REGULATION OF HUMAN PAPILLOMAVIRUSES IN ORAL MUCOSA

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Human papillomaviruses (HPV) are associated with human oral and cervical cancers. Specific types of HPV are present in the majority of these tumors, and the E6 and E7 genes of these viruses can transform cells to a malignant phenotype. However, the presence of an oncogenic HPV in a cell is not sufficient to make a cell malignant - over expression of the transforming gene must be stimulated by other factors. These factors have not been studied in HPV-related oral cancer. This project will therefore clarify two aspects of the factors that control the transforming genes from HPV-16 and -18; the structure of the long control region (LCR) of the virus, and the role of cellular proteins that interact with the LCR. to study the LCR of HPV-16 or -18, it will be isolated and sequenced from genomic DNA of oral cancer cells and tissues by a PCR method.The cis- regulating elements which probably control the expression of the viral genes will be recognized by examination of the sequence. Their function will be confirmed by construction of a luciferase expression vector and transfection of several cell types. The essential sequences, and those which are over-active or eliminated in cancer calls will be located by mutagenesis experiments, using normal and transformed oral epithelial cells. Cellular factors that interact with the LCR will be detected in cell extracts using in vitro footprinting and detected in cell extracts using in vitro footprinting and electrophoretic mobility shift assays with the aid of UV-crosslinking and immunoblotting. Their role in promoting expression of genes that are adjacent to the LCR will be examined by a quantitative in vitro transcription assay and an in vivo luciferase transient transfection.. At the conclusion of the award period, this work will have provided a description of how papillomaviruses are regulated in normal oral epithelium, and how their expression in increased in oral cancer cells. The identified cell factors could be targets and the specified HPV LCR could be modified as a promoter for gene therapy in the future.