Human papillomavirus infection is a causative factor for the majority of anogenital cancers. As a consequence of the expression of the HPV E6 and E7 genes, normally quiescent epithelial cells proliferate. The ability to cease proliferating in response to exogenous DNA damaging agents, or in response to the genetic alterations that occur during senescence or crisis in culture, is lost in the HPV containing cells. This likely results in the appearance of preneoplastic lesions or squamous intraepithelial lesions (SIL) in vivo, or immortalized cells in culture, though SIL and immortalized cell lines may not be entirely analogous. We propose that the E7 gene is responsible for inducing proliferation by freeing E2F from Rb or p107, and that the induction of proliferation is independent of the state of p53 since E7 expression alone induces proliferation and high levels of p53, or E7 together with E6 induces proliferation despite the low levels of p53 resulting from the ability of E6 to target p53 for ubiquitin-mediated degradation. The ability to proliferate in the presence of high levels of p53 is an interesting paradox and specific aim one asks whether the p53 that is post- translationally stabilized by E7 expression is structurally and functionally equivalent to the growth repressive form of p53. The ability of p53 to transactivate or repress promoters, bind to DNA or TBP, and its conformation and phosphorylation state will be examined. Both E7 and E6 are able to abrogate the normal G1 checkpoint that prevents the transit of DNA damaged cells into S phase.
Specific aim 2 will use mutants of E7 to determine whether the ability of E7 to arrest in G1 is dependent on the ability to bind Rb/P107 rather than the ability to stimulate proliferation or p53 stabilization. Our preliminary data suggest that p53, that is activated in response to DNA damage, inhibits the phosphorylation of Rb which is required for transit into S; E6 disrupts the pathway by eliminating p53; and E7 acts downstream of p53 bypassing the inhibition of Rb phosphorylation.
Specific aim 3 will examine the expression and activity of the G1 cyclins, D1-3 and E, cyclin dependent kinases (cdks), cdk2 and cdk4, and kinase inhibitory proteins (kips), p27 and p21, that are involved in the phosphorylation of Rb to determine how p53 inhibits phosphorylation and how E7 bypasses the p53 signals.
Specific aim 4 will use differential display of RNAs, from pairs of DNA damaged keratinocytes with and without E6 or E7, as a complementary approach to identify steps in the p53-mediated G1 arrest of DNA damaged keratinocytes. Taken together these approaches will provide important insights into interactions of the HPV oncogenes with the p53 pathway.
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