Validation of rTS as a Molecular Target

本实验室已发现、克隆并测序了RTS基因。RTS基因与胸苷合成酶(TS)基因重叠，编码一种自然产生的反义RNA(RTSA MRNA)和两种蛋白质(rTSalpha和RTSP)。有人认为，RTS蛋白参与了信号分子的合成，通过类似于细菌群体感应功能的机制来调节细胞生长。RTS蛋白与RSPA同源，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慢和TS水平降低有关，这是细胞种群密度的函数。过度产生RTS的人结肠肿瘤细胞(H630-1)分泌一种分子，该分子可以下调其他细胞中的TS，而不是细胞与细胞之间的接触，这表明RTS介导了信号分子的合成。这些细胞分泌一种分子，可以在基于细菌的生物检测中影响群体感应反应。化学合成的类似物(酰基同型半胱氨酸硫代内酯，AHTS)被认为是由RTS蛋白合成的，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，rTSbeta蛋白在显著数量(5/14)的结肠癌中表达下调，这表明体内肿瘤对生长抑制功能具有选择性。我们建议将RTS基因产物作为药物开发的靶点进行评估。为了验证RTS作为可能的化疗靶点，我们提出了两个特定的目标：1)建立高通量的检测方法来鉴定激活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.