SV40 Large T Antigen Targets Multiple Cellular Pathways to Elicit Cellular Transformation

SV40 large T antigen targets multiple cellular pathways to elicit cellular transformation

Deepika Ahuja1,2, M Teresa Sa´ enz-Robles1,2 and James M Pipas*,1

1Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA

DNA tumor viruses such as simian virus 40 (SV40) three proteins that are structural components of the express dominant acting oncoproteins that exert their virion (VP1, VP2, VP3) and two nonstructural proteins effects by associating with key cellular targets and termed large T antigen (T antigen) and small T antigen altering the signaling pathways they govern. Thus, tumor (t antigen). In addition, some members of the Polyoma- viruses have proved to be invaluable aids in identifying viridae encode additional proteins that are virus specific proteins that participate in tumorigenesis, and in under- or are encoded by a subset of viruses in the family. For standing the molecular basis for the transformed pheno- example, SV40 encodes three such proteins: agnopro- type. The roles played by the SV40-encoded 708 amino- tein, 17K T, and small leader protein. The genomes of acid large T antigen (T antigen), and 174 amino acid small all polyomaviruses can be divided into three elements: T antigen (t antigen), in transformation have been an early coding unit, a late coding unit, and a regulatory examined extensively. These studies have firmly estab- region (Figure 1). In a productive infection, expression lished that large T antigen’s inhibition of the p53 and Rb- of the early coding unit is effected by the cellular family of tumor suppressors and small T antigen’s action transcription apparatus and results in expression of the on the pp2A phosphatase, are important for SV40-induced large, small and 17K T antigens. Expression of the late transformation. It is not yet clear if the Rb, p53 and pp2A coding unit, which encodes VP1, VP2, VP3, and the proteins are the only targets through which SV40 trans- agnoprotein requires an active T antigen in conjunction forms cells, or whether additional targets await discovery. with the cellular transcriptional apparatus. In addition Finally, expression of SV40 oncoproteins in transgenic to activating transcription from the viral late promoter, mice results in effects ranging from hyperplasia to invasive T antigen also functions in viral DNA replication and carcinoma accompanied by metastasis, depending on the virion assembly. tissue in which they are expressed. Thus, the consequences Little is known about the natural history of SV40 of SV40 action on these targets depend on the cell type infection in the wild. In its natural host, the Rhesus being studied. The identification of additional cellular macaque, SV40 is thought to infect the terminally targets important for transformation, and understanding differentiated, growth-arrested epithelial cells of the the molecular basis for the cell type-specific action of the kidney. Since these cells are quiescent they do not viral T antigens are two important areas through which express proteins involved in DNA replication, nucleo- SV40 will continue to contribute to our understanding of tide metabolism, or chromatin assembly, proteins that cancer. are used by SV40 to replicate viral DNA and assemble Oncogene (2005) 24, 7729–7745. doi:10.1038/sj.onc.1209046 new virions. However, the large and small T antigens cooperate to drive infected cells into S-phase thus Keywords: SV40; polyomavirus; transformation; cell- providing the raw materials needed for progeny virus type specificity 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 neutra- lize or ameliorate these defenses allowing productive SV40 infection to proceed. Shortly after its discovery SV40 was shown to induce Simian virus 40 (SV40) is a member of the Polyoma- tumors when injected into newborn hamsters or viridae, a family of viruses characterized by small immunocompromised mice (Eddy et al., 1962; Girardi icosahedral virions and a circular double-stranded et al., 1962). The ability to induce tumors and the types DNA genome of about 5 kb. The SV40 genome consists of tumors observed depended on the route of infection, of 5243 bp and like other members of the family encodes 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, *Correspondence: JM Pipas, Department of Biological Sciences, University of Pittsburgh, 559 Crawford Hall, Pittsburgh, PA 15260, and choroid plexus neoplasia (reviewed by Arrington USA; E-mail: [email protected] and Butel, 2001). SV40 also induces neoplastic trans- 2These two authors contributed equally to this work formation in cell culture as assessed by a number of SV40 large T antigen targets multiple cellular pathways D Ahuja et al 7730 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 Figure 1 Structure of SV40. (a) SV40 genomic DNA is composed lost due to dilution as the cells divide or due to of three elements: the early and late coding units and the regulatory region. The early unit encodes large T antigen (LT), small t antigen degradation of viral DNA. Thus, descendents of these (sT), 17K T antigen (17KT). The late unit encodes the three infected cells have a nontransformed phenotype and do structural proteins (VP1, VP2, and VP3) and the agnoprotein not contain viral DNA. (agno) and a pre-microRNA (miRNA). The regulatory region (ori) Sublines of stably transformed cells do arise from contains sequences for the early and late promoter and the origin of replication. (b) Several domains and motifs make up the SV40 T infections of nonpermissive cells, but with a low antigens: J domain (J), Rb-protein-binding motif (LXCXE), frequency (Figure 2c). The rate-limiting step to stable nuclear localization signal (NLS), phophatase pp2A-binding transformation is thought to be the integration of viral domain (pp2A binding), origin-binding domain (OBD), Zn domain DNA into the cellular genome by nonhomologous (Zn), ATPase domain (AAA þ ), variable region (VR), and host- recombination. Integration occurs at random sites with range domain (HR). Residue numbers are indicated below the domain structure 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 different assays. Genetic analysis has shown that its subsequent descendants are transformed. 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 What is transformation? expression is required for transformation. Neoplastic transformation refers to the acquisition of expanded proliferation and/or survival potential by a SV40 transforms cells that are nonpermissive for viral cell. Several different assays are used to distinguish a productive infection ‘normal’ cell from transformed cells. In each of these assays cells are placed in an environment that is growth Infection of permissive cells, such as the CV1 or BSC40 restrictive for normal cells whereas transformed cells lines of African green monkey kidney cells, results in cell proliferate and survive. In these assays, to score as

Oncogene SV40 large T antigen targets multiple cellular pathways D Ahuja et al 7731

Figure 2 SV40 effects in different cellular environments. (a) Infection of permissive cells results in cell death and virion production. (b) SV40 infection of rodent cells induces S-phase but does not result in cell death or virus production. (c) Integration of viral DNA occurs in a very low percentage of nonpermissive cells, which then become stably transformed transformed requires that the rate of cell proliferation be covered with a monolayer of cells. Growth arrest is greater than the rate of cell loss. Thus, each assay thought to be initiated by contact with adjacent cells so measures the ability of cells to escape specific growth that when a monolayer is obtained most cells exit the and/or survival restrictions. The assays commonly used cycle. This is a stable growth arrest and the cells can be to assess transformation by SV40 are: maintained in this quiescent state for weeks. Saturation density is defined as the maximum number of cells per Immortalization unit area of culture surface. SV40-transformed cells fail to arrest when they reach monolayer and thus reach Primary cells can be passaged a limited number of times much higher or indefinite saturation densities. before they undergo growth arrest and irreversible senescence. MEFs or rat embryo fibroblasts (REFs) Focus formation expressing large T antigen are immortal and can be propagated in culture indefinitely; however, the situa- SV40-transformed cells can proliferate on the surface of tion is more complicated in other cell types. For a growth arrested monolayer of untransformed cells. example, human fibroblasts expressing large T antigen Experimentally, this can be assessed in two ways. SV40- can be propagated for an extended period in culture but transformed cells can be mixed with an excess of do eventually senesce. An active telomerase is required untransformed cells and then maintained in culture to escape senescence in these cells. dishes. The untransformed cells will growth arrest when they reach monolayer while the SV40-transformed cells Growth in low serum will continue to proliferate. This results in the appear- ance of dense regions of multilayered cells, called foci, Normal fibroblasts require serum-supplemented med- on the surface of the monolayer. The same result can be ium to proliferate and survive. SV40-transformed cells achieved by plating SV40-transformed cells on a can proliferate and survive in medium with little or no preformed monolayer of untransformed cells. serum. Anchorage independence Saturation density Untransformed cells remain viable for weeks when Fibroblasts typically proliferate and spread across the suspended in a slurry of agarose supplemented with surface of a culture dish until the entire surface is medium and serum. However, they do not proliferate as

Oncogene SV40 large T antigen targets multiple cellular pathways D Ahuja et al 7732 this requires contact with the culture vessel surface and tumorigenic in immunocompromised mice. Do each of components of serum that coat the surface. In contrast, these assays measure the same molecular event? Does SV40-transformed cells proliferate in the absence of SV40 regulate some key cellular pleiotropic switch that such contact and grow as multicellular spheres when coordinately confers all these transformed properties on suspended in agarose. cells? Or, does SV40 target multiple cellular switches, each of which controls a subset of the transformed Ability to form tumors in animals following injection phenotype? The answer to this question appears to be of transformed cells ‘both’. The evidence that SV40 targets multiple pathways each resulting in pleiotropic effects on cell Untransformed cells are nontumorigenic when injected behavior, is outlined below. into test animals such as immunocompromised mice. SV40-transformed cells form tumors when injected into mice and other test animals. Cellular uncertainty principle

Role of cell type in transformation The Heisenberg Uncertainty Principle is among the most important concepts to emerge from quantum Cell culture transformation assays are carried out using mechanics. With apologies to our physics colleagues either primary cells or established cell lines. Each of we shamelessly borrow and paraphrase a lesson evoked these systems carries inherent advantages and draw- by that Principle, namely, the act of experimental backs. Primary cells, such as MEFs, are useful because manipulation alters the behavior of the experimental they have not been passaged extensively in culture and system. In quantum physics this refers to the inability to thus these cells carry the same genetic complement as the simultaneously determine the position and momentum animal from which they were derived. Since established of a photon. Experimental manipulation also alters the cell lines, by definition, are immortal, immortalization behavior of cell culture systems in unpredictable ways. assays can only be carried out using primary cells. Furthermore, cell culture manipulations impinge on Furthermore, primary cells can be cultured from specific many of the very pathways that we wish to study: those strains of mice, such as gene knockout or transgenic involved in regulating cell cycle, apoptosis, and stress lines, thus allowing the study of the influence of specific responses. Therefore, animal models are essential to alleles on transformation. One of the disadvantages of examine the influence of oncogenic signals in native using primary cell cultures is that they consist of a environment. mixture of cell types that may respond differently to In practice, the cellular uncertainty principle means oncogenic signals. The diversity and properties of these that cultured cells are not necessarily in a native state cell populations change dramatically during embryogen- when they are cultured on plastic surfaces in medium esis. Even single cell types within this population may supplemented with fetal bovine serum or the equivalent exhibit significantly different properties. For example, (Figure 3). In fact, the process of generating a primary fibroblasts collected from different anatomical sites of cell culture results in the death of the vast majority of the same individual display different patterns of global cells in the tissue or embryo, and the outgrowth of a gene expression (Chang et al., 2002). Therefore, the age small population of cells capable of proliferating in this of the animal at the time of embryo extraction, method environment. Still, there is little doubt these systems are of cell collection, and culture conditions can be useful, even powerful. Genes that are active in the assays important influences on the outcome of transformation and cell systems described above are often found assays. mutated in human cancer. Thus, these systems success- Established cell lines have the advantage of being fully identify proteins that participate in tumorigenesis. clonal. This allows molecular studies on a large number What is unclear is how each of the cell behaviors of cells that respond similarly to oncogenic stimuli. assessed in these transformation assays relates to a given However, establishment results from genetic changes step in tumorigenesis, or to specific properties of tumor that select for cell subpopulations that are immortal. cells. This means that cell culture systems are very good The specific genetic changes carried by a given cell line for understanding the molecular aspects of regulating influence the response to oncogenic and death stimuli, signaling pathways, but not so good at predicting how and thus the results of transformation assays. The perturbing specific pathways alters cellular behavior in important conclusion from these observations is that the the context of a tissue. choice of cell system has major consequences in governing the outcome of transformation assays. T antigens elicit transformation by targeting cellular proteins Key questions: how are these different transformation assays related to each other? Dense focus assay screens have identified four SV40 functions, three in large T and one in small t, that clearly SV40 transforms multiple cell types as measured by each contribute to transformation (reviewed by Ali and of the assays described above. For example, SV40- DeCaprio, 2001; Saenz-Robles et al., 2001; Sullivan transformed MEFs proliferate in low serum and in soft and Pipas, 2002). In each case, the SV40-transforming agar, grow to a high saturation density, and are function correlates with the ability of one of the

Oncogene SV40 large T antigen targets multiple cellular pathways D Ahuja et al 7733

Figure 3 The cellular uncertainty principle. Multiple manipulations are required to establish an immortal cell line, from killing the donor animal to dissecting the appropriate tissues or cell source, to culturing the cells in vitro through numerous passages. Each step can induce genetic and/or morphological alterations in response to the environment and procedure used

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 Figure 4 Cellular pathways affecting cell proliferation. By missed if redundant large T antigen functions can blocking both pRB- and p53-dependent responses T antigen is independently induce foci. able to drive quiescent cells to re-enter S-phase and to escape apoptosis. The net result is entry into the cell cycle

Large T antigen action on Rb proteins activity of E2F transcription factors. There are eight Large T antigen contains an LXCXE motif (residues known E2F proteins (E2F1-8), all of which possess a 103–107) that is essential for its interaction with the pRb DNA-binding domain that governs interaction with a family of tumor suppressors. Mutants that alter this common consensus sequence present in the promoters of motif are defective for transformation in nearly all assay E2F-regulated genes (reviewed by Attwooll et al., 2004; systems (Chen and Paucha, 1990; Christensen and Frolov and Dyson, 2004; Dimova and Dyson, 2005). Imperiale, 1995; Zalvide and DeCaprio, 1995; Sriniva- The active forms of E2F1-5 consist of a heterodimer san et al., 1997). The importance of this sequence in containing one E2F polypeptide associated with a mediating the interaction with Rb-proteins, and in dimerization partner, DP1 or DP2. Rb-E2F interactions transformation was ﬁrst demonstrated for the adeno- regulate entry and exit into the cell cycle (Figure 4). In virus E1A protein (Whyte et al., 1988a). Subsequently, this scenario, E2F-regulated genes are not expressed in SV40 large T antigen was shown to bind Rb-proteins quiescent cells because their promoters are occupied through this motif (DeCaprio et al., 1988; Ewen et al., primarily with p130/E2F4 complexes which repress 1989). Large T antigen binds to all three Rb-proteins, transcription. Inactivation of Rb-proteins by phosphor- but as will be discussed below, the consequences of this ylation results in the replacement of these complexes by association are different in each case. ‘activating’ E2F1-3. This leads to the transcription of Most, if not all, of large T antigen’s effects on the Rb- E2F-regulated genes many of which encode proteins proteins are thought to be exerted by regulating the required for DNA replication, nucleotide metabolism,

Oncogene SV40 large T antigen targets multiple cellular pathways D Ahuja et al 7734 DNA repair and cell cycle progression. Thus, cell cycle themselves, but cells expressing both mutants are entry and growth arrest are, at least in part, governed by immortal (Tevethia et al., 1998). signals that ultimately dictate the phosphorylation state of Rb-proteins, and consequently their ability to repress Large T antigen is a DnaJ molecular chaperone E2F-dependent transcription. Large T antigen short- circuits this pathway by binding Rb-proteins and The first 70 amino acids of large T antigen and small t blocking their ability to regulate E2Fs (Figure 4). antigen have sequence identity with the J domain of the This model is consistent with transfection experiments DnaJ class of molecular chaperones (Kelley and Landry, indicating that a functional LXCXE motif is required 1994). Structural studies with SV40 T antigen or with for large T antigen to override Rb-induced growth related T antigens from murine polyomavirus show that arrest, and to release E2F from Rb-mediated repression this region folds as a J domain (Berjanskii et al., 2000; (Zalvide and DeCaprio, 1995). Furthermore, unlike Kim et al., 2001). DnaJ chaperones work in concert with their normal counterparts, cell lines transformed with a partner chaperone of the DnaK class (reviewed by large T antigen do not accumulate p130/E2F4 and other Mayer and Bukau, 2005). DnaK chaperones consist of Rb/E2F complexes when they reach confluence, or if an ATPase domain linked to a substrate-binding they are maintained in low serum (Zalvide et al., 1998; domain. The J domain of DnaJ chaperones binds to Sullivan et al., 2000b). Thus, large T antigen disrupts the ATPase domain of its DnaK partner, thereby repressive Rb-E2F complexes, allowing transcription of stimulating the DnaK ATPase activity. Subsequent to E2F-dependent genes and progression into S-phase. binding ATP, DnaK proteins form a tight association The LXCXE motif is required to allow growth to a with substrate proteins, and then the energy derived high density in low and high serum concentrations and from DnaK-mediated ATP hydrolysis is used to modify anchorage independence growth of MEFs both in the substrate in some way. a normal and Rb null background (Zalvide and Large T antigen binds to hsc70, the major DnaK DeCaprio, 1995). Similarly mutations in this sequence homologue present in mammalian cells and this binding render T antigen defective in these assays when tested in is dependent upon the J domain and ATP hydrolysis MEFs lacking both p130 and p107 (Stubdal et al., 1997). (Sullivan et al., 2001). Furthermore, T antigen stimu- This indicates that loss of pRb function itself is not lates the ATPase activity of hsc70 and stimulates the sufficient to induce transformation and that large T release of unfolded peptides from the substrate-binding antigen must inactivate at least two, and possibly all domain of hsc70 (Srinivasan et al., 1997). A hybrid three, retinoblastoma proteins to induce transformation DnaJ protein, in which the J domain of DnaJ has been as measured by this assay. Interestingly, the requirement replaced by the J domain from SV40, functions in of the LXCXE motif for anchorage-independent Escherichia coli (Kelley and Georgopoulos, 1997), while growth is ameliorated in p19ARF null MEFs (Chao mutants of the SV40 J domain do not function in E. coli et al., 2000). (Genevaux et al., 2003). Similarly, a hybrid DnaJ These data strongly indicate that the Rb-proteins are protein, in which the J domain of Ydj1p, a yeast DnaJ an important target for large T antigen in mediating chaperone, has been replaced by the J domain from transformation. However, they do not rule out the SV40, functions in Saccharomyces cerevisiae (Fewell possibility that T antigen interacts with other targets et al., 2002). Thus, T antigen is a DnaJ molecular important for transformation through the LXCXE chaperone. motif. Many cellular proteins have LXCXE motifs and The J domains of SV40 T antigen and of mammalian are thought to bind Rb-proteins via the same surface as DnaJ chaperones appear to have functions absent in the large T antigen (Dick et al., 2000). Thus, in some cases T yeast and E. coli DnaJ proteins since hybrid viruses, in antigen can be expected to displace cellular proteins which the J domain of large T antigen is replaced by the bound to Rb-proteins, while in other cases T antigen J domain of either E. coli DnaJ or Ydj1p from yeast, binding to Rb-proteins might be sterically blocked by bind hsc70, but are defective for productive infection the presence of an Rb-associated protein. (Sullivan et al., 2000b). In contrast, the DnaJ domains While an intact LXCXE motif is required for T from the JCV large T antigen, or from human DnaJ antigen to induce high-density cell growth and ancho- homologues do functionally replace the SV40 T antigen rage independence, it appears to be dispensable for J domain (Stubdal et al., 1997; Sullivan et al., 2000b). extending the lifespan of primary cells MEFs and REFs et al in culture (Chen and Paucha, 1990; Thompson ., Role of the J domain in the disruption of Rb–E2F 1990; Zalvide and DeCaprio, 1995). In fact, a carboxy- complexes terminal fragment missing the first 250 amino acids, which includes the J domain, Rb-binding sequences and Several observations indicate that, at least in some cases, the DNA-binding domain of large T antigen, is still the J domain acts in concert with the LXCXE motif to capable of extending primary cell lifespan (Thompson disrupt Rb–E2F complexes. For example, growth et al., 1990). Interestingly, a truncated large T antigen arrested MEFs display a prominent p130–E2F complex that consists of the first 147 amino acids, including a in EMSA experiments, while this complex is not present functional J domain and Rb-binding sites, is also in cells expressing T antigen. However, the presence of capable of extending primary cell lifespan. Neither of this complex is maintained in cells expressing either these T antigen fragments induces immortalization by J domain mutants or LXCXE motif mutants (Zalvide

Oncogene SV40 large T antigen targets multiple cellular pathways D Ahuja et al 7735

Figure 5 The chaperone model. Large T antigen recruits hsc70 in order to disrupt Rb/E2F complexes

et al., 1998). In addition, the J domain is required for T can use energy from ATP hydrolysis to free E2F from antigen to overcome p130 or pRb-mediated repression p130. of E2F-dependent transcription in transient transfection T antigen does not treat all Rb–E2F complexes in the assays (Zalvide and DeCaprio, 1995). same way. The obvious manifestation of this is in Purified T antigen binds stably to preformed p130– MEFs, where transformation by T antigen leads to the E2F4–DP1 complexes in vitro (Sullivan et al., 2000a). proteosome-dependent degradation of p130, but does Thus, T antigen does not dislodge p130 from E2F4 by not alter the levels of pRb and leads to increased levels affinity displacement. The coexistence of large T of p107 (Stubdal et al., 1997). Similarly, during SV40 antigen, and perhaps other LXCXE motif-containing infection in BSC40 cells, T antigen expression results in proteins, and E2F on the same p130 molecule is loss of p130–E2F complexes, while some pRb–E2F consistent with structural studies that indicate that complexes remain intact (Sullivan et al., 2004). Finally, LXCXE motif proteins, including T antigen, bind to a the J domain is required to confer a growth advantage to groove on the surface of the B domain of pRb, while MEFs expressing functional p130 and p107 proteins, E2Fs bind a separate surface formed at the intersection even in the absence of a functional pRb (Zalvide and of the A and B domains (Kim et al., 2001; Xiao et al., DeCaprio, 1995). In contrast, the J domain is not 2003). Disruption of the p130–E2F4 complex in vitro required to confer this growth advantage to MEFs occurs upon the addition of hsc70 and ATP, and derived from p130, p107 double null embryos (Stubdal requires both a functional J domain and LXCXE motif. et al., 1997). One interpretation of these data is that the It is important to note that this in vitro chaperone J domain is needed for T antigen to inactivate p130 and reaction does not indiscriminately break apart protein p107, but not for pRb inactivation. How T antigen complexes, since functional E2F4–DP1 dimers remain discriminates different Rb–E2F complexes, as well as intact during this reaction (Sullivan et al., 2000a). The the biological significance of this differential action, is first 136 amino acids of T antigen are sufficient to unclear. disrupt p130–E2F complexes both in vivo and in vitro (unpublished results). Thus, the J domain and LXCXE How does the J domain contribute to transformation? motif are sufficient to disrupt Rb–E2F complexes, but they must reside in cis, that is, on the same T antigen In-frame deletion mutants of T antigen missing part or molecule (Srinivasan et al., 1997). Thus, J domain all of the J domain coding sequences are defective for mutants do not complement LXCXE motif mutants the induction of dense foci despite being able to bind either in productive infection or transformation. These Rb-proteins and p53 (Pipas et al., 1983; Srinivasan et al., observations give rise to a model (Figure 5) in which 1997). Thus, the J domain per se is required for focus large T antigen recruits p130–E2F complexes via its formation. Furthermore, genetic complementation tests LXCXE motif so that hsc70 bound to the J domain indicate that the J domain cannot function in trans.In

Oncogene SV40 large T antigen targets multiple cellular pathways D Ahuja et al 7736 fact, the J domain must be in cis with at least two other ras cooperation is J domain and LXCXE motif T antigen elements to effect transformation; namely, the dependent. LXCXE motif, and an unidentified function that One interpretation of this data is that amino-terminal requires sequences carboxy-terminal to amino acid 136 fragments of T antigen that include a functional J (Srinivasan et al., 1997). Oddly, the ability of J domain domain and LXCXE motif are sufficient to inactivate deletion mutants to induce anchorage-independent Rb-proteins. However, is the action of truncated T growth has not been reported. Deletions of the J domain antigens, like N121 or N136, really equivalent to the are also capable of immortalizing primary cells (Hahn simultaneous loss of pRb, p107, and p130? One piece of et al., 2002). However, this result is complicated by the evidence suggests that the answer to this question is no. fact that T antigen has at least two independent Genetic ablation of pRb, p107, and p130 from MEFs functions that can extend the lifespan of primary cells: results in immortalization and anchorage-independent one residing in the first 121 amino acids that includes the growth (Sage et al., 2000). In contrast, the expression of J domain and LXCXE motif, and the other residing in N136, or similar fragments, results in MEFs that have sequences in the carboxy-terminal half of the molecule an extended lifespan, but are not immortal, and do not (Tevethia et al., 1998). induce anchorage-independent growth (Thompson In contrast to J domain deletions, amino-acid et al., 1990). Perhaps, sequences in the carboxy-terminus substitution mutants, such as D44N, do induce focus of T antigen contribute to the inhibition of Rb-protein formation and anchorage-independent growth, although tumor suppressor function. Alternatively, the amino- at a somewhat reduced frequency compared to wild type terminal T antigen fragments may possess activities, in (Peden and Pipas, 1992; Stubdal et al., 1997; Hahn et al., addition to hsc70- and Rb-protein binding, that inter- 2002). However, D44N, as well as other alleles, are fere with the action of T antigen on Rb-proteins. defective for inducing growth to a high saturation et al density and for growth in low serum (Stubdal ., The J domain cooperates with a function(s) in the 1997). Thus, the fact that D44N is clearly defective for carboxy-terminal portion of T antigen hsc70 binding and for the disruption of p130–E2F4 complexes appears to dissociate the T antigen chaperone As mentioned above, the J domain is required in cis with function from focus formation and anchorage-indepen- an unknown activity that maps carboxy-terminal to dent growth, but links it to growth in low serum amino acid 136 and is required to induce focus (Sullivan et al., 2000a, b, 2001). formation (Srinivasan et al., 1997). In addition to its Why are some J domain alleles defective for focus role in transformation, the T antigen J domain is also formation while others are not? One possibility is that required for viral DNA replication and virion assembly the interaction of the J domain with hsc70 is allele (Peden and Pipas, 1992; Spence and Pipas, 1994; specific. For example, perhaps a given mutant J domain Campbell et al., 1997). The J domain acts in cis with can stimulate hsc70 enough to achieve partial action on carboxy-terminal sequences of T antigen in each of these all or some of its substrates leading to a subset of cases as well. This suggests that the J domain recruits phenotypes observed with the wild-type protein. An- hsc70 for action on targets in addition to Rb–E2F other possibility is that the SV40 J domain binds cellular complexes, and that this action is required for several proteins in addition to hsc70. In this scenario, some aspects of viral infection, as well as for transformation. alleles would disrupt T antigen interaction with the The simplest model that explains these results is that unknown cellular target, thus losing the phenotypes T antigen binds a target(s) through sequences that are resulting from this interaction, while maintaining inter- carboxy-terminal to residue 136, and that the J domain action with hsc70, or vice versa. Still other alleles, such must act on this target to elicit transformation. While as deletions, might result in the loss of both binding this target or targets are unknown, we raise two functions. possibilities. First, is the interaction of T antigen with It is clear that truncated T antigen mutants that the histone acetyltransferase activator complex, CBP/ terminate after the LXCXE motif but before the p300 (Eckner et al., 1996; Lill et al., 1997). CBP/p300 do origin-binding domain (OBD), carry potent biological not bind large T antigen directly, but rather are activities. The best characterized of these truncation recruited into association via p53 (Poulin et al., 2004). mutants are dl1137 (N121) and N136. These, and In the case of the adenovirus, the E1A protein must similar fragments, induce foci in many established interact with both Rb-proteins and CBP/p300 to induce cell lines (Srinivasan et al., 1989) and hyperplasia transformation (Egan et al., 1988, 1989; Whyte et al., in transgenic mice (Fromm et al., 1994; Kim et al., 1988b). Wild-type SV40 large T antigen can complement 1994; Tevethia et al., 1997). In most of these cases, the transformation defect of E1A mutants that do not biological activity requires both the J domain and the bind CBP/p300, while T antigen J domain mutants do LXCXE motif. Furthermore, N136 can rescue neuronal not (Yaciuk et al., 1991). Thus, it is intriguing to stem cell lines from apoptosis induced by growth-factor speculate that the T antigen J domain must act on the T withdrawal, and this rescue requires both an active J antigen–p53–p300 complex to induce transformation, domain and LXCXE motif (Slinskey et al., 1999) but no hard evidence has been forthcoming to indicate Finally, amino-terminal fragments (residues 1–127 and this is the case. A second potential target that 1–121) of T antigen cooperate with oncogenic ras to exemplifies the cooperation between the J domain and transform primary REFs (Beachy et al., 2002). Again, carboxy-terminus of T antigen is cyclin A. Cyclin A–cdk

Oncogene SV40 large T antigen targets multiple cellular pathways D Ahuja et al 7737 complexes phosphorylate and inactivate pRb, thus 1981). However, while T antigen blocks some p53 overcoming its growth-suppressive effects. T antigen functions, an alternative hypothesis is that other p53 transactivates the cyclin A promoter, thereby sending activities may not be inhibited by T antigen and in fact, the cells into S-phase. This activity is independent of the might be dependent upon the interaction of p53 with T LXCXE motif, but requires both the J domain and antigen (Deppert et al., 1989). This gain-of-function sequences carboxy-terminal to amino acid 127 (Beachy scenario is made more plausible by recent reports that et al., 2002). some p53 alleles found in cancer express p53 protein that contributes to neoplasia (Lang et al., 2004; Olive T antigen interaction with the tumor suppressor p53 et al., 2004). At present, there is not enough evidence to tip this argument one way or another. However, MEFs The p53 tumor suppressor was discovered as a cellular derived from p53 null embryos are immortal, while protein bound to large T antigen in SV40-transformed MEFs expressing carboxy-terminal fragments of T cells (Lane and Crawford, 1979; Linzer and Levine, antigen, capable of binding p53, have an extended 1979). Under normal circumstances the steady-state lifespan, but are not immortal (Tevethia et al., 1998). levels of p53 in a cell are very low. This is because p53 Thus, either the interaction of full-length T antigen with binds to the promoter of the mdm2 gene and stimulates p53 is required to fully inhibit its tumor suppressor its transcription (reviewed by Bond et al., 2005; Coutts functions, or T antigen action on p53 is not equivalent and La Thangue, 2005). As its levels rise, mdm2 binds to a p53 null cell. p53 and induces its polyubiquitination and subsequent degradation. This feedback loop is disrupted by several Small t antigen targets the cellular phosphatase pp2A kinds of stress, including DNA damage, depletion of nucleotide pools, anoxia, and abnormal inhibition of The 174 amino-acid small t antigen consists of a J Rb-proteins. Under these conditions phosphorylation of domain identical to the one found in large T antigen, p53 results in inhibition of the mdm2–p53 interaction, and a unique carboxy-terminus that directs its interac- and consequently in an increase in p53 steady-state tion with the cellular phosphatase, pp2A. Small t levels. p53 is a potent transcriptional activator, and as antigen does not usually score in any cell culture its steady-state levels rise it upregulates a number of transformation assays when expressed alone. However, genes, some of which mediate cell cycle arrest or under many circumstances where T antigen expression is apoptosis (see Figure 4). not sufficient for transformation, coexpression of large Large T antigen interacts with the DNA-binding T antigen and small t antigen leads to transformation. surface of p53, blocking its ability to bind promoters Small t antigen’s contribution to transformation re- and thus to regulate gene expression (Bargonetti et al., quires its binding to the catalytic subunit of pp2A 1992; Jiang et al., 1993). The T antigen–p53 interaction (Mungre et al., 1994; Hahn et al., 2002). This binding occurs through amino acids on the solvent-exposed results in displacement of the regulatory B subunit of surface of the T antigen ATPase domain (Li et al., 2003). pp2A and consequent inhibition of phosphatase activity, Carboxy-terminal fragments of T antigen, such as or change in substrate specificity. C351–708 are sufficient to bind p53 and block its The contribution of small t antigen to SV40 transfor- growth-suppressive functions (Cavender et al., 1995). mation is discussed by Arroyo and Hahn in a separate The carboxy-terminal variable region/HR domain (ami- chapter of this volume. no acids 627–708) of T antigen is not required for interaction with p53. Furthermore, an internal deletion Are there additional T antigen targets? that removes residues 451–532 is capable of p53 binding (Kierstead and Tevethia, 1993). This suggests that The importance of the large T antigen interaction with a bipartite-binding region consisting of amino acids Rb-proteins and with p53 in SV40 transformation is well 351–450 and 533–626 govern the T antigen–p53 inter- established. However, is this the whole story, or do other action. T antigen activities contribute to the tumorigenic SV40 appears to block p53 function by multiple phenotype? Large T antigen was identified as one of redundant mechanisms. Thus, T antigen can also block four oncogenes required to transform normal human p53-dependent transcriptional activation and growth- epithelial and fibroblast cells to tumorigenic cells (Hahn arrest independent of p53 binding (Quartin et al., 1994; et al., 1999, 2002). In these experiments large T antigen Rushton et al., 1997). The amino-terminal 121 residues acted along with an oncogenic allele of ras, hTERT and of T antigen are sufficient for this effect, which requires small t antigen to induce transformation. Furthermore, both the J domain and LXCXE motif. Small t antigen large T antigen can be functionally replaced by also participates in blocking p53 function (Pipas and combinations of viral and cellular proteins that inacti- Levine, 2001). vate the pRb and p53 pathways. For example, over- Is T antigen action on p53 equivalent to the total loss expression of cyclin D, an INK4a-resistant CDK4 or the of p53 activity, or does the interaction with T antigen human papillomavirus E7 protein combined with either inhibit some p53 activities, while sparing others? Large a dominant negative p53 or the human papillomavirus T antigen binding results in p53 stabilization and thus proteins E6 protein, could replace large T antigen in SV40-transformed cells contain large amounts of p53 these assays. Similarly, short interfering (si) RNA that is thought to be functionally inactive (Oren et al., directed towards pRb and p53 appear to replace T

Oncogene SV40 large T antigen targets multiple cellular pathways D Ahuja et al 7738 antigen in this system (Voorhoeve and Agami, 2003). maintain a normal interaction with p53 would be of Taken together, these observations suggest that T great help. antigen’s contribution to tumorigenesis can be attri- buted solely to its inhibition of pRb and p53 pathways Cul7 (Hahn et al., 2002). On the other hand, genetic studies suggest that Imperiale and colleagues first noticed a protein, termed inactivation of pRb and p53 may not account for the p185, associated with large T antigen in transformed full transformation potential of T antigen (Cavender cells (Kohrman and Imperiale, 1992). The p185-binding et al., 1995; Sachsenmeier and Pipas, 2001; Wei site is within the first 121 amino acids of T antigen and et al., 2003). Furthermore, several cellular T antigen- binding is independent of the LXCXE motif and the J binding proteins have been identified that, based on domain. This same protein, here termed p193, was their known functions, have a potential to contribute to independently shown to bind T antigen in cardiomyo- transformation. cytes (Tsai et al., 2000). Subsequently, the T antigen associated protein was shown to be a cullin, Cul7, which p300/CBP is part of an E3 ligase complex involved in the ubiquitin degradation of proteins (Ali et al., 2004). Mutagenesis The transcriptional adapter proteins p300 and CBP play studies indicate that sequences carboxy-terminal to the J roles in multiple biological processes, including cell domain but amino-terminal to the LXCXE motif are growth and transformation (reviewed in Goodman and required for Cul7 association with T antigen. Smolik, 2000). The interaction of E1A with p300/CBP is Cul7-binding mutants of T antigen are unable to essential for adenovirus transformation (Egan et al., induce anchorage-independent growth of MEFs and fail 1988, 1989; Whyte et al., 1988b). Genetic studies to support growth in low serum (Ali et al., 2004). These demonstrated that SV40 T antigen can complement mutants can bind to pRb and inhibit pRb-mediated p300/CBP-binding deficient mutants of E1A to restore repression of E2F-dependent transcription. They transformation (Yaciuk et al., 1991). Several studies are also able to bind to and stabilize p53, although suggested that p300/CBP associates with large T antigen the authors did not specifically show that p53 was but it was unclear whether or not this interaction was inactivated. direct (Avantaggiati et al., 1996; Eckner et al., 1996; Lill These observations suggest that binding to Cul7 could et al., 1997). Recently, it was demonstrated clearly that be required in addition to inactivation of pRb and p53 p300/CBP associates with large T antigen indirectly, for T antigen-mediated transformation. However, how through its interaction with p53 (Poulin et al., 2004). does the interaction of Cul7 contribute to transforma- The functional consequences of T antigen binding to tion? One possibility is that T antigen recruits Cul7 to p300/CBP are not understood fully, and the role of this target some other T antigen-associated protein for interaction in transformation has not yet been estab- degradation. Alternatively, the elimination of Cul7 lished clearly. T antigen may enhance the p300/CBP- function might be a prerequisite for SV40 transforma- associated histone acetyltransferase activity (Valls et al., tion. In this model, which is favored by the authors, 2003). In addition, CBP acetylates T antigen on K697 in Cul7 functions as a tumor suppressor (Ali et al., 2004). a p53-dependent manner (Poulin et al., 2004). While the This hypothesis predicts that T antigen mutants, role of this acetylation in virus growth or transforma- deficient for Cul7 binding, will transform MEFs derived tion is not clear, it is interesting to note that this from Cul7 null embryos. In any event, the link between acetylation site is conserved on T antigens of the human T antigen–Cul7 binding and transformation is poten- polyomaviruses, JC and BK, and the baboon poly- tially of great interest since it could indicate a pathway, omavirus, SA12 (Cantalupo et al., 2005 submitted; independent of p53 and Rb-proteins, that plays an Poulin et al., 2004). Finally, some experiments suggest essential role in tumorigenesis. that p300/CBP is required for E2F transcriptional in vitro et al activity (Ait-Si-Ali ., 2000). Thus, one might Bub1 speculate that T antigen increases cell proliferation not only by inactivating pRb, but also by delivering p300/ A yeast-two hybrid analysis led to the identification of CBP to E2F-dependent promoters. This hypothesis is Bub1, a mitotic spindle checkpoint protein, as a T consistent with the observation that activation of E2F- antigen interacting protein (Cotsiki et al., 2004). dependent promoters by the murine polyomavirus large Mutations, such as W94A or W95A, lead to loss of T antigen requires both the inhibition of Rb-proteins the T antigen–Bub1 interaction. These mutants are and an interaction with p300/CBP (Nemethova et al., unable to induce focus formation in Rat-1 cells, 2004). suggesting that Bub1–T antigen interaction may play a Despite these provocative observations, definitive role in transformation (Cotsiki et al., 2004). evidence that the interaction of large T antigen and Mutations in Bub1 have been found in certain types p300/CBP plays a role in transformation has not been of human cancers, such as colorectal cancer and reported. Of course, the fact that p300/CBP associates leukemia (Cahill et al., 1998; Ru et al., 2002). with T antigen through p53 greatly complicates these Furthermore, the interaction between T antigen and studies. Clearly, T antigen mutants that are defective for Bub1 results in the perturbation of the spindle T antigen binding to, or action on, p300/CBP, but checkpoint (Cotsiki et al., 2004) and may be one

Oncogene SV40 large T antigen targets multiple cellular pathways D Ahuja et al 7739 explanation for how T antigen induces aneuploidy and competitive inhibitor of Fbw7, reducing the turnover of genetic instability (Woods et al., 1994; Chang et al., physiological substrates, like cyclin E. The role of this 1997). However, several key questions need to be interaction in T antigen-mediated transformation has addressed before the importance of Bub1 to T antigen- not yet been determined. 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, Cell type specificity of T antigen action including primary cells. Additionally, the T antigen– Bub1 interaction needs to be established as either Tumorigenesis is thought to occur as a progression playing a role in the inactivation of Rb-proteins or through different stages (Vogelstein et al., 1988). In this p53, or as an independent transforming function. model, the progression from one stage to the next is Finally, while the link between the T antigen–Bub1 thought to be driven by genetic changes that enhance association and genetic instability is provocative, it has cell growth and/or survival. Thus, neoplasias occur in not yet been shown that this function actually con- different histopathological states such as: hyperplasia/ tributes to transformation. dysplasia, benign or noninvasive tumor, invasive tumor, and metastasis. One of the major challenges of cancer research is to associate changes in specific molecular TEF-1 pathways with progression from one stage to another. T antigen modulates the transcription of both cellular What can SV40 tell us about the role of the Rb and p53 and viral genes by targeting various components of the tumor suppressor pathways in tumor progression? eukaryotic transcriptional machinery (Gruda et al., The current paradigm stems largely from the pioneer- 1993; Martin et al., 1993; Johnston et al., 1996). One ing work of Van Dyke and colleagues who have studied of its targets is the transcription factor TEF-1, which the effects of T antigen expression in the choroid plexus. appears to play a role in the early-to-late switch in viral The choroid plexus epithelium (CPE) is a single layer transcription (Berger et al., 1996). TEF-1 binds to the T protecting and detoxifying the brain by maintaining the antigen OBD. S189N, a TEF-1-binding deficient mutant cerebro-spinal fluid–blood barrier. Ectopic expression of of T antigen, is seven- to eight-fold less efficient than large T antigen in the CPE results in renewed prolifera- wild-type T antigen in focus formation assays (Dick- tion and tumor formation of this epithelial tissue (Chen manns et al., 1994). The S189N mutant is able to bind and Van Dyke, 1991). The first 121 amino acids of T p53 but its ability to block p53 function has not been antigen are sufficient to induce CPE tumors, but the rate tested. Similarly, it is not known if S189N is fully of tumor growth is much slower than that of tumors capable of inactivating Rb-proteins. Thus, until the expressing wild-type T antigen (Chen et al., 1992). The ability of TEF-1 mutants to transform in different same truncation mutant of T antigen is unable to induce systems is assessed, and the T antigen–TEF-1 interac- tumors in the CPE when mutated in the LXCXE motif, tion is shown to be independent of T antigen–pRb and – indicating that the inactivation of the Rb pathway is p53 interactions, the relevance of TEF-1 to SV40- required for these cells to enter S-phase (Chen et al., mediated transformation will remain unclear. 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 Nbs1 antigens, unlike tumors induced by wild-type T antigen, T antigen has been shown to form a complex with Nbs1, CPE tumors induced by the truncated T antigen exhibit a component of the MRN complex that functions in large amounts of apoptosis. Rapid tumor growth can DNA repair (Wu et al., 2004). The OBD of T antigen is be restored by expressing the truncated T antigen in a required to bind to Nbs1, and this interaction is p53-deficient background (Symonds et al., 1994). independent of T antigen binding to p53, and pRb This suggests a simple model in which cell prolifera- and does not require the J domain. The T antigen tion is induced by inactivation of Rb-proteins, while cell interaction with Nbs1 is associated with endoreduplica- death is blocked by inhibiting p53 function. Consistent tion and thus, could conceivably contribute to genomic with this model, the expression of amino-terminal instability. However, at present there is no known role fragments of large T antigen in transgenic mice induces for this interaction in transformation. 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 expres- Fbw7 sion in different tissues also reveals dramatic cell –type- Recently, T antigen was shown to interact with Fbw7, a specific effects (Table 1). Here we discuss a selected set component of the ubiquitination machinery (Welcker of studies that illustrate this point, emphasizing the and Clurman, 2005). Fbw7 binds to its substrates via a relevance of these observations to cancer progression. phosphorylated epitope, the Cdc4 phospho-degron. This Inactivation of p53 is not always required for motif is present in the host-range domain at the tumorigenesis carboxy-terminus of large T antigen. However, unlike substrates of Fbw7, T antigen is not degraded. Instead The expression of the N121 truncated T antigen in the authors hypothesize that T antigen functions as a astrocytes results in their re-entry into the cell cycle

Oncogene SV40 large T antigen targets multiple cellular pathways D Ahuja et al 7740 Table 1 Cell-type-speciﬁc effects Organ Cell type Oncogene Consequences Invasiveness Metastases Citation

Brain Endocrine Large T, small t No effects No No Lee et al. (1992) Neuroblast Large T No effects No No Efrat et al. (1988) Choroid plexus Large T Hyperplasia, aggressive tumors Yes No Chen and Van Dyke (1991) epithelium

Eye Fiber cells of Large T, small t Increased proliferation, lenses Yes No Mahon et al. (1987) lenses tumors, brain tumors Cone photo- Large T, small t Increased proliferation, retinal Yes No al-Ubaidi et al. (1992a) receptors tumors, brain tumors. Rod photo- Large T, small t Increased DNA synthesis, No No al-Ubaidi et al. (1992b) receptors photoreceptor degeneration

Intestine Paneth Large T, small t Loss of mature Paneth cells No No Hauft et al. (1992); Garabedian et al. (1997)

Enterocyte Large T, small t Hyperplasia, dysplasia No No Kim et al. (1993); Chandrasekaran et al. (1996)

Goblet Large T, small t Entry in S-phase but death by No No Gum et al. (2001) apoptosis Endocrine Large T, small t Endocrine tumors No No Upchurch et al. (1996); Ratineau et al. (2000)

Smooth muscle Large T, small t Hyperproliferation, hyper- No No Herring et al. (1999) contractility, obstruction and megacolon.

Pancreas Acinar Large T, small t Dysplasia, neoplasia, acinar Yes Yes Ornitz et al. (1987); Bell et al. carcinomas, metastases (1990); Ceci et al. (1991); Ramel et al. (1995)

Endocrine (a) Large T, small t Hyperplasia, tumor No No Lee et al. (1992) Endocrine (a) Large T Hyperplasia, tumor No No Efrat et al. (1988) Endocrine (b) Large T Hyperplasia, vascularization Yes No Hanahan (1985); (10%), solid islet tumors Christofori et al. (1994); (1–2%). Berkovich and Efrat (2001) Prostate Endocrine Large T, small t Neoplasia, metastases to lymph Yes Yes Maroulakou et al. (1994); and liver. Garabedian et al. (1998)

Epithelium Large T, small t Hyperplasia, adenocarcinoma, Yes Yes Greenberg et al. (1995); metastases to lymph and liver Gingrich et al. (1996)

Epithelium Large T Hyperplasia, low-grade dys- Yes Yes Kasper et al. (1998); plasia, metastatic potential Masumori et al. (2001) depending on age/expression.

The expression of large T antigen and small t antigen in different tissues leads to different outcomes, ranging from no effects to metastases

accompanied by extensive apoptosis. However, unlike Taken together these results suggest that inactivation of CPE tumorigenesis this apoptosis is not alleviated by the the tumor suppressor p53 is not a prerequisite for elimination of p53. Rather astrocyte apoptosis is transformation in all cell types. dependent on a functional PTEN protein (Xiao et al., 2002). Similarly, both wild-type T antigen and N121 The expression of wild-type T antigen leads to different induce S-phase and hyperplasia when expressed in outcomes in different tissues intestinal enterocytes (Hauft et al., 1992; Kim et al., 1994). In this case, inactivation of Rb-proteins by N121 T antigen has been expressed in a large number of does not result in increased apoptosis. Molecular studies different issues of transgenic mice. Some of these indicate that enterocytes do not express signiﬁcant systems have been studied in detail while other await amounts of p53 and that there are no detectable T further analysis. However, it is clear that the con- antigen–p53 complexes present in the intestines of these sequences of T antigen expression are cell type speciﬁc. transgenic mice (Markovics et al., 2005). Finally, Here we summarize the effects of T antigen expression in expression of a similar amino-terminal fragment of T cells from four different tissues. antigen (N147) in the acinar cells of the pancreas induces tumors that are indistinguishable from those Eye The eye is a sensory organ composed of the iris, induced by wild-type T antigen. The tumors induced by lens, the cornea and two superimposed layers, the N147 still express a wild-type p53 (Tevethia et al., 1997). pigmented retina, capable of producing melanin, and

Oncogene SV40 large T antigen targets multiple cellular pathways D Ahuja et al 7741 the neural retina, containing light sensitive photorecep- gut lumen and transporting them to the underlying tors, glia, interneurons and ganglion cells. Through capillaries; the goblet cells, scattered between the embryogenesis, retinal progenitor cells divide and absorptive cells, secrete mucus (Paulus et al., 1993); generate postmitotic transition cells that commit to and the enteroendocrine cells are scattered throughout different neural and glial cell fates (ganglion cells, cone the epithelial layer and release hormones (Roth et al., cells, horizontal cells, amacrine cells, rod cells, Mu¨ ller 1992; Hocker and Wiedenmann, 1998). These three cell glia, and bipolar cells) and differentiate in conserved lineages migrate from the crypts to the villi, where they order, forming the different layers of the adult retina. are finally exfoliated when they reach an extrusion zone The lens contains three regions, and anterior zone of located near the villus tip. On the other hand, Paneth dividing epithelial cells, an equatorial zone of cellular cells, responsible for secreting a number of antimicrobial elongation, and a posterior and central zone of molecules when exposed to bacteria or bacterial anti- differentiated, crystalline-containing fiber cells. This gens, differentiate while they move to the bottom of the structure persists throughout life, as fibers are crypt, where they reside (Ayabe et al., 2000). continuously being laid down. Expression of both large T antigen and small t antigen T antigen expression in the fiber cells of the lens results in ectopic proliferation of all of these cell types. results in their re-entry into the cell cycle and lens Both enterocytes and smooth muscle cells are induced to tumors (Mahon et al., 1987). The induction of proliferate by T antigen expression with no detectable proliferation requires an intact LXCXE motif but does effect on differentiation (Hauft et al., 1992; Kim et al., not require the presence of small t antigen (Fromm 1993; Herring et al., 1999). While T antigen expression et al., 1994; Chen et al., 2004). Furthermore, expression results in hyperplasia and dysplasia in both of these of a truncated T antigen (N191) stimulates fiber cell cases, no distinct tumors are observed. In contrast, proliferation, but this is accompanied by increased expression of large and small T antigens in Paneth or apoptosis resulting in microphtalmia, microlentia and goblet cells results in S-phase entry followed by lens ablation (Fromm et al., 1994). While it is has not elimination of the specific cell type (Garabedian et al., been established that this apoptosis is p53 dependent, 1997; Gum et al., 2001, 2004). In fact, T antigen is as these results are consistent with a model in which the effective as cholera toxin in ablating Paneth and goblet amino-terminal portion of T antigen induces prolifera- cells from the intestine. Finally, T antigen expression in tion by inhibiting Rb-proteins while carboxy-terminal endocrine cells results in multiple endocrine tumors sequences of T antigen inhibit apoptosis, most likely by (Upchurch et al., 1996; Ratineau et al., 2000). blocking p53 activity. Interestingly, T antigen also blocks fiber cell differentiation through a mechanism Pancreas The mammalian pancreas is a compound that is LXCXE-motif independent and requires se- gland composed of endocrine and exocrine tissues. The quences carboxy-terminal to amino acid 191 (Fromm four endocrine cell types that produce insulin (a-cells), et al., 1994). glucagon (b-cells), somatostatin (g-cells), and pancreatic T antigen also induces tumors when expressed in cone polypeptide (PP-cells) are contained in the islets of photoreceptor cells (al-Ubaidi et al., 1992a). In contrast, Langerhans, comprising 1–2% of the cellular mass of expression of T antigen in rod photoreceptors induces the adult pancreas. The exocrine tissue is organized into DNA synthesis but the photoreceptors degenerate acini, which synthesize digestive hydrolases, and ducts, (al-Ubaidi et al., 1992b). Thus, T antigen appears to be which secrete a bicarbonate fluid that flushes the acinar able to induce S-phase and proliferation in three different secretions to the intestine. differentiated cell types of the eye. However, expression T antigen expression in the beta-islet cells results in in fiber cells and cone cells results in tumorigenesis, while S-phase entry and about 50% of the islets become expression in rod cells leads to tissue ablation. hyperplastic. Subsequently, about 10% of the islets become vascularized and 1–2% progress to carcinoma (Hanahan, 1985; Christofori et al., 1994). Thus, it Intestine The intestine is composed of three basic appears that T antigen initiates tumorigenesis by layers. On the outside, a layer of loose connective tissue stimulating b-cell proliferation and that this is followed followed by a double layer of smooth muscle (mesench- by cellular mutations that drive tumor progression. yma); in the middle, a layer of connective tissue Interestingly, the expression of IGF2 appears to be an containing blood and lymphatic vessels (stroma); and important cofactor for tumor progression in this system the innermost surface (mucosa) is composed of epithelial (Christofori et al., 1994). Similarly, T antigen expres- cells lining the actual lumen of the intestinal tract and sion in a-cells leads to hyperplasia with a minority of the organized as proliferative (crypts) and differentiated islets progressing to noninvasive tumors (Efrat et al., regions (villi). Multipotent stem cells are located near 1988; Lee et al., 1992; Asa et al., 1996). Production of the base of the crypt, and give rise to progenitor cells both large and small T antigen in the acinar cells results that eventually exit the cell cycle and differentiate into in acinar carcinomas some of which show metastasis one of four different cell types, one absorptive and (Ornitz et al., 1987; Bell et al., 1990; Ceci et al., 1991; forming the majority of the epithelium (enterocytes), Ramel et al., 1995). Surprisingly, the N147 truncation and three secretory (enteroendocrine, goblet, and mutant of T antigen induces carcinomas and metastasis Paneth cells). The enterocytes are responsible for with the same frequency as wild-type T antigen absorbing nutrients, water and electrolytes from the (Tevethia et al., 1997).

Oncogene SV40 large T antigen targets multiple cellular pathways D Ahuja et al 7742 Prostate 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 com- mitted 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). Figure 6 T antigen effects in vivo. Animal tissues are made of T antigen expression does not always result in cell multiple cell types characterized by different molecular properties proliferation and growth capabilities. Studies on transgenic mice expressing T antigen in a variety of tissues indicate that the elicited response The studies reviewed above suggest that the expression depends on the target cell type 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 our understanding of cancer (Van Dyke and Jacks, some cases where T antigen expression fails to stimulate 2002). cell proliferation. For example, transgenic mice expres- The observation that T antigen expression in different sing T antigen in neuroblasts of the midbrain and cell types leads to different histopathological outcomes forebrain remain quiescent (Efrat et al., 1988; Lee et al., is of fundamental importance to cancer research. Much 1992). Similarly, T antigen does not induce proliferation emphasis has been placed on defining the genetic in some enteroendocrine cells of the small intestine (Asa changes that govern the tumorigenic phenotype. How- et al., 1996). These very interesting cases warrant further ever, the cell type in which these changes occur appears study. Is T antigen unable to inhibit Rb-protein to be of equal, or perhaps greater importance, in functions in these cell types, or, alternatively, is there a determining tumor behavior. The observation that T block to proliferation downstream of Rb-protein antigen induces outcomes ranging from hyperplasia to inactivation? carcinoma and frequent metastasis emphasizes this point. In fact, in most cases T antigen has been expressed in terminally differentiated cells. Such cells Conclusions generally pass through several predifferentiated states before reaching permanent cell cycle exit. Cells in each The interaction of large T antigen with p53 and with the of these predifferentiated states have different potentials Rb family of tumor suppressor proteins clearly con- for proliferation, migration, and death, and each tributes to SV40 transformation. T antigen possesses respond to different growth and survival signals (see multiple additional activities that could also potentially Figure 6). Thus, the regulatory state of the cell in which contribute to cellular transformation, but their roles oncogenesis is initiated may play as important a role as have yet to be clearly established. Cell culture studies genetics in determining properties such as tumor will continue to be invaluable for identifying T antigen aggressiveness and metastatic potential. targets and for understanding the cellular pathways these targets regulate. However, the acts of establishment and maintenance of these in vitro systems alter Acknowledgements many of the regulatory networks that we wish to study. This work was supported by Grants CA40586 and CA098956 Thus, the development of animal model systems that to JMP. The authors thank the members of the Pipas Lab for more closely mimic human tumorigenesis are essential to their helpful comments.

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