Cervical cancer and its precursor lesions cause significant morbidity and mortality for women, both in the United States and elsewhere. The association of cervical cancer and infection with certain types of human papillomaviruses (HPVs) has been recognized for some time; however, it is clear that HPV infection alone is insufficient to bring about the fully malignant phenotype. Clues to the mechanisms by which HPVs contribute to cervical tumorigenesis have been elucidated by studies demonstrating interaction between HPV oncoproteins (E6 and E7) and tumor suppressor gene products (p53 and pRB). However, the functional consequences of these interactions in the HPV-infected cell remain poorly understood. Furthermore, additional genetic alterations required for cervical tumor development and progression have yet to be fully characterized. Genetic and molecular approaches will he used to accomplish the following specific aims: 1) To map functional domains of the E6 proteins of high- and low-risk HPVs with respect to p53 binding and degradation in living cells. E6 genes mutated in the regions encoding p53 binding and degradation functions will be expressed in mammalian cells and analyzed for effects on the p53- dependent DNA damage response. 2) To use HPV E6-53 and E7-pRB interactions as tools to identify additional genes in the DNA damage response pathway. mRNA Differential Display (mDD) technology will be used to identify and clone candidate genes underlying p53 dependent G1 arrest, by comparison of gene expression patterns in cells with an intact response and in cells in which the pathway has been disrupted by HPV16 E6 and/or E7 expression. 3) To examine the effect of HPV E6 and E7 expression on integration of exogenously introduced DNA as a model of viral integration during cervical tumorigenesis. Cell lines expressing E6 and E7 proteins of high-vs. low- risk HPVs will be transfected with a reporter plasmid (pHyGal) encoding hygromycin resistance and scored for differences in frequency of stable integration of the test plasmid. 4) To identify other genes involved, in cervical tumor progression. DNA sequences involved in a rearrangement associated with progression from the well to poorly differentiated state in a primary cervical carcinoma will be cloned and characterized. mDD will he used to identify genes differentially expressed in tumors with well and poorly differentiated components and in pre-invasive versus invasive neoplasms. The proposed studies should provide new insights into the molecular pathogenesis of cervical cancer. Because HPV infection is likely to be an initiating event in the development of most cervical cancers, understanding the functional consequences of HPV infection in cervical epithelial cells may eventually lead to more effective treatment strategies for cervical cancer patients.
