The experiments described in this proposal are designed to investigate, at the molecular level, the events which lead to immortalization of human keratinocytes (HKc) in vitro, after transfection with human papillomavirus type 16 (HPV16) DNA. Immortalization, (a first step in the carcinogenetic process) is a reproducible effect of the transfection with HPV16 DNA in HKc. Due to their frequent association with human genital cancer (80% of the cases), some types of human papillomavirues are strongly suspected to play an important role in the pathogenesis of these diseases. In particular, HPV16 is associated with the highest number of cases (50%) of premalignant and malignant lesions. The importance of defining the exact role of the virus in this process is obvious; a better understanding of its interactions with its host cell will lead to more rational approaches for the diagnosis and therapy of papillomavirus infections of the genital tract, and for a more precise assessment of the risk connected with infection by specific HPV types. Furthermore, new information on this subject may lead to new insights on the factors and mechanisms which control growth and differentiation in epithelial cells, a subject of great interest under the point of view of carcinogenesis, in general. The following specific aims are being addressed: 1. To analyze the transforming ability of different viral gene products, a) by using cloned subgenomic fragments of the viral DNA, representing specific viral genes, in a well established transformation assay on normal HKc. b) by analyzing viral gene expression in the immortal cell lines already established, which contain the whole viral DNA. This will involve the construction and analysis of cDNA libraries form these cells, to identify the viral gene products constantly expressed in lines from different individuals, and the """"""""consensus"""""""" structure of the transforming proteins, necessary for transformation, by comparing the lines obtained with isolated specific HPV16 genes to the ones established with the entire HPV16 genome. The production of suitable amounts of the transforming protein (by expressing its cDNA in bacteria and in mammalian cells) for structural and biochemical characterization will follow. 2. To identify, at the molecular level, the integration sites of the viral sequences in the cellular DNA, by cloning the viral/cellular DNA junctions and comparing the flanking sequences in different lines. The goal is to assess if specific sequences in the cellular DNA are necessary for integration. 3) To investigate the relationship between viral gene expression and altered growth and differentiation properties observed in the immortalized lines, by studying viral gene expression under various differentiation-inducing conditions.
