GLUTATHIONE BIOSYNTHESIS AND DRUG RESISTANCE

烷化剂最常见的生化相关物之一 耐药性是细胞内普遍存在的 非蛋白巯基，谷胱甘肽（GSH）。 的总体目标 建议的研究是评估GSH水平 在耐药肿瘤细胞中增加。 我们的假设是，耐药肿瘤细胞中GSH的增加是 可归因于药物诱导的关键酶活性改变 或参与该重要三肽生物合成的底物。 因此，我们计划将初步调查的重点放在 我们认为有三个主要候选者：GCS， 谷氨酰转肽酶（GGT），谷胱甘肽合成的起始酶， GSH补救途径中的一个步骤;以及细胞内半胱氨酸，一个关键的氨基 酸前体 我们在过去的12个月里开发了 必要的测定和克隆和测序的cDNA， γ-谷氨酰半胱氨酸合成酶（GCS），并认为我们 现在处于一个独特的位置来启动这种类型的调查。 具体来说，我们将比较这些酶的活性， 敏感和烷化剂抗性细胞系对;相关 GCS和GGT基因表达的酶活性差异; 确定转染GCS或GGT cDNA是否可以改变GSH水平 和对烷化剂的反应;并检查丁噻呋胺的作用 亚砜亚胺（BSO），GSH合成的特异性抑制剂，正在评估 临床上作为肿瘤GSH水平的潜在调节剂， 和这些重要的GSH生物合成调节剂的活性。 这些 评价将包括评估淋巴细胞中的这些参数 来自参与我们BSO一期临床试验的患者。 这项对分子和生化机制的全面研究 负责GSH的升高和它们对GSH调节的反应 代理人可以大大有助于了解分子和 这种重要的巯基在细胞对 抗肿瘤剂。

One of the most common biochemical correlates of alkylating agent resistance is an elevation of intracellular levels of the ubiquitous nonprotein thiol, glutathione(GSH). The overall objective of the proposed studies is to evaluate possible mechanism(s) by which GSH levels are increased in drug resistant tumor cells. It is our hypothesis that increased GSH in resistant tumor cells is attributable to drug-induced alterations in the activity of key enzymes or substrates involved in biosynthesis of this important tripeptide. Consequently we plan to focus our initial investigations on changes in what we consider to be three prime candidates: GCS, the rate limiting enzyme in GSH synthesis; gamma-glutamyl transpeptidase (GGT), the initial step in a GSH salvage pathway; and intracellular cysteine, a key amino acid precursor. We have spent the past 12 months developing the requisite assays and cloning and sequencing the cDNA for gamma-glutamylcysteine synthetase (GCS) from human kidney and believe we are now in an unique position to initiate this type of investigation. Specifically, we will compare the activity of these enzymes in select pairs of sensitive and alkylating agent resistant cell lines; correlate differences in enzyme activity with the expression of GCS and GGT genes; determine whether transfection of GCS or GGT cDNAs can alter GSH levels and response to alkylating agents; and examine the effect of buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis being evaluated clinically as a potential modifier of tumor GSH levels, on the regulation and activity of these important regulators of GSH biosynthesis. These evaluations will include assessment of these parameters in lymphocytes from patients participating in our Phase I clinical trial of BSO. This comprehensive investigation of molecular and biochemical mechanisms responsible for the elevation of GSH and their response to GSH modulating agents could contribute significantly to understanding the molecular and cellular pharmacology of this important thiol in the response of cells to anti-neoplastic agents.