Cell, Vol. 75, 1107-1117,
December
17, 1993, Copyright
0 1993 by Cell Press
Tumor Suppression by RNA from the 3’ Untranslated Region of a=Tropomyosin Fanan Rastinejad, Michael J. Conboy, Thomas A. Rando, and Helen M. Blau Department of Molecular Pharmacology Stanford University School of Medicine Stanford, California 94305-5332
Summary NMUP, a nondifferentiating mutant myogenic cell line, gives rise to rhabdomyosarcomas in mice. We show that constitutive expression of RNA from 0.2 kb of the a-tropomyosin (Tm) 3’ untranslated region (UTR), but not control YllTRs, suppresses anchorage-independent growth and tumor formation by NMU2 cells. When fl-galactosidase (f.I-gal)-labeled cells were implanted into muscles of adult mouse hindllmbs, Tm 3’UTR expression suppressed the proliferation, invasion, and destruction of muscle tissues characteristic of NMUS. In the rare tumors that developed from Tm 3lJTR transfectants, RNA expression was extinguished. These results suggest that suppression of tumorlgenicity is dependent on the continued expression of Tm transcripts lacking a coding region. We conclude that untranslated RNAs can function as regulators (rlboregulators) that suppress tumor formation. Introduction A well defined genetic alteration that bypasses normal growth control and gives rise to cancer is the inactivation of tumor suppressor genes (Knudson, 1971). Cell fusion experiments first showed that loss of tumor suppressor gene activity could lead to neoplastic transformation. In hybrids formed between normal and transformed cells, the normal state is dominant (Harris et al., 1969). The prediction that tumor suppressor genes are inactivated in malignant cells was borne out by the discovery that in naturally occurring human tumors a defective allele is inherited, and the remaining allele is inactivated (Cavenee et al., 1963). Definitive evidence for tumor suppressor genes was obtained first by the introduction into tumorigenie cells of entire chromosomes bearing presumed tumor suppressor genes (Weissman et al., 1967) and more recently by expression of the cloned genes themselves (Huang et al., 1966; Finlay et al., 1989). The protein products of tumor suppressor genes inhibit transformation by a multiplicity of mechanisms including blocking angiogenesis (Rastinejad et al., 1989), enhancing responsiveness to growth inhibitors such as TGF-8 (Kimchi et al., 1968; Gerwin et al., 1992) disrupting the cell cycle (Baker et al., 1990; Hinds et al., 1992; Nigro et al., 1992) or inducing cell differentiation (Mechler et al., 1985; Rosengard et al., 1989). All tumor suppressor genes described to date are thought to act through their encoded proteins. Transformation is often associated with a change in cell shape and cytoskeletal architecture due to altered expres-
sion and organization of microfilament and cell adhesion proteins (Cooper et al., 1987; Chan et al., 1989). Actins, a-actinin, vimentin, and tropomyosins are among the structural proteins that are expressed at diminished levels in tumor-derived or virally transformed cells (Leavitt et al., 1982; Matsumura et al., 1983; Cooper et al., 1985; Leavitt et al., 1985; Lin et al., 1985). Especially well documented are changes in tropomyosins. Certain Tm isoforms decrease in neoplastic cells and increase when the cells revert to normal (Cooper et al., 1985). In addition, upon constitutive expression of single cDNAs encoding adhesion or cytoskeletal proteins including tropomyosin, tumorigenicity of cell lines is suppressed (Eiden et al., 1991; Rodriguez Fernandez et al., 1992; Gliick et al., 1993; Prasad et al., 1993). It should be noted that in each of these cases theexpressed cDNAconstruct included both coding and noncoding sequences. We showed previously that noncoding regions of messenger RNAs can act in trans to control growth and differentiation (Rastinejad and Blau, 1993). When a cDNA expression library was introduced into the nondifferentiating myogenic cell line, NMU2, threeof the cDNAs that partially complemented the mutant phenotype derived from genes encoding cytoskeletal proteins: a-actin, tropomyosin, and troponin I. The activity of these cDNAs mapped to the 3’UTR.s. The Tm 3’UTR was particularly effective in promoting expression of differentiation-specific genes in myogenie cell lines. In fibroblasts, expression of the Tm 3’UTR markedly inhibited growth without inducing muscle-specific gene expression (Rastinejad and Blau, 1993). Given these findings and the evidence for tumor suppressor activity by full-length cytoskeletal cDNAs cited above, we examined whether the noncoding region of the a-Tm mRNA could play a role independent of the protein coding region in suppressing transformation. Here we show that an untranslated region of messenger RNA can function as a tumor suppressor. Specifically, we demonstrate that a 0.2 kb RNA transcribed from the a-Tm 3’UTR in the absence of protein coding sequence can inhibit anchorage-independent growth of neoplastic NMUP cells. Moreover, upon continuous expression of the Tm 3’UTR, P-gal-labeled NMU2 cells implanted in the muscles of mice no longer invade and destroy the tissue or form tumors. These novel findings provide evidence that an RNA from an untranslated region of a transcript can act as a tumor suppressor. Results Stable Transfectants Assayed for Transformation Suppression We used the differentiation-defective myogenic mutant, NMU2, to test the potential of the Tm 3’UTR to suppress transformation and promote differentiation in vivo. NMU2 was isolated following low dose mutagenesis of myoblasts of the C2C12 line (subclone C2F3) and selection for growth in soft agar (Rastinejad and Blau, 1993). Unlike its C2F3
Cell 1108
parent, NMU2 cells do not express several gene products characteristic of the myogenic lineage, including sarcomerit actins, myosin heavy chain, tropomyosins, and troponin I. In culture, NMUP cells remain rounded and refractile, do not fuse to form myotubes, and proliferate even in low serum medium. Thus, like many neoplastic cell types, NMU2 cells are capable of anchorage-independent growth, have reduced serum requirements for growth in liquid medium, and have largely lost the potential to differentiate. Genetic complementation experiments yielded four cDNAs, all of which contained 3’UTR sequences capable of promoting muscle-specific gene expression in NMU2 cells (Rastinejad and Blau, 1993). Of the 3’UTRs isolated, tropomyosin was selected for this study because loss of Tm isoforms is frequently correlated with tumorigenicity (Cooper et al., 1986). Moreover, the Tm 3’lJTR exhibited profound growth-inhibitory activity in fibroblasts, and to a lesser extent in NMUP cells (Rastinejad and Blau, 1993). To assess the effect of Tm 3’UTR expression on the transformed phenotype of NMU2 cells, the Fl cDNA, which includes 214 bp of human a-tropomyosin initiating 380 bp downstream of the translation termination codon, was constitutively expressed from the pCDM8 vector (Rastinejad and Blau, 1993). Controls included stable transfectants expressing an irrelevant cDNA, the 0.7 kb subunit 8 of an ATPase (A8), and two 3’UTR cDNAs, the 0.5 kb histone H3.3 (H3.3) 3’UTR and the 0.4 kb junS 3’UTR. All were cloned into pCDM8 as described previously (Rastinejad and Blau, 1993). A8 was used as a control because, like Tm, A8 was isolated by polymerase chain reaction (PCR) from a genetically complemented NMUP clone together with several other cDNAs, but unlike Tm, A8 proved to be inactive upon secondary transfection (Rastinejad and Blau, 1993). H3.3 and jun0 were selected because they are 3’UTRs of similar size to the Tm 3’UTR and are widely expressed in numerous cell types. H3.3 and junB were obtained by PCR from the same human myoblast expression library from which the A8and Tm 3’UTR cDNAs were obtained (Rastinejad and Blau, 1993). A8, H3.3, and junB sewed as controls for nonspecific cDNA or 3lJTR effects. They also controlled for potential effects of the expression vector that utilizes the CMV promoter and the S/40-small t splice and polyadenylation signals (Seed and Aruffo, 1987). NMU2 cells were cotransfected with a plasmid containing the gene conferring resistance to puromycin (pSV2puro) or hygromycin (pSV2hygro) and a cDNA expression plasmid containing either the tropomyosin 3’UTR, or control A6, H3.3 3’UTR, or junB 3’UTR cDNAs. Stable transfectants were selected for growth in puromycin or hygromycin. Northern analysis with probes specific to each cDNA allowed a determination of the relative levels of expression obtained in clones stably transfected with each type of cDNA. Differences in RNA accumulation were evident among clones, presumably due to copy number and site of integration of the plasmid (Figure 1). However, this analysis did not allow a direct comparison of the relative expression levels among clones containing different
Tm u
1
A6 2
3
U234
--
H U3
J u
5
Figure 1. Northern Analysis of RNA Accumulated in stably Transfected NMUP Cells NMUP cells were cotransfected with 8 ug of cDNA expression plasmid and 2 ug of either SV24ygm or SVP-pure. Total RNA was isolated following selection of clones in media containing either hygromycin or puromycin and analyzed by electrophoresis through formaldehydeagarose gels (10 @lane). RNA was transferred to a nylon membrane and hybridized with “P-labeled tropomyosin S’UTR (Tm), ATPase 8 (A8), histone 3’UTR (H), or junS 3’UTR (J) probes. Numbers above each lane indicate clone number, whereas U is RNA from untransfected NMU2 cells. Relative amounts of RNA loaded in each lane are visualized in lower panels by staining with ethidium bromide.
cDNAs, since different probes that were labeled to different specific activities were used for hybridization. To ensure that RNA accumulation in control A6, H3.3, and junB transfectants was comparable to that in Tm transfectants, RNAs from a representative transfectant of each cDNA were analyzed together using a probe specific to transcribed pCDM8 vector sequences common to all transcripts (Figure 2A). Hybridization to RNA from untransfected cells was not observed, demonstrating the specificity of the probe for transcripts from the pCDM8 vector. This Northern analysis allowed identification of control transfectants in which the levelsof RNA accumulation were equal to or exceeded those of Tm transfectants. To rule out the possibility that suppression of transformation could be due to toxicity resulting from overexpression of the Tm 3’UTR, we compared the amount of tropomyosin 3’UTR RNA that accumulated in stable transfectants with that in cultured muscle cells. Northern blots were hybridized with the Tm 3lJTR probe, Fl, which recognizes human but not mouse 3’UTR transcripts. The NMUP transfectant expressing the highest level of Tm 3’UTR RNA approached, but did not exceed, the endogenous level of expression of a-Tm RNA found in primary human myoblasts at confluence just prior to myotube formation (Figure 28). To address further the question of toxicity, the growth rates of NMUP cells expressing different 3lJTR cDNAs were compared in tissue culture. Cells were plated in 96 well plates in six replicates, and cell number was assessed at 12 hr intervals using a standard calorimetric assay(Mosmann, 1983). As shown in Figure 3, cells plated in 10% calf serum grew exponentially and at a similar rate irrespective of the 3’UTR they expressed. The myogenic parental C2F3 and nondifferentiating mutant NMU2 cells also grew at similar rates under these culture conditions. It should be noted that the reduced growth of fibroblasts
Tumor 1109
Suppression
A.
by RNA
B.
pCDM6 U
Tm, Tm, As
Figure 2. Comparison Transfectants, Control
H
J
Tm M
1
u
of Levels of Accumulated RNA in Tm 3’UTR cDNA, and in Muscle Cells
(A) Northern blot analysis showing relative amounts of transcripts initiated from the pCDM6 promoter in different stable transfectants. Total RNA was isolated from NMUP transfectants Tm clones 1 and 2 (Tm 1, Tm 2). A6 clone 4 (A6), histone clone 3 (H), and jun6 clone 5 (J) (see Figure 1). Lane U is RNA from untransfected NMUP cells. The Northern blot was probed with a transcribed vector sequence that is common to all transcripts from the pCDM6 vector. (6) Northern blot analysis showing relative abundance of Tm 3’UTR transcripts in transfected NMU2 cells and in cultured muscle cells. Total RNA was isolated from cultured primary myoblasts (Lane M), transfected NMUP Tm clone 1 (Lane 1). and untransfected NMUP cells (Lane U) and hybridized with the tropomyosin 31JTR probe. Relative amounts of RNA loaded in each lane are visualized in lower panels by staining with ethidium bromide.
Figure 3. Comparison of Growth pressing NMU2 Clones
Rates of Tm and Control
3’UTR Ex-
For each stable transfectant, 2000 cells were plated in 10% calf serum in DMEM medium in 6 replicate wells of 96 well plates. At half day intervals cell growth was assayed using the MTT cell proliferation assay, where the cell number is proportional to ODem. The standard deviatttn was less than 0.06 in two separate experiments. NMUP, open diamonds; NMUP histone 3’LJTR clone 3, open squares; junEI 3’UTR clone 5, open circles; A6 clone 4, open triangles; Tm clone 1, cfosed circles; Tm clone 2, closed triangles; and Tm clone 3, closed diamonds. When plated at high density in a mitogen rich medium, no significant differences in growth rate are observed.
and NMU2 cells reported prevk sly was observed at low cell density in a low serum, mitugen-poor medium (Rastinejad and Blau, 1993). These data indicate that at high cell density in a mitogen-rich medium, Tm 3’UTR expression does not significantly inhibit theviabilityor the proliferation of NMU2 cells.
Suppression of Anchorage Independence Anchorage-independent growth in the semisolid medium of soft agar is a strong indicator of the transformed phenotype in rodent cell lines. To assess the effect of Tm 3’UTR RNA expression on the transformed phenotype, stably transfected NMUP cells were analyzed for growth in soft agar. Untransfected controls included nontransformed C2F3 cells, NMUP, and a subclone NMUP HCA-gpt.3 (Rastinejad and Blau, 1993). Cells were seeded in parallel in liquid tissue culture medium and in medium containing soft agar. To assess viability, the number of colonies that grew in 1 O%I calf serum in liquid culture medium wasdetermined in each case. Under these conditions, Tm transfectants were morphologically indistinguishable from and grew as well as untransfected NMU2 cells (Figure 3). To assess relative anchorage independence, for each cell type the number of colonies that grew in soft agar was determined and normalized to the frequency of colony formation in liquid medium. In three separate experiments, transformed NMU2 cells produced anchorage-independent colonies at a much greater frequency than the nontransformed C2F3 cells (Table 1). Like C2F3 myoblasts, the four Tm 3’UTR expressing clones tested did not grow well in soft agar. By contrast, three independently derived stable clones expressing the control cDNA A6, or clones expressing the control 3’UTR cDNAs, jun6, and histone H3.3, grew as well as NMU2 cells in soft agar. Northern analysis of the levels of mRNA expression for these stable transfectants are shown in Figures 1 and 2A, and the relative RNA levels of all clones are indicated in Table 1. The data show a marked change in the anchorage-independent phenotype upon transcription of Tm but not control cDNAs expressed from the pCDM8vector. Taken together, these results suggest that suppression of anchorage independence is specific to the tropomyosin 3UTR cDNA. Suppression of Subcutaneous Tumor Formation To determine whether NMU2 cells could be used in assays of malignant transformation in vivo, cells were injected subcutaneously into athymic nude mice. The tumors that arose were analyzed histochemically and shown by a number of criteria to be rhabdomyosarcomas (Enzinger and Weiss, 1988; Hiti et al., 1989). The malignant neoplasm6 contained occasional myofibers with distinct cross-striations, and an abundance of cells that expressed musclespecific proteins such as desmin (data not shown) and sarcomeric actin (Figure 4). In addition, the tumors contained poorly differentiated multinuclear giant cells with darklyeosinophiliccytoplasm. In the central regionof large tumors, mitotic figures were rare, whereas other regions
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Table 1. Suppression by Tm 3UTR Cells Untransfected C2F3 NMU2 HCA-gpt.9 Transfected NMUP Tropomyosin 3’UTR Tm clone 1 Tm clone 2 Tm clone 34 Tm clone 40 Control cDNA.s A6 clone 2 A6 clone 3 A6 clone 4 jun6 clone 5 Histone clone 3
of Anchorage
independence
RNA Level’
of NMUP Growth in Soft Agap (%I 0.02 26.3
+++ ++ ++ +++ ++ ++++ +++ ++++ +++++
0.4 0.03 1.1 0.01 35.0 20.2 20.6 14.0 21.3
’ Relative amounts of accumulated RNA are based on Northern blot analysis shown in Figures 1 and 2A. b Colony formation in soft agar was corrected for the number of viable colony-forming cells in liquid medium. The results represent the average of three experiments for C2F3 and NW2 HCA-gpf.3 and of two experiments for each of the transfected NW2 clones. c NMUP HCA-gpt.3 is a subclone of NMU2 that was cotransfected with a silent acardiac actin-gpt (HCA+pf) construct and SM-neo and used previousfy in genetic compfementation experiments (Rastinejad and Blau, 1993).
of the tumor had a characteristically embryonal rhabdomyoblastic appearance. To assess the effect of the 3lJTFl on tumor formation in nude mice, NMUP cells stably transfected with Tm 3lJTR, A6, or junl3 3’UTR cDNAs were compared. The levels of RNA accumulation for these clones are shown in Figures 1 and 2A. Untransfected NMU2 cells, the NMU2 HCA-gpt.3 subclone, and wild-type C2F3 cells served as controls. Various inoculum sizes of NMU2 cells were tested, and a minimum number of 1 x 105 cells was found to be required to produce tumors consistently within 4 weeks after injection (data not shown). The data for six separate experiments show that tumor suppression was correlated with expression of the Tm 3lJTR (Table 2). Injection of 1 x 105 C2F3 cells did not produce tumors (0 tumors/l0 sites) for up to 10 weeks, at which time the experiments were terminated. By contrast, both a stable NMUP transfectant expressing the control cDNA A6 (clone 4, Figure 1) (12 tumors/l2 sites) and a stable transfectant expressing the junB 3’UTR (12 tumors/ 12 sites) produced tumors in all of the injected sites within 4 weeks, a time course and efficiency consistent with that for tumor formation by NW2 cells (26 tumors/27 sites). Two stable transfectants that expressed relatively high levels of Tm cDNA (clones 1 and 2, Figure 1) showed profound suppression of tumor formation, 2 tumors/l4 sites and 3 tumors/l 6 sites, respectively. Moreover, most tumors arising from injection of Tm-expressing clones were delayed in appearance by 2-4 weeks beyond the typical latency period seen for tumors that expressed the control cDNAs. Tm clone 3, which produced tumors in 2 of 4 sites, expressed the lowest level of Tm 3lJTR. These
Figure 4. Formation of Rhabdomyosanxwna by Subcutan~us Injection of NW2 Cells After 5 weeks, 1 x 105NMUP cells that had been injected subcutaneously into the ffanks of athyrnfc
nude mice gave rise to tumors
(5 mm
dieter) that were characteristic of rhabdomyosarcomas. Forrnalinfixed, paraffin-embedded sections of tumor tissue (5 pm) were stained wfth hemotoxylin-eosin (upper and lower panels; 106x and420x)to~myofibers,orwithantibodiegtothemusde
rnagnificatfons
are
specific protein, sarwmeric actin (middle panel, magnification lo5 x ). Arrow indicates mature satwnnem with striations present in muftinucieatedcelllypicalofafhabdomyosarmma. results indicate that suppression of tumorigenicity, like the suppression of anchorage independence, is specific to the Tm 3’UTR. Absence of Tm 3YTR RNA Expreaekn Condates withLossofTumorSuppw&on The long latency period of tumors arising from Tm 321TR transfectants suggested that these tumors were derived from rare variants in which transcription of the cDNA had
Tumor 1111
Suppression
Table 2. Suppression
by RNA
of Tumorgenicity
Ceils Subcutaneous Untransfected C2F3 NMUP NMUP HCA-gpf.3 Transfected NMU2 Tm clone 1 Tm clone 2 Tm clone 3 A6 clone 4 (control) JunB clone 5 (control) Intramuscular Untransfected NMUP HCA-gof.3 NW2 Transfected Tm clone 1 Tm clone 2
Tm
of NMUP by Tm 3UTR
RNA Level’
Tumor Incidence (Tumors/Site)b
1
1T
A6 3
3T
4
4T
O/IO 6i6 20/21 +++ -H + ++++ ++++
2/14” 3/16 2l4’ 12il2 12il2
13113 919 O/l3 o/9
a Relative amounts of accumulated RNA are based on Northern blot analysis shown in Figures 1 and 2A. b Cells (1 x 1 OS) were injected subcutaneously in the flanks of athymic nude mice or implanted intramuscularly in hindlimbs of scid and nude mice. Tumors were first detected at 3-4 weeks postinjection. Values shown indicate tumor incidence at 10 weeks postinjection for subcutaneous and 5 weeks postinjection for intramuscular sites. Data are the combined results from up to six separate experiments (subcutaneous) or two experiments (intramuscular). c One tumor each that arose from Tm clone 1 and Tm clone 3 were analyzed and shown in Figure 5 to have extinguished mRNA expression. Tumors derived from Tm clones were generally delayed in appearance.
ceased. To test this possibility, two tumors arising following injection of Tm clone 1 and Tm clone 3 ceils were examined for retention and expression of cDNA sequences. Tumors were dissected, and the dissociated cells were cultured in vitro in the presence of puromycin to restrict the analysis to the injected cells. DNA and RNA were isolated from the cultured tumorderived cells and examined for the presence of Tm cDNA by Southern analysis and for Tm RNA expression by Northern analysis. The numbers and sizes of the hybridizing fragments in the restriction digests of the DNA analyzed by Southern blot were not altered (Figure 5, lower panel). Thus, the cells recovered from the tumors contained Tm 3’UTR cDNA sequences that did not appear to be lost or rearranged. By contrast, Tm RNA was undetectable in the recovered cells (Figure 5, upper panel). These findings suggest that although the transfected Tm 3’lJTR cDNA was present, its expression was extinguished in these cells. For comparison, a tumor derived from cells transfected with the control cDNA A6 was analyzed. The A6 RNA accumulated as efficiently in the cells recovered from the tumor as in the cells that had been injected. These data suggest that the extinction of Tm cDNA expression in tumors is specific to that cDNA and not due to nonspecific silencing of the pCDM6 CMV promoter in tumors. Moreover, the maintenance of integrated Tm cDNA in the genome of transfectants does not appear to be sufficient for tumor suppression. Instead, it appears that the suppression of
Figure 5. Loss of RNA Expression by Tm 3’UTR-Transfected NMUL Cells That Form Tumors Analysis of stably transfected NMUP clones (Tm clones 1 and 3 and A6 clone 4) that gave rise to tumors when injected subcutaneousfy into athyrnic nude mice. RNA isolated from the cells prior to injection (Tm 1, Tm 3. A6 4) and from cells recovered from the tumors (Tm lT, Tm 3T, A6 4T) are compared by Northern analysts (top panel). The relative amounts of RNA loaded in each lane are visualized by staining with ethidium bromide (middle panel). DNA isolated from cells was analyzed for the presence of the transfected DNA by Southern hybrldizatton (lower panel). Hindllldigeated DNA was probed with Tm 3’UTR or A6 probes. The patterns of hybridizing fragments indicate no significant loss of transfected DNA.
tumorigenicity is dependent of Tm 3UTR RNA.
on the continued
expression
Suppreaaion of Tumor Formation In Muscle To determine the fate of NMUP cells expressing the Tm 3IJTR in the environment of the muscle, the tissue from which the cells were derived, cells were labeled with the /acZgene and implanted into the hindlimb muscles of nude or scid mice. Within 1 week of implantation, both NW2 cells and NMUP cells expressing the Tm 3’UTR were detectable as 5galactosidasepositive cells in a small area of the muscle. At this time, differences between legs injected with the two cell types were negligible. Three weeks after implantation, NMU2 cells had extensively invaded the host muscle. At the site of implantation in the center of the muscle, nearly all muscle fibers had been replaced by a tumor mass. Peripheral to this site, tumor cells circumscribed virtually every muscle fiber, and tissue destruction was extensive. By four weeks, tumors were detectable by palpation, and after six weeks tumors were readily evident as large masses (Figure 6). In contrast, at similar time points, Tm 3’UTR-expressing NMUP cells remained highly localized at the site of implantation (Figure 7). Growth was minimal, and the size of the area encompassed by Bgalactosidase-labeled cells after 3 weeks had not changed significantly from that observed 1 week after implantation. In addition, there was evidence of muscle regeneration with new fiber formation in the area of cell implantation. In all of the 22 sites analyzed, tumors
Cell 1112
were either undetectable or substantially delayed in appearance after injection of Tm 3’UTRexpressing cells. The finding that the NMU2 cells expressing the 3IJTR survive indicates that suppression of tumor formation is due to growth inhibition, not cell death. Neither NMUP cells nor NMU2 cells expressing Tm 3’UTR are capable of fusion in vitro even in the presence of fusion-competent cells (data not shown), but when implanted into muscle tissue both cell types were incorporated into myofibers. One week after implantation, 8galactosidasepositive myofibers were evident at implantation sites. Eventually large diameter labeled fibers ofsimilarsizetothe musclefibersofthesurroundingtissue were also apparent. The incorporation into host fibers may be due to the disruption of muscle fiber membranes at the site of implantation. These areas of muscle regeneration persisted where S’UTR-expressing cells had been injected (Figure 7, right). By contrast, 3 weeks after injectIon, the site of implantation of NMU2 cells was obscured by tumor growth (Figure 7, left). Discussion
Figure
6. Tumor
Suppression
by Tm 3’UTR
in Mouse
Leg Muscle
One of the eight scid mice analyzed is shown 6 weeks after injection of NW2 cells into left hindlimb muscles and NMUP Tm clone 1 ceils into right hindlimb muscles. A large tumor mass is readily apparent on the left side but not on the right.
Genetic complementation experiments revealed that untranslated regions of mRNAs in the absence of coding sequence can act in trans as regulators of growth and differentiation (Rastinejad and Blau, 1993). When a cDNA expression library was introduced into a nondifferentiating myogenic cell line, NMUP, the sequences of three of the cDNAs that partially complemented the mutant phenotype derived from cytoskeletal genes, and their activity mapped to the 3’UTR (Rastinejad and Blau, 1993). Expression of the 3’UTRs promoted the expression of muscle-specific genes in both mutant and wild-type myogenic lines. In fibroblasts, expression of the 3’UTR of Tm markedly inhibited growth without inducing muscle gene expression. Inhibition of the growth of transformed NMUP cells was also detectable when the cells were plated at low density in low serum medium. Given the known dichotomy between growth and differentiation in muscle cells, these observations suggested that the 3’UTR of Tm mRNA functions as a regulator in a pathway that inhibits growth and promotes differentiation. Since the growth controls that accompany differentiation and tumor suppression are thought to be closely associated (Mintz and Fleischman, 1981; Sachs, 1981, 1984; Harris, 1985) we examined the effects of Tm 3’UTR expression on the transformed state of NMU2 cells. The results showed that 0.2 kb of the Tm 3lJTR was sufficient to inhibit anchorage-independent growth and tumorigenicity (Tables 1 and 2 and Figures 8 and 7). In the rare tumors that developed, 3UTR DNA sequences, although present in the genome, were not expressed, suggesting that sup pression was not mediated by the integrated DNA itself but by its transcribed product (Figure 5). Perhaps most compelling were the profound changes in cell behavior observed in vivo (Figures 8 and 7). @gal-labeled Tm 3’UTRexpressing NMU2 cells remained localized at the injection site, often present in regenerating host muscle
Tumor 1113
Suppression
by RNA
NMU2
NMU2 + Tm 3’UTR
Figure 7. Suppression by Tm 3’UTR of NMUP Proliferation, Invasion, and Tissue Destruction in Mouse Muscle NMUP cells and NMU2 Tm clone 2 cells expressing Tm S’UTR were each labeled with a retrovirus encoding the marker enzyme (5-galactosidase and injected into muscles of nude mice analyzed two weeks later. &galactosidase-labeled NMU2 cells (left) are shown invading the entire muscle surrounding each muscle fiber, causing massive tissue destruction (upper panel 40x. middle panel 160x). By contrast, Tm S’UTR-transfected cells (right) remain localized and do not exhibit uncontrolled proliferation or tissue destruction. Indeed, local tissue regeneration is apparent, and implanted cells have become incorporated into regenerating fibers (upper panel magnification 40 x , middle panel magnification 160 x). Lower panels show sections immediately adjacent to those in middle panels, stained with hematoxylin-eosin to reveal muscle morphology and cytoarchitecture in the region of cell injection.
fibers, whereas the majority of their untransfected counterparts proliferated and invaded the entire muscle, surrounding each fiber and destroying the tissue. These experiments show that expression of the Tm S’UTR inhibits properties characteristic of neoplastic cells. Since loss of Tm 3UTR expression accompanies malignant transfor-
mation of NMUP, and tumor growth is suppressed upon Tm 3’UTR introduction, this untranslated region of mRNA meets the criteria for a tumor suppressor. Several experiments were carried out to control for potential nonspecific effects of overexpression of the Tm 3’UTR. First, we analyzed the relative levels of Tm 3’UTR
Cdl 1114
transcripts in stable NMUP transfectants and in primary myogenic cells. The levels of accumulated Tm 3’UTR transcripts necessary to inhibit transformation in NMUP cells approached but did not exceed levels of Tm transcripts detected in confluent primary myogenic cells just prior to fusion (Figure 28). Second, to determine that the effects were specific to Tm 3’UTR expression, we assessed the relative levels of mRNA expression for Tm and control transfectants. Using a probe to vector sequences common to all transcripts produced from the pCDM8 vector, we determined that the clones analyzed expressed control A8, junS 3lJTR, and histone H3.3 3UTR RNAs at levels equal to or in excess of the Tm BUTR-expressing clones (Figure 2A). Third, we showed that Tm 3’UTR expression was not responsible for growth inhibition per se. When plated at high density in mitogen-rich media, no significant differences in proliferation among Tm 3UTR and control CDNA-expressing clones were evident (Figure 3). Finally, P-gal-labeled Tm 3’UTR-expressing cells were found to persist at the injection site in regenerating fibers and ceased to be tumorigenic (Figure 7). Taken together, it appears that the findings reported here are unlikely to result from nonspecific toxicity due to Tm 3’UTR RNA overexpression. Several findings have suggested that expression of cytoskeletal proteins like tropomyosins may be incompatible with neoplasia. Tropomyosin isoforms are frequently missing from spontaneously arising tumors (Gabbiani et al,, 1978). Cells transformed experimentally by expression of various oncogenes or by viral infection also cease expressing tropomyosin isoforms (Cooper et al., 1988) whereas revertants that are no longer transformed reexpress them. Other structural proteins that cease to be expressed by tumors also have a role in cell shape (keratin, vimentin, a-actinin, and certain actins) or in cell surface interactions (thrombospondin, vinculin, collagen, and acatenin) (Matsumura et al., 1983; Cooper et al., 1988; Harris, 1985; Leavitt et al., 1988; Chan et al., 1989; Good et al., 1990; Zajchowski et al., 1990; Shimoyama et al., 1992). Elimination of one protein, for example, vinculin or tropomyosin by antisense RNA expression, induces transformation in 3T3 cells or supB- cells, respectively (Rodriguez Fernandezet al., 1993; J. Boyd, personal communication). Finally, constitutive expression in tumorigenic cells of a single cDNA encoding either vimentin, vinculin, a-actinin, acatenin, or tropomyosin leads to suppression of the transformed state (Eiden et al., 1991; Rodriguez Fernandez et al., 1992; Gliick et al., 1993; Prasad et al., 1993). A common interpretation of these transfection experiments is that expression of any one of these protein products that cause changes in cell morphology and adhesion can lead to tumor suppression. A caveat in each case is that the cDNA constructs contained coding as well as noncoding regions such as 3’UTRs or, in the case of vimentin, an unspliced intron. The findings reported here suggest that such untranslated regions may significantly contribute to the observed inhibition of transformation. Untranslated RNAs may play a fundamental regulatory role in development. Indeed, thedisruption of untranslated RNA sequences is known to be associated with both con-
genital disease and malignancy. Myotonic dystrophy, an autosomal dominant degenerative disease of muscle tissue, results from adefect in the 3lJTR of a muscle-specific kinase (Brook et al., 1992). Overexpression of the product of the H79 gene, a muscle transcript with no apparent open reading frame, leads to prenatal lethality in transgenie mice (Brannan et al., 1990). Interestingly, a genetic complementation study designed to isolate suppressors of K-Ras-mediated transformation led to the isolation of a small cDNA with only a very short putative open reading frame (Liu et al., 1992). This sequence appears to be identical to the 3UTR of NF-IL8 reported by Kinoshita et al. (1992). Translocations leading to cancer may not only involve alterations in the protein coding region, but also the disruption of untranslated regions of RNA. The trk oncogene, isolated from a human carcinoma, was created by a translocation that juxtaposes tropomyosin and a cellular kinase and eliminates the tropomyosin 3’UTR (Martin-Zanca et al., 1988). We suggest that the tumorigenic potential of the trk oncogene may be due not only to the well-documented activation of the kinase (Oskam et al., 1988) but also to the absence of Tm 3’UTR tumor suppressor activity from the hybrid WC RNA. Other human malignancies such as lymphomas and leukemias often result from translocations that disrupt the 3’LtTR of the SC/, bc12, or myosin heavy chain transcripts (Begley et al., 1989; Liu et al., 1993; Zelenetz et al., 1993). As a result of the translocation, these 3lJTRs may cease to be expressed and, like the Tm 3UTR, may encode RNAs with the potential to inhibit tumorigenicity. Together with our results, these findings suggest that untranslated RNAs may contain sequences with an important function in regulating cell growth both in development and in cancer. The mechanism by which RNAs may exert tumorsuppressive activity in trans remains to be elucidated. Untranslated regions of mRNAs may sequester and limit access of regulators of cell growth to their targets. Alternatively, the RNAs could alter growth factor concentrations indirectly by titrating or binding proteins that affect their metabolism or stability. On the other hand, the RNAs could suppress cell proliferation through a general perturbation of the translation machinery. In this case, the effect must be either specific or transient and self-limiting. A transient suppression of protein synthesis may suffice to shift the balance from relatively labile regulators of growth to relatively stable regulators of differentiation. The RNAs may play a role in regulating initiation of or progression through the cell cycle by other mechanisms. The ~53 protein binds covalently to an RNA molecule, suggesting that RNAs may act as ancillary factors to tumor suppressor proteins (Fontoura et al., 1992). RNAs are also known to interact with cellular kinases with a role in growth control (Koromilas et al., 1992). As ribozymes or as antisense, regulatory RNAs may alter the function of other RNAs and proteins leading to changes in cell proliferation. We propose the term riboregulators for RNAs with a regulatory effect that is not mediated by an encoded protein. Although the regulatory RNA studied here, a-Tm 3IJTR, was isolated from a muscle cDNA expression li-
Tumor 1115
Suppression
by RNA
brary, its activity is unlikely to be restricted to muscle. a-tropomyosin is particularly abundant in muscle, but multiple alternative transcripts of this gene are relatively<biquitous (Lees-Miller and Helfman, 1991). In fibroblasts transformed by Ras, the constitutive expressiotiof a B-tropomyosin cDNA suppresses tumorigenicity (Prasad et al., 1993). As in the examples of tumor suppression by other cDNAs cited above, both the coding region and 3’UTR were present in the @tropomyosin construct. It seems likely that such a profound change in cell function as tumorigenicity would require a more global change than can be provided by the presence or absence of a single protein such as tropomyosin or any of the other structural proteins previously reported to suppress tumor formation (Chan et al., 1989; Eiden et al., 1991; Rodriguez Fern&ldez et al., 1992; Gliick et al., 1993). Our results indicate that this global regulatory effect could be mediated by untranslated regions of RNA. We speculate that a family of genes may generate RNAs with noncoding regions that function in trans to regulate growth. Further experiments which combine mutagenesis, protein binding, and functional assays will be required to determine the molecular mechanism through which such untranslated RNA sequences exert both their differentiation-promoting and tumor-suppressive activity. Experimental
Procedures
Cell Culture and Transfection C2F3 is a subclone of the C2C12 mouse myoblast line (Blau et al., 1983; Yaffe and Saxel, 1977) and was chosen for its low frequency of generating anchorage-independent cells. NMUP was isolated as an anchorage-independent mutant following exposure of C2F3 cells to 12.5 rglml nitrosomethylurea (Rastinejad and Blau, 1993). NMU2 HCA-gpt.3 cells were derived by stable cotransfection of NMUP cells with pSV2neo and a construct that included 486 bp of the human cardiac actin promoter driving the expression of the bacterial gpt coding region. The HCA-gpt construct is silent in these cells, allowing genetic complementation (Rastinejad and Blau, 1993). All cells were routinely grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% calf serum in 8% COz at 38OC. Cells were passaged once every 2 days, before reaching confluence. For selection of drug-resistant stable transfectants with SV2-hygro or SVZ-puro plasmids, the medium was supplemented with hygromycin (200 rg/ml, Calbiochem) or puromycin (1 &ml, Sigma). Human embryonic week 7 myoblasts were obtained from thigh muscle samples described in Cho et al. (1993). NMU2 Tm clone I, A6 clones 2, 3, and 4, histone 3’lJTR clone 3, and junB 3’UTR clone 5 were derived by transfecting NMUP HCAgpt.3. Other NMUP transfectants were obtained by transfeding the parental NMUP cell line. The cotransfected selectable marker for NMUP Tm clone 1 was SVP-hygru. Other clones were cotransfected with SVP-pure and selected in the presence of puromycin. Transfections were performed using the calcium phosphate method of Graham and van der Eb (1973). Cells were plated at approximately 1 x 105 per 60 mm dish between 4 and 16 hr prior to transfection. DNA precipitate containing 2 ug of selectable marker plasmid (SV2-hygro or SV2pure) and 8 pg of cDNA expression plasmid (in pCDM8 vector) was added to each dish. The medium was replaced 8 hr later, and following an additional 24-36 hr of incubation, cells were harvested and replated at clonal density under the selection conditions described above. Clones of stable transfectants were isolated after 7-10 days using glass cloning cylinders. Cell Growth Assay Cell proliferation was assayed by a modification of the method described by Mosmann (1983). Cells were plated in 150 PI of medium
(10% calf serum in DMEM) in 6 replicate wells of 96 well tissue culture clusters. Cells were assayed at 12 hr intervals over a period of 3-4 days. To assay cell number, 25 pl of s[4,5dimeulylthiezol-2-yl1-2,5 diphenyltetrazolium bromide (MTT) substrate (3 kg/ml in phosphatebuffered saline [PBS], Sigma) was added to each well. After a 3 hr incubation, 100 pl of solubilization solution containing 50% dimethylformamide (Bigma) and 20% SDS was added, and absorbance was read on a Bio-Rad ELISA plate reader at 570 nm and 630 nm. Anchorage Independence Aaaay To assess anchorageindependent growth, cells were suspended in agar (Difco) according to the procedure described by Bouck and di Mayorca (1982). Each cell type was tested in duplicate for growth in liquid medium and growth in soft agar. To assess viability, 200 cells were plated in duplicate dishes of liquid medium. This assessment of cloning efficiency of each cell type in liquid medium allowed correction for viability of the cells at the time of plating in all assays of cloning efficiency in agar. For determining cbning efficiency in agar, 1000 or 50,000 cells were plated in 1.5 ml of DMEM containing 8%-l 0% calf serum and 0.34% agar. This suspension was layered over 5 ml of a base layer of solidified medium of DMEM containing 10% calf serum and 0.5% agar. After each week of culture, 0.5 ml of liquid medium was layered over the agar cultures to prevent desiccation. The number of large colonies (approximately x.1 mm in diameter) with dense centers was scored for each plate after 3 weeks. Tumorlgenlclty Assay To assay for tumor formation in vivo, cultures of cells were harvested, rinsed with PBS, and resuspended in PBS. For subcutaneous injection, an inoculum of 1 x 106 cells in 50 pl of PBS was injected into the flanks of athymic nude mice. For intramuscular injectiin. 1 x I@ or 2 x 105 cells in 5 gl of 0.5% bovine serum albumin in PBS were injected into the tibialis anterior muscles of nude mice. In independent experiments, either all male or all female mice were used, and the animals ranged from 5-10 weeks in age. Tumor formation was asseesed twice a week for IO weeks. In all cases shown, tumors continued to grow until the animal was sacrificed. When necessary, tumors of approximately 5 mm diameter were removed in the course of the experiment after sacrificing the animals. Tumor cells were recovered by extensively mincing and triturating the tumor tissue. The minced tissue was cultured in medium containing either puromycin or hygromycin as appropriate for selecting the particular injected cells. Northern and Southern Blot Analysis For Northern analysis, total RNA was isolated by centrifugation of the cell lysates over a cesium chloride cushion (Chirgwin et al., 1979). RNA samples were electrophoresed through 1% agarose-formalda hyde gels and transferred to Nytran hybridization membranes (Micron Separations, Incorporated) using a vacuum blotter (LKB Pharmacia). The RNA was cross-linked to the membrane by exposure to ultraviolet light in a Stratalinker (Stratagene) and hybridized to the probes in a phosphate buffer as previously described (Peterson et al., 1990). For Southern analysis, DNA was isolated from cells in culture according to the method of Fukui et al. (1982) and digested with Hindlll. DNA (10 vg) waselectrophoresed through a 1% agarosegel and transferred to Nytran membrane. Southern blots were prehybridized for 8 hr in 5x SSC, 5 x Denhardt’s solution, 0.1% SDS, and 100 &ml sheared salmon sperm DNA. Tm 3’lJTR. A6, histone 3’UTR, and junf3 JIJTR cDNA probes were isolated from the pCDM8 vector by Xhol digestion and separation of the insert fragment on a 2% low-melting temperature agarose gel (Rastinejad and Blau, 1993). The Tm probe is human-specific and does not recognize mouse Tm sequences. The probe common to all pCDM&initiated transcripts was the 1.6 kb Hincll fragment of pCDM8, which includes the transcribed vector sequences present at the 3’and 5’ ends of the transcripts. Approximately 25 ng of DNA were labeled with [a-J2P]dCTP (Amersham), using random hexamers as primer (Multiprime kit, Amersham). Hlstobgy of Muscle Impfanted with Bgal-Expresslng Cells TO analyze the fate of the cells histologically, the /acZ gene was introduced by retroviral infection into both NMUP cells and NMUP cells stably transfected with the Tm 3’UTR. The MFG retrovirus (provided
Cell 1116
by Paul Robbins, University of Pittsburgh) drives expression of lac.2 from the MoMuLV long terminal repeat. Virus-containing supernatant was obtained from confluent amphotropic packaging cells. Repeated rounds of retroviral infection were carried out until more than 95% of NMUP cells and stable transfectants expressed &alactosidase, as determined by histochemical assay of enzyme activity (Hughes and Blau, 1992). In nude mice 5-6 weeks of age, 1 x 106 to 2 x 105 cells were implanted into the interior of tibialis anterior muscles, directly visualized by making an incision in the overlying skin. This method of cell delivery overcomes the problems associated with blind injection, in which cells are often implanted between two muscles rather than within a given muscle. In general, myoblasts injected between muscles do not have access to muscle fibers. Mice were sacrificed at different times up to 3 weeks after implantation. The muscles were removed and frozen, and serial sections were obtained by cryostat for histochemical analysis. The fate of implanted cells was determined by staining fixed 30 pm sections for j+galactosidase activity (Hughes and Blau, 1992). Adjacent IO pm sections were stained with hematoxylin and eosin for histological analysis of tissue morphology and architecture in the same regions in which injected 6galactosidasblabeled cells resided. Immunohlstochemical Analysis of Subcutsneous Rhabdomyosarcomas Formalin-fixed and paraffin-embedded tumor sections (5 pm) were assayed for expression of muscle-specific markers, using monoclonal antibodies to desmin (1:20) (clone D33, Dako) and to sarcomeric actin (1:1600) (clone HHF35, Dako). Staining was visualized by an indirect biotin-avidin method, using an automated immunohistochemistry instrument (Ventana Medical Systems model 320) as described by van de Rijn et al. (1993). Acknowladgments We thank our colleagues for critical discussions of the manuscript, Dr. Matthijs van de Rijn for providing expertise in rhabdomyosarcoma histology, Dr. David Gelfand for invaluable advice regarding PCR, Marilyn Travis and Sarah Elson for aiding in the preparation of figures, and Elizabeth Mancuso for expert secretarial assistance. The myogenin promoter used in the initial genetic complementation studies wasgenerouslyprovided by Dr. EricOlson. Wegratefullyacknowledge support from a National Research Service Award from the National Institutes of Health (GM 14566) to F. R., from a Neonatology and Developmental Biology Training Grant of the National Institutes of Health (HD 07249-11) to M. J. C., from a Howard Hughes Medical Institute Physician Research Fellowship toT. A. A., and grants from the National Institutes of Health (HD 20203 and AG 09521) the National Science Foundation (DCB 6417069) and the Muscular Dystrophy Association to H. M. B. Received
July 6, 1993; revised
October
16, 1993.
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