Genital papillomaviruses are associated with a spectrum of changes in the squamous epithelium, including condylomata, precancers (intraepithelial neoplasms) and invasive carcinomas. Specific HPV types, such as type 16, are associated with genital precancers, which, in contrast to condyloma, have a high risk of progressing to invasion. In this project, we will investigate HPV 16 gene expression in genital precancers to determine if HPV 16 codes unique transcripts or translation products in precancers which may account for differences in their biology vis a vis condylomata. To accomplish this, we will analyze transcription of HPV 16 open reading frames (ORF's) in precancers by RNA-RNA in-situ hybridization and cDNA cloning. The former will make it possible to localize transcription of individual ORF's in the epithelium and the latter will more precisely identify the structure of the transcripts produced. Specific early ORF's of HPV 16 which are known to be expressed in precancers, including those which are analogous to transforming genes in bovine papillomavirus (E4, E5, E6) will be cloned into Path vectors and the Tryp-fusion proteins produced will be used to generate polycolonal antibodies in rabbits. These will be reacted with tissue samples by western blot analysis and immunohistochemistry. These studies will aid in defining precisely the HPV gene products produced in the early stages of genital squamous neoplasia and will provide reagents which will be useful for further analysis of their function. Finally, to elucidate the host response to HPV infection, we will attempt to transform established cell lines (NIH 3T3, CREF) with high molecular weight DNA from precancers. Transforming sequences will be characterized in an effort to establish a profile of genes which are necessary to the process of HPV mediated neoplastic transformation in genital squamous epithelium.
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