Significance of GGT Expression in Tumors

描述(申请人提供)：顺铂是一种有效的化疗药物，也是一种肾毒素。由这笔赠款资助的研究表明，伽马-谷氨酰转肽酶对于顺铂的肾毒性是必不可少的，但会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是通过铂-谷胱甘肽结合物代谢成肾毒素的。这是首次证明顺铂可以对比母体化合物毒性更大的代谢物进行酶激活。我们的假设是，顺铂杀死肾脏中未分裂的细胞的机制与其杀死分裂中的肿瘤细胞的机制不同。这份续签申请有三个具体目标。第一个是鉴定催化顺铂肾毒性铂-谷胱甘肽结合物形成的谷胱甘肽-S转移酶(GSTs)。高效液相色谱法将被用来鉴定和监测催化形成肾毒性铂-谷胱甘肽结合物的GSTs的纯化。将进行siRNA和转基因研究相结合的研究，以确认GST在顺铂肾毒性中的作用。GSTs在肿瘤中的表达与顺铂耐药有关。这种抗性可能是由于它们作为结合蛋白的能力所致。结合顺铂的GST也将被识别出来。第二个特殊目的是鉴定催化铂-半胱氨酸偶合物与毒素代谢的肾酶。体内和体外研究表明，5‘-磷酸吡哆醛依赖的酶催化这一反应。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降低顺铂的肾毒性。这种保护作用的机制尚不清楚。细胞培养系统将被用来研究这种反应的分子基础。将对肿瘤细胞进行分析，以确定钠应激反应是否在顺铂耐药细胞中有结构性表达。顺铂是最常用的化疗药物之一。这项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.