Validation of rTS as a Molecular Target

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Our laboratory has discovered, cloned and sequenced the rTS gene. The rTS gene overlaps the TS (thymidylate synthase) gene and codes for a naturally occurring antisense RNA (rTSa mRNA) and two proteins (rTSalpha and rTSP). It is proposed that the rTS proteins are involved in the synthesis of signal molecules that modulate cell growth through a mechanism similar to quorum sensing functions in bacteria. The rTS proteins have homology to RspA, a bacterial protein that can interfere with quorum sensing. Expression of the rTS gene is linked to slowed growth of cells and diminished TS levels as a function of cell population density. Human colon tumor cells (H630-1) that overproduce rTS secrete a molecule that can down-regulate TS in other cells without cell-to-cell contact, suggesting rTS mediates synthesis of a signal molecule. These same cells secrete a molecule that can effect a quorum sensing response in a bacteria-based bioassay. Analogs (acyl homocysteine thiolactones, AHTs) of the molecules hypothesized to be synthesized by rTS proteins) have been chemically prepared and been found to alternatively stimulate and inhibit growth and colony formation of cultured human colon tumor cells. In patient materials we have found that expression of rTSbeta protein is down regulated in a significant number (5/14) of colon tumors compared to paired normal mucosa, suggesting the growth inhibitory function is selected against by tumors in vivo. We propose to evaluate the rTS gene products as targets for drug development. To validate rTS as a possible chemotherapeutic target we propose two Specific Aims: 1) Develop a high-throughput assay to identify compounds that activate rTS function; 2) Synthesize and evaluate a variety of potential rTS ligands to validate rTS as a potential chemotherapeutic target.