even though p53 is a short-lived protein, it stabilizes and accumulates when the cell undergoes damage (Ko and Prives, 1996). The precise signal transduction pathway that activates p53 has not been elucidated but is likely to include genes like ATM (mutated in ataxia telangiectasia) (Enoch and Norbury, 1995). The p53 protein acts as a transcription factor and cancer related mutations cluster in its binding domain (Ko and Prives, 1996). Targeting the cell cycle control proteins is a possible approach to eliminate tumor growth.

Tsatsanis and Spandidos: Signal transduction pathways in cancer cells

VI. Apoptosis related proteins Programmed cell death occurs when a cell has suffered DNA damage that cannot be repaired, is under environmental stress or receives extracellular apoptotic signals. Stress-induced apoptosis is regulated by a mechanism that involves cytochrome c release from the mitochondria and subsequent activation of several proteolytic molecules termed caspases that lead to degradation of cellular components, DNA cleavage (â&#x20AC;&#x2DC;ladderingâ&#x20AC;&#x2122;) and death (Green, 1998). Receptor-mediated apoptosis, such as Fas or the TNF-" receptors, initiate signals that lead to caspase 8 activation, cytochrome c release from the cytoplasm, activation of caspase 9 and the APAF complex and subsequent cleavage and activation of caspase 3, caspase 6 or caspase 7 (Alnemri, 1999; Qin et al, 1999). Caspases also translocate into the nucleus triggering their pro-apoptotic effects (Alnemri, 1999). In cancer cells an anti-apoptotic mechanism is often activated to rescue the transformed cell from programmed cell death. The most common mechanism is activation of the bcl-2 family of proteins (Bcl-2, Bcl-xL, Bcl-W) that are able to inhibit cytochrome c release from the mitochondria and rescue the cell from apoptosis. Inactivation of the pro-apoptotic molecules Bax, Bak, Bid or Bim also contributes to rescuing the cell from apoptosis. Activation of oncogenic kinases such as Akt-1 protects cells from apoptosis by inhibiting the proapoptotic molecule Bad (Khwaja, 1999). Several antiapoptotic signals such as growth factors (PDGF, EGF etc) lead to the activation of signaling pathways including the PI3Kinase or MAPK pathways that can also be activated by oncogenic kinases like Akt and Tpl-2. Thus, activation of these oncogenic kinases rescues the cell from the apoptotic signals and promotes survival. Activation of the apoptotic mechanism is, therefore, a key stage where therapeutic agents could interfere. Gene therapy approaches could be used by introducing proapoptotic molecules into tumor cells, whereas pharmacological inhibitors of anti apoptotic molecules such as the bcl-2 family of proteins may be a therapeutic approach in tumors where these molecules are activated. Several conventional chemotherapeutic agents induce apoptosis in tumor cells and drug resistant tumors exhibit activated anti-apoptotic mechanism. The fact that proapoptotic and anti-apoptotic molecules are Important in maintaining the homeostasis in all tissues may be a potential drawback for such pharmacological treatments.

VII. Conclusions Human tumors are a result of accumulation of two or more mutations in a cell. These mutations alter the protein profile of the cell and lead to faster proliferation and transformation. The mutant or the overexpressed proteins can be targeted with therapeutic agents to inhibit their action and kill the tumor cells. Gene therapy approaches

can be used, introducing mutant proteins that compete or inhibit the transforming ones. In the case of a deleted protein a gene therapy approach is beneficial to reintroduce the gene that is deleted. Thus, understanding of the signal transduction pathways altered in tumor cells is important for detecting novel targets for cancer therapy.

References Agnantis, N. J., Mahera, H., Maounis, N., and Spandidos, D. A. (1992). Immunohistochemical study of ras and myc oncoproteins in apocrine breast lesions with and without papillomatosis. Eur. J. Gynaecol Oncol 13, 309-15. Ahmed, N. N., Grimes, H. L., Bellacosa, A., Chan, T. O., and Tsichlis, P. N. (1997). Transduction of interleukin-2 antiapoptotic and proliferative signals via Akt protein kinase. Proc. Nat. Ac. Sc. USA 94, 3627-32. Alnemri, E. S. (1999). Hidden powers of the mitochondria [news]. Nature Cell Biol. 1, E40-2. Baker, S. J., Fearon, E. R., Nigro, J. M., Hamilton, S. R., Preisinger, A. C., Jessup, J. M., vanTuinen, P., Ledbetter, D. H., Barker, D. F., Nakamura, Y., and et al (1989). Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science 244, 217-21. Ballester, A., Tobena, R., Lisbona, C., Calvo, V., and Alemany, S. (1997). Cot kinase regulation of IL-2 production in Jurkat T cells. J. Immunol. 159, 1613-8. Bargmann, C. I., and Weinberg, R. A. (1988). Oncogenic activation of the neu-encoded receptor protein by point mutation and deletion. EMBO J. 7, 2043-52. Beals, C. R., Sheridan, C. M., Turck, C. W., Gardner, P., and Crabtree, G. R. (1997). Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3. Science 275, 1930-4. Belich, M. P., Salmeron, A., Johnston, L. H., and Ley, S. C. (1999). TPL-2 kinase regulates the proteolysis of the NFkappaB-inhibitory protein NF-kappaB1 p105. Nature 397, 363-8. Bellacosa, A., de Feo, D., Godwin, A. K., Bell, D. W., Cheng, J. Q., Altomare, D. A., Wan, M., Dubeau, L., Scambia, G., Masciullo, V., and et al (1995). Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas. Int. J. Cancer 64, 280-5. Bellacosa, A., Testa, J. R., Staal, S. P., and Tsichlis, P. N. (1991). A retroviral oncogene, akt, encoding a serinethreonine kinase containing an SH2-like region. Science 254, 274-7. Ben-Neriah, Y., Daley, G. Q., Mes-Masson, A. M., Witte, O. N., and Baltimore, D. (1986). The chronic myelogenous leukemia-specific P210 protein is the product of the bcr/abl hybrid gene. Science 233, 212-4. Berkowicz, M., Rosner, E., Rechavi, G., Mamon, Z., Neuman, Y., Ben-Bassat, I., and Ramot, B. (1991). Atypical chronic myelomonocytic leukemia with eosinophilia and translocation (5;12). A new association [letter]. Cancer Genet. & Cytogen. 51, 277-8. Bouchard, C., Staller, P., and Eilers, M. (1998). Control of cell proliferation by Myc. Trends in Cell Biol. 8, 202-6. Brandt-Rauf, P. W., and Pincus, M. R. (1998). Molecular markers of carcinogenesis. Pharmacol. & Therapeutics 77, 135-48. Bukowski, R. M., Rayman, P., Uzzo, R., Bloom, T., Sandstrom, K., Peereboom, D., Olencki, T., Budd, G. T., McLain, D.,

Gene Therapy and Molecular Biology Vol 5, page 117

Elson, P., Novick, A., and Finke, J. H. (1998). Signal transduction abnormalities in T lymphocytes from patients with advanced renal carcinoma: clinical relevance and effects of cytokine therapy. Clin. Cancer Res. 4, 2337-47. Chakravarthy, D., Green, A. R., Green, V. L., Kerin, M. J., and Speirs, V. (1999). Expression and secretion of TGF-beta isoforms and expression of TGF-beta-receptors I, II and III in normal and neoplastic human breast. Int. J. Oncol. 15, 187-94. Chan, T. O., Rittenhouse, S. E., and Tsichlis, P. N. (1999). AKT/PKB and other D3 phosphoinositide-regulated kinases: Kinase activation by phosphoinositide-dependent phosphorylation [Review]. Ann. Rev. Biochem. 68, 9651014. Cheng, J. Q., Godwin, A. K., Bellacosa, A., Taguchi, T., Franke, T. F., Hamilton, T. C., Tsichlis, P. N., and Testa, J. R. (1992). AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas. Proc. Nat. Ac. Sc. USA 89, 9267-71. Cooper, J. A., and Howell, B. (1993). The when and how of Src regulation. Cell 73, 1051-4. Daley, G. Q., McLaughlin, J., Witte, O. N., and Baltimore, D. (1987). The CML-specific P210 bcr/abl protein, unlike v-abl, does not transform NIH/3T3 fibroblasts. Science 237, 532-5. Denhardt, D. T. (1996). Oncogene-initiated aberrant signaling engenders the metastatic phenotype: synergistic transcription factor interactions are targets for cancer therapy. Crit. Rev. in Oncogen. 7, 261-91. Dittmer, J., and Nordheim, A. (1998). Ets transcription factors and human disease. Biochim. et Biophys. Acta 1377, F1-11. el-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993). WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817-25. Enoch, T., and Norbury, C. (1995). Cellular responses to DNA damage: cell-cycle checkpoints, apoptosis and the roles of p53 and ATM. Trends in Biochem. Sci. 20, 426-30. Fearon, E. R. (1997). Human cancer syndromes: clues to the origin and nature of cancer. Science 278, 1043-50. Franke, T. F., Kaplan, D. R., and Cantley, L. C. (1997). PI3K: downstream AKTion blocks apoptosis. Cell 88, 435-7. Franke, T. F., Yang, S. I., Chan, T. O., Datta, K., Kazlauskas, A., Morrison, D. K., Kaplan, D. R., and Tsichlis, P. N. (1995). The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81, 727-36. Golub, T. R., Barker, G. F., Lovett, M., and Gilliland, D. G. (1994). Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation. Cell 77, 307-16. Golub, T. R., Goga, A., Barker, G. F., Afar, D. E., McLaughlin, J., Bohlander, S. K., Rowley, J. D., Witte, O. N., and Gilliland, D. G. (1996). Oligomerization of the ABL tyrosine kinase by the Ets protein TEL in human leukemia. Mol. Cell. Biol. 16, 4107-16. Golub, T. R., McLean, T., Stegmaier, K., Carroll, M., Tomasson, M., and Gilliland, D. G. (1996). The TEL gene and human leukemia. Biochim. et Biophys. Acta 1288, M7-10. Green, D. R. (1998). Apoptotic pathways: the roads to ruin. Cell 94, 695-8. Groffen, J., Stephenson, J. R., Heisterkamp, N., de Klein, A., Bartram, C. R., and Grosveld, G. (1984). Philadelphia

chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell 36, 93-9. Hall, M., and Peters, G. (1996). Genetic alterations of cyclins, cyclin-dependent kinases, and Cdk inhibitors in human cancer. Adv. in Cancer Res. 68, 67-108. Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57-70. Heisterkamp, N., Stam, K., Groffen, J., de Klein, A., and Grosveld, G. (1985). Structural organization of the bcr gene and its role in the Ph' translocation. Nature 315, 758-61. Hermans, A., Heisterkamp, N., von Linden, M., van Baal, S., Meijer, D., van der Plas, D., Wiedemann, L. M., Groffen, J., Bootsma, D., and Grosveld, G. (1987). Unique fusion of bcr and c-abl genes in Philadelphia chromosome positive acute lymphoblastic leukemia. Cell 51, 33-40. Kennedy, S. G., Wagner, A. J., Conzen, S. D., Jordan, J., Bellacosa, A., Tsichlis, P. N., and Hay, N. (1997). The PI 3kinase/Akt signaling pathway delivers an anti-apoptotic signal. Gen. & Dev. 11, 701-13. Khwaja, A. (1999). Akt is more than just a Bad kinase. Nature 401, 33-4. Kinzler, K. W., and Vogelstein, B. (1996). Lessons from hereditary colorectal cancer. Cell 87, 159-70. Knudson, A. G., Jr. (1971). Mutation and cancer: statistical study of retinoblastoma. Proc. Nat. Ac. Sc. USA 68, 820-3. Ko, L. J., and Prives, C. (1996). p53: puzzle and paradigm. Gen. & Dev. 10, 1054-72. Kolibaba, K. S., and Druker, B. J. (1997). Protein tyrosine kinases and cancer. Biochim. et Biophys. Acta 1333, F21748. Lee, M. H., Reynisdottir, I., and Massague, J. (1995). Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution. Gen. & Dev. 9, 639-49. Lerza, R., Castello, G., Sessarego, M., Cavallini, D., and Pannacciulli, I. (1992). Myelodysplastic syndrome associated with increased bone marrow fibrosis and translocation (5;12)(q33;p12.3). Brit. J. Haematol. 82, 4767. Lin, X., Cunningham, E. T., Jr., Mu, Y., Geleziunas, R., and Greene, W. C. (1999). The proto-oncogene Cot kinase participates in CD3/CD28 induction of NF-kappaB acting through the NF-kappaB-inducing kinase and IkappaB kinases. Immunity 10, 271-80. Lugo, T. G., Pendergast, A. M., Muller, A. J., and Witte, O. N. (1990). Tyrosine kinase activity and transformation potency of bcr-abl oncogene products. Science 247, 1079-82. Masciullo, V., Scambia, G., Marone, M., Giannitelli, C., Ferrandina, G., Bellacosa, A., Benedetti Panici, P., and Mancuso, S. (1997). Altered expression of cyclin D1 and CDK4 genes in ovarian carcinomas. Int. J. Cancer 74, 3905. Morgan, D. O. (1995). Principles of CDK regulation. Nature 374, 131-4. Nakatani, K., Thompson, D. A., Barthel, A., Sakaue, H., Liu, W., Weigel, R. J., and Roth, R. A. (1999). Up-regulation of Akt3 in estrogen receptor-deficient breast cancers and androgenindependent prostate cancer lines. J. Biol. Chem. 274, 21528-32. Nesbit, C. E., Tersak, J. M., and Prochownik, E. V. (1999). MYC oncogenes and human neoplastic disease. Oncogene 18, 3004-16. Nigro, J. M., Baker, S. J., Preisinger, A. C., Jessup, J. M., Hostetter, R., Cleary, K., Bigner, S. H., Davidson, N.,

Tsatsanis and Spandidos: Signal transduction pathways in cancer cells

Baylin, S., Devilee, P., and et al (1989). Mutations in the p53 gene occur in diverse human tumour types. Nature 342, 7058. Nobori, T., Miura, K., Wu, D. J., Lois, A., Takabayashi, K., and Carson, D. A. (1994). Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 368, 753-6. Ohara, R., Hirota, S., Onoue, H., Nomura, S., Kitamura, Y., and Toyoshima, K. (1995). Identification of the cells expressing cot proto-oncogene mRNA. J. Cell Sci. 108, 97-103. Okuda, K., Matulonis, U., Salgia, R., Kanakura, Y., Druker, B., and Griffin, J. D. (1994). Factor independence of human myeloid leukemia cell lines is associated with increased phosphorylation of the proto-oncogene Raf-1. Exp. Hematol. 22, 1111-7. Ozes, O. N., Mayo, L. D., Gustin, J. A., Pfeffer, S. R., Pfeffer, L. M., and Donner, D. B. (1999). NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401, 82-5. Palmero, I., and Peters, G. (1996). Perturbation of cell cycle regulators in human cancer. Cancer Surveys 27, 351-67. Papadopoulos, P., Ridge, S. A., Boucher, C. A., Stocking, C., and Wiedemann, L. M. (1995). The novel activation of ABL by fusion to an ets-related gene, TEL. Cancer Res. 55, 34-8. Patriotis, C., Makris, A., Chernoff, J., and Tsichlis, P. N. (1994). Tpl-2 acts in concert with Ras and Raf-1 to activate mitogenactivated protein kinase Proc. Nat. Ac. Sc. USA 91, 97559. Pendergast, A. M., Quilliam, L. A., Cripe, L. D., Bassing, C. H., Dai, Z., Li, N., Batzer, A., Rabun, K. M., Der, C. J., Schlessinger, J., and et al (1993). BCR-ABL-induced oncogenesis is mediated by direct interaction with the SH2 domain of the GRB-2 adaptor protein. Cell 75, 175-85. Porter, M. J., Field, J. K., Leung, S. F., Lo, D., Lee, J. C., Spandidos, D. A., and van Hasselt, C. A. (1994). The detection of the c-myc and ras oncogenes in nasopharyngeal carcinoma by immunohistochemistry. Acta Oto-Laryngol. 114, 105-9. Qin, H., Srinivasula, S. M., Wu, G., Fernandes-Alnemri, T., Alnemri, E. S., and Shi, Y. (1999). Structural basis of procaspase-9 recruitment by the apoptotic proteaseactivating factor 1. Nature 399, 549-57. Rodrigues, G. A., and Park, M. (1994). Oncogenic activation of tyrosine kinases. Curr. Op. in Genetics & Dev. 4, 15-24. Romashkova, J. A., and Makarov, S. S. (1999). NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling. Nature 401, 86-90. Ruggeri, B. A., Huang, L., Wood, M., Cheng, J. Q., and Testa, J. R. (1998). Amplification and overexpression of the AKT2 oncogene in a subset of human pancreatic ductal adenocarcinomas. Mol. Carcinogen. 21, 81-6. Salmeron, A., Ahmad, T. B., Carlile, G. W., Pappin, D., Narsimhan, R. P., and Ley, S. C. (1996). Activation of MEK-1 and SEK-1 by Tpl-2 proto-oncoprotein, a novel MAP kinase kinase kinase. EMBO J. 15, 817-26. Sawyers, C. L., and Denny, C. T. (1994). Chronic myelomonocytic leukemia: Tel-a-kinase what Ets all about. Cell 77, 171-3. Schmidt, C. A., Oettle, H., Ludwig, W. D., Serke, S., PawlaczykPeter, B., Wilborn, F., Binder, L. T., Huhn, D., and Siegert, W. (1994). Overexpression of the Raf-1 proto-oncogene in human myeloid leukemia. Leuk. Res. 18, 409-13. Schmidt, E. V. (1999). The role of c-myc in cellular growth control. Oncogene 18, 2988-96.

Sherr, C. J. (1996). Cancer cell cycles. Science 274, 1672-7. Shtivelman, E., Lifshitz, B., Gale, R. P., and Canaani, E. (1985). Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature 315, 550-4. Sourvinos, G., Tsatsanis, C., and Spandidos, D. A. (1999). Overexpression of the Tpl-2/Cot oncogene in human breast cancer. Oncogene 18, 4968-73. Sovak, M. A., Bellas, R. E., Kim, D. W., Zanieski, G. J., Rogers, A. E., Traish, A. M., and Sonenshein, G. E. (1997). Aberrant nuclear factor-kappaB/Rel expression and the pathogenesis of breast cancer. J. Clin. Invest. 100, 2952-60. Spandidos, D. A., and Anderson, M. L. (1990). A role of ras oncogenes in carcinogenesis and differentiation. Adv. in Exp. Med. & Biol. 265, 127-31. Spandidos, D. A., Karayiannis, M., Yiagnisis, M., Papadimitriou, K., and Field, J. K. (1991). Immunohistochemical analysis of the expression of the c-myc oncoprotein in human stomach cancers. Digestion 50, 127-34. Sumitomo, K., Shimizu, E., Shinohara, A., Yokota, J., and Sone, S. (1999). Activation of RB tumor suppressor protein and growth suppression of small cell lung carcinoma cells by reintroduction of p16INK4A gene. Int. J. of Oncol. 14, 1075-80. Toyoshima, H., and Hunter, T. (1994). p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 78, 67-74. Tsatsanis, C., Patriotis, C., Bear, S. E., and Tsichlis, P. N. (1998). The Tpl-2 protooncoprotein activates the nuclear factor of activated T cells and induces interleukin 2 expression in T cell lines. Proc. Nat. Ac. Sc. USA 95, 3827-32. Tsatsanis, C., Patriotis, C., and Tsichlis, P. N. (1998). Tpl-2 induces IL-2 expression in T-cell lines by triggering multiple signaling pathways that activate NFAT and NF-kappaB. Oncogene 17, 2609-18. Wang, H. G., Pathan, N., Ethell, I. M., Krajewski, S., Yamaguchi, Y., Shibasaki, F., McKeon, F., Bobo, T., Franke, T. F., and Reed, J. C. (1999). Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science 284, 339-43. Wang, W., Abbruzzese, J. L., Evans, D. B., Larry, L., Cleary, K. R., and Chiao, P. J. (1999). The nuclear factor-kappa B RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells. Clin. Cancer Res. 5, 11927. Weiner, D. B., Liu, J., Cohen, J. A., Williams, W. V., and Greene, M. I. (1989). A point mutation in the neu oncogene mimics ligand induction of receptor aggregation. Nature 339, 230-1. Zachos, G., and Spandidos, D. A. (1997). Expression of ras proto-oncogenes: regulation and implications in the development of human tumors. Critical Rev. in Oncol.Hematol. 26, 65-75. Zhang, S. Y., Klein-Szanto, A. J., Sauter, E. R., Shafarenko, M., Mitsunaga, S., Nobori, T., Carson, D. A., Ridge, J. A., and Goodrow, T. L. (1994). Higher frequency of alterations in the p16/CDKN2 gene in squamous cell carcinoma cell lines than in primary tumors of the head and neck. Cancer Res. 54, 5050-3. Zhu, X., Ohtsubo, M., Bohmer, R. M., Roberts, J. M., and Assoian, R. K. (1996). Adhesion-dependent cell cycle progression linked to the expression of cyclin D1, activation of cyclin E-cdk2, and phosphorylation of the retinoblastoma protein. J. Cell Biol. 133, 391-403.

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Zimmermann, S., and Moelling, K. (1999). Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 286, 1741-4. Zong, C. S., Poon, B., Chen, J., and Wang, L. H. (1993). Molecular and biochemical bases for activation of the transforming potential of the proto-oncogene c-ros. J. Virol. 67, 6453-62.

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Gene Ther Mol Biol Vol 5, 121-130, 2000

Control of pre-mRNA processing by extracellular signals: emerging molecular mechanisms Review Article

Rossette Daoud, Peter Stoilov, Oliver Stoss, Mark HĂźbener, Maria da Penha Berzaghi, Annette M. Hartmann, Manuela Olbrich, and Stefan Stamm* Max-Planck Institute of Neurobiology, Am Klopferspitz 18a, D-82152 Martinsried, Germany

__________________________________________________________________________________ *Correspondence: Stefan Stamm, Ph.D., Institute of Biochemistry, Iahastrasse 17, Erlaujan 91054, Germany; Phone: +49 89 8578 3625, Fax: +49 89 8578 3749, E-mail: stefan@stamms-lab.net Key words: acetylcholinesterase, tra-2: transformer-2, N-methyl-D-aspartart receptor, 1, serine-arginine-rich protein, heterogenous ribonuclear protein, kinase G-1 Received: 9 November 2000; accepted: 11 December 2000

Summary Alternative splicing is an important mechanism to regulate gene expression. At least 30% of all human genes are alternatively spliced. This process can be regulated by extracellular signals that include stress and cellular activity. Splice site selection is regulated by a multiprotein complex Its composition can be regulated by either releasing proteins from nuclear storage sites or by changing protein:protein, as well as protein:RNA Interactions by serine and tyrosine phosphorylation.

I. Introduction

elements (Cooper and Mattox, 1997), for example in tauopathies (Gao et al, 2000) or spinal muscular atrophy (Lorson et al, 1999). A recent survey of disease-associated genes suggested that as much as a third of them might be alternatively spliced, suggesting that more pathologies might be associated with splicing defects (Hanke et al, 1999). Alternative splicing pathways are not static, because an organism can dynamically change its splicing patterns, e.g. during development and/or in response to extracellular stimuli such as insulin (Smith et al, 1999), nerve growth factor (Varani and Nagai, 1998), cytokines (Reddy, 1989; Eissa et al, 1996), and neuronal activity (Vezzani et al, 1995; Daoud et al, 1999). Some changes of splicing patterns require protein synthesis and may be based on the differential transcriptional control of splicing factor expression (Shifrin and Neel, 1993). One prominent example for a protein dependent change in splice site selection are the changes in the development of cancer. Here, the processing of CD44 changes during the transition of preneoplasias to neoplasias and their metastases which is associated with de novo synthesis of several SR proteins (Stickeler et al 1999) In addition, there is growing evidence that alternative splicing may also be regulated by transient covalent modifications of proteins implicated in mRNA splicing. For example, inclusion of CD44 exon v5 is independent of de novo protein synthesis and is coupled to a kinase downstream of Ras (KĂśnig et al, 1998).

The expression of genetic information is controlled at several stages, such as DNA structure, transcription, premRNA processing, translation and protein stability. To date, the most studied control mechanism is transcription. However, recent studies emphasized the importance of RNA metabolism in regulation of gene expression: RNA plays a crucial role in epigenetic regulation (Woiffe and Matzke, 1999), RNA editing is important for proper brain function (Sprengel et al, 1999), information stored in RNA can flow back into the genome (Cousineau et al, 2000) and almost all human genes are spliced by at least two splicing systems, and about 30% of them are alternatively spliced (Hanke et al, 1999; Mironov et al, 1999). The regulation of splicing was probably crucial for the evolution of eukaryotes (Herbert and Rich, 1999). Splice site recognition is helped by auxiliary proteins (trans factors) binding to short degenerate sequences on the RNA (exonic sequence elements). The fine tuned concentration of these trans factors governs splice site selection, both in vivo and in vitro (Black, 1995; Manley and Tacke, 1996; Grabowski, 1998). Proper splicing regulation is important for an organism, as it has been estimated that up to 15% of genetic defects caused by point mutations in humans manifest themselves as pre- mRNA splicing defects caused by changing splice site sequences (Krawczak et al, 1992; Nakai and Sakamoto, 1994). In addition, it became apparent that point mutations in exons can cause missplicing by changing exonic sequence

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Figure lA: The effect of pilocarpine on alternatively spliced genes (A) Experimental paradigm. Rats are injected intraperitoneally with pilocarpine, a cholinergic muscarinic agonist that can cross the blood brain barrier. As a result the drug causes strong neuronal activity in neurons of the hippocampus resulting in seizures that resemble an epileptic episode. The diagram shows a coronal cross section of the brain.

Figure 1 B-C: The effect of pilocarpine on alternatively spliced genes. RT-PCR analysis of hippocampal RNA frompilocarpine treated rats. Pilocarpineincreases neuronal activity in the brain thatstarts in the hippocampal formation.Removal of tissue after pilocarpine injection was at the time indicated. C: untreated control. A statistical evaluation for three experiments is given on the right. Standard deviations are indicated. Location of primers and eDNA structure are (B) RT PCR for fos B and its statistical schematically indicated for each gene.evaluation (C) RT PCR for clk2 and its statistical evaluation

Specific examples of changes in pre-mRNA processing after external stimuli have been compiled in this issue (Stoss et al, 2000). Here, we summarize changes of alternative splicing in response to stress and discuss the possible regulatory mechanisms.

when mice are subjected to acute stress by being forced to swim, this read through variant is upregulated. In contrast to the dominant "synaptic" variant (AchE-S), the AchE-R variant is soluble and monomeric. Physiologically, this switch is seen as a means to prevent neurodegenration, caused by excess activity of AchE-S (Sternfeld et al, 2000). Similar effects have been seen with potassium channels, where the effect is most likely hormone mediated (Xie and McCobb, 1998) Another system studied is a change in neuronal activity evoked by pilocarpine (Daoud et al, 1999). Pilocaipine is a cholinergic muscarinic agonist that crosses the blood-brain barrier. This system has been used as model for human temporal lobe epilepsy (Turski, 1983, 1984). After neurons were stimulated with this drug, the pre-mRNA processing of the splicing factor transformer2beta (tra2-beta) is changed (Daoud et al, 1999). The tra2beta gene generates at least five isoforms that can encode three open reading frames (Nayler et al, 1998a) from which two, htra2-betal and beta2, are translated into protein (Daoud et al, 1999). Neuronal stimulation

II. Change of splice site selection evoked by cellular activity The influence of cellular stimulation on alternative splice site selection has been mostly studied in the brain, because acute stress that is reflected in neuronal activity promotes neuroanatomic changes and increases the risk for neurodegeneration (MeEwen, 1999). In several model systems a change in alternative splice site selection after cellular stress has been observed. The gene of acetyleholinesterase generates two isoforms by alternative usage of an intron located at the end of the open reading frame (Kaufer et al, 1998). In normal brains, this "readthrough" (AchE-R) variant is hardly detectable. However,

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F i g u r e 2 A - D : The effect of whisker removal on alternative splice site selection in the rat barrel cortex. The experimental system. (A) shows the rat's face and its whisker pad. Some whiskers are already removed. (B) is a diagram of the whisker pad. (C) is a drawing of a rat brain, in which the somatosensory representation of the rat's body surface is indicated. The representation of the whisker pad is indicated. (D) is a cytochrome oxidase staining of the region in the somatosensory cdrtex S1. Each of the barrel shaped structures represents a single whisker. Removal of a whisker decreases the neuronal activity in this region ("barrel").

Figure 2E-I: Effect of whisker removal on allernadve splicing patterns in the barrel cortex. RNA was isolated after the whiskers were repeate4ly removed (deprived) for the time body indicated (4, 7, 14 days). As a control, the colateral side was used. Whiskers corresponding to this side were not removed. A statistical evaluation for three experiments is given on the right. Standard deviations are indicated. Location of primers and eDNA structure are schematically indicated for each gene. The altematively splice genes were: (E) Clathrin light chain B, exon EN (Stamm et al, 1992) (F) GABA A receptor, gamma2 subunit, alternative exon (Wang and Grabowski, 1996) (G) NMDA receptori, exon 21, (Hollmann et al, 1993; Zimmer et al, 1995) (H) NMDA receptori, exon 5, (Hollmann et al, 1993; Zimmer et al, 1995) (I) Doparnin D3 receptor, (Giros et al, 1991)

( F i g u r e 1 ) . A change of the splicing pattern of the NMDA receptorl has also been observed in a kindling model, in which neuronal activity is evoked by repeated electrical stimulation (Vezzani et al, 1995). Finally, induction of long term potentiation was shown to regulate syntaxin 3 isofomrs (Rodger et al, 1998). Together, these data indicate that after a strong burst of neuronal activity, different isoforms of splicing regulatory proteins are generated, which changes the processing of a number of genes. In both of these systems a change in splice site selection was observed after an increase of neuronal activity. We used the rat barrel cortex as a third model, in which neuronal activity is decreased. In

causes a shift from the beta1 to the beta3 isoform. Since both transformer proteins have different abilities to regulate splice site selection when tested in cotransfection experiments (Stamm et al, 1999), it is likely that this switch changes the alternative splicing patterns of several still unknown target genes. In agreement with this hypothesis, the splicing patterns of clathrin light chain B, exon EN, NMDA receptor1, exon 22 and the neuron specific exon of c-src are changed after pilocarpine treatment (Daoud et al, 1999). In addition, the splicing patterns of the fosB gene and the SR protein kinase clk2 change after pilocarpine induced neuronal activity as well

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rodents, the facial whiskers indirectly project to a region in the primary somatosensory cortex Sl (Figure 2A) (Woolsey and Van der Loos, 1970; Van der Loos and Woolsey, 1973). Each whisker is represented by an arrangement of cortical neurons that resembles a barrel. Stimulation of a whisker increases the activity of the neurons corresponding to this whisker, which can be detected histologically by cytochrome oxidase staining (Worig-Riley and Welt, 1980). When whiskers are completely removed on one side of the animal's snout, alternative splice site selection in several genes of the corresponding barrel cortex are changed (Figure 2B, C). This indicates that a change in cellular activity, not just an increase, results in differences of pre-mRNA processing. Cellular activity is not the only stress condition that alters splice site selection, as a change of alternative splicing patterns is also seen in response to temperature shock. Alternative splicing of the human neurofibromatosis type 1(NFI) gene (Ars et al, 2000) and the potato invertase gene (Bournay et al, 1996) are induced by cold shock, whereas a rise in temperature changes the splicing pattern of heat shock protein 47 (Takechi et al 1994) and the collagen Al gene of a Danlos syndrome patient (Weil et al, 1989). Finally, osmotic stress changes the pre mRNA processing of an adenovirus reporter gene (van Oordt et al, 2000). Together, these results show that a change in pre-mRNA splicing patterns is a common cellular adaptation to stress and cellular activity. This raises the question how the signal is transduced to the spliceosome.

Weiner, 1986).

III. Mechanisms of alternative splice site

Figure 3 : Model for a l t e r n a t i v e s p l i c e s i t e regulation (A) An exon (box) is recognized by binding of multiple proteins (A, B) on splicing enhancers located on the pre-mRNA (t, 2). Splicing of this exon is initiated by contact with components of the constitutive splicing machinery, shown here as the Ul snRNP (Ul). The formation of this complex is cooperative and involves protein:protein interactions, RNA:protein interaction and RNA: RNA interactions that are indicated by different colors. This complex can be regulated by several ways: (B) A protein C with higher affinity to the enhancer sequences can repress the exon usage by competing with proteins A and B for binding to the exon. (C) The recognition is concentration dependent, as an increase of the concentration of protein A could compete for binding with protein B. (D) Likewise protein B could be sequestered by a different protein D (E) Phosphorylation can influence the binding of individual factors assembling around exon enhancers (F) Tissue specific factors can recognize a protein in a cell type or tissue specific way.

Although there has been tremendous progress in elucidating the mechanisms regulating constitutive splicing, the rules governing alternative splice site selection still remain elusive. Since the general mechanism has been reviewed in this volume (Stoss et al, 2000), we concentrate on the question bow the recognition of an alternative exon can be modulated. All the elements on the RNA that govern splice site selection are only weakly conserved (Breitbart et al, 1987; Berget, 1995; Stamm et al, 2000). The high by fidelity observed in splicing is therefore achieved by the formation of a protein-RNA complex, that involved cooperative binding of several molecules (Figure 3). On the pre-mRNA, sequences known as exonic enhancers or silencers have been identified and were shown to bind to splicing regulatory proteins, such as SR proteins and hnRNPs (reviewed in: (Manley and Tacke, 1996; Cooper and Mattox, 1997; Hertel et al, 1997; Stamm et al, 2000; Stoss et al, 2000). SR proteins multimenze and can bind to components of the spliceosome, e.g. to the Ul snRNP particle (Wu and Maniatis, 1993). The 5' end of the Ul snRNA present in this particle hybridizes to the 5' site, which initiates the recognition of an exon (Zhuang and

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Figure 4: Subnuclear compartments visualized with endogenous antibodies of as GFPfusion proteins (ataxin-1) Subnuclear compartments include SC35-speckles (Fu and Maniatis, 1992) YTbodies containing YT521-B (Nayler et al, 2000) coiled bodies (Carmo-Fonseca et al, 1992) containing coilin, PML bodies (Grande et al, 1996) nuclear inclusion, such as the ones formed by ataxin-l (Skinner et al, 1997) and gems (Liu and Dreyfuss, 1996) formed by SMN (survival of motoneuron).

Figure 5 : Stress evoked by osmotic shock changes the intracellular localization of the splicing regulatory proteins htra2-betal. Cells were subject to 3 hours of hyperosmotic medium and then stained for htra2-betal (left). When compared to untreated cells, a translocation of splicing regulatory proteins is apparent.

As a result the initial recognition of an exon is regulated by protein:protein interaction (e.g. between different SR proteins), by protein:RNA interactions (e.g. between an SR protein and a splicing enhancer) and RNA:RNA interactions (e.g. between Ut snRNA and the 5' splice site) (Figure 3A). In vitro models suggest that the formation of this multi-protein:RNA complex involves cooperative binding of the individual components (Hertel and Maniatis, 1998) As a result, splice site recognition is dependent on the relative concentration of regulatory proteins and can be influenced change in concentration of a constitutively expressed factor can alter the composition of the protein complex forming around an enhancer, which can either decrease (Figure 3C) or increase exon usage (Mayeda and Kramer, 1992; Cรกceres et al, 1994; Wang and Manley, 1995; Coulter et al, 1997; Hanamura et al, 1998; Caputi et al, 1999) (iii) factors necessary for recognition can be sequestered by binding to a different protein (Figure 3D) (Nayler et al, 1998c; Hartmann et al, 1999) (iv) the interaction of proteins can be regulated by phosphorylation (Figure 3E) (Fu, 1995; Colwill et al, 1996; Duncan et al, 1997; Prasad et al, 1999) (v) tissue specific factors can recognize exons in a cell type specific way (Figure 3F) (Jensen et al, 2000; Polydorides et al, 2000). A number of studies have revealed that transcription and splice site selection (Cramer et al, 1999) are occurring

concomitantly in a large complex that was termed 'RNA factory' (McCracken et al, 1997) or transcriptosomal complex (Corden and Patturajan, 1997), which is probably associated with components the nuclear matrix (Nayler et al, 1998c; Bode et al, 2000). Various components of this complex are stored in subnuclear compartments (Figure 4) and can be released into the nucleoplasma by regulatory mechanisms, such a phosphorylation. One of the best studied example of this domains are nuclear speckles in which splicing factors are stored until they are released e.g. by phosphorylation (Spector, 1993; ltuang and Spector, 1996; Misteli et al, 1997; Misteli et al, 1998). Nuclear factories and storage compartments are dynamically linked to RNA polymerase activity. Speckles change their morphology under the influence of transcriptional inhibitors (Carmo- Fonseca et al, 1992; Misteli et al, 1998; Nayler et al, 1998b; Misteli and Spector, 1999). This suggests the existence of a dynamically regulated nuclear architecture The importance of proper regulation is apparent under supporting the compartmentalization of the nucleus (Nakayasu and Berezney, 1989; Jackson et al, 1993; Ma et al, 1998) cellular stress conditions. Activation of the MKK(3/6)-p38 pathway changes the subcelluar localization of several factors involved in premRNA processing and ultimately results in a change in splice site selection (van Oordt et al, 2000).

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Figure 6 S i g n a l t r a n s d u c t i o n a n d s p l i c i n g : A m o d e l An overview of possible signal transduction pathways and their links to the spliceosome is shown. Extracellular stimuli and factors are indicated on the top and are boxed. Some of the receptors are shown below. Their phosphorylation (YP) leads to activation of src tyrosine kinases, which likely stimulate nuclear tyrosine kinases (nPTK), such as abi, Rak, Fes, Fer, Weel and SiklBrk (Pendergast, 1996). These proteins phosphorylate nuclear proteins, which affects splice site selection by changing the composition of splice site enhancer complexes. Similar, a change in serine phosphorylation can change the composition of splice site enhancer complexes. Several nuclear kinases and phosphatases have been identified, but the upstream kinases and signals renainn to be determined (Stojdl and Bell, 1999). Nitric oxide (NO) and natriuretic peptides activate guanylyl cyclase either in the plasma membrane (GC-P) or in the cytosol (GC-S), which leads to the activation of phospho-kinase G-I (PKG-I) mat phosphorylates splicing factor 1 (SF 1) (Wang et al, 1999) PTKR: phosoph-tyrosine receptor; MIRR: multichain immune recognition receptor; FAK: focal adhesion kinase; OPCR: G-protein coupled receptor; GC-P: OTP cyclase, plasma membrane bound; GC-S GTP cyclase, soluble; PKG-l: Phospho kinase G-l; 505: son of sevenless; clk:cdc2 like kinase; SRPK: SR protein kinase; PP: protein phosphatase; SAF-B: scaffold attachment factor B; rSLM-2: rat 5AM68 like molecule; polIl: RNA polymerase II; SR: protein: serine-arginine-rich protein; Ul, U2: Ul, U2: U1-U4 snRNP; U2AF: U2 auxiliary factor

Similar results are observed when primary neuronal cultures are subjected to stress evoked by osmotic shock. In this system, the splicing factor htra2-betal (Beil et al, 1997) is translocated into the neurites and the alternative splicing patterns of several genes change (Figure 5).

was shown that pre-mRNA processing is influenced by the tyrosine kinase activity of src (Neel et al, 1995; Gondran and Dautry, 1999). Since src is anchored to the cell membrane, the question how the phosphorylation signal reaches the nucleus needs to be addressed. Only a limited number of tyrosine kinases have been identified in the nucleus, among them AbI, Rak, Fes, Fer, Weel and SiklBrk (reviewed in (Wang, 1994; Pendergast, 1996). Most of these kinases shuttle between nucleus and cytosol and it is possible that they phosphorylate proteins participating in pre-mRNA processing. For example, Sik/BRK was shown to phosphorylate Sam68, a process which regulates the RNA binding activity of Sam68 (Derry et al, 2000) and most likely the composition of the protein complex forming around Sam68 (Figure 3E)

IV. How are signals transduced to the spliceosome? Despite the accumulating evidence that cellular activity influences splice site selection, the molecular mechanism that underline this phenomenon remain to he determined. It is clear however, that phosphorylation plays a fundamental role in regulation. The majority of proteins regulating alternative splicing are phosphorylated and it

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Transcriptional augrnentation, modulation of gene expression by secifold/matrixattached regions (S/MAR elernents). Crit Rev. Eukar. Gene Exp. 10, 73-90. Bournay, AS., Hedley PE., Maddison A., Waugh R., and Machnay. G.C. (1 9 9 6 ). Exon skipping induced by cold stsess in a potato mvemse gene transcript. N u c l . Aci ds R e s . 24, 2347-2351. Breirbat, RE., Anderadis A., and Nalal-Ginard. B. (1 9 8 7 ). Alternative splicing, a ubiquitous meebanism for tae generation of multiple protein isoforms from single genes. A n n u . R e v . B i o c h e m 56, 467-495. Cáceres, JS. Stamm, DM. Helfinan, and Kralner. AR. (1 9 9 4 ) Regulation of alternative splicing in viso by overexpression of antagonistic splicing isetors. S c i e n c e . 265, 1706-1709. Caputi, M, Mayeda A., Kedner AR., and Zahler. AM. (1 9 9 9 ) hnRNP A/B proteins ane required for inhibition of HIV-l pre-mRNA splicing. EMBO J. 18, 40604067. Carrno-Fonseea, M, Pepperkol R., Carvalno M.T., and Larnond A.. (1 9 9 2 ) Transcription dependent cebealization of the Ut, U2, U4/5 and US snRNPs in coiled bathes. J . C e l l B i o l 117, 1-14. Chan, RC, and Black. DL, (1 9 9 7 ) The polypynnadine tact binding protein binds upsteam of neural cell-specific c-src exon N1 to repress the splicing of the intron downstream. M o l . C e l l . B i o l 17, 4667-4676. Cern, T, Damaj B.B, Herrera C, Lasko P, and Ricbard S, (1 9 9 7 ) Self association of the single-KIl-domain family members Sam68, GRP33, and Qk1, role of the KH domain. M o l . C e l l . B i o l 17, 5707-5718. Colwill, K, Pawson T, Andrews B, Prasad J, Manley JL, Bell J.C, and Duncan PI, (1 9 9 6 ). The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution. EMBO J. 15, 265-275. Cooper, TA, and Mattox W, (1 9 9 7 ) The regulation of splicesite selection, and its role in human disease. Am J. Hum Genet 61, 259-266. Comen, J.L, and Pattunajan. M. (1 9 9 7 ) A CJD function linking transcription to splicing. Trends B i o c h e m S c i , 413-419. Coulter, L.R, Landree MA, and Cooper. TA, (1 9 9 7 ) Identification of a new class of exonic splicing enhancers by in vivo selection [published ematum appears in Mol Cell Biol 1997 Jun;] 7(6)34681. M o l . C e l l . B i o l 17, 2143-2150. Cousineau, B, Lawrence S, Smith D, and Belfort M, (2 0 0 0 ) Retrorsansposition of a bacterial group 11 intron. Nature. 404, 1018-1021. Cramer, P, Cáceres J.F, Cazalla D, Ksdener S, Mum A.F, Baralle FE, and Kornblihtt A.R, (1 9 9 9 ) Coupling of ranseription with alternative splicing, RNA polII promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer. M o l C e l l . 4, 251-258. Daoud, R, Berzaghi M, Siedler F, Hübener M, and Stamm. S, (1 9 9 9 ) Activity dependent regulation of alternative splicing patterns in therat brain. E u r . J . N e u r o s c i 11, 788-802. Derry, JJ, Richard S, Valderrama Carvajal H, Ye X, Vasiouluan V, Coclarane A.W, Chen T, and Tyner A.L, (2 0 0 0 ) Sik (BRK) phosphorylates Sam68 in the nucleus and negatiely regulates its RNA binding ability. M o l C e l l B i o l 20, 6114-6126. Duncan, PI , Stojdl D.F, Marius R.M, and Bell. J.C. (1 9 9 7 ) In vivo regulation of alternative pre-mRNA splicing by the Clk1 protein kinase. M o l . C e l l . B i o l . 17, 5996-6001. Eissa, NJ, Stauss AJ, Haggerty C.M, Coon E.K, Chu S.C, Moss. J, (1 9 9 9 ). Alternative splicing of human inducible

(Chen et al, 1997; Hartmann et al, 1999). The composition of this complex will most likely influence splice site seleclion by controlling the recognition of exon enhancers (Figure 3). Furthermore, serine phosphotylation of SF1 (Berglund et al, 1998; Rain et al, 1998), a factor that recognizes the branch point and is therefore important for the formation of the spliceosomal A complex, was shown to be regulated by PKG-I (Wang et al, 1999). This kinase is activated by cGMP. The cGMP level itself can be regulated by a membrane bound guanyl cyclase receptor that is activated by natriuretic peptides or by a cytoplasmic guanyl cyclase which is activated by nitric oxide (NO). Phosphorylation of SF1 on Ser2O inhibits the SF1 -U2AF65 interaction, leading to a block of pre-spliceosome assembly. One of the best studied class of splicing regulatory proteins are SR proteins hat are regulated by serine phosphorylation. Several SR ) protein kinases have been identified (reviewed by Stoss et al, 2000, this volume) and the identification of their ipstream regulatory kinases will help to identify the signal -transduction pathways regulating pre-mRNA processing.

IV. Conclusions The regulation of transcription by signal transduction pathways is well documented. Since at least 30% of all human genes are subject to alternative splicing, regulation of splice site selection after an extracellular signal seems to be another important mechanism to regulate gene expression. Although it is clear that phosphorylation events are involved in mediating this signal, the identification of the molecular players remains the challenge of the future.

Acknowledgments We thank Athena Andreadis, Tom Cooper, Konstantin Hossrnann, Otinther Mies, Oliver Nayler, and Brunhilde Wirth for helpful discussions and support. Supported by the Max-Planck Societey, the DFG (Sta 399/2- 1), the HSFP (RG 562/96), the EU (Bio4-98-0259) and the Theodore and Vada Stanley Foundation.

References Ars, E., Sena E., de la Luna S., Estivill X., and Laxen. C. (2 0 0 0 ) . Cold shock induoes the insertion of a cryptic exon in the neorofibronatosis type 1 (NF1) mRNA. N u c l . A c i d s R e s . 28, 1307-1312. Beil, B., Screaton G, and Stamm S. (1 9 9 7 ) Molecular cloning of htra2-beta-l and htra2-beta-2, two human homologues of tra-2 generated by alternative splicing. DNA and C e l l B i o l 16, 679690. Berget SM. (1 9 9 5 ). Exon Recugnition in vertebrate splicing. J . B i o l C h e m . 270, 2411-2414. Betglund, JA., Flerning M.L., and Rosbath M. (1 9 9 8 ) The KH domain of the branclipoint sequence binding protein determines specificity for the pre-mRNA branchpnjnt sequence. RNA. 4, 998-1006. Black, D.L. (1 9 9 5 ) Finding splice sites within a wilderness of RNA. RNA. 1, 763-771. Bode, J., Benham CA.K., and Mielke. C. (2 0 0 0 )

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Daoud et al: RNA splicing and signal transduction

nitric-oxide synthase messenger-RNA – tissue specific regulation and induction by cytokines. J . B i o l . C h e m . 271, 27184-27187. Fu, XD, (1 9 9 5 ) The superfainily of aginine/serinerich splicing factors. RNA 1, 663-680. Fu, XD, and Mattiatis. T. (1 9 9 2 ). Isolation of a complementary DNA that encodes the mammalian splicing factor 5C35. S c i e n c e . 256, 535-538. Gao.Q.S, Memmott J, Lafyatis R, Stamm S, Screaton G. and Andreadis A, (2 0 0 0 ) Complex regulation of tau exon 10, whose misaplicing causes frontotemporal dementia. J . Neurochem. 74, 490-500. Gims, B, Mantres MP, Pllon C, Sokoloff P, and Schwariz JC.(1 9 9 1 ). Shorter vatiants of the D3 dopamine receptor produced through various patterns of altemative splicing. B i o c h e m B i o p h y s . R e s . C o m . 176, 1584-1592. Gondran, P, and Dauty. F, (1 9 9 9 ) Regulation of mRNA splicing and transport by the tyrosine kinase activity of src. O n c o g e n e . 18, 2547-2555. Grabowski, P. (1 9 9 8 ) Splicing regulation in neurons, tinkering with cell-specific control. C e l l . 92, 709-712. Grande, MA, van der Kraan I, van Steensel B, Schul W, de The H, van der Voort H.T, de Jong L, and van Driel R. (1 9 9 9 ) PMLcontaining nuclear bodies, their spatial distribution in relation to other nuclear components. J. C e l l . B i o c h e m . 63, 280291. Guo, W, Mulligan GJ, Wormsley S, and Helfman. D.M. (1 9 9 1 ) Alternative splicing of !-tropomyosin premRNA, cis acting elements and cellular factors that block the use of a skeletal muscle exon in nonmuscle cells. G e n e s D e v . 5, 2096-2107. Hanamura, A, Cáceres J.F, Mayeda A, Franza B.R, and Krainer. A.R. (1 9 9 8 ) Regulated tissue-specific expression of antagonistic pre-mRNA splicing factors. RNA. 4, 430444. Hanke, J, Brett D, Zastrow I, Aydin A, Delbruck S, Lehmann G, Luft F, Reich J, and Bork. P. (1 9 9 9 ) Alternative splicing of human genes, more the role than the exception? Trends Genet 15, 389-390. Hattmann, AM, Nayler O, Schwaiger F.W, Obermeier A, and Stamm. S. (1 9 9 9 ) The interaction and colocalisation of SAM68 with the splicing associated factor YT52 1-B in nuclear dots is regulated by the Src family kinase p59fyn . M o l B i o l C e l l , 3909-3926. Herbert A, and Rich. A. (1 9 9 9 ) RNA processing and the evolution of eukaryotes. Nat Genet 21, 265-269. Hertel, K.J, Lynch K.W, and Maniatis. T. (1 9 9 7 ). Common themes in the function of transcription and splicing enhancers. C u r r . O p i n C e l l B i o l 9, 350-357. Henel, KJ, and Maniatis T. (1 9 9 8 ) The function of multisite splicing enhancers. M o l . C e l l . 1, 449455. Holimano, M, Boulter J, Maron C, Beasley L, Sullivan J, G. Pecht and Heinemann. S. (1 9 9 3 ). Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor. Neuron. 10, 943-954. Huang, S, and Spector. DL. (1 9 9 6 ) Intron-dependent recruitment of pre mRNA splicing factors to sites of transcription. J . C e l l B i o l . 133, 719-732. Jackson, DA, Hassan A.B, Errington RI, and Cook PR. (1 9 9 3 ). Visualization of focal Sites of transcription within human nuclei. EMBO J. 12, 1059-1065. Jensen, K.B, Drege B.K, Stefani G, Zhong R, Buchanovich RJ, Okano H.J, Yang Y.Y, and Darnell. RB. (2 0 0 0 ) Nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability. Neuron. 25, 359-371. Kaufer, O, Friedman A, Seidman S, and Soreq. H. (1 9 9 8 )

Acute stress facilitates long-lasting changes in choinergic gene expression. Nature. 393, 373-377. König, H, Ponta H, and Herrlich P. (1 9 9 8 ) Coupling of signal transduction to alternative pre-mRNA splicing by a composite splice regulator. EMBO J. 10, 2904-2913. Kawesak, M, Reiss I, and Cooper D.N. (1 9 9 2 ) The mutational sponrurn of single base-pair substitutions in mRNA splice junctions of human genes, causes and consequences. Hum. Genet 90, 41-54. Liu, Q, and Dreyfuss G. (1 9 9 9 ) A novel nuclear structure containing the survival of motor neurons protein. EMBO J. 15, 3555-3565. Lorson, CL, Hahnen E, Androphy EJ, and Wirth B. (1 9 9 9 ) A single nucleutide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. P r o c . N a t Acad. Sci. USA . 96, 6307-6311. Ma H, Samarabandu J, Devdhar R.S, Acharya R, Cheng P.C, Meng C, and R. Berezney. (1 9 9 8 ) Spatial and temporal dynamics of DNA replication sites in mammalian cells. J . C e l l B i o l 143, 1415-1425. Manley, JL, and Tacke. R. (1 9 9 6 ) SR proteins and splicing control. Genes and Dev. 10, 1569-1579. Mayeda, A, and Krainer. A.R. (1 9 9 2 ) Regulation of alternative pre mRNA splicing by hnRNP A1 and splicing factor SF2. C e l l . 68, 365-375. McCracken, S, Fong N, Yankulov K, Ballantyne S, Pan G, Greenblatt J, Patterson S.D, Wickens M, and Bentley DL. (1 9 9 7 ) The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature. 385, 357-361. McEwen, B.S. (1999) Stress and hippocampal plasticity. Annu. R e v . Neurosci. 22, 105-122. Mironov, A.A, Fickett J.W, and Gelfand MS. (1 9 9 9 ) Frequent alternative splicing of human genes. G e n o m e R es, 9, 1288-1293. Misteli, T, Cáceres JF, Clement J.Q, Krainer AR, Wilkinson M.F, and Spector D.L. (1 9 9 8 ) Serine phosphorylation of SR proteins is required for their recruitment to sites of transcription in vivo. J . C e l l . B i o l 143, 297-307. Misteli, T, Cáceres JF, and Spector D.L. (1 9 9 7 ) The dynamics of a pre-mRNA splicing factor in living cells. Nature. 387, 523-527. Misteli, T, and Spector. DL. (1 9 9 9 ) RNA polymerase II targets pre-mRNA splicing factors to transcription sites in vivo. M o l . C e l l . 3, 697-705. Nakai, K, and Sakamoto. H. (1 9 9 4 ) Construction of a novel database containing aberrant splicing mutations of mammalian genes. Gene.14, 171-177. Nakayasu, H, and Berezney. R. (1 9 8 9 ) Mapping replicational Sites in the eucatyotic cell nucleus. J . C e l l B i o l 108, 111. Nayler, O, Cap C, and Stamm S. (1 9 9 8 a ) Human transformer2-beta gene: Complete nucleotide sequence, chromosomal localisation and generation of a tissue specific isoform. G e n o m i c s . 53, 191-202. Nayler, O, Haitmann A.M, and Stamm S. (2 0 0 0 ) The ERrepeat protein YT521-B localizes to a novel subnuclear compartment J . C e l l B i o l 150, 949-961. Nayler, O, Schnorrer F, Stamm S, and Ullrich A. (1 9 9 8 b ) The cellular localization of the murine serine/arginine rich protein kinase CLK2 is regulated by serine 141 autophosphorylation. J B i o l . Chem. 273, 3434134348. Nayler, O, Strätling W, Bousquin JP, Slagljar L, Undernann L, Jasper H, Hartrmann A.M, Fackelmayer F.O, Ullrich A, and Stamm S. (1 9 9 9 ) SAF-B couples transcription and pre-mRNA splicing to SAR/MAR elements. N u c l e i c

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A c i d s R e s 26, 3542-3549. Neel, H, Gondran P, Weil D, and Dautry F. (1 9 9 5 ) Regulation of pre-mRNA processing by src. Curr. B i o l , 413422. Pendergast, A.M. (1 9 9 6 ) Nuclear tyrosine kinases: from Abl to WEE1. C u r r . O p i n . C e l l . B i o l 8, 174-181 Polydorides AD, Okano HJ, Yang Y.YL, Stefani G, and Darnell R.B. (2 0 0 0 ) A brain-enriched polypyrimidine tractbinding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing. P r o c N a t l Acad. Sci USA. 97, 63506355. Prasad, J, Colwill K, Pawson T, and Manley J. (1 9 9 9 ) The protein kinase Clk/Sty directly modulates SR protein activity, both hyper and hypophosphorylation inhibit splicing. M o l . C e l l . B i o l . 19, 6991-7000. Rain, JC, Rafi Z, Rhani Z, Legrain P, and Krämer. A. (1 9 9 8 ) Conservation of functional domains involved in RNA binding and protein-protein interactions in human and Saccharomyces cerevisiae pre-mRNA splicing factor SF1. RNA. 4, 551-565. Reddy, R. (1 9 8 9 ) Compilation of small nuclear RNA sequences. Meth. E n z y m o l 180, 521-532. Rodger, I, Davis S, Laroche S, Mallet I, and Hicks. A. (1 9 9 8 ) Induction of Long-Term Potentiation in vivo regulates alternative splicing to alter syntaxin 3 isoform expnession in rat dentate gyros. J . Neurochem 71, 666675. Shifrin, VI, and Neel. B.G. (1 9 9 3 ) Growth factor-inducible alternative splicing of nontransmembrane phosphotyrosine phosphatase PTP-1B pre-mRNA. J . B i o l . C h e m 268, 25376-25384. Skinner, PJ, Koshy B.T, Cummings CJ, Klement IA, Helin K, Servadio A, Zoghbi H.Y, and Orr H.T. (1 9 9 7 ) Ataxin-1 with expanded glutamine tract alters nuclear matrixassociated structure [published erratum appears in Nature 1998 Jan 15;391(6664), 30 Nature. 389, 971-974. Smith, K.P, Moen P.T, Wydner KL, Coleman JR, and Lawrence JB. (1 9 9 9 ) Processing of endogenous premRNAs in association with SC-35 domains is gene specific. J . C e l l B i o l 144, 617629. Spector. DL. (1 9 9 3 ) Macromolecular domains within the cell nucleus A n t R e v . C e l l Biol 9, 265-315. Sprengel, R, Higuchi M, Monyer H, and Seeburg PH, (1 9 9 9 ) Glutamate receptor channels, a possible link between RNA n in the brain and epilepsy. A d v . N eu r o l 79, 525534. Stamm, S, Casper D, Dinsmore I, Kaufmann CA, Brosius I, Helfman A. (1 9 9 2 ) Clathrin light chain B, gene structure and neuron-specific splicing. N u c l . Acids R e s 20, 5097-5l03 Stamm, S, Casper D, Hanson V, and Helfman. D.M, (1 9 9 9 ) Iteg of the neuron-specilic exon of clathrin light chain B. M o l . B r a i n R e s 64, l08-l18. Stamm, S, Zhu I, Nakai K., Stoilov P., Stoss O., and Zhang M.Q. (2 0 0 0 ) An alternative exon database (AFDB) and its statistical in press. 19. Sternfeld, M, Shoham S, Klein O, Flores- Flores C, Evron T, Idelson GH, Kitsberg D, Paniek JW, and Soreq. H. (200 0) Excess "read-through" acetyleholinestease attenuates but the variant intensifies neurodeterioration correlates. Proc Natl Acad USA. 97, 8647-8652. Stickeler, E, Kittrell F, Medina D. and Berget S.M. (1 9 9 9 ) Stage-specific changes in SR splicing factors and alternative mammary tumorgenesis. O n c o g e n e . 18, 3574-3582. Stojdl, D.F, and Bell JC. (1 9 9 9 ) SR protein kinases, the splice of life. Biochem Cell Biol 77, 293-298. Stoss, O, Stoilov P, Daoud R, Doniat U, Hartmann A.M,

Olbrich M, and Stamm S. (2 0 0 0 ) Misregulation of premRNA splicing that causes human diseases. Gene Ther M o l B i o l 3: in press Taltechi, H, Hosokawa N, Hiraynsli K, Nagata K, (1 9 9 4 ) Alternative 5' splice site selection induced by heat shock B i o l . 14, 567-575. Turski, W.A, Cavalheiro, EA, Schwartz, M, Czuczwar SJ, Kleinrok Z and Turski, L. (1 9 8 3 ) Limbic seizures produced by pilocarpine in rats. Behavioral, electronencephalographic and neurophathological study. Brain Res 321, 237-253. Turski, WA, BA. Cavaiheso, ZA. Bortolloto, L. Sehwtirtz, and L Turski. (1 9 8 4 ) . Seizures produced by pilocarpine in mice. A behavioral, electroencephalographic and morphological analysis. Brain Ret 321, 237-253. Van der Loos, H, and Woolsey TA. (1 9 7 3 ) Somatosensory Cortex, Structural Alterations following early injury to sense organs. S c i e n c e . 179, 395-398. van Oordt W, Diaz-Meco M.T, Lozano J, Krainer AR, Moscat J. and Cáceres. J.F. (2 0 0 0 ) The MKK 3/6-p38-signaling cascades alters the subcellular distribution of hnRNPA1 and modulates alternative splicing regulation. J . C e l l B i o l 149, 307-316. Varani, G, and Nagal K. (1 9 9 8 ) RNA recognition by RNP proteins during RNA processing. Annu. Rev. B i o p h y s B i o m o l S t r u c t 27, 407445. Vezzani, A, Speciale C, Vedova ED, Tamburin M, and Benatti L. (1 9 9 5 ) Alternative splicing at the C-terminal but not the N-terminal domain of the NMDA receptor NRI is altered in the kindled hippocampus. E u r . J . N e u r o s c i . 7, 2513-2517. Wang, I, and Manley IL. (1 9 9 5 ) Overexpression of the SR proteins ASFISF2 and SC3S influences alternative splicing in vivo in diverse ways. RNA. 1, 335-346. Wang, IY. (1994) Nuclear protein tyrosine kinases. Trends B i o c h e m S c i 19, 373-376. Wang, X, Bruderer S, Rafi Z, Xue I, Milburn P.J, Krämer A, and Robinson PJ. (1 9 9 9 ) Phosphorylation of splicing factor SF1 on Ser20 by cGMP-dependent protein kinase regulates spliceosome assembly. EMBO J. 18, 45494559. Wang, Z, and Grabowski. P. (1996) Cell- and stage specific splicing events resolved in specialized neurons of the tat cerebellum. RNA. 2, 1241-1253. Weil, D, D’Alessio M, Ramirez F, Steinmann B, Wirtz R.W. M.K, Glanville, and Hollister. D.W. (1 9 8 9 ) Temperaturedependent expression of a collagen splicing defect in the fibroblasts of a patient with Ehlers-Danlos syndrome type VII. J . B i o l C h e m . 204, 16804-16809. Wollfe, A.P, and Matzke. M.A. (1 9 9 9 ) Epigenetics: Regulation through repression. S c i e n c e . 268, 481-486. Wong-Riley, M.T.T, and Welt C. (1 9 8 0 ) Histoehernical iltanges in cyteebrom oxidase of conical barrels after vibrissal removal in neonatal and adult mice. P r o c . N a t l Acad. Sci USA 77, 2333-2337. Woolsey, TA, and Van der Loos H. (1 9 7 0 ) The structural organization of layer IV in the somatosensory region (S1) of mouse cerebral cortex. Brain R e s 17, 205-242. Wu, IY, and Maniatis. T. (1 9 9 3 ) Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. C e l l . 75, 1061-1070. Xie, I, and McCobb D.P, (1 9 9 8 ) Contml of alternative splicing of potassium channels by stress hormones. S c i e n c e . 280, 443446. Zhang, LI, Liu W, and Grabowski. P. (1 9 9 9 ) Coordinate repression of a trio of neuron-specific splicing events by the splicing regulator PTB. RNA. 5, 117-130.

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Daoud et al: RNA splicing and signal transduction

Zhuang, Y, and Weiner. A.M. (1 9 8 6 ) A compensatory base change in Ul snRNA suppresses a 5' splice site mutation. C e l l , 827-835. Zimmer, M, Fink T.M, Franke Y, Lichter P, and Spiess J.

(1 9 9 5 ) Cloning and structure of the gene encoding the human N-methyl-D aspartate receptor (NMDAR1) Gene. 159, 219-223.

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Herpes Simplex Virus vector-based gene therapy for malignant glioma Review Article

Edward A. Burton and Joseph C. Glorioso* University of Pittsburgh School of Medicine, Department of Molecular Genetics and Biochemistry, Pittsburgh, Pennsylvania 15261, USA _________________________________________________________________________________________________ * Correspondence: Joseph C. Glorioso, Ph.D., Professor and Chariman, Department of Molecular Genetics and Biochemistry, University of Pittsburgh, School of Medicine, E1240 Biomedical Science Tower, Pittsburgh, PA 15261; Tel: (412) 648 8105; Fax: (412) 624 8997; e-mail: glorioso@pitt.edu Key Words: Herpes Simplex Virus vector, gene therapy, malignant glioma, HSV-TK, bystander effect, TNF!

Received: 3 October 2000; accepted: 9 October 2000

Summary Conventional therapies have made little impact on the poor prognosis associated with malignant glioma. Recent advances in the construction of replication-defective Herpes Simplex Virus (HPV)-based vectors have offered an opportunity to explore the therapeutic affects of simultaneous multiple transgene delivery to these tumours. Identification of co-operative molecular targets has enabled the rational selection of therapeutic transgene combinations. Exploiting the large capacity of HSV for the insertion of multiple transgenes, the high infectivity of HSV for many cell types and the ability to manufacture vectors of high tire and purity, a series of combination gene therapy vectors have been developed and tested in animal models of malignant glioma. Recent work has been established the principle that multi-modal therapies, including both radiosurgery and combination multi-gene therapy, are superior to single molecular interventions. Eradication of some experimental gliomas has been possible using a multi-modal approach, which provides optimism that further developments may yield reagents that prove therapeutically useful in the neuro-oncology clinic.

intervention represents a valid and logical approach to developing novel anti-cancer therapeutics. Gliomas are attractive targets for delivery of therapeutic transgenes by genetically engineered vectors; the tumours are highly localised as, although they are usually invasive locally at the tumour margin (McComb and Bigner, 1984), they only metastasise under unusual circumstances (al Rikabi et al, 1997; Hsu et al, 1998). This enables direct inoculation of the tumour or post-operative tumour cavity with recombinant vector, circumventing many challenges currently associated with systemic transgene delivery. In this review, we consider advances in the construction of herpes simplex-based gene therapy vectors, discuss the types of therapeutic transgenes whose deliver to tumours may be desirable, and review the results from pre-clinical experimental treatment trials using these approaches.

I. Introduction Primary CNS neoplasms of adults affect approximately 8.2/100,000 population annually in the USA (Walker et al, 1985). About half of these tumours are highly aggressive malignant gliomas, and are associated with a median survival of four to twelve months following diagnosis (Jubelirer, 1996; Lopez Gonzalez and Sotelo, 2000). Treatment is palliative; despite recent advances in surgery, radiotherapy and chemotherapy, little impact has been made on the poor prognosis associated with this malignancy (West et al, 1983; Kelly et al, 1984; Jubelirer, 1996; Lang et al, 1999; Lopez Gonzalez and Sotelo, 2000). Exploration of novel treatment strategies is therefore of importance. Cancer is a genetic disease (Collins, 1998; Hill et al, 1999; Hanahan and Weinberg, 2000), oncogenic mutations usually being acquired rather than inherited. Genetic

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encoding structural components of the virion (Honess and Roizman, 1974; Honess and Roizman, 1975; Roizman and Sears, 1996). Only ICP4 and ICP27 are essential for expression of E and L genes, and hence viral replication. ICP4 is the major regulatory protein of the virus. It functions as a repressor or activator of viral and cellular transcription by contacts with multiple basal factors; it is necessary for the transition of viral transcription from the IE to the E phase (O'Hare et al, 1988; Smith et al, 1993; Gu et al, 1995; Kuddus et al, 1995; Carrozza and DeLuca, 1996). ICP27 regulates the processing of many viral and host mRNAs, and modulates the activity of ICP0 and ICP4. It contributes to efficient E and L gene expression (Hardy and Goldin, 1994; Hardwicke and Goldin, 1994; Brown et al, 1995; Soliman et al, 1997; Mears and Rice, 1998; Sandri Goldin, 1998). The promiscuous transactivator ICP0 contributes to high expression levels of viral genes but is not essential for viral replication in vitro (Jordan and Schaffer, 1997; Samaniego et al, 1997). ICP22 contributes to efficient L gene expression in a cell-type dependent manner and has multiple biochemical functions (Poffenberger et al, 1994; Rice et al, 1995; Prod'hon et al, 1996; Leopardi et al, 1997; Bruni and Roizman, 1998). ICP47 does not have a transcriptional regulatory role, but rather has been reported to interfere with the function of a transporter that is responsible for loading MHC class I molecules with antigenic peptides (York et al, 1994; Fruh et al, 1995; Hill et al, 1995; Lacaille and Androlewicz, 1998). Toxicity associated with lytic wild-type HSV infection in the brain can be prevented by blocking viral replication. As E and L gene expression, and therefore replication, is fully dependent upon the expression of IE genes, generation of replication-incompetent vectors can be accomplished by disruption of one or other essential IE gene, ICP4 or ICP27. For example, an ICP4 null mutant is unable to replicate in non-complementing cells in culture (DeLuca et al, 1985). However, the IE gene products, with the exception of ICP47, are all toxic to host cells (Johnson et al, 1994; Wu et al, 1996; Samaniego et al, 1998). Infection with an ICP4 null mutant results in extensive cell death in the absence of viral replication, which is caused by over-expression of other IE gene products, some of which are negatively regulated by ICP4 (DeLuca et al, 1985; Krisky et al, 1998; Moriuchi et al, 2000). To prevent cytotoxicity, a series of vectors has been generated that are multiply deleted for IE genes. Quintuple mutants, null for ICP0, ICP4, ICP22, ICP27 and ICP4, have been produced, are entirely non-toxic to cells and are able to persist for long periods of time (Samaniego et al, 1998). However, these vectors grow poorly in culture and express transgenes at very low levels in the absence of ICP0. Retention of the trans-activator ICP0 allows efficient expression of viral genes and transgenes (Jordan and Schaffer, 1997), and allows the virus to be prepared to high titre. Recent work has shown that the

II. HSV as a gene therapy vector Herpes Simplex is an enveloped double-stranded DNA virus (reviewed in Roizman and Sears, 1996). It is an attractive candidate gene therapy vector relating to multiple applications, for the following reasons: (i) It has a broad host cell range; the cellular entry receptors HveA (Montgomery et al, 1996) and HveC (Geraghty et al, 1998) are widely expressed cell surface proteins of unknown function. (ii) It is highly infectious - it is possible to transduce 70% cells in vitro at a low multiplicity of infection (1.0), with a replication-defective vector (Moriuchi et al, 2000). (iii) Non-dividing cells may be efficiently transduced and made to express transgenes (iv) Of the 84 known viral genes contained within the 152kilobase pair genome, approximately half are non-essential for growth in tissue culture. This means that multiple therapeutic transgenes can be accommodated, by replacing dispensable viral genes (Krisky et al, 1998). In the majority of circumstances, this does not adversely affect the ability of the virus to replicate to high titre in vitro. (v) Recombinant HSV may readily be prepared to high titre and purity without contamination from wild-type recombinants (vi) The virus can exist in a latent state within nuclei of infected neurons for the lifetime of the host. During latency, the virus adopts a circular or concatemerised configuration, remains episomal, and has minimal effects on host cell metabolism. A limited number of viral genes are chronically expressed during latency; this phenomenon could potentially be exploited for the stable long-term expression of therapeutic transgenes in neurons (Wolfe et al, 1999). The viral genome is organised into long (UL) and short (US) unique segments flanked by inverted repeats (Figure 1; reviewed in Roizman and Sears, 1996). Genes contained within the unique segments are present at one copy per genome; genes present within the repeats (including ICP0 and ICP4 â&#x20AC;&#x201C; see below) are present at two copies per genome. During infection, viral genes are expressed in a tightly regulated, interdependent temporal sequence (Honess and Roizman, 1974; Honess and Roizman, 1975; Roizman and Sears, 1996) (Figure 2). Transcription of the five immediate-early (IE) genes, ICP0, ICP4, ICP22, ICP27 and ICP47 commences on viral DNA entry to the nucleus. Expression of these genes is regulated by promoters that are responsive to VP16, a viral structural protein that is transported to the host cell nucleus with the viral DNA. VP16 is a potent transactivator that associates with cellular transcription factors and binds to cognate motifs within the IE promoter sequences. Expression of IE genes initiates a cascade of viral gene expression, resulting first in transcription of early (E) genes, which primarily encode enzymes involved in DNA replication, followed by late (L) genes mainly

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Figure 1 A. A schematic representation of the HSV-1 genome (not to scale). The inverted repeats flanking the unique long (UL) and short (US) segments of the genome are indicated as ab – b’a’ and a’c’ – ca, respectively. The approximate positions and orientations of those HSV1 genes discussed in the text are shown. B. A series of engineered viruses deleted for immediate early genes and expressing anti-tumour transgenes were generated. The name of each virus is shown to the left of the schematic; the viruses used in the studies reviewed here are referred to by name throughout the text. The diagrammatic genomic map of each vector is aligned with that of the HSV-1 genome in Figure 1A to facilitate comparison between viruses. Each schematic depicts the positions and types of foreign transgenes inserted into each construct, and which subset of immediate-early genes has been inactivated.

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Figure 2. Flow chart depicting the cascade of regulatory events that result in ordered sequential expression of HSV-1 genes during wildtype infection. In order to proceed to E and L gene expression from IE gene expression, both ICP4 and ICP27 must be expressed. Inactivation of either results in loss of E and L gene products and failure to produce infectious virus. Suitable ICP4 and ICP27 expressing cell lines may complement these gene products in trans. Full details of the construction of replication-deficient viruses may be found in the text and references.

restores the expression of MHC class I molecules to the surface of the cells. This may potentially confer advantages in the gene therapy of malignancy, although the utility of this modification is unclear at present. For the majority of work discussed here, triple mutants (ICP4-: ICP22-: ICP27-) were used (Figure 1B). These vectors show minimal cytotoxicity in vitro and in vivo, are efficient vehicles for transgene delivery and can be grown efficiently in cells that complement the absence of ICP4 and ICP27 in trans (Wu et al, 1996; Wolfe et al, 1999). Safety is an important consideration in the development of therapeutic reagents; in this regard, a

post-translational processing of ICP0 in neurons is different to that in glia (Chen et al, 2000). It appears that, although ICP0 mRNA is efficiently expressed in both cell types, ICP0 undergoes proteolytic degradation in neurons. It might be predicted that the use of a vector carrying an intact ICP0 gene would not be toxic to neurons, but may confer additional therapeutic benefit in the treatment of glial-derived malignancy through the differential expression of ICP0 and transgenes in the two cell types. Our current view is that ICP0 expression will be advantageous for oncological applications where intratumoural toxicity is not an issue and transient highlevel gene expression is desirable. Deletion of ICP47

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number of beneficial features are intrinsic to the vector system described here: (i) The viruses are produced in cell lines that contain minimal gene sequences in common with the defective vector - it is extremely unlikely that replication-competent revertants will be inadvertently generated during manufacture (thus far, replication-competent virus has not been detected after repeat passaging of vector stock on complementing cells). (ii) Formation of a transgene-expressing replicationcompetent strain in vivo would require both the presence of replicating wild-type virus and gene therapy vector in the same cell, and multiple recombination events to restore the deleted essential genes. Furthermore, insertion of the transgene at an essential locus in the gene therapy vector prevents its acquisition by wild-type virus; the recombination event necessary to transfer the transgene to the wild-type virus would delete an essential gene and destroy the capacity for replication. These considerations suggest that the generation of a replicating transgeneexpressing virus would be extremely unlikely. (iii) If a replication-competent mutant were generated, expression of the early gene thymidine kinase would enable appropriate treatment with the antiviral agent acyclovir (see below). In addition to deletions of multiple IE genes, which achieve safety and minimise toxicity, it is possible to delete multiple non-essential E and L genes. This enables the insertion of several exogenous sequences. Vectors that express up to five independent expression cassettes have been generated; the expression level of each product seems little affected by the addition of further transgenes (Krisky et al, 1998). This property may have important implications for the gene therapy of cancer, as it seems likely that multiple therapeutic transgenes will be necessary to effectively deal with a disease that is heterogeneous and constantly evolving within an individual patient. The selection of an appropriate cis-acting regulatory domain to drive expression of each transgenic expression cassette is an important issue for a number of gene therapy applications. For example, in diseases where long-term transgene expression is required, the HSV latency active promoter complex (Goins et al, 1994, 1995; Soares et al, 1996) has been successfully used to effect chronic sustained transcription of the desired therapeutic gene (Lachmann and Efstathiou, 1997). In the context of gene therapy for malignant disease, the goal in many circumstances is transient high level expression of a toxic gene. In this setting, use of viral IE promoters is appropriate, for example the ICP4 promoter or the IE promoter from human cytomegalovirus. The possibility of exploiting glioma-specific promoters to restrict expression of toxic genes to target cells is attractive, but will depend on the identification of appropriate elements.

III. Therapeutic transgenes for cancer Many different transgenes have been considered for therapeutic application in cancer. This section outlines general principles that are common to the various groups of molecular targets that have been identified, and provides specific examples of each. Types of genes that may be delivered to cancers for therapeutic purposes are conveniently considered in the following categories, although the distinctions are arbitrary and some genes fall into more than one group.

A. Suicide genes These are genes encoding a product that is toxic to the cells within which it is expressed. An example is herpes simplex thymidine kinase (HSV-TK). The enzyme is encoded by the UL23 gene of HSV1, and functions to phosphorylate deoxypyrimidines with broad substrate specificity. This latter property allows the conversion of a pro-drug ganciclovir into its active form by HSV-TK, but not by its cellular counterpart. The active form of ganciclovir acts as a defective nucleoside analogue that becomes incorporated into replicating DNA and causes premature strand termination. Activated ganciclovir is toxic only to cells undergoing DNA replication. A degree of cytolytic selectivity is therefore inherent in this approach, with toxicity towards actively dividing tumour cells being much greater than to neurons or quiescent glia. In the context of replication-deficient HSV vectors, it is important to note that the HSV-TK expression cassette is placed under the transcriptional control of the ICP4 promoter or another IE promoter. This is necessary, as expression of IE genes is necessary to allow transcription of the E gene TK from its native promoter. Essential IE genes are deleted from replication-deficient vectors, which do not express the unmodified forms of any early genes, including HSV-TK. It is not necessary to transduce all tumour cells with the HSV-TK gene, as in many cases cells surrounding transduced cells are killed following ganciclovir administration. This phenomenon is referred to as â&#x20AC;&#x2DC;bystander lysisâ&#x20AC;&#x2122; (Carroll et al, 1997; Marconi et al, 2000). In vitro, bystander lysis is largely attributable to uptake of activated ganciclovir by HSV-TK negative cells (Rubsam et al, 1999; Marconi et al, 2000). The mechanisms responsible for bystander lysis in vivo are complex, and involve the passage of activated ganciclovir from HSV-TK positive to HSV-TK negative cells in addition to effects attributable to necrosis-induced inflammation and disruption of vasculature (Ram et al, 1994; Hamel et al, 1996; Dilber et al, 1997). Activated ganciclovir may pass from cell to cell through gap junctions (Dilber et al, 1997; Andrade Rozental et al, 2000; Robe et al, 2000). These are intercellular channels formed by a number of proteins including connexin-43 (Nagy and Rash, 2000). As gliomas are often defective in connexin expression (Shinoura et al,

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1996) and intercellular gap junctions (Naus et al, 1999), the expression of connexin-43 in these tumours represents one potential strategy whereby the bystander lysis effect may be enhanced (see Figure 5 and later section).

(iv) Limitless replicative potential (v) Sustained angiogenesis (vi) Tissue invasion and metastasis The biochemical mechanisms of many of these properties are currently being elucidated, and it may soon be possible to define individual tumours in terms of the four to seven molecular events that govern their development. Indeed, mutations linked to malignant behaviour in glial cells have been identified in several genes, including those for growth factors and their receptors, intracellular messengers, cell cycle proteins, transcription factors, tumour suppresser genes and their regulators. The prospect of identifying the responsible molecular defects in an individual tumour and then stably transducing all of the tumour cells with corrective genes is a daunting one, and not necessarily desirable. Certain types of genetic intervention, however, might be effective following short-term transgene expression in limited numbers of tumour cells. Examples might include the promotion of physiological death of cells bearing severely mutated genomes by introduction of p53 (Lang et al, 1999), or disruption of neovascularisation (Wesseling et al, 1997) by antisense directed against VEGF (Im et al, 1999; Machein et al, 1999).

B. Genes whose products enhance the susceptibility of the tumour to radiotherapy Examples of this type of gene include TNF-! (Moriuchi et al, 1998; Niranjan et al, 2000), which is discussed below, and ATM, which is mutated in the hereditary disease ataxia telangiectasia. The phenotype of the disease includes ataxia, dilated loops of capillaries, lymphoreticular malignancy and susceptibility to radiation-induced cell death (Smith and Conerly, 1985). The latter is caused by absence of the ATM protein, which has a pivotal role in the intermediate signalling events linking double strand DNA breaks to cell cycle arrest and subsequent DNA repair (Rotman and Shiloh, 1998). Abolition of ATM expression in a glioma cell line results in enhanced sensitivity to gamma irradiation (Guha et al, 2000). Transient reduction of ATM expression could be achieved during radiotherapy using antisense or ribozyme RNA molecules delivered by a gene therapy vector. This is a potentially useful way of reducing the dose of radiation that is fatal to the tumour cells, enabling reduction of the dose that is received by surrounding tissue.

IV. Combination treatment of experimental glioma using HSV vectors A major advantage of HSV as a gene therapy vector is its ability to accommodate multiple transgenes. In a series of studies using in vitro and in vivo models of glioblastoma, we have demonstrated the feasibility of using HSV as an anti-cancer gene therapy vector, and have started to examine the optimal requirements for combination gene therapy of malignancy. The various vectors used in the studies described here have been listed in Figure 1B. By studying the responses of tumour cells to these different vectors, the following principles have been established:

C. Genes that encode immunomodulatory proteins Several such genes have been used in anti-cancer vectors, most of which fall into the following groups: (i) Cell surface receptors or ligands that activate immune surveillance mechanisms to induce lysis of transduced cells â&#x20AC;&#x201C; e.g. CD80 (Krisky et al, 1998) (ii) Soluble mediators that recruit immunocompetent cells to the tumour or activate immunocompetent cells â&#x20AC;&#x201C; e.g TNF! (Moriuchi et al, 1998; Niranjan et al, 2000), GMCSF (Krisky et al, 1998), IL-2 (Colombo et al, 1997), interferon-" (Kanno et al, 1999), IL12 (Parker et al, 2000).

A. Expression of therapeutic transgenes is increased by non-toxic vectors, and results in enhanced anti-tumour properties (Moriuchi et al, 2000)

D. Genes that correct the molecular defects present within the tumour cell

The in vitro and in vivo cytolytic properties of HSVTK expressing replication-defective viruses deleted for ICP4 alone (SOZ.1) were compared with those of ICP4, ICP22 and ICP27 triple null mutants (T1, TOZ.1, THZ.1) (Moriuchi et al, 2000) (Figure 1B). All viruses were able to infect rat 9L glial cells efficiently in vitro; 100% of the cells were transduced at a multiplicity of infection (MOI) of 10, and 70% of the cells at MOI 1. At MOI 10, cells infected with TOZ.1 showed a reduction in proliferation but normal morphology; 25 % of the cells were seen to be

It has recently become possible to formulate a unifying framework that describes the essential alterations in cell physiology that dictate malignant behaviour (Hanahan and Weinberg, 2000): (i) Self-sufficiency in growth signals (ii) Insensitivity to anti-growth signals (iii) Evasion of apoptosis

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inoculated intracerebrally with 105 x 9L cells and a tumour was established over the subsequent 5 days. The tumours were then stereotactically injected with equivalent doses of the different viruses. Animals were treated with ganciclovir following viral injection and survival monitored.

undergoing apoptosis. In contrast, 98% of cells infected with SOZ.1 showed apoptosis; extensive cytopathic changes and cell loss were evident (Figure 4A). In a parallel series of in vivo experiments, the ability of the viruses to affect the clinical outcome of an experimental model of glioblastoma was assessed (Figure 3). Rats were

Figure 3. Orthotopic transplant model of glioma. Athymic (nude) mice were inoculated with U87 (human glioma) cells into the striatum on day 1. A tumour was allowed to establish for 3 days, following which the same co-ordinates were inoculated with the gene therapy vector under study, or with a negative control. Ganciclovir was given daily for 10 days following viral delivery via intraperitoneal injection. In protocols involving radiosurgery, this was given 2 days after viral inoculation. The survival of at least 8 animals in each treatment group was monitored and is presented as a Kaplan-Meier survival curves in Figures 5B, 6B and 7. The data shown in Figure 4B were generated using a similar protocol, except that rats were inoculated with 9L (rat glioma) cells on day 1, and the tumour was allowed to establish over 5 days prior to gene therapy and ganciclovir treatment.

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Figure 4. A. A single IE gene deleted virus (SOZ.1) was compared with two triple mutants (T.1 and TOZ.1) for cytotoxicity in vitro. Relative to T.1 and TOZ.1, SOZ.1 causes a substantially higher number of cells to undergo apoptosis at any given multiplicity of infection. This is attributable to the inherent toxicity of ICP22 and ICP27, which are not expressed following infection with the triple mutants. Details of the viruses are shown in Figure 1B. B. Survival of rats in an orthotopic transplant model of glioma (see Figure 3). Rats were inoculated intracerebrally with 9L glioma cells, then treated with either SOZ.1 or T.1 vector, or negative control medium. Untreated rats died from brain tumour within 30 days. Survival was not affected by administration of vector alone. However, there was significant prolongation of survival in rats treated with ganciclovir, only after T.1 inoculation. As both SOZ.1 and T.1 express the same HSV-TK expression cassette, the superior anti-tumour action of T.1 must have been attributable to differences in the expression of HSVTK secondary to differences in the toxicity of the two vectors. Thus, the less toxic vector allows better in situ expression of TK and an augmented therapeutic effect following ganciclovir treatment

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Figure 5 A. The bystander lysis effect is mediated, in part, by cell to cell spread of activated ganciclovir through gap junctions. (i) Transduction of a small proportion of cells with HSV-TK in the absence of gap junctions leads to the activation of ganciclovir only within the transduced cells. Non-transduced cells are able to escape the toxicity of ganciclovir in the absence of its conversion into the active nucleoside analogue. (ii) In vitro studies indicate that, provided a very low basal level of connexin-43expression is present on HSV-TK- (nontransduced) recipient cells, enhanced expression of connexin-43 on HSV-TK+ (transduced) donor cells encourages the formation of gap junctions between TK- and TK+ cells and augments the passage of activated ganciclovir between cells. This results in an enhanced bystander lysis effect. This observation constitutes a rational basis for attempts to deliver both HSV-TK and connexin-43 simultaneously to a sub-population of tumour cells using single vectors expressing both genes. B. The therapeutic effect of simultaneous TK and Cx43 expression was examined in the mouse orthotopic xenotransplant model described in Figure 3. Negative controls were compared with triple IE mutant vectors expressing either HSV-TK alone or in combination with connexin-43. Details of the viruses are shown in Figure 1B. Untreated animals died within 30 days from brain tumour. Survival was unaffected by treatment with TK vector alone (TOZ) but was enhanced by treatment with TOZ + ganciclovir. Expression of connexin-43 from TOCX had an anti-tumour action approximately equivalent to that seen with TOZ + ganciclovir. A combination of connexin-43 and HSV-TK/ganciclovir therapies (TOCX + ganciclovir) resulted in substantial survival benefit.

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animals were alive (Figure 5). Thus, bystander lysis from suicide gene therapy can be augmented substantially and with a clinically therapeutic effect by co-expression of connexin-43 from the same vector.

Rats injected with SOZ.1 showed no survival advantage over controls or following treatment with ganciclovir. Animals treated with T1 showed a clear therapeutic response to ganciclovir, which increased survival time by up to 50% in some animals (Figure 4B). Histological examination showed that a localised area of necrosis was evident within tumours infected with the single mutant, but that the area of necrosis was inadequate to affect tumour progression. Thus, bystander lysis from enhanced transgene expression consequent to reduced toxicity is a superior anti-cancer strategy than direct viral toxicity.

C. Co-expression of an antitumour cytokine enhances lysis by TK-ganciclovir (Moriuchi et al, 1998). Tumour necrosis factor alpha (TNF!) is a potent antitumour cytokine that demonstrates a range of actions against malignant cells, including the induction of apoptosis via activation of TNF! receptors, enhancement of HLA antigen expression in tumours, and immunomodulatory effects such as induction of NK- and CTL- mediated tumour lysis (Scheurich et al, 1986; Pfizenmaier et al, 1987; Ostensen et al, 1989; Rosenblum and Donato, 1989; Cao et al, 1997; Mueller, 1998). The molecule is too toxic to deliver systemically (Rosenblum and Donato, 1989; Mueller, 1998), but the ability of HSV vectors to accommodate multiple transgenes readily enables its incorporation into a locally administered suicide gene therapy paradigm. The hypothesis that TNF! might enhance tumour lysis mediated by TK-ganciclovir was tested by comparison of a replication-deficient HSVTK expressing virus (THZ.1) with an isogenic vector containing an expression cassette for TNF! at the ICP22 locus (TH:TNF) (Moriuchi et al, 1998) (Figure 1B). TH:TNF was shown to express biologically active TNF!, by ELISA and viability assays of TNF!-sensitive cells following infection. Ganciclovir-mediated lysis of TNF!sensitive cells in vitro following infection was enhanced by the presence of the TNF! expression cassette when a low proportion of the cells was infected (mimicking the situation in vivo after a single dose of vector). When the majority of cells were infected, the cultures were rapidly killed by the expression of TNF!. Many gliomas, however, are TNF!-resistant. It was of interest, therefore, to observe that TNF!-mediated enhancement of HSVTK/ganciclovir lysis was observed in a TNF!-insensitive glioma cell line; the mechanism was unclear, but presumably arose from sensitisation of the cells to one agent as a consequence of exposure to the other (Figure 6A). In vivo studies using the athymic mouse orthotopic xenotransplant tumour model (Figure 3) confirmed the in vitro observations in tumours composed of TNF!sensitive cells, but showed no additional benefit from TNF! expression in tumours derived from resistant cells. Although a substantial and significant prolongation of survival was seen with the TNF!/HSV-TK/ganciclovir regimen compared with negative control, the effect was also observed with the HSV-TK/ganciclovir treatment alone (Figure 6B).

B. Bystander lysis from TK-ganciclovir is enhanced by simultaneous expression of connexin-43 from the same vector (Marconi et al, 2000) It was hypothesised that enhanced gap junction intercellular communication might promote the passage of activated ganciclovir from transduced cells to neighbours within the tumour, thereby augmenting the bystander lysis effect from the HSV-TK/ganciclovir system (Figure 5A). To test this hypothesis, the anti-tumour effect of TOZ.1 was compared with that of an isogenic vector containing a connexin-43 expression cassette at the UL41 locus, TOCX (Marconi et al, 2000) (Figure 1B). First, it was shown by western blot hybridisation that TOCX gave rise to connexin-43 expression in a connexin-43 negative cell line. In vitro, significant enhancements in the levels of bystander lysis were observed in cells transduced with TOCX compared with TOZ. Interestingly, a series of experiments using cells with different levels of endogenous connexin-43, that were transduced with either of the viruses and then mixed with non-transduced cells, showed that the magnitude of the bystander lysis effect was a function of connexin expression in the TK-positive cell. Thus, above a certain threshold, connexin-43 expression in cells able to activate ganciclovir was sufficient to promote transfer of the nucleoside analogue to cells expressing connexin-43 at very low levels (Figure 5A). These observations provided a basis for the rational examination of HSV-TK/connexin-43 vectors in vivo. Nude (athymic, immunodeficient) mice were subject to intracerebral inoculation with 105 human glioma cells, and tumours were allowed to establish for 3 days (Figure 3). The tumours were then stereotactically injected with either TOZ or TOCX, or with medium alone. Half of each treatment group was treated with ganciclovir to assess the therapeutic effect of suicide gene therapy with or without connexin-43 expression. All animals in the control, nonganciclovir treated or TOZ groups were dead by 50 days into the study, at which point 50% of the TOCX/ganciclovir animals were still alive. At the end of the study (70 days) one-third of the TOCX/ganciclovir

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Figure 6 A. Anti-tumour action of TNF-!. A number of gliomas are resistant to the anti-tumour actions of TNF-!. The role of TNF-! in combination therapy with HSV-TK/ganciclovir was examined in vitro using triple IE mutant viruses expressing HSV-TK alone or with TNF-!. Details of the viruses are shown in Figure 1B. (i) As previously reported, survival of U87MG cells was unaffected by expression of HSV-TK alone, or with TNF-! at low MOI (0.1). Ganciclovir treatment, however, caused a substantial degree of cell death in samples transduced with HSV-TK. This effect was greatly augmented by concomitant expression of TNF-!, implying that one treatment modality had sensitised the cells to the effects of the other. (ii) Interestingly, this effect was not recapitulated by administration of exogenous TNF-!. It appears that sensitisation to HSV-TK/ganciclovir requires the endogenous expression and intracellular synthesis of TNF-!. These observations constitute a rational basis for attempts to simultaneously deliver HSV-TK and TNF-! to tumour cells using single vectors that express both genes. B. In vivo therapeutic efficacy of TNF-! expressing viruses in (i) mouse flank tumour model and (ii) mouse orthotopic glioma model. (i) L929 cells were chosen because of their intrinsic sensitivity to TNF-!. In this model, there is a clear survival advantage in the group treated with the TNF-! co-expressing virus. (ii) In contrast, there was no clear benefit of TNF! co-expression over an HSVTK/ganciclovir regime in the mouse glioma model.

clinical cure in the majority of animals (Niranjan et al, 2000).

Possible explanations include the observation that the low proportion of tumour cells that were transduced in vivo may have favoured detection of the bystander lysis effect from HSV-TK/ganciclovir treatment. Additionally, the direct receptor-mediated lysis effect would have been absent from the TNF! resistant cell lines, and the immunomodulatory effects of TNF! would not be seen in athymic mice.

Fractionated radiotherapy has been shown to confer a small but significant survival benefit on patients with glioblastoma. Unfortunately, the dose of radiotherapy that may be tolerated by the brain (about 60 Gray) is inadequate for tumour eradication. To circumvent inherent toxicity problems, techniques have been developed that allow focussing of radiation on the tumour bed, allowing a higher dose to be delivered (radiosurgery). This enables eradication of the central portion of the tumour, but does not allow augmented radiation doses to be delivered to the tumour periphery where malignant cells are seen invading

D. Co-expression of TNF! and TK enhance the therapeutic efficacy of gamma knife radiosurgery, enabling histological and

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the surrounding normal tissue, often migrating along normal white matter tracts. This feature of glioma is largely responsible for the inability to effect a surgical cure by resection, and the correspondingly poor prognosis. It is therefore of interest to examine ways in which the response to radiotherapy may be enhanced by gene delivery in the hope that malignant cells invading the tumour periphery may be eradicated. It was known that TNF! could enhance the therapeutic effect of gamma knife irradiation in athymic mice. Studies were therefore designed to examine whether the individual anti-tumour efficacies of suicide gene therapy, TNF! and radiotherapy were additive in combination (Niranjan et al, 2000). The athymic mouse orthotopic xenotransplant tumour model (Figure 3) and the vectors described in the previous section were used to study this question. A detailed examination of all combinations of the three treatment modalities was undertaken, with or without the administration of ganciclovir, and the reader is referred to the original reference for the full analysis. The most important points that emerged, however, were that (Figure 7): (i) Suicide gene therapy (with or without TNF!) and radiotherapy alone increased survival relative to no treatment, or administration of either vector alone. (ii) TNF! enhanced the effect of radiotherapy, whereas suicide gene therapy did not. (iii) A combination of the three treatment modalities led to 89% long-term (75 days) survivors, of which 75% were tumour-free. The next best treatment protocol led to 50% survival, and 0% of controls survived beyond 35 days. Combination of treatment modalities thus results in improved outcome in this model. Further studies have been undertaken using a combination of radiotherapy with a vector expressing HSV-TK, connexin-43 and TNF! (Nurel-C). Again, the general principal to emerge from these studies is that combination treatment protocols are superior to single interventions (manuscript in preparation).

tumours, which have evolved over a number of years in an immunocompetent host and are heterogeneous by the time of diagnosis. These initial studies, however, provide some optimism that tumour cells may be targeted in vivo, and form a basis for the continued investigation of this general strategy. Further studies will be directed at developing more advanced molecular therapeutic agents, and addressing the efficacy issue in models more closely resembling the human disease. Current work is focussed on improving the vector system further and identifying new molecular targets that might rationally be combined with the strategies described above to disrupt additional aspects of the oncogenic process. The possibility of further enhancing the combinatorial effects of TNF! and radiosurgery by downregulation of ATM is being actively pursued, as is the generation of more severely disabled vectors that are

V. Conclusions: future directions Our experience with experimental models of glioma indicates that combination multi-modality therapies are superior to single interventions, which is not surprising in view of the nature of basic cancer biology. A vector system allowing simultaneous delivery of multiple genes is ideally suited to this application. We have developed replication-defective HSV vectors with many favourable properties for use in tumour gene therapy, including absence of cytotoxicity, effective transgene delivery and expression, and ability to accommodate multiple therapeutic cassettes. Recent studies have established the principle that it is possible to eradicate experimental tumours in laboratory animals by using combination HSV gene therapy-based approaches. There remains uncertainty regarding the applicability of these models to human

Figure 7. Combination HSV-TK/ganciclovir, TNF-! and radiotherapy treatment is superior to any single intervention or combination of two treatment modalities. The TNF-! coexpressing virus (TH:TNF) was examined in the mouse glioma model shown in Figure3, with or without radiotherapy and/or ganciclovir. Untreated mice died within 3 days from cerebral tumour. Both radiotherapy and ganciclovir treatment conferred a survival advantage in the context of tumour infection with TH:TNF. Combining all three treatments led to long-term, tumour-free survival in most of the animals.

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simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4. J Virol 56, 558-70. Dilber, M. S., Abedi, M. R., Christensson, B., Bjorkstrand, B., Kidder, G. M., Naus, C. C., Gahrton, G., and Smith, C. I. (1997). Gap junctions promote the bystander effect of herpes simplex virus thymidine kinase in vivo. Cancer Res 57, 1523-8. Fruh, K., Ahn, K., Djaballah, H., Sempe, P., van Endert, P. M., Tampe, R., Peterson, P. A., and Yang, Y. (1995). A viral inhibitor of peptide transporters for antigen presentation. Nature 375, 415-8. Geraghty, R. J., Krummenacher, C., Cohen, G. H., Eisenberg, R. J., and Spear, P. G. (1998). Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor. Science 280, 1618-20.

likely to be less toxic and better able to deliver their payload. It is envisaged that glioma therapy of the future might involve cytoreductive surgery followed by vector inoculation into the tumour cavity and radiotherapy. The vector might encode genes that allow pharmaceutical lysis of actively dividing cells and their neighbours, enhance the radiosensitivity of tumour cells at the invasive margin, and inhibit neovascularisation of potential areas of recurrence at the tumour margin. Whether this approach will have a major impact on the prognosis of malignant glioma is uncertain, but in the absence of other promising experimental approaches it seems well worth pursuing.

References:

Goins, W. F., Sternberg, L. R., Croen, K. D., Krause, P. R., Hendricks, R. L., Fink, D. J., Straus, S. E., Levine, M., and Glorioso, J. C. (1994). A novel latency-active promoter is contained within the herpes simplex virus type 1 UL flanking repeats. J Virol 68, 2239-52.

al Rikabi, A. C., al Sohaibani, M. O., Jamjoom, A., and al Rayess, M. M. (1997). Metastatic deposits of a high-grade malignant glioma in cervical lymph nodes diagnosed by fine needle aspiration (FNA) cytology--case report and literature review. Cytopathology 8, 421-7.

Gu, B., Kuddus, R., and DeLuca, N. A. (1995). Repression of activator-mediated transcription by herpes simplex virus ICP4 via a mechanism involving interactions with the basal transcription factors TATA-binding protein and TFIIB. Mol Cell Biol 15, 3618-26. Guha, C., Guha, U., Tribius, S., Alfieri, A., Casper, D., Chakravarty, P., Mellado, W., Pandita, T. K., and Vikram, B. (2000). Antisense ATM gene therapy: a strategy to increase the radiosensitivity of human tumors. Gene Ther 7, 852-8.

Andrade Rozental, A. F., Rozental, R., Hopperstad, M. G., Wu, J. K., Vrionis, F. D., and Spray, D. C. (2000). Gap junctions: the "kiss of death" and the "kiss of life". Brain Res Brain Res Rev 32, 308-15. Brown, C. R., Nakamura, M. S., Mosca, J. D., Hayward, G. S., Straus, S. E., and Perera, L. P. (1995). Herpes simplex virus trans-regulatory protein ICP27 stabilizes and binds to 3' ends of labile mRNA. J Virol 69, 7187-95. Bruni, R., and Roizman, B. (1998). Herpes simplex virus 1 regulatory protein ICP22 interacts with a new cell cycleregulated factor and accumulates in a cell cycle-dependent fashion in infected cells. J Virol 72, 8525-31. Cao, G., Kuriyama, S., Du, P., Sakamoto, T., Kong, X., Masui, K., and Qi, Z. (1997). Complete regression of established murine hepatocellular carcinoma by in vivo tumor necrosis factor alpha gene transfer [see comments]. Gastroenterology 112, 501-10. Carroll, N. M., Chase, M., Chiocca, E. A., and Tanabe, K. K. (1997). The effect of ganciclovir on herpes simplex virusmediated oncolysis. J Surg Res 69, 413-7.

Hamel, W., Magnelli, L., Chiarugi, V. P., and Israel, M. A. (1996). Herpes simplex virus thymidine kinase/ganciclovirmediated apoptotic death of bystander cells. Cancer Res 56, 2697-702. Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57-70. Hardwicke, M. A., and Sandri Goldin, R. M. (1994). The herpes simplex virus regulatory protein ICP27 contributes to the decrease in cellular mRNA levels during infection. J Virol 68, 4797-810. Hardy, W. R., and Sandri Goldin, R. M. (1994). Herpes simplex virus inhibits host cell splicing, and regulatory protein ICP27 is required for this effect. J Virol 68, 7790-9. Hill, A., Jugovic, P., York, I., Russ, G., Bennink, J., Yewdell, J., Ploegh, H., and Johnson, D. (1995). Herpes simplex virus turns off the TAP to evade host immunity. Nature 375, 4115. Hill, J. R., Kuriyama, N., Kuriyama, H., and Israel, M. A. (1999). Molecular genetics of brain tumors. Arch Neurol 56, 43941. Honess, R. W., and Roizman, B. (1974). Regulation of herpesvirus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral proteins. J Virol 14, 8-19.

Carrozza, M. J., and DeLuca, N. A. (1996). Interaction of the viral activator protein ICP4 with TFIID through TAF250. Mol Cell Biol 16, 3085-93. Chen, X., Li, J., Mata, M., Goss, J., Glorioso, J. C., and Fink, D. J. (2000). The ICP0 protein of herpes simplex virus 1 does not accumulate in the nucleus of primary neurons in culture. J. Virol. (in press). Chen, X., Schmidt, M. C., Goins, W. F., and Glorioso, J. C. (1995). Two herpes simplex virus type 1 latency-active promoters differ in their contributions to latency-associated transcript expression during lytic and latent infections. J Virol 69, 7899-908. Collins, V. P. (1998). Gliomas. Cancer Surv 32, 37-51. Colombo, F., Zanusso, M., Casentini, L., Cavaggioni, A., Franchin, E., Calvi, P., and Palu, G. (1997). Gene stereotactic neurosurgery for recurrent malignant gliomas. Stereotact Funct Neurosurg 68, 245-51.

Honess, R. W., and Roizman, B. (1975). Regulation of herpesvirus macromolecular synthesis: sequential transition of polypeptide synthesis requires functional viral polypeptides. Proc Natl Acad Sci U S A 72, 1276-80. Hsu, E., Keene, D., Ventureyra, E., Matzinger, M. A., Jimenez, C., Wang, H. S., and Grimard, L. (1998). Bone marrow metastasis in astrocytic gliomata. J Neurooncol 37, 285-93.

DeLuca, N. A., McCarthy, A. M., and Schaffer, P. A. (1985). Isolation and characterization of deletion mutants of herpes

143

Burton and Glorioso: HSV vectors in gene therapy of malignant glioma

Im, S. A., Gomez Manzano, C., Fueyo, J., Liu, T. J., Ke, L. D., Kim, J. S., Lee, H. Y., Steck, P. A., Kyritsis, A. P., and Yung, W. K. (1999). Antiangiogenesis treatment for gliomas: transfer of antisense-vascular endothelial growth factor inhibits tumor growth in vivo. Cancer Res 59, 895-900.

Lopez Gonzalez, M. A., and Sotelo, J. (2000). Brain tumors in Mexico: characteristics and prognosis of glioblastoma. Surg Neurol 53, 157-62. Machein, M. R., Kullmer, J., Fiebich, B. L., Plate, K. H., and Warnke, P. C. (1999). Vascular endothelial growth factor expression, vascular volume, and, capillary permeability in human brain tumors. Neurosurgery 44, 732-40. Marconi, P., Tamura, M., Moriuchi, S., Krisky, D. M., Niranjan, A., Goins, W. F., Cohen, J. B., and Glorioso, J. C. (2000). Connexin 43-enhanced suicide gene therapy using herpesviral vectors. Mol Ther 1, 71-81. McComb, R. D., and Bigner, D. D. (1984). The biology of malignant gliomas--a comprehensive survey. Clin Neuropathol 3, 93-106. Mears, W. E., and Rice, S. A. (1998). The herpes simplex virus immediate-early protein ICP27 shuttles between nucleus and cytoplasm. Virology 242, 128-37.

Johnson, P. A., Wang, M. J., and Friedmann, T. (1994). Improved cell survival by the reduction of immediate-early gene expression in replication-defective mutants of herpes simplex virus type 1 but not by mutation of the virion host shutoff function. J Virol 68, 6347-62. Jordan, R., and Schaffer, P. A. (1997). Activation of gene expression by herpes simplex virus type 1 ICP0 occurs at the level of mRNA synthesis. J Virol 71, 6850-62. Jubelirer, S. J. (1996). A review of the treatment and survival rates of 138 patients with glioblastoma multiforme. W, 18690. Kanno, H., Hattori, S., Sato, H., Murata, H., Huang, F. H., Hayashi, A., Suzuki, N., Yamamoto, I., Kawamoto, S., Minami, M., Miyatake, S., Shuin, T., and Kaplitt, M. G. (1999). Experimental gene therapy against subcutaneously implanted glioma with a herpes simplex virus-defective vector expressing interferon-gamma. Cancer Gene Ther 6, 147-54. Kelly, K. A., Kirkwood, J. M., and Kapp, D. S. (1984). Glioblastoma multiforme: pathology, natural history and treatment. Cancer Treat Rev 11, 1-26.

Montgomery, R. I., Warner, M. S., Lum, B. J., and Spear, P. G. (1996). Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell 87, 427-36. Moriuchi, S., Krisky, D. M., Marconi, P. C., Tamura, M., Shimizu, K., Yoshimine, T., Cohen, J. B., and Glorioso, J. C. (2000). HSV vector cytotoxicity is inversely correlated with effective TK/GCV suicide gene therapy of rat gliosarcoma. Gene Ther 7, 1483-90.

Krisky, D. M., Marconi, P. C., Oligino, T. J., Rouse, R. J., Fink, D. J., Cohen, J. B., Watkins, S. C., and Glorioso, J. C. (1998). Development of herpes simplex virus replicationdefective multigene vectors for combination gene therapy applications. Gene Ther 5, 1517-30.

Moriuchi, S., Oligino, T., Krisky, D., Marconi, P., Fink, D., Cohen, J., and Glorioso, J. C. (1998). Enhanced tumor cell killing in the presence of ganciclovir by herpes simplex virus type 1 vector-directed coexpression of human tumor necrosis factor-alpha and herpes simplex virus thymidine kinase. Cancer Res 58, 5731-7. Mueller, H. (1998). Tumor necrosis factor as an antineoplastic agent: pitfalls and promises. Cell Mol Life Sci 54, 1291-8.

Krisky, D. M., Wolfe, D., Goins, W. F., Marconi, P. C., Ramakrishnan, R., Mata, M., Rouse, R. J., Fink, D. J., and Glorioso, J. C. (1998). Deletion of multiple immediate-early genes from herpes simplex virus reduces cytotoxicity and permits long-term gene expression in neurons. Gene Ther 5, 1593-603. Kuddus, R., Gu, B., and DeLuca, N. A. (1995). Relationship between TATA-binding protein and herpes simplex virus type 1 ICP4 DNA-binding sites in complex formation and repression of transcription. J Virol 69, 5568-75.

Nagy, J. I., and Rash, J. E. (2000). Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS. Brain Res Brain Res Rev 32, 29-44. Naus, C. C., Bani Yaghoub, M., Rushlow, W., and Bechberger, J. F. (1999). Consequences of impaired gap junctional communication in glial cells. Adv Exp Med Biol 468, 37381.

Lacaille, V. G., and Androlewicz, M. J. (1998). Herpes simplex virus inhibitor ICP47 destabilizes the transporter associated with antigen processing (TAP) heterodimer. J Biol Chem 273, 17386-90.

Niranjan, A., Moriuchi, S., Lunsford, L. D., Kondziolka, D., Flickinger, J. C., Fellows, W., Rajendiran, S., Tamura, M., Cohen, J. B., and Glorioso, J. C. (2000). Effective Treatment of Experimental Glioblastoma by HSV Vector-Mediated TNFalpha and HSV-tk Gene Transfer in Combination with Radiosurgery and Ganciclovir Administration. Mol Ther 2, 114-120.

Lachmann, R. H., and Efstathiou, S. (1997). Utilization of the herpes simplex virus type 1 latency-associated regulatory region to drive stable reporter gene expression in the nervous system. J Virol 71, 3197-207. Lang, F. F., Yung, W. K., Sawaya, R., and Tofilon, P. J. (1999). Adenovirus-mediated p53 gene therapy for human gliomas. Neurosurgery 45, 1093-104.

Niranjan, A., Moriuchi, S., Lunsford, L. D., Kondziolka, D., Flickinger, J. C., Fellows, W., Rajendiran, S., Tamura, M., Cohen, J. B., and Glorioso, J. C. (2000). Effective treatment of experimental glioblastoma by HSV vector-mediated TNFalpha and HSV-tk gene transfer in combination with radiosurgery and ganciclovir administration [In Process Citation]. Mol Ther 2, 114-20.

Leopardi, R., Ward, P. L., Ogle, W. O., and Roizman, B. (1997). Association of herpes simplex virus regulatory protein ICP22 with transcriptional complexes containing EAP, ICP4, RNA polymerase II, and viral DNA requires posttranslational modification by the U(L)13 proteinkinase. J Virol 71, 11339.

O'Hare, P., Goding, C. R., and Haigh, A. (1988). Direct combinatorial interaction between a herpes simplex virus regulatory protein and a cellular octamer-binding factor mediates specific induction of virus immediate-early gene expression. EMBO J 7, 4231-8.

144

Gene Therapy and Molecular Biology Vol 5, page 145

Ostensen, M. E., Thiele, D. L., and Lipsky, P. E. (1989). Enhancement of human natural killer cell function by the combined effects of tumor necrosis factor alpha or interleukin-1 and interferon-alpha or interleukin-2. J Biol Response Mod 8, 53-61.

transcription from the viral genome and infected-cell survival in the absence of ICP4 and ICP27. J Virol 71, 4614-25. Sandri Goldin, R. M. (1998). ICP27 mediates HSV RNA export by shuttling through a leucine-rich nuclear export signal and binding viral intronless RNAs through an RGG motif. Genes Dev 12, 868-79.

Parker, J. N., Gillespie, G. Y., Love, C. E., Randall, S., Whitley, R. J., and Markert, J. M. (2000). From the cover: engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors. Proc Natl Acad Sci U S A 97, 2208-13.

Scheurich, P., Kronke, M., Schluter, C., Ucer, U., and Pfizenmaier, K. (1986). Noncytocidal mechanisms of action of tumor necrosis factor-alpha on human tumor cells: enhancement of HLA gene expression synergistic with interferon-gamma. Immunobiology 172, 291-300. Shinoura, N., Chen, L., Wani, M. A., Kim, Y. G., Larson, J. J., Warnick, R. E., Simon, M., Menon, A. G., Bi, W. L., and Stambrook, P. J. (1996). Protein and messenger RNA expression of connexin43 in astrocytomas: implications in brain tumor gene therapy. J Neurosurg 84, 839-45. Smith, C. A., Bates, P., Rivera Gonzalez, R., Gu, B., and DeLuca, N. A. (1993). ICP4, the major transcriptional regulatory protein of herpes simplex virus type 1, forms a tripartite complex with TATA-binding protein and TFIIB. J Virol 67, 4676-87. Smith, L. L., and Conerly, S. L. (1985). Ataxia-telangiectasia or Louis-Bar syndrome. J Am Acad Dermatol 12, 681-96. Soares, K., Hwang, D. Y., Ramakrishnan, R., Schmidt, M. C., Fink, D. J., and Glorioso, J. C. (1996). cis-acting elements involved in transcriptional regulation of the herpes simplex virus type 1 latency-associated promoter 1 (LAP1) in vitro and in vivo. J Virol 70, 5384-94. Soliman, T. M., Sandri Goldin, R. M., and Silverstein, S. J. (1997). Shuttling of the herpes simplex virus type 1 regulatory protein ICP27 between the nucleus and cytoplasm mediates the expression of late proteins. J Virol 71, 9188-97. Walker, A. E., Robins, M., and Weinfeld, F. D. (1985). Epidemiology of brain tumors: the national survey of intracranial neoplasms. Neurology 35, 219-26. Wesseling, P., Ruiter, D. J., and Burger, P. C. (1997). Angiogenesis in brain tumors; pathobiological and clinical aspects. J Neurooncol 32, 253-65.

Pfizenmaier, K., Scheurich, P., Schluter, C., and Kronke, M. (1987). Tumor necrosis factor enhances HLA-A,B,C and HLA-DR gene expression in human tumor cells. J Immunol 138, 975-80. Poffenberger, K. L., Idowu, A. D., Fraser Smith, E. B., Raichlen, P. E., and Herman, R. C. (1994). A herpes simplex virus type 1 ICP22 deletion mutant is altered for virulence and latency in vivo. Arch Virol 139, 111-9. Prod'hon, C., Machuca, I., Berthomme, H., Epstein, A., and Jacquemont, B. (1996). Characterization of regulatory functions of the HSV-1 immediate-early protein ICP22. Virology 226, 393-402. Ram, Z., Walbridge, S., Shawker, T., Culver, K. W., Blaese, R. M., and Oldfield, E. H. (1994). The effect of thymidine kinase transduction and ganciclovir therapy on tumor vasculature and growth of 9L gliomas in rats. J Neurosurg 81, 256-60. Rice, S. A., Long, M. C., Lam, V., Schaffer, P. A., and Spencer, C. A. (1995). Herpes simplex virus immediate-early protein ICP22 is required for viral modification of host RNA polymerase II and establishment of the normal viral transcription program. J Virol 69, 5550-9. Robe, P. A., Princen, F., Martin, D., Malgrange, B., Stevenaert, A., Moonen, G., Gielen, J., Merville, M., and Bours, V. (2000). Pharmacological modulation of the bystander effect in the herpes simplex virus thymidine kinase/ganciclovir gene therapy system: effects of dibutyryl adenosine 3',5'cyclic monophosphate, alpha-glycyrrhetinic acid, and cytosine arabinoside. Biochem Pharmacol 60, 241-9. Roizman, B., and Sears, A. E. (1996). Herpes Simplex Viruses and Their Replication. In Fields Virology, B. N. Fields, D. M. Knipe and P. M. Howley, eds. (Philadelphia: LippincottRaven), pp. 2231-2295.

West, C. R., Avellanosa, Hong, C. I. (1983). nitrosourea (BCNU) malignant gliomas: a 420-6.

Rosenblum, M. G., and Donato, N. J. (1989). Tumor necrosis factor alpha: a multifaceted peptide hormone. Crit Rev Immunol 9, 21-44. Rotman, G., and Shiloh, Y. (1998). ATM: from gene to function. Hum Mol Genet 7, 1555-63.

A. M., Barua, N. R., Patel, A., and Intraarterial 1,3-bis(2-chloroethyl)-1and systemic chemotherapy for follow-up study. Neurosurgery 13,

Wolfe, D., Goins, W. F., Yamada, M., Moriuchi, S., Krisky, D. M., Oligino, T. J., Marconi, P. C., Fink, D. J., and Glorioso, J. C. (1999). Engineering herpes simplex virus vectors for CNS applications. Exp Neurol 159, 34-46. Wu, N., Watkins, S. C., Schaffer, P. A., and DeLuca, N. A. (1996). Prolonged gene expression and cell survival after infection by a herpes simplex virus mutant defective in the immediate-early genes encoding ICP4, ICP27, and ICP22. J Virol 70, 6358-69.

Rubsam, L. Z., Boucher, P. D., Murphy, P. J., KuKuruga, M., and Shewach, D. S. (1999). Cytotoxicity and accumulation of ganciclovir triphosphate in bystander cells cocultured with herpes simplex virus type 1 thymidine kinase-expressing human glioblastoma cells. Cancer Res 59, 669-75.

York, I. A., Roop, C., Andrews, D. W., Riddell, S. R., Graham, F. L., and Johnson, D. C. (1994). A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell 77, 525-35.

Samaniego, L. A., Neiderhiser, L., and DeLuca, N. A. (1998). Persistence and expression of the herpes simplex virus genome in the absence of immediate-early proteins. J Virol 72, 3307-20. Samaniego, L. A., Wu, N., and DeLuca, N. A. (1997). The herpes simplex virus immediate-early protein ICP0 affects

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Gene Ther Mol Biol Vol 5, 147-156, 2000

Viral vectors carrying a marker-suicide fusion gene (TK-GFP) as tools for TK/GCV –mediated cancer gene therapy Research Article

Sami Loimas1,2, Tiina Pasanen1, Andreia Gomes1, Susana Bizarro1, Richard A. Morgan3, Juhani Jänne1 and Jarmo Wahlfors1,2* 1

A.I.Virtanen Institute for Molecular Sciences, University of Kuopio and 2Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland, 3Clinical Gene Therapy Branch/National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892 __________________________________________________________________________________________________ *Correspondence: Dr. Jarmo Wahlfors, Gene Transfer Techology Group, A.I.Virtanen Institute for Molecular Sciences; University of Kuopio, P.O.Box 1627 (street address: Neulaniementie 2), FIN-70211 Kuopio, Finland; Tel. +358-17-163121; Fax +358-17-163030; email jarmo.wahlfors@uku.fi Key words: gene transfer, cancer, viral vectors, GFP, HSV-TK, fusion construct

Received: 6 November 2000; accepted: 15 November 2000

Summary A herpes simplex thymidine kinase – green fluorescent protein (TK-GFP) fusion gene was constructed to couple a marker gene to a therapeutic gene. For testing the utility of the fusion gene, it was cloned into four different viral vectors: Semliki forest virus (SFV), Sindbis virus, adenovirus and lentivirus. The produced viral TK-GFP vectors were then used to test their transduction efficiency on different tumor cells and especially the relationship between TK gene transfer and treatment result with prodrug ganciclovir (GCV). When the efficiency of the other three viral vectors were compared to adenoviral vector, a commonly used tool in cancer gene therapy, the alphaviral vectors SFV and Sindbis performed better on glioma cells, but were less efficient on renal carcinoma cells. On the other hand, an HIV-1 –based, VSV-G pseudotyped lentiviral vector was efficient on all cell lines tested, suggesting the potential of this vector type for cancer gene therapy. GCV-sensitivity studies revealed that regardless of the vector type used, the treatment result was directly proportional to the gene transfer efficiency (not to the multiplicity of infection). However, good gene transfer rate alone is not always sufficient: twenty- percent efficiency was enough to cause adequate cytotoxicity with 5 µg/ml GCV in both of the glioma cell lines, whereas even higher than 80% transduction was not enough to successfully treat Caki-2 cells under the same conditions. When the bystander effect was examined, Caki-2 cells were shown to display a much weaker effect than the glioma cells. Our results demonstrate the benefit of TK-GFP fusion gene in cancer gene therapy research and emphasize the importance of finding an appropriate vector type for each tumor, as well as testing the presence of bystander effect before continuing with TK/GCV approach. 1986). Another desired feature of this gene therapy approach is that some tumor cells display so called bystander effect (Moolten, 1986;Culver et al, 1992). This means that affected cells can share the cytotoxic effect of the treatment with their neighbors and therefore enhance the initial cytotoxicity. In addition to a vast number of preclinical studies in vitro and in vivo, the TK/GCV approach has been tested several times in clinical trials as

I. Introduction Suicide gene therapy with herpes simplex virus type 1 thymidine kinase (HSV-TK) and ganciclovir (GCV) has been one of the most widely used and well-studied paradigm in cancer gene therapy research. In this approach, HSV-TK gene is transferred to cancer cells, followed by destruction of the TK-positive cells through administration of non-toxic doses of GCV (Moolten,

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the preparation was 1 x 109 pfu/ml. LentiTGL was produced in 293T cells, yielding titers of 1-2 x 106 iu/ml (as measured by the FACS method). SinTGL and SFVTGL production was carried out in BHK cells as described in (Wahlfors et al, 2000). The titers obtained by FACS were 1 x 107 – 1 x 108 iu/ml for both vectors. According to our earlier results (Wahlfors et al, 2000), it is possible that formation of replication competent alphaviruses (RC!V) occur at a high frequency. We therefore tested our SFVTGL and SinTGL stocks by serial passaging on BHK cells. As observed before, SFVTGL was devoid of any RC!V, whereas SinTGL contained approximately one RC!V per 104 biologically active vector particles. We have found that RC!V, when low MOI is used, can spread efficiently and kill BHK cells, but the effect is much more subtle in BT4C or 9L cells and cannot be detected in human cells at all (Wahlfors et al, unpublished results). Therefore, it is unlikely that replication competent viruses in SinTGL preparations would distort our results from 9L, BT4C and Caki-2 cells.

well (for review, see (Morris et al, 1999)). Despite the extensive use of this modality and encouraging results from many in vivo studies, we are still waiting for an actual clinical breakthrough. A fundamental problem in gene therapy is insufficient gene transfer and it is likely to be the major reason for modest clinical success with TK/GCV suicide gene therapy. Numerous attempts have been carried out to increase the gene transfer efficiency, for example by making the TK-carrying vectors replication competent (Wildner et al, 1999). Even though improved vector systems may provide better gene transfer efficiency, the amount of gene transfer required to achieve specific results is largely unknown. Smiley and co-workers stated that, especially in the context of TK/GCV therapy, the inability to accurately quantitate the transgene transfer efficiency is “a consistent barrier to understanding the mechanisms of retroviral action” (Smiley et al, 1997). This is certainly true also in case of other vector types and many times it is not known, how extensive gene transfer efficiency is needed for a sufficient treatment result with GCV. We wanted to study this relationship and directly demonstrate the efficiency of gene transfer under various conditions and its correlation to the treatment result achieved. The most reliable way to determine this would a therapeutic protein that is easy to detect. Since antibody based detection methods of HSV-TK are not very practical, we reasoned that by coupling a marker gene to TK could solve the problem. To directly couple a marker gene to HSV-TK, we constructed a fusion between TK and green fluorescent protein (GFP). This construct produced a functional fusion protein that had the activities of both components, sensitizing the target cells to GCV and emitting green fluorescence under UV light (Loimas et al, 1998). After this proof-of-principle phase, we wanted to test the utility of TK-GFP by using it in viral vectors for studying the features that affect the outcome of TK/GCV cancer gene therapy. Three different TK-GFP -carrying viral vectors, Sindbis virus-, Semliki forest virus (SFV)- and lentivirus vectors were compared to respective adenovirus vector by transducing three different tumor cell lines, followed by analysis of gene transfer efficiency and its relationship to treatment result with GCV. This paper shows the utility of TK-GFP fusion construct and emphasizes the fact that the gene transfer efficiency has the major impact on the success of TK/GCV –mediated cancer gene therapy.

B. Characterization and comparison of the inducible promoter expression vector To get a rough idea of the utility of SFV-, Sindbisand lentiviral vectors compared to the commonly used adenoviral vector in cancer cells, gene transfer efficiency and level of TK-GFP expression were determined on BT4C- and 9L cells (rat brain tumor cell lines), and Caki-2 cells (human kidney tumor cell line). Only low multiplicities of infection (MOI 0.3 and 3) were used to mimic the situation in in vivo gene transfer studies, where transduction efficiencies are usually quite modest. Cells were incubated for either 20 hours (SinTGL and SFVTGL) or 96 hours (AdenoTGL and LentiTGL) after transduction, followed by FACS analyses.

1. Transduction efficiency (Figure 1) All viral vectors tested were capable to transduce all three cell lines to some extent, but the difference between rat and human cells was clear in the case of adenoviral vectors. As expected, AdenoTGL did not perform well in rat cells. MOI 0.3 yielded only 1.4 % positive BT4C cells and < 1 % positive 9L cells. On the other hand, the human renal carcinoma cell line Caki-2 was a very good target for AdenoTGL vector, which transduced 24 % of the cells, when MOI 0.3 was used. SFVTGL was relatively efficient on all three cell lines and was the most efficient vector type in 9L cells. SinTGL was poor on the human cell line Caki-2, but showed better performance in the rat cell lines and was the best vector for BT4C cells (13 % efficiency at MOI 0.3). LentiTGL was equally good for all three cell lines, being almost comparable to the best vector type in each case, especially when MOI 3 was used (51% for BT4C cells and 68% for both 9L and Caki-2 cells).

II. Results A. Viral TK-GFP vector production After cloning the improved version of TK-GFP construct (Wahlfors et al, 2000) into different vector plasmids, viral vector preparations were produced following the standard protocols described in Materials and Methods. AdenoTGL was produced in 293 cells and the final clone obtained by plaque purification. The titer of

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2. Expression level of TK-GFP.

C. Ganciclovir sensitivity of the cells transduced with TK-GFP vectors

Data from flow cytometer was also analyzed in terms of the mean fluorescence of the TK-GFP –positive population, indicating the level of transgene expression. In all three cell lines, the pattern was similar: both alphaviral vectors displayed a very high (yet transient) gene expression, whereas lentiviral vector yielded much lower mean fluorescence that intensified during the first week after transduction and was stable over time (results not shown). AdenoTGL-based expression was not as high as that of SFVTGL or SinTGL in the human renal carcinoma cell line Caki-2, and even lower than LentiTGL –based expression in rat glioma cells. The expression pattern of each vector type is demonstrated with FACS data from BT4C cells in Figure 2.

To study the relationship between gene transfer efficiency and treatment result, we selected the adenoviral vector, since it was shown to yield varying transduction rates on different target cell types (see the previous section). This experiment was carried out by transducing 9L, BT4C and Caki-2 cells with different amounts of AdenoTGL (MOI 0.1 – 10), followed by incubation in the presence of GCV (from 0.1 to 1000 µg/ml) and cell viability analysis by MTT-assay. The use of TK-GFP made possible to determine gene transfer efficiencies accurately and measure the proportion of positive cells obtained at each MOI. Therefore, we were able to precisely determine the transduction percentage needed for cancer cell destruction under clinically relevant GCV concentrations (Figure 3).

Figure. 2. TK-GFP expression levels with different viral vectors. BT4C cells transduced with different viral vectors were analyzed by FACSCalibur flow cytometer to obtain dot blot: forward scatter (X-axis) versus GFP fluorescence (Y-axis). Control = untreated BT4C cells. The numeric value of the mean fluorescence intensity of the GFP positive population (Fl.) is indicated in each panel.

Figure 1. Transduction efficiencies with different viral vectors. BT4C (A), 9L (B) and Caki-2 (C) cell lines were transduced with Sindbis-, SFV-, adenoviral- and lentiviral vector carrying TKGFP gene. Two different multiplicities of infection, 0.3 and 3, were used. Proportion of GFP –positive cells (%) was determined by FACS analysis 20 h (SinTGL, SFVTGL) or 96 h (AdenoTGL, LentiTGL) post-transduction. The results are mean of three different transductions, standard deviation markers are indicated.

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As demonstrated in the previous experiment, low MOIs of AdenoTGL transduced rat glioma cells, especially 9L cells at low rate. This can be seen as low or moderate transduction efficiency, even at MOI 3 or 10 (25% or 61% in BT4C cells and 5% or 15% in 9L cells, respectively). On the other hand, Caki-2 cells were very permissive to adenoviral vectors and the respective efficiencies on these cells were 72% and 89%. As shown in Figure 3., the cytotoxicity of GCV correlates well with the actual transduction efficiency. In order to determine the sufficient gene transfer efficiency for satisfactory treatment result, we used the following criteria: what is the gene transfer percentage, where 5 µg/ml GCV destroys 60 – 80% of the transduced cells? This ganciclovir concentration is comparable to the standard human dose 5 mg/kg/day and thus is relevant to the dose response for in vivo settings. In BT4C cells, this was achieved at MOI 2 - 3, yielding about 20% positive cells. Nine L cells were showing barely satisfactory cytotoxicity only at MOI 10, which was due to the fact that these cells are somewhat resistant to adenoviruses (the transduction efficiency was 15%, although 5 times more AdTGL was used for 9L cells than for BT4C cells). Nevertheless, in both rat glioma cell lines, the cell destruction was remarkably higher than the actual transduction percentage, suggesting an efficient bystander effect present in these cells. Despite high adenovirus vector –mediated gene transfer efficiency, Caki-2 cells were not killed effectively with 5 µg/ml GCV. These cells were clearly more resistant to GCV, since the control (non-transduced) cells are not fully destroyed in 1000 µg/ml GCV. This may reflect lower proliferation activity of Caki-2 cells compared to the glioma cell lines. When observing cytotoxicity with GCV concentration 10 µg/ml (Figure 3, panel C), it appears that only about 20% more cells were destroyed than expected on basis of the proportion of TK-GFP positive cells. For example, MOI 0.6 yielded 24% positive cells and about 40% of the cells died (the respective numbers at MOI 3 in BT4C were 25% and >90%). These results indicate that Caki-2 cells displayed a weak bystander effect in comparison with the rat glioma cells. We also tested the ability of other viral vector types to induce cell death upon GCV administration. BT4C or Caki-2 cells were subjected to low MOI (0.3) of LentiTGL, SFVTGL or SinTGL followed by treatment with increasing concentrations of GCV. This experiment revealed similar, transduction efficiency-dependent and vector-independent cytotoxic effect that was again weaker in Caki-2 cells than in BT4C cells (results not shown).

extent of bystander effect in a way where the proportion of TK-GFP positive cells remains the same and the density of the cells changes. In this experiment, when certain threshold density is reached, the cell-cell contacts start to form, manifesting the bystander effect. In comparison with the “classical” bystander analysis (where increasing percentage of TK-positive cells at the same density is used), this method is only qualitative and tells, whether the bystander effect exists or not. However, this assay can also reveal the presence of a cell contact -independent effects,

Figure. 3. Ganciclovir sensitivity of the cells transduced with TK-GFP vectors. BT4C (A), 9L (B) and Caki-2 (C) cell lines were transduced with indicated multiplicities of infection (MOI) of AdenoTGL, followed by determination of transduction rate (Td rate, percent of GFP positive cells). Transduced cells were then subjected to increasing concentrations of ganciclovir (GCV) for 5 days, followed by cell viability analysis using MTT-assay. The results are shown as percent of the A595nm –value from the assay, the negative control (non-transduced cells) being 100%. Each data point is mean of three different analyses, standard deviation markers have been omitted for clarity.

D. Bystander effect in BT4C, 9L and Caki-2 cells Results from GCV sensitivity experiments indicated that there are differences in the level of bystander effect present in the cell lines studied. We decided to test the

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that these cells displayed the classical, cell contact –dependent bystander effect. In addition, 9L cells displayed a strong bystander effect, but it was detected also in low cell densities, where there were no cell-cell contacts. As shown in panel B of Figure 4, the cell destruction was slightly amplified after the threshold cell density (2000-4000 cells) was reached, yet the difference was not statistically significant. This indicates that the dominant form of bystander effect in 9L cells is cell contact -independent, but the cell contact –mediated effect may be present as well. As opposed to BT4C and 9L rat glioma cells, Caki-2 human renal carcinoma cells didn't show any significant increase of cell death, when the number of cell-cell contacts increased (Figure 4, panel C). These data confirm that the bystander effect in Caki-2 cells is weak in comparison to rat glioma cells.

III. Discussion To assess the extent of gene transfer efficiency sufficient to provide a satisfactory TK/GCV –mediated cancer gene therapy, we created a TK-GFP fusion construct, TGL. This fusion was shown to make the target cells fluorescent under UV light and sensitize them to GCV at concentrations similar to native TK (Loimas et al, 1998). After the functionality of the fusion construct was verified, it was cloned into several viral vectors. In this paper, we report how these TGL –carrying viral vectors were used in vitro to study the features of TK/GCV suicide gene therapy approach. We had two major tasks in this research: 1. To get a rough idea how well three viral vector types (SFV, Sindbis and lentivirus) perform on model tumor cell lines compared to first generation adenoviral vector. 2. With the aid of TK-GFP –carrying viral vectors to find out, what is the true transduction efficiency needed for sufficient destruction of different cancer cells with GCV and what is the exact role of bystander effect in the treatment outcome. Our results from transduction efficiency analyses were in line with existing published data. First, type 5 human adenoviruses are good vectors for human cells but for many rodent cells, high MOIs were needed to obtain similar transduction efficiency. Secondly, alphaviral vectors (especially Sindbis virus) may not be promising vectors for all human tumor cells based on low transduction efficiency of Caki-2 in this report and similar results with HeLa (human cervical carcinoma) and HepG2 (human hepatoma) cells (Wahlfors et al, 2000). Thirdly, lentiviruses appeared to be promising vectors for cancer gene therapy, as they worked equally well on every tumor cell type tested. As many solid tumors contain large masses of quiescent or very slowly dividing cells, lentiviral vectors could be suitable for in vivo cancer gene therapy studies. The actual reasons for different transduction rates with different cell lines were not studied, but it is likely that the viral receptor status of the cells plays a role.

Figure 4. Bystander effect in the cell lines. BT4C (A), 9L (B) and Caki-2 (C) cell lines, containing 20% of TK-GFP positive cells, were split in 96-well plates using the indicated densities (X-axis). The cells were then grown with (AdTGL+GCV) or without (AdTGL) 5 µg/ml GCV for five days, followed by cell viability analysis using MTT-assay. The results are shown as A595nm –values from the assay and they are means of three different analyses, standard deviation markers are indicated. The presence of bystander effect is demonstrated as higher proportion of cytotoxicity than the original 20% (of TK-GFP cells present in the cell population). The threshold density of all the cells (i.e. cells starting to form physical contacts) was between 2000 and 4000 cells per well.

where the GCV-induced cytotoxicity is amplified by indirect contact. The idea of this type of analysis has been described before (Samejima and Meruelo, 1995). In this experiment (Figure 4), the proportion of TKGFP positive cells (created by AdenoTGL transduction) in each population was set to 20% and the cells were incubated in different cell densities in the presence of 5µg/ml GCV for five days. The negative controls (nontransduced cells treated with GCV) did not reveal any significant cytoxicity of ganciclovir per se (results not shown). As expected, the bystander effect was obvious in BT4C cells, where the amount of living cells decreased when the cell density increased. This effect was not seen before the cells grew close enough to each other (between 2000 and 4000 cells per 96-well plate well), suggesting

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It is highly unlikely that the differences in transduction efficiency in the tested cell lines are relevant to in vivo situation. The purpose of these experiments was solely to map the potential of certain viral vector types that have not been tried on tumor cells extensively and may be useful for further cell culture and animal work. We are aware of the fact, for example, that different viral vectors are titrated using different target cells and transduction protocols. Therefore, their efficiencies cannot be compared directly and readers are asked to be cautious about the conclusions that can be made based on the results shown in Figure 1. Varying gene expression levels (for example, in rat glioma cell line BT4C, see Figure 2) apparently reflect the nature of the vectors and the strength of the promoter used. Alphaviral vectors typically display a very powerful expression due to the replication cycle that amplifies the subgenomic sequence (which is transgene in recombinant vectors). Therefore, alphaviral expression is usually significantly higher than from any known promoter that utilizes eukaryotic RNA polymerase II. Promoters present in the adenoviral- and lentiviral vectors (hEF1! and CMV, respectively) are considered strong, but when low MOIs are used, multiple transduction events per cells are rare and most of the positive cells contain a few or only one TK-GFP expression unit. This is especially relevant explanation for the adenoviral vector (MOI 0.3) that yields only a few percent transduction efficiency on rat BT4C cells. The studies that determine transduction efficiency of different viral vectors can naturally be carried out with vectors that contain GFP alone. Therefore, the genuine utility of the TKGFP fusion gene system is demonstrated in GCV –sensitivity assays (Figure 3). In this experiment, similar multiplicity of infection with the TK-GFP vector on different cell lines yielded very different transduction efficiencies. If this kind of analysis was carried out using a vector carrying TK only, the actual gene transfer efficiency would have been difficult to determine and the reasons for treatment outcome would be more difficult to explain. The ganciclovir sensitivity experiments revealed a strict correlation between gene transfer efficiency and cytotoxicity, but the strength of bystander effect also appeared to play major role. AdenoTGL -transduced BT4C glioma cells were destroyed efficiently with low concentrations of GCV, because the transduction rate was high enough and bystander effect strong in these cells. The other glioma cell line 9L did not respond as well, although the bystander effect was strong. This was clearly attributable to modest transduction efficiency of adenoviral vectors. The third case was human renal carcinoma cell line Caki-2, which was very permissive to adenoviral vectors (almost 90% transduction rate with MOI 10) and displayed a weak bystander effect. In addition, these cells were responding poorly to low GCV concentrations, regardless that most of the cells were TK-

GFP positive. It is possible that this is due to low proliferative activity of this cell line. Adenoviral vector was used in the GCV sensitivity experiment to demonstrate the differences in treatment efficacy caused by different gene transfer rates. In order to test whether TK-GFP behaves differently in other viral vectors, SFVTGL, SinTGL and LentiTGL were tested in BT4C and Caki-2 cells using the same GCV sensitivity protocol. It turned out that regardless of the vector type carrying TK-GFP, the GCV-induced cytotoxicity was dependent on the gene transfer efficiency. We didn’t observe an effect of the higher TK-GFP expression level from alphaviral vectors or lower levels from the lentiviral vector. This in accordance with a report about variable effect of TK/GCV approach in different tumor cells. Beck and co-workers show that TK expression level is insignificant to the treatment result and there are other factors governing the susceptibility of the cell to TK/GCV effect (Beck et al, 1995). Another study by Chen et al (Chen et al, 1995) that used retrovirus-TK -transduced 9L cells, demonstrated that cell clones containing different TK enzyme levels give a similar GCV sensitivity. On the other hand, a study with C6 glioma cells (Shewach et al, 1994) concluded that increasing MOI (of retrovirus TK) increases the GCV sensitivity of the target cells. Alltogether, these findings suggest that the level of TK expression plays a role in the efficiency of this form of cancer gene therapy, but it is limited to situations, where GCV concentration and the proportion of TK-positive cells are low. We think that TK expression level is unlikely to be a factor in the primary TK-transduced cells, but may plays a major role in the fate of the neighboring cells i.e. the effect of the TK expression level is transmitted through the bystander effect. The bystander effect in our experiments was detected in changing the cell density instead of the proportion of TK positive cells. This method can reveal both cell contact –mediated and cell contact –independent effects. In our studies (see Figure 4. ), we found three different types of bystander effect: a strong, cell contact -mediated (BT4C cells), a weak, cell contact -mediated (Caki-2 cells), and a mixed type, with both cell contact -dependent and cell contact –independent components (9L cells). Although Princen and co-workers have recently shown that bystander effect in 9L is strictly dependent of cell-cell contacts (Princen et al, 1999), their experimental approach was different and does not rule out the possibility of mixed-type bystander effect in these cells. In addition, Bai et al. have shown the presence of cell contact –independent, apoptosis-mediated bystander mechanism in another rat glioma cell line C6 (Bai et al, 1999). Nine L cells have been studied extensively as targets for TK/GCV suicide gene therapy and their bystander effect has been reported. Based on the dye transfer results by Princen et al, it is likely that at least part of the effect is gap junction -mediated (Princen et al, 1999). Also, connexins 26 and 43 have been shown to be expressed in

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Life Technologies, respectively) or the original publication: adenoviral vectors (Graham and Prevec, 1992), lentiviral vectors with VSV-G envelope and pCMV_R8.2 helper construct –derived virus particles (Naldini et al, 1996; Zufferey et al, 1997). Sindbis viruses were produced using DH-BB packaging RNA and SFV viruses with conditionally infective helper2 packaging RNA (Berglund et al, 1993). For details of alphaviral vector production, see (Wahlfors et al, 2000). AdenoTGL vector preparation was titered on 293 cells, using the standard plaque assay method (Graham and Prevec, 1992). Titers of alphaviral and lentiviral vectors were determined with FACS analysis (FACSCalibur, Becton Dickinson, San Jose, CA, USA) by counting the proportion of GFP positive cells after viral transduction. Briefly, 1, 10 and 100 µl of viral supernatant was used for transduction of known number of BHK cells (alphaviral vectors) or 293T cells (lentiviral vectors) as described below. After appropriate incubation period, the transduced cells were analyzed by flow cytometry (triplicate samples, 10 000 events per sample). The titer was calculated from the percentage of fluorescent cells, using the value from transduction volume that yields 10-20% positives.

9L cells, yet overexpression of connexin 43 further enhanced the bystander effect in these cells (Estin et al, 1999). There are some published TK gene transfer studies on BT4C cells (Poptani et al, 1998;Sandmair et al, 1999) and the most recent one is indicating that there is a strong bystander effect in these cells (Sandmair et al, 2000). However, none of the papers indicated, whether the effect is connexin dependent. The same applies to Caki-2 cells that have not been studied as targets for TK/GCV –mediated gene therapy. Altogether, our results demonstrate that it is important to select the correct viral vector for each tumor type, and that the bystander effect must be present in the target cancer for optimal cell killing. Based on the data presented here, we suggest that TK/GCV approach should be considered only if the tumor cells have an efficient bystander effect and the viral vector in use can give at least 20% gene transfer efficiency. As shown in Caki-2 cells with a weak bystander effect, transduction efficiency as high as 90% is not enough at clinically relevant GCV concentration ranges. More importantly, our studies demonstrate the utility of TK-GFP as a tool in cancer gene therapy. In this paper, we have shown that viral vectors carrying this fusion construct can give valuable information about the features of TK/GCV –mediated cancer cell destruction in cell cultures. We are currently carrying out experiments in an animal model to seek answer to the most crucial question: how much gene transfer can we get in vivo with different viral vectors and is that enough for a sufficient GCV treatment result?

B. Cell lines BT4C rat glioma cells (Laerum et al, 1977), 9L rat gliosarcoma cells (ATCC CRL 2200) and Caki-2 human renal carcinoma cells (ATCC HTB-47) were grown in high-glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, 2 mM sodium pyruvate and 50 µg/ml gentamicin at 37 °C in the presence of 5 % CO2. BHK and 293T cells were grown as described in (Wahlfors et al, 2000).

C. Transduction of cultured cells Viral transductions were carried out as follows: the target cells were split into 24-well plates (50 000 / well). After 20 hours of incubation, the cells were washed once with PBS and incubated with viral vectors (MOI 0.3 or 3.0) for 1 hour, followed by addition of fresh medium. Flow cytometry was carried out 20 hours (SinTGL and SFVTGL) or 96 hours (AdenoTGL and LentiTGL) post-transduction to determine the number of positive cells and the level of TK-GFP expression.

IV. Materials and Methods A. Plasmid constructs and viral vectors The plasmid pETLGB containing TK-GFP fusion gene has been described earlier (Loimas et al, 1998). Due to a suboptimal emission wavelength for FACS analyses, the construct was modified by replacing the original GFP by the improved GFP from plasmid pGreenLantern (Life Technologies, Gaithersburg, MD) (Wahlfors et al, 2000). Shortly, the stop codon of the HSV1-TK was removed and a sequence encoding 11 amino acid linker (LEU ARG ASP PRO MET ALA ARG ALA ALA ALA THR) was added in-frame between the TK and GFP open reading frames. The second generation TK-GFP fusion gene (TGL) was subsequently cloned into following viral vectors using standard procedures: adenoviral vector pAVC2 (Ramsey et al, 1998) (the resulting vector: AdenoTGL), HIV-1 based lentiviral vector pHR' (Naldini et al, 1996) (the resulting vector: LentiTGL), Sindbis virus pSinRep5 (Hahn et al, 1992) (the resulting vector: SinTGL) and Semliki forest virus vector pSFV1 (Liljestrom and Garoff, 1991) (the resulting vector: SFVTGL). The expression of TGL is driven by 26S subgenomic promoter in both alphaviral vectors SFV and Sindbis, whereas the promoter in the adenoviral vector is hEF1! (from human elongation factor 1 alpha –gene) and CMV (cytomegalovirus early promoter) in the lentiviral vector. All the vectors were grown and purified according to manufacturers instructions (Sindbis and SFV, Invitrogen and

D. GCV sensitivity of transduced cells Alphaviral vectors: The cells were split into 96-well plates (2 500 / well), incubated for 20 hours, washed once with PBS (containing Ca2+ and Mg2+), incubated with viruses for 1 hour and exposed to different concentrations of GCV (0.1-1000µg/ml) for five days. To determine cell viability, MTT-assay was performed according to manufacturer’s instructions (Cell Proliferation Kit I, Roche Diagnostics, Indianapolis, IN). Adeno- and lentiviral vectors: The cells were split into 6-well plates (200 000 / well), incubated for 20 hours, washed once with PBS and incubated with viruses for 1 hour. After 3 days incubation the cells were split into 96-well plates (2500 / well), incubated for 20 hours and exposed to GCV. FACS analysis was performed to determine the proportion of GFP positive cells i.e. the transduction efficiency with

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Estin, D, Li, M W, Spray, D & Wu, J K. (1999) Connexins are expressed in primary brain tumors and enhance the bystander effect in gene therapy, Neurosurgery. 44, 361-368. Graham, F L & Prevec, L. (1992) Adenovirus-based expression vectors and recombinant vaccines, Biotechnology. 20, 363390. Hahn, C S, Hahn, Y S, Braciale, T J & Rice, C M. (1992) Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation, Proc Natl Acad Sci U S A. 89, 2679-2683. Laerum, O D, Rajewsky, M F, Schachner, M, Stavrou, D, Haglid, K G & Haugen, A. (1977) Phenotypic properties of neoplastic cell lines developed from fetal rat brain cells in culture after exposure to ethylnitrosourea in vivo, Z Krebsforsch Klin Onkol Cancer Res Clin Oncol. 89, 273295. Liljestrom, P & Garoff, H. (1991) A new generation of animal cell expression vectors based on the Semliki Forest virus replicon., Biotechnology (N Y). 9, 1356-1361. Loimas, S, Wahlfors, J & Janne, J. (1998) Herpes simplex virus thymidine kinase green fluorescent protein fusion gene: New tool for gene transfer studies and gene therapy, Biotechniques. 24, 614-618. Moolten, F L. (1986) Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy, Cancer Res. 46, 52765281. Morris, J C, Touraine, R., Wildner, O. and Blaese, R. M. (1999) Suicide Genes: Gene Therapy Applications Using Enzyme/Prodrug Strategies in The Development of Human Gene Therapy (Friedmann, T, ed) pp. 477-526, CSHL Press, Cold Spring Harbor, NY. Naldini, L, Blomer, U, Gallay, P, Ory, D, Mulligan, R, Gage, F H, Verma, I M & Trono, D. (1996) In-Vivo Gene Delivery and Stable Transduction Of Nondividing Cells By a Lentiviral Vector, Science. 272, 263-267. Poptani, H, Puumalainen, A M, Grohn, O H, Loimas, S, Kainulainen, R, Yla-Herttuala, S & Kauppinen, R A. (1998) Monitoring thymidine kinase and ganciclovir-induced changes in rat malignant glioma in vivo by nuclear magnetic resonance imaging, Cancer Gene Ther. 5, 101-109. Princen, F, Robe, P, Lechanteur, C, Mesnil, M, Rigo, J M, Gielen, J, Merville, M P & Bours, V. (1999) A cell typespecific and gap junction-independent mechanism for the herpes simplex virus-1 Thymidine kinase gene/ganciclovirmediated bystander effect, Clin. Cancer Res. 5, 3639-3644. Ramsey, W J, Caplen, N J, Li, Q, Higginbotham, J N, Shah, M & Blaese, R M. (1998) Adenovirus vectors as transcomplementing templates for the production of replication defective retroviral vectors, Biochem. and Biophys. Res. Commun. 246, 912-919. Samejima, Y & Meruelo, D. (1995) 'Bystander killing' induces apoptosis and is inhibited by forskolin, Gene Ther. 2, 50-58. Sandmair, A M, Loimas, S, Poptani, H, Vainio, P, Vanninen, R, Turunen, M, Tyynela, K, Vapalahti, M & Yla-Herttuala, S. (1999) Low efficacy of gene therapy for rat BT4C malignant glioma using intra-tumoural transduction with thymidine kinase retrovirus packaging cell injections and ganciclovir treatment, Acta Neurochir. 141, 867-872. Sandmair, A M, Turunen, M, Tyynela, K, Loimas, S, Vainio, P, Vanninen, R, Vapalahti, M, Bjerkvig, R, Janne, J & YlaHerttuala, S. (2000) Herpes simplex virus thymidine kinase gene therapy in experimental rat BT4C glioma model: Effect of the percentage of thymidine kinase-positive glioma cells

each MOI used. After five days incubation with GCV, MTT-assay was carried out. E. Bystander effect studies For bystander experiment, the cells were transduced with a high MOI of AdenoTGL as described above and FACS analysis was performed three days post-transduction. Parental (nontransduced) 9L cells with or without GCV treatment were used as negative controls. Based on flow cytometry, the proportion of TK-GFP positive cells in each transduced culture was set to 20%. The cells were the divided into 96-well plates in different densities (500-32000 cells/well) in order to obtain cultures with varying extent of cell-cell contacts. After incubation with 5Âľg/ml GCV for 5 days, cell viability was measured by MTT assay. The bystander effect was demonstrated by a higher degree of cytotoxicity than the 20% that was based on the presence of positive cells in the culture.

F. Statistical analysis Bystander effect experiments were analyzed with twotailed t test. Differences were considered significant when the probability (P) was <0.05.

Acknowledgements We thank Ms. Marjo-Riitta Toppinen for alphavirus vector preparation and analysis, Dr. Jay Ramsey (Clinical Gene Therapy Branch, NHGRI, NIH) for help with adenovirus vector preparation and Dr. Didier Trono (University of Geneva) for providing the lentiviral vector plasmids. This work was financially supported by grants from Saastamoinen Foundation and Finnish Cultural Foundation of Northern Savo to S.L.

References: Bai, S C, Du, L P, Liu, W Q, Whittle, I R & He, L. (1999) Tentative novel mechanism of the bystander effect in glioma gene therapy with HSV-TK/GCV system, Biochem. and Biophys. Res. Commun. 259, 455-459. Beck, C, Cayeux, S, Lupton, S D, Dorken, B & Blankenstein, T. (1995) The Thymidine Kinase Ganciclovir-Mediated Suicide Effect Is Variable In Different Tumor-Cells, Hum. Gene Ther. 6, 1525-1530. Berglund, P, Sjoberg, M, Garoff, H, Atkins, G, Sheahan, B & Liljestrom, P. (1993) Semliki Forest virus expression system: production of conditionally infectious recombinant particles., Biotechnology (N Y). 11, 916-920. Chen, C Y, Chang, Y N, Ryan, P, Linscott, M, McGarrity, G J & Chiang, Y L. (1995) Effect Of Herpes-Simplex Virus Thymidine Kinase Expression Levels On GanciclovirMediated Cytotoxicity and the Bystander Effect, Hum. Gene Ther. 6, 1467-1476. Culver, K W, Ram, Z, Wallbridge, S, Ishii, H, Oldfield, E H & Blaese, R M. (1992) In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors, Science. 256, 1550-1552.

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on treatment effect, survival time, and tissue reactions, Cancer Gene Ther. 7, 413-421. Shewach, D S, Zerbe, L K, Hughes, T L, Roessler, B J, Breakefield, X O & Davidson, B L. (1994) Enhanced cytotoxicity of antiviral drugs mediated by adenovirus directed transfer of the herpes simplex virus thymidine kinase gene in rat glioma cells, Cancer Gene Ther. 1, 107112. Smiley, W R, Laubert, B, Howard, B D, Ibanez, C, Fong, T C, Summers, W S & Burrows, F J. (1997) Establishment Of Parameters For Optimal Transduction Efficiency and Antitumor Effects With Purified High-Titer HSV-TK Retroviral Vector In Established Solid Tumors, Hum. Gene Ther. 8, 965-977.

Wahlfors, J J, Zullo, S A, Loimas, S, Nelson, D M & Morgan, R A. (2000) Evaluation of recombinant alphaviruses as vectors in gene therapy, Gene Ther. 7, 472-480. Wildner, O, Blaese, R M & Morris, J C. (1999) Therapy of colon cancer with oncolytic adenovirus is enhanced by the addition of herpes simplex virus thymidine kinase, Cancer Res. 59, 410-413. Zufferey, R, Nagy, D, Mandel, R J, Naldini, L & Trono, D. (1997) Multiply Attenuated Lentiviral Vector Achieves Efficient Gene Delivery In-Vivo, Nat. Biotechnol. 15, 871875.

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Gene Ther Mol Biol Vol 5, 157-162, 2000

Aberrant DNA methylation of p16 oncosuppressor gene in human cervical carcinoma Research Article

Luciano Mariani3, Giuseppe Zardo2, Cesare Rapone1, Anna Reale1, Giuseppe Netri5, Serena Buontempo1, Adriana de Capoa4 and, Paola Caiafa1* 1

Department of Cellular Biotechnologies and Haematology, University “La Sapienza” Rome, Italy Department of Biomedical Sciences and Technologies, University of L’Aquila, 67100 L’Aquila, Italy 3 Department of Gynecological Oncology, «Regina Elena» Cancer Institute, 00185 Rome, Italy 4 Department of Genetics and Molecular Biology, University of Rome «La Sapienza», Rome, Italy 5 Catholic University «Sacro Cuore», Rome, Italy. 2

__________________________________________________________________________________________________ *Correspondence: Prof. Paola Caiafa, Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Biochimica Clinica, Facoltà di Medicina e Chirurgia, Università di Roma "La Sapienza", Viale Regina Elena, 324 (Policlinico), 00161, Roma, Italia; Tel:06 49910900, Fax: 06 4440062; e-mail caiafa@bce.med.uniroma1.it Key words: DNA methylation, p16 gene, cervical carcinoma

Received: 23 October 2000; accepted: 12 November 2000

Summary The aim of this paper is to investigate the role played by DNA methylation in uterine cervical carcinoma. Since in a subset of human cancers the onco-suppressor CDKN2/p16 gene is an essential target for malignant transformation, its first and second exon regions were investigated for their DNA methylation levels by PCR methylation-dependent analysis. Our results on purified DNAs from cervical tissues show that, in spite of the diffuse DNA hypomethylation which characterizes neoplastic cells, specific DNA methylation of HpaII and CfoI sequences of the first exon occurs and increases with the grade of neoplastic transformation. These data support the idea that the p16 oncosuppressor gene is directly involved in malignant transformation through the methylation process.

housekeeping genes and there is evidence that transcription of genes correlated with CpG islands is inhibited when these regions are methylated (Bird, 1986; 1987). CpG islands have also been found to overlap part of the gene but in this case their methylation is not related to gene silencing (Jones, 1999). The DNA methylation process, through DNA methylase (Bestor and Ingram, 1983; Okano et al, 1998; Okano et al, 1999) and demethylase activities (Bhattacharya et al, 1999; Ramchandani et al, 1999), is involved in carcinogenesis in a paradoxical way. It is in fact possible to identify the development of two contrasting events in the same tumor sample: a general pattern of DNA hypomethylation (Feinberg and Vogelstein, 1983; Gama-Sosa et al, 1983; Goelz and Vogelstein, 1985; Feinberg et al, 1988; Kim et al, 1994; Laird and Jaenisch, 1994; Jurgens et al, 1996; Bernardino et al, 1997; de Capoa et al, 1999; Soares et al, 1999) in which specific onco-suppressor genes become methylated

I. Introduction DNA methyltransferase(s) by transferring methyl groups from S-adenosyl methionine to cytosines (Adams and Burdon, 1985) - which are essentially located in the CpG dinucleotides - encodes epigenetic information on DNA of great importance for the control of gene expression (Ng and Bird, 1999, Bird AP and Wolffe AP, 1999). The distribution of cytosines and 5-methylcytosines is non-random in genomic DNA and the methylation pattern, defined in early stages of embryonic development (Brandeis et al, 1993), is that in which methylated cytosines are present in the bulk of DNA while some DNA regions (1-2%) of about 1-2 Kbp in size, termed CpG islands, result in being highly protected from methylation despite their enrichment in dinucleotide CpG by about 5-10 times. The maintenance of this unmethylated state is extremely important since these DNA regions are located in the promoter regions of

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(Herman et al, 1994, 1995; Merlo et al, 1995; Fueyo et al, 1996; Nuovo et al, 1999; Baylin and Herman, 2000; Costello et al, 2000). Presently the high level of DNA demethylase found in neoplastic cells (Ramchandani et al, 1999; Bhattacharya et al, 1999) explains the spreading of DNA hypomethylation during tumorigenesis. However the aberrant hypermethylation of onco-suppressor genes, which would seem to be one of the most important events involved in tumorigenesis, still cannot be explained (Bird and Wolffe, 1999). An interesting – but as yet undemonstrated model – has been put forward (Baylin, 1997) to account for this hypermethylation of onco-suppressor genes in tumor cells. The fact that interaction between PCNA and DNMT1 is found in transformed cells (Chuang et al, 1997), is the basis of this model which supposes that a cellular event – to be identified – allows the DNMT1 to bind PCNA in an early replication phase and this would make it possible for the enzyme to methylate the DNA when CpG rich DNA regions replicate themselves (Selig et al, 1992). Thinking about this paradoxical behaviour of DNA methylation in neoplastic cells, the purpose of our preliminary studies was to analyze the DNA methylation pattern in human cervical tumors, taking the oncosuppressor gene CDKN2/p16 (Bonetta, 1994; Marx, 1994) as a model. The CDKN2 gene, located in chromosome 9p21 (Kamb et al, 1994), is likely to be a tumor suppressor gene since lack of its expression is frequently observed in many types of cancers (Nobori et al, 1994; Gonzalgo et al, 1995, 1997; Maesawa et al, 1996). This gene encodes the p16 protein (16 KDa), which plays in normal cells a critical role in cell-cycle regulation. In fact the p16, through its association with the cyclin-dependent kinase-4, inhibits kinase activity and consequently the progression from G1 to the S phase of cell cycle.

the low level of amplification product depends on the fact that not all sites are totally methylated. As shown in Figure 1B (1, 2), when digestions were carried out on DNA samples from healtly tissues no PCR amplification of DNA fragment either from the first or the second exon of p16 gene was found. These data show that in normal tissues and at least for the specific sequences examined, both exons were unmethylated. A different result was obtained by performing the same experiments on DNA purified from tumor samples. In Figure 1B (3, 4) the electrophoretic pattern of one tumor sample (C4), corresponding to an advanced stage (IIA) of cervical

II. Results and Discussion A. Evaluation of p16 gene methylation pattern The evaluation of p16 gene methylation level was carried out by the PCR-based methylation assay (Gonzalgo et al, 1998). This method foresees the digestion of DNA samples by methylation-sensitive restriction enzymes before PCR amplification, so that the DNA fragment will be amplified only if the enzymes do not cut inside the sequence. Enzymes used in the experiments were HpaII, CfoI and MspI. By comparing the electrophoretic pattern of non-digested amplified fragment to those obtained following enzymatic digestion we came up with information about the state of methylation of CpG moieties in the restriction sites. It is important to remember that more than one HpaII and CfoI site (Figure 1A), is located in the first and second p16 exon so that, since amplification occurs only if all sites are methylated

Figure 1: A - maps showing the HpaII (MspI) and CfoI sites in the first and the second exon of p16 gene. B - PCR amplification pattern of the first (1, 3) and the second exon (2, 4) of p16 gene from healthy (F2) and carcinogenic (C4) samples: UN (undigested control), M (digestion by MspI), H (digestion by HpaII), C (digestion by CfoI). C - table showing the results of densitometric scanning (Bio Image, Millipore) of the bands of p16 gene first exon amplified from tumor tissues (C3, C4, C5, C6) following digestion by methylation sensitive restriction enzymes. The average value of four indipendent experiments is presented.

cancer is shown. In these experiments DNA samples were digested by three different restriction enzymes before PCR

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amplification. Following HpaII and CfoI digestion a partial amplification was observed in the first but not in the second exon. When stage IB samples were examined result was different because there was either no amplification (C1, C2) or a lower amplification level (C3, C6). Figure 1C shows densitometric scanning of the electrophoretic bands obtained following digestion and PCR amplification of tumor samples (C3, C4, C5, C6). These data are indicative of an anomalous methylation pattern in the first exon of p16 gene in tumor DNA. As far as the second exon is concerned, since the methylation dependent assay is a method in which the amplification occurs only if all sites are methylated, we cannot exclude that some of the numerous HpaII or CfoI sites in the second exon are partially methylated.

inducing cell transformation or if they play a supporting role in the progression of carcinogenesis.

IV. Materials and Methods Experiments were performed on tissue samples from six patients affected by pathologically proven cervical squamous carcinoma. Mean age was 60 years (range: 50 - 75). According to the current FIGO staging system 4 patients were assigned to IB (3 IB1 and 1 IB2) and the other two women to IIA stage of disease. An adequate amount of cervical tissue was biopsed directly from the tumor mass: half of the specimen was referred to the pathologist to confirm the diagnosis, while the other half was processed. A control group of 5 healthy women, all having cytological and colposcopical negative tests, underwent cervical punch biopsy. Tissue samples were divided into two parts, one was analysed by the pathologist and the other was processed in our experiments. Each DNA sample (2 µg in 160 µl), from normal and malignant tissues, was pre-digested with 10 units of EcoRI at 37°C for 6 hours with the aim to improve the efficiency of enzymic digestion. Subsequently each sample was divided in four aliquots (40 µl) and DNAs digested by adding methylation independent enzyme as MspI and methylation dependent enzymes as HpaII and CfoI; the fourth aliquot was not added of any enzyme representing the undigested control. The enzymic digestion was carried out at 37°C for 36 hours and 10 units of enzyme were added every twelve hours for a total amount of 30 units of enzyme. PCR amplification was performed with the aim

B. Evaluation of DNA methylation level by methyl-accepting ability assay The methylation level which characterizes the neoplastic vs normal epitelial cervical cells, was evaluated by DNA methyl-accepting ability assay carried out on purified DNAs. As shown in Figure 2, all tumor samples showed higher levels of methyl-accepting ability, about 2.5-5 times more than in normal samples, thereby indicating lower levels of pre-existing methyl groups onto DNA. Our results have shown that despite the general DNA hypomethylation that characterizes the neoplastic cells, in 66% of the neoplastic samples examined there is an anomalous presence of some methyl groups on the HpaII and CfoI sensitive sequences located in the first exon of CDKN2/p16 gene. Indeed, it is interesting to note the coexistence in the same DNA sample of a general hypomethylation and a specific onco-suppressor gene hypermethylation. However it must be remembered that these two phenomena do not co-exist in the DNA from all types of tumors since while diffuse DNA hypomethylation has been shown to be a general state of neoplastic cells (Feinberg and Vogelstein, 1983; Gama-Sosa et al, 1983; Goelz and Vogelstein, 1985; Feinberg et al, 1988; Kim et al, 1994; Laird and Jaenisch, 1994; Jurgens et al, 1996; Bernardino et al, 1997; de Capoa et al, 1999; Soares et al, 1999) the anomalous hypermethylated state of specific onco-suppressor genes has not been identified in all kinds of tumors (Soares et al, 1999). For example in human urothelial carcinoma, the co-existence of hypo and hypermethylation, observed in tumor cell lines, is not present in tumor tissues where only general DNA hypomethylation is evidenciable (Soares et al, 1999). Therefore although the correlation between DNA methylation and neoplastic evolution would seem to be evident (Jones et al, 1992; Laird and Jaenisch, 1994; Laird and Jaenish, 1996; Schmutte and Jones, 1998; Jones and Laird, 1999; Jones, 1999) it is still difficult to establish whether these changes in methylation level are involved in

Figure 2: Methyl-accepting ability of DNAs extracted and purified from differently staged tumor samples (C1 IB, C2 IB, C3 IB, C4 IIA, C5 IIA, C6 IB) and from normal samples (F1, F2, F3, F4).

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of amplifying the first and the second exon of onco-suppressor gene p16 (Gonzalgo et al, 1998). Primers used for amplification of the first exon were: forward 5’ GAAGAAAGAGGAGGGGCT 3’ and reverse 5’ GCGCTACCTGATTCCAAT 3’. Primers used for amplification of the second exon were: forward 5’ GGAAATTGGAAACTGGAA 3’ and reverse 5’ AAAATGAATGCTCTGAGC 3’. The PCR mixture contained 100 ng of DNA (8 µl of digestion solution), primers (50 pmol both in forward and in reverse), dNTPs (final concentration of 0.2 mM) and 2.5 units of Taq DNA polymerase in Qiagen amplification buffer without MgCl2. The final concentration of MgCl2 was of 1.4 mM and it was added into reaction mixture from a 25 mM stock of MgCl2 taking into account the drawed volume and the concentration of MgCl2 present into buffer we used for restriction enzymic digestions. The reaction (50 µl) was carried out under the following conditions for both exons: denaturation at 95°C for 5 min; 95°C for 1 min, 55°C for 1 min, 72°C for 1 min for 30 cycles and a final segment at 72°C for 6 min. The amplified fragments were evidenced by 2% agarose gel. DNAs purified from different sample tissues were used as substrate for evaluating their methyl accepting ability. In a final volume of 100 µl 2.5 µg of DNA were incubated in presence of 1.5 units of bacterial SssI methylase using as methyl donor 80µM S-adenosyl-methionine plus 30 µCi /ml [3H] S-adenosylmethionine for 1 hour at 37ºC. The reaction was stopped by addition of 1% SDS and 250 µg/ml of proteinase K at 37°C for 30 mim. After cooling on ice 100 µg/ml of salmon sperm DNA were added as carrier and DNA was precipitated at 0°C with 20% of trichloroacetic acid (final concentration) and centrifugated at 7000 rpm for 10 min. Pellets were washed with 5% trichloroacetic acid and resuspended in 0.5 ml of 0.5 M NaOH and heated at 60°c for 30 min. After cooling in ice , DNAs were precipitated with 15% trichloroacetic acid and then each DNA sample was recovered on a glass fiber paper (GF/C Whatman). The filters were repeatedly washed by 5% trichloroacetic acid and by 95% ethanol. The incorporated radioactivity was measured in a Beckman Ls-6800 liquid scintillation spectrometer.

Bernardino J, Roux C, Almeida A, Vogt N, Gibaud A, GerbaultSeureau M, Magdelenat H, Bourgeois CA, Malfoy B and Dutrillaux B (1997) DNA hypomethylation in breast cancer: an independent parameter of tumor progression? Cancer Genet Cytogenet 97, 83-89. Bestor TH and Ingram VM (1983) Two DNA methyltransferases from murine erythroleukemia cells: purification, sequence specificity and mode of interaction with DNA, Proc Natl Acad Sci USA 80, 5559-5563. Bhattacharya SK, Ramchandani S, Cervoni N and Szyf M ( 1999) A mammalian protein with specific demethylase activity for mCpG DNA. Nature 397, 579-483. Bird AP (1986) CpG-rich islands and the function of DNA methylation. Nature, 321, 209-213. Bird AP (1987) CpG islands as gene markers in the vertebrate nucleus. Trends Genet, 3, 342-347. Bird AP and Wolffe AP (1999) Methylation-Induced RepressionBelts, Braces, and Chromatin. Cell 99, 451-454. Bonetta L (1994) Open questions on p16. Nature 370, 180. Brandeis M, Ariel M and Cedar H (1993) Dynamics of DNA methylation during development. Bioessays 15, 709-713. Chuang LS-H, Ian H-I, Koh T-W, Ng H-H- Xu G and Li BFL (1997) Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21 WAF1. Science 26, 1996-2000. Costello JF, Fruhwald MC, Smiraglia DJ et al. (2000) Aberrant CpG-island methylation has non-random and tumor-typespecific patterns. Nature Genet 25, 132-138. de Capoa A, Grappelli C, Febbo FR, Span_ A, Niveleau A, Cafolla A, Cordone I and Foa R (1999) Methylation levels of normal and chronic lymphocytic leukemia B lymphocytes: computer-assisted quantitative analysis of anti5methylcytosine antibody binding to individual nuclei. Cytometry 36, 157-159. Feinberg AP and Vogelstein B (1983) Hypomethylation distinguished genes of some human cancers from their normal counterparts. Nature 301, 89-92. Feinberg AP, Gehrke CW, Kuo KC and Ehrlich M (1988) Reduced genomic 5-methylcitosine content in human colonic neoplasia. Cancer Res 48, 1159-1161. Fueyo J, Gomez-Manzano C, Bruner JM, Saito Y, Zhang B, Zhang W, Levin VA, Yung WKA and Kyritsis AP (1996) Hypermethylation of the CpG of the p16-CDKN2 correlates with gene inactivation in gliomas. Oncogene 13, 1615-1619. Gama-Sosa MA, Slagel VA, de Bustros A, Trewyn RW, Oxenhandler R, Kuo KC, Gehrke CW and Ehrlich M (1983) The 5-methylcytosine content of DNA from human tumors. Nucleic Acids Res 11, 6883-6894. Goelz SE and Vogelstein B (1985) Hypomethylation of DNA from benign and malignant human colon neoplasms. Science 228, 187-190. Gonzalgo ML, Bender CM, Edward H, You J, Glendenin JM, Flores JF, Walker GJ, Hayward NK, Jones PA and Fountain J-W (1997) Low frequency of p16/CDKN2A methylation in sporadic melanoma: comparative approaches for methylation analysis of primary tumors. Cancer Res 57, 5336-5347. Gonzalgo ML, Hayashida T, Bender CM, Pao MM, Tsai YC, Gonzales FA, Nguyen HD, Nguyen TT and Jones PA (1998) The role of DNA methylation in expression of the p19/p16 locus in human bladder cancer cell lines. Cancer Res 58, 1245-1252. Herman JG, Latif F, Weng J, Lerman MI, Zbar B, Liu S, Samid D, Duan DSR, Gnarra JR, Linehan WM and Baylin SB (1994) Silencing of the VHL tumor-suppressor gene by DNA

Acknowledgements We thank Alessandra Spanò for technical assistance. This work was supported by the Italian Ministry of University and Scientific and Technological Research (40% Progetti di Interesse Nazionale, 60% Ricerca Scientifica Università di L’Aquila e di Roma, «La Sapienza») and by the Consiglio Nazionale delle Ricerche (CNR).

References: Adams RLP and Burdon RH (1985) Molecular Biology of DNA Methylation. Chapter 7. Springer Series in Molecular Biology. Springer Verlag, New York. Baylin SB (1997) Tying it all together: epigenetics, genetics, cell cycle and cancer. Science 277, 1948-1949. Baylin SB and Herman JG (2000) DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet 16, 168-174.

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methylation in renal carcinoma. Proc Natl Acad Sci USA 91, 9700-9704. Herman J G, Merlo A, Mao L, Lapidus RG, Issa J-PJ, Davidson NE, Sidransky D and Baylin SB (1995) Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res 55, 4525-4530. Jones PA, Rideout WM, Shen JC, Spruck CH and Tsai YC (1992) Methylation, mutation and cancer. Bioessays 14, 3336 Jones PA (1999) The DNA methylation paradox. Trends Genet 15, 34-37. Jones PA and Laird PW (1999) Cancer epigenetics comes of age. Nature Genet 21, 163-166. Jurgens B, Schimtz-Drager BJ and Schulze W (1996) Hypomethylation of L1 LINE sequences prevailing in human urothelial carcinoma. Cancer Res 56, 5698-5703. Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, Stockert E, Day RSIII, Johnson BE and Skolnick MH (1994) A cell cycle regulator potentially involved in genesis of many tumor types. Science 264, 436440. Kim YI, Giuliano A, Hatch KD, Schneider A, Nour MA, Dallal GE, Selhub J and Mason JB (1994). Global DNA hypomethylation increases progressively in cervical dysplasia and carcinoma. Cancer 74, 893-899. Laird PW and Jaenisch R (1994) DNA methylation and cancer Hum Mol Genet 3, 1487-1495. Laird PW and Jaenisch R (1996) The role of DNA methylation in cancer genetics and epigenetics. Annu Rev Genet 30, 441464. Maesawa C, Tamura G, Satoshi N, Ogasawara S, Ishida K, Terashima M, Sakata K, Sato N, Saito K and Satodate R (1996) Inactivation of CDKN2 gene by homozygous deletion and de novo methylation is associated with advanced stage esophageal squamous cell carcinoma. Cancer Res 56, 38754878. Marx J (1994) A challenge to p16 gene as a major tumor suppressor. Science 264, 1846.

Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, Burger PC, Baylin SB and Sidransky D (1995) 5â&#x20AC;&#x2122; CpG island methylation is associated with transcriptional silencing of the tumor suppressor p16/CDKN2/MTS1 in human cancer. Nature Med 1, 686-692. Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K and Carson DA (1994) Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 368, 753756. Ng HH and Bird A (1999) DNA methylation and chromatin modification. Curr Opin Genet 9, 158-163. Nuovo GJ, Plaia TW, Belinsky SA, Baylin SB and Herman JG (1999) In situ detection of the hypermethylation-induced inactivation of the p16 early event in oncogenesis. Proc Natl Acad Sci USA 96, 12754-12759. Okano M (1998) Cloning and characterization of a family of novel mammalian DNA(Cytosine-5) methyltransferase. Nature Genet 19, 219-220. Okano M, Bell DW, Haber DA, and Li E (1999) DNA methyltransferase Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247-257. Ramchandani S, Bhattacharya SK, Cervoni N and Szyf M ( 1999) DNA methylation is a reversible biological signal. Proc Natl Acad Sci USA 96, 6107-6112. Schmutte C and Jones PA (1998) Involvment of DNA methylation in human carcinogenesis. Biol Chem 379, 377388. Selig S., Okumura K., Ward D.C. and Cedar H (1992) Delineation of DNA replication time zones by fluorescence in situ hybridization. EMBO J 11, 1217-1225. Soares J, Pinto AE, Cunha CV, Barao AS, Sousa JM and Cravo M (1999) Global DNA hypomethylation in breast carcinoma. Cancer 85, 112-118.

161

Mariani et al: DNA methylation of p16 gene in cervical carcinoma

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