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.     

p27kip1 protein levels reflect a nexus of oncogenic signaling during cell transformation

SV40 small t-antigen (ST) collaborates with SV40 large T-antigen (LT) and activated rasv12 to promote transformation in a variety of immortalized human cells. A number of oncogenes or the disruption of the general serine-threonine phosphatase protein phosphatase 2A (PP2A) can replace ST in this paradigm. However, the relationship between these oncogenes and PP2A activity is not clear. To address this, we queried the connectivity of these molecules in silico. We found that p27 was connected to each of those oncogenes that could substitute for ST. We further determined that p27 loss can substitute for the expression of ST during transformation of both rodent and human cells. Conversely, knock-in cells expressing the degradation-resistant S10A and T187A mutants of p27 were resistant to the transforming activities of ST. This suggests that p27 is an important target of the tumor-suppressive effects of PP2A and likely an important target of the multitude of cellular oncoproteins that emulate the transforming function of ST.     

Reintroduction of a normal retinoblastoma gene into retinoblastoma and osteosarcoma cells inhibits the replication associated function of SV40 large T antigen

The product of the retinoblastoma (Rb) gene can form complexes with the transforming proteins of small DNA tumor viruses, including SV40 large T antigen (Tag), adenovirus E1A, and the human papilloma virus E7. The strong correlation between their ability to transform and their ability to bind Rb protein suggests that these oncoproteins exert their effect through blocking the Rb function. SV40 Tag causes oncogenic cell transformation of rodent cells, and it is also required for viral DNA replication. In this paper, we investigated the effect of the Rb protein on the SV40 replication associated function of Tag. We present evidence suggesting that the complex formation between Rb and Tag interferes with the viral DNA replication. In Y79 retinoblastoma and Saos-2 osteosarcoma cells, which lack functional Rb protein, a SV40 based plasmid vector, pSVEpR4, replicates well. In the same cells reconstituted for Rb expression with an intact Rb gene introduced by retroviral mediated gene transfer, pSVEpR4 replicates to a considerably lower level. The inhibitory effect of Rb protein was surmounted by increasing the intracellular level of Tag. Increasing amounts of Tag in wild-type Rb negative Y79 cells had virtually no effect on SV40 replication. Furthermore, the overexpression of Tag in Rb reconstituted Y79 cells did not alter the growth rate of the cells. These data suggest that Rb protein interacts with Tag and modulates its ability to promote SV40 DNA replication.     

Overexpression of simian virus 40 small-T antigen blocks centrosome function and mitotic progression in human fibroblasts

Recombinant adenoviruses that express high levels of the simian virus 40 (SV40) small-t (ST) antigen have been used to study the requirement for ST to drive cell cycle proliferation of confluent human diploid fibroblasts. This occurs when either large-T (LT) antigen or serum is added to provide a second signal. While cells readily completed S phase in these experiments, they were found to accumulate with 4N DNA content. Cellular and nuclear morphology, as well as the biochemical status of cyclin B complexes, showed that these cells entered mitosis but were blocked prior to mitotic metaphase. The defect appears to reflect an inability of cells overexpressing ST to form organized centrosomes that duplicate and separate normally during the cell cycle and, therefore, the absence of a mitotic spindle. The ability of ST to bind protein phosphatase 2A was required for this pattern, suggesting that altered phosphorylation of key centrosomal components may occur when ST is overexpressed. Although the possible significance of ST effects on the centrosome cycle is not fully understood, these findings suggest that ST could influence chromosomal instability patterns that are a hallmark of SV40-transformed cells and LT expression.     

"Transformation" of human endothelial cells by SV40 virions

Human endothelial cells derived from the umbilical vein were transformed with SV40 virions. A cell line subcultured for over 60 serial passages was characterized in comparison with its untransformed counterpart which was culturable for less than five passages. The SV40-transformed human endothelial cells, designated SV-HUVEC, were positive not only for tumor (T) antigen specific to the SV40-transformed cell, but also for two markers of endothelial cells, Factor VIII-related antigen and a receptor for Ulex europaeus agglutinin I. By transformation the growth potential of the human endothelial cells was increased and their serum requirement was decreased. The SV40-transformed endothelial cells were, however, unable to form colonies in soft agar or to form tumors in athymic nude mice, although a small nodule was produced at the site of inoculation. Subcultivation of these cells up to the 62nd passage eventually resulted in crisis and loss of further cell division. Thus, the human endothelial cells were transformed by SV40 while retaining certain normal functions but without showing tumorigenicity.     

Simian Virus 40 Small T Antigen Activates AMPK and Triggers Autophagy To Protect Cancer Cells from Nutrient Deprivation

As tumors grow larger, they often experience an insufficient supply of oxygen and nutrients. Hence, cancer cells must develop mechanisms to overcome these stresses. Using an in vitro transformation model where the presence of the simian virus 40 (SV40) small T (ST) antigen has been shown to be critical for tumorigenic transformation, we investigated whether the ST antigen has a role to play in regulating the energy homeostasis of cancer cells. We find that cells expressing the SV40 ST antigen (+ST cells) are more resistant to glucose deprivation-induced cell death than cells lacking the SV40 ST antigen (−ST cells). Mechanistically, we find that the ST antigen mediates this effect by activating a nutrient-sensing kinase, AMP-activated protein kinase (AMPK). The basal level of active, phosphorylated AMPK was higher in +ST cells than in −ST cells, and these levels increased further in response to glucose deprivation. Additionally, inhibition of AMPK in +ST cells increased the rate of cell death, while activation of AMPK in −ST cells decreased the rate of cell death, under conditions of glucose deprivation. We further show that AMPK mediates its effects, at least in part, by inhibiting mTOR (mammalian target of rapamycin), thereby shutting down protein translation. Finally, we show that +ST cells exhibit a higher percentage of autophagy than −ST cells upon glucose deprivation. Thus, we demonstrate a novel role for the SV40 ST antigen in cancers, where it functions to maintain energy homeostasis during glucose deprivation by activating AMPK, inhibiting mTOR, and inducing autophagy as an alternate energy source.The localization of most mammalian cells within a 100- to 150-μm distance from blood vessels ensures a continuous supply of oxygen and nutrients, a prerequisite for cell survival. However, tumors often grow beyond this limit, thereby experiencing oxygen and nutrient deprivation (28). Tumors overcome this barrier by initiating neoangiogenesis, a process that supplies new blood vessels (44). However, before neoangiogenesis can set in, incipient tumors must survive the stresses of nutrient deprivation. Therefore, an understanding of the molecular mechanisms that regulate cancer cell survival under conditions of nutrient deprivation is fundamental in cancer biology. Additionally, targeting the ability of cancer cells to survive under nutrient-deprived conditions can be exploited for designing novel cancer therapeutics.Glucose is the major source of energy for mammalian cells. Several types of cancer cells exhibit marked resistance to cell death upon glucose deprivation (22). In this study we have attempted to delineate the mechanisms that allow cancer cells to survive under conditions of glucose deprivation by using human foreskin fibroblasts that have been transformed by the serial introduction of the simian virus 40 (SV40) early region (coding for the large T [LT] and small T [ST] antigens), the catalytic subunit of human telomerase (hTERT), and an oncogenic allele of H-Ras (H-Ras V12) (referred to below as +ST cells) (32). In this model, human cells lacking the ST antigen but expressing the rest of these genetic elements (referred to below as −ST cells) are nontumorigenic (16, 32), highlighting the importance of the ST antigen in human cell transformation. However, little is known about the specific cellular functions moderated by the ST antigen that aid in transformation (3).Since glucose is the major source of energy for mammalian cells, and cancer cells experience glucose deprivation when they are beyond the diffusion limit, we investigated whether the ST antigen has any role to play under conditions of glucose deprivation. We report here a novel link between the ST antigen and AMP-activated protein kinase (AMPK) activation that enables cancer cell survival under glucose deprivation via inhibition of protein synthesis and activation of autophagy as an alternate energy source.     

PP2A inactivation is a crucial step in triggering apoptin-induced tumor-selective cell killing

Apoptin (apoptosis-inducing protein) harbors tumor-selective characteristics making it a potential safe and effective anticancer agent. Apoptin becomes phosphorylated and induces apoptosis in a large panel of human tumor but not normal cells. Here, we used an in vitro oncogenic transformation assay to explore minimal cellular factors required for the activation of apoptin. Flag-apoptin was introduced into normal fibroblasts together with the transforming SV40 large T antigen (SV40 LT) and SV40 small t antigen (SV40 ST) antigens. We found that nuclear expression of SV40 ST in normal cells was sufficient to induce phosphorylation of apoptin. Mutational analysis showed that mutations disrupting the binding of ST to protein phosphatase 2A (PP2A) counteracted this effect. Knockdown of the ST-interacting PP2A–B56γ subunit in normal fibroblasts mimicked the effect of nuclear ST expression, resulting in induction of apoptin phosphorylation. The same effect was observed upon downregulation of the PP2A–B56δ subunit, which is targeted by protein kinase A (PKA). Apoptin interacts with the PKA-associating protein BCA3/AKIP1, and inhibition of PKA in tumor cells by treatment with H89 increased the phosphorylation of apoptin, whereas the PKA activator cAMP partially reduced it. We infer that inactivation of PP2A, in particular, of the B56γ and B56δ subunits is a crucial step in triggering apoptin-induced tumor-selective cell death.     

Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.     

Transformation of a continuous rat embryo fibroblast cell line requires three separate domains of simian virus 40 large T antigen.

Mouse C3H 10T1/2 cells and the established rat embryo fibroblast cell line REF-52 are two cell lines widely used in studies of viral transformation. Studies have shown that transformation of 10T1/2 cells requires only the amino-terminal 121 amino acids of simian virus 40 (SV40) large T antigen, while transformation of REF-52 cells requires considerably more of large T antigen, extending from near the N terminus to beyond residue 600. The ability of a large set of linker insertion, small deletion, and point mutants of SV40 T antigen to transform these two cell lines and to bind p105Rb was determined. Transformation of 10T1/2 cells was greatly reduced by mutations within the first exon of the gene for large T antigen but was only modestly affected by mutations affecting the p105Rb binding site or the p53 binding region. All mutants defective for transformation of 10T1/2 cells were also defective for transformation of REF-52 cells. In addition, mutants whose T antigens had alterations in the Rb binding site showed a substantial reduction in transformation of REF-52 cells, and the degree of this reduction could be correlated with the ability of the mutant T antigens to bind p105Rb. There was a tight correlation between the ability of mutants to transform REF-52 cells and the ability of their T antigens to bind p53. These results demonstrate that multiple regions of large T antigen are required for full transformation by SV40.     

Changes in HL-A antigen profiles on SV40-transformed human fibroblasts

The histocompatibility antigen profile of human fibroblasts transformed with SV40 virus has been investigated by determining their ability to specifically absorb HL-A alloantisera. Four out of four human adult skin fibroblast lines acquired, after SV40 transformation, the ability to absorb the HL-A alloantiserum VICTOR directed against the specificities HL-A5, W5. On the contrary, none of six embryonic fibroblast lines showed any qualitative or quantitative change of their HL-A antigenic profile. Similarly, murine, monkey and hamster cells could not absorb the activity of the HL-A alloantiserum VICTOR after SV40 transformation. It is suggested that the ‘new HL-A antigen’ specificity on human fibroblasts after SV40 transformation may be due to either a cross-reaction between HL-A specificities and antigenic structures present on glycoprotein(s) coded by the viral genome and expressed on the cell or to a change in the regulating mechanism of the expression of histocompatibility antigens on the cell surface, should the genetic information for all the HL-A specificities be present in the cell genome.     

Expression of SV40 tumor antigen in SV40 transformed teratocarcinoma-derived cell lines

The relative importance of viral tumor antigen expression and the cellular background in the maintenance of a transformation phenotype was examined in five SV40-transformed teratocarcinoma-derived cell lines. These cell lines show qualitative differences in growth characteristics associated with transformation, and vary in their state of differentiation. Viral T antigen expression was evaluated by two criteria: 1) the amount of immunoprecipitated antigen in growing cells, and 2) the amount and rate of antigen synthesis in density-inhibited cells. There was no direct correlation found between retention, or rate of synthesis, of the viral T antigen and the degree of transformation. These findings imply that the cellular environment has a more important influence on the growth properties of a stably transformed cell than the quantitative levels of viral T antigen expression.     

Simian virus 40 deletion mutants that transform with reduced efficiency.

We have constructed two simian virus 40 (SV40) early-region deletion mutants that lack a significant portion of the sequences normally used to encode the SV40 large tumor antigen. Despite these deletions, the mutants were able to transform mouse cells in a focus assay, although with a frequency that was drastically reduced relative to wild-type SV40. Cell lines expanded from the mutant-transformed foci contained integrated mutant DNA, expressed an SV40 tumor antigen (small-t), and exhibited a range of transformed phenotypes, which included the ability to grow while suspended in soft agar. We also present evidence that these mutants are defective for abortive transformation in an assay that tested the transient loss of anchorage dependence. Their ability to stably transform, contrasted with their inability to abortively transform at detectable levels, raises the possibility that the mechanism by which these mutants transform may be different from that of wild-type SV40.     

Development of antigens in human cells infected with Simian virus 40

Bissett, Marjorie L. (University of Michigan, Ann Arbor), and Francis E. Payne. Development of antigens in human cells infected with simian virus 40. J. Bacteriol. 91:743-749. 1966.-An explanation for the apparent infrequency with which human cells transform in response to exposure to simian virus 40 (SV40) was sought by following the development of virus-induced antigens in human euploid cells, strain CR. For about 8 weeks after exposure to a high multiplicity of SV40, only a small proportion of the cells produced tumor (T) or viral (V) antigen detected by immunofluorescence. Double-tracer staining techniques revealed that the development of T and V antigen in about 1% of the CR cells resembled that in green monkey kidney cells, strain BS-C-1, in which SV40 replicates and destroys all the cells. T antigen was detected before V antigen; both antigens were detected in the nucleus, but only V antigen appeared later in the cytoplasm. All intact cells that contained V antigen also contained T antigen. Infected CR cell cultures, before and after transformation or when in "crisis," contained only 0.1 to 1.0% of cells with both V and T antigen. Some CR cells contained only T antigen, and by 8 days after exposure to virus these cells were present as loose foci associated with an occasional cell containing V antigen. The proportion of CR cells with only T antigen increased from about 1% during the first 4 weeks to 8% at 7 weeks, and to nearly 100% at 11 weeks, when essentially all of the cells were epithelioid. Foci of epithelioid cells were first recognized in the 9th week. It was concluded that those CR cells that contained T antigen at any given time represented (i) a few cells that subsequently produced V antigen and lysed, and (ii) a progressively increasing population that produced only T antigen. If the latter population, in whole or in part, gave rise to the epithelioid transformed cells, then its initial size could account, at least in part, for the apparent infrequency with which human cells transform in response to SV40.     

Transformation of precrisis human cells by the simian virus 40 cytoplasmic-localization mutant pSVCT3 is accompanied by nuclear T antigen.

pSVCT3 is a cytoplasmic-localization mutant of simian virus 40 (SV40) isolated from the SV40 adenovirus 7 hybrid virus (PARA) and cloned into plasmid PBR. The large T antigen of pSVCT3 accumulates in the cytoplasm of infected monkey cells instead of being transported to the nucleus. The sole change in CT3 large T antigen is amino acid residue 128 (Lys----Asn). Transformation of precrisis rodent cells by pSVCT3 is negligible, whereas the frequency of transformation of established rodent cell lines by pSVCT3 is comparable to that of wild-type SV40. According to the model, in which transformation of precrisis cells involves the combined oncogenic action of both nuclear and cytoplasmic gene products, we predicted that pSVCT3 would localize in the cytoplasm of human cells and would therefore at most only partially and rarely transform precrisis human cells. We have found that pSVCT3 is able to transform precrisis human cells at high frequency. Furthermore, pSVCT3-transformed human precrisis cells relocalized T antigen to their nuclei. The relocalization of large T antigen was not dependent on cell growth. Wild-type and pSVCT3-transformed human cell lines both have about five copies of integrated SV40 DNA. SV40 virus-specific proteins, including the 100,000-molecular-weight super large T antigen, were expressed in pSVCT3-transformed human cells. Our results suggest that molecules in precrisis human cells, but not cells of other species, are able to complement the cytoplasmic-localization defect of the CT3 mutant large T antigen.     

The transforming proteins of simian virus 40

Simian virus 40 (SV40) is a small DNA tumor virus whose early gene products, large T and small t antigens, efficiently immortalize and transform primary rodent cells, transform rodent cell lines and extend the lifespan of primary human cells. Mutational analysis has revealed that the transforming and lifespan extension properties of large T antigen correlate with binding to and disruption of the normal functions of the human tumor suppressor proteins pRb and p53. Small t antigen contributes to cell proliferation through inactivation of protein phosphatase 2A and subsequent activation of the MAP kinase pathway. By disrupting key cell growth control mechanisms, SV40 transforming proteins provide a valuable system for analysis of cellular growth control mechanisms.