Significance of GGT Expression in Tumors

描述（由申请人提供）：顺铂是一种强效化疗药物和肾毒素。由该基金资助的研究表明，γ-谷氨酰转肽酶对顺铂的肾毒性至关重要，但在快速生长的肿瘤细胞中诱导对顺铂的耐药性。这些数据导致发现顺铂通过铂-谷胱甘肽缀合物代谢为肾毒素。这是第一次证明顺铂可以经过酶激活产生比母体化合物毒性更大的代谢物。我们的假设是，顺铂通过一种与其杀死分裂肿瘤细胞的机制不同的机制杀死肾脏中的非分裂细胞。这次更新申请有三个具体目标。第一个是确定谷胱甘肽-S-转移酶（GST），催化顺铂的肾毒性铂-GSH结合物的形成。将使用HPLC测定法鉴别和监测催化肾毒性铂-GSH结合物形成的GST的纯化。将进行siRNA和转染研究的组合以确认GST在顺铂肾毒性中的作用。GST在肿瘤中的表达与顺铂耐药相关。这种抗性可能是由于它们作为结合蛋白的能力。还将鉴定结合顺铂的GST。第二个具体目标是鉴定催化铂-半胱氨酸结合物代谢为毒素的肾酶。体内和体外研究表明，吡哆醛5 '-磷酸依赖性酶催化该反应。第三个具体目标是确定钠应激反应阻断顺铂肾毒性的机制。氯化钠在临床上用于减少顺铂的肾毒性。这种保护作用的机制尚不清楚。将使用细胞培养系统研究这种反应的分子基础。将分析肿瘤细胞，以确定钠应激反应是否在顺铂耐药细胞中组成型表达。顺铂是最常用的化疗药物之一。本研究的目的是了解顺铂杀死细胞的分子机制以及细胞对顺铂产生耐药性的机制。

DESCRIPTION (provided by applicant): Cisplatin is a potent chemotherapy drug and a nephrotoxin. The studies funded by this grant have revealed that the enzyme gamma-glutamyl transpeptidase is essential for the nephrotoxicity of cisplatin, but induces resistance to cisplatin in rapidly growing tumor cells. These data have led to the discovery that cisplatin is metabolized to a nephrotoxin via a platinum-glutathione conjugate. This is the first demonstration that cisplatin can undergo enzymatic activation to a metabolite that is more toxic than the parent compound. Our hypothesis is that cisplatin kills the non-dividing cells in the kidney by a mechanism that is distinct from the mechanism by which it kills the dividing tumor cells. This renewal application has three specific aims. The first is to identify the glutathione-S-transferases (GSTs) that catalyze the formation of the nephrotoxic platinum-GSH conjugate of cisplatin. An HPLC assay will be used to identify and monitor the purification of GSTs that catalyze the formation of the nephrotoxic platinum-GSH conjugate. A combination of siRNA and transfection studies will be done to confirm the role of the GST in cisplatin nephrotoxicity. Expression of GSTs in tumors has been associated with resistance to cisplatin. The resistance may be due to their ability to serve as a binding protein. GSTs that bind cisplatin will also be identified. The second specific aim is to identify the renal enzyme that catalyzes the metabolism of the platinum-cysteine conjugate to a toxin. In vivo and in vitro studies have shown that a pyridoxal 5'- phosphate-dependent enzyme catalyzes this reaction. The third specific aim is to identify the mechanism by which the sodium stress response blocks cisplatin nephrotoxicity. Sodium chloride is used clinically to reduce cisplatin nephrotoxicity. The mechanism of this protective effect is not known. A cell culture system will be used to investigate the molecular basis of this response. Tumor cells will be analyzed to determine if the sodium stress response is constitutively expressed in cisplatin-resistant cells. Cisplatin is one of the most commonly used chemotherapy drugs. The goal of this research is to understand the molecular mechanisms by which cisplatin kills cells and the mechanism by which cells become resistant to cisplatin.