Use of isogenic human cancer cells for high-throughput screening and drug discovery

Cell-based screening for novel tumor-specific drugs has been compromised by the lack of appropriate control cells. We describe a strategy for drug scr eening based on isogenic human cancer cell lines in which key tumorigenic g enes have been deleted by targeted homologous recombination, As a test case , a yellow fluorescent protein (YFP) expression vector was introduced into the colon cancer cell line DLD-1, and a blue fluorescent protein (BFP) expr ession vector was introduced into an isogenic derivative in which the mutan t K-Ras allele had been deleted. Co-culture of both cell lines allowed faci le screening for compounds with selective toxicity toward the mutant Ras ge notype. Among 30,000 compounds screened, a novel cytidine nucleoside analog was identified that displayed selective activity in vitro and inhibited tu mor xenografts containing mutant Ras. The present data demonstrate a broadl y applicable approach for mining therapeutic agents targeted to the specifi c genetic alterations responsible for cancer development.