We have demonstrated a suppression of ras oncogene induced transformation of NIH3T3 cells by several protease inhibitors, including antipain. We propose to investigate cellular and molecular mechanisms responsible for this activity of protease inhibitors. These studies will include reconstitution and other experiments to evaluate the role of growth conditions and cellular interactions in transformation suppression. We will test the hypothesis that the action of protease inhibitors is related to effects on endogenous oncogene expression, and determine whether the expression or incorporation of the transfected gene is affected by these agents. New techniques such as polymerase chain reaction will be employed to examine these parameters in the fraction of cells that have undergone stable transfection. Both sustained and transient alterations in gene expression will be measured. We will expand our initial studies to examine the effects of protease inhibitors on cell transformation induced by enhanced expression of proto-ras, as well as the NIH3T3 transforming genes neu, raf, met, and hst. We will also use alternative cell systems such as C3H/10T 1/2 and primary rat embryo fibroblasts to determine whether protease inhibitor suppression of transformation by oncogenes can be observed in cell types less sensitive to ras than NIH3T3. These experiments will provide important mechanistic information, and will define the generality of our initial findings. Finally, we will test other protease inhibitors such as Bowman-Birk inhibitor and potato inhibitor to develop an activity spectrum of protease inhibitors in this assay. It is anticipated that the experiments described in this application will provide important information regarding the mechanisms of action of an important class of anti-carcinogens - the protease inhibitors - at a molecular level using a defined target gene. Increased knowledge of oncogene transformation pathways, and development of a useful assay for the anti-neoplastic activity of protease inhibitors and other agents, are also likely to come from this research.
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