Cancer Investigation, 10(1), 85-92 (1992)
MINISERIES IN MOLECULAR BIOLOGY John C. Bell, Ph.D. and Nahum Sonenberg, Ph.D., Guest Editors
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Polyomavirus-Mediated Transformation: A Model of Multistep Carcinogenesis Marcel Bastin, D.Sc. Department of Microbiology Faculty of Mediclne University of Sherbrooke Sherbrooke, Quebec J1 H 5N4 Canada
INTRODUCTION
GENETIC STRUCTURE OF POLYOMAVIRUS
There is overwhelmingevidence that carcinogenesis occurs as a progressiveprocess, involving multiple independent events. The process has been formally divided into two steps, as it requires at least two distinct types of stimulus (1). The first, initiation, is thought to be an absolute requirement involving mutational events in as-yet unknown genes. The second, promotion or completion, involves subsequentaltemtions that genemte heterogeneity and results, in combination with the selectivityof the host environment, in the emergence of subpopulationsof cells with increased malignancy and metastatic potential (2). It is likely that activated proto-oncogenes play a major role not only as initiators but also as completion genes in multistep carcinogenesis(3,4). The role of specific viral and cellular oncogenes in different stages of neoplastic development has been addressed in several laboratories. This review examines how studies on viral oncogenes, particularly the polyomavirus oncogenes, have helped establish the concept that carcinogenesis is a multistep process at the molecular level.
Polyomavirusprovides a useful model of multistep carcinogenesis. It is a papovavirus that can transform the growth properties of rodent cells in culture and form tumors in animals. Transformation is the result of stable integration of the viral DNA sequences into the host genome and subsequent expression of the viral early region (5). This region, which is about 3 kb in length, corresponds to the part of the genome transcribed during the early phase of the lytic cycle in permissive mouse cells (Fig. 1). It encodes three distinct proteins, in alternate translational reading frames, via the production of distinct mRNAs by means of different splicing processes operating on a common primary transcript (6). They are referred to as the large T (MW = lOOK), middle T (MW = 56K),and small T (MW = 22K) antigens. The role of the T antigens in oncogenic transformation has been difficult to establish because they are encoded by extensively overlapping DNA sequences. However, the properties of all three proteins can now be studied individually thanks to the cloning of the three cDNAs and
85 Copyright 0 1592 by Marcel Dekker, Inc.
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Figure 1. Map of the polyomavirus early region. The nucleotide numbers are shown on the top line, and the three differentspliced m R N h are aligned with the map. Boxes represent the encoding sequences for the three T antigens with different symbols according to the reading frame used. Nontrmlated regions are represented by continuous lines. Upward deflections denote the intervening sequences.
the construction of modified genomes that separately encode the T antigens (7,8). The polyomavirus large T antigen is a nuclear phosphoprotein of 785 amino acids. Like the SV40 T antigen, it exists as multiple molecular species and its activity has been implicated in several processes that occur after infection, includingthe stimulation of host DNA synthesis, the initiation of viral DNA replication, the repression of early gene expression, the switch from early to late transcription, and the events that lead to integration, excision, and amplification of the virus DNA within the host chromosome (see refs. 9 and 10 for reviews). Although the SV40 T antigen is sufficient to initiate and maintain transfonnation, the polyoma large T antigen does not cause tumorigenic transformation. It can reduce the serum growth factor requirements of established cell lines an confer on primary rat embryo fibroblasts the ability to grow in long-term culture without entering crisis, a process called establishmentor immortalization(11-1 5 ) . Both polyomavirus and SV40 large T antigens have been described as promiscuous activators of eucaryotic promoters in transient expression experiments (16- 18). Many of the so-called “immortalizingoncogenes” have in common the property to encode proteins that are localized in the cell nucleus and the ability to stimulate transcription from certain viral promoters. It has been suggested that these tram-activating oncogenes immortalizecells because they bind to the promoter region of specific cellular genes and activate or repress their transcription (19). However, recent studies fail to support this hypothesis (10,20-22). The oncogenicpotential of polyomavirus is largely due to the middle T antigen, since the middle T gene alone is capable of transforming murine cell lines in vitro (7)
as well as primary avian cells (23,24). Unlike the large T antigen, which is located exclusively in the nucleus, the middle T antigen is a membrane protein. Its transforming activity is thought to result from its association with cellular tyrosine kinases, principally pp60C-src (25,26). The association has been shown to activate the tyrosine kinase activity of pp6OesrC(27), most likely by preventing negative regulation of pp60c-src bY phosphorylation of residue 527 (28-30). The role of the small T antigen is not clear. The protein is found in the soluble cytoplasmic fraction, and microinjection studies have indicated that it could be required for the characteristic disruption of actin cables in transformed cells (31). A recent study has reported an intracellular function of the SV40 small T antigen: the capacity to trans-activate selected RNA polymerase II and 111-requiringpromoters (32). It is likely that small T transactivates, at least in part, by modifying the activity of selected transcription factors.
MORE THAN ONE ONCOGENE IS REQUIRED FOR EXPRESSION OF A FULLY TRANSFORMED PHENOTYPE An important brealahrough in the analysis of the respective roles of the polyoma early proteins in transformation resulted from the construction in 1981, by R. h e n and his colleagues, of modified polyoma genomes expressing only one of the T antigens (7,8). Transfection of the middle T gene into established rat fibroblast lines (F2408 and FR3T3) conferred on cells loss of topoinhibition and anchorage dependency, which are characteristic of transformed cells. However, different results were obtained when primary cultures of rat embryo fibroblasts were used instead of established cell lines (33). Transfer of the middle T plasmid into primary cells did not produce stable transfonnants, even in the presence of high serum concentrations. Similar results were obtained in our laboratory using an in vivo assay developed to directly test the oncogenicity of both polyoma and SV40 DNA by injecting recombinant DNA into newborn rodents (34). Injection of 0.2-2.0 pg of linear polyoma genomic DNA induced the development of subcutaneous liposarcomas and fibrosarcomas at the site of inoculation. However, injection of the plasmid carrying middle T alone did not produce any tumor (35). Cells of established cell lines transformed in vitro by middle T alone grew into tumors when transplanted in nude mice or syngeneic rats. Thus, tumor induction by injecting DNA into newborn rats provided an in vivo equivalent to the transformation assay
Polyomavirus Transformation but appeared to be a more stringent and rigorous criterion of oncogenictransformation. These experimentsindicated that functions other than those expressed by the middle T antigen were required for the elaboration of all the properties associated with transformation of cells in culture as well as tumor formation in animals.
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COMPLEMENTATION STUDIES The failure of the middle T gene to fully transform prompted the laboratories of F. Cuzin and R. Kamen, as well as our own, to investigate whether a complementary function could be exerted by one of the other two viral early proteins, namely the large and small T antigens. Earlier work by Cuzin and his colleagues had shown that at least the N-terminal region of the large T gene was essential to maintainthe transformed state in lines derived from FR3T3 by infection with the ts-A mutant of polyomavirus (36). These investigators asked whether an immortalization function might by exerted by either the large T or the small T antigen. Immortality which is defined as the unlimited growth potential in culture is exhibited by many tumor cell lines and by nontumorigenic established cell lines such as 3T3 fibroblastic lines. The pioneer work of Vogt and Dulbecco (37) had already demonstratedthat immortality was conferred on primary cells by polyomavirus transformation. In 1983, Rassoulzadegan et al. showed that transfer of the large T plasmid into primary rat embryo fibroblasts enabled a fraction of the cells to grow in sparse culture, whereas neither middle T nor small T had any effect above the background frequency observed with untreated cells (11). Most importantly, the authors also showed that cell lines established by transfer of large T into primary fibroblasts could subsequentlybe transformed by transfer of the middle T plasmid. A paradoxical result was obtained when they tried to transform rat embryo fibroblasts by the simultaneoustransfer of the large and middle T genes (38). No stable transformantcould be isolated by focus formation under these conditions. Combinations of middle T and small T or large T and small T were similarly inefficient. Since efficient transformation could only be achieved by transfer of the polyoma genomic DNA, it was suggested that the small T antigen was also required for transformation of primary rat embryo fibroblasts. Our laboratory, which had taken a different approach, obtained somewhat different results. The failure of middle T alone to induce tumors when injected into newborn rats indicated that functions other than those expressed by the middle T antigen were required for the tumorigenic
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process. To determine whether a complementary function could be exerted by the small T antigen, we evaluated the tumorigenic properties of bc1051, a mutant constructed in G. Magnusson's laboratory (39). Because of a base change (guanine adenine) at nucleotide 410, bc1051 which prevents the splicing of the large T -A, does not express the large T antigen, but does produce the small and middle T antigens. Surprisingly, injection of bc1051 DNA induced tumors in rats with an efficiency comparable to that of wild-type polyoma genomic DNA (40). Although middle T alone was not oncogenic in newborn rats, it induced some tumors when injected into newborn hamsters. The tumors were reduced in numbers and appeared after long latencies (34). Thus, under certain conditions, middle T was suffficient to induce the tumorigenicprocess. This was consistent with the notion that the middle T antigen was primarily responsible for tumor induction by polyomavirus. However, unlike the v-src sequence which did not require other viral genes to induce sarcomas in vivo (41), the polyoma middle T gene was far more tumorigenic in the presence of small T and required the cooperation from the latter to induce tumors in rats. These results contrasted with those of Cuzin's laboratory claiming that all three polyoma early proteins must be simultaneously expressed in order to transform primary cells. It is possible that this disagreement reflected the fact that in vitro transformation, unlike tumorigenesis, required an additional step, provided by the large T antigen, which enabled the cells to grow efficiently in culture. Our studies provided evidence that the polyoma large T antigen could be dispensable in the tumorigenic process. Nevertheless, several attempts were made to complement middle T with large T. We constructed a hybrid plasmid encoding both polyoma middle T and large T, but repeated injections of this recombinant into newborn rats failed to induce tumors (40). It seemed therefore that the experimental conditions did not allow simultaneous expression of both genes in vivo, or that polyoma large T was not sufficient to complement middle T. However, surprising results were obtained with constructs encoding polyoma middle T antigen together with other early proteins from related DNA tumor viruses. Injection of a recombinant carrying polyoma middle T linked to the SV40 genome, induced tumors in newborn rats within the same time and as efficiently as did injection of wiid-type polyoma DNA or bc1051 DNA (40). When inoculated alone, SV40 DNA was not tumorigenic in rats. The plasmids carrying polyoma middle T and SV40 large T (mutant A2005, Ref. 42), or only half of the SV40 early region (deletion between 0.17 and 0.37 map units) were
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also active, although at a lower efficiency. A recombinant encoding middle T and the N-terminal fragment of SV40 large T was equally active. Surprisingly, a complementary effect was also observed with the ElA region of adenovirus DNA (Ad2 or Ad5) (40) as well as with the c-myc oncogene (43). All tumors induced by either wild-type polyoma DNA or bc1051 expressed the middle and small T antigens. The tumors induced by polyomaSV40 or polyoma-adenovirusrecombinants expressed the middle T antigen as well as one or several proteins from the complementing oncogenes (40).
COMPLEMENTATION GROUPS The concept of cooperation or complementation between oncogenes of different classes was put forward in 1983 by Land et al. (44)and Ruley (45) who used two different oncogenes (ras and myc or ras and adenovirus E1A) to transform primary rat embryo fibroblasts in vitro. Like polyoma middle T, the ras oncogene was unable to transform primary cells efficiently,but it could transform after cotransfer of either polyoma large T, ad2-E1A, or the myc oncogene. Thus, some viral and cellular transforming genes could be classified operationally into two complementation groups, each group defining a different step required for transformation. One group contained genes thought to be involved only in immortalization and included myc, ElA, and polyoma and SV40 large T antigens. The second group included genes involved in the tumorigenic conversionof immortalized cells, such as ras and polyoma middle T. It became clear therefore that the in vivo steps in polyomavirus-mediated tumorigenesis depended on additional cellular alterationsbeyond the acquisition of the polyoma transforming gene. Such alterations could be achieved by either polyoma small T or other early gene products from related DNA tumor viruses. Some of these genes, such as SV40 large T and ElA, appeared to be associated with functions that confer on primary cells the ability to grow indefinitely in culture. Small T, however, could not be implicated in immortalization.
COMPLEMENTATION BY POLYOMA LARGE T Although polyoma large T was capable of immortalizing primary cells, the combination of the large and
middle T genes failed to induce tumors in newborn rats (see above). To determine whether this was due to certain experimental conditions that did not allow simultaneous expression of both genes in vivo, or to a failure of large T to complement middle T in tumorigenesis, we attempted to construct large T mutants with increased immortalization potentials. Unlike its function in viral DNA replication, the effect of large T antigen in immortalization could be exerted by truncated forms deleted from the C-terminal part of the protein (36). To localize more precisely the functional domains of large T involved in immortalization, various deletions were generated in both polyoma and SV40 large T genes (12). The region of the SV40 large T antigen involved in immortalization was localized within the first 137 amino acid residues; that of polyoma within the first 150-220 residues. Thus, the domains were encoded by the first large T exon and a small portion of the second exon which was shown later on to include a putative binding site for plO5-RB (see below). A polyoma large T mutant, d197, with a 30 base-pair deletion (nucleotides 1367 through 1396) was of special interest. When introduced into primary rat embryo fibroblastsby DNA transfection, the mutant exhibited an increased immortalization potential (46).Unlike the wildtype large T, dl-97 could fully complement polyoma middle T in the tumorigenic process (in vivo assays) as well as in the transformation of primary cells in vitro. Thus, our work showed that polyoma large T was sufficient to complementmiddle T in transformation, even when both genes were transferred simultaneously into cells. However, the full-sized large T appeared to be very inefficient in the process. Land et al. (44) could not study the activity of the intact protein because of a toxic effect after gene transfer at high multiplicity. This toxicity was not observed when we studied the effect of large T in immortalization, but it was apparent when both middle and large T were transfected with the neo marker in transformation assays. Subsequent studies showed that the activity of dl-97 was due to its inability to replicate and, hence, its inability to exert a cytopathic effect (47). Finally, an important observation that emerged from this study was that only two of the three viral early gene functions were required for polyomavirus-mediatedtransformation. It had been reported that a function of the small T antigen was necessary, in addition to and in conjunction with, the functions of the large and middle T antigens for transformation of primary cells (38). Our results showed that this was not the case.
Polyomavirus Transformation
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ANOMALIES TO THE MODEL OF MULTISTEP CARCINOGENESIS While it has become evident that more than one oncogene was required for full transformation, there were several anomalies in the scheme of multistage carcinogenesis. A first question arose with the classification of oncogenes. Many oncogenes could be grouped into functional classes on the basis of their effects on cellular phenotype, suggesting that there was only a small number of action mechanisms for the oncogene-encodedproteins (3). Some of these proteins mediate distinct and perhaps cooperative transformation-relatedfunctions that appear to correlate with their particular location within the cell (44,45). In the cytoplasm, they may regulate levels of critical second messenger molecules, whereas in the nucleus they probably modulate the activity of the transcriptional machinery (3). The problem with this model can be illustrated by the function of the polyoma small T antigen. Although operationally very similar in tumorigenesis to immortalizationfactors, polyoma small T cannot be implicated in establishmentand immortalization functions. This suggests that all genes which complement the transforming function of middle T do not create identical changes in cellular behavior but may, on the contrary, interact with different cellular targets. Other exceptions include certain pairs of nuclear oncogenes that can collaborate with one another (fos and polyoma large T) (48), and certain nuclear oncogenes that transform spontaneously immortalized cells (49,50). Thus, classificationof oncogenes on the basis of the cellular location of their products is somewhat arbitrary. A second question regarding the requirement of multiple oncogenes arises from observations that a single oncogene can be sufficient for full transformation. Many retrovirusescarrying a single oncogene can induce tumors in vivo (reviewed in Ref. 4). The mutant T24 H-rasl oncogene has been shown to transform early passage rodent cells (51), and middle T alone can induce tumors in newborn hamsters (34). However, as pointed out by Spandidos (52), these results do not really contradict the multistage model of carcinogenesis when both aspects of quantitative (caused by the enhancers) and qualitative (caused by the T24 mutation) changes in the expression of the rm gene are considered. Similarly, the delay in tumor development observed by injecting the middle T gene alone in newborn hamsters is compatible with a multistep process of carcinogenesis. Expression of middle T in the cells of the newborn is not sufficient to induce transformationbecause these cells lack the other viral complementary functions. However, subsequent acquisi-
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tion of a genetic alteration enables cells already producing middle T to express the malignant phenotype. It seems therefore that the temporal order of the two events has little or no importance. Another factor that may provide an explanation for transformation by single oncogenes is the environment of the cell that initially acquires an oncogene. Weinberg (4) points out that cells bearing a single oncogene will not grow into a large mass unless the normal neighboring cells are removed by killing with a cytotoxic drug or by recruiting them into the tumor mass by viral spread in vivo. Thus, the environment of a cell may strongly influence its responsiveness to a given oncogene. Finally, the SV40 large T oncogene provides another anomaly in the scheme of multistage carcinogenesis. Through the actions of a single protein, large T is able to induce nuclear functions (immortalization) and cytoplasmic functions such as anchorage-independent growth (53-55). Lanford and Butel (56) have described a mutation (cT) that inhibits the transport of the T antigen into the nucleus. Transformation of established cell lines by the cT mutant is not significantly reduced under normal culture conditions, but transformationof primary cells does not occur in the absence of detectable levels of nuclear T antigen (57). Thus, it seems that transformation by SV40 is regulated by both nuclear and plasma membrane-associatedT antigen. This model predicts that the cT antigen would be very ineffective both in immortalization and in complementing polyoma middle T in tumorigenesis. However, this was not the case. The cT antigen had the capacity to complement middle T in tumorigenesis and to immortalize primary rat embryo fibroblasts provided it was transfected along with pSV2". These observations did not completely exclude the possibility that undetectable yet biologically functional amounts of cT antigen accumulated in the nucleus. However, they did not confirm the claim (16) that the capacity of the cT antigen to immortalize was dependent upon the ability of a subpopulationof the transfected cells to transport the antigen into the nucleus. If both the immortalization of primary cells and the complementation of polyoma middle T in tumorigenesis were due to undetectable levels of nuclear cT antigen, one would expect the cT mutant to be less active in these processes than was the wild-type genome. This assumption was based upon other experiments with polyomavirus large T showing that the ability of primary cells containing the large T gene to become immortalized was related to the level of large T expression (12). Thus, under certain conditions, such as those involved in cotransfection with a dominant selection marker or in DNA-mediated tumor
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induction, the cellular alterations achieved by nonnuclear oncogenes, for instance small T and SV40 (cT), could be sufficientto complement polyoma middle T in transformation and tumorigenesis. It should be pointed out, however, that the nonkaryophilic T antigen described by Fischer-Fantuzzi and Vesco (58) has yielded ambiguous results. This mutant, which lacks amino acids 110 to 152 and has no DNA binding activity, has been reported to immortalizeprimary cells (59). However, when tested in our laboratory, it failed to exhibit any activity in immortalization or in complementing middle T (Vass-Marengo, unpublished results). The reason for this discrepancy is not clear. It may be due to a difference in the stringency of immortalizationassays. Whatever the reason, it seems that the amino acids surrounding LYSlzs have another role besides that of providing a nuclear location signal. The amino terminal domain consisting of the first 121 amino acids can transform established cells quite efficiently even though the polypeptide does not bind p53 (60). A possible clue concerning the role of this region in immortalization is provided by studies showing that both E1A and SV40 large T antigen form stable complexes with p 105-RB, the retinoblastoma susceptibility gene product (61,62) (see below), and that this interaction is important for transformation. Thus, it is tempting to speculate that the nonnuclear T antigen functions in transformation by its capacity to complex with and inactivate plO5-RB in the cytoplasm.
INTERACTIONS WITH pRB The binding sites for plO5-RB include a portion of conserved regions 1 and 2 of E1A which show considerable similarity to small regions of amino acid sequences conserved among papovaviruses such as human papillomavirus-16, SV40and polyomavirus (63-65). Recent studies indicate that RB inactivation may also be the mechanism whereby polyoma large T exerts its action in transformation. Deletion of amino acids 141 to 146, the putative RB-binding site on polyoma large T antigen, inactivates its immortalizationpotential (22). The pRB binding properties of various large T mutants have been assessed using an in vitro coimmunoprecipitation assay (66). pRB binding is readily detected with wild-type large T but coprecipitation is completely abolished by as little as a single amino acid substitution (asp 141 glu or glu 146 asp) in region 2 of the polyoma large T antigen. Mutants defective in pRB binding are unable to immortalize primary rat embryo fibroblasts, suggesting that
association with pRB is an important component of immortalization mediated by polyoma large T. The mutations in region 1 affect pRB binding only marginally; yet some of them severely impair immortalization, indicating that pRB binding may be essential but not sufficient for immortalization. A recent report by Chen and Paucha (67) claims that some SV40 mutants with deletions affecting the pRB binding site are able to rescue primary rat and mouse embryo fibroblasts from senescence without conferring upon them a typical transformed phenotype. These results imply that the ability of SV40 to immortalize is independent of its ability to bind to the Rl3 gene product. The domain responsiblefor this activity has not been formally identified but it is conceivable that it is the p53-binding domain. This suggests that the function specified by either the N-terminal or the carboxy-terminal domain of SV40 T-Ag can be sufficient to elicit a partially transformed phenotype, such as immortalization, but that both domains are required for full transformation.
CONCLUDING REMARKS To date, as many as 40 distinct oncogenes of viral and cellular origin have been identified and divided into a small number of functional classes on the basis of their effects on cellular phenotype, suggesting a rather small number of action mechanisms of the oncogene products. Of particular significance is the discovery that the polyomavirus middle T antigen mediates transformation through an interaction with a proto-oncogene @p60c-src), and that the complementing viral oncogenes are found complexed with other cellular proteins @53or plO5-RB, or both), which have been identified as antioncogenes or tumor suppressors (reviewed in Refs. 4 and 68). It is fascinating that different DNA tumor viruses have developed oncoproteins that use a common molecular mechanism for disrupting cell growth control. Obviously, this is indicative of the key position plO5-RB must occupy in the control of cell growth, and the nature of the cooperation between cellular oncogenes and antioncogenes to create the fully malignant phenotype. When cancer researchers, having identified viral and cellular oncogenes, moved into a new phase of investigationsto confront mechanistic problems, they did not expect to find a link betweeen DNA tumor viruses, the retinoblastoma and other forms of human cancer. This is particularly rewarding for those studying tumor viruses as models of carcinogenesisbecause it offers great hope for further insight into the cause of human cancer,
Polyomavirus Transformation
ACKNOWLEDGMENTS Research in the author's labmatory is sponsoredby the National Cancer Institute and the Medical Research Council of Canada. The author thanks C. Asselin, L. Bouchard, C. Gelinas, A. Larose, C. Roberge, L. StOnge, M. Sullivan, and J. Vass-Marengo for stimulating discussions.
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1986. 47. Roberge C, Bastin M: Site-directed mutagenesis of the polyomavirus genome: replication-rkfectivelarge T mutants with increased immortalization potential. Virology 162:144-150, 1988. 48. Jenuwein T, Muller D, Curran T et al: Extended life span and tumorigenesis of nonestablished m u s e connective tissue cells transformed by thefos oncogeneof FBR-MuSV. Cell 416294537,
1985. 49. Eliyahu D, Michalovitz D, Oren M: Overproductionof p53 antigen makes established cells highly tumorigenic. Nature
316:158-160,1985. 50. Keath El, Caimi PG, Cole MD: Fibroblast lines expressing activated c-myc oncd$enes are tumorigenic in nude mice and syngeneic animals. Cell 39:339-348. 51. Spandidos DA, Wilkie NM: Malignant transformation of early passage rodent cells by a single mutated human oncogene. Nature
310:469-475,1984. 52. Spandidos DA: Ras oncogenes in cell transformation. IS1 Atlas of Science. Immunology 1:l-6, 1988. 53. Colby WW, Shenk T: Fragments of the simian virus 40 transforming gene facilitate transformation of rat embryo cells. Proc Natl Acad Sci (USA) 795189-5193, 1982.
54. Kriegler M, Perez CF, Hardy C et al: Transformation mediated by the SV40 T antigens: Separation of the overlapping SV40 early genes with a retroviral vector. Cell 38:483-491, 1984. 55. Petit CA, Gardes M, Feunteun J: Immortalization of rodent embryo fibroblasts by SV40 is maintained by the A gene. Virology
127~74-82,1983. 56. Landford RE,Butel JS: Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen. Cell 37:801-813,1984. 57. Landford RE, Wong C, Butel JS: Differentialability of a T-antigen transportdefective mutant of simian v h 40 to transform primary and established rodent cells. Mol Cell Biol5: 1043-1050, 1985. 58. Fischer-Fantuzzi L, Vesco C: Deletion of 43 amino acids in the NH2-terminal half of the large tumor antigen of simian virus 40 results in a nonkaryophilic protein capable of transforming established cells. Proc Natl Acad Sci (USA) 82:1891-1895,
1985. 59. Fischer-Fantuzzi L,Vpsco C: A nonkaryophilic T antigen of SV40 can either i m r t a l i z e or transform rodent cells, and cooperates
better with cytoplasmicthan with nuclear oncoproteins. Oncogene Res 1:229-242,1987. 60. Srinivasan A, M e n KW, Pipas JM: The large tumor antigen of simian virus 40 encodes at least two distinct transforming functions. J Virol 635459-5463, 1989. 61. Whyte P, Buchkovich KJ, Horowitz JM et al: Associationbetween an oncogene and an antioncogene: the adenovirus ElA proteins bind to the retinoblastoma gene product. Nature 334:124-129, 1988. 62. DeCaprio JA, Ludlow JW, Figge J et al: SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell 54275-283, 1988. 63. Moran E: A region of SV40 large T antigen can substitute for a transforming domain of the adenovirus ElA product. Nature 334:168-170,1988. 64. Dyson N, Howley PM, Munger K et al: The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243:934-936, 1989. 65. Dyson N, Bemards R, Friend SH et al: Large T antigens of many polyomaviruses are able to form complexes with the retinoblastoma protein. J Vird 64:1353-1356, 1990. 66. Larose A, Dyson N, Sullivan M et al: Polyoma large T mutants affected in pRB binding are defective in immortalization. J Virol 65:2308-2313,1991. 67. Chen S, Paucha E: Identification of a region of simian virus 40 large T antigen required for cell transformation. J Virol 64:3350-3357,1990. 68. Lane D, Benchimol S: p53: oncogene or anti-oncogene? Genes D ~ 4:1-8, v 1990.
MINISERIES IN MOLECULAR BIOLOGY John C. Bell, Ph.D. and Nahum Sonenberg, Ph.D., Guest Editors
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Polyomavirus-Mediated Transformation: A Model of Multistep Carcinogenesis Marcel Bastin, D.Sc. Department of Microbiology Faculty of Mediclne University of Sherbrooke Sherbrooke, Quebec J1 H 5N4 Canada
INTRODUCTION
GENETIC STRUCTURE OF POLYOMAVIRUS
There is overwhelmingevidence that carcinogenesis occurs as a progressiveprocess, involving multiple independent events. The process has been formally divided into two steps, as it requires at least two distinct types of stimulus (1). The first, initiation, is thought to be an absolute requirement involving mutational events in as-yet unknown genes. The second, promotion or completion, involves subsequentaltemtions that genemte heterogeneity and results, in combination with the selectivityof the host environment, in the emergence of subpopulationsof cells with increased malignancy and metastatic potential (2). It is likely that activated proto-oncogenes play a major role not only as initiators but also as completion genes in multistep carcinogenesis(3,4). The role of specific viral and cellular oncogenes in different stages of neoplastic development has been addressed in several laboratories. This review examines how studies on viral oncogenes, particularly the polyomavirus oncogenes, have helped establish the concept that carcinogenesis is a multistep process at the molecular level.
Polyomavirusprovides a useful model of multistep carcinogenesis. It is a papovavirus that can transform the growth properties of rodent cells in culture and form tumors in animals. Transformation is the result of stable integration of the viral DNA sequences into the host genome and subsequent expression of the viral early region (5). This region, which is about 3 kb in length, corresponds to the part of the genome transcribed during the early phase of the lytic cycle in permissive mouse cells (Fig. 1). It encodes three distinct proteins, in alternate translational reading frames, via the production of distinct mRNAs by means of different splicing processes operating on a common primary transcript (6). They are referred to as the large T (MW = lOOK), middle T (MW = 56K),and small T (MW = 22K) antigens. The role of the T antigens in oncogenic transformation has been difficult to establish because they are encoded by extensively overlapping DNA sequences. However, the properties of all three proteins can now be studied individually thanks to the cloning of the three cDNAs and
85 Copyright 0 1592 by Marcel Dekker, Inc.
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Figure 1. Map of the polyomavirus early region. The nucleotide numbers are shown on the top line, and the three differentspliced m R N h are aligned with the map. Boxes represent the encoding sequences for the three T antigens with different symbols according to the reading frame used. Nontrmlated regions are represented by continuous lines. Upward deflections denote the intervening sequences.
the construction of modified genomes that separately encode the T antigens (7,8). The polyomavirus large T antigen is a nuclear phosphoprotein of 785 amino acids. Like the SV40 T antigen, it exists as multiple molecular species and its activity has been implicated in several processes that occur after infection, includingthe stimulation of host DNA synthesis, the initiation of viral DNA replication, the repression of early gene expression, the switch from early to late transcription, and the events that lead to integration, excision, and amplification of the virus DNA within the host chromosome (see refs. 9 and 10 for reviews). Although the SV40 T antigen is sufficient to initiate and maintain transfonnation, the polyoma large T antigen does not cause tumorigenic transformation. It can reduce the serum growth factor requirements of established cell lines an confer on primary rat embryo fibroblasts the ability to grow in long-term culture without entering crisis, a process called establishmentor immortalization(11-1 5 ) . Both polyomavirus and SV40 large T antigens have been described as promiscuous activators of eucaryotic promoters in transient expression experiments (16- 18). Many of the so-called “immortalizingoncogenes” have in common the property to encode proteins that are localized in the cell nucleus and the ability to stimulate transcription from certain viral promoters. It has been suggested that these tram-activating oncogenes immortalizecells because they bind to the promoter region of specific cellular genes and activate or repress their transcription (19). However, recent studies fail to support this hypothesis (10,20-22). The oncogenicpotential of polyomavirus is largely due to the middle T antigen, since the middle T gene alone is capable of transforming murine cell lines in vitro (7)
as well as primary avian cells (23,24). Unlike the large T antigen, which is located exclusively in the nucleus, the middle T antigen is a membrane protein. Its transforming activity is thought to result from its association with cellular tyrosine kinases, principally pp60C-src (25,26). The association has been shown to activate the tyrosine kinase activity of pp6OesrC(27), most likely by preventing negative regulation of pp60c-src bY phosphorylation of residue 527 (28-30). The role of the small T antigen is not clear. The protein is found in the soluble cytoplasmic fraction, and microinjection studies have indicated that it could be required for the characteristic disruption of actin cables in transformed cells (31). A recent study has reported an intracellular function of the SV40 small T antigen: the capacity to trans-activate selected RNA polymerase II and 111-requiringpromoters (32). It is likely that small T transactivates, at least in part, by modifying the activity of selected transcription factors.
MORE THAN ONE ONCOGENE IS REQUIRED FOR EXPRESSION OF A FULLY TRANSFORMED PHENOTYPE An important brealahrough in the analysis of the respective roles of the polyoma early proteins in transformation resulted from the construction in 1981, by R. h e n and his colleagues, of modified polyoma genomes expressing only one of the T antigens (7,8). Transfection of the middle T gene into established rat fibroblast lines (F2408 and FR3T3) conferred on cells loss of topoinhibition and anchorage dependency, which are characteristic of transformed cells. However, different results were obtained when primary cultures of rat embryo fibroblasts were used instead of established cell lines (33). Transfer of the middle T plasmid into primary cells did not produce stable transfonnants, even in the presence of high serum concentrations. Similar results were obtained in our laboratory using an in vivo assay developed to directly test the oncogenicity of both polyoma and SV40 DNA by injecting recombinant DNA into newborn rodents (34). Injection of 0.2-2.0 pg of linear polyoma genomic DNA induced the development of subcutaneous liposarcomas and fibrosarcomas at the site of inoculation. However, injection of the plasmid carrying middle T alone did not produce any tumor (35). Cells of established cell lines transformed in vitro by middle T alone grew into tumors when transplanted in nude mice or syngeneic rats. Thus, tumor induction by injecting DNA into newborn rats provided an in vivo equivalent to the transformation assay
Polyomavirus Transformation but appeared to be a more stringent and rigorous criterion of oncogenictransformation. These experimentsindicated that functions other than those expressed by the middle T antigen were required for the elaboration of all the properties associated with transformation of cells in culture as well as tumor formation in animals.
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COMPLEMENTATION STUDIES The failure of the middle T gene to fully transform prompted the laboratories of F. Cuzin and R. Kamen, as well as our own, to investigate whether a complementary function could be exerted by one of the other two viral early proteins, namely the large and small T antigens. Earlier work by Cuzin and his colleagues had shown that at least the N-terminal region of the large T gene was essential to maintainthe transformed state in lines derived from FR3T3 by infection with the ts-A mutant of polyomavirus (36). These investigators asked whether an immortalization function might by exerted by either the large T or the small T antigen. Immortality which is defined as the unlimited growth potential in culture is exhibited by many tumor cell lines and by nontumorigenic established cell lines such as 3T3 fibroblastic lines. The pioneer work of Vogt and Dulbecco (37) had already demonstratedthat immortality was conferred on primary cells by polyomavirus transformation. In 1983, Rassoulzadegan et al. showed that transfer of the large T plasmid into primary rat embryo fibroblasts enabled a fraction of the cells to grow in sparse culture, whereas neither middle T nor small T had any effect above the background frequency observed with untreated cells (11). Most importantly, the authors also showed that cell lines established by transfer of large T into primary fibroblasts could subsequentlybe transformed by transfer of the middle T plasmid. A paradoxical result was obtained when they tried to transform rat embryo fibroblasts by the simultaneoustransfer of the large and middle T genes (38). No stable transformantcould be isolated by focus formation under these conditions. Combinations of middle T and small T or large T and small T were similarly inefficient. Since efficient transformation could only be achieved by transfer of the polyoma genomic DNA, it was suggested that the small T antigen was also required for transformation of primary rat embryo fibroblasts. Our laboratory, which had taken a different approach, obtained somewhat different results. The failure of middle T alone to induce tumors when injected into newborn rats indicated that functions other than those expressed by the middle T antigen were required for the tumorigenic
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process. To determine whether a complementary function could be exerted by the small T antigen, we evaluated the tumorigenic properties of bc1051, a mutant constructed in G. Magnusson's laboratory (39). Because of a base change (guanine adenine) at nucleotide 410, bc1051 which prevents the splicing of the large T -A, does not express the large T antigen, but does produce the small and middle T antigens. Surprisingly, injection of bc1051 DNA induced tumors in rats with an efficiency comparable to that of wild-type polyoma genomic DNA (40). Although middle T alone was not oncogenic in newborn rats, it induced some tumors when injected into newborn hamsters. The tumors were reduced in numbers and appeared after long latencies (34). Thus, under certain conditions, middle T was suffficient to induce the tumorigenicprocess. This was consistent with the notion that the middle T antigen was primarily responsible for tumor induction by polyomavirus. However, unlike the v-src sequence which did not require other viral genes to induce sarcomas in vivo (41), the polyoma middle T gene was far more tumorigenic in the presence of small T and required the cooperation from the latter to induce tumors in rats. These results contrasted with those of Cuzin's laboratory claiming that all three polyoma early proteins must be simultaneously expressed in order to transform primary cells. It is possible that this disagreement reflected the fact that in vitro transformation, unlike tumorigenesis, required an additional step, provided by the large T antigen, which enabled the cells to grow efficiently in culture. Our studies provided evidence that the polyoma large T antigen could be dispensable in the tumorigenic process. Nevertheless, several attempts were made to complement middle T with large T. We constructed a hybrid plasmid encoding both polyoma middle T and large T, but repeated injections of this recombinant into newborn rats failed to induce tumors (40). It seemed therefore that the experimental conditions did not allow simultaneous expression of both genes in vivo, or that polyoma large T was not sufficient to complement middle T. However, surprising results were obtained with constructs encoding polyoma middle T antigen together with other early proteins from related DNA tumor viruses. Injection of a recombinant carrying polyoma middle T linked to the SV40 genome, induced tumors in newborn rats within the same time and as efficiently as did injection of wiid-type polyoma DNA or bc1051 DNA (40). When inoculated alone, SV40 DNA was not tumorigenic in rats. The plasmids carrying polyoma middle T and SV40 large T (mutant A2005, Ref. 42), or only half of the SV40 early region (deletion between 0.17 and 0.37 map units) were
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also active, although at a lower efficiency. A recombinant encoding middle T and the N-terminal fragment of SV40 large T was equally active. Surprisingly, a complementary effect was also observed with the ElA region of adenovirus DNA (Ad2 or Ad5) (40) as well as with the c-myc oncogene (43). All tumors induced by either wild-type polyoma DNA or bc1051 expressed the middle and small T antigens. The tumors induced by polyomaSV40 or polyoma-adenovirusrecombinants expressed the middle T antigen as well as one or several proteins from the complementing oncogenes (40).
COMPLEMENTATION GROUPS The concept of cooperation or complementation between oncogenes of different classes was put forward in 1983 by Land et al. (44)and Ruley (45) who used two different oncogenes (ras and myc or ras and adenovirus E1A) to transform primary rat embryo fibroblasts in vitro. Like polyoma middle T, the ras oncogene was unable to transform primary cells efficiently,but it could transform after cotransfer of either polyoma large T, ad2-E1A, or the myc oncogene. Thus, some viral and cellular transforming genes could be classified operationally into two complementation groups, each group defining a different step required for transformation. One group contained genes thought to be involved only in immortalization and included myc, ElA, and polyoma and SV40 large T antigens. The second group included genes involved in the tumorigenic conversionof immortalized cells, such as ras and polyoma middle T. It became clear therefore that the in vivo steps in polyomavirus-mediated tumorigenesis depended on additional cellular alterationsbeyond the acquisition of the polyoma transforming gene. Such alterations could be achieved by either polyoma small T or other early gene products from related DNA tumor viruses. Some of these genes, such as SV40 large T and ElA, appeared to be associated with functions that confer on primary cells the ability to grow indefinitely in culture. Small T, however, could not be implicated in immortalization.
COMPLEMENTATION BY POLYOMA LARGE T Although polyoma large T was capable of immortalizing primary cells, the combination of the large and
middle T genes failed to induce tumors in newborn rats (see above). To determine whether this was due to certain experimental conditions that did not allow simultaneous expression of both genes in vivo, or to a failure of large T to complement middle T in tumorigenesis, we attempted to construct large T mutants with increased immortalization potentials. Unlike its function in viral DNA replication, the effect of large T antigen in immortalization could be exerted by truncated forms deleted from the C-terminal part of the protein (36). To localize more precisely the functional domains of large T involved in immortalization, various deletions were generated in both polyoma and SV40 large T genes (12). The region of the SV40 large T antigen involved in immortalization was localized within the first 137 amino acid residues; that of polyoma within the first 150-220 residues. Thus, the domains were encoded by the first large T exon and a small portion of the second exon which was shown later on to include a putative binding site for plO5-RB (see below). A polyoma large T mutant, d197, with a 30 base-pair deletion (nucleotides 1367 through 1396) was of special interest. When introduced into primary rat embryo fibroblastsby DNA transfection, the mutant exhibited an increased immortalization potential (46).Unlike the wildtype large T, dl-97 could fully complement polyoma middle T in the tumorigenic process (in vivo assays) as well as in the transformation of primary cells in vitro. Thus, our work showed that polyoma large T was sufficient to complementmiddle T in transformation, even when both genes were transferred simultaneously into cells. However, the full-sized large T appeared to be very inefficient in the process. Land et al. (44) could not study the activity of the intact protein because of a toxic effect after gene transfer at high multiplicity. This toxicity was not observed when we studied the effect of large T in immortalization, but it was apparent when both middle and large T were transfected with the neo marker in transformation assays. Subsequent studies showed that the activity of dl-97 was due to its inability to replicate and, hence, its inability to exert a cytopathic effect (47). Finally, an important observation that emerged from this study was that only two of the three viral early gene functions were required for polyomavirus-mediatedtransformation. It had been reported that a function of the small T antigen was necessary, in addition to and in conjunction with, the functions of the large and middle T antigens for transformation of primary cells (38). Our results showed that this was not the case.
Polyomavirus Transformation
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ANOMALIES TO THE MODEL OF MULTISTEP CARCINOGENESIS While it has become evident that more than one oncogene was required for full transformation, there were several anomalies in the scheme of multistage carcinogenesis. A first question arose with the classification of oncogenes. Many oncogenes could be grouped into functional classes on the basis of their effects on cellular phenotype, suggesting that there was only a small number of action mechanisms for the oncogene-encodedproteins (3). Some of these proteins mediate distinct and perhaps cooperative transformation-relatedfunctions that appear to correlate with their particular location within the cell (44,45). In the cytoplasm, they may regulate levels of critical second messenger molecules, whereas in the nucleus they probably modulate the activity of the transcriptional machinery (3). The problem with this model can be illustrated by the function of the polyoma small T antigen. Although operationally very similar in tumorigenesis to immortalizationfactors, polyoma small T cannot be implicated in establishmentand immortalization functions. This suggests that all genes which complement the transforming function of middle T do not create identical changes in cellular behavior but may, on the contrary, interact with different cellular targets. Other exceptions include certain pairs of nuclear oncogenes that can collaborate with one another (fos and polyoma large T) (48), and certain nuclear oncogenes that transform spontaneously immortalized cells (49,50). Thus, classificationof oncogenes on the basis of the cellular location of their products is somewhat arbitrary. A second question regarding the requirement of multiple oncogenes arises from observations that a single oncogene can be sufficient for full transformation. Many retrovirusescarrying a single oncogene can induce tumors in vivo (reviewed in Ref. 4). The mutant T24 H-rasl oncogene has been shown to transform early passage rodent cells (51), and middle T alone can induce tumors in newborn hamsters (34). However, as pointed out by Spandidos (52), these results do not really contradict the multistage model of carcinogenesis when both aspects of quantitative (caused by the enhancers) and qualitative (caused by the T24 mutation) changes in the expression of the rm gene are considered. Similarly, the delay in tumor development observed by injecting the middle T gene alone in newborn hamsters is compatible with a multistep process of carcinogenesis. Expression of middle T in the cells of the newborn is not sufficient to induce transformationbecause these cells lack the other viral complementary functions. However, subsequent acquisi-
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tion of a genetic alteration enables cells already producing middle T to express the malignant phenotype. It seems therefore that the temporal order of the two events has little or no importance. Another factor that may provide an explanation for transformation by single oncogenes is the environment of the cell that initially acquires an oncogene. Weinberg (4) points out that cells bearing a single oncogene will not grow into a large mass unless the normal neighboring cells are removed by killing with a cytotoxic drug or by recruiting them into the tumor mass by viral spread in vivo. Thus, the environment of a cell may strongly influence its responsiveness to a given oncogene. Finally, the SV40 large T oncogene provides another anomaly in the scheme of multistage carcinogenesis. Through the actions of a single protein, large T is able to induce nuclear functions (immortalization) and cytoplasmic functions such as anchorage-independent growth (53-55). Lanford and Butel (56) have described a mutation (cT) that inhibits the transport of the T antigen into the nucleus. Transformation of established cell lines by the cT mutant is not significantly reduced under normal culture conditions, but transformationof primary cells does not occur in the absence of detectable levels of nuclear T antigen (57). Thus, it seems that transformation by SV40 is regulated by both nuclear and plasma membrane-associatedT antigen. This model predicts that the cT antigen would be very ineffective both in immortalization and in complementing polyoma middle T in tumorigenesis. However, this was not the case. The cT antigen had the capacity to complement middle T in tumorigenesis and to immortalize primary rat embryo fibroblasts provided it was transfected along with pSV2". These observations did not completely exclude the possibility that undetectable yet biologically functional amounts of cT antigen accumulated in the nucleus. However, they did not confirm the claim (16) that the capacity of the cT antigen to immortalize was dependent upon the ability of a subpopulationof the transfected cells to transport the antigen into the nucleus. If both the immortalization of primary cells and the complementation of polyoma middle T in tumorigenesis were due to undetectable levels of nuclear cT antigen, one would expect the cT mutant to be less active in these processes than was the wild-type genome. This assumption was based upon other experiments with polyomavirus large T showing that the ability of primary cells containing the large T gene to become immortalized was related to the level of large T expression (12). Thus, under certain conditions, such as those involved in cotransfection with a dominant selection marker or in DNA-mediated tumor
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induction, the cellular alterations achieved by nonnuclear oncogenes, for instance small T and SV40 (cT), could be sufficientto complement polyoma middle T in transformation and tumorigenesis. It should be pointed out, however, that the nonkaryophilic T antigen described by Fischer-Fantuzzi and Vesco (58) has yielded ambiguous results. This mutant, which lacks amino acids 110 to 152 and has no DNA binding activity, has been reported to immortalizeprimary cells (59). However, when tested in our laboratory, it failed to exhibit any activity in immortalization or in complementing middle T (Vass-Marengo, unpublished results). The reason for this discrepancy is not clear. It may be due to a difference in the stringency of immortalizationassays. Whatever the reason, it seems that the amino acids surrounding LYSlzs have another role besides that of providing a nuclear location signal. The amino terminal domain consisting of the first 121 amino acids can transform established cells quite efficiently even though the polypeptide does not bind p53 (60). A possible clue concerning the role of this region in immortalization is provided by studies showing that both E1A and SV40 large T antigen form stable complexes with p 105-RB, the retinoblastoma susceptibility gene product (61,62) (see below), and that this interaction is important for transformation. Thus, it is tempting to speculate that the nonnuclear T antigen functions in transformation by its capacity to complex with and inactivate plO5-RB in the cytoplasm.
INTERACTIONS WITH pRB The binding sites for plO5-RB include a portion of conserved regions 1 and 2 of E1A which show considerable similarity to small regions of amino acid sequences conserved among papovaviruses such as human papillomavirus-16, SV40and polyomavirus (63-65). Recent studies indicate that RB inactivation may also be the mechanism whereby polyoma large T exerts its action in transformation. Deletion of amino acids 141 to 146, the putative RB-binding site on polyoma large T antigen, inactivates its immortalizationpotential (22). The pRB binding properties of various large T mutants have been assessed using an in vitro coimmunoprecipitation assay (66). pRB binding is readily detected with wild-type large T but coprecipitation is completely abolished by as little as a single amino acid substitution (asp 141 glu or glu 146 asp) in region 2 of the polyoma large T antigen. Mutants defective in pRB binding are unable to immortalize primary rat embryo fibroblasts, suggesting that
association with pRB is an important component of immortalization mediated by polyoma large T. The mutations in region 1 affect pRB binding only marginally; yet some of them severely impair immortalization, indicating that pRB binding may be essential but not sufficient for immortalization. A recent report by Chen and Paucha (67) claims that some SV40 mutants with deletions affecting the pRB binding site are able to rescue primary rat and mouse embryo fibroblasts from senescence without conferring upon them a typical transformed phenotype. These results imply that the ability of SV40 to immortalize is independent of its ability to bind to the Rl3 gene product. The domain responsiblefor this activity has not been formally identified but it is conceivable that it is the p53-binding domain. This suggests that the function specified by either the N-terminal or the carboxy-terminal domain of SV40 T-Ag can be sufficient to elicit a partially transformed phenotype, such as immortalization, but that both domains are required for full transformation.
CONCLUDING REMARKS To date, as many as 40 distinct oncogenes of viral and cellular origin have been identified and divided into a small number of functional classes on the basis of their effects on cellular phenotype, suggesting a rather small number of action mechanisms of the oncogene products. Of particular significance is the discovery that the polyomavirus middle T antigen mediates transformation through an interaction with a proto-oncogene @p60c-src), and that the complementing viral oncogenes are found complexed with other cellular proteins @53or plO5-RB, or both), which have been identified as antioncogenes or tumor suppressors (reviewed in Refs. 4 and 68). It is fascinating that different DNA tumor viruses have developed oncoproteins that use a common molecular mechanism for disrupting cell growth control. Obviously, this is indicative of the key position plO5-RB must occupy in the control of cell growth, and the nature of the cooperation between cellular oncogenes and antioncogenes to create the fully malignant phenotype. When cancer researchers, having identified viral and cellular oncogenes, moved into a new phase of investigationsto confront mechanistic problems, they did not expect to find a link betweeen DNA tumor viruses, the retinoblastoma and other forms of human cancer. This is particularly rewarding for those studying tumor viruses as models of carcinogenesisbecause it offers great hope for further insight into the cause of human cancer,
Polyomavirus Transformation
ACKNOWLEDGMENTS Research in the author's labmatory is sponsoredby the National Cancer Institute and the Medical Research Council of Canada. The author thanks C. Asselin, L. Bouchard, C. Gelinas, A. Larose, C. Roberge, L. StOnge, M. Sullivan, and J. Vass-Marengo for stimulating discussions.
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