The tumor antigens and the early functions of polyoma virus

Summary Polyoma virus (Py) tumor (T) antigens are the proteins specified by the early region of the viral genome. They are responsible for most biological effects caused by this oncogenic virus, i.e. induction of tumors, cell transformation and most of the virus-induced events observed in productive and transforming infection. By immunoprecipitation with antitumor serum followed by gel electrophoresis three major Py T-antigens have been characterized: large Tantigen (IT) with an apparent M_T of about 100 000, middle T-antigen (mT) of about 55 000 Mr and small T-antigen (sT) of about 23 000 Mr. In addition, there may exist one or more minor species by Py T-antigens. Analysis of the tryptic peptides showed that IT, mT and sT have a common N-terminal amino acid sequence, but differ from each other in the size and the sequence of the C-terminal part of the molecule as a consequence of different splicing of their mRNAs. With the nucleotide sequence of the Py genome being known, the coding regions for each of the Py T-antigens have been identified and consequently the amino acid sequence of IT, mT and sT was deduced. Cell fractionation experiments showed that the major part of 1T is located in the nucleus, mT was found in plasma membranes and sT is mainly present in the cytoplasm. Large T is a phosphoprotein and undergoes posttranslational modification. Two-dimensional gel electrophoresis of Py T-antigens revealed considerable charge heterogeneity particularly for mT and sT.All Py transformed cell lines analyzed contained mT and sT. Large T was not detected in virtually all Py transformed mouse cell lines and in about one third of Py transformed rat and hamster cell lines. Instead of 1T often new immunoreactive proteins were found which are probably truncated forms of 1T. These and other recent results suggest that IT is required neither for initiation nor for maintenance of cell transformation. For tumor induction in hamsters, similar conclusions were reached from analysis of Py T-antigens and viral DNA sequences in cell lines derived from tumors that had been induced either by virus or by viral DNA digested with various restriction enzymes. Experiments done with several deletion mutants indicated that mT is required for cell transformation by Py. In a protein kinase assay done in vitro with Py T-antigen immunoprecipitates, a kinase activity associated with Py mT was found which phosphorylates tyrosine residues mainly of mT and less frequently of 1T and of rat immunoglobulins. In all transformation defective mutants, kinase activity measured by this assay was absent or strongly reduced.In a concluding chapter I discuss the events occurring in wild-type virus and mutant infected cells trying to attribute specific functions to each of the three Py T-antigens. At least two functions are known for 1T, one is initiation of viral DNA replication, the other induces a mitotic response of the host cell, i.e. the events leading to and including host chromatin duplication. Middle T-antigen is certainly involved in cell transformation, possibly by its presence in the membrane. No function has been defined yet for sT. Since there are more virus-induced events observed in infected cells than Py T-antigens at least one of them must be a multifunctional protein.     

Small tumor antigen of polyomaviruses: role in viral life cycle and cell transformation

The regulatory proteins of polyomaviruses, including small and large T antigens, play important roles, not only in the viral life cycle but also in virus-induced cell transformation. Unlike many other tumor viruses, the transforming proteins of polyomaviruses have no cellular homologs but rather exert their effects mostly by interacting with cellular proteins that control fundamental processes in the regulation of cell proliferation and the cell cycle. Thus, they have proven to be valuable tools to identify specific signaling pathways involved in tumor progression. Elucidation of these pathways using polyomavirus transforming proteins as tools is critically important in understanding fundamental regulatory mechanisms and hence to develop effective therapeutic strategies against cancer. In this short review, we will focus on the structural and functional features of one polyomavirus transforming protein, that is, the small t-antigen of the human neurotropic JC virus (JCV) and the simian virus, SV40.     

Inositol trisphosphate levels in cells expressing wild-type and mutant polyomavirus middle T antigens: evidence for activation of phospholipase C via activation of pp60c-src.

The transforming protein of polyomavirus, middle T (mT), forms a complex with two cellular enzymes: the protein tyrosine kinase pp60c-src and a phosphatidylinositol (PtdIns) 3-kinase. A mutant virus, Py1178T, encodes an mT protein which associates with and activates pp60c-src to the same extent as the wild type but fails to associate with PtdIns 3-kinase. To investigate relationships between activation of pp60c-src, association of PtdIns 3-kinase, and cellular levels of the second messenger inositol 1,4,5-trisphosphate (InsP3), we examined the effects of wild-type and mutant mT proteins on inositol metabolism in rat and mouse fibroblasts. Expression of either wild-type or 1178T mT caused a 300 to 500% increase in the InsP3 level. Cells transformed by Rous sarcoma virus also showed similar increases in InsP3 levels. Mutant mT proteins which failed to activate pp60c-src (NG59 and 1387T) had no effect on InsP3 levels. Pulse-chase experiments with [3H]inositol showed that the turnover of phosphoinositides was increased in cells transformed by either wild-type polyomavirus or Py1178T as compared with the normal parent cell line. The turnover of inositol phosphates was unchanged upon transformation. These data indicate that cells expressing either wild-type or mutant 1178T mT or pp60v-src exhibit elevated levels of InsP3 because of activation of phospholipase C. This activation appears to depend, directly or indirectly, upon activation of pp60src protein kinase activity. Activation of pp60c-src and elevation of InsP3 content are not sufficient for full transformation. Full transformation also requires the association of mT-pp60c-src complexes with PtdIns 3-kinase.     

Cell transformation by pp60c-src mutated in the carboxy-terminal regulatory domain

We introduced two mutations into the carboxy-terminal regulatory region of chicken pp60c-src. One, F527, replaces tyrosine 527 with phenylalanine. The other, Am517, produces a truncated pp60c-src protein lacking the 17 carboxy-terminal amino acids. Both mutant proteins were phosphorylated at tyrosine 416 in vivo. The specific activity of the Am517 mutant protein kinase was similar to that of wild-type pp60c-src whereas that of the F527 mutant was 5- to 10-fold higher. Both mutant c-src genes induced focus formation on NIH 3T3 cells, but the foci appeared at lower frequency, and were smaller than foci induced by polyoma middle tumor antigen (mT). The wild-type or F527 pp60c-src formed a complex with mT, whereas the Am517 pp60c-src did not. The results suggest that one, inability to phosphorylate tyrosine 527 increases pp60c-src protein kinase activity and transforming ability; two, transformation by mT involves other events besides lack of phosphorylation at tyrosine 527 of pp60c-src; three, activation of the pp60c-src protein kinase may not be required for transformation by the Am517 mutant; and four, the carboxyl terminus of pp60c-src appears to be required for association with mT.     

Regulation of cellular phenotype and expression of polyomavirus middle T antigen in rat fibroblasts.

Polyoma middle T antigen (mT) was expressed in rat F-111 cells under control of the dexamethasone-regulatable mouse mammary tumor virus promoter. Graded phenotypic responses to levels of mT induction by the hormone were seen, with morphological transformation, focus formation, and anchorage-independent growth requiring increasing levels of mT expression. The ability of different clones to form tumors reflected their maximum level of induction of mT-associated kinase and their ability to grow in soft agar. Expression of transformation parameters and tumorigenicity correlates with the level of mT phosphorylated by pp60c-src in immune complexes and not with the total amount of mT determined by metabolic labeling. We suggest that cellular factors regulate mT activity by forming a kinase-active fraction of mT molecules that controls the transformed state.     

Studies of partially transforming polyomavirus mutants establish a role for phosphatidylinositol 3-kinase in activation of pp70 S6 kinase.

Infection of mouse fibroblasts by wild-type polyomavirus results in increased phosphorylation of ribosomal protein S6 (D.A. Talmage, J. Blenis, and T.L. Benjamin, Mol. Cell. Biol. 8:2309-2315, 1988). Here we identify pp70 S6 kinase (pp70S6K) as a target for signal transduction events leading from polyomavirus middle tumor antigen (mT). Two partially transforming virus mutants altered in different mT signalling pathways have been studied to elucidate the pathway leading to S6 phosphorylation. An upstream role for mT-phosphatidylinositol 3-kinase (PI3K) complexes in pp70S6K activation is implicated by the failure of 315YF, a mutant unable to promote PI3K binding, to elicit a response. This conclusion is supported by studies using wortmannin, a known inhibitor of PI3K. In contrast, stable interaction of mT with Shc, a protein thought to be involved upstream of Ras, is dispensable for pp70S6K activation. 250YS, a mutant mT which retains a binding site for PI3K but lacks one for Shc, stimulates pp70S6K to wild-type levels. Mutants 315YF and 250YS induce partial transformation of rats fibroblasts with distinct phenotypes, as judged from morphological and growth criteria. Neither mutant induces growth in soft agar, indicating that an increase in S6 phosphorylation, while necessary for cell cycle progression in normal mitogenesis, is not sufficient for anchorage-independent cell growth. In the polyomavirus systems, the latter requires integration of signals from mT involving both Shc and PI3K.     

Concomitant targeting of EGF receptor, TGF-beta and SRC points to a novel therapeutic approach in pancreatic cancer

To test the hypothesis that concomitant targeting of the epidermal growth factor receptor (EGFR) and transforming growth factor-beta (TGF-β) may offer a novel therapeutic approach in pancreatic cancer, EGFR silencing by RNA interference (shEGFR) was combined with TGF-β sequestration by soluble TGF-β receptor II (sTβRII). Effects on colony formation in 3-dimensional culture, tumor formation in nude mice, and downstream signaling were monitored. In both ASPC-1 and T3M4 cells, either shEGFR or sTβRII significantly inhibited colony formation. However, in ASPC-1 cells, combining shEGFR with sTβRII reduced colony formation more efficiently than either approach alone, whereas in T3M4 cells, shEGFR-mediated inhibition of colony formation was reversed by sTβRII. Similarly, in vivo growth of ASPC-1-derived tumors was attenuated by either shEGFR or sTβRII, and was markedly suppressed by both vectors. By contrast, T3M4-derived tumors either failed to form or were very small when EGFR alone was silenced, and these effects were reversed by sTβRII due to increased cancer cell proliferation. The combination of shEGFR and sTβRII decreased phospho-HER2, phospho-HER3, phoshpo-ERK and phospho-src (Tyr416) levels in ASPC-1 cells but increased their levels in T3M4 cells. Moreover, inhibition of both EGFR and HER2 by lapatinib or of src by SSKI-606, PP2, or dasatinib, blocked the sTβRII-mediated antagonism of colony formation in T3M4 cells. Together, these observations suggest that concomitantly targeting EGFR, TGF-β, and src may constitute a novel therapeutic approach in PDAC that prevents deleterious cross-talk between EGFR family members and TGF-β-dependent pathways.     

静磁场对小鼠黑色素瘤细胞B16生长和氧化应激的影响

目的:利用小鼠黑色素瘤细胞B16,研究静磁场对肿瘤细胞生长和氧化应激的影响,探讨氧化应激介导静磁场影响肿瘤细胞生长的机制,为磁场在肿瘤疾病的治疗中的应用提供理论依据。方法:采用MTT法测定磁场对B16细胞活力的影响;利用流式细胞仪测定静磁场暴露对B16细胞周期分布的影响;利用生物化学方法测定磁场暴露对细胞氧化防御系统相关蛋白酶活性的影响。结果:24 h内50 m T-200 m T静磁场暴露可以抑制B16生长,但超过24 h的磁场暴露可以促进B16生长;100 m T和200 m T静磁场暴露对B16的细胞周期分布没有影响;B16暴露于100 m T和200 m T静磁场48 h,GST活性和GSH/GSSG水平表现为先上升后下降,SOD活性和T-AOC水平先下降后上升,CAT活性没有受到影响。结论:50 m T-200 m T静磁场可以抑制小鼠黑色素瘤细胞B16的生长,诱导肿瘤细胞产生氧化应激。     

Identification of FAM111A as an SV40 Host Range Restriction and Adenovirus Helper Factor

The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT.     

Accelerated mammary tumor development in mutant polyomavirus middle T transgenic mice expressing elevated levels of either the Shc or Grb2 adapter protein

The Grb2 and Shc adapter proteins play critical roles in coupling activated growth factor receptors to several cellular signaling pathways. To assess the role of these molecules in mammary epithelial development and tumorigenesis, we have generated transgenic mice which individually express the Grb2 and Shc proteins in the mammary epithelium. Although mammary epithelial cell-specific expression of Grb2 or Shc accelerated ductal morphogenesis, mammary tumors were rarely observed in these strains. To explore the potential role of these adapter proteins in mammary tumorigenesis, mice coexpressing either Shc or Grb2 and a mutant form of polyomavirus middle T (PyV mT) antigen in the mammary epithelium were generated. Coexpression of either Shc or Grb2 with the mutant PyV mT antigen resulted in a dramatic acceleration of mammary tumorigenesis compared to parental mutant PyV mT strain. The increased rate of tumor formation observed in these mice was correlated with activation of the epidermal growth factor receptor family and mitogen-activated protein kinase pathway. These observations suggest that elevated levels of the Grb2 or Shc adapter protein can accelerate mammary tumor progression by sensitizing the mammary epithelial cell to growth factor receptor signaling.     

12-O-tetradecanoylphorbol-13-acetate stimulates phosphorylation of the 58,000-Mr form of polyomavirus middle T antigen in vivo: implications for a possible role of protein kinase C in middle T function.

The 58,000-Mr form (58K form) of the polyomavirus middle T antigen (mT) is a minor species distinguished by its phosphorylation in vivo on serine and by its efficient phosphorylation on tyrosine in immune complexes (B.S. Schaffhausen and T.L. Benjamin, J. Virol. 40:184-196, 1981). Here we report that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, rapidly stimulates phosphorylation of this mT species when added to cultures of wild-type polyomavirus-infected or polyomavirus-transformed 3T3 cells. Incubation with TPA leads to an accumulation of the 58K mT species to levels 1.5- to 5-fold higher than that in untreated cells within 15 min. TPA specifically stimulates phosphorylation of the 58K mT species without affecting that of the 56K species. Mapping by partial proteolysis shows that TPA-stimulated phosphorylation occurs at or near the site in 58K mT that is normally phosphorylated in the absence of TPA. A synthetic diacyl glycerol, 1-oleoyl-2-acetyl-glycerol, also specifically stimulates phosphorylation of 58K mT in vivo, while an inactive phorbol analog does not. TPA fails to induce phosphorylation of a 58K mT species encoded by certain nontransforming virus mutants with altered mT proteins that normally fail to undergo phosphorylation at the 58K site. These results indicate that the 58K form of mT is phosphorylated by or through the action of protein kinase C. TPA treatment of infected cells also leads to increased levels of 58K mT as measured in the immune complex kinase reaction, in which mT becomes phosphorylated on tyrosine by pp60c-src. These results are discussed in terms of a possible role for protein kinase C in activating mT function(s), including the formation of stable complexes with pp60c-src.     

Enumeration of the simian virus 40 early region elements necessary for human cell transformation

While it is clear that cancer arises from the accumulation of genetic mutations that endow the malignant cell with the properties of uncontrolled growth and proliferation, the precise combinations of mutations that program human tumor cell growth remain unknown. The study of the transforming proteins derived from DNA tumor viruses in experimental models of transformation has provided fundamental insights into the process of cell transformation. We recently reported that coexpression of the simian virus 40 (SV40) early region (ER), the gene encoding the telomerase catalytic subunit (hTERT), and an oncogenic allele of the H-ras gene in normal human fibroblast, kidney epithelial, and mammary epithelial cells converted these cells to a tumorigenic state. Here we show that the SV40 ER contributes to tumorigenic transformation in the presence of hTERT and oncogenic H-ras by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation of human cells requires the additional perturbation of protein phosphatase 2A by ST. Expression of ST in this setting stimulates cell proliferation, permits anchorage-independent growth, and confers increased resistance to nutrient deprivation. Taken together, these observations define the elements of the SV40 ER required for the transformation of human cells and begin to delineate a set of intracellular pathways whose disruption, in aggregate, appears to be necessary to generate tumorigenic human cells.     

Inactivation of retinoblastoma gene product RB or an RB-related protein by SV40 T antigen in MDCK epithelial cells results in massive apoptosis

SV40 T antigen (LT) transformation of renal MDCK epithelial cells resulted in massive apoptosis in the presence of serum. Cell death was dependent on the ability of LT to bind RB or a related protein, since MDCK cells expressing LT mutants unable to bind RB did not die. Apoptosis could be rescued by treatment of cells with EGF and TPA, a property linked to their ability to promote cell growth. Our results indicate an inverse correlation between proliferation and apoptosis. Thus LT transformation induced survival-factor dependence in epithelial cells, in contrast to its effect in fibroblasts. RB inactivation also resulted in a strong down-regulation of c-myc and c-fos, which were previously found to be highly and constitutively expressed in epithelial cells. RB gene transfer in MDCK(LT) cells restored cell viability and high c-myc expression. C-myc gene transfer in these cells also resulted in a significant survival effect. These results suggest that RB anti-cell death activity is at least partly mediated by up-regulation of c-myc. Overexpression of Bcl2 also protected cells against apoptosis. The role of RB and c-myc in cell survival is discussed and related to maintenance of the differentiation state rather than to their properties in cell cycle progression.     

Functional asymmetry of the regions juxtaposed to the membrane-binding sequence of polyomavirus middle T antigen.

The functional importance of the two clusters of positively charged amino acids which flank the hydrophobic membrane-anchoring sequence of polyomavirus middle T (mT) protein has been investigated by using site-directed mutagenesis. A clear asymmetry was apparent. No effect on transformation was seen following multiple alterations or complete removal of the cluster at the carboxyl end of the protein. In contrast, a single substitution replacing the first arginine amino terminal to the hydrophobic stretch with glutamic acid, but not with lysine, histidine, or methionine, produced a partially transformation-defective mutant with a novel phenotype. This mutant failed to confer anchorage-independent growth on F111 established rat embryo fibroblasts but induced foci with altered morphology compared with wild-type mT. Biochemical studies on this mutant revealed that F111 clones expressing levels of mutant mT equivalent to those of wild-type controls showed a 65% reduction in pp60c-src activation and an 87% reduction in mT-associated phosphatidylinositol 3-kinase activity. However, F111 clones expressing seven times more mutant mT than did wild-type controls showed equal or greater levels of kinase activities yet remained incompletely transformed. Possible mechanisms involving this transformation-sensitive region of mT are discussed.