Ionizing radiation is a universal carcinogen which has been implicated in the development of malignant phenotypes in both human and animal model systems. The development of an in vitro transformation system using cells of human and epithelial origin would be useful for confirming and extending the results already obtained with rodent fibroblasts. Using immortalized human epidermal keratinocytes in vitro, we have demonstrated the induction of a malignant phenotype by the concerted action of a DNA tumor virus and ionizing radiation. this process involved multiple events. First, was the acquisition of unlimited growth potential as a result of infection with the AD12-SV40 hybrid virus. the remaining steps involved successive exposures to ionizing radiation. This radiation-induces transformation resulted in morphological alterations, anchorage-independent growth and the formation of poorly differentiated squamous cell carcinomas in athymic mice. These observations indicate that immortalized human epidermal keratinocytes will provide a good in vitro model system to study both radiation carcinogenesis and its suppression by protease inhibitors. Accordingly, the goal of this proposal is to evaluate this system as a reproducible in vitro model for the study of the role of protease inhibitors in the suppression of radiation carcinogenesis in a human epithelial system. Further, using subtracted cDNA expressions libraries, identify the cellular genes or proto-oncogenes that have been activated or inactivated during the suppression of transformation following treatment with protease inhibitors. Finally, we propose to identify by 2-dimensional electrophoresis the protein species which are the substrates of the endogenous proteases that have been inactivated by the protease inhibitors. The studies will provide a molecular basis for the role of protease inhibitors in the suppression of radiation-induces malignant transformation of human cells in vitro. further, they may provide a molecular basis for the development of chemo-preventative therapies for human subjects at risk.
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