We have established a unique system to induce pre-neoplastic transformation of normal human mammary epithelial cells (MECs) with a single oncogene, the human papilloma virus (HPV) 16 E6. Use of natural papilloma virus E6 variants and mutagenesis of HPV-16 E6 directly demonstrated that p53 degradation was necessary for MEC immortalization. Surprisingly, selective elimination of p53 function by introduction of dominant-negative mutant p53 genes or SV-40 large T gene failed to immortalize MECs. These results suggested that E6 must interact with and disrupt the function of additional protein(s) that complement p53 in maintaining the untransformed state of MECs. Here, we propose to directly demonstrate and characterize the biochemical basis of additional oncogenic function of E6. We will introduce p53 degradation-defective HPV-16 E6 mutants, or inefficiently immortalizing HPV-6 or BPV-1 E6 genes, together with dominant-negative p53 genes into 76N normal MECs. The ability of co- transfected non-immortalizing genes to complement each other and induce MEC immortalization will demonstrate an additional oncogenic function of E6. We demonstrated that defective E6 mutants retained binding to a subset of E6-binding cellular polypeptides (E6-BPs) which represent putative mediators of additional E6 function. We will isolate monoclonal antibodies against E6-BPs, purify them on GST-E6 or antibody affinity columns and clone cDNAs encoding these proteins by expression library screening. We will demonstrate in vitro and in vivo association between E6 and E6-BPs, and assess their role in the regulation of cell growth. Finally, we will examine the ability of E6-BPs to physically or functionally interact with p53 tumor suppressor gene product. Elucidation of the biochemical nature of novel oncogenic targets in mammary cells should further our understanding of the biology of early breast cancer.
|Effective start/end date||1/28/95 → 12/31/00|
- National Institutes of Health: $187,925.00
- National Institutes of Health: $203,260.00
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