Validation of rTS as a Molecular Target

本实验室已发现rTS基因，并对其进行了克隆和测序。rTS基因与TS（胸苷酸合成酶）基因重叠，并编码天然存在的反义RNA（rTSa mRNA）和两种蛋白质（rTSalpha和rTSP）。有人提出，rTS蛋白参与信号分子的合成，通过类似于细菌中的群体感应功能的机制来调节细胞生长。rTS蛋白与RspA具有同源性，RspA是一种可以干扰群体感应的细菌蛋白。rTS基因的表达与细胞生长减缓和TS水平降低有关，TS水平是细胞群体密度的函数。过量产生rTS的人结肠肿瘤细胞（H630-1）分泌一种分子，该分子可以在没有细胞与细胞接触的情况下下调其他细胞中的TS，这表明rTS介导信号分子的合成。这些相同的细胞分泌一种分子，可以在基于细菌的生物测定中影响群体感应反应。假设由rTS蛋白合成的分子的类似物（酰基高半胱氨酸硫代内酯，AHT）已经被化学制备，并且被发现交替地刺激和抑制培养的人结肠肿瘤细胞的生长和集落形成。在患者材料中，我们发现与配对的正常粘膜相比，rTS β蛋白的表达在显著数量（5/14）的结肠肿瘤中下调，表明生长抑制功能被体内肿瘤选择。我们建议评估rTS基因产物作为药物开发的靶点。为了验证rTS作为可能的化疗靶点，我们提出了两个具体目标：1）开发高通量测定以鉴定激活rTS功能的化合物; 2）合成和评估各种潜在的rTS配体以验证rTS作为潜在的化疗靶点。

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.