The DNA damage response of rodent fibroblasts expressing all or part of simian virus 40 large T antigen

The Simian Virus 40 large tumor (T) antigen is sufficient to transform cells in culture and induce tumors in experimental animals.; Among the many functions of T antigen are stimulation of cell DNA synthesis, binding the growth control protein p300/CBP, and binding and inactivating the tumor suppressor proteins pRb and p53. Both pRb and p53 have been implicated in growth arrest and apoptosis.; The study presented here examines whether specific regions of T antigen would either render cells susceptible to, or protect from DNA damage induced apoptosis. The purpose of this study was to more closely define the T antigen activities involved in induction or inhibition of apoptosis.; Since T antigen binds p53, p300, pRb and contains a Bcl-2 homology domain, the hypothesis underlying the research described here was that rat embryo fibroblasts expressing this protein would be resistant to DNA damage induced apoptosis. Additionally, rat embryo fibroblasts expressing a C-terminal portion of T antigen, containing the p53/p300 binding region and the bcl-2 homology domain, would also be resistant to apoptosis following DNA damage.; In contrast to the expected result, rodent fibroblasts expressing either full-length T antigen (amino acids 1–708) or a C-terminal portion (amino acids 251–708) undergo apoptosis following exposure to the chemotherapeutic agent 5-fluorouracil (5-FU) through a p53-dependent pathway. These data demonstrate that neither the p53-binding region nor the Bcl-2 homology region of T antigen is sufficient to prevent cell death induced by DNA damaging agents. Specific mutations within the first 128 amino acids of T antigen can result in a variable apoptosis response following 5-FU treatment suggesting that T antigen mediated chemosensitivity is dependent, in part, on the integrity of the J-domain and subcellular localization. Finally, rat embryo fibroblasts expressing the C-terminal fragment 251–708 are protected from 5-FU-induced apoptosis only when this fragment is targeted to the nucleus. These data suggest that the sensitization function(s) of T antigen is located in the N-terminus and is dominant. The apoptosis response in cells expressing full-length T antigen following 5-FU treatment demonstrates that while T antigen can inhibit p53-mediated apoptosis in response to the hyperproliferative signals T antigen provides, other p53-mediated response pathways, such as the DNA damage response, are still active. (Abstract shortened by UMI.)...