Effects of SV40 T-antigen expression on DNA damage in murine mesothelial cells

Diffuse malignant mesothelioma is a uniformly fatal tumor that is highly resistant to current therapies and is strongly, but not exclusively, associated with asbestos exposure. Simian virus 40 (SV40) has emerged as a potential co-factor with asbestos in the development of mesothelioma, but its precise role in mesothelioma pathogenesis remains unclear. SV40 large (Tag) and small (tag) t-antigens are known to bind and inactivate cellular proteins involved in DNA damage, apoptosis, and senescence, including p53 and pRb. We hypothesize that Tag expression will alter mesothelial cell responses to DNA damage induced by asbestos fibers or chemotherapeutic agents. We are investigating the effects of Tag expression in both preneoplastic mesothelial cells and mesothelioma cells. Expression of Tag correlated with stabilization of wild-type p53, decreased doubling time, and anchorage-independent growth in both cell lines. Tag enhanced spontaneous DSB in preneoplastic and mesothelioma cell lines, as shown by formation of foci containing phospho-histone H2AX (gamma H2AX), and potentiated micronucleus formation in mesothelioma cells following exposure to asbestos or bleomycin sulfate. Prolonged DNA damage produced morphological alterations characteristic of senescence that correlated with elevated senescence-associated beta-galactosidase activity, reduced BrdU incorporation, and reduced colony formation. Tag expression appeared to abrogate the senescent phenotype, as transfected cell lines did not undergo these alterations following prolonged DNA damage. These studies suggest that SV40 Tag enhances spontaneous and induced DNA damage and prevents entry of mesothelial cells into a senescent-like state of growth arrest.