The DNA damage response of rodent fibroblasts expressing all or part of Simian Virus 40 large T antigen
Open Access
- Author:
- Cole, Sara L.
- Graduate Program:
- Microbiology and Immunology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 13, 2001
- Committee Members:
- John Warren Wills, Committee Member
Anita Klein Hopper, Committee Member
Craig Matthew Meyers, Committee Member
Mary Judith Tevethia, Committee Chair/Co-Chair
Satvir Singh Tevethia, Committee Member - Keywords:
- cell stress
Rb
Bcl-2
J-domain
SV40
5-fluorouracil
p53
apoptosis
DNA damage
large t antigen
transformation - Abstract:
- ABSTRACT The Simian Virus 40 large tumor (T) antigen is sufficient to transform cells in culture and induce tumors in experimental animals. T antigen has also been implicated in the development of human tumors. By virtue of its ability to transform cells in experimental systems and its association with human tumors, T antigen serves as an important model for studying carcinogenesis. Transformation of primary cells in culture requires both overcoming growth arrest by stimulating the cell cycle and blocking apoptosis (cell death signals presumably activated by oncogene-mediated hyperproliferative signals). 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 regions of T antigen that contain the functions described above have been defined and are genetically separable. T antigen also contains a domain homologus to the anti-apoptosis protein, Bcl-2. Therefore, T antigen has activities that stimulate cell cycle progression and activities that can inhibit apoptosis. However, the role of each T antigen activity in modulating apoptosis has not previously been determined. Studies have shown that transformed cells can be susceptible to p53-dependent apoptosis following treatment with DNA-damaging agents while primary cells are resistant. 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. The N-terminus of T antigen binds pRb and contains a DNA J-domain (amino acids 1-82) which act in concert to inactivate Rb functions. The C-terminus contains the p53 and p300 binding regions and contains a Bcl-2 homology domain. 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. In addition, rodent fibroblasts expressing N-terminal fragments of T antigen (amino acids 1-127, 1-136 or 1-147) are also susceptible, demonstrating that there are two independent regions of T antigen which sensitize cells to 5-FU induced apoptosis. 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.