This proposal investigates at the molecular level the role of the human papillomavirus type 16 (HPV16) transforming gene products E6, E7, and E5, in combination with changes in the expression of epidermal growth factor receptor (EGFR) and transforming growth factor-alpha (TGF-alpha), in the following two initial steps of HPV16-mediated human cell carcinogenesis in vitro: 1. HPV16-induced immortalization of normal human keratinocytes (HKc), and 2. The subsequent acquisition of growth factor-independence (GFI) in cell lines (HKc/GFI) selected from the HPV16-immortalized HKc (HKc/HPV16) by culture in the absence of epidermal growth factor (EGF) and bovine pituitary extract (BPE). In the in vitro model for HPV16-initiated human carcinogenesis used in these studies, malignancy is achieved through at least four reproducible and phenotypically-defined """"""""steps"""""""": immortalization, GFI, differentiation-resistance, then malignant conversion. Preliminary data indicate that changes in EGFR and TGF-alpha (a ligand for the EGFR) expression play an important role in both the immortalization and the GFI step: EGFR mRNA expression is consistently increased in HKc/HPV16 compared to their parental normal HKc. EGFR numbers are further increased in HKc/GFI compared with HKc/HPV16, with no additional increase in EGFR mRNA levels. EGFR in HKc/GFI (but not HKc/HPV16 or normal HKc) are tyrosine-phosphorylated in the absence of EGF. HKc/HPV16 and HKc/GFI overexpress TGF-alpha mRNA by about 4-fold over normal HKc, but secrete into the medium about 100-fold less mature TGF-alpha. In addition, HPV16 E5 is required for the acquisition of the GFI phenotype. In these proposed studies defective retroviruses expressing single HPV16 open reading frames (ORFs: E6, E7, and E5) or the EGFR will be used to infect normal HKc, to investigate the role of each ORF, as well as the interactions o the viral oncoproteins with the EGFR and TGF-alpha genes that lead to immortalization and GFI. The role of E5 at the GFI step will also be investigated. The turnover, tyrosine kinase activity, phosphorylation state, and dimerization of the EGFR in HKc/GFI, in comparison with their parental HKc/HPV16 lines, will be studied. EGFR- mediated signal transduction in the context of HPV-initiated transformation will be investigated. Changes in TGF-alpha processing in HKc/HPV16 and HKc/GFI which may contribute to the GFI phenotype, and factors that may determine these changes will also be studied. Taken together, the results of this work will help clarify the mechanism of HPV16-mediated human carcinogenesis, and tumor progression in general.
