Simian virus 40 (SV40)-like DNA sequences not detectable in finnish mesothelioma patients not exposed to SV40-contaminated polio vaccines.

Occupational asbestos exposure can be demonstrated in 80% of mesothelioma cases. A possible role of simian virus 40 (SV40) in the etiology of mesothelioma was raised because several studies reported the presence and expression of SV40-like DNA sequences in human mesotheliomas. It is also known that expression of SV40 large T antigen inhibits cellular Rb and p53. This suggests that SV40 might render infected cells more susceptible to asbestos carcinogenicity. The SV40-like sequences are suggested to have arisen from contaminated polio vaccines. Millions of people in the United States and most European countries were inoculated with SV40-contaminated polio vaccine in 1955-1963. However, in Finland, where polio vaccination started in 1957, no SV40-contaminated vaccine was used. We used a polymerase chain reaction-based method to test for the presence of SV40-like sequences in DNA extracted from the frozen tumor tissues of 49 Finnish mesothelioma patients, most of whom had been occupationally exposed to asbestos. All of the Finnish tumor tissues tested negative for SV40-like sequences. The results suggest that the SV40-like sequences detected in mesothelioma tissue in some previous studies may indeed originate from SV40-contaminated polio vaccines. It is a matter of speculation whether the absence of SV40 infection has contributed to the relatively low incidence of mesothelioma in Finland (1/10(5) in 1990-1995).     

Strategies to circumvent SV40 oncoprotein expression in malignant pleural mesotheliomas

Although nearly 60% of mesotheliomas contain SV40 early region DNA sequences, the role of T/t antigens in initiating and maintaining the transformed state of mesothelioma cells remains unclear. The majority of mesothelioma cells which contain SV40 early region sequences exhibit extremely low basal expression of SV40 oncoproteins; however, T/t antigen expression can be induced under conditions of cellular stress. Abrogation of SV40 T/t expression by antisense techniques induces apoptosis in part via restoration of p53 function, and enhances chemosensitivity in SV40 (+) MPM cells by mechanisms which have not been fully elucidated. This review briefly summarizes our ongoing efforts to define the role of SV40 oncoproteins in modulating the malignant phenotype of mesothelioma cells, and highlights strategies which may prove efficacious in vivo for circumventing SV40 T/t antigen expression in mesotheliomas.     

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.     

Infrequent existence of simian virus 40 large T antigen DNA in malignant mesothelioma in Japan

Malignant mesothelioma is the most common primary pleural neoplasm. Association of simian virus 40 (SV40) with malignant mesothelioma has been reported, suggesting that SV40 plays an important role in the origin of a subset of these tumors. However, significant geographic variation is present as to how often this association occurs. As no study concerning SV40 in malignant mesothelioma has been reported from Japan, we examined the frequency of SV40 infection in Japanese malignant mesothelioma cases. In pleural malignant mesothelioma tissue from 35 patients in Japan, we sought the presence of SV40 large T antigen DNA using real-time polymerase chain reaction (PCR), as well as expression of the viral protein using immunohistological methods. Real-time PCR demonstrated that two of 35 mesotheliomas contained DNA sequences encoding portions of SV40 large T antigen. None of the 35 malignant mesothelioma specimens showed immunoreactivity for SV40 large T antigen. SV40 infection does not appear to have a major role in the development of malignant mesothelioma in Japan.     

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.     

Association between SV40 and non-Hodgkin's lymphoma

Millions of people worldwide were inadvertently exposed to live simian virus 40 (SV40) between 1955 and 1963 through immunization with SV40-contaminated polio vaccines. Although the prevalence of SV40 infections in humans is not known, numerous studies suggest that SV40 is a pathogen resident in the human population today. SV40 is a potent DNA tumor virus that is known to induce primary brain cancers, bone cancers, mesotheliomas, and lymphomas in laboratory animals. SV40 oncogenesis is mediated by the viral large tumor antigen (T-ag), which inactivates the tumor suppressor proteins p53 and pRb. During the last decade, independent studies using different molecular biology techniques have shown the presence of SV40 DNA, T-ag, or other viral markers in primary human brain and bone cancers and malignant mesotheliomas. Evidence suggests that there may be geographic differences in the frequency of these virus-positive tumors. Recent large independent controlled studies have shown that SV40 T-ag DNA is significantly associated with human non-Hodgkin's lymphoma (NHL). In our study, we analyzed systemic NHL from 76 HIV-1-positive and 78 HIV-1-negative patients, and nonmalignant lymphoid samples from 79 HIV-1-positive and 107 HIV-1-negative patients without tumors; 54 colon and breast carcinoma samples served as cancer controls. We used polymerase chain reaction (PCR) followed by Southern blot hybridization and DNA sequence analysis to detect DNAs of polyomaviruses and herpesviruses. SV40-specific DNA sequences were detected in 64 (42%) of 154 NHL, none of 186 nonmalignant lymphoid samples, and none of 54 control cancers. For NHL from HIV-1-positive patients, 33% contained SV40 DNA and 39% Epstein Barr virus (EBV) DNA, whereas NHLs from HIV-1-negative patients were 50% positive for SV40 and 15% positive for EBV. Few tumors were positive for both SV40 and EBV. Human herpesvirus type 8 was not detected. SV40 sequences were found most frequently in diffuse large B cell and follicular-type lymphomas. We conclude that SV40 is significantly associated with some types of NHL and that lymphomas should be added to the types of human cancers associated with SV40.     

SV40 and cell cycle perturbations in malignant mesothelioma

Although epidemiological findings have established that exposure to asbestos fibers is the major cause of malignant mesothelioma (MM), recent studies have implicated simian virus 40 (SV40) in the etiology of some of these tumors. Cytogenetic and molecular genetic evidence suggests that multiple somatic genetic events are required for tumorigenic conversion of a mesothelial cell. As with many other types of cancer, in MM critical oncogenic events exert their action via perturbations of the cell cycle. Interactions between the retinoblastoma (Rb) family of proteins and oncoproteins encoded by SV40 lead to cell cycle alterations. Likewise, inhibition of the p53 tumor suppressor by SV40 can inactivate a crucial cell cycle checkpoint, thereby permitting cells to undergo mitosis regardless of the presence of DNA damage. Many MMs exhibit loss and/or inactivation of the tumor suppressors p16(INK4a)and p14(ARF), components of the pRb and p53 cell cycle regulatory pathways, respectively. Recent investigations have demonstrated that SV40 large T antigen, isolated from frozen biopsies of human MM specimens, binds to and inactivates various tumor suppressor gene products such as pRb and p53. In this review, we discuss how SV40-oncosuppressor interactions can lead to functional alterations of the pRb- and p53-dependent cell cycle regulatory pathways and thereby contribute to neoplastic transformation of human mesothelial cells.     

Simian virus 40 and malignant mesothelioma (Review)

Simian virus 40 (SV40) was recognized as a contaminant of early poliovirus vaccines that were provided to millions of individuals in Europe and in the USA between 1955 and 1963. SV40, a DNA virus of the family of papovaviridae, was proven to be oncogenic in rodents and able to transform human and animal cells in vitro. In 1993 SV40 was accidentally discovered to produce mesotheliomas in hamsters when it was injected in visceral cavities. Afterwards, SV40 DNA sequences were detected with significative frequency in human pleural mesotheliomas by using polymerase chain reaction (PCR) and then SV40 DNA oncogenicity was associated with its large T antigen (Tag). This finding was confirmed by many laboratories, while a few research groups failed to replicate these data and argued that the SV40 DNA detection might be a PCR contamination artefact. In this review the dispute is examined in the light of recent experiments performed to identify molecular and cellular aspects of carcinogenicity and/or co-carcinogenicity of SV40 in human mesothelioma.     

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.     

Immunohistochemical investigation of SV40 large T antigen in Hodgkin and non-Hodgkin's lymphoma

DNA sequences from simian virus 40 (SV40) have been detected in various human tumors, including non-Hodgkin's lymphomas (NHLs), by highly sensitive PCR techniques. However, there is a strong debate as to whether SV40 is present in lymphoma cells. Using immunohistochemistry and tissue microarrays, we investigated a series of French and Canadian cases of Hodgkin's lymphomas (HLs) and NHLs and tried to detect the SV40 large T antigen using routine paraffin sections with standard and highly sensitive catalyzed system amplification methods. None of the cases of HLs (n = 250) or NHLs (n = 232) were found to contain a single large T antigen-positive cell, whereas 2 positive controls were repeatedly stained. Therefore, our results do not support the hypothesis that SV40 is implicated in the etiology of human lymphomas.     

Detection and quantification of SV40 large T-antigen DNA in mesothelioma tissues and cell lines

SV40 viral DNA sequences, particularly large T-antigen (T-ag) DNA, have been reported in malignant pleural mesothelioma (MPM) and suggested to play a role in the tumorigenesis of this cancer. These results remain somewhat controversial owing to variability among a number of laboratories in reported SV40 DNA and protein detection in MPM tissues. This could be explained in part if SV40 DNA is present in relatively low abundance in many MPM tissues determined to contain viral sequences and is therefore difficult to detect. To this end, we investigated the efficacy of real time quantitative PCR in detecting low copy number SV40 DNA sequences, then we quantified SV40 copy number in MPM tissues and cell lines at our institution. Quantitative PCR demonstrated consistent precision and accuracy in detecting SV40 DNA sequences over a large logarithmic range of viral copy number. In addition, SV40 sequences were found in 2/35 tissues and 3/7 cell lines in relatively low abundance. We conclude that SV40 is not a contributing factor in the pathobiology of the majority of MPM tumors from patients at our institution.     

Simian virus 40 and its association with human lymphomas

Simian virus 40 (SV40) is a potent DNA tumor virus that is known to induce cancer in laboratory animals. The neoplasias induced by SV40 in animal models are brain cancers, mesothelioma, bone cancers, and systemic lymphomas. SV40 oncogenesis is mediated primarily by the viral large tumor antigen, which inactivates the tumor suppressor proteins p53 and pRb family members. Evidence indicates that SV40 is an emergent human pathogen and that a significant excess risk of SV40 is associated with primary human brain cancers, malignant mesothelioma, bone cancers, and non-Hodgkin’s lymphoma. Therefore, the major types of tumors induced by SV40 in laboratory animals are the same as those human malignancies found to contain SV40 markers. Experimental and clinical data indicate that SV40 may be functionally important in the development of some of those malignancies. Recently, the Institute of Medicine of the National Academies concluded that SV40 infections could lead to cancer in humans under natural conditions (based on moderate strength biologic evidence). This review examines the data implicating SV40 in the pathogenesis of human lymphomas and discusses future directions to define the causative role for SV40 in these malignancies.     

Association of SV40 with human tumors

In 1994, PCR and protein studies suggested that SV40 DNA sequences and proteins were present in 29/48 (60%) USA human mesothelioma samples. Sequence analysis confirmed that the sequences were homologous to SV40. One year later, SV40 was also found in 5/9 human mesotheliomas, and in 1996 SV40 was also reported to be present in 1/3 of the tumor specimens examined. These reports, in combination with an earlier study in 1992 which had detected SV40 in human brain tumors, raised concerns that SV40 was associated with certain types of human tumors, specifically mesothelioma, bone, and brain tumors. These findings raised concerns, because these tumor types are the same malignancies that had been observed in animals injected with SV40. However, a study in 1996 and a presentation made at the International Mesothelioma Interest Group, IMIG in 1997 failed to detect SV40 in mesotheliomas, suggesting the possibility that laboratory artifacts, such as PCR contamination, had caused the previous positive findings. In 1997, the FDA, the NIH, and the CDC organized an international conference in Bethesda to review the literature and to address the possibility that SV40 was present in, and was possibly the cause of, some human tumors. The results of that conference were reported the same year in a meeting review in Oncogene by Carbone and colleagues. Briefly, the consensus was that before accepting the possibility that SV40 was present in human tumors, a multi-laboratory study needed to be conducted. It was recommended that a blinded multi-laboratory study be directed by an independent scientist not previously associated with the controversial reports of SV40 in human specimens. It was also recommended that this study include laboratories that had reported positive findings as well as laboratories that had failed to detect SV40 in human specimens. Since 1997, about 30 independent reports have been published on this topic, including the multi-laboratory study. Evidence in favor and against a possible association of SV40 with human cancer was reviewed at an international consensus meeting at the University of Chicago on 20, 21 April 2001, entitled "Malignant Mesothelioma: Therapeutic Options and the Role of SV40, 2001". The main focus was the association of SV40 with mesothelioma and other human tumors. At the end of the meeting, a panel discussion, which included independent experts who had not published on this topic, critically reviewed the evidence presented at the meeting. The results of the meeting and of the final panel discussion are outlined below.     

Different simian virus 40 genomic regions and sequences homologous with SV40 large T antigen in DNA of human brain and bone tumors and of leukocytes from blood donors

BACKGROUND: Many studies found only a small fragment of the large T-antigen coding sequences in human tumors, raising doubts on authenticity of SV40 sequences detected in these samples. METHODS: Five different regions of SV40 DNA were investigated in 106 fresh human tumor biopsies (25 brain, 69 bone, 12 Wilms' tumors), 71 tumor-derived cell cultures (38 from brain and 33 from bone tumors) and normal tissues (5 fresh bone biopsies and 38 buffy coats) by polymerase chain reaction (PCR) techniques and filter hybridization with specific oligoprobes. Expression of SV40 Tag sequences was analyzed in human tumor specimens by RT-PCR. RESULTS: SV40 large T-antigen sequences were detected at high prevalence, in human biopsies of primary brain (37-44%) and bone (21-37%) tumors, in cell cultures derived from brain (30-54%) and bone (53-80%) tumors. SV40 Tag sequences were detected in 29% of buffy coats of blood donors. However, only four brain tumor cell lines showed all the five regions of the SV40 genome investigated. Expression of SV40 Tag sequences was found in 11 of 27 (41%) human tumor samples. DNA sequence analysis indicated that the PCR-amplified products belong to the SV40 wild type. Polymerase chain reaction products of Tag middle portion from 20 of 78 (26%) samples showed a 97% homology with telomeric sequences of human chromosomes 10 and 11. CONCLUSIONS: Authentic SV40 sequences were detected in human samples. The expression of SV40 Tag sequences indicates that SV40 could play a role, as a cofactor, in the onset/progression of specific human cancers. The inability to detect some regions of the virus genome may suggest that those regions are not required for tumor persistence or growth and have been lost or, alternatively, may be the result of assay conditions that were unable to PCR-amplify those regions in the tumors.     

p53 and Kirsten-ras mutations in human mesothelioma cell lines.

Twenty cell lines from 17 individuals with malignant mesothelioma have been examined for p53 alterations by direct sequencing of genomic DNA, by evaluation of mRNA expression levels, and by immunocytochemical analysis of p53 protein expression in comparison with normal human pleural mesothelial cells. The results of this study show p53 abnormalities in cell lines from 3 individuals. These include 2 point mutations and one null cell line. Interestingly, while both cell lines with point mutations exhibit high levels of p53 protein, normal mesothelial cells as well as 12 of the mesotheliomas evaluated express low but significant levels. In addition, sequencing of K-ras at codons 12, 13, and 61 reveals wild-type sequence in all 20 mesothelioma cell lines. The capacity to induce tumors in athymic nude mice did not correlate with the presence of a p53 mutation or elevated p53 protein levels. These data suggest that neither p53 alteration nor K-ras activation constitutes a critical step in the development of human mesothelioma.     

Role of T antigen interactions with p53 in tumorigenesis

SV40 induces neoplastic transformation by disabling several key cellular growth regulatory circuits. Among these are the Rb- and p53-families of tumor suppressors. The multifunctional, virus-encoded large T antigen blocks the function of both Rb and p53. Large T antigen uses multiple mechanisms to block p53 activity, and this action contributes to tumorigenesis, in part, by blocking p53-mediated growth suppression and apoptosis. Since the p53 pathway is inactivated in most human tumors, T antigen/p53 interactions offer a possible mechanism by which SV40 contributes to human cancer.     

Medulloblastomas and primitive neuroectodermal tumors rarely contain polyomavirus DNA sequences

To address the hypothesis that medulloblastoma or supratentorial primitive neuroectodermal tumor (sPNET) can arise through infection by polyomaviruses, we examined genomic DNA isolated from 15 primary medulloblastoma and 5 sPNET biopsy specimens and from 2 medulloblastoma cell lines for the presence of DNA sequences from the polyomaviruses simian virus 40 (SV40), JC virus, and BK virus. These polyomaviruses have oncogenic potential in animals, and their DNA sequences have been detected in other surveys of various solid tumors, including childhood brain tumors. The tumor DNA samples were analyzed by Southern blot hybridization of polymerase chain reaction products that employed probes designed to detect specific polyomavirus sequences. Neither JC virus nor BK virus DNA sequences were detected in any of the specimens. None of the primary medulloblastoma or sPNET specimens contained SV40 sequences. However, SV40 DNA coding and noncoding sequences were detected in the D283-Med (medulloblastoma) cell line. Immunocytochemical studies of D283-Med revealed nuclear expression of SV40 large T antigen. In contrast to childhood ependymomas and choroid plexus tumors, medulloblastomas and sPNETs infrequently express evidence of polyomavirus infection.     

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.     

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.