Gene Therapy & Molecular Biology Volume 7 Issue A by Niko Boulikas

GENE THERAPY & MOLECULAR BIOLOGY FROM BASIC MECHANISMS TO CLINICAL APPLICATIONS

Volume 7 2003 Published by Gene Therapy Press

GENE THERAPY & MOLECULAR BIOLOGY FREE ACCESS www.gtmb.org

!!!!!!!!!!!!!!!!!!!!!!!! ! Editor

Teni Boulikas Ph. D., CEO Regulon Inc. 715 North Shoreline Blvd. Mountain View, California, 94043 USA Tel: 650-968-1129 Fax: 650-567-9082 E-mail: teni@regulon.org

Teni Boulikas Ph. D., CEO, Regulon AE. Gregoriou Afxentiou 7 Alimos, Athens, 17455 Greece Tel: +30-210-9853849 Fax: +30-210-9858453 E-mail: teni@regulon.org

!!!!!!!!!!!!!!!!!!!!!!!! ! Assistant to the Editor Maria Vougiouka B.Sc., Gregoriou Afxentiou 7 Alimos, Athens, 17455 Greece Tel: +30-210-9858454 Fax: +30-210-9858453 E-mail: maria@cancer-therapy.org

!!!!!!!!!!!!!!!!!!!!!!!! ! Associate Editors

Aguilar-Cordova, Estuardo, Ph.D., AdvantaGene, Inc., USA Berezney, Ronald, Ph.D., State University of New York at Buffalo, USA Crooke, Stanley, M.D., Ph.D., ISIS Pharmaceuticals, Inc, USA Crouzet, Joël, Ph.D. Neurotech S.A, France Gronemeyer, Hinrich, Ph.D. I.N.S.E.R.M., IGBMC, France Rossi, John, Ph.D., Beckman Research Institute of the City of Hope, USA Shen, James, Ph.D., Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China & University of California at Davis, USA. Webb, David, Ph.D., Celgene Corporation, USA Wolff, Jon, Ph.D., University of Wisconsin, USA

!!!!!!!!!!!!!!!!!!!!!!!! ! Editorial Board Akporiaye, Emmanuel, Ph.D., Arizona Cancer Center, USA Anson, Donald S., Ph.D., Women's and Children's Hospital, Australia Ariga, Hiroyoshi, Ph.D., Hokkaido University, Japan Baldwin, H. Scott, M.D Vanderbilt University Medical Center, USA Barranger, John, MD, Ph.D., University of Pittsburgh, USA Black, Keith L. M.D., Maxine Dunitz Neurosurgical

Institute, Cedars-Sinai Medical Center, USA Bode, Jürgen, Gesellschaft für Biotechnologische Forschung m.b.H., Germany Bohn, Martha C., Ph.D., The Feinberg School of Medicine, Northwestern University, USA Bresnick, Emery, Ph.D., University of Wisconsin Medical School, USA Caiafa, Paola, Ph.D., Università di Roma “La Sapienza”, Italy Chao, Lee, Ph.D., Medical University of South Carolina, USA

Cheng, Seng H. Ph.D., Genzyme Corporation, USA Clements, Barklie, Ph.D., University of Glasgow, USA Cole, David J. M.D., Medical University of South Carolina, USA Chishti, Athar H., Ph.D., University of Illinois College of Medicine, USA Davie, James R, Ph.D., Manitoba Institute of Cell Biology;USA DePamphilis, Melvin L, Ph.D., National Institute of Child Health and Human, National Institutes of Health, USA Donoghue, Daniel J., Ph.D., Center for Molecular Genetics, University of California, San Diego, USA Eckstein, Jens W., Ph.D., Akikoa Pharmaceuticals Inc, USA Fisher, Paul A. Ph.D., State University of New York, USA Galanis, Evanthia, M.D., Mayo Clinic, USA Gardner, Thomas A, M.D., Indiana University Cancer Center, USA Georgiev, Georgii, Ph.D., Russian Academy of Sciences, USA Getzenberg, Robert, Ph.D., Institute Shadyside Medical Center, USA Ghosh, Sankar Ph.D., Yale University School of Medicine, USA Gojobori, Takashi, Ph.D., Center for Information Biology, National Institute of Genetics, Japan Harris David T., Ph.D., Cord Blood Bank, University of Arizona, USA Heldin, Paraskevi Ph.D., Uppsala Universitet, Sweden Hesdorffer, Charles S., M.D., Columbia University, USA Hoekstra, Merl F, Ph.D., Epoch Biosciences, Inc., USA Hung, Mien-Chie, Ph.D., The University of Texas, USA Johnston, Brian, Ph.D., Somagenics, Inc, USA Jolly, Douglas J, Ph.D., Advantagene, Inc.,USA Joshi, Sadhna, Ph.D., D.Sc., University of Toronto Canada Kaltschmidt, Christian, Ph.D., Universität Witten/Herdecke, Germany Kiyama, Ryoiti, Ph.D., National Institute of Bioscience and Human-Technology, Japan Krawetz, Stephen A., Ph.D., Wayne State University School of Medicine, USA Kruse, Carol A., Ph.D., La Jolla Institute for Molecular Medicine, USA Kuo, Tien, Ph.D., The University of Texas M. D. Anderson Cancer USA Kurachi Kotoku, Ph.D., University of Michigan Medical School, USA Kuroki, Masahide, M.D., Ph.D., Fukuoka University School of Medicine, Japan Lai, Mei T. Ph.D., Lilly Research Laboratories USA

Latchman, David S., PhD, Dsc, MRCPath University of London, UK Lavin, Martin F, Ph.D., The Queensland Cancer Fund Research Unit, The Queensland Institute of Medical Research, Australia Lebkowski, Jane S., Ph.D., GERON Corporation, USA Li, Jian Jian, Ph.D., City of Hope National Medical Center, USA Li, Liangping Ph.D., Max-Delbrück-Center for Molecular Medicine, Germany Lu, Yi, Ph.D., University of Tennessee Health Science Center, USA Lundstrom Kenneth, Ph.D. , Bioxtal/Regulon, Inc. Switzerland Malone, Robert W., M.D., Aeras Global TB Vaccine Foundation, USA Mazarakis, Nicholas D. Ph.D., Oxford BioMedica, UK Mirkin, Sergei, M. Ph.D., University of Illinois at Chicago, USA Moroianu, Junona, Ph.D., Boston College, USA Müller, Rolf, Ph.D., Institut für Molekularbiologie und Tumorforschung, Phillips-Universität Marburg, USA Noteborn, Mathieu, Ph.D., Leiden University, The Netherlands Papamatheakis, Joseph (Sifis), Ph.D., Institute of Molecular Biology and Biotechnology Foundation for Research and Technology Hellas, USA Platsoucas, Chris, D., Ph.D., Temple University School of Medicine, USA Rockson, Stanley G., M.D., Stanford University School of Medicine, USA Poeschla, Eric, M.D., Mayo Clinic, USA Pomerantz, Roger, J., M.D., Tibotec, Inc., USA Raizada, Mohan K., Ph.D., University of Florida, USA Razin, Sergey, Ph.D., Institute of Gene Biology Russian Academy of Sciences, USA Robbins, Paul, D, Ph.D., University of Pittsburgh, USA Rosenblatt, Joseph, D., M.D, University of Miami School of Medicine, USA Rosner, Marsha, R., Ph.D., Ben May Institute for Cancer Research, University of Chicago, USA Royer, Hans-Dieter, M.D., (CAESAR), Germany Rubin, Joseph, M.D., Mayo Medical School Mayo Clinic, USA Saenko Evgueni L., Ph.D., University of Maryland School of Medicine Center for Vascular and Inflammatory Diseases, USA Salmons, Brian, Ph.D., (FSG-Biotechnologie GmbH), Austria Santoro, M. Gabriella, Ph.D., University of Rome Tor Vergata, USA Sharrocks, Andrew, D., Ph.D., University of Manchester, USA

Shi, Yang, Ph.D., Harvard Medical School, USA Smythe Roy W., M.D., Texas A&M University Health Sciences Center, USA Srivastava, Arun Ph.D., University of Florida College of Medicine, USA Steiner, Mitchell, M.D., University of Tennessee, USA Tainsky, Michael A., Ph.D., Karmanos Cancer Institute, Wayne State University, USA Sung, Young-Chul, Ph.D., Pohang University of Science & Technology, Korea Taira, Kazunari, Ph.D., The University of Tokyo, Japan Terzic, Andre, M.D., Ph.D., Mayo Clinic College of Medicine, USA Thierry, Alain, Ph.D., National Cancer Institute, National Institutes of Health, France

Trifonov, Edward, N. Ph.D., University of Haifa, Israel Van de Ven, Wim, Ph.D., University of Leuven, Belgium Van Dyke, Michael, W., Ph.D., The University of Texas M. D. Anderson Cancer Center, USA White, Robert, J., University of Glasgow, UK White-Scharf, Mary, Ph.D., Biotransplant, Inc., USA Wiginton, Dan, A., Ph.D., Children's Hospital Research Foundation, CHRF , USA Yung, Alfred, M.D., University of Texas, USA Zannis-Hadjopoulos, Maria Ph.D., McGill Cancer Centre, Canada Zorbas, Haralabos, Ph.D., BioM AG Team, Germany

!!!!!!!!!!!!!!!!!!!!!!!! ! Associate Board Members

Aoki, Kazunori, M.D., Ph.D., National Cancer Center Research Institute, Japan Cao, Xinmin, Ph.D., Institute of Molecular and Cell Biology, Singapore Falasca, Marco, M.D., University College London, UK Gao, Shou-Jiang, Ph.D., The University of Texas Health Science Center at San Antonio, USA Gibson, Spencer Bruce, Ph.D., University of Manitoba, USA Graâ&#x20AC;˘a, Xavier, Ph.D., Temple University School of Medicine, USA

For submission of manuscripts and inquiries: Editorial Office Teni Boulikas, Ph.D./ Maria Vougiouka, B.Sc. Gregoriou Afxentiou 7 Alimos, Athens 17455 Greece Tel: +30-210-985-8454 Fax: +30-210-985-8453 and electronically to maria@cancer-therapy.org

Gu, Baohua, Ph.D., The Jefferson Center, USA Hiroki, Maruyama, M.D., Ph.D., Niigata University Graduate School of Medical and Dental Sciences, Japan MacDougald, Ormond A, Ph.D., University of Michigan Medical School, USA Rigoutsos, Isidore, Ph.D., Thomas J. Watson Research Center, USA

Instructions to authors: Gene Therapy and Molecular Biology (GTMB) FREE ACCESS www.gtmb.org Scope Gene Therapy and Molecular Biology, bridging various fields is one of the most rapid with free access at gtmb.org. The scope of Gene Therapy and Molecular Biology is to promote interaction between researchers in the fields of Gene Therapy and Molecular Biology providing rapid publication of review articles and research papers. Articles (both invited and submitted) review or report novel findings of importance to a general audience in gene therapy, molecular medicine, gene discovery, and molecular biology with emphasis to molecular mechanisms. The journal will accept papers on all aspects of gene therapy, including gene delivery systems, gene therapy of cancer and other diseases (e.g. CFTR, hemophilia, AIDS, restenosis) at the clinical, preclinical or cell culture stage, gene discovery, cancer immunotherapy, DNA vaccines, use of DNA regulatory elements in gene transfer, cell therapy and transplantation, arraying technologies & DNA chips, peptide libraries and drug discovery related to gene therapy, cell targeting, gene targeting, therapy with oligonucleotides (antisense, ribozymes, triplex). The authors are encouraged to elaborate on the molecular mechanisms that govern a gene therapy approach. Gene Therapy and Molecular Biology will also publish articles on, transcription factors, DNA replication, recombination, repair, chromatin, nuclear matrix, DNA regulatory regions, locus control regions, protein phosphorylation, signal transduction, development, and on molecular mechanism of human disease. To make the publication attractive authors are encouraged to include color figures.

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Please submit an electronic version of full text and figures preferably in jpeg format. The electronic version of the figures will be used for the rapid reviewing process. High quality prints or photograph of the figures and the original with one copy should be sent via express mail to the Editorial Office. Editorial Office Teni Boulikas, Ph.D./ Maria Vougiouka, B.Sc. Gregoriou Afxentiou 7 Alimos, Athens 17455 Greece Tel: +30-210-985-8454 Fax: +30-210-985-8453 and electronically to maria@cancer-therapy.org The free electronic access to articles published in "GTMB" to a big general audience, the attractive journal title, the speed of the reviewing process, the no-charges for page numbers or color figure reproduction, the 25 complimentary reprints, the rapid electronic publication, the embracing of many fields in cancer, the anticipated high quality in depth reviews and first rate research articles and most important, the eminent members of the Editorial Board being assembled are prognostic factors of a big success for the newly established journal.

Table of contents

Gene Therapy and Molecular Biology Vol 7, December 2003 Pages

Type of Article

Article title

Authors (corresponding author is in boldface)

1-14

Review Article

Dynamic histone acetylation and its involvement in transcription

Virginia A. Spencer and James R. Davie

15-23

Review Article

Tumor therapy using radiolabelled antisense oligomers- aspects for antiangiogenetic strategy and positron emission tomography

Kalevi JA Kairemo, Mark Lubberink, Mikko Tenhunen, Antti P Jekunen

25-35

Review Article

Strategy of sensitizing tumor cells with adenovirus-p53 transfection

Jekunen Antti, Miettinen Susanna, Mäenpää Johanna, Kairemo Kalevi

37-42

Review Article

Antigenicity and immunogenicity of HIV envelope gene expressed in baculovirus expression system

Alka Arora, Pradeep Seth

43-59

Review Article

Characterization of genes transcribed in an Ixodes scapularis cell line that were identified by expression library immunization and analysis of sequence tags

Consuelo Almazan, Katherine M. Kocan, Douglas K. Bergman, Jose C. GarciaGarcia, Edmour F. Blouin and José de la Fuente

61-68

Research Article

Delayed intratracheal injection of manganese superoxide dismutase (MnSOD)-plasmid/liposomes provides suboptimal protection against irradiationinduced pulmonary injury compared to treatment before irradiation

Michael W. Epperly, Hongliang Guo, Michael Bernarding, Joan Gretton, Mia Jefferson, Joel S. Greenberger

69-73

Mini Review

Regulation of vascular endothelial growth factor by hypoxia

Ilana Goldberg-Cohen, Nina S Levy, Andrew P Levy

75-89

Review Article

Gene therapy antiproliferative strategies against cardiovascular disease.

Marisol Gasc!n-Ir"n, Silvia M. SanzGonz#lez and Vicente Andrés

91-98

Review Article

Regulation of the Sp/KLF-family of transcription factors: focus on posttranscriptional modification and proteinprotein interaction in the context of chromatin

Toru Suzuki, Masami Horikoshi, and Ryozo Nagai

99-102

Research Article

Detection of MET oncogene amplification in hepatocellular carcinomas by comparative genomic hybridization on microarrays

W.L. Robert Li, Nagy A. Habib, Steen L. Jensen, Paul Bao, Diping Che, Uwe R. Müller

103-111

Research Article

HMG-CoA-reductase inhibitiondependent and independent effects of statins on leukocyte adhesion

Triantafyllos Chavakis, Thomas Schmidt-Wรถll, Peter. P. Nawroth, Klaus T. Preissner, Sandip M. Kanse

113-133

Review Article

Current progress in adenovirus mediated gene therapy for patients with prostate carcinoma

Ahter D. Sanlioglu,, Turker Koksal, Mehmet Baykara, Guven Luleci, Bahri Karacay and Salih Sanlioglu

135-151

Review Article

Gene therapy for vascular diseases

Sarah J. George, Filomena de Nigris, Andrew H. Baker, Claudio Napoli

153-165

Review Article

Angiogenic gene therapy for improving islet graft vascularization.

Nan Zhang, Karen Anthony, Katsunori Shinozaki, Jennifer Altomonte, Zachary Bloomgarden and Hengjiang Dong

167-172

Research Article

G-CSF Receptor-mediated STAT3 activation and granulocyte differentiation in 32D cells.

Ruifang Xu, Akihiro Kume, Yutaka Hanazono, Kant M. Matsuda, Yasuji Ueda, Mamoru Hasegawa, Fumimaro Takaku, and Keiya Ozawa

173-179

Research Article

Calcium induces apoptosis and necrosis in hematopoetic malignant cells: Evidence for caspase-8 dependent and FADDautonomous pathway

Christof J. Burek Malgorzata Burek, Johannes Roth, and Marek Los

181-209

Review Article

The current status and future direction of fetal gene therapy

Anna L David, Michael Themis, Simon N Waddington, Lisa Gregory, Suzanne MK Buckley, Megha Nivsarkar, Terry Cook, Donald Peebles, Charles H Rodeck, Charles Coutelle

211-219

Research Article

The role of EBV and genomic sequences in gene expression from extrachromosomal gene therapy vectors in mouse liver

Stephanie M. Stoll, Leonard Meuse, Mark A. Kay, and Michele P. Calos

221-228

Review Article

Site-specific kidney-targeted plasmid DNA transfer using nonviral techniques

Hiroki Maruyama, Noboru Higuchi, Shigemi Kameda, Gen Nakamura, Junichi Miyazaki, and Fumitake Gejyo

229-238

Research Article

Hepatocyte-targeted delivery of Sleeping Beauty mediates efficient gene transfer in vivo

Betsy T. Kren, Siddhartha S. Ghosh, Cheryle L. Linehan, Namita RoyChowdhury, Perry B. Hackett, Jayanta Roy-Chowdhury, and Clifford J. Steer

239-243

Research Article

PRL-3 as a target for cancer therapy

Koh Vicki, Fu Jianlin, Guo Ke, Lip Kuo Ming, Li Jie and Zeng Qi

245-254

Review Article

Protective effect of heat shock proteins: potential for gene therapy

David S. Latchman

255-272

Review Article

Lung cancer gene therapy

273-289

Review Article

Advances in cationic lipid-mediated gene delivery

Kexia Cai, Mai Har Sham, Paul Tam, Wah Kit Lam and Ruian Xu Benjamin Martin, Abderrahim Aissaoui, Matthieu Sainlos, Noufissa Oudrhiri, Michelle Hauchecorne, Jean-Pierre

291-298

Research Article

Unusual chemical hypersensitivity of the d(GA)nâ&#x20AC;˘ d(TC)n repeat in vivo dependent on functional lactose repressor

Vigneron, Jean-Marie Lehn and Pierre Lehn Gerald L. Buldak and Sergei M. Mirkin

Gene Therapy and Molecular Biology Vol 7, page 1 Gene Ther Mol Biol, Vol 7, 1-13, 2002

Dynamic histone acetylation and its involvement in transcription Review Article

Virginia A. Spencer and James R. Davie! Manitoba Institute of Cell Biology, 675 McDermot Avenue, Winnipeg, Manitoba, R3E 0V9 CANADA

__________________________________________________________________________________ Correspondence: Dr. J.R. Davie; Manitoba Institute of Cell Biology; 675 McDermot Avenue Winnipeg, MB, R3E 0V9; Tel: (204) 7872391; Fax: (204) 787-2190; E-mail: Davie@cc.umanitoba.ca Key words: histone acetylation, histone acetyltransferases and deacetylases, transcription Received: 10 January 2002; accepted: 22 January, 2002; electronically published: July 2003

Summary Histones are subject to a variety of posttranslational modifications, the most studied being acetylation of the N terminal lysine residues. Acetylation is a dynamic event mediated by the actions of histone acetyltransferases and deacetylases. The exact function of this event in transcription has remained an enigma for several reasons. The enzymes that catalyze this dynamic event act on histones, but are also capable of affecting the properties of nonhistone proteins including transcription factors. Also, some histone acetyltransferases can acetylate the histones along a specific gene, while simultaneously acetylating the histones over an entire region of the genome. More confusing are the observations that the acetylation pattern of histones along one transcriptionally active gene may differ significantly from that along another. Perhaps some of these discrepancies can be explained by the dynamic interplay between histone acetyltransferases and deacetylases, and the proximity of a gene to these enzymes. Thus, to fully appreciate the role of acetylation in transcription, we must further understand the dynamic nature of this event. with proteins or DNA (Hansen et al, 1998). The DNA extending from one nucleosome to another varies in length and is referred to as linker DNA (Spencer and Davie, 1999). A fifth type of histone called linker histone H1 contains a central globular domain surrounded by N and C-terminal tails. Histone H1 binds to the regions of linker DNA that enter and exit the nucleosome, as well as to nucleosomal DNA near the dyad axis of symmetry (Spencer and Davie, 1999).

I. The organization of nuclear DNA The DNA within a cell is packaged into chromatin. The basic structural repeating unit of chromatin is the nucleosome which is composed of an octamer of two histone H2A-H2B dimers bound to a histone H3 and H4 tetramer (Spencer and Davie, 1999). During nucleosome assembly, histones H3 and H4 first associate with DNA, followed by histones H2A and H2B (Kimura and Cook, 2001). The association of H3 and H4 with nucleosomal DNA appears to be more stable than that for H2A and H2B. The end result is the wrapping of one hundred and forty-six base pairs of DNA around each histone octamer. The four core histones have a basic N terminal tail, a central globular domain organized into a histone fold and a C terminal tail (Figure 1). The central histone fold domain is involved in histone-histone and histone-DNA interactions, and, therefore is important in histone octamer and nucleosome formation (Spencer and Davie, 1999). The histone N terminal tails protrude from the core particle in all directions and vary in length from 16 to 44 amino acids (Davie and Spencer, 2001). Evidence showing that H3 and H4 display "-helical structures in their N terminal domains when bound to nucleosomal DNA has lead to the belief that these domains fold upon contact

II. Chromatin structure & organization At physiological ionic strength, chromatin assumes the form of a 30 nm fiber and higher order structures (Davie, 1995). This fiber is a dynamic structure that is continually condensing and unfolding. For example, a chromatin fiber composed of nucleosomes spaced at physiological intervals is in equilibrium between an unfolded, moderately folded, highly folded and oligomerized conformation (Annunziato and Hansen, 2000). The proteolytic removal of the N terminal domains does not significantly change nucleosome structural integrity, and, instead, prevents the formation of the 30 nm fiber (Davie and Spencer, 2001). Thus, the stability of this

Spencer and Davie: Dynamic histone acetylation and its involvement in transcription 30 nm fiber is maintained by the N terminal tails (Davie and Spencer, 2001). The chromatin fiber becomes moderately folded by the H3 and H4 N terminal tails at physiological ionic strength. However, the N terminal tails of the four core histones are required for the chromatin fiber to undergo extensive folding (Tse and Hansen, 1997; Logie et al, 1999). At low ionic strength, the chromatin fiber assumes a three-dimensional irregular shape that is stabilized by the globular domain of H1 and either the H1 tails or the H3 N terminal tail (Zlatanova et al, 1998; Leuba et al, 1998a). The N terminal tails from histones H2A, H2B and H4 do not have the same effect as H3 on the chromatin fiber. However, the N terminal tail of H3 is 44 amino acids long, whereas histones H4, H2B and H2A have N terminal tails that are only 26, 32, and 16 amino acids long, respectively. As a result, the N terminal tail of histone H3 can extend over a significantly larger portion of linker DNA compared to the other core histones (Leuba et al, 1998b). The H3 N terminus is also positioned close to the point where linker DNA enters and exits the nucleosome, and, therefore, it can undergo extensive interactions with the linker DNA (Zlatanova et al, 1998). The chromatin fibers within a cell interdigitate with neighboring fibers into a higher order fibrous mass that impedes the access of transcription factors to their target sequences, thereby preventing transcription initiation (Schwarz et al, 1996). At physiological ionic strength, the interaction of these neighboring fibers with one another is partly dependent on either the H2A and H2B or the H3 and H4 core histone N terminal tails (Davie and Spencer, 2001). These fibrous masses are then further organized into compact chromosome territories within interphase nuclei (Verschure et al, 1999).

In addition to binding linker DNA, the histone N terminal tails are capable of interacting with other histones and non-histone chromosomal proteins. The N terminus of H4 binds to the H2A-H2B dimer of neighboring nucleosomes, and, as such, is thought to assist in chromatin folding (Luger et al, 1997). In yeast, the transcriptional repressors Sir3, Sir4, and Ssn6/Tup1 interact with the H3 and H4 N terminal domains, causing the associated chromatin to become transcriptionally repressed (Grunstein, 1998). Likewise, the Drosophila Groucho and its mammalian homologues bind to the N terminal domain of H3 and repress transcription (Palaparti et al, 1997; Fisher and Caudy, 1998). These domains also interact with non-histone proteins such as HMG-14 and HMG-17 that promote the unfolding of higher order chromatin structures (Bustin, 1999).

III. Acetylation of the histone N terminal tails The N terminal tails can undergo a series of posttranslational modifications at specific amino acids including acetylation, phosphorylation, ubiquitination and methylation (Spencer and Davie, 1999) (Figure 1). The most extensively studied of these modifications is dynamic acetylation, a reversible process catalyzed by acetyltransferases and deacetylases which mediate the transfer of acetyl groups on to and off of the #-amino group of N terminal lysine residues, respectively (Kuo and Allis, 1998).

Figure 1. General structure of the core histones and their sites of post-translational modifications. The central globular domain of each histone is depicted as a circle with the N and C terminal tails extending towards the left and right sides, respectively. Me, Ac, P, and Ub represent methylation, acetylation, phosphorylation, and ubiquitination, respectively. HAT A (histone acetyltransferase) and HDAC (histone deacetylase) represent the enzymes that catalyze the reversible acetylation of lysine residues along the histone N terminal tails. H3 kinase and PP1 (protein phosphatase 1) represent the enzymes responsible for the reversible phosphorylation of H3 serine residue.

Gene Therapy and Molecular Biology Vol 7, page 3 HDACs 1,2,3 and 8. These class I members are nuclear transcriptional co-repressors with homology to the yeast Rpd3 deacetylase. The class II histone deacetylases are larger proteins of approximately 1000 amino acids with structural homology to yeast Hda1 and include HDACs 4,5,6,7,9 and 10 (Davie and Moniwa, 2000; Bertos et al, 2001; Guardiola and Yao, 2002). Class III histone deacetylases are encoded by genes similar to the yeast silent information regulator (Sir 2) gene (Afshar and Murnane, 1999; Frye, 1999). These deacetylases are dependent on NAD+ and ADP-ribosylase activity (Frye, 2000; Imai et al, 2000; Landry et al, 2000). Class I deacetylases are ubiquitously expressed, while class II deacetylases are tissue-, cell-and differentiation-specific (Davie and Moniwa, 2000). Both classes of deacetylases can deacetylate the four core histones, however, each deacetylase has a site preference (Davie and Spencer, 2001). Similar to histone acetyltransferases, the yeast Rpd3 and Hda1 deacetylases exist in distinct multi-protein complexes, suggesting that class I and II deacetylases have distinct biological functions. Furthermore, the components of these complexes influence the substrate specificity of these enzymes (Davie and Moniwa, 2000). For example, the free form of avian HDAC1 preferentially deacetylates free but not nucleosomal H3. When assembled into a multi-protein complex, this deacetylase preferentially deacetylates free H2B and histones assembled into a nucleosome (Sun et al, 1999). Class I deacetylases reside in the nucleus (Davie and Moniwa, 2000). However, the sub-cellular distribution of class II deacetylases is not as straight forward. HDACs 4 and 5 shuttle between the cytoplasm and the nucleus (Bertos et al, 2001). HDAC7 is predominantly nuclear but binds to the membrane-associated endothelin receptor A and most likely functions in the cytoplasm (Lee et al, 2001). HDAC6 is strictly cytoplasmic, and HDAC9 appears to be both nuclear and cytoplasmic (Zhou et al, 2001). HDACs 4,5, and 7 are transcriptional co-repressors that interact with MEF2 transcription factors, as well as the co-repressors N-CoR, BCoR, and CtBP (Bertos et al, 2001; Guardiola and Yao, 2002). Similarly, HDAC9 interacts with MEF-2 and represses MEF-2-mediated transcription (Zhou et al, 2001). HDAC10 resides in the nucleus and the cytoplasm (Guardiola and Yao, 2002). In the nucleus, this deacetylase functions as a transcriptional repressor when tethered to a promoter (Guardiola and Yao, 2002). Interestingly, HDAC6 can interact with ubiquitin. As well, the mammalian homologue of UFD3, a yeast protein involved in protein ubiquitination, is part of the cytoplasmic mammalian HDAC6 complex (SeigneurinBerny et al, 2001).

This modification typically occurs on up to five lysine residues along the H3 and H4 N terminal tails, four residues along H2B, and one residue along H2A (Davie and Spencer, 1999). Whether a histone is hypo- or hyperacetylated depends on the net activities of neighboring histone acetyltransferases and deacetylases.

IV. Histone acetyltransferases The following is only a brief summary of the histone acetyltransferases identified to date. For a more detailed description of histone acetyltransferases and their substrates, please refer to the following reviews (Sterner and Berger, 2000; Davie and Spencer, 2001; Marmorstein and Roth, 2001; Bertos et al, 2001). Numerous transcription co-activators including yGcn5, P/CAF, CBP/p300, Esa1, NuA4, and ACTR/SRC-1 have been identified as having intrinsic histone acetyltransferase activity (Sterner and Berger, 2000; Davie and Spencer, 2001; Klochendler-Yeivin and Yaniv, 2001; Marmorstein and Roth, 2001). In addition, the DNA-binding transactivator ATF-2, the general transcription factors TAFII250 and Nut1, and the elongation factor Elp3 are histone acetyltransferases (Marmorstein and Roth, 2001). Histone acetyltransferases generally exist in large complexes (Spencer and Davie, 1999). Each histone acetyltransferase has a different target substrate, and the specificity for this substrate depends on the proteins associated with the histone acetyltransferase (Grant et al, 1999). For example, the free full-length form of yeast Gcn5 preferentially acetylates H3 in vitro and H3 and H4 in vivo (Zhang et al, 1998; Sterner and Berger, 2000; Davie and Spencer, 2001). However, the acetylating efficiency of yeast Gcn5 for nucleosomal histones increases when assembled into high molecular weight, multi-protein complexes referred to as SAGA (Spt-AdaGcn5-acetyltransferase) and Ada (Grant et al, 1999). In addition, the pattern of histone acetylation for Gcn5 assembled into the SAGA complex is distinct from that exhibited by Gcn5 when assembled into Ada (Grant et al, 1999). Similarly, the histone substrate specificity of individual human PCAF and yeast Esa1 acetyltransferases becomes altered when these enzymes are assembled into multi-protein complexes (Davie and Spencer, 2001). The phosphorylation of CBP by ERK1 enhances the activity of this acetyltransferase, suggesting that the function of histone acetyltransferases may be regulated by phosphorylation events (Liu et al, 1999).

V. Histone deacetylases As many as 10 histone deacetylases have been identified to date (Bertos et al, 2001). Refer to the following reviews (Sterner and Berger, 2000; Bertos et al, 2001; Davie and Spencer, 2001; Marmorstein and Roth, 2001) for a more detailed description of histone deacetylases. These deacetylases are divided into 3 classes defined by their size and sequence homologies to yeast deacetylases. The class I histone deacetylases are approximately 400-500 amino acids in length and include

VI. The acetylation

dynamics

histone

Studies of histone acetylation dynamics indicate that both acetylation and deacetylation occur at more than one rate (Covault and Chalkley, 1980; Zhang and Nelson, 1988a). In human fibroblasts and mature avian

Spencer and Davie: Dynamic histone acetylation and its involvement in transcription erythrocytes, there are two populations of acetylated histones. The first population, which accounts for approximately 15% of acetylated core histones in hepatoma tissue culture cells, is rapidly hyperacetylated (t1/2= 7 to 15 min for monoacetylated H4) and rapidly deacetylated (t1/2= 3 to 7 min). The second population, which accounts for up to 50% of acetylated histones, is slowly acetylated (t1/2= 140-300 min for monoacetylated H4) and then slowly deacetylated (t1/2= 30 min) (Covault and Chalkley, 1980; Zhang and Nelson, 1988a). Similarly, MCF-7 human breast cancer cells also display two populations of acetylated H3, H4 and H2B histones: a rapidly acetylated one comprising 10% of the total nuclear acetylated histones and a slowly acetylated one that includes approximately 50% of acetylated histones (Sun et al, 2001). In immature chicken erythrocytes, approximately 2% of the genome is dynamically acetylated, while the rest is either frozen in a state of mono- or di-acetylation or unacetylated (Zhang and Nelson, 1988a). The acetylated histones in immature avian erythrocytes are divided into two populations. In contrast to mature avian erythrocytes, both populations within the immature erythrocytes display the same rate of histone acetylation (t1/2=12 min for monoacetylated H4). However, in the case of H4, one population is hyperacetylated to tri- or tetra-acetylated isoforms and then rapidly deacetylated (t1/2= 5 min) (referred to as class I). The other population, however, is only mono- or di-acetylated, and subsequently deacetylated at a slower rate (t1/2=90 min) (referred to as class II)(Zhang and Nelson, 1988a; Zhang and Nelson, 1988b). Histones H3 and H2B are also class I acetylated since butyrate-treated immature chicken erythrocytes display a drastic and rapid decline in tri- and tetraacetylated H3 and H2B within 10 minutes of incubation in

the absence of butyrate (Spencer and Davie, 2001) (Figure 2).

VII. The effect of histone acetylation on chromatin structure Histone acetylation affects chromatin structure in several ways. One theory suggests that histone acetylation alters nucleosome structure and weakens the interaction of histone N terminal tails with DNA (Turner, 1991; Norton et al, 1989). Histone acetylation also maintains the open conformation of the transcriptionally active nucleosome (Walia et al, 1998). Thus, histone acetylation may neutralize the positive charges on the N terminal lysine residues, and loosen the contacts between histones and DNA. However, Gcn5 similarly affects transcription and cell growth whether H3 contains a lysine, arginine, or glutamine at position 14 of its N terminal tail. Similarly, replacement of lysine 8/16 residues with arginine or glutamine does not alter the affect of Gcn5 on transcription or cell growth (Zhang et al, 1998). This suggests that histone acetylation may influence transcription by mechanisms other than the neutralization of N terminal lysine residues. Histone acetylation is also thought to disrupt the higher order folding of chromatin fibers (Garcia-Ramirez et al, 1995; Moore and Ausio, 1997; Hansen, 1997). At physiological salt concentrations, acetylated chromatin fibers are salt-soluble, while unacetylated fibers are insoluble (Ridsdale et al, 1990). However, these fibers are incapable of interacting with other fibers by the process of oligomerization, and, therefore, are unable to form higher order structures (Annunziato and Hansen, 2000).

Figure 2. Immunoblot analyses of H2B deacetylation. Avian immature erythrocytes were incubated with sodium butyrate for 1 h, and then incubated in the absence of butyrate for 0, 5, 10, 15 or 30 min. The total nuclear histones from erythrocytes at each time point were extracted. Twenty Âľg of acid-extracted histones were electrophoresed on an Acid-Urea-Triton 15% polyacrylamide gel. The resolved proteins were then transferred to nitrocellulose and immunostained with an antibody to hyperacetylated H2B (Serotec, UK). 0, 1, 2, 3, and 4 designate un-, mono-, di-, tri-, and tetra-acetylated histone isoforms, respectively .

Gene Therapy and Molecular Biology Vol 7, page 5

VIII. The effect of histone acetylation on ATP-dependent chromatin remodeling

The acetylation of only 12 out of 28 lysine residues per histone octamer promotes transcription approximately 15 fold in vitro, and affects chromatin similar to the proteolytic removal of the core histone N terminal tails (Tse et al, 1998; Annunziato and Hansen, 2000). As a result, acetylation of the histone N terminal tails is thought to facilitate transcription by disrupting the folding of the chromatin fiber, as well as inter-fiber interactions. Such an event would allow transcription factors access to their target DNA binding sites. In support of this, the treatment of estrogen-responsive cells with estrogen induces H3 and H4 acetylation along the TATA sequence of the PS2 promoter, subsequently, exposing the TATA binding site and allowing the TATA binding protein to bind to this site (Sewack et al, 2001). In addition, chromatin immunoprecipitation studies show an enrichment of hyperacetylated H3 and H4 along the promoter regions of several genes including the vitamin A and vitamin D genes when transcriptionally activated (Chen et al, 1999; Kadosh and Struhl, 1998; Parekh and Maniatis, 1999; Krebs et al, 1999). As well, the binding of estrogen to its receptor leads to the recruitment of p300/CBP to the promoter of estrogen-responsive genes (Chen et al, 1999). In addition to disrupting chromatin fiber-fiber interactions, histone acetylation disrupts the interactions between the histone N terminal tails and non-nucleosomal proteins or DNA. For example, H3 and H4 hyperacetylation abolish Ssn6-Tup1-mediated transcriptional repression (Watson et al, 2000). The histone N terminal domains display "-helical structures when assembled into the nucleosome (Annunziato and Hansen, 2000). This "-helical character increases upon acetylation (Wang et al, 2000). Histone acetyltransferases may positively influence transcription by altering the structure of the N terminal tails and perturbing the interactions of these tails with proteins that repress transcription. However, histone acetylation may also be associated with transcriptional repression since the heterochromatin of several organisms contains H4 acetylated at lysine 12 (Turner, 2000; Turner et al, 1992). As well, loss of the yeast RPD3 histone deacetylase causes an increase in the silencing of telomeric DNA (De Rubertis et al, 1996). It has also been suggested that histone acetylation plays a role in marking the state of genetic activity or inactivity from one cell generation to the next, thereby epigenetically determining the long-term transcriptional competence of a gene (Turner, 1998). However, recent evidence shows that catalytically active histone acetyltransferases and histone deacetylases are unable to acetylate or deacetylate chromatin in situ during mitosis (Kruhlak et al, 2001). Moreover, these enzymes become spatially reorganized and displaced from condensing chromosomes. Instead, it appears that the spatial organization of these enzymes relative to euchromatin and heterochromatin plays an important role in determining the post-mitotic activation of a gene (Kruhlak et al, 2001).

Besides playing a role in transcription factor binding, histone acetylation may also be fundamental for ATPdependent chromatin remodeling. These type of complexes use ATP hydrolysis as a source of energy to alter nucleosome and chromatin structure and enhance transcription factor binding to nucleosomal DNA-binding sites (Davie and Moniwa, 2000). For a more detailed description of ATP-dependent chromatin remodeling factors refer to the following reviews (Kingston and Narlikar, 1999; Davie and Moniwa, 2000). While these complexes can alter the chromatin structure of transactivator binding sites, they are unable to activate transcription alone (Gregory et al, 1999). The recruitment of the SWI/SNF chromatin remodeling complex to nuclear receptor and BRCA1-regulated genes is thought to increase nucleosome fluidity, and facilitate the subsequent binding of transcription factors to affected regions (Singh et al, 2000). In the case of the yeast HO gene, the binding of the chromatin remodeling factor, SWI/SNF leads to the recruitment of the SAGA histone acetyltransferase complex (Krebs et al, 1999). These two complexes facilitate the binding of a second activator, SBF, which most likely recruits TBP and other components of the preinitiation complex. ATP-dependent chromatin remodeling are also involved in transcription repression (Davie and Moniwa, 2000). Because of this, ATP-dependent chromatin remodeling complexes may increase the rate at which a chromatin region fluctuates between an active and repressed structure (Kingston and Narlikar, 1999). If factors are present that stabilize chromatin structure and promote transcriptional repression, then the remodeling complex will drive the chromatin into a repressed state by allowing the transcriptional repressors to associate with the chromatin. However, if transcriptional activators bind to the remodeled chromatin instead, then the remodeling complexes will drive the chromatin structure to a transcriptionally active state. The subsequent binding of histone acetyltransferases and activating complexes to this chromatin structure will then â&#x20AC;&#x153;fixâ&#x20AC;? it in an active state (Kingston and Narlikar, 1999). In support of this, the elimination of SAGA acetyltransferase activity prevents proper chromatin remodeling at the PHO8 promoter in vivo (Gregory et al, 1999). However, ATP-dependent chromatin remodeling complexes do not always bind chromatin before histone acetyltransferases. In the case of the interferon $ promoter, the enhanceosome assembles at a nucleosome-free enhancer region of this gene and initially recruits Gcn5 to acetylate the nucleosome positioned over the TATA box and transcription start site (Agalioti et al, 2000). This leads to the recruitment of the CBP-PolII holoenzyme complex, and CBP subsequently recruits SWI/SNF. Therefore, in some cases, the SWI/SNF complex prefers acetylated chromatin as a substrate (Agalioti et al, 2000). The BRG1 sub-unit of the SWI/SNF complex contains a bromodomain, and this type of domain can interact with acetylated histones (Winston and Allis, 1999; Cairns et al, 5

Spencer and Davie: Dynamic histone acetylation and its involvement in transcription 1999). The presence of acetylated histones along a promoter may increase the affinity of the SWI/SNF complex to this gene region. In support of this, SWI/SNF was recruited to a promoter by a transactivator, however, its retention was enhanced when the histones along this region were acetylated (Hassan et al, 2001). Incubation of these nucleosomal arrays with SAGA and NuA4 increased this retention (Hassan et al, 2001). Furthermore, histone acetyltransferases have been shown to increase the rate of gene induction by accelerating ATP-dependent chromatin remodeling (Barbaric et al, 2001). The order of recruitment for chromatin-remodeling activities and the function of these complexes in gene activation or repression is most likely gene-specific, and dependent on the combination of transcription factors bound to the promoter.

1 by p300 reduces its ability to bind DNA, as well as its nuclease activity, while acetylation of importin-alpha by CBP promotes its interaction with importin-beta in vitro (Hasan et al, 2001; Bannister et al, 2000). Furthermore, the acetylation of ACTR by another acetyltransferase suggests that acetylation may be a cascading event involved in signal transduction (Kouzarides, 2000; Marmorstein and Roth, 2001).

X. Global versus targeted histone acetylation Numerous studies have displayed an enrichment of acetylated H3 and H4 along the promoter regions of transcriptionally active genes. For example, activation of the human interferon gene induces H3 and H4 hyperacetylation over 2-3 nucleosomes within the promoter region (Parekh and Maniatis, 1999). Likewise, the yeast Gcn5 histone acetyltransferase complex acetylates histones only in the HO gene promoter (Krebs et al, 1999). Hormone-mediated transcriptional activation also involves the H3 and H4 hyperacetylation over the promoter regions of hormone-responsive genes (Chen et al, 1999; Sewack G.F. et al, 2001). A similar scenario occurs for histone deacetylation where the yeast Sin3-Rpd3 histone deacetylase complex deacetylates histones over a 1-2 nucleosome range within the promoter of a repressed gene (Kadosh and Struhl, 1998). In a recent study, the CpG island of the transcriptionally active chicken carbonic anhydrase gene was associated with higher levels of acetylated histones compared to the near-by promoter region (Myers et al, 2001). The acetylation of H3 and H4 along this gene was greatest at the CpG island and showed a drastic drop at approximately 1.5 kilobases into the transcribed region. Similarly, the chicken thymidine kinase gene displayed elevated levels of hyperacetylated histones along its CpG island (Crane-Robinson et al, 1999). High levels of hyperacetylated histones were also mapped to the chicken GAPDH promoter, which is located within a CpG island (Myers et al, 2001). The regions downstream of this promoter that do not contain CpG islands displayed a sharp drop in the levels of hyperacetylated H3 and H4. As well, chromatin fragments containing CpG islands are enriched in highly acetylated H3 and H4 isoforms (Tazi and Bird, 1990). These findings suggest that histone hyperacetylation is a feature of CpG islands. In a recent study, acetylated histones were mapped to CpG islands located both within the promoter and regions downstream from the transcription start site of a reporter gene (Cervoni and Szyf, 2001). The significance of histone acetylation along CpG islands is not known. However, when associated with acetylated histones, a methylated DNA sequence will become demethylated (Cervoni and Szyf, 2001). Because the interaction of demethylase with DNA is thought to be the limiting step in DNA demethylation, the acetylation of histones associated with CpG islands may increase the accessibility of demethylase to its target DNA sequence (Cervoni and Szyf, 2001). However, histone hyperacetylation does not always appear to be promoter- or CpG island-targeted. H4

IX. The effect of acetylation on nonhistone proteins Histone acetyltransferases can also acetylate transcription factors (p53, ACTR, EKLF, estrogen receptor, MyoD, GATA-1, E2F1), non-histone chromosomal proteins (HMG), components of the transcription machinery (TFIIE, TFIIF), the nuclear import protein importin, tubulin, and flap endonuclease-1 (Fen-1), an enzyme involved in DNA metabolism (Bannister et al, 2000; Chen et al, 1999; Imhof et al, 1997; Munshi et al, 1998; Hasan et al, 2001; Wang et al, 2001; Polesskaya et al, 2000; Herrera et al, 1999; Zhang and Bieker, 1998; Hung et al, 1999; L'Hernault and Rosenbaum, 1985; Martinez-Balbas et al, 2000). The acetylation of p53 and MyoD increases their binding affinity for DNA (Gu and Roeder, 1997; Polesskaya et al, 2000). As well, acetylation of E2F1 extends the half-life of this protein (MartinezBalbas et al, 2000). Thus, along with modifying chromatin structure, acetyltransferases may function in transcription by altering the DNA-binding properties of transcription factors or enhancing the stability of transcription factors. The acetylation of HMGI(Y) plays an important role in viral-induced interferon $ gene activation as well as the inactivation of this event (Parekh and Maniatis, 1999). Upon infection, the enhanceosome assembles at the interferon gene promoter with the help of HMGI(Y). At the same time, CBP and P/CAF are recruited to the interferon $ gene promoter where they acetylate H3 and H4 and, in combination with the enhanceosome, activate transcription of the interferon $ gene. Following induction, CBP acetylates HMGI(Y) which decreases its DNA binding affinity and causes the disruption of the enhanceosome complex. In addition, p300 binds to estrogen receptor " in the absence of estrogen and acetylates lysine residues within the hinge/ligand binding domain of this receptor. This event suppresses the sensitivity of the receptor to ligand (Wang et al, 2001). The evidence from these studies suggests that the theory of acetylation stimulating transcriptional activity is not always true. Acetyltransferases may also function in other biological processes. The acetylation of flap endonuclease-

Gene Therapy and Molecular Biology Vol 7, page 7 acetylated at lysine 16 (H4Ac16) is distributed along the entire length of X-linked genes targeted by the malespecific lethal dosage compensation. The promoter regions of these genes are associated with lower levels of H4Ac16 compared to the middle and 3â&#x20AC;&#x2122; regions (Smith et al, 2001). Similarly, pol I- and pol II-transcribed genes contain elevated levels of H4Ac16, while the levels of H4Ac12 are significantly elevated in yeast and Drosophila heterochromatin (Johnson et al, 1998; Braunstein et al, 1996). As well, the chicken $A-globin gene does not contain a CpG island, but displays high levels of widespread H3 and H4 acetylation (Myers et al, 2001). Acetylated lysine residues are also located throughout the c-myc gene, as well as the entire adult chicken $-globin domain (Hebbes et al, 1994; Madisen et al, 1998; Myers et al, 2001). While a particular histone acetyltransferase can be recruited to and acetylate the histones along a specific gene, recent evidence suggests that some histone acetyltransferases can also globally affect the acetylation of many genes in a non-targeted manner. Depletion of Esa1, an acetyltransferase specifically recruited to the ribosomal protein and heat shock promoters, causes a dramatic decrease in H4 acetylation over many regions of the genome without affecting the transcription of many genes (Reid et al, 2000). Similarly, the acetylation of the yeast PHO5 promoter by Esa1 and Gcn5, and the subsequent deacetylation of this region by HDA1 and Rpd3 also results in the widespread histone acetylation/deacetylation of three separate chromosomal regions making to 22 kb of DNA (Vogelauer et al, 2000). Thus, the promoter-targeted acetylation activity of some histone acetyltransferases and deacetylases may occur in a background of non-targeted histone acetylation that is mediated by these same enzymes and not required for transcription. However, this global acetylation can, in some cases, be targeted to particular regions of the genome. The expression of the C/EBP" transcription factor in GHFT1-5 pituitary cells causes an increase in the levels of acetylated H3 at pericentromeric chromatin domains (Zhang et al, 2001). CBP may be the histone acetyltransferase associated with C/EBP", since this enzyme concentrates at pericentromeric chromatin during C/EBP" expression (Schaufele et al, 2001). The global activity of these enzymes may maintain the balance of acetylated and deacetylated histones throughout the genome or regions of the genome and prevent the histones along a gene from becoming transiently or permanently fully acetylated. The hyperacetylation of histones on regions downstream from the promoter suggests that histone acetylation may function in transcriptional elongation. For example, Elp3, a 60-kilodalton subunit of the elongator/RNAPII holoenzyme has histone acetyltransferase activity and is able to acetylate all four core histones in vitro (Wittschieben et al, 1999). This histone acetyltransferase activity is essential for the elongator function of Elp3 in vivo (Wittschieben et al, 2000). Furthermore, the removal of Gcn5 and Elp3 acetyltransferase activity from yeast cells causes

widespread transcription defects (Wittschieben et al, 2000). Gcn5 functions in the transcription of only a subset of genes. Therefore, Elp3 histone acetyltransferase activity must be important for the transcription of a significant number of genes. Other evidence suggesting a role for histone acetylation in transcriptional elongation comes from observations that transcription by T7 RNA polymerase through a nucleosome occurs at a similar rate on nucleosomal templates containing either tailless or hyperacetylated histones (Protacio et al, 2000). As well, H3 and H4 hyperacetylation is necessary to maintain the transcriptionally active nucleosome in an open conformation for transcriptional elongation (Walia et al, 1998). As a result, a cell may contain two types of histone acetyltransferases with respect to the transcriptional process: those involved in initiation, and those involved in elongation. Histone acetyltransferases required for the initiation process would either enhance transcription factor binding to promoter/enhancer target regions by one or several of the mechanisms previously described, while acetyltransferases required for elongation would increase the accessibility of elongation factors to the DNA within coding regions. In support of this theory, the p300 histone acetyltransferase interacts specifically with initiationcompetent form of RNA polymerase II, while PCAF interacts with the elongation-competent form (Cho et al, 1998). Furthermore, p300 associates with the promoter region of an estrogen-responsive gene only during immediate exposure to estrogen when transcription is initiated rather than during subsequent re-initiation stages of transcription (Shang et al, 2000). Salt-soluble chromatin fragments enriched in active genes are associated with several unidentified histone acetyltransferases (Hebbes and Allen, 2000). Whether these acetyltransferases function in initiation and/or elongation remains to be determined. Different histone acetyltransferases have different histone substrates along certain regions of specific target genes. The histone deacetylase Rpd3 preferentially acetylates lysine 5 of H4 at only a select number of genes (Rundlett et al, 1998). As well, the yeast histone acetyltransferase, Esa1, interacts only with the promoter regions of ribosomal protein genes (Reid et al, 2000). Histone deacetylases along with nuclear receptor corepressors can exist in discrete nuclear bodies (Downes et al, 2000). Similarly, nuclear matrix-associated promyelocytic leukemia bodies contain PML proteins that bind and concentrate CBP into discrete domains (Boisvert et al, 2001). The differential levels of hyperacetylated histones observed on different regions of active genes may be explained by the proximity of histone acetyltransferases and deacetylases to specific regions of these genes. Regions situated close to regions of high acetyltransferase activity are more frequently acetylated than deacetylated, while regions close to deacetylases are deacetylated more often than acetylated. As well, cellular context may influence the acetylation status of histones along specific gene regions. Histone acetyltransferases and deacetylases exist in large

Spencer and Davie: Dynamic histone acetylation and its involvement in transcription multi-protein complexes, and the types of proteins associated with these enzymes can determine their substrate specificity (Grant et al, 1999). For example, in one cell type a specific histone acetyltransferase may exist in a complex capable of acetylating H4, while, in another cell type this same enzyme may be associated with different proteins and have a substrate specificity for H3. In some cases, the ability of histone acetyltransferases and deacetylases to occupy a particular gene region may be transient (Shang et al, 2000). Within 15-20 minutes following estradiol exposure, the histone acetyltransferases AIB1 and p300 within MCF-7 human breast cancer cells associate with the estrogen-responsive cathepsin D promoter. RNA polymerase associates shortly following this event. This association most likely initiates transcription since significant levels of transcription are observed 45 min after estrogen stimulation. The association of these factors then starts to decline 60 min from the initial time of estrogen treatment. A few minutes before these acetyltransferases are removed, the levels of CBP and PCAF histone acetyltransferases associated with the cathepsin D promoter starts to rise and peak between 60 and 75 minutes. However, the levels of cathepsin D transcription are significantly reduced after 75 minutes. The levels of CBP and PCAF and the rate of transcription then drop sharply at 90 minutes. Approximately 100 minutes after estrogen stimulation, the AIB1, CBP and PCAF acetyltransferases all assemble on the promoter in the same order as before, and the rate of transcription simultaneously increases. Similar results were also observed for the PS2 estrogen-responsive promoter in MCF-7 cells, and the cathepsin D promoter in ECC-1 endometrial cells, showing that estrogen-induced transcription involves the cyclical assembly of histone acetyltransferases along the promoters of estrogenresponsive genes. Even though the association of histone acetyltransferases with estrogen-responsive promoters is cyclical after estrogen stimulation, the levels of acetylated histones along the promoter region never drop to the levels observed in estrogen-deplete conditions when the acetyltransferases are displaced. Once transcription has been initiated, histone acetylation may maintain the open structure of an entire gene, and increase the accessibility of the promoter and downstream regions to the RNA polymerase complex for subsequent rounds of initiation and elongation. Such an event may increase the rate of transcription (Orphanides and Reinberg, 2000). Determining the structure of chromatin after initiation, but before and after elongation will help elucidate the function of acetylation in elongation.

(Hendzel et al, 1991). The majority of histone acetyltransferase and deacetylase activity, class I acetylated histones, and transcriptionally active $-globin and histone H5 genes are located in the insoluble nuclear material which contains the nuclear matrix (Hendzel et al, 1991). As well, the nuclear matrix is the site of transcription (Davie, 1995). We recently showed that intronic regions of the transcriptionally active $-globin gene, and transcriptionally competent, DNAse I-sensitive but inactive #-globin genes are associated with class I acetylated histones (Spencer and Davie, 2001). This association was shown for chromatin fragments in both salt-soluble and nuclear matrix-containing nuclear fractions. Of the two sequences, the $-globin intron appeared to have a higher concentration of class I acetylated histones, while the #-globin intron was associated with a mosaic of class I and class II acetylated histones. These findings suggest that the N terminal tails of the core histones situated on transcriptionally active genes contact nuclear-matrix associated histone acetyltransferases and deacetylases in a rapid and transient manner, while the frequency of contact between these enzymes and the histones along transcriptionally competent genes is less. In support of this, the entire chicken $-Aglobin gene, which has a high rate of transcription, was associated with higher levels of H3 and H4 acetylation when compared to genes transcribed at slower rates (GAPDH, carbonic anhydrase (Myers et al, 2001). As well, multiple histone acetyltransferases are associated with chromatin fragments enriched in transcriptionally active genes (Hebbes and Allen, 2000). Thus, dynamic histone acetylation may function to selectively retain transcriptionally active genes at sites of transcription within the nuclear matrix (Spencer and Davie, 2001). In fact, evidence from a recent study on estrogenresponsive human breast cancer cells suggests that exposure to estrogen changes the dynamics of histone acetylation by altering the balance of histone acetyltransferases and deacetylases along different regions of estrogen-responsive genes (Sun et al, 2001). In human breast cancer cells, exposure to estradiol causes the recruitment of acetyltransferases and the subsequent hyperacetylation of histones at the promoter region of estrogen-responsive genes (Chen et al, 1999). In addition, exposure of hormone-responsive human breast cancer cells to estrogen reduces the rate of histone deacetylation without affecting the rate of histone acetylation, or the sub-nuclear location, level or activity of class I and II histone deacetylases (Sun et al, 2001). Instead, exposure to estrogen alters the distribution of the estrogen receptor and histone acetyltransferases (SRC-1 and SRC-3) by causing both types of factors to become tightly associated with the nuclear matrix (Stenoien et al, 2001; Sun et al, 2001). Thus, the binding of estrogen to the estrogen receptor may cause the estrogen receptor to recruit histone acetyltransferases from other nuclear regions to the promoter region of estrogen-responsive genes (Figure 3). At present, a large emphasis is placed on the role of

XII. Transcription and the dynamics of histone acetylation The exact function of dynamic histone acetylation in transcription is unknown. Nuclear fractionation studies indicate that the nuclear distribution of class I, but not class II, acetylated histones closely follows that of the transcriptionally active $-globin and histone H5 genes

Gene Therapy and Molecular Biology Vol 7, page 9 histone acetyltransferases in transcriptional initiation and elongation. However, as previously mentioned, histone acetylation is a dynamic event resulting from the combined activities of histone acetyltransferases and deacetylases. Thus, more attention must be given to understanding how acetyltransferases and deacetylases function together at specific sites along transcriptionally active genes to fully appreciate the role of dynamic histone acetylation in transcription.

Moreover, Gcn5 preferentially associates with a Ser10 phosphorylated form of H3 over a non-phosphorylated form (Cheung et al, 2000). Recently, the phosphorylation of H3 Ser10 by the Snf1 kinase was shown to lead to Gcn5-mediated acetylation at the INO1 promoter (Lo et al, 2001). Thus, the recruitment of a kinase complex to specific promoters may cause Ser10 phosphorylation and either increase the affinity of histone acetyltransferase complexes for nucleosomes or increase acetyltransferase catalytic activity (Lo et al, 2000). However, the affect of one post-translational modification on another may not always be positive. Heterochromatic silencing requires the methylation of Lys9 on H3 by the lysine methyltransferase Su(var)39 (Rea et al, 2000). The methylation of Lys9 inhibits phosphorylation of H3 at Ser10 possibly by hindering the access of kinases to this serine residue (Rea et al, 2000). Thus, methylation of Lys9 may impair transcription by inhibiting phosphorylation events required for transcriptional stimulation (Berger, 2001). This finding, however, needs to be further investigated since immunoprecipitation studies have identified an association between CBP and a histone methyltransferase that specifically targets lysines 4 and 9 of H3 without significantly affecting the ability of CBP to efficiently acetylate other H3 lysine residues (Vandel and Trouche, 2001).

XII. The histone code The histone N terminal tails undergo several posttranslational modifications mediated by a variety of enzymes. Research in the field of gene expression has focussed primarily on determining the function of each modification in transcription. However, a new concept has emerged referred to as the â&#x20AC;&#x153;histone codeâ&#x20AC;? (Strahl and Allis, 2000; Jenuwein and Allis, 2001). This term proposes that the different post-translational modifications occurring on one or more histone tails act either together or in sequence to form recognition sites for specific proteins involved in distinct cellular functions. Furthermore, these modifications may positively or negatively influence the affect of one another on specific cellular functions. Evidence from several recent studies suggests that histone phosphorylation and acetylation may function together to promote gene expression. For example, the stimulation of mammalian cells by epidermal growth factor causes the sequential phosphorylation of Ser10, and acetylation of Lys14 on H3 (Cheung et al, 2000).

Figure 3. Proposed model for the effect of estradiol on the distribution of histone acetyltransferases and histone deacetylases in human breast cancer cells. In the absence of estradiol (left), histone acetyltransferases (HAT) such as CBP, SRC-1, SRC-3, and PCAF occupy the same chromatin regions as histone deacetylases (HDAC) such as HDAC1, and HDAC2. Upon addition of estradiol (right), the estrogen receptor (ER) is recruited to nuclear matrix sites and associates with the estrogen response element of estrogen responsive genes. When bound to estradiol, the ER recruits histone acetyltransferases from other nuclear regions, thereby altering the balance of histone acetyltransferases and deacetylases along specific chromatin regions.

Spencer and Davie: Dynamic histone acetylation and its involvement in transcription distinct forms of the RSC nucleosome-remodeling complex, containing essential AT hook, BAH, and bromodomains. Mol Cell 4, 715-723. Cervoni N and Szyf M (2001) Demethylase activity is directed by histone acetylation. J Biol Chem 276, 40778-40787. Chen H, Lin RJ, Xie W, Wilpitz D, and Evans RM (1999) Regulation of hormone-induced histone hyperacetylation and gene activation via acetylation of an acetylase. Cell 98, 675686. Cheung P, Tanner KG, Cheung WL, Sassone-Corsi P, Denu JM, and Allis CD (2000) Synergistic coupling of histone H3 phosphorylation and acetylation in response to epidermal growth factor stimulation. Mol Cell 5, 905-915. Cho H, Orphanides G, Sun X, Yang XJ, Ogryzko V, Lees E, Nakatani Y, and Reinberg D (1998). A human RNA polymerase II complex containing factors that modify chromatin structure. Mol Cell Biol 18, 5355-5363. Covault J and Chalkley R (1980) The identification of distinct populations of acetylated histone. J Biol Chem 255, 91109116. Crane-Robinson C, Myers FA, Hebbes TR, Clayton AL, and Thorne AW (1999). Chromatin immunoprecipitation assays in acetylation mapping of higher eukaryotes. Methods Enzymol 304, 533-547. Davie JR (1995) The nuclear matrix and the regulation of chromatin organization and function. Int Rev Cytol 162A, 191-250. Davie JR and Moniwa M (2000) Control of chromatin remodeling. Crit Rev Eukaryot Gene Expr 10, 303-325. Davie JR and Spencer VA (1999) Control of histone modifications. J Cell Biochem Suppl 32-33, 141-148. Davie JR and Spencer VA (2001) Signal transduction pathways and the modification of chromatin structure. Prog Nucleic Acid Res Mol Biol 65, 299-340. De Rubertis F, Kadosh D, Henchoz S, Pauli D, Reuter G, Struhl K, and Spierer P (1996) The histone deacetylase RPD3 counteracts genomic silencing in Drosophila and yeast. Nature 384, 589-591. Downes M, Ordentlich P, Kao HY, Alvarez JG, and Evans RM (2000) Identification of a nuclear domain with deacetylase activity. Proc Natl Acad Sci USA 97, 10330-10335. Fisher AL and Caudy M (1998) Groucho proteins: transcriptional corepressors for specific subsets of DNA-binding transcription factors in vertebrates and invertebrates. Genes Dev 12, 1931-1940. Frye RA (1999) Characterization of five human cDNAs with homology to the yeast SIR2 gene: Sir2-like proteins (sirtuins) metabolize NAD and may have protein ADPribosyltransferase activity. Biochem Biophys Res Commun 260, 273-279. Frye RA (2000) Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins. Biochem Biophys Res Commun 273, 793-798. Garcia-Ramirez M, Rocchini C, and Ausio J (1995) Modulation of chromatin folding by histone acetylation. J Biol Chem 270, 17923-17928. Grant PA, Eberharter A, John S, Cook RG, Turner BM, and Workman JL (1999) Expanded lysine acetylation specificity of Gcn5 in native complexes. J Biol Chem 274, 5895-5900. Gregory PD, Schmid A, Zavari M, Munsterkotter M, and Horz W (1999) Chromatin remodeling at the PHO8 promoter requires SWI-SNF and SAGA at a step subsequent to activator binding. EMBO J 18, 6407-6414.

A recent study mapping the distribution of di-methylated lysine 9 on H3 across the chicken $-globin domain during erythropoiesis showed that regions enriched in methylated lysine 9 were depleted of di-acetylated H3 (K9 and K14). However, H3 acetylation correlated with lysine 4 methylation, suggesting that transcriptional activation is associated with H3 methylated at K4, as well as with acetylated H3 and H4 isoforms (Litt et al, 2001). Likewise, in Tetrahymena, methylated Lys4 of H3 is found only in transcriptionally active macronuclei (Strahl et al, 1999).

Acknowledgments Research supported by grants from the Canadian Institutes of Health Research (CIHR) (MT-9186,RO15183), CancerCare Manitoba, and the U.S. Army Medical and Materiel Command Breast Cancer Research Program (#DAM17-00-1-0319), and the National Cancer Institute of Canada with funds from the Canadian Cancer Society. A CIHR Senior Scientist Award to J.R.D. and a U.S. Army Medical and Materiel Command Fellowship to V.A.S. are gratefully acknowledged.

References Afshar G, and Murnane JP (1999) Characterization of a human gene with sequence homology to Saccharomyces cerevisiae SIR2. Gene 234, 161-168. Agalioti T, Lomvardas S, Parekh B, Yie J, Maniatis T, and Thanos D (2000) Ordered recruitment of chromatin modifying and general transcription factors to the IFN-$ promoter. Cell 103, 667-678. Annunziato AT, and Hansen J C (2000) Role of histone acetylation in the assembly and modulation of chromatin structures. Gene Expr 9, 37-61. Bannister AJ, Miska EA, Gorlich D, and Kouzarides T (2000) Acetylation of importin-alpha nuclear import factors by CBP/p300. Curr Biol 10, 467-470. Barbaric S, Walker J, Schmid A, Svejstrup JQ, and Horz W (2001) Increasing the rate of chromatin remodeling and gene activation--a novel role for the histone acetyltransferase Gcn5. EMBO J 20, 4944-4951. Berger SL (2001) An embarrassment of niches: the many covalent modifications of histones in transcriptional regulation. Oncogene 20, 3007-3013. Bertos NR, Wang AH, and Yang XJ (2001) Class II histone deacetylases: structure, function, and regulation. Biochem Cell Biol 79, 243-252. Boisvert FM, Kruhlak MJ, Box AK, Hendzel MJ, and BazettJones DP (2001) The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body. J Cell Biol 152, 1099-1106. Braunstein M, Sobel RE, Allis CD, Turner BM, and Broach JR (1996) Efficient transcriptional silencing in Saccharomyces cerevisiae requires a heterochromatin histone acetylation pattern. Mol Cell Biol 16, 4349-4356. Bustin M ( 1999) Regulation of DNA-dependent activities by the functional motifs of the high-mobility-group chromosomal proteins. Mol Cell Biol 19, 5237-5246. Cairns BR, Schlichter A, Erdjument-Bromage H, Tempst P, Kornberg RD, and Winston F (1999) Two functionally

Gene Therapy and Molecular Biology Vol 7, page 11 Grunstein M (1998) Yeast heterochromatin: regulation of its assembly and inheritance by histones. Cell 93, 325-328. Gu W and Roeder RG (1997) Activation of p53 sequencespecific DNA binding by acetylation of the p53 C-terminal domain. Cell 90, 595-606. Guardiola AR and Yao TP (2002) Molecular cloning and characterization of a novel histone deacetylase HDAC10. J Biol Chem 277, 3350-3356. Hansen JC (1997) The core histone amino-termini: combinatorial interaction domains that link chromatin structure with function. Chemtracts Biochem Mol Biol 10, 56-69. Hansen JC, Tse C, and Wolffe AP (1998) Structure and function of the core histone N-termini: more than meets the eye. Biochemistry 37, 17637-17641. Hasan S, Stucki M, Hassa PO, Imhof R, Gehrig P, Hunziker P, Hubscher U, and Hottiger MO (2001) Regulation of human flap endonuclease-1 activity by acetylation through the transcriptional coactivator p300. Mol Cell 7, 1221-1231. Hassan AH, Neely KE, and Workman JL (2001) Histone acetyltransferase complexes stabilize swi/snf binding to promoter nucleosomes. Cell 104, 817-827. Hebbes TR and Allen SC (2000) Multiple histone acetyltransferases are associated with a chicken erythrocyte chromatin fraction enriched in active genes. J Biol Chem 275, 31347-31352. Hebbes TR, Clayton AL, Thorne AW, and Crane-Robinson C (1994) Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken $-globin chromosomal domain. EMBO J 13, 1823-1830. Hendzel MJ, Delcuve GP, and Davie JR (1991) Histone deacetylase is a component of the internal nuclear matrix. J Biol Chem 266, 21936-21942. Herrera JE, Sakaguchi K, Bergel M, Trieschmann L, Nakatani Y, and Bustin M (1999) Specific acetylation of chromosomal protein HMG-17 by PCAF alters its interaction with nucleosomes. Mol Cell Biol 19, 3466-3473. Hung HL, Lau J, Kim AY, Weiss MJ, and Blobel GA (1999) CREB-Binding protein acetylates hematopoietic transcription factor GATA-1 at functionally important sites. Mol Cell Biol 19, 3496-3505. Imai S, Armstrong CM, Kaeberlein M, and Guarente L (2000) Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403, 795-800. Imhof A, Yang XJ, Ogryzko VV, Nakatani Y, Wolffe AP, and Ge H (1997) Acetylation of general transcription factors by histone acetyltransferases. Curr Biol 7, 689-692. Jenuwein T and Allis CD (2001) Translating the histone code. Science 293, 1074-1080. Johnson CA, O'Neill LP, Mitchell A, and Turner BM (1998) Distinctive patterns of histone H4 acetylation are associated with defined sequence elements within both heterochromatic and euchromatic regions of the human genome. Nucleic Acids Res 26, 994-1001. Kadosh D and Struhl K (1998) Targeted recruitment of the Sin3Rpd3 histone deacetylase complex generates a highly localized domain of repressed chromatin in vivo. Mol Cell Biol 18, 5121-5127. Kimura H and Cook PR (2001) Kinetics of core histones in living human cells: little exchange of H3 and H4 and some rapid exchange of H2B. J Cell Biol 153, 1341-1353. Kingston RE and Narlikar GJ ( 1999) ATP-dependent remodeling and acetylation as regulators of chromatin fluidity. Genes Dev 13, 2339-2352.

Klochendler-Yeivin A and Yaniv M (2001). Chromatin modifiers and tumor suppression. Biochim Biophys Acta 1551, M110. Kouzarides T (2000) Acetylation: a regulatory modification to rival phosphorylation? EMBO J 19, 1176-1179. Krebs JE, Kuo MH, Allis CD, and Peterson CL (1999) Cell cycle-regulated histone acetylation required for expression of the yeast HO gene. Genes Dev 13, 1412-1421. Kruhlak MJ, Hendzel MJ, Fischle,W, Bertos NR, Hameed S, Yang XJ, Verdin E, and Bazett-Jones DP (2001) Regulation of global acetylation in mitosis through loss of histone acetyltransferases and deacetylases from chromatin. J Biol Chem 276, 38307-38319. Kuo MH and Allis CD (1998) Roles of histone acetyltransferases and deacetylases in gene regulation. Bioessays 20, 615-626. L'Hernault SW and Rosenbaum JL (1985) Chlamydomonas alpha-tubulin is posttranslationally modified by acetylation on the epsilon-amino group of a lysine. Biochemistry 24, 473-478. Litt MD, Simpson M, Gaszner M, Allis CD, and Felsenfeld G (2001) Correlation between histone lysine methylation and developmental changes at the chicken $-globin locus. Science 293, 2453-2455. Landry J, Slama JT, and Sternglanz R (2000) Role of NAD(+) in the deacetylase activity of the SIR2-like proteins. Biochem Biophys Res Commun 278, 685-690. Lee,HJ, Chun M, and Kandror KV (2001) Tip60 and HDAC7 interact with the endothelin receptor a and may be involved in downstream signaling. J Biol Chem 276, 16597-16600. Leuba SH, Bustamante C, van Holde K, and Zlatanova J (1998a) Linker histone tails and N-tails of histone H3 are redundant: scanning force microscopy studies of reconstituted fibers. Biophys J 74, 2830-2839. Leuba SH, Bustamante C, Zlatanova J, and van Holde K (1998b) Contributions of linker histones and histone H3 to chromatin structure: scanning force microscopy studies on trypsinized fibers. Biophys J 74, 2823-2829. Liu YZ, Thomas NS, and Latchman DS (1999) CBP associates with the p42/p44 MAPK enzymes and is phosphorylated following NGF treatment. Neuroreport 10, 1239-1243. Lo WS, Duggan L, Tolga NC, Emre, Belotserkovskya R, Lane WS, Shiekhattar R, and Berger SL. (2001) Snf1--a histone kinase that works in concert with the histone acetyltransferase Gcn5 to regulate transcription. Science 293, 1142-1146. Lo WS, Trievel RC, Rojas JR, Duggan L, Hsu JY, Allis CD, Marmorstein R, and Berger SL (2000) Phosphorylation of serine 10 in histone H3 is functionally linked in vitro and in vivo to Gcn5-mediated acetylation at lysine 14. Mol Cell 5, 917-926. Logie C, Tse C, Hansen JC, and Peterson CL (1999) The core histone N-terminal domains are required for multiple rounds of catalytic chromatin remodeling by the SWI/SNF and RSC complexes. Biochemistry 38, 2514-2522. Luger K, Mader AW, Richmond RK, Sargent DF, and Richmond TJ (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251-260. Madisen L, Krumm A, Hebbes TR, and Groudine M (1998) The immunoglobulin heavy chain locus control region increases histone acetylation along linked c-myc genes. Mol Cell Biol 18, 6281-6292.

Spencer and Davie: Dynamic histone acetylation and its involvement in transcription Marmorstein R and Roth SY (2001) Histone acetyltransferases: function, structure, and catalysis. Curr Opin Genet Dev 11, 155-161. Martinez-Balbas MA, Bauer UM, Nielsen SJ, Brehm A, and Kouzarides T (2000) Regulation of E2F1 activity by acetylation. EMBO J 19, 662-671. Moore SC and Ausio J (1997) Major role of the histones H3-H4 in the folding of the chromatin fiber. Biochem Biophys Res Commun 230, 136-139. Munshi N, Merika M, Yie J, Senger K, Chen G, and Thanos D (1998) Acetylation of HMG I(Y) by CBP turns off IFN-$ expression by disrupting the enhanceosome. Mol Cell 2, 457-467. Myers FA, Evans DR, Clayton AL, Thorne AW, and CraneRobinson C (2001) Targeted and extended acetylation of histones H4 and H3 at active and inactive genes in chicken embryo erythrocytes. J Biol Chem 276, 20197-20205. Norton VG, Imai BS, Yau P, and Bradbury EM (1989) Histone acetylation reduces nucleosome core particle linking number change. Cell 57, 449-457. Orphanides G and Reinberg D (2000) RNA polymerase II elongation through chromatin. Nature 407, 471-475. Palaparti A, Baratz A, and Stifani S (1997) The Groucho/transducin-like enhancer of split transcriptional repressors interact with the genetically defined aminoterminal silencing domain of histone H3. J Biol Chem 272, 26604-26610. Parekh BS and Maniatis T (1999) Virus infection leads to localized hyperacetylation of histones H3 and H4 at the IFN$ promoter. Mol Cell 3, 125-129. Polesskaya A, Duquet A, Naguibneva I, Weise C, Vervisch A, Bengal E, Hucho F, Robin P, and Harel-Bellan A (2000) CREB-binding protein/p300 activates MyoD by acetylation. J Biol Chem 275, 34359-34364. Protacio RU, Li G, Lowary PT, and Widom J (2000) Effects of histone tail domains on the rate of transcriptional elongation through a nucleosome. Mol Cell Biol 20, 8866-8878. Rea S, Eisenhaber F, O'Carroll D, Strahl BD, Sun ZW, Schmid M, Opravil S, Mechtler K, Ponting CP, Allis CD, and Jenuwein T (2000) Regulation of chromatin structure by sitespecific histone H3 methyltransferases. Nature 406, 593599. Reid JL., Iyer VR, Brown PO, and Struhl K (2000) Coordinate regulation of yeast ribosomal protein genes is associated with targeted recruitment of Esa1 histone acetylase. Mol Cell 6, 1297-1307. Ridsdale JA, Hendzel MJ, Delcuve GP, and Davie JR (1990) Histone acetylation alters the capacity of the H1 histones to condense transcriptionally active/competent chromatin. J Biol Chem 265, 5150-5156. Rundlett SE, Carmen AA, Suka N, Turner BM, and Grunstein M (1998) Transcriptional repression by UME6 involves deacetylation of lysine 5 of histone H4 by RPD3. Nature 392, 831-835. Schaufele F, Enwright JF, Wang X, Teoh C, Srihari R, Erickson R, MacDougald OA, and Day RN (2001) CCAAT/Enhancer Binding Protein alpha Assembles Essential Cooperating Factors in Common Subnuclear Domains. Mol Endocrinol 15, 1665-1676. Schwarz PM, Felthauser A, Fletcher TM, and Hansen JC (1996) Reversible oligonucleosome self-association: dependence on divalent cations and core histone tail domains. Biochemistry 35, 4009-4015.

Seigneurin-Berny D, Verdel A, Curtet S, Lemercier C, Garin J, Rousseaux S, and Khochbin S (2001) Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways. Mol Cell Biol 21, 8035-8044. Sewack GF, Ellis TW, and Hansen U (2001) Binding of TATA binding protein to a naturally positioned nucleosome is facilitated by histone acetylation. Mol Cell Biol 21, 14041415. Shang Y, Hu X, DiRenzo J, Lazar MA, and Brown M ( 2000) Cofactor dynamics and sufficiency in estrogen receptorregulated transcription. Cell 103, 843-852. Singh H, Sekinger EA, and Gross DS (2000) Chromatin and cancer: causes and consequences. J Cell Biochem Suppl 35, 61-68. Smith ER, Allis CD, and Lucchesi JC (2001) Linking global histone acetylation to the transcription enhancement of Xchromosomal genes in Drosophila males. J Biol Chem 276, 31483-31486. Spencer VA and Davie JR ( 1999) Role of covalent modifications of histones in regulating gene expression. Gene 240, 1-12. Spencer VA and Davie JR (2001) Dynamically acetylated histone association with transcriptionally active and competent genes in the avian adult $-globin gene domain. J Biol Chem 276, 34810-34815. Stenoien DL, Patel K, Mancini MG, Dutertre M, Smith CL, O'Malley BW, and Mancini MA (2001) FRAP reveals that mobility of oestrogen receptor-" is ligand- and proteasomedependent. Nat Cell Biol 3, 15-23. Sterner DE and Berger SL (2000) Acetylation of histones and transcription-related factors. Microbiol Mol Biol Rev 64, 435-459. Strahl BD and Allis CD (2000) The language of covalent histone modifications. Nature 403, 41-45. Strahl BD, Ohba R, Cook RG, and Allis CD (1999) Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in Tetrahymena. Proc Natl Acad Sci USA 96, 14967-14972. Sun JM, Chen HY, and Davie JR (2001) Effect of Estradiol on histone acetylation dynamics in human breast cancer cells. J Biol Chem 276, 49435-49442. Sun JM, Chen HY, Moniwa M, Samuel S, and Davie JR (1999) Purification and characterization of chicken erythrocyte histone deacetylase 1. Biochemistry 38, 5939-5947. Tazi J and Bird A (1990) Alternative chromatin structure at CpG islands. Cell 60, 909-920. Tse C and Hansen JC (1997) Hybrid trypsinized nucleosomal arrays: identification of multiple functional roles of the H2A/H2B and H3/H4 N-termini in chromatin fiber compaction. Biochemistry 36, 11381-11388. Tse C, Sera T, Wolffe AP, and Hansen JC (1998) Disruption of higher-order folding by core histone acetylation dramatically enhances transcription of nucleosomal arrays by RNA polymerase III. Mol Cell Biol 18, 4629-4638. Turner BM (1991) Histone acetylation and control of gene expression. J Cell Sci 99 ( Pt 1), 13-20. Turner BM (1998) Histone acetylation as an epigenetic determinant of long-term transcriptional competence. Cell Mol Life Sci 54, 21-31. Turner BM (2000) Histone acetylation and an epigenetic code. Bioessays 22, 836-845.

Gene Therapy and Molecular Biology Vol 7, page 13 Turner BM, Birley AJ, and Lavender J (1992) Histone H4 isoforms acetylated at specific lysine residues define individual chromosomes and chromatin domains in Drosophila polytene nuclei. Cell 69, 375-384. Vandel L and Trouche D (2001) Physical association between the histone acetyl transferase CBP and a histone methyl transferase. EMBO Rep 2, 21-26. Verschure PJ, van Der Kraan, I, Manders EM, and van Driel R (1999) Spatial relationship between transcription sites and chromosome territories. J Cell Biol 147, 13-24. Vogelauer M, Wu J, Suka N, and Grunstein M (2000) Global histone acetylation and deacetylation in yeast. Nature 408, 495-498. Walia H, Chen HY, Sun JM, Holth LT, and Davie JR (1998) Histone acetylation is required to maintain the unfolded nucleosome structure associated with transcribing DNA. J Biol Chem 273, 14516-14522. Wang C, Fu M, Angeletti RH, Siconolfi-Baez L, Reutens AT, Albanese C, Lisanti MP, Katzenellenbogen BS, Kato S, Hopp T, Fuqua SA, Lopez GN, Kushner PJ, and Pestell RG (2001). Direct acetylation of the estrogen receptor " hinge region by p300 regulates transactivation and hormone sensitivity. J Biol Chem 276, 18375-18383. Wang X, Moore SC, Laszckzak M, and Ausio J (2000) Acetylation increases the "-helical content of the histone tails of the nucleosome. J Biol Chem 275, 35013-35020. Watson AD, Edmondson DG, Bone JR, Mukai Y, Yu Y, Du W, Stillman DJ, and Roth SY (2000) Ssn6-Tup1 interacts with class I histone deacetylases required for repression. Genes Dev 14, 2737-2744. Winston F and Allis CD (1999) The bromodomain: a chromatintargeting module? Nat Struct Biol 6, 601-604. Wittschieben BO, Fellows J, Du W, Stillman DJ, and Svejstrup JQ (2000) Overlapping roles for the histone acetyltransferase activities of SAGA and elongator in vivo. EMBO J 19, 3060-3068. Wittschieben BO, Otero G, de Bizemont T, Fellows J, Erdjument-Bromage H, Ohba R, Li Y, Allis CD, Tempst P, and Svejstrup JQ (1999) A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme. Mol Cell 4, 123-128. Zhang DE and Nelson DA (1988b) Histone acetylation in chicken erythrocytes. Rates of acetylation and evidence that histones in both active and potentially active chromatin are rapidly modified. Biochem J 250, 233-240.

Zhang DE and Nelson DA (1988a) Histone acetylation in chicken erythrocytes. Rates of deacetylation in immature and mature red blood cells. Biochem J 250, 241-245. Zhang W and Bieker JJ (1998) Acetylation and modulation of erythroid Kruppel-like factor (EKLF) activity by interaction with histone acetyltransferases. Proc Natl Acad Sci USA 95, 9855-9860. Zhang W, Bone JR, Edmondson DG, Turner BM, and Roth SY (1998) Essential and redundant functions of histone acetylation revealed by mutation of target lysines and loss of the Gcn5p acetyltransferase. EMBO J 17, 3155-3167. Zhang WH, Srihari R, Day RN, and Schaufele F (2001) CCAAT/enhancer-binding protein alpha alters histone H3 acetylation at large subnuclear domains. J Biol Chem 276, 40373-40376. Zhou X, Marks PA, Rifkind RA, and Richon VM (2001) Cloning and characterization of a histone deacetylase, HDAC9. Proc Natl Acad Sci USA 98, 10572-10577. Zlatanova J, Leuba SH, and van Holde K (1998) Chromatin fiber structure: morphology, molecular determinants, structural transitions. Biophys J 74, 2554-2566.

Virginia A. Spencer and James R. Davie

Spencer and Davie: Dynamic histone acetylation and its involvement in transcription

Gene Therapy and Molecular Biology Vol 7, page 15 Gene Ther Mol Biol Vol 7, 15-23, 2002

Tumor therapy using radiolabelled antisense oligomers- aspects for antiangiogenetic strategy and positron emission tomography Review Article 1*

Kalevi JA Kairemo , Mark Lubberink , Mikko Tenhunen , Antti P Jekunen

Department of Nuclear Medicine1 and Hospital Physics2 Uppsala University, Uppsala Sweden Department of Oncology3 and Department of Clinical Pharmacology4, Helsinki University Central Hospital, Helsinki, Finland

__________________________________________________________________________________ *Correspondence: Kalevi J A Kairemo, MD, PhD, MSc (Eng); Professor, Department of Nuclear Medicine, Uppsala University Hospital, Sweden; Tel. +46-18-611 1006; Fax. +46-18-611 4124; e-mail: kalevi.kairemo@onkologi.uas.lul.se Key words: antisense therapy, oligonucleotides, phosphorus radioisotopes, sulphur radioisotopes, AIDS, cancer, dosimetry, positron emission tomography Received: 17 January 2002; accepted: 29 January, 2002; electronically published: July 2003

Summary Angiogenesis provides a putative target for radiochemotherapy as endothelial cells on vascular wall are sensitive for radiation and by destructing of one endothelial cell may lead to death hundred of tumor cells. Endothelial cells in the angiogenic vessels within solid tumors express several proteins that are absent or faintly expressing in established blood vessels, including !v integrins (Hammes, 1996) and receptors for certain angiogenic growth factors (Hanahan, 1997) (Risau, 1997). Recently, vascular endothelial cell growth factor (VEGF)-induced invasiveness has been inhibited specifically by ETS-1 antisense oligonucleotide. ETS-1 gene expression can be induced, while there are several other systems with constant expression. In this paper, we extent use of oligos from conventional biokinetic studies to therapeutic use by comparing radioactive oligos to peptide counterparts. Radiolabelled oligos have a potential of having both direct antisense inhibition and radiation effects. Previously we have shown theoretically that oligonucleotide therapy may be effective with internally labelled (P-32, P-33 and S-35) oligodeoxynucleotide phosphorothioates. This has also been demonstrated in vitro using P-33 (Kairemo et al, 1999). We investigate also the possibility of using 15-mer oligodeoxynucleotide phosphorothioates (oligos) or oligomers in which the phosphate-ribose backbone has been replaced with polyamide backbone (peptide nucleic acids). The absorbed organ doses of these radiolabelled compounds were estimated from biodistribution data. Subcellular biodistribution was used in evaluation of the best targeting inside the cell with one oligomer. Our results indicate that oligos can give significantly up to 130-fold higher absorbed organ doses in oligos than in peptides. Mainly this is due to slower biokinetics of oligos (35-fold slower half-lives). For imaging, positron emitters such as F-18 and Br-76, offer an advantage for radiopharmacokinetic studies (Wu at al., 2000). We have therefore calculated the subcellular dosimetry for these isotopes in different cell dimensions (nuclear diameter 6-16Âľm, cellular diameter 12-20Âľm). angiogenetic factors in our model; tie tyrosine kinase receptor and ets, representing a factor participating and inducing angiogenesis On the basis of amino acid sequence and structural similarities, receptor tyrosine kinases can be divided into several families (Ullrich and Schlessinger, 1990). Tie is the protein product of a recently described receptor tyrosine kinase cDNA, which together with tek defines a new subfamily. The tie gene is mandatory for the normal growth and differentiation of endothelial cells during fetal

I. Introduction Angiogenesis is a cascade of processes involving both soluble angiogenic factors and insoluble extracellular matrix factors (Jekunen and Kairemo, 1997). Soluble multiple molecules, that induce angiogenesis, are released by both tumor cells and host cells, including endothelial cells, epithelial cells, mesothelial cells, and leukocytes. These processes provide several targets for development of angiogenesis inhibitors. We have used two

Kairemo et al: Oligonucleotide radiotherapy development (Korhonen, 1992). It is abundantly expressed in vascular endothelia during development, and in some megakaryoblastic and erythroleukemia cell lines; as well as tieRNA accumulates in the epithelium of local vessels during ovulation and wound healing (Korhonen, 1992). Tie receptor has an important role in the angiogenesis associated with melanoma metastasis (Kaipainen, 1994). Radioantibodies against tie receptor have been used in targeting studies in vivo with success (Kairemo et al, 1996). As the location of tie receptor is at the outer cell membrane, receptor is easily reachable and effects of radiation and receptor blocking should occur immediately, which may be beneficiary for the radioantibody treatment. For further development of these receptors the crucial point is to find inducers for normally low levels. Ligands for endothelial cell receptors tyrosine kinases, Tie-1 and Tie-2 are not known. Ligands with agonistic and antagonistic activities for Tie-2 have now been identified: angiopoetin 1 is an activating ligand for Tie 2 and regulates blood vessel maturation (Suri, 1996), while angiopoetin 2 serves as antagonist (Maisonpierre, 1997). The ETS family proteins are transcription factors that bind to the regulatory control region of certain genes via ETS binding motif, which has been found in numerous genes including proteases and receptor tyrosine kinases (Wasylyk, 1993). ETS regulates the expression of proteases and migration of endothelial cells, and in fact, the induction of ETS-1 mRNA is a mutual phenomenon in endothelial cells stimulated with angiogenic growth factors (Iwasaka, 1996). It has also been shown that ETS 1 antisense oligo markedly reduced the DNA- ETS complex diminishing the responsiveness to the stimulus of

angiogenic factor (Iwasaka, 1996). Induction of expression of ETS gene is faster and more prominent than protein expression providing better although transient target for therapy. The specificity resides in the sequence of oligo, which interacts with its complementary mRNA, but only minimally with noncomplementary structures. The antisense oligo, through the formation of a mRNA-DNA duplex, specifically prevents the translation of that mRNA into protein (Figure 1). For oligos to be effective antisense agents, they first must enter the cells and achieve appropriate concentration in the correct intracellular compartment. Cellular nucleases are highly potent in digesting phosphodiester oligos. Thus several nuclease resistant oligos have been developed. Phosphorothioate oligo has a non-bridging oxygen atom replacing a sulphur atom. Peptide nucleic acid (PNA) is an oligomer in which the charged phosphate-ribose backbone has been eliminated and replaced with an uncharged backbone (Egholm 1992) and PNAs have been reported to resist nuclease and protease degradation (Egholm 1993). Oligos bind to serum albumin and other proteins with low affinity and distribute to all peripheral tissues with the kidneys and liver accumulating most of the drug. They are cleared by slow metabolism with an elimination half-life up to 50 hrs. The biokinetics of GEM 91 phosphorothioate oligodeoxynucleotide has been evaluated in six AIDS patients, where the plasma mean residence time varied from 24.7 to 49.6 hrs, the mean being 41.7 Âą 3.6 hrs (Zhang, 1995a).

Figure 1. Schematic presentation of radionanotargeting

Gene Therapy and Molecular Biology Vol 7, page 17 Case 1: rapid kinetics compared with physical decay:

Phosphorothioate oligodeoxynucleotides have several advantages: they are relatively resistant to destruction by nucleases; they have good aqueous solubility; they hybridize efficiently with target RNA with relatively high specificity; they are relatively efficiently taken up by cells; and they are widely used in automated oligonucleotide synthesizers (Zhang, 1995b). Phosphorothioate oligodeoxynucleotides labelled internally either with sulphur or phosphorus do not require any extra coupling techniques as in the case with transition metals. The therapeutic possibilities of radiolabelled antisense oligodeoxynucleotides or peptides are still unknown, and one of the basic questions in radiotherapy is the optimal source of radiation. Here we have estimated dosimetric properties of different radiolabels on oligonucleotides and peptides at cellular level, that could be predicted from existing data. The aim of this study was to calculate internal radiation dose from the known data and assess the suitability of different isotopes for the labels. Macroscopic doses were calculated for oligonucleotides labelled with 76 Br, 111In, 90Y and 211At, as examples of positron emitters, Auger-electron emitters, high-energy beta radiation emitters, and alpha emitting nuclides. We have previously shown by using calculations from the biodistribution data of oligonucleotide phosphorothioates in a xenograft model that oligonucleotide radiotherapy can optimally be given with P-32 and P-33 (Kairemo et al, 1996). Calculations can suggest recommendable source of radiation, and thus allow a proper selection of the optimal label. By selecting a radiation source the penetrability of radiation can be controlled and severe side effects may be avoided efficiently.

T f >> Tb1 , Tb 2

TT T + Tb2 D1 Ã1 A01 = = " f b1 " f = D2 Ã2 A02 Tf + Tb1 Tf Tb2 A T = 01 " b1 A02 Tb2

Case 2: rapid kinetics compared with very slow kinetics:

Tb 2 >> T f >> Tb1

Tf Tb1 Tf + Tb2 D1 Ã1 A01 = = " " = D2 Ã2 A02 Tf + Tb1 Tf Tb2 A T = 01 " b1 A02 Tf

(5)

The absorbed dose of P-32, P-33 and S-35 labelled oligonucleotides were estimated using published biodistribution data with several oligonucleotides and mouse models. (Crooke et al, 1996) have investigated pharmacokinetics of a 20-mer oligodeoxynucleotide phosphorothioate (ISIS 3082) and its 2_-propoxy phosphorothioate (ISIS 9045) in mice. This oligodeoxynucleotide inhibits the expression of mouse intercellular adhesion molecule (Crooke et al, 1996). (Dewanjee et al, 1994a) have published the data in mouse for 15-mer oligonucleotide sequence coupled with diethylenetriamine pentaacetate (DTPA)-isothiocyanate. (Mardirossian et al, 1997) have published the pharmacokinetic and stability data for radiolabeled aminederivatized 15-base DNA oligomer in mice. The pharmacokinetics of the compounds were expected not to change depending on P-32, P-33 or S-35 labelling. Here we also studied positron emitters F-18 and Br-76, betaemitter Y-90, Auger-emitter In-111 and alpha-emitter At211. The whole organ uptakes as percent of the injected activity were used.

II. Dosimetric calculations The accumulated dose from radionuclides used internal labelling of oligos, phosphorus-32 (P-32), phosphorus-33 (P-33) and sulphur-35 (S-35) was estimated using the MIRD (Medical Internal Radiation Dose) formalism, the basic equations of which are

D= Ã"S

(4)

(1)

III. Dosimetric data

and x

Ã = # A(t)dt = A0 0

Teff ln2

Table I summarizes actual delivered doses in liver, kidney and tumor. Data was collected from different published reports on pharmacokinetic data with different S-35 labelled oligonucleotides and mouse models. The liver doses in mouse models varied from 0.003 to 30 Gy/MBq. The kidney doses in the same animal models varied from from 0.01 to 35 Gy/MBq. The values in these modelswere all within tolerance limits of radiotoxicity except those for ISIS 9045. In the mammary tumor model the observed kidney dose of 9.1 Gy/MBq for P-32 (not shown) is close to the maximum tolerated dose, whereas for S-35 the absorbed radiation dose in kidneys was acceptable 1.3 Gy/MBq. The tumor dose was 1.0 Gy per administered MBq. Table I shows that oligos deliver up to 130-fold higher organ doses (including tumors) than peptide nucleic

(2)

where D, A, S and Teff refer to absorbed doses, activities, geometric factors and effective half-lives. The effective half-life can be calculated using monoexponential kinetics by

Teff =

Tb T f Tb + T f

(3)

where Tb is the biological half-life of the oligomer and T f physical half-life of the specific radionuclide. Two different situations were investigated to calculate the relative dose. 17

Kairemo et al: Oligonucleotide radiotherapy acids of the same size. The PNAs have rapid biokinetics; the half-lives are approximately 35-fold faster than those of oligo phosphorothiates. The lipophilic oligo phosphorothiate 9045 with is 2´-propoxy modification gives very high organ doses. All other 15-21-mer oligos give identical liver doses. The smallest kidney dose was calculated for the 15-mer oligo, and both ISIS 3082 and 2105 had 3.2-fold higher kidney dose. Despite the heterogeneity of the origin of the input data and used approximations of the time-activity distribution, consistent results were obtained. Subcellular dosimetry was applied in situations as described in Figure 2. The following results were obtained as shown in Figure 3. It demonstrates subcellular dosimetric data in different cell dimensions (nuclear diameter 3-8 µm,cellular diameter 610 µm) for positron emitters F-18 and Br-76 in four different oligodeoxynucleotide target systems. If high nuclear DNA target is used,large variation especially in Br-76 dose can be observed. This means that the cell nuclear dose is very much dependent on cell dimensions. If highly inductable RNA target is used, variation is much smaller as as in less extreme subcellular concentrations of oligodeoxynucleotide. Kinetics of oligonucleotides are highly dependent on the chemistry of the sugar-phosphate backbone of the molecules, and of the length of the molecules. Here, the 20h SUVs and cellular distribution reported by (Wu et al, 2000) for antisense 76Br-phosphorothioate oligonucleotides of length 20 mer was used, combined with octreotide kinetics. For tumour, a SUV of 17.5 was used, as likely for octreoscan, since no oligonucleotide data was found. Cellular uptake values in tumour are assumptions. The only data on oligonucleotide kinetics found was made (Tavitian et al, 1998), describing only the first 90 min after administration of three different oligonucleotides in baboons as measured by PET with 18F.

Macroscopic doses were calculated for oligonucleotides labelled with 76Br, 111In, 90Y and 211At, as examples of positron emitters, Auger-electron emitters, high-energy beta radiation emitters, and alpha emitting nuclides (Table III). Absorbed doses were calculated using the Mirdose 3.1 program by Stabin (Stabin, 1996), except for 211At where gamma radiation was ignored and local absorbtion of all alpha and beta radiation energy was assumed. Kidney, liver, spleen and remainder of the body were used as source organs. Using cellular S-value data (Bolch, 1999), nucleus to nucleus absorbed doses were calculated for the subcellular distributions (Table II, IV), and compared to macroscopic doses. The mean number of decays in each cell was calculated assuming a uniform distribution of the activity within each organ, and assuming spherical cells with a diameter of 14 µm and a nucleus diameter of 10 µm.

IV. Discussion Here, we have emphasized the possible role of radiolabelled antisense oligos in the anti-angiogenetic therapy. It is known that new tumor vessels due to angiogenesis differ from capillaries in normal tissues due to properties of regulation of blood flow and also interstitial fluid pressure in tumors is elevated. Molecules, related to angiogenesis in tumors may retain longer in tumors and thus give for a longer effect for therapeutic agents. The ETS1 gene has a direct role in angiogenesis: the antisense oligonucleotides directed against the ETS1 gene thus altered a cellular property of endothelial cells that is correlated with the ability of the cells to migrate through basement membranes (Chen 1997). While ETS1 regulates the expression of various proteins by endothelial cells related their growth, it is also regulating various proteins affecting coagulation and other factors which perform important endothelial functions.

Table I. The calculated organ doses for different oligomers in mouse models Oligomer

Initial activity (% of Biologic half- Liver dose (S-35) injected dose) life, Tb (hours) Gy/ MBq

Kidney dose (S35) Gy/ MBq

Reference

Peptide nucleic acid, 15-mer

0.19% (liver) 1.45 % (kidney)

5.1% (liver) 4.8 (kidney)

0.79% 0.01 Gy/ MBq

Mardirossian et al, 1997

c-myc, antisense, 15-mer

6.95 % (liver) 5.15 % (kidney)

178.2 (liver) 100 % 170.7 (kidney) 0.4 Gy/ MBq

100 % 1.3 Gy/ MBq

Dewanjee et al, 1994

ISIS 3082 20-mer

18 % (liver) 25 % (kidney)

62 (liver) 112 (kidney)

90% 0.4 Gy/ MBq

320 % 4.0 Gy/ MBq

Crooke et al, 1996

ISIS 9045, 20-mer

45 % (liver) 12 % (kidney)

$ (liver) $ (kidney)

7620 % (S-35) 30 Gy/ MBq

2710 % (S-35) 35 Gy/ MBq

Crooke et al, 1996

ISIS 2105, 21-mer

18 % (liver) 25 % (kidney)

62 (liver) 112 (kidney)

90% 0.4 Gy/ MBq

320 % 4.0 Gy/ MBq

Crooke et al, 1996

c-myc, antisense, 15-mer

11.0 % (tumor)

194 (tumor)

100 % (tumor) 1.0 Gy/ MBq

0.078 % 0.003 Gy/ MBq

Dewanjee et al, 1994

Gene Therapy and Molecular Biology Vol 7, page 19 Figure 2: Schematic model for cellular calculations in real and extreme situations. Subcellular dosimetry was applied in these situations

Dose calculations

Figure 3. It demonstrates subcellular dosimetric data in different cell dimensions (nuclear diameter 3-8 Âľm, cellular diameter 6-10 Âľm) for positron emitters F-18 and Br-76 in four different oligodeoxynucleotide target systems.

Kairemo et al: Oligonucleotide radiotherapy Table II. Shows subcellular distributions calculated by the nucleus to nucleus absorbed doses

The following SUVs at 20h after injection were given by Wu et al, 1999: Kidney Liver Spleen

6 mer 53.1 0.5 0.5

12 mer 13.3 0.5 0.5

20 mer 17.8 8.6 3.4

30 mer 1.9 12.3 5.1

The following subcellular distribution was assumed for 20 mer, approximately as in Wu et al, 1999: Nucleus 30% 30% 80%, 50%

Kidney Liver Tumour

Rest 70% 70% 20%, 50%

Table III shows the calculated absorbed doses for a number of organs and tumours. Macroscopic absorbed doses (mGy/MBq) Organ Liver Spleen Kidney Whole body (mGy/MBq)

111

In 0.63 0.43 0.95 0.13

Y 4.41 3.59 10.1 0.57

Br 1.29 0.96 2.35 0.23

211

Tumour, 100g Tumour, 0.01g Organ Liver

1.03 0.54 111 In 0.63

12.0 3.29 90 Y 4.41

2.65 0.59 76 Br 1.29

10.9 10.9 211 At 4.90

At 4.90 1.91 8.74 0.54

Table IV shows the cellular doses Average nucleus self-dose (mGy/MBq), and percentage of average nucleus absorbed dose 111 90 76 211 Organ In Y Br At Liver 0.03 (4.0%) 0.004 (0.09%) 0.006 (0.5%) 0.21 (4.4%) Kidney Tumour, 100g Tumour, 20g

0.05 (4.8%) 0.15 (14.5%), 0.09 0.15 (27.8%), 0.09

0.007 (0.04%) 0.011 (0.5%) 0.023 (0.19%), 0.015 0.036 (1.4%), 0.023 0.023 (0.71%), 0.015 0.036 (6.1%), 0.023

0.38 (4.4%) 1.26 (11.6%), 0.79 1.26 (11.6%), 0.79

transplanted into nude mice: growth of the antisenseVEGF cell lines was inhibited compared to control cells, despite the fact that they have a faster division time in vitro. These tumors had fewer blood vessels and a higher degree of necrosis explaining the reduced tumor size (Saleh 1996). Also, human melanoma cells transfected with sense vascular permeability factor (VPF)/VEGF expressed and secreted large amounts of mouse VPF/VEGF and formed well-vascularized tumors with hyperpermeable blood vessels and minimal necrosis in nude/SCID mice (Claffey, 1996).

Furthermore, ETS1 has expression in B and T lymphocytes and thymus. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that promotes angiogenesis in solid tumors. The VEGFinduced invasiveness was inhibited by ETS1 antisense oligonucleotides but not by a sense control (Chen 1997). Antisense-VEGF has been successfully used to control tumor growth and it may provide another basis for the development of antiangiogenic gene therapy (Saleh 1996). Rat glioma cells were transfected with a eukaryotic expression vector bearing an antisense-VEGF cDNA and 20

Gene Therapy and Molecular Biology Vol 7, page 21 VPF/VEGF promoted melanoma growth by stimulating angiogenesis and constitutive VPF/VEGF expression dramatically promoted tumor colonization in the lung up to 50-fold of that of controls (Claffey, 1996). Minimal sequence information required for high-affinity binding to VEGF is contained in 29-36-nucleotide motifs for the development of potent and specific VEGF antagonists (Jellinek, 1994). Transforming growth factor alpha (TGF-alpha) has been shown to induce VEGF/VPF in normal human epidermal keratinocytes in vitro (Smyth, 1997). By using a 19-mer antisense phosphorothioate oligodeoxynucleotide complementary to bases 6-24 relative to the translational start site of the VEGF/VPF mRNA, modulation of VEGF/VPF induction by TGFalpha was examined in vitro. The anti-sense oligo was capable of inhibiting VEGF/VPF RNA and protein to near-basal levels providing an antiangiogenetic strategy (Smyth, 1997). Previously, it was shown that phosphorothioate antisense oligonucleotides directed against basic fibroblast growth factor (bFGF) mRNA inhibited both the growth of Kaposi's sarcoma (KS) cells derived from different patients and the angiogenic activity associated with these cells, including the induction of KS-like lesions in nude mice (Ensoli, 1994). These effects were due to the block of the production of bFGF which is required by AIDS-KS cells to enter the cell cycle and which, after release, mediates angiogenesis (Ensoli, 1994). We describe oligos to be superior to peptide oligos in vehicle characteristics of radiation. While phosphorothioate oligos have rapid disappearance from plasma within an hour, and a biexponential elimination, their half lives apparently longer than in the peptide oligos. Although a phosphorothioate oligo leaves plasma rapidly, it requires days to leave the whole body. There is also significant extravascular accumulation of greater than 50 % of the injected dose over a period of 3 to 12 hr. Furthermore, uptake into tissues is not saturated, as some uptake is happening even at 28 days during continuos infusion (Iversen et al, 1994). The oligos are extensively eliminated in the urine over first 3 days after bolus injection. Distribution to, and tissue accumulation and distribution is tissue-specific (Iversen et al, 1992, 1994). It can be addressed that the behavior of the radiation at small distancies is crucial. This would be crucial in oligoradiotherapy with highest possible uptake in the target cell and minimal radiation toxicity to surrounding normal cells. Here, oligos are transferring radioactive source inside the cell and finally to close contact with target RNA macromolecule. We have shown earlier that for subcellular targeting internal labels give the lowest variation in estimated absorbed nuclear doses in our cell model with given dimensions (nuclear diameter 6-16 Âľm, cellular diameter 12-20 Âľm) (Kairemo et al, 1996). From the published data (Crooke et al, 1995) for ISIS 2105,21-mer oligonucleotide the following subcellular distribution was obtained: the nuclearuptake 0.2 %, cytoplasmic uptake 1.3 %, and cell surface uptake 0.3 % of injected dose. In this anti-human papilloma virus (HPV) model these uptakes as % cell

volume are 11 % for nucleus, 72% for cytoplasm and 17 % for cell surface. We calculated concentration distributions including the uniform distribution and published biodistribution. We normalized the results relative to the uniform distribution and the effect of the activity outside the cell was not taken into account, which assumption lead to the maximal possible inhomogeneity in absorbed dose distribution within a single cell. We have also calculated in vivo subcellular tissue distribution for oligodeoxynucleotide phosphorothioates with some Auger emitting radionuclides. Auger emittersare low-range electrons with high biological efficiency with a tendency of becoming more and more frequently used, at least theoretically. The doses vary considerably depending on cellular dimensions when using Auger-emitting isotopes; however, in small cells they may give a high dose. In tumors cell dimensions may vary and therefore these Auger-emitting isotopes should be applied only when nuclear target circumstances are well characterized. High energy %-emitter P-32 gives the nuclear dose closest to uniform distribution in cell sizes, but this is due to high energy. We have previously shown (Kairemo) that when using P-32 labelled oligos other than target cells will be destroyed because of long range. This is not the case when using %-emitters, P-33 and S-35, which are optimal when targets are smaller than 300 Âľm in diameter. P-33 was not studied here separately because its characteristics are very close to those of S-35. Now we also demonstrate that calculations related to positron emitters F-18 and Br-76, beta-emitter Y-90, Auger-emitter In-111 and alpha-emitter At-211 add substantial information to radionanotargeting dosimetry. Calculations using Br-76 demonstrate up to 5-fold differences in cell nuclear dose only in different cellular dimensions. This indicates the importanc of careful selection of a proper radionuclide. It is possible to use a mixture of radioisotopes to ensure a complete coverage of targets in more than one locations, e.g. targeting nuclear related and cellular RNA at the same time. In addition, modern imaging technique allows visual control over kinetic events. Dual labelling may provide therapeutic benefits when treating smaller and larger targets simultaneously. Further in vivo development, especially with various labels for oligos is highly indicated.

References Agrawal S, Temsamani J, Galbraith W, Tang J. (1995) Pharmacokinetics of antisense oligonucleotides. Clin Pharmacokinet 28, 7-16. Agrawal S, Temsamani J, Tang JY. (1991) Pharmacokinetics, biodistribution and stability of oligodeoxynucleotide phosphorothioates in mice. Proc Natl Acad Sci USA 88, 7595-7599. Bolch WE, Bouchet LG, Robertson JS, Wessels BW, Siegel JA, Howell RW, Erdi AK, Aydogan B, Costes S, Watson EE, Brill AB, Charkes ND, Fisher DR, Hays MT, Thomas SR. (1999) MIRD pamphlet No. 17, the dosimetry of nonuniform activity distributions--radionuclide S values at the voxel

Kairemo et al: Oligonucleotide radiotherapy level. Medical Internal Radiation Dose Committee. J Nucl Med 40, 11S-36S. Chen Z, Fisher RJ, Riggs CW, Rhim JS, Lautenberger JA. (1997) Inhibition of vascular endothelial growth factor-induced endothelial cell migration by ETS1 antisense oligonucleotides. Cancer Res 57, 2013-9 Claffey KP, Brown LF, del Aguila LF, Tognazzi K, Yeo KT, Manseau EJ, Dvorak HF. (1996) Expression of vascular permeability factor/vascular endothelial growth factor by melanoma cells increases tumor growth, angiogenesis, and experimental metastasis. Cancer Res 56, 172-81 Crooke RM, Graham MJ, Cooke ME, Crooke ST. (1995) In vitro pharmacokinetics of phosphorothioate antisense oligonucleotides. J Pharmacol Exp Ther 275, 462-473. Crooke ST, Graham MJ, Zuckerman JE, Brooks D, Conklin BS, Cummins LL, Greig MJ, Guinosso CJ, Kornburst D, Manorahan M, Sasmor HM, Schleich T, Tivel KL, Griffey RH. (1996) Pharmacokinetic properties of several novel oligonucleotide analogs in mice. J Pharmacol Exp Ther 277, 923-937 Dewanjee M.K, Ghafouripour A.K, Kapadvanjwala M, Dewanjee S, Serafini AN, Lopez DM, and Sfakianakis GN. (1994a) Noninvasive imaging of c-myc oncogene messenger RNA with indium-111-antisense probes in a mammary tumor-bearing mouse model. J. Nucl. Med. 35, 1054-1063. Egholm M, Buchardt O, Christensen L, Behrens C, Freier SM, Driver DA, Berg RH, Kim SK, Norden B, Nielsen PE. (1993) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365, 566-568. Egholm M, Burchardt O, Nielsen PE, Berg RH. (1992) Peptide nucleic acids (PNA), oligonucleotide analogs with an achiral peptide backbone. J Am Chem Soc 114, 1895-1897. Ensoli B, Markham P, Kao V, Barillari G, Fiorelli V, Gendelman R, Raffeld M, Zon G, Gallo RC. (1994) Block of AIDSKaposi's sarcoma (KS) cell growth, angiogenesis, and lesion formation in nude mice by antisense oligonucleotide targeting basic fibroblast growth factor. A novel strategy for the therapy of KS. J Clin Invest 94, 1736-46 Geselowitz DA, Neckers LM. (1992) Analysis of oligonucleotide binding, internalization and intracellular trafficking utilizing a novel radiolabeled crosslinker. Antisense Res Dev 2, 1725. Hammes HP, Brownlee M, Jonczyk A, Sutter A, Preissner KT. (1996) Subcutaneous injection of a cyclic peptide antagonist of vitronectin receptor-type integrins inhibits retinal neovascularization. Nat Med. 2, 529-33. Hanahan D. (1997) Signaling vascular morphogenesis and maintenance. Science 277, 48-50. Iversen PL, Mata J, Tracewell WG, and Zon G. (1994) Pharmacokinetics of an antisense phosphorothioate oligodeoxynucleotide against rev from human immunodeficiency virus type 1 in the adult male rat following single injections and continuos infusion. Antisense Res. Dev 4, 43-52. Iversen PL, Shu S, Meter A, and Zon G. (1992) Cellular uptake and subcellular distribution of phosphorothioate oligonucleotides into cultured cells. Antisense Res. Dev 2, 211-222. Iwasaka C, Tanaka K, Abe M, Sato Y. (1996) Ets-1 regulates angiogenesis by inducing the expression of urokinase-type plasminogen activator and matrix metalloproteinase-1 and migration of vascular endothelial cells. J Cell Physiol 169, 522-531

Jekunen AP,Kairemo KJA. (1997) Inhibition of malignant angiogenesis. Cancer Treat Rev 23, 263-86. Jellinek D, Green LS, Bell C, Janjic N. (1994) Inhibition of receptor binding by high-affinity RNA ligands to vascular endothelial growth factor. Biochemistry 33, 10450-6 Kaipainen A, Vlaykova T, Hatva E, Bรถhling T, Jekunen A, Pyrhรถnen S, Alitalo K. (1994) Enhanced expression of the tie receptor tyrosine kinase messenger RNA in the vascular endothelium of metastatic melanomas. Cancer Res 54, 6571-6577 Kairemo KJA, Jekunen A, Karnani P. (1996) Modulation of antibody kinetics by the cell membrane active agent Tween 80 in vivo.Anticancer Res 16, 3542-3550 Kairemo KJA, Jekunen AP, Tenhunen M. (1999) Essentials of radionanotargeting using oligodeoxynucleotides. Gene Ther Mol Biol4, 171-176 Kairemo KJA, Tenhunen M, Jekunen AP. (1996) Oligoradionuclidetherapy using radiolabelled antisense oligodeoxynucleotide phosphorothioates. Anti-Cancer Drug Design 11, 439-449 Kairemo KJA,Tenhunen M, Jekunen AP. (1996) Dosimetry of radionuclide therapy using radiophosphonated antisense oligodeoxynucleotide phosphorothioates based on animal pharmacokinetic and tissue distribution data. Antisense Nucl Acid Drug Dev 6, 215-220. Kairemo KJA, Thorstensen K,Mack M, Tenhunen M, Jekunen AP. (1999) Ets-1 mRNA as target for antisense radiooligonucleotide therapy in melanoma cells. Gene Ther Mol Biol 4, 177-182 Korhonen J, Partanen J, Armstrong E, Vaahtokari A, Elenius K, Jalkanen M, Alitalo K. (1992) Enhanced expression of the tie receptor tyrosine kinase in cells during neovascularization. Blood 20, 2548-2555 Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand SJ, Radziejewski C, Compton D, McClain J, Aldrich TH, Papadopoulos N, Daly TJ, Davis S, Sato TN, Yancopoulos GD. (1997) Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277, 55-60. Mardirossian G, Lei K, Rusckowski M, Chang F, Qu T, Egholm M, Hnatowich DJ. (1997) In vivo hybridization of technetium-99m-labeled peptide nucleic acid (PNA). J Nucl Med 38, 907-913 Masood R, Cai J, Zheng T, Smith DL, Naidu Y, Gill PS. (1997) Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma. Proc Natl Acad Sci U S A. 94, 979-84 Risau W. (1997) Mechanisms of angiogenesis Nature 386, 6714. Saleh M, Stacker SA, Wilks AF. (1996) Inhibition of growth of C6 glioma cells in vivo by expression of antisense vascular endothelial growth factor sequence. Cancer Res 56, 393-401 Sands H, Gorey-Feret LJ, Cocuzza AJ, Hobbs FW, Chidester D, Trainor GL. (1994) Biodistribution and metabolism of internally 3H-labeled oligonucleotides. I. Comparison of a phosphodiester and a phosphorothioate. Mol Pharmacol 45, 932-943. Shoji Y, Akhtar S, Periasamy A. (1991) Mechanism of cellular uptake of modified oligonucleotides methylphosphonate linkage. Nucl Acid Res 19, 5543-5550. Smyth AP, Rook SL, Detmar M, Robinson GS. (1997) Antisense oligonucleotides inhibit vascular endothelial growth factor/vascular permeability factor expression in normal

Gene Therapy and Molecular Biology Vol 7, page 23 human epidermal keratinocytes. J Invest Dermatol 108, 523-6 Stabin MG. (1996) MIRDOSE, personal computer software for internal dose assessment in nuclear medicine. J Nucl Med 37,538-46. Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S, Sato TN, Yancopoulos GD. (1996) Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87, 1171-80. T Wu J, Zhou L, Tonissen K, Tee R, Artzt K. (1999) The quaking I-5 protein (QKI-5) has a novel nuclear localization signal and shuttles between the nucleus and the cytoplasm. J Biol Chem 274,29202-10. Tavitian B, Terrazzino S, Kuhnast B, Marzabal S, Stettler O, Dolle F, Deverre JR, Jobert A, Hinnen F, Bendriem B, Crouzel C, Di Giamberardino L. (1998) In vivo imaging of oligonucleotides with positron emission tomography. Nat Med 4,467-71. Ullrich A, Schlessinger J. (1990) Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203-212

Wasylyk B, Hahn SL, Giovane A. (1993) The ets family of transcription factors. Eur J Biochem 211, 7-18. Wu F, Yngve U, Hedberg E, Honda M, Lu L, ErikssonB, Watanabe Y, Bergstrรถm M, L_ngstrรถm B. (2000) Distribution of 76Br-labelled antisense oligonucleotides of different lengthdetermined ex vivo in rats. Eur J Pharm Sci 10, 179-186 Zhang R, Diasio RB, Lu Z, Liu T, Jiang Z, Galbraith WM, and Agrawal S. (1995) Pharmacokinetics and tissue distribution in rats of an oligodeoxynucleotide phosphorothioate (GEM 91) developed as a therapeutic agent for human immunodeficiency virus type-1. Biochem Pharmacol 49, 929-939. Zhang R, Yan J, Shahinian H, Amin G, Lu Z, Liu T, Saag MS, Jiang Z, Temsamani J, Martin RR, et al (1995) Pharmacokinetics of an anti-human immunodeficiency virus antisense oligodeoxynucleotide phosphorothioate (GEM 91) in HIV-infected subjects. Clin Pharmacol Ther 58, 44-53.

Kairemo et al: Oligonucleotide radiotherapy

Gene Therapy and Molecular Biology Vol 7, page 25 Gene Ther Mol Biol Vol 7, 25-35, 2003

Strategy of sensitizing tumor cells with adenovirusp53 transfection Review Article

Jekunen Antti1*, Miettinen Susanna2, M채enp채채 Johanna3, Kairemo Kalevi4 1

Department of Clinical Pharmacology, Helsinki University, and Department of Oncology, Turku University, and Aventis Pharma Finland, Finland. 2Department of Anatomy, Tampere University, Finland. 3Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Tampere University Hospital and Tampere University, Finland. 4 Department of Nuclear Medicine, Uppsala University Hospital, Sweden

__________________________________________________________________________________ *Correspondence: Antti Jekunen, MD, PhD, PL96, 00241 Helsinki, Finland; Tel. +358400 755208; Fax. +3589 47638140; email:antti.jekunen@aventis.com Received: 29 January 2002; accepted: 06 March 2002; electronically published: July 2003

Summary Loss or malfunction of the p53-mediated apoptotic pathway has been proposed as one mechanism by which tumors become resistant to chemotherapy. While it may be the most frequently mutated gene in human tumor samples, the function of p53 is critical for maintaining the integrity of the cellular genome in its responses to treatment with cytotoxic agents. Intact p53 protein in nuclei of normal cells acts as a transcriptional activator for a group of genes involved in cell cycle arrest, DNA repair and apoptosis. The transfection of adenovirus p53 (adeno-p53) alone has been shown in ovarian cancer cell culture models to inhibit cell growth and to promote apoptosis regardless of the endogenous p53 status of the cells. Both mutant p53 in the tumor cells and the loss of p53 function were associated with resistance to chemotherapeutic agents. There are various reports of at least additive interactions between adeno-p53 and several chemotherapeutic agents in a number of cancers, e.g. bladder cancer, NSCLC, prostate cancer, breast cancer, and ovarian cancer both in vitro and in vivo. The mechanisms of these interactions are unknown, but they may depend on the chemotherapeutic agents used, the targets and critical tissues, and the intracellular signal transduction pathways affected.Results obtained with a speculative treatment regimen consisting of oligonucleotide therapy and p53 transfection suggest that p53 expression in tumor cells may improve their sensitivity to routine chemotherapy, e.g. docetaxel and irinotecan, which are efficacious drugs possessing different modes of action: prevention of depolymerization of tubulin and specific DNA topoisomerase I inhibition, respectively. It is known, however, that even these new agents cannot achieve responses in all tumors, and that in some tumors the efficacy, once established, diminishes along with the treatment. In these cases of resistant tumors or recurrences and relapses, combined treatment with adeno-p53 and chemotherapeutic agents may be an attractive strategy for inhibiting the progression of local cancers. In fact, the ground is ready for a rapid practical development of adeno-p53, which itself causes only minimal side-effects after administration, e.g. injection site rashes and fever, and an immunostimulation that seems to be quite mild and transient in nature. Future cancer therapy strategies may consist of effective chemotherapy coupled to molecular medicine specifically targeting tumor cells. So far, we do not have proper means in molecular medicine for achieving high enough tumor access with any of the current systemic virus vectors having the proper level of selectivity between tumor and normal cells. We have already some clinical experience, however, with intratumoral approaches that ensure the highest possible concentrations inside NSCLC, ovarian cancer and head and neck cancer tumors. It seems that there is clear evidence of good tolerability at non-maximal doses, but unfortunately, only modest activity when the construct is used alone. We review here the published data on the use of adenovirus p53 for sensitizing tumors to chemotherapeutic agents and outline perspectives for the future. Its basic function is to control the entry of the cell into the S phase of the cell cycle. p53 extends the time available for DNA repair before S phase entry (Fan et al, 1995). The wild- type gene product regulates cell growth and division negatively. Although not essential for progression of the

I. Introduction A. Function of p53 The p53 protein, a nuclear phosphoprotein, is indispensable for genomic integrity and cell cycle control. 25

Jekunen et al: Strategy of sensitizing tumor cells with adenovirus-p53 transfection cell cycle, it is critical as a checkpoint that blocks uncontrolled cell division (Levine, 1992). In the nuclei of normal cells, the intact p53 protein acts as a transcriptional activator for a group of genes involved in cell cycle arrest (p21cip1/waf1), DNA repair (GADD45), and apoptosis (Bax) (O'Connor et al, 1997; Sugrue et al, 1997; Yin et al, 1997; Carrier et al, 1999). In addition to this, p53 is a potent inducer of programmed cell death (apoptosis) within a cell in which the DNA has been damaged. Normally, the p53 gene is inactive. When, after DNA damage, the normal p53 is activated, the levels of p21, p27, and GADD 45 may become very high (Sherr, 1994). DNA damage in cells induces expression of p53 and interruption of the cell cycle in both G1 and G2 (Chu and DeVita, 2001). If DNA repair is successful, the cell continues its cycle. If repair does not succeed, the cell undergoes apoptosis.

to chemotherapeutic agents (Lowe et al, 1994; Righetti et al, 1996; Blandino et al, 1999). A recent study of ovarian cancer shows that women with tumors having the p53 null mutation have a survival disadvantage over those with p53 missense mutations (Shahin et al, 2000).

II. Evidence of the role of p53 in chemosensitizing A. p53 and chemotherapeutic agents Dysregulation of the p53 pathway may lead to drug resistance due to overproduction of the gene products responsible for entry into the S phase and rapid cell growth (Figure 1). Activation of these genes could theoretically increase the resistance of cells to the following chemotherapeutic agents: methotrexate, 2-chlorodeoxyadenosine, hydroxyurea, fludarabine, cytosine arabinoside, and 5fluorouracil. Under some experimental circumstances, cell death in response to exposure to DNA-damaging agents may require an intact p53-dependent apoptotic mechanism. Some of the genes that are transcriptionally activated by p53 belong to a class of proteins known to inhibit cyclin-dependent kinases (cdk). p21 forms a complex with proliferating cell nuclear antigen or inhibits cdkâ&#x20AC;&#x2122;s, e.g. cdk4 (Polyak et al, 1997). Activated p53 can cause a G1 cell cycle arrest by increasing the transcription of the cdk inhibitor p21 (Figure 2), which block cdk4 activity, preventing reitinoblastoma gene product (RB) phosphorylation (Sherr, 1994) and release of E2F blocking the transcription of a number of genes, and inhibiting entry into S phase (Kirsch, 1998). The E2F family of transcription factors bind to the regulatory regions of a number of genes that participate in the synthesis of DNA (Figure 2).

B. Mutation of p53 Mutations in the p53 gene are among the most common genetic alterations observed in human tumor samples (Oren, 1992). The specific cytotoxic treatment, the conditions of treatment, the p53 status, and other elements of cell-cycle regulation may all contribute to the outcome of exposure of a cell to DNA-damaging agents (Chu and DeVita, 2001). p53 can activate an apoptotic response to DNA damage, especially in hematopoietic and lymphoid cells, which often overrides the G1 checkpoint response (Fan et al, 1995). In cell types programmed for apoptosis, loss of p53 function decreases their sensitivity to a wide variety of DNA-damaging agents, while in cell apoptosis, it has been more difficult to establish a clear relationship between p53 gene status and chemosensitivity types of some solid tumors not inherently programmed for (Fan et al, 1995). If the DNA is damaged, the cell with intact p53 function will undergo p53-dependent apoptosis (Chu and DeVita, 2001). In tumor cells with mutated p53, the loss of p53 function, is thought to result in resistance

Figure 1. Effect of chemotherapy via p53 pathway. After chemotherapy has induced DNA damage, p53 protein is activated and transcription of many genes is increased, resulting in cell cycle arrest and apoptosis. For apoptotically sensitive cells, genotoxic damage can signal an immediate apoptotic response, while for apoptotically insensitive cells, the primary apoptotic decision point is disabled. Cells that avoid apoptotic or necrotic death after DNA repair can survive and grow. (Kirsch 1998; Brown and Wouters 1999)

Gene Therapy and Molecular Biology Vol 7, page 27 vivo and may strongly suggest the presence of synergy in vivo. Nielsen et al, used three-dimensional statistical modeling to evaluate the presence of synergistic, additive, or antagonistic efficacy between adenovirus-mediated p53 gene transfer and paclitaxel in a panel of human tumor cell lines, including those for ovarian, head and neck, prostate, and breast cancer (Nielsen et al, 1998). Cells were either pretreated with paclitaxel 24 h or not, before proliferation was measured 3 days later. Paclitaxel had synergistic or additive efficacy with p53 transfer, independently of whether the cells expressed mutant p53 protein or no p53 protein at all. Cell cycle analysis demonstrated that, prior to apoptotic cell death, p52 transfection arrested cells in the G0/G1 stage, whereas paclitaxel arrested cells in the G2-M stage. When combined, the relative concentrations of the two agents determined the dominant cellular response. The observed synergy remained unexplained; however, some speculations were offered. P53 has been shown to down regulate the expression of the antiapoptotic bcl-2 gene and up regulate the expression of the proapoptotic bax gene in other tumor cells (Selter and Montenarh 1994). Thus, p53 and paclitaxel may potentiate each other in stimulating the apoptotic pathway in neoplastic cells (Nielsen et al, 1998). It may also be that paclitaxel increased the number of cells transfected by the adenovirus. Particularly, the concentrations of paclitaxel responsible for increased adenovirus transduction are lower than the concentrations required for microtubule condensation. Moreover, the rate of change in the number of cells transduced by adenovirus appears to be independent of paclitaxel-induced cell death. The authors also determined the efficacy of the combination therapy in vivo. In some instances, it seems that loss of p53 may increase resistance to one agent, while simultaneously increasing sensitivity to another. Bunz et al, (1999) have reported that deletion of p53 in colorectal cancer cell lines maintained the cells that were resistant to 5-fluorouracil, but increased the sensitivity to doxorubicin and radiation in vitro. If the compound exerts it effects by apoptosis, as does 5-fluorouracil, loss of the apoptotic pathway may lead to resistance.

These genes include ribonucleotide reductase, dihydrofolate reductase, DNA-dependent RNA polymerase, thymidylate synthase, c-myc, c-fos, and cmyb. Activation of these gene products facilitates the entry of the cell into the S phase. There is much evidence in support of the idea that a mutation in p53 may lead to resistance to cytotoxic agents. In premenopausal women with node negative breast cancer, it has been shown by immunohistochemistry that p53(+) tumors are less sensitive to treatment with a regimen including 5-fluorouracil, doxorubicin, and cyclophosphamide than p53 (-) tumors. (Clahsen et al, 1998). Under in vitro conditions Koechli et al, have shown that mutant p53 can increase chemoresistance to 5fluorouracil, cyclophosphamide, and methotrexate (Koechli, 1994). Cisplatin resistance seems to be connected with p53 mutations, and in advanced ovarian cancer, the p53 mutational status is a predictor of the responsiveness to platinum-based chemotherapy (Calvert, 1999). However, there are also reports that apparently disagree with the chemoresistance effect of p53 (Fan et al, 1995; Stal 1995; Hawkins et al, 1996). Human fibroblasts lacking functional p53 were more sensitive to cisplatin, carboplatin, paclitaxel, nitrogen mustard or melphalan than cells with functional p53 (Hawkins et al, 1996). Similar results, loss of p53 function and the sensitizing effect of cisplatin, have been demonstrated in MCF-7 breast cancer cells and RKO methotrexate, and 5-fluorouracil have been reported in colon cancer cell lines with or without disruption of p53 function by a dominant negative p53 transgene (Fan et al, 1995). Increased rates of response to cyclophosphamide, patients with breast cancer who were determined to be immunohistochemically p53(+) (Stal,1995).

B. In vitro interactions Synergy between two chemical agents in vitro is an empirical phenomenon, in which the observed effect of the combination is greater than would be predicted from the effect of each agent working alone. While synergy is not directly measurable in clinical practice, it may predict a favorable outcome when two treatments are combined in

Figure 2. Two examples of cell cycle arrest via p53 activation. P53 mediated cell-cycle arrest is demonstrated with two examples: A) inhibition of cdk4 and cdk2 resulting G1-S and G2-M arrest, respectively. B) p53 activation increases the transcription of the cyclindependent kinase (cdk) inhibitor p21. Increase levels of p21 protein prevent cdkâ&#x20AC;&#x2122;s from phosphorylating their substrates, such as the retinoblastoma protein (RB) and thus block cell-cycle progression from G1 into S phase. (Kirsch 1998; Brown and Wouters 1999)

Jekunen et al: Strategy of sensitizing tumor cells with adenovirus-p53 transfection enhanced in cells that expressed wild-type p53 and were able to trigger their own cell death program. In cell culture models, adenovirus-mediated p53 gene transfer alone inhibits cell growth and promotes apoptosis, regardless of the endogenous p53 status of the ovarian cancer cells (Santoso et al, 1995). In tumor cells, mutated p53 and also loss of p53 function were associated with resistance to chemotherapeutic agents. There are several reports of at least an additive interaction between adenop53 and cisplatin in bladder cancer (Miyake et al, 2000), between adeno-p53 and cisplatin, SN-38 (a metabolite of irinotecan), 5-fluorouracil, taxanes, bleomycin, and cyclophosphamide in NSCLC (Fujiwara et al, 1994) (Horio et al, 2000), and between adeno-p53 and paclitaxel in ovarian cancer (Nielsen et al, 1998). In the ovarian cancer model, enhanced efficacy has been reported in a three-drug combination of adeno-p53, cisplatin, and paclitaxel (Gurnani et al, 1999). There is some evidence that chemosensitivity can be increased by replacement of the p53 gene. Roth (Roth, 1996) reported that recombinant-adenovirus-mediated transfer of the wild-type p53 gene into several human cells with homozygous deletions of p53 markedly increased cellular chemosensitivity to the major chemotherapeutic drugs. An additive antiproliferative effect was reported in p53null H358 lung cancer cells when cultured with cisplatin for 24 h before transduction with adeno-p53 (Fujiwara et al, 1994). Enhanced apoptosis, detected by DNA fragmentation, was reported for the combination compared with each agent alone. A viability assay demonstrated that a replicationdefective adenovirus encoding the wild-type p53 gene (INGN 201, Introgen Therapeutics, Inc.) suppresses growth and enhances sensitivity to DNA-damaging chemotherapeutic drugs (5-fluorouracil, doxorubicin, cisplatin) in p53-mutant-expressing cell lines (Gjerset and Mercola, 2000). These cells lines represent DLD-1 colon cancer, T47D breast cancer, PC-3 prostate cancer, and T98G glioblastoma. Transfection efficiencies were 6070%. It seems that restoration of the wild-type p53 to mutant p53-expressing or p53null cells results in marked enhancement of sensitivity to several DNA damaging agents. This enhancement of sensitivity was not observed in two wild-type p53-expressing cell lines, MCF7 and LS174T, suggesting that, in this model, wild-type p53 gene transfer is effective as therapy sensitization only in tumors that have lost wild-type p53 function.

Recently, a report using isobologram modelling have showed that the combination of adeno-p53 + radiation produced significantly synergistic effects in NSCL cell lines, whereas the combination of docetaxel + adeno-p53 and docetaxel + radiation produced mixed effects ranging between additive and synergistic (Nguyen al., 1996). The three-agent combination also produced significantly synergistic effects. Brown and Wouters have criticized the sensitizing results obtained in cell cultures. They have pointed out the need for further evidence in relating p53 to the sensitivity of anticancer agents (Brown and Wouters, 1999). Because apoptosis, particularly p53窶電ependent apoptosis, can occur rapidly after drug exposure, short-term growth rate assays tend to underestimate overall death of cells with mutant p53 or of cells not undergoing apoptosis. This may result in a situation where short-term assays may incorrectly assess overall cell death in tumor cells with different probabilities of undergoing early apoptosis. Thus, results may have a bias toward increased cell death in wild-type p53 cells and decreased cell kill in mutant p53 cells. Results of experiments with normal cells transformed with dominant oncogenes have often been extrapolated to tumor cells, instead of initially using cancer cell models. Transformed normal cells are usually apoptotically more sensitive than cancer cells. Therefore, in sensitizing experiments, both long term clonogenic assays and tumor cell models with solid tumors should be used rather than growth rate assays and transformed normal cells. However, the more widely accepted conclusion drawn from studies conducted in cancer cell lines and tumors of different origin is still that restoration of normal p53 function in tumors restores the apoptotic pathway and leads to an increased response to chemotherapy (Peller, 1998; Ferreira, 1999; Chang, 2000).

C. Transfection of cell cultures with the adenovirus p53 gene construct Adenovirus vectors have many advantages over other viral and non-viral vectors. Their transfection efficacy is high, in both dividing and resting cells, and they show high expression levels (Hwu, 2001). As adenoviral DNA is not incorporated into the cell genome, expression of the transgene is transient, but adenoviral vectors can be produced at high titers. Introduction of wild-type p53 into tumors with non functional p53 offers a novel strategy for treating cancer, by inducing apoptotic death in neoplastic cells. Genomic instability accompanied by loss of p53mediated apoptosis can also lead to therapy resistance. The support for this rationale is that loss of p53 could desensitize cells to the damaging effects of drugs. Normal transgenic hematopoetic cells (Lotem and Sachs, 1993), E1A-expressing transgenic fibroblasts (Lowe et al, 1993), and transformed transgenic fibroblasts (Lowe et al, 1994) were all more resistant to apoptosis following treatment with any of a wide variety of anticancer agents, than were comparable cells from the parental strain of mice, which expressed wild-type p53. Apoptosis seemed to be

1. Glioma and pancreatic cancer Somatic gene therapy based on the reintroduction of p53 limits the proliferation of human malignant glioma cells, but is unlikely to induce clinically relevant sensitization to chemotherapy in these tumors. Wild-type p53 failed to sensitize glioma cells to cytotoxic drugs including BCNU, cytarabine, doxorubicin, teniposide, and vincristine. The combined effects of the wild-type p53 gene transfer and drug treatment were less than additive rather than synergistic, suggesting that the intracellular cascades activated by p53 and chemotherapy were redundant. Unexpectedly, forced expression of mutant28

Gene Therapy and Molecular Biology Vol 7, page 29 p53 reduced 3H-thymidine incorporation by about 90% at 48 hr, cell viability at 6 days was reduced by only about 50% relative to controls. Although apoptosis is detectable in the adeno-p53-treated cultures, these results suggest that a large fraction of adeno-p53-treated cells merely undergo reversible cell cycle arrest. Combined treatment with adeno-p53 and doxorubicin results in a greater than additive loss of viability in vitro and increased apoptosis. These data indicate an additive to synergistic effect of adeno-p53 and doxorubicin for the treatment of primary and metastatic breast cancer. However, in breast cancer cell lines results without any clear cut link between transfection of p53 and a sensitizing effect have been reported. Two human breast cancer cell lines, MDA-MB-231 and MDA-MB-435, both with p53 mutations, were transduced with adenoviral vectors containing wild-type p53 and the effects on growth were determined by clonogenic assays (Parker et al, 2000). Combining VP-16 and paclitaxel with Ad5CMV-p53 did not consistently or significantly decrease clonogenic survival.

p53-modulated drug sensitivity enhanced the toxicity of some drugs but attenuated the effects of others (Trepel et al, 1998). Likewise, in p53-null pancreatic carcinoma cells, wild-type p53 gene transduction had no effect on in vitro chemosensitivity to cisplatin, etoposide, 5fluorouracil and paclitaxel (Kimura et al, 1997). Moreover, in anaplastic thyroid cancer cells, adeno-p53 increased the sensitivity to doxorubicin with a 10-fold decrease in IC50 values.

2. Hepatocellular cancer One of the goals of gene therapy for treating cancer is selective expression of cytotoxic gene products in tumor cells. When replication-defective retroviruses were constructed containing p53 cDNA that was transcriptionally regulated by the human hepatocellularcarcinoma-associated alpha-fetoprotein gene transcriptional control elements, the expression of exogenous wild-type p53 from this retroviral vector was limited to the cells producing alpha-fetoprotein. Introduction of wild-type p53 into alpha-fetoprotein positive human hepatocellular carcinoma cells by retroviral infection markedly inhibited their clonal growth in a monolayer and increased the sensitivity of these cells to the chemotherapeutic drug cisplatin (Xu et al, 1996).

5. Bladder cancer Combined treatment with Ad5CMV-p53 and cisplatin could be an attractive strategy for inhibiting progression of bladder cancer. In human bladder cancer KoTCC-1 cells, transfer of an adenovirus-mediated p53 gene enhances cisplatin cytotoxicity in vitro, and Ad5CMV-p53 and cisplatin synergistically inhibit growth and metastasis in vivo. Ad5CMV-p53 substantially enhances cisplatin chemosensitivity in a dose-dependent manner, reducing the median IC50 by more than 50%. Furthermore, orthotopic injection of adeno-p53 combined with cisplatin therapy synergistically inhibits growth of subcutaneous KoTCC-1 tumors and the incidence of metastasis (Miyake et al, 2000). In contrast, p21cip1/waf1 gene therapy had no effect on in vitro or in vivo chemosensitivity to cisplatin (Miyake et al, 1998).

3. Ovarian cancer In cell culture models adenovirus-mediated p53 gene therapy is one way to inhibit cell growth and promotes apoptosis, regardless of the endogenous p53 status of the ovarian cancer cells (Santoso et al, 1995) (Wolf et al, 1999). Adeno-p53 gene transfer, combined with cisplatin, doxorubicin, 5-fluorouracil, methotrexate, or etoposide, inhibited cell proliferation more effectively than chemotherapy alone in head and neck, ovarian, prostate and breast tumor cell lines. Of particular significance, in an ovarian cancer model enhanced efficacy was noted when using the three-drug combination of adeno-p53, cisplatin, and paclitaxel (Gurnani et al, 1999). In human head and neck, ovarian, prostate, and breast cancer cells, low concentrations of paclitaxel also increase the number of cells transduced by recombinant adeno-p53 in a dosedependent manner (Nielsen et al, 1998). The concentration of paclitaxel responsible for increased adenovirus transduction is lower than that required for microtubule condensation.

6. Lung cancer Recombinant adenovirus-mediated transfer of the wild-type p53 gene into monolayer cultures or multicellular tumor spheroids of the human NSCLC cell line H358, in which there is homozygous deletion of p53, markedly increased the cellular sensitivity of these cells to cisplatin (Fujiwara et al, 1994). In a study made by Osaki et al,(Osaki et al, 2000), an alteration in drug chemosensitivity caused by the adenovirus-mediated transfer of the wild-type p53 gene in human lung cancer cells was tested on a human pulmonary squamous cell carcinoma cell line, NCI-H157, and a human pulmonary large-cell carcinoma cell line, NCI-H1299. Based on isobologram data, a supra-additive effect was observed for 5-fluorouracil and SN-38 on NCI-H157 cells. An additive effect was also observed for cisplatin, paclitaxel, bleomycin, and cyclophosphamide on NCI-H157 cells. Cisplatin, paclitaxel, 5-fluorouracil, and SN-38 had an additive effect on NCI-H1299 cells. No drug showed any subadditive or protective effects. These findings suggest

4. Breast cancer Transduction of cells using replication-deficient adenovirus vectors can induce endogenous p53 expression in cells containing the wild-type p53 gene and this response is different from the p53 induction observed after DNA damage (McPake et al, 1999). Lebedeva et al, have examined the effects of a replication-defective adenovirus encoding p53 (INGN 201, Ad5CMV-p53), alone or in combination with the breast cancer therapeutic doxorubicin, in suppressing growth and inducing apoptosis in breast cancer cells in vitro (Lebedeva et al, 2001). They found that whereas in vitro treatment of cells with adeno29

Jekunen et al: Strategy of sensitizing tumor cells with adenovirus-p53 transfection that CPT-11 and 5-fluorouracil may be useful as anticancer agents for use in a combination therapy regimen, using wild-type p53 gene transfer. These results indicate that CPT-11, as well as cisplatin, is a candidate for the combination of chemotherapy and gene therapy for NSCLC. Adeno-p53 and DNA-damaging agents, cisplatin, etoposide and CPT-11 showed synergistic effects in NSCLC, but, in contrast had additive effects with antitubulin agents such as paclitaxel and docetaxel (Horio, Hasegawa et al, 2000). Perdomo et al, (Perdomo et al, 1998) have demonstrated that human NSCLC cells having a mutant form of p53 grow faster in vivo than wild-type p53 cell lines and the treatment with cisplatin or radiation does not reduce the size of mutant p53 tumors, although wild-type p53 tumors regress markedly. Apoptosis occurred in mutant p53 cell types only at high cisplatin doses and not at the magnitude detected in wild-type tumors.

III. In vivo evidence chemosensitization by adenovirus p53

later by doxorubicin or mitomycin-C, but not by vincristine (Blagosklonny and El-Deiry 1996). In the p53 null SK-OV-2 xenograft model of ovarian cancer, a dosing schedule of the p53 therapy that, by itself, had a relatively minimal effect on the tumor burden (16%) caused a much greater decrease in tumor burden (55%) when combined with paclitaxel (Nielsen et al, 1998). Further, in nude mice implanted intraperitoneally with 2774 human ovarian cancer cells (mutated p53), the response to adeno-p53 gene therapy showed significant survival duration, with a survival time greater than that of untreated animals. However, no statistically significant survival advantage was observed between adeno-p53- and adenovirus-!gal-treated mice (von Gruenigen et al, 1998). In another ovarian cancer study using nude mice, the adeno-p53 treatment effectively suppressed the growth of peritoneal tumors and prolonged the survival of the treated group, especially when the tumor burden was small (Kim et al, 1999). Greater combined efficacy was observed in the p53null DU-145 prostate, p53Mut MDA-MB-468 breast, and p53met MDA-MB-231 breast cancer xenograft models in vivo. The authors concluded that their data, taken together, offer the possibility of enhanced antitumor activity with lower than normal doses of paclitaxel and adenovirus p53, when the two drugs are administered in combination (Nielsen et al, 1998). They noted that this could potentially decrease the chemotherapy-induced side effects, increasing the quality of life of the patients and, perhaps, reducing the overall expense of a complete course of cancer treatment.

These observations have been extended to in vivo models. Tumors have been treated in vivo with replication-defective p53 adenovirus and chemotherapy. Nguyen et al, have reported convincing in vivo studies, in which p53null H1299 lung tumor xenografts were given i.p. cisplatin before, concurrently with, or after intratumoral adenovirus p53 (Nguyen et al, 1996). The most effective dosing regimen was cisplatin given two days before p53 therapy. Cisplatin and CPT-11 had a significant antitumoral effect on lung cancer H157 cell xenografts of nude mice in vivo. Human head and neck cancer and colon cancer (Gjerset et al, 1997) and prostate cancer (Gjerset and Mercola 2000) in nude mice models in vivo have been found to exhibit a similar sensitization effect with adenovirus plus cisplatin as in studies in vitro. Gjerset et al, demonstrated increased sensitivity to cisplatin cytotoxicity in p53mut T98G glioblastoma and p53 mut H23 small cell lung carcinoma cells transduced with p53 expression vectors one or two days before exposure to cisplatin (Gjerset et al, 1995). These results are consistent with other in vivo studies in animal models showing a combined benefit of p53 and chemotherapy (Badie et al, 1998), (Fujiwara et al, 1994), (Miyake et al, 1998), (Nielsen et al, 1998), (Nguyen et al, 1996). Gjerset and Mercola are convinced that these results support the clinical application of adenovirus p53 combination approaches to tumors expressing mutant p53 (Gjerset and Mercola 2000). Chemosensitization by p53 has also been studied using ex vivo modified cells in an orthotopic model of glioblastoma in Fisher rats (Dorigo et al, 1998). The combination of p53 with 5-fluorouracil and topotecan has been studied in p53mut SW480 colorectal tumor cells transfected with an inducible p53 construct (Yang et al, 1996). Dose-dependent enhancement of cytotoxicity was observed with these drugs by the concurrent expression of wild-type p53. Increased cytotoxicity has been reported in p53mut SkBr3 mammary tumor cells when transduction with p53 was followed 8 hr

IV. Clinical results of adenovirus p53 transfection with chemotherapy The first evidence of the efficacy of p53 gene therapy for cancer was given by a pilot study in which retroviral p53 expression vectors were directly injected into small endobronchial lesions of NSCLC patients (Roth et al, 1996). Tumor regression was noted in three patients out of nine, and tumor growth stabilized in three other patients. The safety and feasibility of the intratumoral injection of adenoviral wild-type p53 expression vectors have been established in NSCLC patients, with clear evidence for transgenic expression, and possibly induction of apoptosis (Swisher et al, 1999; see Table 1). The antitumor activity in this trial was consistent with the activity of retroviral p53 injection in NSCLC patients. Twenty-four patients received intratumor injections of adenovirus p53 and two patients achieved a partial response, while 17 patients achieved stable disease as the best clinical response. A nonrandomized, phase I, dose-escalating study by Clayman et al expanded these findings into head and neck squamous cell carcinoma (Clayman et al, 1998). Patients with incurable recurrent local or regionally metastatic HNSCC received multiple intratumoral injections of adeno-p53, either with or without tumor resection. P53 expression was detected in tumor biopsies despite antibody responses after injections. prevent the appearance of adeno-p53 in blood and urine. were seen in the study As expected, almost Neither dose-limiting effects nor serious

Gene Therapy and Molecular Biology Vol 7, page 31 adverse events all the patients developed anti-adenovirus antibodies in the course of treatment, but this immune response did not treatment. The most common treatmentrelated adverse event was pain at injection site. Other reported adverse events were transient fever, headache, pain, and edema. No evidence of systemic hypersensitivity or allergic reactions was seen, despite the fact that patients received many repeated courses of treatment. In some patients, adenovirus p53 administration led to objective antitumor activity. Two out of 17 patients showed objective tumor regressions greater than 50% and six patients showed stable disease for up to 3.5 months. In addition, one patient showed a complete pathologic response. The median survival for responding patients was 13.6 months, and the overall median survival was 267 days, which is about 60% longer than that reported in chemotherapy trials with a similar patient profile (Schornagel et al, 1995). Of course, it is impossible, for a phase-one study with limited numbers of patients to state anything more than that these results are promising and that further studies are needed, and are underway, to determine the actual role of adenovirus-mediated p53 intratumoral injections as a treatment option for HNSCC. The next step in the development of p53 treament is to include combination therapy with cytotoxic agents. There is also a negative trial published by Schuller and coworkers (2001). Twenty-five patients with nonresectable NSCLC were enrolled in an open-label, multicenter, phase II study of three cycles of chemotherapeutics with intratumoral injection of recombinant adenovirus p53. The main idea of this small study was to compare the isolated responses of a tumor lesion treated by transfer of the adenoviral wild-type p53 gene with a comparable lesion not receiving any injections in patients undergoing first-line chemotherapy for NSCLC. In the 13 patients receiving carboplatin and paclitaxel, there was no obvious difference between the mean response of gene-therapy-treated and the reference lesions. In contrast, the mean regression of the reference lesions in patients treated with cisplatin and vinorelbine was 15%, whereas it amounted to 55% in lesions that were additionally injected with the gene construct. There was no difference between the responses of lesions treated with p53 gene therapy in addition to chemotherapy (52%) and those of lesions treated with chemotherapy alone (48%). The authors concluded that, in these patients

the therapy appears to provide no additional benefit. However, there were several possible shortcomings in the clinical set-up: no injections to the reference lesions, highly restrictive inclusion criteria may result in selection bias, a higher response rate (50%) than is normally achieved in this disease, a chance of having a biologically inactive virus construct, and insufficient spreading of the replication-defective adenoviral vectors within the tumors after only one central intralesional injection. Recently, attemps have been made to overcome the problem of ineffective vector spreading by administration of replication-competent adenoviruses (Heise, Sampson et al, 1997) and encouraging clinical results have been reported (Khuri et al, 2000). There were concerns about the safety, which, however, turned out to be exaggerated. Khuri et al, (2000) demonstrated an acceptable safety pattern with no sign of any dissemination to the environment. A Phase II trial of a combination of intratumoral ONYX-015 injection with cisplatin and 5fluorouracil was carried out with patients having recurrent squamous cell cancer of the head and neck. Only pain at the injection site (45%), mucous membrane disorder (21%), syncope (5%), kidney failure (5%), and anorexia (3%) could not be ruled out as attributable to Onyx-015. In addition, the injected tumors achieved objective responses at a substantially higher rate (9 of the 11) than the non-injected tumors (3 of the 11) within the same patients. In six patients, the injected tumor responded and the non injected tumor did not respond. The time to tumor progression was also longer for the injected tumors than for the non-injected tumors. There was no correlation between the response and the baseline tumor size, baseline neutralizing antibody titer, p53 gene status, or prior treatment. It was also clear that the efficacy of the intratumoral injection was not prevented by neutralizing antibodies. There has been discussion about whether or not enough evidence about viral replication of ONYX-015 in patients, as along experience based on 190 patients treated by a replication-defective adenovirus demonstrating similar biodistribution (Clayman et al, 1998; ConstenlaFigueiras et al, 1999). It may simple be that Taqman realtime polymerase chain reaction technology is not sufficient to prove that viral reproduction is taking place (Yver et al, 2001).

Table 1. Sensitising effect of adenovirus-p53 on chemotherapeutic agents, major clinical treatment results Disease

Phase

Combination

Treatment responses

Reference (first author year)

NSCLC

2 PR, 17 SD_

(Swisher et al, 1999)

Head & neck

1CR, 2 PR, 6 SD_

(Clayman et al, 1998)

NSCLC Heach & neck (_) on patients

II II

Cisplatin + vinorelbin Cisplatin +5-FU

25 11

13 PR* 9 PR*

(Schuler et al, 2001) (Khuri et al, 2000)

(*) on measurable lesions

Jekunen et al: Strategy of sensitizing tumor cells with adenovirus-p53 transfection strategy for inhibiting progression of local cancers. It is clear that even a modest change in drug sensitivity may bring some refractory tumors within a range that is treatable with conventional chemotherapy. Future therapy might couple standard cytotoxic agents with new biologic agents that attack specific molecular targets to reregulate the cell-cycle checkpoint. Human data supporting the effect of sensitizing chemotherapy with adenovirus p53 is still maturing, although we have not found a way to use systemic administration. We know that is s safe to perform intratumoral gene therapy with adenovirus either with a replication non-competent or replication competent vector. As yet, there is no clinical evidence to support a definite conclusion that adenovirus p53 provides a clinically meaningful improvement on conventional chemotherapy. However, it is clear that in some trial set ups it has been possible to demonstrate encouraging results and the possibility of a clinical sensitizing effect of p53 gene therapy on the chemotherapy used when specifically indicated. Intratumoral expression of transgenes and tumor-selective tissue destruction have been documented in phase I and phase II clinical trials of adenovirus p53 mediated gene therapy. However, durable responses and the clinical benefit seen have been limited, with of 10-15% response rates. The rationale of combining p53 gene therapy with a chemotherapeutic agent in the clinical setting has been noted to be as follows: combinations of agents with different toxicologic profiles can result in increased efficacy without increased overall toxicity, they may thwart the development of resistance to the single agents, they may offer a solution to the problem of heterogeneous tumor cell populations with different drug sensitivity profiles and they allow the physician to take advantage of possible synergies between drugs, resulting in increased anticancer efficacy in patients (Nielsen, Lipari et al, 1998). Several phase III clinical trials with adenovirus p53 therapy in head and neck cancer, NSCLC, and ovarian cancer, will be completed in the near future, and the role of gene therapy may become routine a part of treatment regimens.

V. Conclusion Several subsequent studies have confirmed that various malignant cell lines and tumors expressing mutant or deleted p53 are chemoresistant to a wide range of anticancer agents. However, other studies disagree suggesting that cells with impaired p53 function can become sensitized to various anticancer agents. Thus, the relationship between p53 status and chemosensitivity is complex and presumably depends on a number of factors, including the specific cytotoxic stimuli, tissue-specific differences, and the specific cellular context that incorporates the overall genetic machinery and the various intracellular signaling pathways (Chu and DeVita 2001). The relationship between p53 and chemotherapy depends on the chemotherapeutic agents used, the target and the critical tissues, and the intracellular signal transduction pathways affected. The theoretical basis of the sensitizing effect of chemotherapeutic agents in combination with adenovirus p53 has been presented and so have a number of supportive data. As adenovirus p53 has its own activity, there seems to be a possibility that the cytotoxicity may be enhanced at least in some cell lines by transfer of the gene into the tumor cells. This concept has reached the level of proof in some, although not all, experimental conditions. This leaves a room for doubt, as all spontaneous solid tumors are heterogeneous and there may always remain cell clones that fail to obey the sensitizing principle. It is clear that more evidence is needed to support this principle, especially clonogenic assays and classical interaction studies. Although the in vivo experiments are convincing and strongly positive, it may not be altogether correct to extrapolate these results into clinical practice. There is a relative lack of pharmacokinetic studies and pharmacokinetic interaction studies in adenovirus p53 gene therapy. Several strategies may be used to develop p53-based anticancer therapies, with the goal of resensitizing tumor cells to conventional chemotherapy (Chang 2000). These include reintroduction of the gene encoding wild-type p53 and methods for restoring normal p53 function to mutant p53. In addition, methods are being developed that target the p53-mdm-2 interaction of using lack of wild-type p53 in tumors to protect normal tissue from the adverse effects of chemotherapy. Replacement of the wild-type p53 by intratumoral transfection has already reached the phase III stage of clinical trials. Transfection of p53 can be combined with radioimmunotherapy as part of a tumor manipulation scheme (Kairemo, Jekunen et al, 1999). Increasing suppressor gene p53 expression in tumor cells improves the sensitivity of the tumor cells to routine chemotherapy. In a variety of tumor types, docetaxel and irinotecan are efficacious drugs with a new mode of action: prevention of depolymerization of tubulin and inhibition of specific DNA topoisomerase I, respectively. But we cannot obtain responses from all tumors, and in some tumors the efficacy, although established, diminished with time. In these cases of resistant tumors or recurrences and relapses, combined treatment with adenop53 and chemotherapeutic agents may be an attractive

Acknowledgments We would like to thank Aventis Pharma Finland for supporting this work.

References Badie B, Kramar MH, Lau R, Boothman DA, Economou JS, Black KL. (1998). “Adenovirusmediated p53 gene delivery potentiates the radiation-induced growth inhibition of experimental brain tumors.” J Neurooncol 37: 217-222. Blagosklonny, M. V. and W. S. El-Deiry (1996). “In vitro evaluation of a p53-expressing adenovirus as an anti-cancer drug.” Int J Cancer 67: 386-392. Blandino G, Levine AJ, Oren M. (1999). “Mutant p53 gain of function: differential effects of different p53 mutants on resistance of cultured cells to chemotherapy.” Oncogene 18(2): 477-85.

Gene Therapy and Molecular Biology Vol 7, page 33 Brown, M. J. and B. G. Wouters (1999). “Apoptosis, p53, and tumor cell sensitivity to anticancer agents.” Cancer Res 59: 1391-1399. Bunz, F. ( 1999). “Disruption of p53 in human cancer cells alters the responses of chemotherapeutic agents.” J Clin Invest 104: 263-269. Calvert, A. H. (1999). “Carboplatin and paclitaxel, alone and in combination: dose escalation, measurement of renal function, and role of the p53 tumor supressor gene.” Semin. Oncol. 29: 90-94. 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, A.J., Jr. (1999). “Gadd45, a p53-responsive stress protein, modifies DNA accessibility on damaged chromatin.” Mol Cell Biol 19(3): 1673-85. Chang, E. H. (2000). “Tp53 gene therapy: akey to modulating resistance to anticancer therapies?” Molecular Medicine Today 6: 358-364. Chu, E. and V. T. J. DeVita (2001). Apoptosis, cell-cycle control, and resistance to chemotherapy. Cancer. Principles & Practice of Oncolgy. V. T. J. DeVita, S. Hellman and S. A. Rosenberg. Philadelphia, Lippincott Williams & Wilkins. 6: 289-306. Clahsen PC, van de Velde CJ, Duval C, Pallud C, Mandard AM, Delobelle-Deroide A, van den Broek L, Sahmoud TM, van de Vijver MJ. (1998). “p53 protein accumulation and response to adjuvant chemotherapy in premenopausal women with node-negative early breast cancer.” J Clin Oncol 16(2): 470-3945. Clayman GL, el-Naggar AK, Lippman SM, Henderson YC, Frederick M, Merritt JA, Zumstein LA, Timmons TM, Liu TJ, Ginsberg L, Roth JA, Hong WK, Bruso P, Goepfert H. (1998). “Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head and neck squamous cell carcionoma.” JCO 16: 2221-2232. Constenla-Figueiras M, Betticher DC, DelCampo JM, Hitt R, Rochlitz C, Dhondt V, Gautier E, Saulnier P, Yver A, Badri N. (1999). “A phase II trial with Ad5CMV-p53 as a single agent in recurrent/refractory SCCHN looking at vector biodistribution and horizontal trnasmission under normal life conditions.” Proc Am Soc Clin Oncol 18: 444a. Dorigo O, Turla ST, Lebedeva S, Gjerset RA. (1998). “Sensitization of rat gliblastoma multiforme to cisplatin in vivo following restoration of wild-type p53 function.” J Neurosurg 88: 535-540. Fan S, Smith ML, Rivet DJ 2nd, Duba D, Zhan Q, Kohn KW, Fornace AJ Jr, O'Connor PM. (1995). “Disruption of p53 function sensitizes breast cancer MCF-7 cells to cisplatin and pentoxifylline.” Cancer Res 55(8): 1649-907. Ferreira, C. G. (1999). “p53 and chemosensitivity.” Ann Oncol 9: 1011-1021. Fujiwara T, Grimm EA, Mukhopadhyay T, Zhang WW, OwenSchaub LB, 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(9): 2287-91. Gjerset, R. A., O. Dorigo, et al, (1997). “Tumor regression in vivo following p53 combination therapy.” Cancer Gene Therapy 4: 0-2. Gjerset, R. A. and D. Mercola (2000). “Sensitizing of tumors to chemotherapy through gene therapy.” Cancer Gene Therapy 465: 273-291. Gjerset RA, Turla ST, Sobol RE, Scalise JJ, Mercola D, Collins H, Hopkins PJ. (1995). “Use of wild-type p53 to achieve

complete treatment sensitization of tumor cells expressing endogenous mutant p53.” Mol Carcinog 14: 275-285. Gurnani M, Lipari P, Dell J, Shi B, Nielsen LL. (1999). “Adenovirus-mediated p53 gene therapy has greater efficacy when combined with chemotherapy against human head and neck, ovarian, prostate, and breast cancer.” Cancer Chemother Pharmacol 44(2): 143-51. Gurnani M, Lipari P, Dell J, Shi B, Nielsen LL. (1998). “Adenovirus-mediated p53 gene therapy and paclitaxel have synergistic efficacy in models of human head and neck, ovarian, prostate, and breast cancer.” Clin Cancer Res 4: 835-846. Hawkins DS, Demers GW, Galloway DA. (1996). “Inactivation of p53 enhances sensitivity to multiple chemotherapeutic agents.” Cancer Res 56: 892-898. Heise C, Sampson-Johannes A, Williams A, McCormick F, Von Hoff DD, Kirn DH. (1997). “ONYX-015, an E1B geneattenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficaacy that can be augmented by standard chemotherapuetic agents.” Nature Medicine 3: 639-645. Horio Y, Hasegawa Y, Sekido Y, Takahashi M, Roth JA, Shimokata K. (2000). “Synergistic effects of adenovirus expressing wild-type p53 on chemosensitivity of non-small cell lung cancer cells.” Cancer Gene Ther 7(4): 537-44. Hwu, P. (2001). Gene therapy. Cancer. Principles & Practice of Oncology. V. T. J. DeVita, S. Hellman and S. A. Rosenberg. Philadelphia, Lippincott Williams & Wilkins. 6: 3161-3180. Kairemo KJ, Jekunen AP, Tenhunen M. (1999). Dosimetry and optimization of in vivo targeting with radiolabeled antisense oligonucleotides: oligonucleotide radiotherapy. Methods of Enzymology. New York, Academic Press. 314: 506-525. Khuri FR, Nemunaitis J, Ganly I, Arseneau J, Tannock IF, Romel L, Gore M, Ironside J, MacDougall RH, Heise C, Randlev B, Gillenwater AM, Bruso P, Kaye SB, Hong WK, Kirn DH. (2000). “A controlled trial of intraumoral ONYX015, a selectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer.” Nature Medicine 6: 879-885. Kim J, Hwang ES, Kim JS, You EH, Lee SH, Lee JH. (1999). “Intraperitoneal gene therapy with adenoviral-mediated p53 tumor suppressor gene for ovarian cancer model in nude mouse.” Cancer Gene Ther 6(2): 172-8. Kimura M, Tagawa M, Takenaga K, Yamaguchi T, Saisho H, Nakagawara A, Sakiyama S. (1997). “Inability to induce the alteration of tumorigenicity and chemosensitivity of p53-null human pancreatic carcinoma cells after the transduction of wild-type p53 gene.” Anticancer Res 17(2A): 879-83. Kirsch, D., Kastan, M.B. (1998). “Tumor-suppressor p53: implications for tumor development and prognosis.” JCO 16: 3158-3168. Koechli, O. (1994). “Mutant p53 protein associated with chemosensitivity in breast cancer specimens.” Lancet 344: 1647-1648. Lebedeva S, Bagdasarova S, Tyler T, Mu X, Wilson DR, Gjerset RA. (2001). “Tumor suppression and therapy sensitization of localized and metastatic breast cancer by adenovirus p53.” Hum Gene Ther 12(7): 763-772. Levine, A. J. (1992). “The p53 tumour suppressor gene and product.” Cancer Surveys 12: 59-79. Lotem, J. and L. Sachs (1993). “Hematopoietic cells from mice deficient in wild-type p53 are more resistant to induction of apoptosis by some agents.” Blood 82: 1092-1096.

Jekunen et al: Strategy of sensitizing tumor cells with adenovirus-p53 transfection Lowe SW, Bodis S, McClatchey A, Remington L, Ruley HE, Fisher DE, Housman DE, Jacks T. (1994). “p53 status and the efficacy of cancer therapy in vivo.” Science 266(5186): 807-10. Lowe SW, Ruley HE, Jacks T, and Housman DE. (1993). “p53mediated apoptosis modulates the cytotoxicity of anticancer agents.” Cell 74: 957-967. McPake CR, Shetty S, Kitchingman GR, Harris LC.. (1999). “Wild-Type p53 Induction Mediated by Replication-deficient Adenoviral Vectors.” Cancer Res 59(17): 4247-907. Miyake H, Hara I, Gohji K, Yamanaka K, Arakawa S, Kamidono S. (1998). “Enhancement of chemosensitivity in human bladder cancer cells by adenoviral-mediated p53 gene transfer.” Anticancer Res 18(4C): 3087-92. Miyake H, Hara I, Hara S, Arakawa S, Kamidono S. (2000). “Synergistic chemosensitization and inhibition of tumor growth and metastasis by adenovirus-mediated P53 gene transfer in human bladder cancer model.” Urology 56(2): 332-6. Nguyen DM, Spitz FR, Yen N, Cristiano RJ, Roth JA (1996). “Gene therapy for lung cancer: enhancement of tumor suppression by a combination of sequential systemic cisplatin and adenovirus-mediated p53 transfer.” J Thorac Cardiovasc Surg 112: 1372-1376. O'Connor PM, Jackman J, Bae I, Myers TG, Fan S, Mutoh M, Scudiero DA, Monks A, Sausville EA, Weinstein JN, Friend S, Fornace AJ Jr, Kohn KW. (1997). “Characterization of the p53 tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory potency of 123 anticancer agents.” Cancer Res 57(19): 4285-300. Oren, M. (1992). “p53: the ultimate tumor suppressor gene?” Faseb J 6(13): 3169-76. Osaki S, Nakanishi Y, Takayama K, Pei XH, Ueno H, Hara N. (2000). “Alteration of drug chemosensitivity caused by the adenovirus-mediated transfer of the wild-type p53 gene in human lung cancer cells.” Cancer Gene Ther 7(2): 300-7. Parker LP, Wolf JK, Price JE. (2000). “Adenoviral-mediated gene therapy with Ad5CMVp53 and Ad5CMVp21 in combination with standard therapies in human breast cancer cell lines.” Ann Clin Lab Sci 30(4): 395-405. Peller, S. (1998). “Clinical implications of p53: effect on prognosis, tumor progression and chemotherapy response.” Semin. Cancer Biol. 8: 379-387. Perdomo JA, Naomoto Y, Haisa M, Fujiwara T, Hamada M, Yasuoka Y, Tanaka N. (1998). “In vivo influence of p53 status on proliferation and chemoradiosensitivity in nonsmall-cell lung cancer.” J Cancer Res Clin Oncol 124(1): 10-8. Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B. (1997). “A model for p53-induced apoptosis.” Nature 389: 300. Righetti SC, Della Torre G, Pilotti S, Menard S, Ottone F, Colnaghi MI, Pierotti MA, Lavarino C, Cornarotti M, Oriana S, Bohm S, Bresciani GL, Spatti G, Zunino F. (1996). “A comparative study of p53 gene mutations, protein accumulation, and response to cisplatin-based chemotherapy in advanced ovarian carcinoma.” Cancer Res 56(4): 689-93. Roth, J. A. (1996). “Modification of tumor-suppressor-gene expression and induction of apoptosis in non-small-cell lung cancer (NSCLC) with an adenovirus vector expressing wildtype p53 and cisplatin.” Hum Gene Ther 7: 1013-1030. Roth JA, Nguyen D, Lawrence DD, Kemp BL, Carrasco CH, Ferson DZ, Hong WK, Komaki R, Lee JJ, Nesbitt JC, Pisters KM, Putnam JB, Schea R, Shin DM, Walsh GL, Dolormente

MM, Han CI, Martin FD, Yen N, Xu K, Stephens LC, McDonnell TJ, Mukhopadhyay T, Cai D. (1996). “Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer.” Nature Medicine 2(9): 985991. Santoso JT, Tang DC, Lane SB, Hung J, Reed DJ, Muller CY, Carbone DP, Lucci JA 3rd, Miller DS, Mathis JM. (1995). “Adenovirus-based p53 gene therapy in ovarian cancer.” Gynecol Oncol 59(2): 171-8. Schornagel JH, Verweij J, de Mulder PH, Cognetti F, Vermorken JB, Cappelaere P, Armand JP, Wildiers J, de Graeff A, Clavel M, et al, (1995). “Randomized phase III trial of adatrexate versus methotrexate in patients with metastatic and/or recurrent squamous cell carcinoma of the head and neck: A European Organization for Research and Treatment of Cancer Head and Neck Cancer Cooperative Group study.” JCO 13: 1649-1655. Schuler M, Herrmann R, De Greve JL, Stewart AK, Gatzemeier U, Stewart DJ et al (2001). “Adenovirus-Mediated WildType p53 Gene Transfer in Patients Receiving Chemotherapy for Advanced Non-Small-Cell Lung Cancer: Results of a Multicenter Phase II Study.” J Clin Oncol 19(6): 1750-1758. Selter, H. and M. Montenarh (1994). “The emerging picture of p53.” Int J Biochem 26: 154-154. Shahin MS, Hughes JH, Sood AK, Buller RE. (2000). “The prognostic significance of p53 tumor suppressor gene alterations in ovarian carcinoma.” Cancer 89(9): 2006-17. Sherr, C. J. (1994). “G1 phase progression: cycling on cue.” Cell 79: 55-62. Stal, O. (1995). “p53 expression and the result of adjuvant therapy of breast cancer.” Acta Oncologica 34: 767-770. Sugrue MM, Shin DY, Lee SW, Aaronson SA. (1997). “Wildtype p53 triggers a rapid senescence program in human tumor cells lacking functional p53.” Proc Natl Acad Sci U S A 94(18): 9648-53. Swisher SG, Roth JA, Nemunaitis J, Lawrence DD, Kemp BL, Carrasco CH, Connors DG, El-Naggar AK, Fossella F, Glisson BS, Hong WK, Khuri FR, Kurie JM, Lee JJ, Lee JS, Mack M, Merritt JA, Nguyen DM, Nesbitt JC, Perez-Soler R, Pisters KM, Putnam JB Jr, Richli WR, Savin M, Waugh MK, et al, (1999). “Adenovirus mediated p53 gene tranfer in advanced non-small-cell lung cancer.” J Natl Cancer Inst 91: 763-771. Trepel M, Groscurth P, Malipiero U, Gulbins E, Dichgans J, Weller M. (1998). “Chemosensitivity of human malignant glioma: modulation by p53 gene transfer.” J Neurooncol 39(1): 19-32. Wolf JK, Mills GB, Bazzet L, Bast RC Jr, Roth JA, Gershenson DM. (1999). “Adenovirus-mediated p53 growth inhibition of ovarian cancer cells is independent of endogenous p53 status.” Gynecol Oncol 75(2): 261-6. von Gruenigen VE, Santoso JT, Coleman RL, Muller CY, Miller DS, Mathis JM. (1998). “In vivo studies of adenovirus-based p53 gene therapy for ovarian cancer.” Gynecol Oncol 69(3): 197-204. Xu GW, Sun ZT, Forrester K, Wang XW, Coursen J, Harris CC. (1996). “Tissue-specific growth suppression and chemosensitivity promotion in human hepatocellular carcinoma cells by retroviral-mediated transfer of the wildtype p53 gene.” Hepatology 24(5): 1264-8. Yang B, Eshleman JR, Berger NA, Markowitz SD. (1996). “Wild-type p53 protein potentiates cytotoxicity of

Gene Therapy and Molecular Biology Vol 7, page 35 therapeutic agents in human colon cancer cells.” Clin Cancer Res 1996: 1649-1657. Knudson CM, Tung KSK, Tourtellotte WG, Brown, GAJ, Korsmeyer SJ. (1997). “Bax suppresses tumorigenesis and stimulates apoptosis in vivo.” Nature 385(6617): 637-40.

Yver, A., J. J. Nemunaitis, et al, (2001). “Does detection of circulating ONYX-015 genome by polymerase chain reaction indicate vector replication.” JCO 19(12): 3155-3157.

Jekunen et al: Strategy of sensitizing tumor cells with adenovirus-p53 transfection

Gene Therapy and Molecular Biology Vol 7, page 37 Gene Ther Mol Biol Vol 7, 37-42, 2003

Antigenicity and immunogenicity of HIV envelope gene expressed in baculovirus expression system Research Article

Alka Arora1, Pradeep Seth2* 1 2

Post Doctoral Fellow, Department of Medical Genetics and Microbiology, University of Toronto, Canada. Professor and Head Department of Microbiology, All India Institute of Medical Sciences, India.

__________________________________________________________________________________ *Correspondence: Dr. Pradeep Seth, Professor and Head, Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India -110029.Tel: 91-11-652 6814; Fax: 91-11-686 2663; E mail: pseth@aiims.aiims.ac.in Received: 28 December 2002; Accepted: 5 February 2003; electronically published: July 2003

Summary Human immunodeficiency virus type I (HIV-1) envelope gene was expressed in Spodoptera frugiperda (Sf21) cells. DNA constructs encoding env-tat-rev genes were cloned into the baculovirus expression vector pBacPAK9. Recombinant baculovirus was prepared by cotransfection with linearized wild type virus DNA. Western blotting of cell extracts containing recombinant HIV-1 proteins demonstrated expression of HIV-1 gp160 and its complete cleavage products gp120 and gp41. A time course experiment suggested that the maximum expression was observed at 48-hrs post infection. In order to measure the biological activity recombinant HIV envelope proteins were used for lymphocyte proliferation assay. The results demonstrated that recombinant gp160 and its cleavage products were antigenically and functionally authentic. tend to decrease with progression of clinical symptoms (Lange et al, 1986; Goudsmit et al, 1987). Recombinant antigen based EIAs have been shown to be more sensitive, especially in detecting early seroconverters and specific than peptide or virus lysate based EIAs (Johnson 1992; Galli et al, 1996). The main objective of this study was to obtain large quantities of purified recombinant protein, suitable to be used as an immunogen and for development of HIV-1 detection kit. We used Baculovirus expression vector system for expressing HIV-1 Gp160 as this system results in efficient processing of the protein, posttranslational modifications and is known to give high yields of expressed protein.

I. Introduction HIV genome, like other retroviruses encode for Gag, Pol and Env. In addition, it also encodes for 6 regulatory and accessory proteins Tat, Rev, Nef, Vif, Vpr and Vpu. The major structural protein encoded by env gene of HIV1 consists of a protein of 850-880 amino acids. Extensive glycosylation of this precursor protein results in the production of Gp160 monomers, which then assemble into oligomers for transport from ER to the plasma membrane (Earl et al, 1991). During transport from Golgi, intracellular cleavage of Gp160 yields an outer envelope glycoprotein Gp120 and trans-membrane glycoprotein Gp41 (Kozarsky et al, 1989). Specifically, the HIV viral envelope protein Gp120 is important for virus-receptor interaction and virus entry (Kowalski et al, 1987, Hill et al, 1997). Gp41 is known to play a central role in the envelope glycoprotein oligomerization and fusion function (Poumbourios et al, 1997). HIV infection results in the production of HIV specific antibodies, therefore detection of these antibodies by ELISA and Western blot assay remains the basis of blood donor and patient screening. Serum specimen from HIV infected people regardless of their clinical stage react efficiently with precursor glycoprotein Gp160 or its cleavage product Gp120 and Gp41 (Lange et al, 1986; Goudsmit et al, 1987). Antibodies to gag protein p24 are the earliest protein detectable by Western blot after infection, however, these

II. Materials and Methods A. Plasmids, cells, reagents and peptides pCR-Script SK (+) cloning vector was purchased from Stratagene, LaJolla, CA, USA. pBRU plasmid containing complete genome of BRU strain of HIV-1 cloned in pUC18 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, Bethesda, MD, USA. BacPAK, Baculovirus expression system was purchased from Clontech (BD Biosciences Clontech, Palo Alto, CA). Plasmids were grown in DH5! strains of Escherichia coli (Life Technologies, Gaithesburg, MD, USA), and purified using Wizard miniprep columns (Promega Corp, Madison, WI). TNMFH media for insect cell culture was obtained from HyClone (Genetix, New Delhi, India). TNM-FH medium contains Grace's

Arora and Seth: Antigenicity and immunogenicity of HIV envelope gene medium, lactalbumin hydrolysate and yeast extract. Sf 21 cells were cultured at 27oC in TNM-FH medium supplemented with 10% FBS (TNM-FH/FBS). Vaccinia expressed recombinant gp120 and gp160 (vPE8 and vPE16) were obtained through NIH AIDS Research and Reference Reagent Program.

20µl of 10M-ammonium acetate. Samples were then spotted on to the nitrocellulose membrane by loading on the wells of the dot blot manifold apparatus (Bio Rad Laboratory, Richmond, CA). Vacuum suction was applied to drain off the entire solution. Membrane was dried at room temperature for 5-10 min and then baked for 2 hrs at 80oC. Hybridization was performed using !32P-dCTP labeled envelope probe prepared by random primer labeling using Klenow fragment of DNA polymerase 1 (Amersham Biosciences, Piscataway, NJ). The membrane was then washed and exposed to a Kodak-X film overnight at -70oC.

B. DNA constructs 3kb env-tat-rev gene segment (nt 5352- nt 8354) of HIV-1 subtype B strain, BRU, was PCR amplified using primers API (5352-5390) TTATTCTAGAGAGAAGAGCAAGAAATGGA TCCAGTAGAT and APII (8316-8354) TTTTTGAGCTCTTGCCACCCATTTTAAAGTAAAGACCTT and cloned into pCR-Script (SK+) cloning vector to produce pSBRU-TRE as described earlier (Arora and Seth, 2001). The 3kb HIV-1 env-tat-rev gene segment was released by restriction digestion of pSBRU-TRE with Xba I, Not I and Bgl I. The env, tat and rev gene fragment was then purified from low melting point agarose gel and subcloned into baculovirus transfer vector, pBacPAK9 predigested with Xba I and Not I to generate pBacBRU-TRE. Recombinant clone was screened by colony hybridization followed by restriction enzyme analysis. pCIBRUTRE, mammalian expression vector expressing 3kb HIV-1 envtat-rev gene under the control of Immediate-Early Promoter/Enhancer of CMV, used in this study for immunizing Balb/c mice has been described earlier (Arora and Seth, 2001).

F. In vitro expression A time course experiment was performed to examine the expression of HIV-1 env gene in Sf 21 cells infected with the recombinant virus. Cells were harvested at various time intervals post infection. SDS PAGE, immunofluorescence and Western Blot analysis of cell lysate were conducted to study expression of proteins. SDS-PAGE was performed according to Laemmli. For Western Blot analysis proteins were resolved by SDS-PAGE and transferred onto a nitrocellulose membrane using Trans-blot SD semi-dry electrophoretic transfer Cell (Bio Rad Laboratories) The membrane was treated with non-fat powdered milk in TTBS (Tween 20- Tris buffer Saline) for 1 hr at room temp. and reacted with HIV-1 positive human polyclonal serum (at a dilution of 1:200) in TBS for 1h at room temperature. After washing thrice with TTBS, the membrane was incubated at room temperature for 1 hr. with anti-human IgG conjugated with alkaline phosphatase (1:10,000). Membrane was then washed thrice with TTBS and incubated in the substrate solution (Sigma fast BCIP/NBT tablet dissolved in 10ml of deionized water, Sigma Chemicals Co., St. Louis). For Immunofluorescence, P4 (recombinant baculovirus) infected cells, uninfected cells (control) and AcNPv (wild type virus) infected cells were harvested at different time points and washed thrice with PBS. 1x104 cells were spotted onto the wells of a teflon-coated slide and fixed with acetone: methanol (1:1) at -20oC for 30 min. For staining, cells were allowed to react with HIV-1 positive human polyclonal serum (1:50) for 1h at 37oC. Cells were then washed with PBS and incubated with FITC conjugated anti-human IgG (Sigma) and incubated for 1hr at 37oC. Thereafter, the cells were washed and mounted with glycerol buffer and visualized under fluorescent microscope.

C. Generating a recombinant virus Recombinant virus was prepared as per manufacturer's instructions. Briefly, 35mm tissue culture dishes were seeded with 1x106 Spodoptera frugiperda cells (Sf21) (Vaughn et al, 1977) in 1.5 ml of complete TNM-FH/FBS medium and incubated overnight at 27oC in a humid chamber. 500ng of plasmid pBacBRU-TRE DNA, along with Bsu 361 digested BacPAK6 viral DNA was mixed with 5µg of lipofectin and incubated at room temperature for 15 min. Culture medium in the tissue culture dishes containing Sf21 cells was replaced with 1.5 ml of serum free TNM-FH. Lipofectin-DNA complex was then gently added to Sf21 cells. Plates were incubated at 27oC for 5 hrs. Thereafter, serum free TNM-FH medium was replaced with TNM-FH/FBS medium and the plates were returned for incubation at 27oC for 4 days.

G. T cell proliferation assay

D. Isolation of recombinant virus

H thymidine uptake assay was used to measure the proliferation of splenocytes after antigenic stimulation. Balb/c mice were immunized intramuscularly with pCIBRU-TRE or pCI (control vector) DNA as described earlier (Arora and Seth, 2001). Six groups of Balb/c mice were taken (each group comprising 5 mice) (Table 1). In-group D3 three doses of 100 µg DNA each were given at bi-weekly intervals. In D0P2 group animals were immunized with 2 doses of P4 with no DNA priming. In-group D3P2 animals were immunized with 3 doses of pCIBRU-TRE DNA followed by 2 doses of P4. Group D3V2 consisted of mice immunized with 3 doses of pCIBRU-TRE followed by 2 doses of recombinant vaccinia virus expressed gp120 and gp160 (vPE8 and vPE16). D0V2 group consisted of mice immunized with 2 doses of vPE8 and vPE16 with no priming with DNA construct and control group. Stimulating antigens included vaccinia expressed recombinant gp160/gp120 (vPE16/ vPE8) and baculovirus expressed gp160 (P4). Splenocytes from various groups of mice were harvested and resuspended at a concentration of 2x106 cells/ml in RPMI 1640 medium supplemented with 10% FCS. Cells were stimulated in

Plaque assay was performed using co-transfection supernatant to generate a pure clone of recombinant virus. 1x106 Sf21 cells were seeded in 35mm tissue culture dishes and incubated overnight at 27oC. These cells were then infected with 100µl of neat or 10-1 dilution of co-transfection supernatant. One hour later, the virus inoculum was removed and infected cells were overlaid with 1.5ml of agarose (1.5% in TNM-FH/FBS). After agarose was set 1.5 ml of TNM-FH/FBS medium was added to each dish and incubated for 4 days at 27oC. Plaques were stained with .03% of neutral red solution. 4 plaques were picked up and transferred into an eppendorf tube containing 500µl of TNM-FH/FBS and stored at 4oC overnight.

E. Virus propagation and evaluation The plaque picks were used as a source of virus to infect cells in a 96 well plate. Infections were performed in duplicate. Cells were harvested 4 days following infection and cell lysate was used to perform dot blot analysis to detect the recombinant virus. Each sample was suspended in 200µl of 0.5N NaOH and

Gene Therapy and Molecular Biology Vol 7, page 39 used as control to study the non due to wild type vaccinia/vero baculovirus/Sf21 cell protein in index was calculated by

triplicate. Five Âľg/ml of vPE16/vPE8 infected vero cell lysates/ P4 infected Sf21 cell lysates was used in cell proliferation assay. Lysates of wild type vaccinia virus (WR) infected Vero cells/ wild type baculovirus (AcNPv) infected Sf21 cell lysate was

SI =

specific 3H-thymidine uptake cell proteins or wild type the cell lysates. Stimulation the following formula.

Mean cpm of 3H thymidine incorporated in the presence of stimulating antigen (vPE 16, vPES or P4) Mean cpm of 3H thymidine incorporated in wild type virus (VacWR or AcNPv) control

allows its insertion into the genome of the wild type virus. The BacPAK6 DNA is missing an essential portion of the baculovirus genome, ORF1629, that is essential for viral replication (Possee et al, 1991) When the DNA recombines with the vector (the transfer vector carries the missing ORF1629 sequence), the essential element is restored and the target gene is transferred to the baculovirus genome. Recombinant viruses were collected and selected by plaque purification. Recombinant phenotype of the plaques is verified by Dot-Blot analysis. Two of the plaques were found to be positive by Dot-Blot analysis and were termed as P4 and P5 (Figure 3). Plaque P4 gave the stronger signal and was therefore amplified and used for further infections.

II. Results A. Generating a Recombinant Baculovirus: Complete HIV-1 envelope glycoprotein along with the regulatory protein Tat and Rev were PCR amplified from subtype B, BRU strain of HIV-1 and cloned into pBacPAK9, baculovirus transfer vector, downstream to the baculovirus polyhedrin gene promoter (Figure 1). Recombinant baculovirus transfer vector was screened by colony hybridization followed by restriction enzyme analysis and was termed as pBacBRU-TRE (Figure 2). Following co-transfection, recombinant baculovirus was formed by the homologous recombination between pBacBRU-TRE and Bsu361 digested BacPAK6 viral DNA in the region flanking the chimeric gene, which

Figure 2 a) Autoradiograph showing recombinant colonies as detected by colony hybridization, b) Restriction enzyme analysis of the recombinant plasmid pBacBRU-TRE with different enzymes. Lanes M: Lambda DNA digested with Hind III enzyme. Positions of the molecular weight markers are indicated, 1: uncut; 2: pBacBRU-TRE digested with Bam H1; 3: pBacBRU-TRE digested with Hind III; 4: pBacBRU-TRE digested with Pvu II

Figure 1. a) pBacPAK9 baculovirus transfer vector, b) Recombinant plasmid pBacBRU-TRE. HIV-1 env, tat and rev gene released on digestion of pSBRU-TRE was gel purified and subcloned into baculovirus transfer vector pBacPAK9 predigested with restriction enzymes Xba 1 and Not 1.

Arora and Seth: Antigenicity and immunogenicity of HIV envelope gene

Figure 3. Autoradiograph showing dot blot analysis of cell lysates from plaque picks infected Sf21 cells. 2 plaques labeled as P4 and P5 were found to be positive. Cells infected with wild type baculovirus AcNPv served as the negative control. pBRU plasmid DNA served as the positive control.

Figure 4. The photograph showing Immunofluorescence microscopy of the recombinant baculovirus infected Sf21 cells at 48h-post infection. HIV-1 positive human polyclonal serum served as the source of primary antibody.

B. Expression of HIV-1 Envelope glycoprotein by Recombinant Baculovirus Expression of gp160 in Sf21 cells was examined by indirect immunofluorescence and western blot analysis of infected cells using HIV-1 positive human polyclonal sera. A 3+ fluorescence was observed at 48-hrs post infection on a scale of 0 to 4+ that is from no fluorescence to intense fluorescence (Figure 4). These results were supported by western blot analysis of the infected cells at 48hrs-post infection. Gp160 and its cleavage products, Gp120 and Gp41, could be detected after immunostaining. Since the total carbohydrate load added to the insect cell expressed glycoprotein is marginally less than that added during secretion from a mammalian cell, the baculovirus expressed glycoprotein are correspondingly smaller (105 kDa) than their mammalian counterparts (120 kDa) No corresponding protein bands were detected on from wild type baculovirus (AcNPv) infected cells and uninfected cells (Figure 5).

Figure 5. Western blot analysis of recombinant baculovirus expressed gp160. Lanes M: protein high range molecular weight marker; 1: uninfected cell lysate; 2: cell lysate from AcNPv infected cells; 3 & 4: cell lysate from recombinant baculovirus infected cells.

C. Lymphocyte Proliferation Assay In vitro T cell proliferative activity of splenocytes from animals immunized with DNA vaccine pCIBRU-TRE alone (group D3), boosted with P4 or vPE8/vPE16 (groups D3P2, D3V2) or P4 and vPE8/vPE16 alone (groups D0P2, D0V2) was studied. (Table 1). Splenocytes from all the animal groups showed positive proliferative response on in vitro stimulation (Figure 6). Splenocytes from group D0P2 mice demonstrated proliferation in response to P4 cell lysate (SI-8.16), as well as to vPE8 and vPE16 antigens (SI of 4 and 5.6). Splenocytes from DNA vaccine immunized mice group D3 and D3P2 proliferated with SI of 8.8 on stimulation with vPE8 and with SI of 3.8 and 4.4 respectively on stimulation with P4. Splenocytes from mice immunized with 2 doses of vaccinia expressed recombinant Gp120/Gp160 with no DNA priming (Group D0V2) showed better proliferation with vPE8, as compared with vPE16 and P4. However, splenocytes from mice immunized with 3 doses of DNA followed by 2 doses of vaccinia expressed recombinant Gp120/Gp160 (Group D3V2) gave almost equal proliferation with P4, vPE8 and vPE16 respectively (Figure 6).

Figure 6. In vitro T cell proliferative response to P4, vPE8 & vPE16 (recombinant baculovirus expressed gp160) of splenocytes from Balb/c mice immunized with pCIBRU-TRE (3 doses at biweekly intervals) and boosted with 2 doses of either recombinant baculovirus (P4) or recombinant vaccinia virus (vPE8 & vPE16). These groups of mice were marked as D3P2 or D3V2 respectively. Animals from groups D0P2 and D0V2 were injected only with recombinant baculovirus or recombinant vaccinia virus (no DNA priming).

Gene Therapy and Molecular Biology Vol 7, page 41 Table 1. Different groups of mice primed with pCIBRUTRE DNA and boosted with baculovirus expressed (P4) or vaccinia expressed (vPE8 and vPE16) recombinant gp160. Group

pCIBRU-TRE

3 doses

D0P2 D3P2

vPE8 and vPE16

Acknowledgments 2 doses

3 doses

D0V2 D3V2

baculovirus expressed envelope protein was also demonstrated by lymphocyte proliferation assays. Largescale protein purification is being pursued for further studies.

The Department of Biotechnology, Ministry of Science and Technology, Government of India has provided financial support for this research. Ms Alka Arora received Research Fellowship from CSIR during this study.

2 doses 2 doses

3 doses

2 doses

IV. Discussion

References

The main objective of this study was to prepare large amounts of HIV-1 envelope protein, which may be used as a source of antigen for studying immune response against HIV-1. HIV-1 gp160 with its signal sequence along with the regulatory genes tat and rev was used to produce recombinant baculovirus (Malim et al, 1989; Ruben et al, 1989 Rosen and Pavlakis; 1990, Roy et al, 1990). This system has several advantages over other systems including high level of protein production and posttranslational modification, which cannot be achieved in bacterial system (Luckow and Summers 1988, 1989). We observed poor expression of envelope proteins following infection of Sf21 cells as no protein was observed after SDS-PAGE of the P4 infected Sf21 cell lysate followed by coommassie blue staining. Several other studies have indicated that env protein is refractory to efficient recombinant expression (Lasky et al, 1986 Hu et al, 1987; Hu et al, 1987). Replacement of the signal sequence of the HIV-1 envelope protein with those of herpes simplex virus glycoprotein or human tPA results in efficient expression (Lasky et al, 1986; Berman et al, 1988). These studies therefore suggest that the signal sequence of HIV-1 envelope gene, which consists of 5 positively charged amino acids, may be responsible for the poor expression. Li et al, (1994), showed that substitution of the gp120 natural signal sequences with the signal sequences from honeybee mellitin or murine interleukin 3 promotes a high level of expression of a glycosylated form of gp120 and efficient secretion. These heterologous signal sequences contain one (mellitin) or no (IL-3) positively charged amino acids. These workers also demonstrated that on stepwise substitution of positively charged amino acids with neutral amino acids resulted in enhanced expression of HIV-1 gp120. Similarly, Golden et al, 1998, compared three different signal sequences [human tissue plasminogen activator (tPA), human placental alkaline phosphatase (pap), or baculovirus envelope glycoprotein (gp67)] and found that the tPA leader yielded the highest level of secreted protein, followed by the gp67 and pap sequences. In this study, however, HIV-1 gp160 and its complete cleavage products were observed on Western Blot analysis using HIV-1 positive human polyclonal sera. Suggesting thereby that the envelope protein retained its antigenicity and may be used as a source of antigen for Western Blot analysis. Immunogenicity as well as antigenicity of this

Arora A and Seth P (2001). Immunization with HIV-1 Subtype B gp160-DNA Induces Specific as well as cross-reactive Immune Responses in Mice. Indian J Med Res 114, 1-9. Berman PW, Nunes WM and Haffar OK (1988) Expression of membrane-associated and secreted variants of gp160 of human immunodeficiency virus type 1 in vitro and in continuous cell lines. J Virol 62, 3135-42. Earl PL, Moss B and Doms RW (1991) Folding, interaction with GRP78-BiP, assembly, and transport of the human immunodeficiency virus type 1 envelope protein. J Virol 65, 2047-55. Galli RA, Castriciano S, Fearon M, Major C, Choi KW, Mahony J and Chernesky M (1996) Performance Characteristics of Recombinant Enzyme Immunoassay To Detect Antibodies to Human Immunodeficiency Virus Type 1 (HIV-1) and HIV-2 and To Measure Early Antibody Responses in Seroconverting Patients. J Clin Microbiol 34, 999â&#x20AC;&#x201C;1002. Golden A, Austen DA, van Schravendijk MR, Sullivan BJ, Kawasaki ES, Osburne MS (1998) Effect of promoters and signal sequences on the production of secreted HIV-1 gp120 protein in the baculovirus system. Protein Expr Purif 14, 812. Goudsmit J, Lange JMA, Paul DA, Dawson GJ (1987) Antigenemia and antibody titers to core and envelope antigens in AIDS, AIDS-related complex, and subclinical human immunodeficiency virus infection. J Infect Dis 155, 558-60. Hill CM, Deng H, Unutmaz D, Kewalramani VN, Bastiani L, Gorny MK, Zolla-Pazner S, Littman DR (1997) Envelope glycoproteins from human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus can use human CCR5 as a co-receptor for viral entry and make direct CD4dependent interactions with this chemokine receptor. J Virol 71, 6296-304. Hu SI, Kosowski SG and Schaaf KF (1987) Expression of envelope glycoproteins of human immunodeficiency virus by an insect virus vector. J Virol 61, 3617-20. Johnson JE (1992) Detection of human immunodeficiency virus type 1 antibody by using commercially available whole-cell viral lysate, synthetic peptide, and recombinant protein enzyme immunoassay systems. J Clin Microbiol 30, 216â&#x20AC;&#x201C;218. Kozarsky K, Penman M, Basiripour L, Haseltine W, Sodroski J and Krieger M (1989) Glycosylation and processing of the human immunodeficiency virus type 1 envelope protein. J Acquir Immune Defic Syndr 2, 163-9. Kowalski M, Potz J, Basiripour L, Dorfman T, Goh WC, Terwilliger E, Dayton A, Rosen C, Haseltine W, Sodroski J

Arora and Seth: Antigenicity and immunogenicity of HIV envelope gene (1987) Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science 237, 1351-5. Lange JM, Paul DA, Huisman HG, de Wolf F, van den Berg H, Coutinho RA, Danner SA, van der Noordaa J, Goudsmit J (1986) Persistent HIV antigenemia and decline of HIV core antibodies associated with transition to AIDS. Brit Med J 293, 1459-62. Lasky LA, Groopman JE, Fennie CW, Benz PM, Capon DJ, Dowbenko DJ, Nakamura GR, Nunes WM, Renz ME, Berman PW (1986) Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein. Science 233, 209-12. Li Y, Luo L, Thomas DY, Kang CY (1994) Control of expression, glycosylation, and secretion of HIV-1 gp120 by homologous and heterologous signal sequences Virology 204, 266-78. Luckow VA and Summers MD (1988) Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors. Virology 167, 56-71 Luckow VA and Summers MD (1989) High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors. Virology 170, 31-9. Malim MH, Hauber J, Le SY, Maizel JV and Cullen BR (1989) The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 338, 254-257. Possee RD, Sun TP, Howard SC, Ayres MD, Hill-Perkins M, Gearing KL (1991) Nucleotide sequence of the Autographa californica nuclear polyhedrosis 9.4 kbp EcoRI-I and -R (polyhedrin gene) region. Virology. 185, 229-41. Poumbourios P, Wilson KA, Center RJ, El Ahmar W and Kemp BE (1997) Human immunodeficiency virus type 1 envelope glycoprotein oligomerization requires the gp41 amphipathic

alpha-helical/leucine zipper-like sequence. J Virol 71, 20419. Rosen CA and Pavlakis GN (1990) Tat and Rev: positive regulators of HIV gene expression. AIDS 4, A51 Roy S, Delling U, Chen CH, Rosen CA, Sonenberg N (1990) A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated trans-activation. Genes Dev 4, 1365-1373. Ruben S, Perkins A, Purcell R, Joung K, Sia R, Burghoff R, Haseltine WA, Rosen CA (1989) Structural and functional characterization of human immunodeficiency virus tat protein. J Virol 63, 1-8. Vaughn JL, Goodwin RH, Tompkins GJ, McCawley P (1977) The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae).In Vitro. 13, 213-7.

Pradeep Seth

Gene Therapy and Molecular Biology Vol 7, page 43 Gene Ther Mol Biol Vol 7, 43-59, 2003

Characterization of genes transcribed in an Ixodes scapularis cell line that were identified by expression library immunization and analysis of expressed sequence tags Research Article

Consuelo Almazán, Katherine M. Kocan, Douglas K. Bergman, Jose C. GarciaGarcia, Edmour F. Blouin and José de la Fuente* Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.

__________________________________________________________________________________ *Correspondence: José de la Fuente, Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078; Phone: (405) 744-0372; Fax: (405) 744-5275; e-mail: jose_delafuente@yahoo.com Key words: tick, vaccine, tick cell culture, cDNA library immunization, EST, expression library immunization Received: 23 May 2003; Accepted: 06 June 2003; electronically published: June 2003

Summary Expression library immunization (ELI) combined with analysis of expressed sequence tags (ESTs) were used to identify genes transcribed in a cell line (IDE8) that was originally derived from embryos of Ixodes scapularis. A cDNA expression library was constructed from the IDE8 cells and cDNA clones were screened by ELI. Mice injected with cDNA clones were then infested with I. scapularis larvae. cDNA clones affecting larval feeding or development were subjected to single pass 5’ sequence analysis and the non-redundant sequences were putatively identified by sequence identity using the protein Basic Local Alignment Search Tool (BLAST) algorithm. Sequences of the clones were grouped according to the predicted function of the encoded proteins. 351 cDNAs that affected larval feeding and/or development were identified, of which 316 cDNA clones contained non-redundant sequences and 101 produced a significant identity to sequences reported previously. Gene ontologies could be assigned to 87 clones. Vaccination of mice with plasmid DNA followed by tick infestation resulted in identification of cDNA clones that inhibited tick infestation or promoted tick feeding. cDNAs that inhibited tick infestation were identical to nucleotidase, heat shock proteins, beta-adaptin, chloride channel, ribosomal proteins, and proteins with unknown function. cDNA clones that promoted tick feeding were identical to beta-amyloid precursor, block of proliferation, mannose-binding lectin, RNA polymerase III, ATPases and a protein of unknown function. Herein, we describe the sequence analysis of I. scapularis ESTs selected by ELI that affected larval tick feeding and/or development. These proteins may be useful for incorporation into vaccine preparations designed to interrupt the life cycle of I. scapularis and/or interfere with transmission of pathogens. garinii, Rickettsia helvetica, R. japonica and R. australis, Babesia divergens, as well as tick-borne encephalitis (TBE) and Omsk Hemorrhagic fever viruses (EstradaPeña and Jongejan, 1999; Parola and Raoult, 2001). Throughout eastern and southeastern United States and Canada, I. scapularis (the black legged tick) is the main vector of B. burgdorferi sensu stricto and A. phagocytophilum (Estrada-Peña and Jongejan, 1999; Parola and Raoult, 2001). Control of tick infestations is difficult, particularly for multi-host ticks such as Ixodes spp. Presently, tick

I. Introduction Ticks are ectoparasites of wild and domestic animals and humans, and are considered to be the most important vector of pathogens in North America (Parola and Raoult, 2001). Ixodes spp. (Acari: Ixodidae) are distributed worldwide and are vectors of human pathogens, including Borrelia burgdorferi (Lyme disease), Anaplasma phagocytophilum (human granulocytic ehrlichiosis), Coxiella burnetti (Q fever), Francisella tularensis (tularemia), B. afzelii, B. lusitaniae, B. valaisiana and B.

Almazán et al: Expressed sequence tags in Ixodes scapularis control is effected by integrated pest management in which different control methods are adapted in a geographic area against one tick species with due consideration to their environmental effects. Recently, development of vaccines against one-host Boophilus spp. has provided new possibilities for identification of protective antigens for use in vaccines for control of tick infestations (Willadsen, 1997; Willadsen and Jongejan, 1999; de la Fuente et al, 1999, 2000a; de Vos et al, 2001). Control of ticks by vaccination would avoid environmental contamination and selection of drug resistant ticks that can result from repeated acaricide application (de la Fuente et al, 1998; Garcia-Garcia et al, 1999). Anti-tick vaccines also allow for inclusion of multiple antigens in order to target a broad range of tick species, as well as pathogenblocking antigens. Development of high throughput DNA sequencing technologies and bioinformatic tools facilitate assignment of provisional function to expressed sequence tags (ESTs; Boguski et al, 1993). This approach has resulted in valuable information for the study of biological systems and for the identification of potential vaccine candidates (Lizotte-Waniewski et al, 2000; Knox et al, 2001; Tarleton and Kissinger, 2001; Touloukian et al, 2001; Kessler et al, 2002). In ticks, construction of EST databases has been reported for B. microplus (Crampton et al, 1998), Amblyomma americanum (Hill and Gutierrez, 2000) and A. variegatum (Nene et al, 2002). The application of EST technology has been used for characterization of gene expression in salivary glands of I. scapularis (Valenzuela et al, 2002), I. ricinus (Valenzuela, 2002), A. americanum and Dermacentor andersoni (Bior et al, 2002), for identification of genes differentially expressed in D. variabilis ovaries in response to rickettsial infection (Mulenga et al, 2003) and in I. ricinus salivary glands in response to blood feeding (Leboulle et al, 2002). A new technique, expression library immunization (ELI), in combination with sequence analysis of ESTs, provides an alternative approach for identification of potential vaccine antigens that is based on rapid screening of the expressed genes without prior knowledge of the antigens encoded by the cDNAs. ELI was first reported for Mycoplasma pulmonis (Barry et al, 1995) and since then has been used for unicellular and multicellular pathogens and viruses (Manoutcharian et al, 1998; Alberti et al, 1998; Brayton et al, 1998; Melby et al, 2000; Smooker et al, 2000; Moore et al, 2001; Singh et al, 2002; Leclercq et al, 2003). Recently, we reported the first application of ELI to arthropods, specifically to I. scapularis (Almazán et al, 2003) in a mouse model system. A combination of cDNA ELI and EST analysis resulted in the selection of 351 cDNA clones affecting tick larval development (Almazán et al, 2003). After grouping the clones according to the putative function of predicted proteins, some cDNA pools resulted in the inhibition of tick infestation and others promoted tick feeding after ELI (Almazán et al, 2003). Herein we describe the sequence analysis and characterization of I. scapularis ESTs that were identified

by Almazán et al. (2003) using cDNA ELI and a mouse model for tick infestation.

II. Materials and methods A. Construction of the I. scapularis expression cDNA library The cDNA library was constructed from I. scapularis cultured embryonic IDE8 cells (Munderloh et al, 1994) as reported previously (Almazán et al, 2003). The expression library was constructed in the vector pEXP1 containing the strong human cytomegalovirus major immediate early promoter/enhancer (CMVIE) (Clontech, Palo Alto, CA). The cDNA library contained 4.4 x 106 independent clones and a titer of approximately 1010 cfu/ml with more than 93% of the clones with cDNA inserts. The average cDNA size was 1.7 kb (0.5-4.0 kb).

B. DNA vaccination and tick infestation Vaccinations with plasmid DNA and tick infestations were done as reported previously for the screening of the expression cDNA library by ELI using the mouse model of I. scapualris infestations (Almazán et al, 2003). Briefly, plasmid DNA was purified (Wizard SV 96 plasmid DNA purification system, Promega, Madison, WI) and used to inject CD-1 female mice, 56 weeks of age at the time of first vaccination. Mice were cared for in accordance with standards specified in the Guide for Care and Use of Laboratory Animals. Mice were injected using a 1 ml tuberculin syringe and a 27!G needle at days 0 and 14. Three to 6 mice per group were each immunized IM in the thigh with 1 µg total DNA/dose in 50 µl PBS. Control mice were injected with 1 µg vector DNA alone. Two weeks after the last immunization, mice were infested with 100 I. scapularis larvae per mouse. For tick infestations, mice were retrained in a small wire cage in a cardboard carton. One hundred larvae were counted and applied to the mice with a brush. Ticks were reared at the Oklahoma State University Tick Rearing Facility by feeding larvae on mice, nymphs on rabbits and adults on sheep. For these experiments, larvae were obtained from the eggs oviposited by sister females. Twelve hours after tick infestation, larvae in the bottom of the cage that did not attach were counted in order to calculate the number of attached larvae per mouse. Mice were then transferred to individual cages in which they were placed on an elevated 1/4” mesh wire platform over water (1/2” deep). Replete larvae dropping from each mouse were collected daily from the water and counted during 7 days. Time for larval development was evaluated from the day of tick infestation to the day in which the maximum number of replete larvae was collected. The inhibition of tick infestation (I) for each test group was calculated with respect to vector-immunized controls as [1-(RLn/RLc x RLic/RLin)] x 100, where RLn is the average number of replete larvae recovered per mouse for each test group, RLc is the average number of replete larvae recovered per mouse for control group, RLic is the average number of larvae attached per mouse for control group, and RLin is the average number of larvae attached per mouse for each test group. Engorged larvae were held in a 95% humidity chamber and allowed to molt. Molting of engorged larvae was evaluated 34 days after the last larval collection by visual examination of ticks under a dissecting light microscope. The inhibition of molting (M) for each test group was calculated with respect to controls as [1-(MLn/MLc x RLc/RLn)] x 100, where MLn is the average number of nymphs for each test group, MLc is the average number of nymphs for the control group, RLc is the average number of larvae recovered for the control group, and RLn is the average number of larvae recovered for each test group.

Gene Therapy and Molecular Biology Vol 7, page 45 constructed based on a sequence distance method utilizing the Neighbor Joining algorithm of Saitou and Nei (1987). BLAST (Altschul et al, 1990) was used to search the NCBI databases to identify previously reported sequences with identity to those that we sequenced. Gene ontology assignments were made according to Ashburner et al. (2000) for non-redundant EST sequence data with the help of GoFish v.1.0 (Berriz et al, 2003).

C. Plasmid DNA preparation and sequencing Bacterial colonies were inoculated in Luria-Bertani with 50 µg/ml ampicillin, grown for 16 hr in a 96-well plate and plasmid DNA purified (Wizard SV 96 plasmid DNA purification system, Promega, Madison, WI) and partially sequenced with a 5’ vectorspecific primer (5’-CGACTCACTATAGGGAG-3’) at the Core Sequencing Facility, Department of Biochemistry and Molecular Biology, Noble Research Center, Oklahoma State University, using ABI Prism dye terminator cycle sequencing protocols developed by Applied Biosystems (Perkin-Elmer Corp., Foster City, CA). In most cases a sequence larger than 700 nucleotides was obtained.

III. Results The screening of the I. scapularis expression cDNA library by ELI and EST analysis resulted in 351 cDNAs affecting larval development in the mouse model of tick infestation (Almazán et al, 2003). Of them, 316 cDNA clones contained non-redundant sequences and 101 (32%) produced a significant identity to previously reported sequences by BLAST analysis of NCBI nucleotide and protein databases (Table 1). Gene ontologies could be assigned to 87 clones (27.5% of non-redundant sequences and 86.1% of clones with identity to sequences reported previously) (Table 2).

D. Data analysis Nucleotide sequences were analyzed using the program AlignX (Vector NTI Suite V 5.5, InforMax, North Bethesda, MD). Multiple sequence alignment was performed using an engine based on the Clustal W algorithm (Thompson et al, 1994). Nucleotides were coded as unordered, discrete characters with five possible character-states; A, C, G, T, or N (missing) and gaps were coded as missing data. Phylogenetic trees were

Table 1. cDNA clones with identity to previously reported sequences. EST clone

Predicted protein

GenBank accession number

4A4

V-ATPase E subunit

CD052508

1C5

Na+/K+ ATPase, alpha subunit

CD052509

2A9

NADH dehydrogenase

CD052510

1D6

NADH dehydrogenase subunit 5 (nad5)

CD052511

1A4

Aldehyde dehydrogenase

CD052512

1C11

Translation initiation factor 5A (eIF5A)

CD052489

1E6

Translation initiation factor 5C (eIF-5C)

CD052490

2D2

Initiate factor 5 (if5)

CD052491

1C8

Virilizer (vir)

CD052513

1C10

Hsp70

CD052514

1A10

Elongation factor 2

CD052492

4F7

Elongation factor 1alpha

CD052493

3F6

Hsp60

CD052515

CD052494

1D1

Nucleotide binding protein 1 (Nubp1)

CD052516

1D8

Identity to D. melanogaster GH03607 full length cDNA coding for a putative membrane protein

CD052517

1F6

Ribosomal protein S4 (RpS4)

2B8

Ribosomal protein S11 (RpS11)

2F8

Laminin receptor 1 (ribosomal protein SA)

CD052496

2F10

Ribosomal protein L3 (RpL3)

3A10

Ribosomal protein L7A (RpL7A)

CD052497

1D11

Putative membrane protein

CD052518

3G9

Ribosomal protein S8 (RpS8)

CD052495

1E7

Sterol carrier protein

CD052519

3G10

Ribosomal protein L27A (RpL27A)

CD052498

1F3

Cyclin C (CycC)

CD052520

3D9

Alpha tubulin

CD052521

3C3

QM homolog (DQM) ribosomal protein

CD052499

2A7

Beta tubulin

CD052522

4D12

Proteasome/Signalosome subunit

CD052500

2A11

Notchless (Nle)

CD052523

4E7

Proteasome subunit

CD052501

2B2

Export factor binding protein 2 (Refbp2)

CD052524

4D11

Proteasome subunit

CD052502

2B7

G protein-coupled receptor

CD052525

3D10

Ribophorin I

CD052503

2B9

Ubiquitin-conjugating enzyme

CD052504

Succinate dehydrogenase B (SdhB)

CD052526

1B12 1D10

Ubiquitin

CD052505

2C12

V-ATPase D subunit Contains microsatellite sequence

CD052506

Beta-amyloid precursor protein (APP)

CD052527

1A9

2D1

CD052528

V-ATPase C subunit

CD052507

Fructose-1,6-bisphosphatase (fbp gene)

2D5

DNA repair protein Rad1 (Rad1)

CD052529

2D6

Identity to S. pombe dim1+, helicase protein 1

CD052530

1B2 EST clone

Predicted protein

GenBank accession number

Almazรกn et al: Expressed sequence tags in Ixodes scapularis 2E8

Esterase

CD052531

4G5

Disulfide isomerase

CD052563

2F9

Identity to AvGI TC255 (A. variegatum) & hypothetical protein FLJ12475 (H. sapiens)

CD052532

4G8

Fumarate hydratase

CD052564

4G10

Rab3D (member of the Ras superfamily of small GTPases)

CD052565

Transmembrane G-proteinresponsive adenylyl cyclase

CD052533

4G11

Chloride channel

CD052566

2G8

Lysyl-tRNA synthetase

CD052534

4H4

Solute carrier protein

CD052567

2H11

Sodium- and chloride-dependent taurine transporter

CD052535

1B7

Mitochondrion

1B8

Mitochondrion

3C12

RNA polymerase III

CD052536

2E9

Mitochondrion

CD052537

2G11

Mitochondrion

3C6

Mitochondrion

3G4

Mitochondrion

4A2

Mitochondrion

2F12

3E1

Beta-adaptin

3E2

Microtubule-associated protein, RP/EB family

CD052538

3E4

Myosin II regulatory light chain

CD052539

3E6

Unknown Zinc finger like protein

CD052540

3E10

Mannose binding lectin (rhea)

CD052541

3E12

Clathrin heavy chain (Chc)

CD052542

3F4

Identity to M. musculus adult male testis cDNA

CD052543

3F10

Identity to D. melanogaster Pelement somatic inhibitor (Psi)

3G11

4E9

Mitochondrion

2A6

Mitochondrion

4G7

NAD-dependent malate dehydrogenase

3D4

Cytochrome c oxidase I (COI)

1C2

Cytochrome c oxidase II (COII)

CD052544

4D2

Cytochrome c oxidase III (COIII)

Identity to D. melanogaster BM40 extracellular basement membrane protein

CD052545

1G4

Cytochrome b (cytb)

2G9

16S ribosomal RNA

4A8

Identity to D. melanogaster regulator of gene transcription (Chi)

CD052546

1F4

CD052568

4A10

Identity to D. melanogaster homeoprotein phtf

CD052547

Unknown Identity to I. scapularis clone AC22 microsatellite sequence (AF331735)

2C7

CD052569

4A12

Amino acid transporter system A (ATA2)

CD052548

Unknown Contains microsatellite sequence

3B6

Calmodulin

CD052549

4B7

Alpha-tubulin

CD052550

Unknown Contains a microsatellite sequence

CD052570

4B2 4C9

Identity to D. melanogaster transducin (G protein)-like enhancer of split 3, homolog of E(spl)

CD052551

4G12

Unknown Contains microsatellite sequence

CD052571

4H2

CD052572

4C11

Intracellular receptor of activated protein kinase C1 (Rack1)

CD052552

Unknown Contains microsatellite sequence

4D6

Identity to D. melanogaster CG10395 cDNA

CD052553

4D7

Identity to D. melanogaster LD23959 cDNA

CD052554

4E6

Identity to D. melanogaster CG13597 cDNA

CD052555

4D8

Identity to H. sapiens hypothetical protein FLJ10342

CD052556

4E1

Pre-mRNA splicing factor

CD052557

4E3

Receptor signaling protein serine/threonine kinase

CD052558

4F8

Nucleotidase

CD052559

4F1

Block of proliferation 1 (Bop1)

CD052560

4G1

Identity to H. sapiens hypothetical protein MGC2404

CD052561

4G2

LRP/alpha-2-macroglobulin receptor

CD052562

NR, Not reported to the EST database for being identical to mitochondrial sequences The majority of clones with gene ontology assigned corresponded to non-nuclear gene products involved in cell growth and maintenance, including genes with ligand binding, carrier or enzymatic activities (Table 2). Seventeen clones contained sequences corresponding to tick mitochondrion and were not submitted to the EST database. Other clones such as 2A9 and 1D6, although probably coding for mitochondrial proteins, were analyzed and submitted to the EST database. Interestingly, 11 clones encoded gene products localized in the cell nucleus (Table 2). The average G + C content of the EST dataset (47,503 bases excluding the poly-A tails with 171 (0.4%) undetermined nucleotide positions) was 54%, but some sequences, such as clone 2A9 which probably codes for a mitochondrial protein, had only a 25% G + C content. 46

Gene Therapy and Molecular Biology Vol 7, page 47 Some short ESTs in clones 1D1 and 2D5 contained a long stretch of T. Vaccination of mice with plasmid DNA followed by tick infestation resulted in some cDNA clones that had an inhibitory effect on tick infestations, while others appeared to promote tick feeding (Table 3 ). The cDNAs inhibiting tick infestation were identical to nucleotidase, heat shock proteins, beta-adaptin, chloride channel, ribosomal proteins and proteins with unknown function. cDNA clones identical to beta-amyloid precursor, block of proliferation, mannose-binding lectin, RNA polymerase III, ATPases and a protein of unknown function enhanced tick feeding. Further characterization of cDNAs that affected larval development (Table 3) was conducted for all clones except for 4D8, 4F8, 4D6 and 4E6, which produced high inhibition of tick infestation and are currently being studied separately as recombinant proteins expressed in Escherichia coli. The pool of heat shock proteins hsp70 and hsp60 cDNAs conferred partial protection against tick

infestations and did not affect molting (Table 3). The cDNA sequences for hsp70 and hsp60 in clones 1C10 and 3F6, respectively, were partial and contained the region coding for the C-terminal of the protein, and were highly identical to other hsp70 sequences (data not shown). The sequence of hsp70 contained a 3â&#x20AC;&#x2122; untranslated region (UTR) of 299 bp before the poly-A tail. The clone 3E1 contained a cDNA identical to the beta-adaptin that produced a 27% inhibition of tick infestation and a 5% inhibition of molting to the nymphal stage after vaccination and tick challenge (Table 3). The complete sequence was determined for the clone 3E1 (Figure 1A), and contained an insert of 1,942 bp encoding for a predicted protein of 191 amino acids. The sequence of this protein was shorter than that for other beta-adaptins (Figure 1B), suggesting that it could encode for a betaadaptin appendage or it may be a partial cDNA sequence because of a long 3â&#x20AC;&#x2122; UTR of 1,334 bp located before the poly-A tail.

Table 2. I. scapularis gene ontology assignments. Category

Number of clones

% of 87 clones with gene ontology assignments

% of 101 clones with identity to reported sequences

Cell

36.78

31.88

Cellular component Mitochondria

15.54

16.83

Cell membrane

16.09

13.86

Nucleus

12.64

10.89

Extracellular

2.30

1.98

Unlocalized

2.30

1.98

Unknown

10.34

8.91

Biological process Cell growth or maintenance

70.11

60.40

Physiological process

9.20

7.92

Developmental process

5.75

4.95

Cell communication

2.30

1.98

Unknown

12.64

10.89

Molecular function Ligand binding or carrier

34.48

29.70

Enzyme

33.33

28.71

Transporter

10.34

8.91

Chaperone

2.30

1.98

Structural molecule

8.05

6.93

Unknown

11.49

9.90

Gene ontology assignments were made according to Ashburner et al. (2000) for non-redundant EST sequence data with the help of GoFish v.1.0 (Berriz et al, 2003). The number of clone sequences falling into each category are listed and then calculated as a percent of clones for which gene ontology was assigned and the total number of clones for which identity was found to previously published sequences.

Almazán et al: Expressed sequence tags in Ixodes scapularis Table 3. Summary of results of DNA vaccination and challenge with I. scapularis larvae in the mouse model of tick infestations. EST cDNA clone

Predicted protein

Inhibition of tick infestation I (%)

Inhibition of molting M (%)

4D8

Identity to H. sapiens hypothetical protein FLJ10342 with unknown function

40 a

4F8

Nucleotidase

50 a

17 a

1C10 b

Hsp70

17 a

3F6

Hsp60

4D6

Identity to D. melanogaster CG10395 cDNA with unknown function

4E6

Identity to D. melanogaster CG13597 cDNA with unknown function

3E1

Beta-adaptin

4G11

Chloride channel

17 clones b

Ribosomal proteins

15 a

2C12

Beta-amyloid precursor protein (APP)

-8

c c

4F1

Block of proliferation Bop1

-39

3E10

Mannose binding lectin

-48 a, c

3C12

RNA polymerase III

-104

2F9 b

Identity to A. variegatum AvGI TC255 & Homo sapiens hypothetical protein FLJ12475 with unknown functions

1A9, 1B2, 4A4 b

ATPase

a, c

-57 a, c

ND ND ND ND

Data reported by Almazán et al. (2003). For all other experiments, mice were immunized with cDNA-containing expression plasmid DNA as described above. I and M were calculated as described in Materials and Methods section. ND, not determined. b Pooled together for vaccination experiments by ELI (Almazán et al, 2003) (1C10 and 3F6, cDNA pool “Heat shock”; 3C12 and 2F9, cDNA pool “Secreted protein”; ribosomal clones, cDNA pool “Ribosomal”; 1A9, 1B2 and 4A4, cDNA pool “ATPase”). c Resulted in enhanced tick feeding after mouse vaccination and tick challenge.

identical to fly and mosquito sequences (Figure 3). Vaccination with this cDNA resulted in 8% enhancement of larval feeding (Table 3). Vaccination with cDNA clone 4F1 resulted in enhanced larval feeding (Table 3). The complete sequence of clone 4F1 cDNA was determined and contained an insert of 2,475 bp with 30 bp and 66 bp of 5’ and 3’ UTR, respectively and a poly-A tail of 114 bases. An open reading frame of 2,265 bp encoded for a protein of 754 amino acids that was identical to mouse block of proliferation (Bop 1) (Figure 4). Similar proteins have been identified in other organisms including Drosophila melanogaster, Anopheles gambiae and humans (Figure 4), suggesting that this protein has been highly conserved during evolution. The clone 3E10 had a pronounced stimulatory effect on larval feeding (Table 3). This clone was completely sequenced and contained an insert of 1,848 bp with 50 bp and 279 bp of 5’ and 3’ UTR, respectively and a short poly-A tail of 24 bases. An open reading frame of 1,494 bp encoded for a protein of 497 amino acids that was identical to mannose-binding lectins found in many eukaryotes (Figure 5). A similar sequence was described in A. variegatum ESTs, which clustered together with the I. scapularis sequence (Figure 5).

The cDNA in clone 4G11 was identical to a chloride channel but it contained only a partial sequence (Figure 2A). This sequence protected against tick infestations and inhibited larval molting (Table 3). Chloride channels have been found in living organisms from bacteria to mammals, with some amino acid positions being conserved in all sequences (Figure 2A). As expected, phylogenetic analysis of chloride channel sequences demonstrated that the I. scapularis sequence comprised a sister group to other insect sequences that have been reported (Figure 2B). Vaccination with ribosomal sequences had some inhibitory effect on tick infestations but did not affect molting (Table 3). The pool of ribosomal cDNAs included EST sequences coding for cellular and mitochondrial ribosomal proteins and translation factors (Table 4), and these genes are highly conserved across species. However, proteins encoded by I. scapularis ESTs were 43% to 95% identical to arachnida or insect sequences and 36% to 85% identical to mouse sequences (Table 4). The cDNA in clone 2C12 that was found to be identical to the betaamyloid precursor protein (APP) contained a fragment encoding for the C-terminal of the protein (Figure 3), suggesting that it contains a partial cDNA with a long (1,400 bp) 3’ UTR. Nonetheless, the C-terminal sequence of the I. scapularis APP contained regions of amino acids 48

Gene Therapy and Molecular Biology Vol 7, page 49 A cgATGCAGGCGATGACGGGCTTTGCGGTGCAGTTCAACAAAAACAGTTTCGGGCTGACTCCAGCTCAGCCGCTGCAGTTGCAGATTCCCCT GCAGCCCAACTTCCCAGCTGATGCGAGCTTGCAGCTGGGAACCAACGGTCCCGTGCAGAAGATGGACCCCCTCACCAACCTTCAGGTGGCC ATCAAGAACAATGTGGACGTGTTCTACTTCAGCTGCCTGGTGCCCATGCACGTGCTGAGCACGGAGGACGGCCTGATGGACAAGCGGGTGT TCCTGGCCACCTGGAAAGACATCCCCGCCCAAAACGAGGTCCAGTACACCCTCGACAACGTCAACCTCACTGCAGACCAAGTTTCCCAGAA GCTGCAGAACAACAACATTTTCACGATAGCCAAGAGGAACGTGGACGGCCAGGACATGCTGTACCAGTCCCTGAAGCTCACCAACGGCATT TGGGTGTTGGCGGAGCTCAAGATACAGCCCGGCAATCCAAGGATCACGTTGTCTTTGAAGACAAGAGCACCTGAAGTGGCAGCAGGTGTAC AACAAACTTACGAACTCATTCTACACAGCTGAggctgctgtgaatgaaactcttctcccacccccttcttttgatggcagtcaatgtctcg tttcattttcttgttttcttttgcggcgtgctacggaacaaggtcctacattcccaagttatatggtgttgtcgcgtagggggcagagtgc cgctgagcccgcgacagccttgtttctgaggagagccgaacgcaccacttcgaaaaagaaaaagtgaaaacggaaaaatgaaaaattttcc agttgcttcaaattaacattcctcgtagtcagtctgtggccgttgagtttggtgtaaagaagaaaaaggtgtctcttttagtgaaaatggt tgctttttattggtatcccctatcacaccgagcacgaacataagaaatcctgacaaggattctcctttagttgtattatggtggctggagc acacgaggcacctgttgccaattcgacccagcaaatgcccaattctcaagatttgagttcattgaggttgttttgctcctccccccccacc ccccaactttgtcgttggattgtctaacagtgtaaatgggcgacgactcgttattctttttttcttcattctttctttttgttgtcacgcg ccccgggggacgcgacacaacttatgtgcataattgattttcacaggctgcgacgcagtctgtaaaagaaggggaagtgaaactctgctcc gccgctgctagtgtcatcacgggacgaccatcgcgttttctctgactatttaaacaaaactgcatagcttagggggcagtctgtgcaaagt ggaacaaccaaactgagccctgccctttcggtgtgtgtacaagcatctctgtgtaacatgaactactttacatgaactacattgcatgaac gggagaagtttagttgtttttttgttttttttttcaggtgactatgtcaacagattagaaccattttttggaacggctggaaagataaccg ctcattttgtttctactaaaagactacgaaaagtgttgactttttgcatcggtttggcaacgtttgtttggcatgcatgtagttgagcgta atggtatcacccctcgtaaacaataacagtgcaatggagcagtactgtagtgtccattaaagagcgagagtttggttaaaggttgttaatt gaggtccgtgttatcctttgagtaggagagcggcactttttgcaaatagcgctgctgggggcgtcatatctgccctccaaaacatgcacat tttaagtgtgaattgttgcggcggcttgtacaagtatgtgtgttatgtgtagaaaaagaactcttaattaaaatatttgtggccaaaacgt caaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

B M. musculus D. melanogaster H. sapiens I. scapularis Consensus

(747) (731) (68) (1) (748)

LQHMTDFAIQFNKNSFGVIPSTPLAIHTPLMPNQSIDVSLPLNTLGPVMK MQPMTNFAIQLNKNSFGLVPASPMQ-AAPLPPNQSIEVSMALGTNGPIQR LQHMTDFAIQFNKNSFGVIPSTPLAIHTPLMPNQSIDVSLPLNTLGPVMK MQAMTGFAVQFNKNSFGLTPAQPLQLQIPLQPNFPADASLQLGTNGPVQK LQHMTDFAIQFNKNSFGLIPATPLQIHTPLMPNQSIDVSLPLNTNGPVQK

M. musculus D. melanogaster H. sapiens I. scapularis

(797) (780) (118) (51)

MEPLNNLQVAVKNNIDVFYFSCLIPLNVLFVEDGKMERQVFLATWKDIPN MEPLNNLQVAVKNNIDIFYFACLVHGNVLFAEDGQLDKRVFLNTWKEIPA MEPLNNLQVAVKNNIDVFYFSCLIPLNVLFVEDGKMERQVFLATWKDIPN MDPLTNLQVAIKNNVDVFYFSCLVPMHVLSTEDGLMDKRVFLATWKDIPA

Consensus (798) MEPLNNLQVAVKNNIDVFYFSCLIPLNVLFVEDGKMDKRVFLATWKDIPN M. musculus D. melanogaster H. sapiens I. scapularis Consensus

(847) (830) (168) (101) (848)

ENELQFQIKECHLNADTVSSKLQNNNVYTIAKRNVEGQDMLYQSLKLTNG ANELQYTLSGVIGTTDGIASKMTTNNIFTIAKRNVEGQDMLYQSLKLTNN ENELQFQIKECHLNADTVSSKLQNNNVYTIAKRNVEGQDMLYQSLKLTNG QNEVQYTLDNVNLTADQVSQKLQNNNIFTIAKRNVDGQDMLYQSLKLTNG ENELQFTIKEVHLTADTVSSKLQNNNIFTIAKRNVEGQDMLYQSLKLTNG

M. musculus D. melanogaster H. sapiens I. scapularis Consensus

(897) (880) (218) (151) (898)

IWILAELRIQPGNPNYTLSLKCRAPEVSQYIYQVYDSILKNIWVLLELKLQPGNPEATLSLKSRSVEVANIIFAAYEAIIRSP IWILAELRIQPGNPNYTLSLKCRAPEVSQYIYQVYDSILKNIWVLAELKIQPGNPRITLSLKTRAPEVAAGVQQTYELILHSIWILAELKIQPGNPNYTLSLKCRAPEVAQYIYQVYDSILKS

Figure 1. Analysis of clone 3E1 identical to beta-adaptin. (A) Nucleotide sequence of complete cDNA. Non-coding sequence is shown in lower case letters and coding sequence is shown in capital letters with translation initiation and termination codons in bold letters. (B) Alignment of M. musculus (GenBank accession number XP_109938), D. melanogaster (CAA53509) and Homo sapiens (AAA35583) protein sequences and the translation product of clone 3E1 identified as I. scapularis beta-adaptin appendage (AY296113). Protein sequences are shown in the single letter amino acid code. Identical amino acids are shown in red and amino acids conserved in 3 of 4 sequences are shown in blue.

A E. coli O. mossambicus X. laevis I. scapularis C. elegans D. melanogaster L. major A. gambiae

(4) (98) (146) (1) (141) (223) (114) (272)

DTPSLETPQAARLRRRQLIRQLLERDKTPLAILFMAAVVGTLVGLAA-VA DLKEGVCLSALWFNH--------EQ----------CCWTSNETTFAERDK DLKEGICLPWFWFNH--------EQ----------CCWQSNNVTFEDRNN DLKEGICPQAFWLNK--------EQ----------CCWASNDTFFKG-DD DLKTGVCADRFWLDH--------EH----------CCWSSNDTFYKD-DD DLKHGICPPAFWFNR--------EQ----------CCYPAKQSVFEE-GN AFRSGICANFFWLGR-------------------------N-MCCVDCRE DLKFGICPQAFWLNR--------EQ----------CCWSSNETSFDS-GN

Almazรกn et al: Expressed sequence tags in Ixodes scapularis M. musculus S. tuberosum S. cerevisiae Consensus

(155) (108) (102) (272)

DLKEGICLSALWYNH--------EQ----------CCWGSNETTFEERDK GFKLLLTSNLMLDGK----------------------------------NWKTGHCQRNWLLNKS-------------------FCCNGVVNEVTSTSN DLK GIC AFWLNR EQ CCW SN T F D

E. coli O. mossambicus X. laevis I. scapularis C. elegans D. melanogaster L. major A. gambiae M. musculus S. tuberosum S. cerevisiae Consensus

(53) (130) (178) (32) (172) (254) (138) (303) (187) (123) (133) (322)

FDKGVAWLQNQRMGALVHTADNYPLLLTVAFLCSAVLAMFGYFLVRKYAP CPQWKSWAELILGQ--AEGPGSYIMNYFMYIYWALSFAFLAVCLVKVFAP CPEWRSWSQLVLGR--SEGAFPYILNYFMYVMWALLFSLLAVLLVRNFAP CKQWYRWPEMFDSGMDKDGAGFYLLSYLLYVMWSVLFATLAVMLVRTFAP CKAWTKWPWMLNYYN-SSSFLFLFLEWIFYIGWAVAMSTLAVLFVKIFAP CSTWKTWPEIFGLD--RNGTGPYIVAYIWYVLWALLFASLSASLVRMFAP CGEYYSWGEFFLGR---DNHVVAFVDFVMYVSFSTMAAVTAAYLCKTYAP CSQWYAWSEIFTSS--REGFGAYVISYFFYIMWAMLFALLAASLVRMFAP CPQWKTWAELIIGQ--AEGPGSYIMNYIMYIFWALSFAFLAVSLVKVFAP ----------------------YFQAFAAFAGCNVFFATCAAALCAFIAP LLLKRQEFECEAQG-LWIAWKGHVSPFIIFMLLSVLFALISTLLVKYVAP C W W EL EG YIL YIMYILWALLFA LA LVK FAP

E. coli O. mossambicus X. laevis I. scapularis C. elegans D. melanogaster L. major A. gambiae M. musculus S. tuberosum S. cerevisiae Consensus

(103) (178) (226) (82) (221) (302) (185) (351) (235) (151) (182) (372)

EAGGSGIPEIEGALE---DQRPVRWWRVLPVKFFGGLGTLGGGMVLGREG YACGSGIPEIKTILSGF-IIRGYLGKWTLMIKTITLVLAVASGLSLGKEG YACGSGIPEIKTILSGF-IIRGYLGKWTLIIKTMTLVLAVSSGLSLGKEG YACGSGIPEIKTILSGF-IIRGYLGKWTLTIKSVCLVLAVGAGLSLGKEG YACGSGIPEIKCILSGF-VIRGYLGKWTFIIKSVGLILSSASGLSLGKEG YACGSGIPEIKTILSGF-IIRGYLGKWTLLIKSVGLMLSVSAGLTLGKEG YASGGGIAEVKTIVSGH-HVKRYLGGWTLITKVVGMCFSTGSGLTVGKEG YACGSGIPEIKTILSGF-IIRSYLGKWTLIIKSVGIMLSVSAGLSLGKEG YACGSGIPEIKTILSGF-IIRGYLGKWTLMIKTITLVLAVASGLSLGKEG AAAGSGIPEVKAYLNG-IDAHSILAPSTLLVKIFGSILGVSAGFVVGKEG MATGSGISEIKVWVSGFEYNKEFLGLLTLVIKSVALPLAISSGLSVGKEG YACGSGIPEIKTILSGF IIRGYLGKWTLIIKSVGLVLAVSSGLSLGKEG

E. coli O. mossambicus X. laevis I. scapularis C. elegans D. melanogaster L. major A. gambiae M. musculus S. tuberosum S. cerevisiae Consensus

(150) (227) (275) (131) (270) (351) (234) (400) (284) (200) (232) (422)

PTVQIGGNIGRMV----------LDIFRLKG--DEARHTLLATGAAAGLA PLVHVACCCGNIF----------SYLFPKYSKNEAKKREVLSAASAAGVS PLIHVACCCGNIL----------CHLFTKYRKNEAKRREVLSAAAAAGVS PLVHVACCIGNIF----------SYLFPKYGKNEAKKREILSAAAAAGVS PMVHLACCIGNIF----------SYLFPKYGLNEAKKREILSASAAAGVS PMVHIASCIGNIF----------SHVFPKYGRNEAKKREILSAAAAAGVS PFVHIGACVGGII----------SGALPSYQQ-EAKERELITAGAGGGMA PMVHIASCIGNIL----------SYLFPKYGRNEAKKREILSAAAAAGVS PLVHVACCCGNIF----------SYLFPKYSTNEAKKREVLSAASAAGVS PMVHTGACIANLLGQGGSRKYHLTWKWLKYFKNDRDRRDLITCGAAAGVA PSVHYATCCGYLL----------TKWLLRDTLTYSTQYEYLTAASGAGVA PLVHIA CIGNIL SYLFPKY KNEAKKREILSAAAAAGVS

E. coli O. mossambicus X. laevis I. scapularis C. elegans D. melanogaster L. major A. gambiae M. musculus S. tuberosum S. cerevisiae Consensus

(188) (267) (315) (171) (310) (391) (273) (440) (324) (250) (272) (472)

AAFNAPLAGILFIIEEMRPQ--FRYTLISIKAVFIGVIMSTIMYRIFNHE VAFGAPIGGVLFSLEEVSYY--FPLKTLWRSFFAALVAAFVLRSINPFGN VAFGAPIGGVLFSLEEVSYY--FPLKTLWRSFFAALVAAFTLRSINPFGN VAFGAPIGGVLFSLEEVSYY--XPLKTLWRSFFCALVAASVLRSINPFGN VAFGAPIGGVLFSLEEASYY--FPLKTMWRSFFCALVAGIILRFVNPFGS VAFGAPIGGVLFSLEEVSYY--FPLKTLWRSFFCALIAAFVLRSLTPFGN VAFGAPVGGVIFALEDVSTS--YNFKALMAALICGVTAVLLQSRVDLWHT VAFGAPIGGVLFSLEEVSYY--FPLKTLWRSFFCALIAAFILRSINPFGN VAFGAPIGGVLFSLEEVSYY--FPLKTLWRSFFAALVAAFVLRSINPFGN AAFRAPVGGVLFALEEIASW--WRSALLWRTFFTTAIVAMVLRSLIQFCR VAFGAPIGGVLFGLEEIASANRFNSSTLWKSYYVALVAITTLKYIDPFRN VAFGAPIGGVLFSLEEVSYY FPLKTLWRSFF ALVAA VLRSINPFGN

E. coli O. mossambicus X. laevis I. scapularis C. elegans D. melanogaster L. major A. gambiae M. musculus S. tuberosum S. cerevisiae Consensus

(236) (315) (363) (219) (358) (439) (321) (488) (372) (298) (322) (522)

VA----------LIDVGKLSDAPL SR----------LVLFYVEYHTPW SR----------LVLFYVEFHAPW DH----------LVMFYVEYDFPW NQ----------TSLFHVDYMMKW EH----------SVLFFVEYNKPW GR----------IVQFSVNYQHNW EH----------SVLFYVEYNKPW SR----------LVLFYVEYHTPW GGNCGLFGQGGLIMFDVNSGVSNY GR----------VILFNVTYDRDW LVLFYVEY PW

Gene Therapy and Molecular Biology Vol 7, page 51 B

Figure 2. Analysis of clone 4G11 identical to chloride channel. (A) Alignment of M. musculus (XP_134186), D. melanogaster (AAM76180), Solanum tuberosum (T07608), Oreochromis mossambicus (AAD56388), A. gambiae (EAA11899), C. elegans (NP_495940), Leishmania major (strain Friedlin) (T02805), Saccharomyces cerevisiae (P37020), Escherichia coli K12 (AAC73266), and Xenopus laevis (CAA71071) protein sequences and the translation product of clone 4G11 identified as a fragment of I. scapularis chloride channel (AY296114). Protein sequences are shown in the single letter amino acid code. Identical amino acids are shown in red and amino acids conserved in 6-10 of 11 sequences are shown in blue. (B) Phylogenetic tree constructed from analysis of chloride channel protein sequences based on a sequence distance method utilizing the Neighbor Joining algorithm of Saitou and Nei (1987).

D. melanogaster I. scapularis A. gambiae Consensus

PHAQGFIEVDQNVTTHHPIVREEKIVPNMQINGYENPTYKYFE PQAQGFVQVDQGALPASPEER---HLASMQVNGYENPTYKYFE PHAQGFVEVDQAVGAPVTPEE--RHVANMQINGYENPTYKYFE PHAQGFVEVDQ V P ER HVANMQINGYENPTYKYFE

Figure 3. Analysis of clone 2C12 identical to beta-amyloid precursor protein. Alignment of D. melanogaster (AF181628) and A. gambiae (EAA07868) protein sequences and the translation product of clone 2C12 identified as I. scapularis beta-amyloid peptide (Ă&#x;AP) (AY296115). Protein sequences are shown in the single letter amino acid code. Identical amino acids are shown in red and amino acids conserved in 2 of 3 sequences are shown in blue.

Table 4. Characterization of I. scapularis ESTs encoding for ribosomal proteins EST clone

Predicted protein

Identical amino acids

Species

GenBank accession number

4F7 1A2

Elongation factor 1-alpha

95% 85%

Neacarus texanus

AAK12660 NP_031932

1A10

Elongation factor-2

88% 80%

Mastigoproctus giganteus Mus musculus

AAK12348 BAC26203

1C11

eIF-5A

65% 59%

Drosophila melanogaster Mus musculus

AAM68297 XP_203336

1F6 2C3

RpS4

79% 75%

Spodoptera frugiperda Mus musculus

AAL26580 AAH09100

2B8

RpS11

92% 80%

Dermacentor variabilis Mus musculus

AAO92287 XP_133477

2F8

Laminin receptor 1 (RpSA)

66% 73%

Anopheles gambiae Mus musculus

EAA00413 NP_035159

2F10

RpL3

70% 68%

Spodoptera frugiperda Mus musculus

AAL62468 AAH09655

3A10

RpL7A

55% 60%

Drosophila melanogaster Mus musculus

NP_511063 A30241

3D10

Ribophorin I

57% 50%

Drosophila melanogaster Mus musculus

AAN71150 BAC26679

3G9

RpS8

70% 71%

Spodoptera frugiperda Mus musculus

AAL62472 XP_134904

3G10

RpL27A

42%

Spodoptera frugiperda

AAK92158

Mus musculus

Almazรกn et al: Expressed sequence tags in Ixodes scapularis 36%

Mus musculus

XP_137118

4D11

Proteasome subunit

60% 55%

Drosophila melanogaster Mus musculus

NP_524115 NP_035315

4D12

Proteasome/Signalosome subunit

43% 56%

Anopheles gambiae Mus musculus

EAA11895 AAC33900

4E7

Proteasome subunit

84% 85%

Anopheles gambiae Mus musculus

EAA10351 NP_036096

The sequences of I. scapularis ESTs identical to ribosomal proteins pooled for DNA vaccination as described in Almazรกn et al. (2003), were compared to all non-redundant sequences in GenBank DNA and protein databases (1,419,727 sequences total; Apr-09-2003) using BLASTX 2.2.6 (Altschul et al, 1997). The percent of identical amino acids to arachnida or insect and mouse sequences are shown together with their corresponding GenBank accession number. The GenBank accession numbers for I. scapualris sequences are shown on Table 1. 1 M. musculus D. melanogaster H. sapiens A. gambiae I. scapularis Consensus

(1) (1) (1) (1) (1) (1)