Structural prerequisites of simian virus 40 large T antigen for the maintenance of cell transformation.

We have investigated the functional roles of two structural subsets of simian virus 40 (SV40) large T antigen, namely homo-oligomers and complexes with the host cellular p53 protein, for the transformed phenotype. We examined T antigen produced in cells transformed by temperature-sensitive SV40 large T mutants: heat-sensitive or unrestricted SV40 tsA58-transformed rat cells and unrestricted tsA1499 transformants. In both unrestricted cell lines, T antigen was temperature-sensitive only for the formation of fast sedimenting homo-oligomers. Corresponding to our recent observations obtained with tsA1499-infected monkey cells, in tsA1499 transformants large T was competent to form stable T-p53 complexes independently of the temperature. However, T antigen coded for by tsA58, which is heat-sensitive for binding to p53, occurred in stable complexes with this protein in unrestricted tsA58 transformants under all conditions. Furthermore, in both unrestricted transformants T-p53 complexes arise in the absence of homo-oligomers of T antigen. In conclusion, T antigen homo-oligomers are not involved in cell transformation, whereas T-p53 complexes may be involved in the maintenance of this phenotype.     

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.     

Role of simian virus 40 gene A function in maintenance of transformation.

Mouse, hamster, and human cells were transformed at the permissive temperature by mutants from simian virus 40 (SV40) complementation group A in order to ascertain the role of the gene A function in transformation. The following parameters of transformation were monitored with the transformed cells under permissive and nonpermissive conditions: morphology; saturation density; colony formation on plastic, on cell monolayers, and in soft agar; uptake of hexose; and the expression of SV40 tumor (T) and surface (S) antigens. Cells transformed by the temperature-sensitive (ts) mutants exhibited the phenotype of transformed cells at the nonrestrictive temperature for all of the parameters studied. However, when grown at the restrictive temperature, they were phenotypically similar to normal, untransformed cells. Growth curves showed that the (ts) A mutant-transformed cells exhibited the growth characteristics of wild-type virus-transformed cells at the permissive temperature and resembled normal cells when placed under restrictive conditions. There were 3-to 51-fold reductions in the levels of saturation density, colony formation, and uptake of hexose when the mutant-transformed cells were the elevated temperature as compared to when they were grown at the permissive temperature. Mutant-transformed cells from the nonpermissive temperature were able to produce transformed foci when shifted down to permissive conditions, indicating that the phenotypically reverted cells were still viable and that the reversion was a reversible event. SV40 T antigen was present in the cells at both temperatures, but S antigen was not detected in cells maintained at the nonpremissive temperature. All of the wild-type virus-transformed cells exhbited a transformed cells exhibited a transformed phenotype when grown under either restrictive or nonrestrictive conditions. Thers results indicate that the SV40 group A mutant-transformed cells are temperature sensitive for the maintenance of growth properties characteristics of transformation. Virus rescued from the mutant-transformed cells by the transfection method was ts, suggesting that the SV40 gene A function, rather than a cellular one, is responsible for the ts behavior of the cells.     

Growth of immortal simian virus 40 tsA-transformed human fibroblasts is temperature dependent.

Simian virus 40 (SV40)-mediated transformation of human fibroblasts offers an experimental system for studying both carcinogenesis and cellular aging, since such transformants show the typical features of altered cellular growth but still have a limited life span in culture and undergo senescence. We have previously demonstrated (D. S. Neufeld, S. Ripley, A. Henderson, and H. L. Ozer, Mol. Cell. Biol. 7:2794-2802, 1987) that transformants generated with origin-defective mutants of SV40 show an increased frequency of overcoming senescence and becoming immortal. To clarify further the role of large T antigen, we have generated immortalized transformants by using origin-defective mutants of SV40 encoding a heat-labile large T antigen (tsA58 transformants). At a temperature permissive for large-T-antigen function (35 degrees C), the cell line AR5 had properties resembling those of cell lines transformed with wild-type SV40. However, the AR5 cells were unable to proliferate or form colonies at temperatures restrictive for large-T-antigen function (39 degrees C), demonstrating a continuous need for large T antigen even in immortalized human fibroblasts. Such immortal temperature-dependent transformants should be useful cell lines for the identification of other cellular or viral gene products that induce cell proliferation in human cells.     

Excess wild-type p53 blocks initiation and maintenance of simian virus 40 transformation.

Wild-type (wt) murine p53 has been tested for its ability to block and reverse the transforming effects of simian virus 40 (SV40) large T antigen. Established and precrisis mouse cells overexpressing exogenously introduced wt p53 became resistant to SV40 transformation. The introduction of excess wt p53 into SV40-transformed precrisis cells reverted their transformed phenotype. However, the phenotype of SV40-transformed established cells was not reverted by excess wt p53. We conclude that an antioncogenic action of wt p53 is exerted during SV40 transformation and that in precrisis cells, the antitransforming action of wt p53 can be exerted both at initiation and during the maintenance of transformation.     

Glycosaminoglycan synthesis by cultured human skin fibroblasts after transformation with simian virus 40.

The synthesis of glycosaminoglycans by human skin fibroblasts derived from normal subjects, Hurler and Marfan patients before and after transformation by SV40 virus has been studied. Virus transformation results in a marked increase in hyaluronic acid synthesis in normal and Hurler fibroblasts and, to a lesser extent, in Marfan fibroblasts which show augmented synthesis of this polysaccharide before transformation. There is also an increase in heparan sulfate synthesis but a moderate decrease in dermatan sulfate synthesis on transformation. Incubation of transformed fibroblasts with 4-methylumbelliferyl-beta-D-xyloside results in a marked increase in synthesis of free chondroitin sulfate chains. The synthesis of hyaluronic acid, but not of dermatan sulfate, is inversely proportional to cell density in normal fibroblasts but not in transformed fibroblasts.     

Resistance of teratocarcinoma stem cells to infection with simian virus 40: early events.

Multipotential stem cells of a murine teratocarcinoma are resistant to typical infection with either polyoma virus (PV) or Simian virus 40 (SV40). Differentiated progeny of the stem cells are susceptible to infection in a manner identical to other mouse somatic cells, i.e., they are permissive for PV and nonpermissive for SV40. The early interactions between the stem cells (embryonal carcinoma or EC cells) and SV40 and PV were studied. Virions adsorbed to and penetrated into the cytoplasm and nucleus of EC cells, but did not induce expression of T antigen in the EC nuclei. Purified SV40 DNA was capable of inducing T antigen in differentiated teratocarcinoma cells but not in EC cells. Virus could not be rescued from EC cells previously exposed to SV40. The resistance of the stem cells to infection apparently involves a block in the infectious cycle after adsorption and penetration but before T antigen induction.     

Establishment of rat pancreatic endocrine cell lines by infection with simian virus 40.

The feasibility of infection and transformation by SV40 (simian virus 40) of primary cell cultures derived from newborn-rat pancreas was investigated. As judged by the presence of intranuclear SV40 T-antigen, exposure to the virus resulted specifically in infection and transformation of epithelioid (predominantly endocrine) cells. The transformed cells were subcultured (more than 64 passages) and cloned. Culture medium and acid/ethanol extracts of the cells did not contain detectable amounts of immunoreactive insulin after the third subculture. However, inoculation of such SV40-transformed pancreatic cells into immunodeficient rats results in tumours in which insulin production was partially restored through the passage in vivo, since the tumour cells contained and synthesized small amounts of immunoreactive insulin which co-migrated with an insulin marker on gel chromatography. Interestingly, the transformed cells maintained under tissue-culture conditions produced a protein immunologically related to insulin, soluble in aqueous buffer but insoluble in acid/ethanol. This 3000-dalton protein is too large to be a translation product of the rat preproinsulin 9S mRNA. SV40-transformed pancreatic cells might prove useful in the investigation of the factors controlling and maintaining insulin biosynthesis.     

Temperature dependency for maintenance of transformation in mouse cells transformed by simian virus 40 tsA mutants.

Mouse embryo fibroblasts and 3T3 cells were transformed by wild-type, tsB4, tsA7, tsA58, and tsA209 simian virus 40. Clones of transformants were generated both in soft agar and in liquid medium by focus formation and at both high and relatively low multiplicities of infection. All transformants were assayed for three phenotypes of transformation: (i) the ability to form highly multinucleated cells in cytochalasin B-supplemented medium, i.e., uncontrolled nuclear division; (ii) the capacity to continue DNA synthesis at increasing cell density; and (iii) the ability to form colonies in soft agar. The great majority of mouse embryo fibroblast transformants generated with tsA mutant virus were temperature sensitive for transformation in all three assays, regardless of the input multiplicity or whether they were generated in liquid medium or soft agar. These transformants exhibited a normal or near-normal phenotype at the nonpermissive temperature of 40 degrees C. All but one of the transformants which appeared transformed at both temperatures were in the A209 group. In contrast to mouse embryo fibroblasts, transformants generated with 3T3 cells and tsA virus were often not temperature sensitive, exhibiting the transformation phenotypes at both temperatures. This phenomenon was more often observed when 3T3 transformants were generated in soft agar. These results, along with other published data, suggest that uncontrolled nuclear division and uncontrolled DNA synthesis are a function of the simian virus 40 A gene. Finally, with the 3T3 transformants, there was often discordance in the expression of transformation among the three phenotypes. Some tsA transformants were temperature sensitive in one of two assays but were transformed at both 33 and 40 degrees C in the remaining assay(s). Other transformants exhibited a normal cytochalasin B response at either temperature but were temperature sensitive in the other assays.     

Roles of the simian virus 40 tumor antigens in transformation of Chinese hamster lung cells: studies with simian virus 40 double mutants.

Simian virus 40 mutants containing both a tsA mutation (rendering the 90,000 molecular weight [90K] T-antigen thermolabile) and a deletion between 0.54 and 0.59 map units (reducing the size and the amount of the 20K t-antigen) were used to transform Chinese hamster lung cells. The frequencies of transformation by the double mutants were comparable to that of the tsA mutant alone by both the focus and agar assays except when the cells were serum depleted before infection. Growth-arrested cells were transformed (using the agar assay) by the deletion mutants at less than 2% the frequency found when the 20K t-antigen was normal. Growth arrest had very little effect on the temperature sensitivity of the resultant transformed cell lines whether or not the deletion was present.     

The t-unique coding domain is important to the transformation maintenance function of the simian virus 40 small t antigen.

The small t antigen (t) of simian virus 40, a 174-amino-acid-containing protein, when present together with the other early viral protein, large T antigen (T), plays an important role in the maintenance of simian virus 40-induced neoplastic phenotype in certain cells. Indeed, each protein functions in a complementary manner in this process. The t coding unit is composed of two segments, a 5' region of 246 nucleotides which is identical to that of the corresponding 5' region of the T coding unit and a 3' segment of 276 nucleotides which is unique. Two mutant, t-encoding genomes, one bearing a missense and the other a nonsense mutation at the same point in the t-unique coding region were constructed in vitro and found to be defective in their ability to dissolve the actin cytoskeleton of rat fibroblasts and to complement T in the growth of mouse fibroblasts in soft agar. Therefore, the unique segment of the t gene encodes a portion of the t molecule which is essential to its transformation maintenance function.     

Association of simian virus 40 T antigen with replicating nucleoprotein complexes of simian virus 40.

An immunoprecipitation assay was established for simian virus 40 T-antigen-bound nucleoprotein complexes by means of precipitation with sera from hamsters bearing simian virus 40-induced tumors. About 80% of simian virus 40 replicating nucleoprotein complexes in various stages of replication were immunoprecipitated. In contrast, less than 21% of mature nucleoprotein complexes were immunoprecipitated. Pulse-chase experiments showed that T antigen was lost from most of the nucleoprotein complexes concurrently with completion of DNA replication. T antigen induced by dl-940, a mutant with a deletion in the region coding for small T antigen, was also associated with most of the replicating nucleoprotein complexes. Once bound with replicating nucleoprotein complexes at the permissive temperature, thermolabile T antigen induced by tsA900 remained associated with the complexes during elongation of the replicating DNA chain at the restrictive temperature. These results suggest that simian virus 40 T antigen (probably large T antigen) associates with nucleoprotein complexes at or before initiation of DNA replication and that the majority of the T antigen dissociates from the nucleoprotein complexes simultaneously with completion of DNA replication.     

Hormonal regulation of the transformation phenotype in simian virus 40-transformed rat embryonic preadipose cell lines.

The regulation of transformed phenotypes was studied in newly isolated preadipose cell lines which were established after infection with simian virus 40 tsA58 dl2009. The clonal cell lines isolated exhibited most of the characteristics typical of transformed cells. The transformants, however, were able to differentiate into adipocytes in the presence of low calf serum (0.5%) and a combination of several hormones, including hydrocortisone and insulin. Treatment with insulin alone stimulated the growth of these cells but did not induce lipid accumulation without added hydrocortisone. The effect of hydrocortisone was accompanied by a restoration of growth control in the transformants after they reached high cell density. The blot hybridization analysis of cellular DNAs digested by restriction enzymes revealed that simian virus 40 genomes were integrated at multiple separate sites at which a head-to-tail oligomeric insertion took place. Large T antigen was synthesized in growing cells but was regulated at high cell density when cells were committed to differentiate by glucocorticoids. These results suggest that the glucocorticoid hydrocortisone is capable of restoring growth regulation at high cell densities to simian virus 40-transformed preadipose cell lines.     

Identification of a region of simian virus 40 large T antigen required for cell transformation.

A series of replication-competent simian virus 40 (SV40) large T antigens with point and deletion mutations in the amino acid sequence between residues 105 and 115 were examined for the ability to immortalize primary cultures of mouse and rat cells. The results show that certain mutants, including one that deletes the entire region, are able to immortalize. However, consistent with previous data, the immortalized cells are not fully transformed, as judged by doubling time, sensitivity to concentrations of serum, and anchorage-independent growth. The region from 106 to 114 has structural features in common with a region involved in transformation by adenovirus E1a protein (J. Figge, T. Webster, T.F. Smith, and E. Paucha, J. Virol. 62:1814-1818, 1988) and influences the binding of the retinoblastoma gene product to large T (J.A. DeCaprio, J.W. Ludlow, J. Figge, J.-Y. Shew, C.-M. Huang, W.-H. Lee, E. Marsilio, E. Paucha, and D.M. Livingston, Cell 54:275-283, 1988). Together, these results imply that the sequence from 106 to 114 forms part of a domain that is essential for transformation of established cells, is dispensable for immortalization, and is not required for SV40 replication. The results also indicate that the ability of SV40 large T to immortalize primary cells is independent of its ability to bind to the retinoblastoma gene product.     

Selectivity of interferon action in simian virus 40-transformed cells superinfected with simian virus 40.

Treatment of African green monkey kidney CV-1 cells with human alpha interferons before infection with simian virus 40 (SV40) inhibited the accumulation of SV40 mRNAs and SV40 T-antigen (Tag). This inhibition persisted as long as the interferons were present in the medium. SV40-transformed human SV80 cells and mouse SV3T3-38 cells express Tag, and interferon treatment of these cells did not affect this expression. SV80 and SV3T3-38 cells which had been exposed to interferons were infected with a viable SV40 deletion mutant (SV40 dl1263) that codes for a truncated Tag. Exposure to interferons inhibited the accumulation of the truncated Tag (specified by the infecting virus) but had no significant effect on the accumulation of the endogenous Tag (specified by the SV40 DNA integrated into the cellular genome). The level of Tag in SV40-transformed mouse SV101 cells was not significantly decreased by interferon treatment. SV40 was rescued from SV101 cells and used to infect interferon-treated and control African green monkey kidney Vero cells. Tag accumulation was inhibited in the cells which had been treated with interferons before infection. Our data demonstrate that even within the same cell the interferon system can discriminate between expression of a gene in the SV40 viral genome and expression of the same gene integrated into a host chromosome.     

Modulation of p53 protein expression during cellular transformation with simian virus 40.

We analyzed the relation of metabolic stabilization of the p53 protein during cellular transformation by simian virus 40 (SV40) to (i) expression of the transformed phenotype and (ii) expression of the large tumor antigen (large T). Analysis of SV40-tsA28-mutant-transformed rat cells (tsA28.3 cells) showed that both p53 complexed to large T and free p53 (W. Deppert and M. Haug, Mol. Cell. Biol. 6:2233-2240, 1986) were metabolically stable when the cells were cultured at 32 degrees C and expressed large T and the transformed phenotype. At the nonpermissive temperature (39 degrees C), large-T expression is shut off in these cells and they revert to the normal phenotype. In such cells, p53 was metabolically unstable, like p53 in untransformed cells. To determine whether metabolic stabilization of p53 is directly controlled by large T, we next analyzed the metabolic stability of complexed and free p53 in SV40 abortively infected normal BALB/c mouse 3T3 cells. We found that neither p53 in complex with large T nor free p53 was metabolically stable. However, both forms of p53 were stabilized in SV40-transformed cells which had been developed in parallel from SV40 abortively infected cultures. Our results indicate that neither formation of a complex of p53 with large T nor large-T expression as such is sufficient for a significant metabolic stabilization of p53. Therefore, we suggest that metabolic stabilization of p53 during cellular transformation with SV40 is mediated by a cellular process and probably is the consequence of the large-T-induced transformed phenotype.