GLUTATHIONE BIOSYNTHESIS AND DRUG RESISTANCE

烷化剂最常见的生化相关因素之一 抗性是无处不在的细胞内水平的升高 非蛋白硫醇，谷胱甘肽(GSH)。《公约》的总体目标 建议的研究是评估谷胱甘肽水平的可能机制(S) 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 可归因于药物引起的关键酶活性的改变 或参与这种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，速率限制 谷胱甘肽合成中的酶；γ-谷氨酰转肽酶，最初 细胞内的半胱氨酸是一种关键的氨基酸 酸性前体。在过去的12个月里，我们一直在开发 鸡传染性支气管炎基因的必要分析及克隆测序 人肾中的γ-谷氨酰半胱氨酸合成酶 现在处于发起这类调查的独特地位。 具体地说，我们将比较这些酶在SELECT 对敏感和抗烷基化剂的细胞系；相互关联 酶活性与GCS和GGT基因表达的差异； 确定GCS或GGT cDNAs的转染是否可以改变GSH水平 以及对烷化剂的反应；并检查丁硫氨酸的效果 正在评估的谷胱甘肽合成的特异性抑制剂亚磺胺(BSO) 临床上可作为肿瘤GSH水平的潜在修饰者，对其进行调节 以及这些GSH生物合成的重要调节因子的活性。这些 评估将包括对淋巴细胞中这些参数的评估 来自参与我们的BSO I期临床试验的患者。 对分子和生化机制的全面研究 负责GSH的升高及其对GSH调节的反应 代理人可以大大有助于理解分子和 这种重要的硫醇在细胞反应中的细胞药理学 抗肿瘤药物。

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.