Evidence against a role for SV40 in human mesothelioma

SV40 has been implicated in the etiology of 40% to 60% of human mesotheliomas. These studies could have important medical implications concerning possible sources of human infection and potential therapies if human tumors are induced by this agent. We did PCR-based analysis to detect SV40 large T antigen DNA in human mesotheliomas. None of 69 tumors in which a single copy gene was readily amplified contained detectable SV40 large T antigen sequences. Under these conditions, it was possible to detect one copy of integrated SV40 DNA per cell in a mixture containing a 5,000-fold excess of normal cells using formalin-fixed preparations. Kidney, a known reservoir of SV40 in monkeys, from some of these individuals were also negative for SV40 large T antigen sequences. A subset of mesotheliomas was analyzed for SV40 large T antigen expression by immunostaining with a highly specific SV40 antibody. These tumors as well as several human mesothelioma cell lines previously reported to contain SV40 large T antigen were negative for detection of the virally encoded oncoprotein. Moreover, mesothelioma cell lines with wild-type p53 showed normal p53 function in response to genotoxic stress, findings inconsistent with p53 inactivation by the putative presence of SV40 large T antigen. Taken together, these findings strongly argue against a role of SV40 by any known transformation mechanism in the etiology of the majority of human malignant mesotheliomas.     

A new anti-p53 monoclonal antibody, previously reported to be directed against the large T antigen of simian virus 40

The PAb1620 antibody belongs to a group of monoclonal antibodies reported to recognize nuclear large T antigen of simian virus 40 (SV40). In the case of PAb1620 there was some indication that the antibody reacted preferentially with large T when it was complexed with p53, the cellular transformation-related antigen. However, we found that PAb1620 does not appear to react with SV40 large T, either free or in complex with p53. In this paper we demonstrate that PAb1620 is, in fact, directed against the p53 protein and recognizes a conformation-dependent epitope. In addition, PAb1620 recognises human p53 from SV80 cells.     

Cooperation of SV40 large T antigen and the cellular protein p53 in maintenance of cell transformation

We analysed large T antigen expression and metabolic stabilisation of the cellular protein p53 in cells of a matched pair of SV40 tsA mutant (tsA58) N-type or A-type transformants, respectively. At the permissive growth temperature (32 degrees C), cells of both transformants, like SV40 wild-type transformed cells, were phenotypically transformed and expressed large T antigen, as well as metabolically stable p53 (both complexed and free p53). At the nonpermissive growth temperature (39 degrees C), cells of the N-type transformant reverted to a normal phenotype, whereas cells of the A-type transformant still displayed a transformed phenotype. Under these growth conditions, the mutant large T antigens in both cell types were no longer able to complex p53 (both in vivo and in vitro), but the metabolic stabilities of the free p53 in these cells correlated with their phenotypes: p53 in cells of the N-type transformant was rapidly degraded, whereas it was metabolically stable in cells of the A-type transformant. This difference in p53 stability correlated with an in vivo functional difference between the mutant large T antigens at the nonpermissive growth temperature: large T antigen in cells of the N-type transformant no longer stably associated with the cellular chromatin and the nuclear matrix, but accumulated in the nucleoplasm. In contrast, large T antigen in cells of the A-type transformant at least partially had retained this ability. Maintenance of SV40 cell transformation thus seems to require both a functional large T antigen and a metabolically stabilised p53.     

T antigen and p53 in pre- and post-crisis simian virus 40-transformed human cell lines.

Infection of normal human diploid fibroblasts (HF) with the DNA tumor virus simian virus 40 (SV) leads to an extension of lifespan and concomitant increase in the levels of the viral large tumor antigen (T antigen) and the cellular protein p53. The intracellular localization of T antigen and p53 was mostly nuclear in both SVpre-crisis and SVpost-crisis cells, however certain population doubling (PD) of the SVpre-crisis cells exhibited some cytoplasmic staining. The DNA content of SVpre-crisis cells shifted to tetraploidy and the SVpost-crisis cells were near-tetraploid. Quantitation of T antigen and p53 in single cells by flow cytometry demonstrated that for all antibodies tested the levels of T antigen were higher in the SVpre-crisis HF than in the SVpost-crisis. The quantity of p53 increased with increasing age of SVpre-crisis HF, and the levels of p53 were higher in the SVpost-crisis HF populations. Immunoprecipitation of p53, T antigen and complexes demonstrated that all p53 was bound to T antigen in SVpre-crisis HF and SVpost-crisis HF. The SVpre-crisis HF cells showed that 33% of all T antigen was bound to p53, while 67% was free, and the SVpost-crisis HF exhibited 50% free T antigen and 50% bound to p53. The half-life of p53 was similar in all SVpre-crisis HF; however, the half-life was 2-3 times greater in SVpost-crisis HF than in SVpre-crisis HF. These results suggest that the interaction of DNA (ploidy), T antigen, p53 and complexes may be involved in formation of a stable SV40-transformed human cell line.     

Role of pescadillo in the transformation and immortalization of mammalian cells

The murine and human homologs of the zebrafish pescadillo protein (Pes1 and PES1, respectively) play important roles in ribosome biogenesis and DNA replication. We investigated the effect of Pes1 on the growth of mouse embryo (3T3-like) fibroblasts and conditionally immortalized human fibroblasts expressing the SV40 T antigen (AR5 cells). Increased expression of Pes1 causes transformation of mouse and human fibroblasts in culture (colony formation in soft agar). Although Pes1 can replace the SV40 T antigen in inducing colony formation in soft agar, it cannot substitute the T antigen in the immortalization of human fibroblasts, indicating that it distinguishes between the two functions. As the biological effects of Pes1 are similar to those of the insulin receptor substrate-1 (IRS-1), we investigated the interactions of Pes1 with IRS-1 itself and with the SV40 T antigen. The Pes1 protein (which localizes to the nuclei and nucleoli of cells) interacts with both IRS-1 and the SV40 T antigen, and markedly decreases the interaction of T antigen with p53. Taken together, these results suggest mechanisms for the ability of Pes1 to transform cells, and its failure to immortalize them.     

Regulation of the level of the oncoprotein p53 in non-transformed and transformed cells

In order to contribute to the understanding of the activation of the oncoprotein p53 we determined the metabolic stability of p53 in a variety of non-transformed, immortalized and SV40- and non-SV40-transformed cell lines. In addition, we analyzed the metabolic stability of the SV40 large T antigen in SV40 transformed cell lines. Pulse-chase experiments revealed a low stability (t1/2 = 20 min) of p53 in non-transformed cells and in cells immortalized by the p53 construct pLTRp53cG9. In cells transformed by an activated ras oncogene and pLTRp53cG9 and in methylcholanthrene induced mouse sarcoma cells p53 proved to be progressively more stable with half-lives ranging from 5.5 h to 7 h. Sequential immunoprecipitation with p53- or T antigen specific monoclonal antibodies allowed us to separate T-p53 complexes, uncomplexed p53 and free T antigen in cell extracts from cells transformed by SV40 and pLTRp53cG9. In these transformed cells uncomplexed p53 showed an increased stability (t1/2 = 2.8 h) when compared to p53 from non-transformed cells. Complex formation with T antigen resulted in an additional stabilization of p53 (t1/2 = 13.3 h). Furthermore, T-p53 complex formation also seems to increase the stability of T antigen nearly sixfold. In transformed cells two immunological variants of p53, a PAb246 precipitable and a non-precipitable form showed distinctly different stabilities, indicating a correlation between the ability of p53 subclasses to bind hsc70 protein and their metabolic stability. Moreover, binding to hsc70 correlated with the stabilization of T antigen in CTM cells also where the mutant T antigen is localized exclusively in the cytoplasm. In abortively infected cells p53, even in complex with T antigen, exhibited a relatively low stability (t1/2 = 87 min) indicating that complex formation per se is not sufficient for fully stabilizing p53.     

The minimal set of genetic alterations required for conversion of primary human fibroblasts to cancer cells in the subrenal capsule assay

Based on previous studies, a minimal set of genetic alterations that is required to convert normal human fibroblasts into cancer cells has been defined. Essential roles for telomere maintenance and alterations in phosphatase 2A activity were inferred from experiments in which tumorigenicity was tested by injecting cells under the skin of immunodeficient mice. However, in the present experiments, the combination of SV40 large T antigen and activated Ras, without hTERT or SV40 small t antigen, was sufficient to convert nine different primary human fibroblast cell strains to a fully malignant state. The malignant behavior of the cells was demonstrated by growth of the cells into invasive tumors when the cells were injected beneath the kidney capsule of immunodeficient mice. Lung metastases and circulating tumor cells were also detected. These tumors were not immortal; cells entered crisis, from which they could be rescued by expression of hTERT. However, the same cell populations were not tumorigenic when they were injected under the skin. In this site, tumorigenicity required the expression of hTERT and SV40 small t antigen as well as SV40 large T antigen and Ras. The cellular pathways targeted by SV40 large T antigen (p53 and pRb) and those targeted by activated Ras represent a minimal set of genetic alterations required for the conversion of normal human fibroblasts into cancer cells.     

Loss of c-abl facilitates anchorage-independent growth of p53- and RB- deficient primary mouse embryonic fibroblasts

The c-abl tyrosine kinase is the proto-oncogene of the v-abl oncogene of the Abelson murine leukemia virus. Although mutational variants of c-Abl can exhibit gain of function and can produce a transformed phenotype, the function of c-Abl in transformation remained unclear. Here, we report that the loss of c-abl facilitates transformation. c-abl-knockout mouse embryonic fibroblasts (MEFs) immortalized by SV40 T antigen acquired anchorage-independent growth, and by constructing mutational variants of T antigen we showed that binding of large T antigen to p53 and RB was necessary to induce anchorage-independent growth. Although c-abl/p53 double-knockout MEFs did not undergo anchorage-independent growth, those expressing human papilloma virus 16 E7, which mainly inactivates RB, did. Our results show that the loss of c-abl facilitates anchorage-independent growth in the context of p53 and RB deficiency, and suggest that loss of function of c-abl facilitates some types of transformation.     

Asbestos induction of extended lifespan in normal human mesothelial cells: interindividual susceptibility and SV40 T antigen.

Normal human mesothelial cells from individual donors were studied for susceptibility to asbestos-induction of apoptosis and generation of an extended lifespan population. Such populations were generated after death of the majority of cells and arose from a subset of mesothelial cultures (4/16) whereas fibroblastic cells (5/5) did not develop extended lifespan populations after asbestos exposure. All mesothelial cultures were examined for the presence of SV40 T antigen to obtain information on (i) the presence of SV40 T antigen expression in normal human mesothelial cells and (ii) the relationship between generation of an extended lifespan population and expression of SV40 T antigen. Immunostaining for SV40 T antigen was positive in 2/38 normal human mesothelial cultures. These cultures also had elevated p53 expression. However, the two isolates expressing SV40 T antigen did not exhibit enhanced proliferative potential or develop an extended lifespan population. Asbestos-generated extended lifespan populations were specifically resistant to asbestos-mediated but not to alpha-Fas-induced apoptosis. Deletion of p16Ink4a was shown in 70% of tumor samples. All mesothelioma cell lines examined showed homozygous deletion of this locus which extended to exon 1beta. Extended lifespan cultures were examined for expression of p16Ink4a to establish whether deletion was an early response to asbestos exposure. During their rapid growth phase, extended lifespan cultures showed decreased expression of p16Ink4a relative to untreated cultures, but methylation was not observed, and p16Ink4a expression became elevated when cells entered culture crisis. These data extend the earlier observation that asbestos can generate extended lifespan populations, providing data on frequency and cell type specificity. In addition, this report shows that generation of such populations does not require expression of SV40 T antigen. Extended lifespan cells could represent a population expressing early changes critical for mesothelioma development. Further study of these populations could identify such changes.     

The pathogenesis of mesothelioma

About 80% of malignant mesotheliomas (MM) in the Western World develop in individuals with higher than background exposure to asbestos. Only a fraction of those exposed to asbestos develop mesothelioma, indicating that additional factors play a role. Simian virus 40 (SV40), a DNA tumor virus that preferentially causes mesothelioma in hamsters, has been detected in several human mesotheliomas. The expression of the SV40 large tumor antigen in mesothelioma cells, and not in nearby stromal cells, and the capacity of antisense T-antigen treatment to arrest mesothelioma cell growth in vitro suggest that SV40 contributes to tumor development. The capacity of T-antigen to bind and inhibit cellular p53 and retinoblastoma (Rb)-family proteins in mesothelioma, together with the very high susceptibility of human mesothelial cells to SV40-mediated transformation in vitro, supports a causative role of SV40 in the pathogenesis of mesothelioma. Asbestos appears to increase SV40-mediated transformation of human mesothelial cells in vitro, suggesting that asbestos and SV40 may be cocarcinogens. p53 mutations are rarely found in mesothelioma; p16, p14ARF, and NF2 mutations/losses are frequent. Recent studies revealed the existence of a genetic factor that predisposes affected individuals to mesothelioma in the villages of Karain and Tuzkoy, in Anatolia, Turkey. Erionite, a type of zeolite, may be a cofactor in these same villages, where 50% of deaths are caused by mesothelioma. Mesothelioma appears to have a complex etiology in which environmental carcinogens (asbestos and erionite), ionizing radiation, viruses, and genetic factors act alone or in concert to cause malignancy.     

p53 Mutations in human immortalized epithelial cell lines

Although rodent cells have been immortalized following transfectionwith a mutant p53 gene, the role of p53 in the immortalizationof human cells is unknown. Therefore, human epithelial celllines were examined for p53 mutations in exons 4–9 whichinclude the evolutionarily conserved regions. A spontaneouslyimmortalized skin keratinocyte cell line, HaCat, and three ras-transfectedclones, have a p53 mutational spectrum that is typical of ultravioletlight induced mutations. A normal finite lifespan cell strain(184) and two benzo[a]pyrene immortalized mammary epithelialcell lines derived from 184 (184A1 and 184B5) contain wild typep53 sequences in exons 4–9, although elevated levels ofnuclear p53 indicate an alteration in the stability of the normallytransient protein. Wild type p53 was found in human bronchial,esophageal and hepatic epithelial cells immortalized by SV40T antigen gene and human renal epithelial cells immortalizedby adenovirus 5. BEAS-2B, an SV40 T antigen immortalized bronchialepithelial cell line and two subclones, have a germline polymorphismat codon 47. Inactivation of p53 by mechanisms such as mutationor complexing with proteins of DNA tumor viruses appears tobe important in the immortalization of human epithelial cells.     

CANCER BIOLOGY: Phosphorylation of T antigen and p53 in carcinogen-treated SV40-transformed Chinese hamster cells

SV40-transfonned Chinese hamster CO631 cells pretreated withN-methyl-N'-nitro-N-nitrosoguanidine (MNNG) display SV40 DNAamplification. This study shows that following MNNG treatmentelevated T antigen synthesis and a 4.5-fold reduction in theextent of its phosphorylation occurred in both pulselabeledand steady-state-labeled cells. The decrease in phosphorylationwas found to be inversely related to carcinogen concentration,i.e. an augmented carcinogen concentration brought about a gradualreduction in T antigen phosphorylation and elevated SV40 DNAamplification. Although the majority of phosphorylation siteson T antigen derived from carcinogen-treated cells were underexpressed,as demonstrated by two-dimensional phosphopeptide mapping, peptide12 bearing phos-phoThr124, which is known to be essential forDNA replication, was overexpressed. Carcinogen-treated cellsshowed no changes in p53 synthesis, but it was phosphoryl-atedto a lesser degree. Two-dimensional mapping revealed that thepredicted N-terminal major phosphopeptide of p53 extracted fromCO631 cells exhibited a lower chromato-graphic mobility thanp53 phosphopeptides from SV40-infected monkey BSC-1 cells. Intreated CO631 cells the R_f value of this phosphopeptide washigher than that of control p53. This finding could be ascribedto the failure to phosphorylate the corresponding amino acidresidue in this peptide. Moreover, treatment did not affectthe half-life of either T antigen or p53 proteins, but causeda dramatic rise in the expression of small t antigen, presumablydue to amplification of SV40 DNA.     

Cell cycle checkpoint status in human malignant mesothelioma cell lines: response to gamma radiation

Knowledge of the function of the cell cycle checkpoints in tumour cells may be important to develop treatment strategies for human cancers. The protein p53 is an important factor that regulates cell cycle progression and apoptosis in response to drugs. In human malignant mesothelioma, p53 is generally not mutated, but may be inactivated by SV40 early region T antigen (SV40 Tag). However, the function of p53 has not been investigated in mesothelioma cells. Here, we investigated the function of the cell cycle checkpoints in six human mesothelioma cell lines (HMCLs) by studying the cell distribution in the different phases of the cell cycle by flow cytometry, and expression of cell cycle proteins, p53, p21(WAF1/CIP1) and p27(KIP1). In addition, we studied p53 gene mutations and expression of SV40 Tag. After exposure to gamma-radiation, HMCLs were arrested either in one or both phases of the cell cycle, demonstrating a heterogeneity in cell cycle control. G1 arrest was p21(WAF1/CIP1)- and p53-dependent. Lack of arrest in G1 was not related to p53 mutation or binding to SV40 Tag, except in one HMCL presenting a missense mutation at codon 248. These results may help us to understand mesothelioma and develop new treatments.     

Two different protein-protein interactions in oligomeric complexes of SV40 large T antigen with the cellular oncoprotein p53

The simian virus 40 (SV40) large T antigen appears in monomers, dimers and various high molecular weight homo-oligomers. EDTA treatment of cell extracts from SV40-infected and -transformed cells leads to a dissociation of the high molecular weight oligomers which can be reconstituted by dialysis against an EDTA free buffer. Hetero-oligomers, composed of T antigen and the oncoprotein p53 become disassembled in the presence of EDTA into forms sedimenting minimally at 7S and maximally at 14S. These low molecular weight T-p53 complexes are resistant to EDTA treatment. Therefore, our results suggest at least two kinds of protein-protein interactions, an EDTA resistant linkage between large T antigen and p53 and an EDTA-sensitive ionic interaction between T antigen molecules in highly oligomeric complexes.     

SV40 oncoproteins enhance asbestos-induced DNA double-strand breaks and abrogate senescence in murine mesothelial cells

SV40 virus has emerged as a potential cofactor with asbestos in the development of diffuse malignant mesothelioma, but its precise role in the pathogenesis of this tumor is unclear. SV40 large T antigen is known to inactivate cellular proteins involved in DNA damage and senescence, including p53 and pRb. We hypothesize that SV40 oncoproteins will sensitize mesothelial cells to DNA damage induced by asbestos or chemotherapeutic agents. SV40 oncoprotein expression in murine mesothelial cell lines enhanced spontaneous and asbestos-induced double-strand breaks, indicated by gamma-H2AX foci, and potentiated micronucleus formation. Mesothelial cells exposed to asbestos or bleomycin for 96 h acquired senescent-like morphology and displayed elevated senescence-associated beta-galactosidase activity, reduced bromodeoxyuridine (BrdUrd) incorporation, and reduced colony formation. SV40 oncoprotein expression abrogated the senescent phenotype, and transfected cell lines showed an increase in both BrdUrd incorporation and colony formation after prolonged DNA damage. Murine mesothelial cell lines lacking wild-type p53 due to a point mutation or gene rearrangement also failed to senesce in response to asbestos or chemotherapeutic agents. In addition, stress-induced senescence in human mesothelial cell lines was impaired by SV40 oncoprotein expression (MeT-5A), p53 small interfering RNA, or spontaneous p53 mutation (REN). These studies suggest that exposure to DNA-damaging agents can induce senescence in both murine and human mesothelioma cell lines and suggest a major, although not exclusive, role for p53 in this response. SV40 virus may contribute to mesothelioma progression by impairing stress-induced senescence, in part through p53 inactivation, thereby favoring survival and proliferation of mesothelial cells that have sustained DNA damage.