MECHANISM BASED SCREENING OF CHEMOPREVENTIVE AGENTS

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Basal cell carcinomas, the commonest human cancer, uniformly have abnormal regulation of hedgehog pathway signaling with consequent changes in transcription of hedgehog target genes. Mutations underlying this aberrant transcription may act through inactivation of the tumor suppressor gene PTC or activation of the protooncogene Smoothened (SMO). REF52 cells transfected with a combination of E1A and SMO carrying the same mutations as found in basal cell carcinomas develop transformed foci, and the cells from these foci grow with anchorage- independence in soft agar and as rapidly-growing tumors when injected subcutaneously into nude mice( Xie et al., Nature 391:90-92, 1998). This prototypic cell system where the mutant SMO, unlike wild type, can cooperate with adenovirus E1A to transform rat embryonic fibroblasts (REF52) appears to fulfill the criteria for an efficient, mechanism based, screen of chemopreventive agents. The objective of this study is to screen a large number of chemopreventive agents using phenotypic and genomic profiling of REF cultures transfected with wild type or mutated SMO. Transformed cells in an in vitro screen shall be used to establish efficacy and mechanism of action of chemopreventive agents utilizing the following endpoints: Modulation of anchorage independent growth is being carried out on REF52 cells transfected with E1A+mut-SMO in a 24-well Linbro tray in the absence and presence of 20 compounds representing all classes of chemopreventive agents. To identify compounds specifically with efficacy against hedgehog (HH) pathway abnormalities, REF52 cells transfected with E1A+ras.