DNA tumor viruses such as simian virus 40 (SV40) express dominant acting oncoproteins that exert their effects by associating with key cellular targets and altering the signaling pathways they govern. Thus, tumor viruses have proved to be invaluable aids in identifying proteins that participate in tumorigenesis, and in understanding the molecular basis for the transformed phenotype. The roles played by the SV40-encoded 708 amino-acid large T antigen (T antigen), and 174 amino acid small T antigen (t antigen), in transformation have been examined extensively. These studies have firmly established that large T antigen's inhibition of the p53 and Rb-family of tumor suppressors and small T antigen's action on the pp2A phosphatase, are important for SV40-induced transformation. It is not yet clear if the Rb, p53 and pp2A proteins are the only targets through which SV40 transforms cells, or whether additional targets await discovery. Finally, expression of SV40 oncoproteins in transgenic mice results in effects ranging from hyperplasia to invasive carcinoma accompanied by metastasis, depending on the tissue in which they are expressed. Thus, the consequences of SV40 action on these targets depend on the cell type being studied. The identification of additional cellular targets important for transformation, and understanding the molecular basis for the cell type-specific action of the viral T antigens are two important areas through which SV40 will continue to contribute to our understanding of cancer.
Simian virus 40 (SV40) is a member of the Polyomaviridae, a family of viruses characterized by small icosahedral virions and a circular double-stranded DNA genome of about 5 kb. The SV40 genome consists of 5243 bp and like other members of the family encodes three proteins that are structural components of the virion (VP1, VP2, VP3) and two nonstructural proteins termed large T antigen (T antigen) and small T antigen (t antigen). In addition, some members of the Polyomaviridae encode additional proteins that are virus specific or are encoded by a subset of viruses in the family. For example, SV40 encodes three such proteins: agnoprotein, 17K T, and small leader protein. The genomes of all polyomaviruses can be divided into three elements: an early coding unit, a late coding unit, and a regulatory region (Figure 1). In a productive infection, expression of the early coding unit is effected by the cellular transcription apparatus and results in expression of the large, small and 17K T antigens. Expression of the late coding unit, which encodes VP1, VP2, VP3, and the agnoprotein requires an active T antigen in conjunction with the cellular transcriptional apparatus. In addition to activating transcription from the viral late promoter, T antigen also functions in viral DNA replication and virion assembly.
Little is known about the natural history of SV40 infection in the wild. In its natural host, the Rhesus macaque, SV40 is thought to infect the terminally differentiated, growth-arrested epithelial cells of the kidney. Since these cells are quiescent they do not express proteins involved in DNA replication, nucleotide metabolism, or chromatin assembly, proteins that are used by SV40 to replicate viral DNA and assemble new virions. However, the large and small T antigens cooperate to drive infected cells into S-phase thus providing the raw materials needed for progeny virus production. Cells often respond by activating multiple defense systems that guard against abnormal cell proliferation and/or virus infection. The T antigen proteins and virus-encoded micro-RNAs act to neutralize or ameliorate these defenses allowing productive infection to proceed.
Shortly after its discovery SV40 was shown to induce tumors when injected into newborn hamsters or immunocompromised mice (Eddy et al., 1962; Girardi et al., 1962). The ability to induce tumors and the types of tumors observed depended on the route of infection, amount of virus, and immune status of the test animals. SV40 was shown to induce an array of different tumor types including osteosarcoma, mesothelioma, lymphoma, and choroid plexus neoplasia (reviewed by Arrington and Butel, 2001). SV40 also induces neoplastic transformation in cell culture as assessed by a number of different assays. Genetic analysis has shown that expression of the large T antigen protein is necessary and often sufficient for transformation by SV40. Under many circumstances both large and small T antigen expression is required for transformation.
SV40 transforms cells that are nonpermissive for viral productive infection
Infection of permissive cells, such as the CV1 or BSC40 lines of African green monkey kidney cells, results in cell death and the production of about 300 infectious progeny virions per infected cell (Figure 2a). As with all viruses, many factors determine whether or not the outcome of infection is successful. First, exposure of the cell to virus particles must result in the attachment of virions to the cell surface. For this to occur the cell must express the appropriate receptors for the virus. Thus, cell types lacking a functional receptor cannot be infected or transformed by SV40 virus particles. Following attachment, virions must penetrate the plasma membrane and be transported to the nucleus. Upon arrival in the nucleus SV40 chromatin is released and transcription from the early promoter is initiated. This results in expression of the large and small T antigen proteins, which act to drive the cells into S-phase. Large T antigen also functions to initiate and maintain viral DNA replication and to activate transcription from the late promoter. As the level of the structural proteins rises, progeny virions are assembled. In culture, cell death occurs about 96 h postinfection.
SV40 infection of rodent cells does not result in cell death and no progeny virions are produced (Figure 2b). The basis for this restriction is poorly understood but the consequences are a failure to initiate viral DNA replication and to activate transcription from the late promoter. On the other hand the early events of virus infection appear to occur normally in these cells. Virus particles attach to the cell surface, are transported to the nucleus and uncoated, and the T antigens are produced. Furthermore, T antigen synthesis results in the infected cells entering S-phase. For example, when quiescent mouse embryo fibroblasts (MEFs) are infected with a high multiplicity of infection of SV40 they are driven into S-phase and the entire cell population appears to become transformed. However, since viral DNA cannot replicate in these cells T antigen expression is eventually lost due to dilution as the cells divide or due to degradation of viral DNA. Thus, descendents of these infected cells have a nontransformed phenotype and do not contain viral DNA.
Sublines of stably transformed cells do arise from infections of nonpermissive cells, but with a low frequency (Figure 2c). The rate-limiting step to stable transformation is thought to be the integration of viral DNA into the cellular genome by nonhomologous recombination. Integration occurs at random sites with respect to both the cellular chromosome and the viral DNA. If this integration occurs such that the early coding sequences are intact and expressed, the cell and its subsequent descendants are transformed.
What is transformation?
Neoplastic transformation refers to the acquisition of expanded proliferation and/or survival potential by a cell. Several different assays are used to distinguish a ‘normal’ cell from transformed cells. In each of these assays cells are placed in an environment that is growth restrictive for normal cells whereas transformed cells proliferate and survive. In these assays, to score as transformed requires that the rate of cell proliferation be greater than the rate of cell loss. Thus, each assay measures the ability of cells to escape specific growth and/or survival restrictions. The assays commonly used to assess transformation by SV40 are:
Primary cells can be passaged a limited number of times before they undergo growth arrest and irreversible senescence. MEFs or rat embryo fibroblasts (REFs) expressing large T antigen are immortal and can be propagated in culture indefinitely; however, the situation is more complicated in other cell types. For example, human fibroblasts expressing large T antigen can be propagated for an extended period in culture but do eventually senesce. An active telomerase is required to escape senescence in these cells.
Growth in low serum
Normal fibroblasts require serum-supplemented medium to proliferate and survive. SV40-transformed cells can proliferate and survive in medium with little or no serum.
Fibroblasts typically proliferate and spread across the surface of a culture dish until the entire surface is covered with a monolayer of cells. Growth arrest is thought to be initiated by contact with adjacent cells so that when a monolayer is obtained most cells exit the cycle. This is a stable growth arrest and the cells can be maintained in this quiescent state for weeks. Saturation density is defined as the maximum number of cells per unit area of culture surface. SV40-transformed cells fail to arrest when they reach monolayer and thus reach much higher or indefinite saturation densities.
SV40-transformed cells can proliferate on the surface of a growth arrested monolayer of untransformed cells. Experimentally, this can be assessed in two ways. SV40-transformed cells can be mixed with an excess of untransformed cells and then maintained in culture dishes. The untransformed cells will growth arrest when they reach monolayer while the SV40-transformed cells will continue to proliferate. This results in the appearance of dense regions of multilayered cells, called foci, on the surface of the monolayer. The same result can be achieved by plating SV40-transformed cells on a preformed monolayer of untransformed cells.
Untransformed cells remain viable for weeks when suspended in a slurry of agarose supplemented with medium and serum. However, they do not proliferate as this requires contact with the culture vessel surface and components of serum that coat the surface. In contrast, SV40-transformed cells proliferate in the absence of such contact and grow as multicellular spheres when suspended in agarose.
Ability to form tumors in animals following injection of transformed cells
Untransformed cells are nontumorigenic when injected into test animals such as immunocompromised mice. SV40-transformed cells form tumors when injected into mice and other test animals.
Role of cell type in transformation
Cell culture transformation assays are carried out using either primary cells or established cell lines. Each of these systems carries inherent advantages and drawbacks. Primary cells, such as MEFs, are useful because they have not been passaged extensively in culture and thus these cells carry the same genetic complement as the animal from which they were derived. Since established cell lines, by definition, are immortal, immortalization assays can only be carried out using primary cells. Furthermore, primary cells can be cultured from specific strains of mice, such as gene knockout or transgenic lines, thus allowing the study of the influence of specific alleles on transformation. One of the disadvantages of using primary cell cultures is that they consist of a mixture of cell types that may respond differently to oncogenic signals. The diversity and properties of these cell populations change dramatically during embryogenesis. Even single cell types within this population may exhibit significantly different properties. For example, fibroblasts collected from different anatomical sites of the same individual display different patterns of global gene expression (Chang et al., 2002). Therefore, the age of the animal at the time of embryo extraction, method of cell collection, and culture conditions can be important influences on the outcome of transformation assays.
Established cell lines have the advantage of being clonal. This allows molecular studies on a large number of cells that respond similarly to oncogenic stimuli. However, establishment results from genetic changes that select for cell subpopulations that are immortal. The specific genetic changes carried by a given cell line influence the response to oncogenic and death stimuli, and thus the results of transformation assays. The important conclusion from these observations is that the choice of cell system has major consequences in governing the outcome of transformation assays.
Key questions: how are these different transformation assays related to each other?
SV40 transforms multiple cell types as measured by each of the assays described above. For example, SV40-transformed MEFs proliferate in low serum and in soft agar, grow to a high saturation density, and are tumorigenic in immunocompromised mice. Do each of these assays measure the same molecular event? Does SV40 regulate some key cellular pleiotropic switch that coordinately confers all these transformed properties on cells? Or, does SV40 target multiple cellular switches, each of which controls a subset of the transformed phenotype? The answer to this question appears to be ‘both’. The evidence that SV40 targets multiple pathways each resulting in pleiotropic effects on cell behavior, is outlined below.
Cellular uncertainty principle
The Heisenberg Uncertainty Principle is among the most important concepts to emerge from quantum mechanics. With apologies to our physics colleagues we shamelessly borrow and paraphrase a lesson evoked by that Principle, namely, the act of experimental manipulation alters the behavior of the experimental system. In quantum physics this refers to the inability to simultaneously determine the position and momentum of a photon. Experimental manipulation also alters the behavior of cell culture systems in unpredictable ways. Furthermore, cell culture manipulations impinge on many of the very pathways that we wish to study: those involved in regulating cell cycle, apoptosis, and stress responses. Therefore, animal models are essential to examine the influence of oncogenic signals in native environment.
In practice, the cellular uncertainty principle means that cultured cells are not necessarily in a native state when they are cultured on plastic surfaces in medium supplemented with fetal bovine serum or the equivalent (Figure 3). In fact, the process of generating a primary cell culture results in the death of the vast majority of cells in the tissue or embryo, and the outgrowth of a small population of cells capable of proliferating in this environment. Still, there is little doubt these systems are useful, even powerful. Genes that are active in the assays and cell systems described above are often found mutated in human cancer. Thus, these systems successfully identify proteins that participate in tumorigenesis. What is unclear is how each of the cell behaviors assessed in these transformation assays relates to a given step in tumorigenesis, or to specific properties of tumor cells. This means that cell culture systems are very good for understanding the molecular aspects of regulating signaling pathways, but not so good at predicting how perturbing specific pathways alters cellular behavior in the context of a tissue.
T antigens elicit transformation by targeting cellular proteins
Dense focus assay screens have identified four SV40 functions, three in large T and one in small t, that clearly contribute to transformation (reviewed by Ali and DeCaprio, 2001; Saenz-Robles et al., 2001; Sullivan and Pipas, 2002). In each case, the SV40-transforming function correlates with the ability of one of the T antigens to bind a cellular protein. Thus, large T antigen binding to the heat shock chaperone, hsc70, the retinoblastoma family (Rb-family) of tumor suppressors, and to the tumor suppressor p53, contribute to transformation. The transforming function of small T antigen is associated with binding to the cellular phosphatase pp2A.
Several biochemical activities of large T antigen, such as DNA binding and ATPase/DNA helicase, can apparently be dissociated from transformation (Prives et al., 1983; Manos and Gluzman, 1984, 1985; Peden and Pipas, 1985). That is, mutants that inactivate these functions still induce foci. Similarly, the carboxy-terminal variable region and host-range domains are dispensable for focus induction (Pipas et al., 1983; Zhu et al., 1992). Surprisingly, even nuclear localization signal (NLS) mutants induce transformation of established cell lines, although these mutants are defective when tested on primary cells (Lanford et al., 1985; Fischer-Fantuzzi et al., 1986). Care must be taken in interpreting these results. In most of these cases the contribution of an activity to transformation would be missed if redundant large T antigen functions can independently induce foci.
Large T antigen action on Rb proteins
Large T antigen contains an LXCXE motif (residues 103–107) that is essential for its interaction with the pRb family of tumor suppressors. Mutants that alter this motif are defective for transformation in nearly all assay systems (Chen and Paucha, 1990; Christensen and Imperiale, 1995; Zalvide and DeCaprio, 1995; Srinivasan et al., 1997). The importance of this sequence in mediating the interaction with Rb-proteins, and in transformation was first demonstrated for the adenovirus E1A protein (Whyte et al., 1988a). Subsequently, SV40 large T antigen was shown to bind Rb-proteins through this motif (DeCaprio et al., 1988; Ewen et al., 1989). Large T antigen binds to all three Rb-proteins, but as will be discussed below, the consequences of this association are different in each case.
Most, if not all, of large T antigen's effects on the Rb-proteins are thought to be exerted by regulating the activity of E2F transcription factors. There are eight known E2F proteins (E2F1-8), all of which possess a DNA-binding domain that governs interaction with a common consensus sequence present in the promoters of E2F-regulated genes (reviewed by Attwooll et al., 2004; Frolov and Dyson, 2004; Dimova and Dyson, 2005). The active forms of E2F1-5 consist of a heterodimer containing one E2F polypeptide associated with a dimerization partner, DP1 or DP2. Rb-E2F interactions regulate entry and exit into the cell cycle (Figure 4). In this scenario, E2F-regulated genes are not expressed in quiescent cells because their promoters are occupied primarily with p130/E2F4 complexes which repress transcription. Inactivation of Rb-proteins by phosphorylation results in the replacement of these complexes by ‘activating’ E2F1-3. This leads to the transcription of E2F-regulated genes many of which encode proteins required for DNA replication, nucleotide metabolism, DNA repair and cell cycle progression. Thus, cell cycle entry and growth arrest are, at least in part, governed by signals that ultimately dictate the phosphorylation state of Rb-proteins, and consequently their ability to repress E2F-dependent transcription. Large T antigen short-circuits this pathway by binding Rb-proteins and blocking their ability to regulate E2Fs (Figure 4).
This model is consistent with transfection experiments indicating that a functional LXCXE motif is required for large T antigen to override Rb-induced growth arrest, and to release E2F from Rb-mediated repression (Zalvide and DeCaprio, 1995). Furthermore, unlike their normal counterparts, cell lines transformed with large T antigen do not accumulate p130/E2F4 and other Rb/E2F complexes when they reach confluence, or if they are maintained in low serum (Zalvide et al., 1998; Sullivan et al., 2000b). Thus, large T antigen disrupts repressive Rb-E2F complexes, allowing transcription of E2F-dependent genes and progression into S-phase.
The LXCXE motif is required to allow growth to a high density in low and high serum concentrations and anchorage independence growth of MEFs both in a normal and Rb null background (Zalvide and DeCaprio, 1995). Similarly mutations in this sequence render T antigen defective in these assays when tested in MEFs lacking both p130 and p107 (Stubdal et al., 1997). This indicates that loss of pRb function itself is not sufficient to induce transformation and that large T antigen must inactivate at least two, and possibly all three, retinoblastoma proteins to induce transformation as measured by this assay. Interestingly, the requirement of the LXCXE motif for anchorage-independent growth is ameliorated in p19ARF null MEFs (Chao et al., 2000).
These data strongly indicate that the Rb-proteins are an important target for large T antigen in mediating transformation. However, they do not rule out the possibility that T antigen interacts with other targets important for transformation through the LXCXE motif. Many cellular proteins have LXCXE motifs and are thought to bind Rb-proteins via the same surface as large T antigen (Dick et al., 2000). Thus, in some cases T antigen can be expected to displace cellular proteins bound to Rb-proteins, while in other cases T antigen binding to Rb-proteins might be sterically blocked by the presence of an Rb-associated protein.
While an intact LXCXE motif is required for T antigen to induce high-density cell growth and anchorage independence, it appears to be dispensable for extending the lifespan of primary cells MEFs and REFs in culture (Chen and Paucha, 1990; Thompson et al., 1990; Zalvide and DeCaprio, 1995). In fact, a carboxy-terminal fragment missing the first 250 amino acids, which includes the J domain, Rb-binding sequences and the DNA-binding domain of large T antigen, is still capable of extending primary cell lifespan (Thompson et al., 1990). Interestingly, a truncated large T antigen that consists of the first 147 amino acids, including a functional J domain and Rb-binding sites, is also capable of extending primary cell lifespan. Neither of these T antigen fragments induces immortalization by themselves, but cells expressing both mutants are immortal (Tevethia et al., 1998).
Large T antigen is a DnaJ molecular chaperone
The first 70 amino acids of large T antigen and small t antigen have sequence identity with the J domain of the DnaJ class of molecular chaperones (Kelley and Landry, 1994). Structural studies with SV40 T antigen or with related T antigens from murine polyomavirus show that this region folds as a J domain (Berjanskii et al., 2000; Kim et al., 2001). DnaJ chaperones work in concert with a partner chaperone of the DnaK class (reviewed by Mayer and Bukau, 2005). DnaK chaperones consist of an ATPase domain linked to a substrate-binding domain. The J domain of DnaJ chaperones binds to the ATPase domain of its DnaK partner, thereby stimulating the DnaK ATPase activity. Subsequent to binding ATP, DnaK proteins form a tight association with substrate proteins, and then the energy derived from DnaK-mediated ATP hydrolysis is used to modify the substrate in some way.
Large T antigen binds to hsc70, the major DnaK homologue present in mammalian cells and this binding is dependent upon the J domain and ATP hydrolysis (Sullivan et al., 2001). Furthermore, T antigen stimulates the ATPase activity of hsc70 and stimulates the release of unfolded peptides from the substrate-binding domain of hsc70 (Srinivasan et al., 1997). A hybrid DnaJ protein, in which the J domain of DnaJ has been replaced by the J domain from SV40, functions in Escherichia coli (Kelley and Georgopoulos, 1997), while mutants of the SV40 J domain do not function in E. coli (Genevaux et al., 2003). Similarly, a hybrid DnaJ protein, in which the J domain of Ydj1p, a yeast DnaJ chaperone, has been replaced by the J domain from SV40, functions in Saccharomyces cerevisiae (Fewell et al., 2002). Thus, T antigen is a DnaJ molecular chaperone.
The J domains of SV40 T antigen and of mammalian DnaJ chaperones appear to have functions absent in the yeast and E. coli DnaJ proteins since hybrid viruses, in which the J domain of large T antigen is replaced by the J domain of either E. coli DnaJ or Ydj1p from yeast, bind hsc70, but are defective for productive infection (Sullivan et al., 2000b). In contrast, the DnaJ domains from the JCV large T antigen, or from human DnaJ homologues do functionally replace the SV40 T antigen J domain (Stubdal et al., 1997; Sullivan et al., 2000b).
Role of the J domain in the disruption of Rb–E2F complexes
Several observations indicate that, at least in some cases, the J domain acts in concert with the LXCXE motif to disrupt Rb–E2F complexes. For example, growth arrested MEFs display a prominent p130–E2F complex in EMSA experiments, while this complex is not present in cells expressing T antigen. However, the presence of this complex is maintained in cells expressing either J domain mutants or LXCXE motif mutants (Zalvide et al., 1998). In addition, the J domain is required for T antigen to overcome p130 or pRb-mediated repression of E2F-dependent transcription in transient transfection assays (Zalvide and DeCaprio, 1995).
Purified T antigen binds stably to preformed p130–E2F4–DP1 complexes in vitro (Sullivan et al., 2000a). Thus, T antigen does not dislodge p130 from E2F4 by affinity displacement. The coexistence of large T antigen, and perhaps other LXCXE motif-containing proteins, and E2F on the same p130 molecule is consistent with structural studies that indicate that LXCXE motif proteins, including T antigen, bind to a groove on the surface of the B domain of pRb, while E2Fs bind a separate surface formed at the intersection of the A and B domains (Kim et al., 2001; Xiao et al., 2003). Disruption of the p130–E2F4 complex in vitro occurs upon the addition of hsc70 and ATP, and requires both a functional J domain and LXCXE motif. It is important to note that this in vitro chaperone reaction does not indiscriminately break apart protein complexes, since functional E2F4–DP1 dimers remain intact during this reaction (Sullivan et al., 2000a). The first 136 amino acids of T antigen are sufficient to disrupt p130–E2F complexes both in vivo and in vitro (unpublished results). Thus, the J domain and LXCXE motif are sufficient to disrupt Rb–E2F complexes, but they must reside in cis, that is, on the same T antigen molecule (Srinivasan et al., 1997). Thus, J domain mutants do not complement LXCXE motif mutants either in productive infection or transformation. These observations give rise to a model (Figure 5) in which large T antigen recruits p130–E2F complexes via its LXCXE motif so that hsc70 bound to the J domain can use energy from ATP hydrolysis to free E2F from p130.
T antigen does not treat all Rb–E2F complexes in the same way. The obvious manifestation of this is in MEFs, where transformation by T antigen leads to the proteosome-dependent degradation of p130, but does not alter the levels of pRb and leads to increased levels of p107 (Stubdal et al., 1997). Similarly, during SV40 infection in BSC40 cells, T antigen expression results in loss of p130–E2F complexes, while some pRb–E2F complexes remain intact (Sullivan et al., 2004). Finally, the J domain is required to confer a growth advantage to MEFs expressing functional p130 and p107 proteins, even in the absence of a functional pRb (Zalvide and DeCaprio, 1995). In contrast, the J domain is not required to confer this growth advantage to MEFs derived from p130, p107 double null embryos (Stubdal et al., 1997). One interpretation of these data is that the J domain is needed for T antigen to inactivate p130 and p107, but not for pRb inactivation. How T antigen discriminates different Rb–E2F complexes, as well as the biological significance of this differential action, is unclear.
How does the J domain contribute to transformation?
In-frame deletion mutants of T antigen missing part or all of the J domain coding sequences are defective for the induction of dense foci despite being able to bind Rb-proteins and p53 (Pipas et al., 1983; Srinivasan et al., 1997). Thus, the J domain per se is required for focus formation. Furthermore, genetic complementation tests indicate that the J domain cannot function in trans. In fact, the J domain must be in cis with at least two other T antigen elements to effect transformation; namely, the LXCXE motif, and an unidentified function that requires sequences carboxy-terminal to amino acid 136 (Srinivasan et al., 1997). Oddly, the ability of J domain deletion mutants to induce anchorage-independent growth has not been reported. Deletions of the J domain are also capable of immortalizing primary cells (Hahn et al., 2002). However, this result is complicated by the fact that T antigen has at least two independent functions that can extend the lifespan of primary cells: one residing in the first 121 amino acids that includes the J domain and LXCXE motif, and the other residing in sequences in the carboxy-terminal half of the molecule (Tevethia et al., 1998).
In contrast to J domain deletions, amino-acid substitution mutants, such as D44N, do induce focus formation and anchorage-independent growth, although at a somewhat reduced frequency compared to wild type (Peden and Pipas, 1992; Stubdal et al., 1997; Hahn et al., 2002). However, D44N, as well as other alleles, are defective for inducing growth to a high saturation density and for growth in low serum (Stubdal et al., 1997). Thus, the fact that D44N is clearly defective for hsc70 binding and for the disruption of p130–E2F4 complexes appears to dissociate the T antigen chaperone function from focus formation and anchorage-independent growth, but links it to growth in low serum (Sullivan et al., 2000a, 2000b, 2001).
Why are some J domain alleles defective for focus formation while others are not? One possibility is that the interaction of the J domain with hsc70 is allele specific. For example, perhaps a given mutant J domain can stimulate hsc70 enough to achieve partial action on all or some of its substrates leading to a subset of phenotypes observed with the wild-type protein. Another possibility is that the SV40 J domain binds cellular proteins in addition to hsc70. In this scenario, some alleles would disrupt T antigen interaction with the unknown cellular target, thus losing the phenotypes resulting from this interaction, while maintaining interaction with hsc70, or vice versa. Still other alleles, such as deletions, might result in the loss of both binding functions.
It is clear that truncated T antigen mutants that terminate after the LXCXE motif but before the origin-binding domain (OBD), carry potent biological activities. The best characterized of these truncation mutants are dl1137 (N121) and N136. These, and similar fragments, induce foci in many established cell lines (Srinivasan et al., 1989) and hyperplasia in transgenic mice (Fromm et al., 1994; Kim et al., 1994; Tevethia et al., 1997). In most of these cases, biological activity requires both the J domain and the LXCXE motif. Furthermore, N136 can rescue neuronal stem cell lines from apoptosis induced by growth-factor withdrawal, and this rescue requires both an active J domain and LXCXE motif (Slinskey et al., 1999) Finally, amino-terminal fragments (residues 1–127 and 1–121) of T antigen cooperate with oncogenic ras to transform primary REFs (Beachy et al., 2002). Again, ras cooperation is J domain and LXCXE motif dependent.
One interpretation of this data is that amino-terminal fragments of T antigen that include a functional J domain and LXCXE motif are sufficient to inactivate Rb-proteins. However, is the action of truncated T antigens, like N121 or N136, really equivalent to the simultaneous loss of pRb, p107, and p130? One piece of evidence suggests that the answer to this question is no. Genetic ablation of pRb, p107, and p130 from MEFs results in immortalization and anchorage-independent growth (Sage et al., 2000). In contrast, the expression of N136, or similar fragments, results in MEFs that have an extended lifespan, but are not immortal, and do not induce anchorage-independent growth (Thompson et al., 1990). Perhaps, sequences in the carboxy-terminus of T antigen contribute to the inhibition of Rb-protein tumor suppressor function. Alternatively, the amino-terminal T antigen fragments may possess activities, in addition to hsc70- and Rb-protein binding, that interfere with the action of T antigen on Rb-proteins.
The J domain cooperates with a function(s) in the carboxy-terminal portion of T antigen
As mentioned above, the J domain is required in cis with an unknown activity that maps carboxy-terminal to amino acid 136 and is required to induce focus formation (Srinivasan et al., 1997). In addition to its role in transformation, the T antigen J domain is also required for viral DNA replication and virion assembly (Peden and Pipas, 1992; Spence and Pipas, 1994; Campbell et al., 1997). The J domain acts in cis with carboxy-terminal sequences of T antigen in each of these cases as well. This suggests that the J domain recruits hsc70 for action on targets in addition to Rb–E2F complexes, and that this action is required for several aspects of viral infection, as well as for transformation.
The simplest model that explains these results is that T antigen binds a target(s) through sequences that are carboxy-terminal to residue 136, and that the J domain must act on this target to elicit transformation. While this target or targets are unknown, we raise two possibilities. First, is the interaction of T antigen with the histone acetyltransferase activator complex, CBP/p300 (Eckner et al., 1996; Lill et al., 1997). CBP/p300 do not bind large T antigen directly, but rather are recruited into association via p53 (Poulin et al., 2004). In the case of the adenovirus, the E1A protein must interact with both Rb-proteins and CBP/p300 to induce transformation (Egan et al., 1988, 1989; Whyte et al., 1988b). Wild-type SV40 large T antigen can complement the transformation defect of E1A mutants that do not bind CBP/p300, while T antigen J domain mutants do not (Yaciuk et al., 1991). Thus, it is intriguing to speculate that the T antigen J domain must act on the T antigen–p53–p300 complex to induce transformation, but no hard evidence has been forthcoming to indicate this is the case. A second potential target that exemplifies the cooperation between the J domain and carboxy-terminus of T antigen is cyclin A. Cyclin A–cdk complexes phosphorylate and inactivate pRb, thus overcoming its growth-suppressive effects. T antigen transactivates the cyclin A promoter, thereby sending the cells into S-phase. This activity is independent of the LXCXE motif, but requires both the J domain and sequences carboxy-terminal to amino acid 127 (Beachy et al., 2002).
T antigen interaction with the tumor suppressor p53
The p53 tumor suppressor was discovered as a cellular protein bound to large T antigen in SV40-transformed cells (Lane and Crawford, 1979; Linzer and Levine, 1979). Under normal circumstances the steady-state levels of p53 in a cell are very low. This is because p53 binds to the promoter of the mdm2 gene and stimulates its transcription (reviewed by Bond et al., 2005; Coutts and La Thangue, 2005). As its levels rise, mdm2 binds p53 and induces its polyubiquitination and subsequent degradation. This feedback loop is disrupted by several kinds of stress, including DNA damage, depletion of nucleotide pools, anoxia, and abnormal inhibition of Rb-proteins. Under these conditions phosphorylation of p53 results in inhibition of the mdm2–p53 interaction, and consequently in an increase in p53 steady-state levels. p53 is a potent transcriptional activator, and as its steady-state levels rise it upregulates a number of genes, some of which mediate cell cycle arrest or apoptosis (see Figure 4).
Large T antigen interacts with the DNA-binding surface of p53, blocking its ability to bind promoters and thus to regulate gene expression (Bargonetti et al., 1992; Jiang et al., 1993). The T antigen–p53 interaction occurs through amino acids on the solvent-exposed surface of the T antigen ATPase domain (Li et al., 2003). Carboxy-terminal fragments of T antigen, such as C351–708 are sufficient to bind p53 and block its growth-suppressive functions (Cavender et al., 1995). The carboxy-terminal variable region/HR domain (amino acids 627–708) of T antigen is not required for interaction with p53. Furthermore, an internal deletion that removes residues 451–532 is capable of p53 binding (Kierstead and Tevethia, 1993). This suggests that a bipartite-binding region consisting of amino acids 351–450 and 533–626 govern the T antigen–p53 interaction.
SV40 appears to block p53 function by multiple redundant mechanisms. Thus, T antigen can also block p53-dependent transcriptional activation and growth-arrest independent of p53 binding (Quartin et al., 1994; Rushton et al., 1997). The amino-terminal 121 residues of T antigen are sufficient for this effect, which requires both the J domain and LXCXE motif. Small t antigen also participates in blocking p53 function (Pipas and Levine, 2001).
Is T antigen action on p53 equivalent to the total loss of p53 activity, or does the interaction with T antigen inhibit some p53 activities, while sparing others? Large T antigen binding results in p53 stabilization and thus SV40-transformed cells contain large amounts of p53 that is thought to be functionally inactive (Oren et al., 1981). However, while T antigen blocks some p53 functions, an alternative hypothesis is that other p53 activities may not be inhibited by T antigen and in fact, might be dependent upon the interaction of p53 with T antigen (Deppert et al., 1989). This gain-of-function scenario is made more plausible by recent reports that some p53 alleles found in cancer express p53 protein that contributes to neoplasia (Lang et al., 2004; Olive et al., 2004). At present, there is not enough evidence to tip this argument one way or another. However, MEFs derived from p53 null embryos are immortal, while MEFs expressing carboxy-terminal fragments of T antigen, capable of binding p53, have an extended lifespan, but are not immortal (Tevethia et al., 1998). Thus, either the interaction of full-length T antigen with p53 is required to fully inhibit its tumor suppressor functions, or T antigen action on p53 is not equivalent to a p53 null cell.
Small t antigen targets the cellular phosphatase pp2A
The 174 amino-acid small t antigen consists of a J domain identical to the one found in large T antigen, and a unique carboxy-terminus that directs its interaction with the cellular phosphatase, pp2A. Small t antigen does not usually score in any cell culture transformation assays when expressed alone. However, under many circumstances where T antigen expression is not sufficient for transformation, coexpression of large T antigen and small t antigen leads to transformation. Small t antigen's contribution to transformation requires its binding to the catalytic subunit of pp2A (Mungre et al., 1994; Hahn et al., 2002). This binding results in displacement of the regulatory B subunit of pp2A and consequent inhibition of phosphatase activity, or change in substrate specificity.
The contribution of small t antigen to SV40 transformation is discussed by Arroyo and Hahn in a separate chapter of this volume.
Are there additional T antigen targets?
The importance of the large T antigen interaction with Rb-proteins and with p53 in SV40 transformation is well established. However, is this the whole story, or do other T antigen activities contribute to the tumorigenic phenotype? Large T antigen was identified as one of four oncogenes required to transform normal human epithelial and fibroblast cells to tumorigenic cells (Hahn et al., 1999, 2002). In these experiments large T antigen acted along with an oncogenic allele of ras, hTERT and small t antigen to induce transformation. Furthermore, large T antigen can be functionally replaced by combinations of viral and cellular proteins that inactivate the pRb and p53 pathways. For example, overexpression of cyclin D, an INK4a-resistant CDK4 or the human papillomavirus E7 protein combined with either a dominant negative p53 or the human papillomavirus proteins E6 protein, could replace large T antigen in these assays. Similarly, short interfering (si) RNA directed towards pRb and p53 appear to replace T antigen in this system (Voorhoeve and Agami, 2003). Taken together, these observations suggest that T antigen's contribution to tumorigenesis can be attributed solely to its inhibition of pRb and p53 pathways (Hahn et al., 2002).
On the other hand, genetic studies suggest that inactivation of pRb and p53 may not account for the full transformation potential of T antigen (Cavender et al., 1995; Sachsenmeier and Pipas, 2001; Wei et al., 2003). Furthermore, several cellular T antigen-binding proteins have been identified that, based on their known functions, have a potential to contribute to transformation.
The transcriptional adapter proteins p300 and CBP play roles in multiple biological processes, including cell growth and transformation (reviewed in Goodman and Smolik, 2000). The interaction of E1A with p300/CBP is essential for adenovirus transformation (Egan et al., 1988, 1989; Whyte et al., 1988b). Genetic studies demonstrated that SV40 T antigen can complement p300/CBP-binding deficient mutants of E1A to restore transformation (Yaciuk et al., 1991). Several studies suggested that p300/CBP associates with large T antigen but it was unclear whether or not this interaction was direct (Avantaggiati et al., 1996; Eckner et al., 1996; Lill et al., 1997). Recently, it was demonstrated clearly that p300/CBP associates with large T antigen indirectly, through its interaction with p53 (Poulin et al., 2004).
The functional consequences of T antigen binding to p300/CBP are not understood fully, and the role of this interaction in transformation has not yet been established clearly. T antigen may enhance the p300/CBP-associated histone acetyltransferase activity (Valls et al., 2003). In addition, CBP acetylates T antigen on K697 in a p53-dependent manner (Poulin et al., 2004). While the role of this acetylation in virus growth or transformation is not clear, it is interesting to note that this acetylation site is conserved on T antigens of the human polyomaviruses, JC and BK, and the baboon polyomavirus, SA12 (Cantalupo et al., 2005 submitted; Poulin et al., 2004). Finally, some experiments suggest that p300/CBP is required for E2F transcriptional activity in vitro (Ait-Si-Ali et al., 2000). Thus, one might speculate that T antigen increases cell proliferation not only by inactivating pRb, but also by delivering p300/CBP to E2F-dependent promoters. This hypothesis is consistent with the observation that activation of E2F-dependent promoters by the murine polyomavirus large T antigen requires both the inhibition of Rb-proteins and an interaction with p300/CBP (Nemethova et al., 2004).
Despite these provocative observations, definitive evidence that the interaction of large T antigen and p300/CBP plays a role in transformation has not been reported. Of course, the fact that p300/CBP associates with T antigen through p53 greatly complicates these studies. Clearly, T antigen mutants that are defective for T antigen binding to, or action on, p300/CBP, but maintain a normal interaction with p53 would be of great help.
Imperiale and colleagues first noticed a protein, termed p185, associated with large T antigen in transformed cells (Kohrman and Imperiale, 1992). The p185-binding site is within the first 121 amino acids of T antigen and binding is independent of the LXCXE motif and the J domain. This same protein, here termed p193, was independently shown to bind T antigen in cardiomyocytes (Tsai et al., 2000). Subsequently, the T antigen associated protein was shown to be a cullin, Cul7, which is part of an E3 ligase complex involved in the ubiquitin degradation of proteins (Ali et al., 2004). Mutagenesis studies indicate that sequences carboxy-terminal to the J domain but amino-terminal to the LXCXE motif are required for Cul7 association with T antigen.
Cul7-binding mutants of T antigen are unable to induce anchorage-independent growth of MEFs and fail to support growth in low serum (Ali et al., 2004). These mutants can bind to pRb and inhibit pRb-mediated repression of E2F-dependent transcription. They are also able to bind to and stabilize p53, although the authors did not specifically show that p53 was inactivated.
These observations suggest that binding to Cul7 could be required in addition to inactivation of pRb and p53 for T antigen-mediated transformation. However, how does the interaction of Cul7 contribute to transformation? One possibility is that T antigen recruits Cul7 to target some other T antigen-associated protein for degradation. Alternatively, the elimination of Cul7 function might be a prerequisite for SV40 transformation. In this model, which is favored by the authors, Cul7 functions as a tumor suppressor (Ali et al., 2004). This hypothesis predicts that T antigen mutants, deficient for Cul7 binding, will transform MEFs derived from Cul7 null embryos. In any event, the link between T antigen–Cul7 binding and transformation is potentially of great interest since it could indicate a pathway, independent of p53 and Rb-proteins, that plays an essential role in tumorigenesis.
A yeast-two hybrid analysis led to the identification of Bub1, a mitotic spindle checkpoint protein, as a T antigen interacting protein (Cotsiki et al., 2004). Mutations, such as W94A or W95A, lead to loss of the T antigen–Bub1 interaction. These mutants are unable to induce focus formation in Rat-1 cells, suggesting that Bub1–T antigen interaction may play a role in transformation (Cotsiki et al., 2004).
Mutations in Bub1 have been found in certain types of human cancers, such as colorectal cancer and leukemia (Cahill et al., 1998; Ru et al., 2002). Furthermore, the interaction between T antigen and Bub1 results in the perturbation of the spindle checkpoint (Cotsiki et al., 2004) and may be one explanation for how T antigen induces aneuploidy and genetic instability (Woods et al., 1994; Chang et al., 1997). However, several key questions need to be addressed before the importance of Bub1 to T antigen-mediated transformation can be established. First, the transforming activity of T antigen mutants deficient for Bub1 binding needs to be assessed in different cell types, including primary cells. Additionally, the T antigen–Bub1 interaction needs to be established as either playing a role in the inactivation of Rb-proteins or p53, or as an independent transforming function. Finally, while the link between the T antigen–Bub1 association and genetic instability is provocative, it has not yet been shown that this function actually contributes to transformation.
T antigen modulates the transcription of both cellular and viral genes by targeting various components of the eukaryotic transcriptional machinery (Gruda et al., 1993; Martin et al., 1993; Johnston et al., 1996). One of its targets is the transcription factor TEF-1, which appears to play a role in the early-to-late switch in viral transcription (Berger et al., 1996). TEF-1 binds to the T antigen OBD. S189N, a TEF-1-binding deficient mutant of T antigen, is seven- to eight-fold less efficient than wild-type T antigen in focus formation assays (Dickmanns et al., 1994). The S189N mutant is able to bind p53 but its ability to block p53 function has not been tested. Similarly, it is not known if S189N is fully capable of inactivating Rb-proteins. Thus, until the ability of TEF-1 mutants to transform in different systems is assessed, and the T antigen–TEF-1 interaction is shown to be independent of T antigen–pRb and –p53 interactions, the relevance of TEF-1 to SV40-mediated transformation will remain unclear.
T antigen has been shown to form a complex with Nbs1, a component of the MRN complex that functions in DNA repair (Wu et al., 2004). The OBD of T antigen is required to bind to Nbs1, and this interaction is independent of T antigen binding to p53, and pRb and does not require the J domain. The T antigen interaction with Nbs1 is associated with endoreduplication and thus, could conceivably contribute to genomic instability. However, at present there is no known role for this interaction in transformation.
Recently, T antigen was shown to interact with Fbw7, a component of the ubiquitination machinery (Welcker and Clurman, 2005). Fbw7 binds to its substrates via a phosphorylated epitope, the Cdc4 phospho-degron. This motif is present in the host-range domain at the carboxy-terminus of large T antigen. However, unlike substrates of Fbw7, T antigen is not degraded. Instead the authors hypothesize that T antigen functions as a competitive inhibitor of Fbw7, reducing the turnover of physiological substrates, like cyclin E. The role of this interaction in T antigen-mediated transformation has not yet been determined.
Cell type specificity of T antigen action
Tumorigenesis is thought to occur as a progression through different stages (Vogelstein et al., 1988). In this model, the progression from one stage to the next is thought to be driven by genetic changes that enhance cell growth and/or survival. Thus, neoplasias occur in different histopathological states such as: hyperplasia/dysplasia, benign or noninvasive tumor, invasive tumor, and metastasis. One of the major challenges of cancer research is to associate changes in specific molecular pathways with progression from one stage to another. What can SV40 tell us about the role of the Rb and p53 tumor suppressor pathways in tumor progression?
The current paradigm stems largely from the pioneering work of Van Dyke and colleagues who have studied the effects of T antigen expression in the choroid plexus. The choroid plexus epithelium (CPE) is a single layer protecting and detoxifying the brain by maintaining the cerebro-spinal fluid–blood barrier. Ectopic expression of large T antigen in the CPE results in renewed proliferation and tumor formation of this epithelial tissue (Chen and Van Dyke, 1991). The first 121 amino acids of T antigen are sufficient to induce CPE tumors, but the rate of tumor growth is much slower than that of tumors expressing wild-type T antigen (Chen et al., 1992). The same truncation mutant of T antigen is unable to induce tumors in the CPE when mutated in the LXCXE motif, indicating that the inactivation of the Rb pathway is required for these cells to enter S-phase (Chen et al., 1992; Symonds et al., 1994). Furthermore, while the amount of proliferation, as assessed by BrdU labeling, is equivalent in CPE expressing the wild-type or mutant T antigens, unlike tumors induced by wild-type T antigen, CPE tumors induced by the truncated T antigen exhibit large amounts of apoptosis. Rapid tumor growth can be restored by expressing the truncated T antigen in a p53-deficient background (Symonds et al., 1994).
This suggests a simple model in which cell proliferation is induced by inactivation of Rb-proteins, while cell death is blocked by inhibiting p53 function. Consistent with this model, the expression of amino-terminal fragments of large T antigen in transgenic mice induces cell proliferation in a number of diverse cell types (Symonds et al., 1994; Tevethia et al., 1997; Xiao et al., 2002). However, the consequences of T antigen expression in different tissues also reveals dramatic cell –type-specific effects (Table 1). Here we discuss a selected set of studies that illustrate this point, emphasizing the relevance of these observations to cancer progression.
Inactivation of p53 is not always required for tumorigenesis
The expression of the N121 truncated T antigen in astrocytes results in their re-entry into the cell cycle accompanied by extensive apoptosis. However, unlike CPE tumorigenesis this apoptosis is not alleviated by the elimination of p53. Rather astrocyte apoptosis is dependent on a functional PTEN protein (Xiao et al., 2002). Similarly, both wild-type T antigen and N121 induce S-phase and hyperplasia when expressed in intestinal enterocytes (Hauft et al., 1992; Kim et al., 1994). In this case, inactivation of Rb-proteins by N121 does not result in increased apoptosis. Molecular studies indicate that enterocytes do not express significant amounts of p53 and that there are no detectable T antigen–p53 complexes present in the intestines of these transgenic mice (Markovics et al., 2005). Finally, expression of a similar amino-terminal fragment of T antigen (N147) in the acinar cells of the pancreas induces tumors that are indistinguishable from those induced by wild-type T antigen. The tumors induced by N147 still express a wild-type p53 (Tevethia et al., 1997). Taken together these results suggest that inactivation of the tumor suppressor p53 is not a prerequisite for transformation in all cell types.
The expression of wild-type T antigen leads to different outcomes in different tissues
T antigen has been expressed in a large number of different issues of transgenic mice. Some of these systems have been studied in detail while other await further analysis. However, it is clear that the consequences of T antigen expression are cell type specific. Here we summarize the effects of T antigen expression in cells from four different tissues.
The eye is a sensory organ composed of the iris, lens, the cornea and two superimposed layers, the pigmented retina, capable of producing melanin, and the neural retina, containing light sensitive photoreceptors, glia, interneurons and ganglion cells. Through embryogenesis, retinal progenitor cells divide and generate postmitotic transition cells that commit to different neural and glial cell fates (ganglion cells, cone cells, horizontal cells, amacrine cells, rod cells, Müller glia, and bipolar cells) and differentiate in conserved order, forming the different layers of the adult retina. The lens contains three regions, and anterior zone of dividing epithelial cells, an equatorial zone of cellular elongation, and a posterior and central zone of differentiated, crystalline-containing fiber cells. This structure persists throughout life, as fibers are continuously being laid down.
T antigen expression in the fiber cells of the lens results in their re-entry into the cell cycle and lens tumors (Mahon et al., 1987). The induction of proliferation requires an intact LXCXE motif but does not require the presence of small t antigen (Fromm et al., 1994; Chen et al., 2004). Furthermore, expression of a truncated T antigen (N191) stimulates fiber cell proliferation, but this is accompanied by increased apoptosis resulting in microphtalmia, microlentia and lens ablation (Fromm et al., 1994). While it is has not been established that this apoptosis is p53 dependent, these results are consistent with a model in which the amino-terminal portion of T antigen induces proliferation by inhibiting Rb-proteins while carboxy-terminal sequences of T antigen inhibit apoptosis, most likely by blocking p53 activity. Interestingly, T antigen also blocks fiber cell differentiation through a mechanism that is LXCXE-motif independent and requires sequences carboxy-terminal to amino acid 191 (Fromm et al., 1994).
T antigen also induces tumors when expressed in cone photoreceptor cells (al-Ubaidi et al., 1992a). In contrast, expression of T antigen in rod photoreceptors induces DNA synthesis but the photoreceptors degenerate (al-Ubaidi et al., 1992b). Thus, T antigen appears to be able to induce S-phase and proliferation in three different differentiated cell types of the eye. However, expression in fiber cells and cone cells results in tumorigenesis, while expression in rod cells leads to tissue ablation.
The intestine is composed of three basic layers. On the outside, a layer of loose connective tissue followed by a double layer of smooth muscle (mesenchyma); in the middle, a layer of connective tissue containing blood and lymphatic vessels (stroma); and the innermost surface (mucosa) is composed of epithelial cells lining the actual lumen of the intestinal tract and organized as proliferative (crypts) and differentiated regions (villi). Multipotent stem cells are located near the base of the crypt, and give rise to progenitor cells that eventually exit the cell cycle and differentiate into one of four different cell types, one absorptive and forming the majority of the epithelium (enterocytes), and three secretory (enteroendocrine, goblet, and Paneth cells). The enterocytes are responsible for absorbing nutrients, water and electrolytes from the gut lumen and transporting them to the underlying capillaries; the goblet cells, scattered between the absorptive cells, secrete mucus (Paulus et al., 1993); and the enteroendocrine cells are scattered throughout the epithelial layer and release hormones (Roth et al., 1992; Hocker and Wiedenmann, 1998). These three cell lineages migrate from the crypts to the villi, where they are finally exfoliated when they reach an extrusion zone located near the villus tip. On the other hand, Paneth cells, responsible for secreting a number of antimicrobial molecules when exposed to bacteria or bacterial antigens, differentiate while they move to the bottom of the crypt, where they reside (Ayabe et al., 2000).
Expression of both large T antigen and small t antigen results in ectopic proliferation of all of these cell types. Both enterocytes and smooth muscle cells are induced to proliferate by T antigen expression with no detectable effect on differentiation (Hauft et al., 1992; Kim et al., 1993; Herring et al., 1999). While T antigen expression results in hyperplasia and dysplasia in both of these cases, no distinct tumors are observed. In contrast, expression of large and small T antigens in Paneth or goblet cells results in S-phase entry followed by elimination of the specific cell type (Garabedian et al., 1997; Gum et al., 2001, 2004). In fact, T antigen is as effective as cholera toxin in ablating Paneth and goblet cells from the intestine. Finally, T antigen expression in endocrine cells results in multiple endocrine tumors (Upchurch et al., 1996; Ratineau et al., 2000).
The mammalian pancreas is a compound gland composed of endocrine and exocrine tissues. The four endocrine cell types that produce insulin (α-cells), glucagon (β-cells), somatostatin (γ-cells), and pancreatic polypeptide (PP-cells) are contained in the islets of Langerhans, comprising 1–2% of the cellular mass of the adult pancreas. The exocrine tissue is organized into acini, which synthesize digestive hydrolases, and ducts, which secrete a bicarbonate fluid that flushes the acinar secretions to the intestine.
T antigen expression in the beta-islet cells results in S-phase entry and about 50% of the islets become hyperplastic. Subsequently, about 10% of the islets become vascularized and 1–2% progress to carcinoma (Hanahan, 1985; Christofori et al., 1994). Thus, it appears that T antigen initiates tumorigenesis by stimulating β-cell proliferation and that this is followed by cellular mutations that drive tumor progression. Interestingly, the expression of IGF2 appears to be an important cofactor for tumor progression in this system (Christofori et al., 1994). Similarly, T antigen expression in α-cells leads to hyperplasia with a minority of the islets progressing to noninvasive tumors (Efrat et al., 1988; Lee et al., 1992; Asa et al., 1996). Production of both large and small T antigen in the acinar cells results in acinar carcinomas some of which show metastasis (Ornitz et al., 1987; Bell et al., 1990; Ceci et al., 1991; Ramel et al., 1995). Surprisingly, the N147 truncation mutant of T antigen induces carcinomas and metastasis with the same frequency as wild-type T antigen (Tevethia et al., 1997).
The prostate is a tubulo-alveolar gland surrounding the urethra and containing three epithelial cell types. The proliferative basal cells form a single layer on the basement membrane underlying the normal prostatic epithelium. They are thought to contain a stem cell population as well as a population of intermediate, amplifying cells, that are in the process of generating differentiated cell populations but have not yet committed to one particular lineage. The secretory cells are the predominant, differentiated group at the luminal part of the prostate, and the rare neuroendocrine cells are scattered throughout acini and ducts, secreting a variety of growth factors that may affect development and maintenance of this tissue.
Expression of large T antigen and small t antigen in the prostate induces hyperplasia with further progression to adenoma, adenocarcinoma, and frequent metastases to other tissues (Greenberg et al., 1995; Gingrich et al., 1996; Kasper et al., 1998). Metastasis is dependent on the age of the animals and the level of expression of the transgene (Kasper et al., 1998). Furthermore, metastatic tumors have been shown to express T antigen by immunohistochemistry (Masumori et al., 2001).
T antigen expression does not always result in cell proliferation
The studies reviewed above suggest that the expression of large and small T antigens, and often the expression of large T antigen alone, is sufficient to initiate S-phase and drive cells to proliferate. The eventual outcome then depends on how specific cell types respond to this unscheduled entry into the cell cycle. However, there are some cases where T antigen expression fails to stimulate cell proliferation. For example, transgenic mice expressing T antigen in neuroblasts of the midbrain and forebrain remain quiescent (Efrat et al., 1988; Lee et al., 1992). Similarly, T antigen does not induce proliferation in some enteroendocrine cells of the small intestine (Asa et al., 1996). These very interesting cases warrant further study. Is T antigen unable to inhibit Rb-protein functions in these cell types, or, alternatively, is there a block to proliferation downstream of Rb-protein inactivation?
The interaction of large T antigen with p53 and with the Rb family of tumor suppressor proteins clearly contributes to SV40 transformation. T antigen possesses multiple additional activities that could also potentially contribute to cellular transformation, but their roles have yet to be clearly established. Cell culture studies will continue to be invaluable for identifying T antigen targets and for understanding the cellular pathways these targets regulate. However, the acts of establishment and maintenance of these in vitro systems alter many of the regulatory networks that we wish to study. Thus, the development of animal model systems that more closely mimic human tumorigenesis are essential to our understanding of cancer (Van Dyke and Jacks, 2002).
The observation that T antigen expression in different cell types leads to different histopathological outcomes is of fundamental importance to cancer research. Much emphasis has been placed on defining the genetic changes that govern the tumorigenic phenotype. However, the cell type in which these changes occur appears to be of equal, or perhaps greater importance, in determining tumor behavior. The observation that T antigen induces outcomes ranging from hyperplasia to carcinoma and frequent metastasis emphasizes this point. In fact, in most cases T antigen has been expressed in terminally differentiated cells. Such cells generally pass through several predifferentiated states before reaching permanent cell cycle exit. Cells in each of these predifferentiated states have different potentials for proliferation, migration, and death, and each respond to different growth and survival signals (see Figure 6). Thus, the regulatory state of the cell in which oncogenesis is initiated may play as important a role as genetics in determining properties such as tumor aggressiveness and metastatic potential.
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This work was supported by Grants CA40586 and CA098956 to JMP. The authors thank the members of the Pipas Lab for their helpful comments.
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Ahuja, D., Sáenz-Robles, M. & Pipas, J. SV40 large T antigen targets multiple cellular pathways to elicit cellular transformation. Oncogene 24, 7729–7745 (2005). https://doi.org/10.1038/sj.onc.1209046
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