The aim of this proposal is to understand at the molecular level the pathogenic mechanics by which human papillomavirus type 16 (HPV-16) transforms epithelial cells. Both HPV16 and 18 are associated with the majority of cases of premalignant and malignant disease of the cervix, while types 6 and 11 are associated with benign disease. When the whole genome of HPV16 was transfected into primary human foreskin keratinocytes it caused the inhibition of normal differentiation when examined in the collagen raft system. the morphology and abnormal cells observed was similar to immortalize and inhibit differentiation, but not as efficiently as the full length genome. HPV6 does not immortalize primary keratinocytes cultures. It is proposed to elucidate which combination of HPV16 genes are important for the immortalization and inhibition of differentiation and characterize their mechanisms of action. 1. We propose to examine which combinations of the three transcripts from the E6 and E7 region are important for immortalization and inhibition of differentiation and which other genes may cooperate. In addition, using an inducible promoter to express either E6 or E7 we hope to confirm that the continued presence of both is necessary for maintenance of the phenotype. 2. Identify important functional regions of the genes involved by site directed mutagenesis. 3. Investigate the effects of site directed mutagenesis at the protein level to see if function or stability is affected. 4. Determine the effects of the over expression of HPV6 genes on keratinocyte immortalization and differentiation. Also, investigate if combinations of HPV6 and 16 genes can immortalize cells. 5. Elucidate the transcription patterns of HPV6 and 16 containing keratinocytes by production of cDNAs. 6. Since papillomaviruses have overlapping reading frames, elucidate which cDNAs code for which proteins and assess the functional role of each cDNA. 7. Examine the effects of estrogens and progestagens in HPV6 and 16 transfected epithelial cells and examine the expression of their receptors. 8. Attempt to elucidate the mechanisms by which HPV16 inhibits epithelial cell differentiation. We will examine: a. the binding of the HPV16 E7 gene with the retinoblastoma gene product and, b. the expression of the retinoic acid receptor.
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