HUMAN MAMMARY EPITHELIAL CELL IMMORTALIZATION

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.