Protease inhibitors have been shown to possess a broad range of transformation-suppressing effects including reduction of tumor formation and blockage of transformation in cell culture systems. In previous work our laboratory has described the stage-specific nature of the process of transformation of human epidermal keratinocytes by the oncogenic virus, SV40. The overall goal of this proposal is to use the viral transformed epithelial cells as a model system to study the mechanism of action of protease inhibitors in antioncogenesis. The studies described in this proposal are divided into two parts. In the first part we will characterize the effects of protease inhibitors on transformation in the viral transformed epithelial cells using four types of parameters of growth and differentiation as stage-specific markers of transformation in vitro: a) clonal growth, b) growth dependence on serum and growth factors, c) anchorage independent growth, d) density dependent regulation of growth and differentiation. These studies will determine aspects of growth control and critical stages in the transformation process which are targets of the transformation suppressing effects of protease inhibitors. In the second part we will investigate the role of gene expression as a molecular mechanism of action of protease inhibitors. These studies will have three components: 1) DNA and RNA blot hybridization analyses to determine whether protease inhibitor treatment can prevent two types of SV40-induced changes in the myc and ras protooncogenes: a) structural polymorphisms and b) altered levels of transcription, 2) Immunochemical analysis and RNA blot hybridization to determine whether protease inhibitor treatment can reverse the transformation-related induction of simple epithelial/fetal keratin genes and/or block the expression of a newly characterized cytoskeletal cDNA marker of transformation and, 3) Isolation of transformation-related sequences from cDNA libraries whose expression is either blocked or induced by protease inhibitors.
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