Carcinomas, which constitute about 80 percent of all human cancers, arise as a malignant transformation of epithelial cells. To elucidate the pathophysiology of epithelial cell transformation, we have established a unique in vitro model, in which a single oncogene, the HPV16 E6, induces a highly efficient immortalization of normally senescent human mammary epithelial cells. Using this model, we have demonstrated that the loss of function of p53 tumor suppressor protein is essential for immortalization but is not sufficient, suggesting that additional cellular targets are involved in E6-induced immortalization. Using the yeast two-hybrid interaction system, we have isolated one such cellular protein that binds to and is targeted for degradation by the immortalization-competent but not immortalization-incompetent HPV16 E6 protein. This novel protein, designated E6TP1 (E6-targeted protein 1) exhibits high homology to GTPase-activating proteins for the Ras-related small G-proteins of the Rap family. We hypothesize that a loss of E6TP1 function constitutes one of the lesions in epithelial cell immortalization. Here, we propose analyses to examine the basis of a functional role of E6TP1 in epithelial cell transformation. We will develop anti-E6TP1 antibodies and use these to characterize the E6TP1 polypeptide(s) in relation to cellular proliferation, senescence and cell cycle and delineate the mechanism of E6-targeted degradation of E6TP1; assess the potential growth inhibitory/tumor suppressor function of E6TP1; perform mutational analyses to define the functionally critical domains of E6TP1 and characterize the GAP activity of E6TP1 protein. Characterization of this novel putative mediator of epithelial oncogenesis represents a step forward in our understanding of the pathophysiology of carcinomas and may aid in future design of novel molecular therapeutics against carcinomas.
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