SIGNIFICANCE OF GGT EXPRESSION IN TUMORS

描述：(逐字改编自研究人员摘要)顺铂是治疗卵巢癌最有效的化疗药物。它在临床上的应用受到剂量限制的肾毒性和肿瘤内耐药性的发展的限制。在之前的资助期间，我们的研究表明顺铂的肾毒性是由于顺铂-谷胱甘肽结合物通过GGT介导的途径在肾脏中代谢所致。我们进一步证明，这种GGT介导的毒性是肾脏特有的。当GGT被导入肿瘤细胞时，它降低了顺铂的毒性，而不是像在肾脏中看到的那样增加了它的毒性。这些发现表明顺铂的毒性有两种不同的机制。这两种机制的阐明将为在不影响顺铂抗肿瘤活性的情况下抑制顺铂的副作用提供机会。目前的建议侧重于确定顺铂发挥毒性作用的机制，并确定肿瘤细胞对顺铂产生耐药性的机制。第一个具体目标是进一步鉴定不稳定的顺铂-谷胱甘肽结合物，它是GGT的底物，并跟踪其在肾脏内的代谢。将测试一系列顺铂-谷胱甘肽结合物作为GGT介导的LLC-PK1细胞和人肾近端小管细胞对肾毒素的激活的底物。采用高效液相色谱分离顺铂-谷胱甘肽结合物，并用质谱法确定其结构。它们的代谢途径将通过在途径中使用选择性的酶抑制剂来确定。在体外阻断顺铂激活的抑制剂将在体内测试其阻断顺铂肾毒性的能力。第二个具体目标是确定肿瘤在体内对顺铂产生耐药性的机制。人卵巢上皮性肿瘤将在裸鼠体内繁殖。一半的小鼠将接受顺铂治疗。顺铂耐药肿瘤将在几个疗程后被分离出来。将从这些肿瘤中分离出mRNA，并通过代表性差异分析来分离差异表达基因。

DESCRIPTION: (adapted verbatim from the investigator's abstract) Cisplatin is the most effective chemotherapy drug for the treatment of ovarian cancer. Its usefulness in the clinic is restricted by dose-limiting nephrotoxicity and the development of drug-resistance within the tumor. During the previous funding period our studies demonstrated that the nephrotoxicity of cisplatin is due to the metabolism of cisplatin-glutathione conjugate through a GGT- mediated pathway in the kidney. We further demonstrated that this GGT-mediated toxicity is specific to the kidney. When GGT was transfected into tumor cells it reduced the toxicity of cisplatin rather than increasing it as was seen in the kidney. These findings indicate that there are two distinct mechanisms of cisplatin toxicity. The delineation of these two mechanisms would provide the opportunity to inhibit the side effects of cisplatin without compromising its antitumor activity. The current proposal focuses on defining the mechanisms by which cisplatin exerts its toxic effects and identifying the mechanism by which tumor cells develop resistance to cisplatin. The first specific aim is to further characterize the unstable cisplatin-glutathione conjugate that is a substrate for GGT and follow its metabolism within the kidney. A series of cisplatin-glutathione conjugates will be tested as substrates for GGT-mediated activation to nephrotoxins by LLC-PK1 cells and human renal proximal tubule cells. Cisplatin-glutathione conjugates will be isolated by HPLC and their structure determined by Mass Spectrometry. The pathway by which they are metabolized will be determined with the use of selective inhibitors of enzymes within the pathways. Inhibitors that block the activation of cisplatin in vitro will be tested for their ability to block the nephrotoxicity of cisplatin in vivo. The second specific aim is to identify the mechanism by which tumors become resistant to cisplatin in vivo. Human epithelial ovarian tumors will be propagated in nude mice. Half the mice will be treated with cisplatin. Cisplatin resistant tumors will be isolated after several courses of treatment. mRNA will be isolated from these tumors and differentially expressed genes will be isolated by representational differential analysis.