Human epithelial cells will be cultured and we will attempt to control their state of differentiation by agents such as calcium, growth factors, and tumor promoting agents in order to provide targets for HPV transformation. As calcium phosphate precipitation of DNA onto the cells may not be the ideal means of introduction, other approaches such as DEAE dextran, protoplast fusion, microinjection and electroporation will be tried. The transformation protocols will include in addition to HPV DNA exposure of the cells to tumor promoting agents, other oncogenes, eg Ha-ras, growth factors, and the HSV transforming fragment. Co-transformation protocols will be used with a dominant selectable marker, eg. neomycin or gpt to select cells which have retained HPV DNA. Another approach will be to link the coding regions of potential transforming regions such as E2 and E5 to strong promoter and enhancer sequences to ask whether, if these genes are efficiently expressed, they can transform cells. We will analyze the activated genes in tumor cells and benign neoplasms which contain various HPV types. DNAs from these tissues will be used to transform NIH3T3 cells and mouse epithelial cells. The transformants will be analyzed for the retention of human and viral sequences. Secondary and tertiary transformants will be obtained and lambda libraries will be constructed to identify the oncogenes transferred in this way. The tumors and other HPV containing tissues will be analyzed for rearrangements, amplification or altered expression of known cellular oncogenes.
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