The applicant has developed a unique in vitro model system to immortalize normally senescent human mammary epithelial cells using a single transforming gene, the human papilloma virus (HPV)16 E6. While HPVs do not appear to be associated with breast cancer, this model provides a powerful tool to delineate the biochemical and molecular basis of preneoplastic transformation in mammary epithelial cells. Here, we will transfect mutant HPV16 E6 genes, and E6 genes of HPV6, HPV11 and BPV-1, into the 76N normal mammary epithelial cells. We will assess the ability of these transfected genes to induce immortalization and other preneoplastic changes, such as clonal growth, reduced growth factor requirements and altered binding to extracellular matrix proteins. Through these analyses we will determine the role of different regions of the E6 gene critical for E6-induced mammary cell transformation. Earlier we observed a marked reduction of p53 protein in E6-immortalized mammary epithelial cells. We will examine the levels of p53 protein and its association with mutant E6 proteins to address if any E6 mutants can be isolated that do not affect p53 yet retain the ability to transform. Further, we will examine if transfection with wild type p53 gene prevents or reverses E6-induced immortalization, or if oncogenic mutant p53 gene can induce immortalization in normal cells. Together, these studies should help determine the role of p53 protein in E6-induced mammary epithelial cell immortalization. We will use co-immunoprecipitation with antibodies (against E6 protein or against an E2 tag engineered into the transfected E6 gene), and direct binding to bacterially expressed E6 protein to examine the association of E6 with cellular proteins. If new E6-binding cellular proteins are identified, they will be biochemically characterized and purified, and attempts will be made to clone the corresponding genes. Functional characterization of such cellular proteins may reveal novel targets of oncogenic transformation. These studies aimed at elucidating the molecular mechanisms of mammary epithelial cell immortalization, should further our understanding of the biology of early breast cancer.
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