Murine polyomavirus T antigens interfere with distinct check-points in G(1) phase of the cell cycle

Abstract

Murine polyomavirus is a DNA tumor virus that transforms cells in culture and induces tumors in mice. The major viral oncoproteins responsible for these phenomena are large T (LT) and middle T (MT) antigen. This thesis examines the ability of the T antigens to abrogate specific cell cycle check-points, which is a fundamental process in neoplastic transformation. We examined the ability of the T antigens to overcome the growth suppression function of p53. As a model system, a temperature sensitive p53 gene was introduced into mouse embryo fibroblasts from a p53 null mouse. At the permissive temperature (32C) a functionally wild-type p53 protein is expressed and arrests the growth of these cells in G1/G0 phase of the cell cycle. We introduced LT and MT antigen into these cells and demonstrated that LT overcame the growth arrest while MT did not. A LT mutant that was defective for binding the retinoblastoma tumor suppressor protein (pRB), was unable to overcome the growth arrest. This suggests that p53 directs the growth arrest through regulation of pRB and inactivation of pRB overcomes the arrest. Although MT did not overcome the arrest, mitogenic signals stimulated by MT were not inhibited by p53. We show that MT still associated with the signaling molecules PI3K and SHC, and increased the transcriptional activity of c-myc and AP1 in the p53 arrested cells. These results suggest that the growth arrest mediated by p53 is dominant over the proliferative signals generated by MT. Serum deprivation of NIH 3T3 cells results in growth arrest in G 0. Expression of MT in serum deprived cells was sufficient to reinitiate cell cycle progression. Following MT expression, levels of cyclin D and E increased and the level of p27 decreased resulting in increased G1 cyclin dependent kinase activity and hyperphosphorylation of pRB. These results indicate that MT abrogates the mitogen dependent check-point in G0. These studies demonstrate that LT overcomes the growth suppressive effects of p53 and MT substitutes for serum growth factors and overcomes growth arrest in G0. We conclude that the ability of the T antigens to overcome these distinct check-points is critical for cell transformation and presumably is also important for virus growth.