GLUTATHIONE BIOSYSTHESIS AND DRUG RESISTANCE

细胞内谷胱甘肽（GSH）水平的升高是最早的 和最常见的生化变化与囚犯或获得 对烷化剂、顺铂和阿霉素类似物的抗性。 尽管GSH升高和药物治疗之间存在长期联系， 抗性，负责改变的分子机制的细节 抗肿瘤细胞中GSH稳态的变化仍不清楚。 我们 已经证明了抗性细胞中GSH浓度的增加 通常与伽马射线活动的增加有关， 谷氨酰半胱氨酸合成酶（GCS），其催化 还原型谷胱甘肽的从头合成反应。 我们还确定， GCS表达的增加涉及到mRNA的稳态水平的增加， 重（催化，GCSh）和轻（调节，GCS 1）亚基 包括GCS全酶。 在重亚基基因的情况下 （轻亚基尚未检查）这种过度表达可归因于 基因转录速率的增加。 此外，我们还克隆了 人GCS催化和调节亚基基因，并对其进行测序 5 '侧翼区。 我们已经进一步证明， 这两个亚基的cDNA导致GSH升高和抗性增强 烷基化剂L-PAM。 我们研究的长期目标是确定 通常在细胞中检测到的与GSH升高相关的途径 抗烷基化剂和铂类似物。 我们假设 在抗性细胞中观察到的GSH水平的频繁增加是 GSH中限速酶（GCS）上调的结果 合成途径，并涉及特定的顺式- 和反式作用元件，其介导对这些挑战的反应， 细胞毒性剂。 拟议调查的具体目标是 定义那些介导上调的顺式和反式作用因子 GCSh和GCSI亚基基因在耐药肿瘤细胞中的表达， GSH水平升高。 为此，我们建议：1。 确定 GCSh和GCS 1 cDNA共转染是否导致GSH增加 使用诱导型免疫抑制剂的水平和对烷化剂和顺铂的抗性 构建了基于大肠杆菌四环素抗性操纵子的表达系统。杆菌以 在GCS活性增加和药物之间建立直接关系 并建立两个亚基之间的关系 表达抗性所需; 2. 确定是否表达 GCS 1在耐药肿瘤细胞中转录上调， 完成了启动子区的测序和表征， 人GCS 1和GCSh亚基基因; 3. 鉴定顺式作用元件 调节细胞中GCSh和GCS 1亚基基因的表达 对烷基化剂或顺铂有抗性;和4.确定具体 耐药细胞中表达的反式作用因子，调节 GCS亚单位基因的转录增加。 一个全面的调查的分子机制负责 GSH的升高将有助于理解 分子和细胞药理学的这一重要硫醇的反应 细胞的抗肿瘤药物及其在药物演变中的作用- 阻力

Elevations of intracellular glutathione (GSH) levels are among the earliest and most common biochemical changes associated with inmate or acquired resistance to alkylating agents, analogs of cisplatinum and doxorubicin. Despite a long-standing association between elevated GSH and drug resistance, details of the molecular mechanisms responsible for alterations of GSH homeostasis in resistant tumor cells have remained undefined. We have demonstrated that an increase in GSH concentration in resistant cells is frequently associated with an increase in the activity of gamma- glutamylcysteine synthetase (GCS), which catalyzes the rate-limiting reaction in the de novo synthesis of GSH. We have also established that increased GCS expression involves increased steady-state levels of mRNA for both the heavy (catalytic, GCSh) and light (regulatory, GCS1) subunits comprising the GCS holoenzyme. In the case of the heavy subunit gene (light subunit not yet examined) this over-expression is attributable to an increase in the rate of gene transcription. Further, we have cloned the human GCS catalytic and regulatory subunit genes and sequenced their 5'flanking regions. We have further demonstrated that co-transfection with cDNAs for both subunits results in GSH elevations and increased resistance to the alkylating agent, L-PAM. The long term objective of our studies is to define the molecular pathway(s) associated with the elevation of GSH commonly detected in cells resistant to alkylating agents and platinum analogs. We hypothesize that the frequent increase in GSH levels observed in resistant cells is the result of up-regulation of the rate-limiting enzyme (GCS) in the GSH synthetic pathway in response to drug exposure and involves specific cis- and trans-acting elements which mediate response to challenge with these cytotoxic agents. The specific objective of the proposed investigations is to define those cis- and trans-acting factors which mediate up-regulation of the GCSh and GCSI subunit genes in drug-resistant tumor cells which express increased levels of GSH. To this end we propose to; 1. determine whether co-transfection with GCSh and GCS1 cDNAs results in increased GSH levels and resistance to alkylators and cisplatin using an inducible expression system based on the tetracycline-resistance operon of E. coli to establish a direct relationship between increased GCS activity and drug resistance and to establish the relationship between the two subunits required for expression of resistance; 2. determine whether expression of GCS1 is transcriptionally up-regulated in drug-resistant tumor cells and complete sequencing and characterization of the promoter region of the human GCS1 and GCSh subunit genes; 3. identify cis-acting elements regulating expression of GCSh and GCS1 subunit gene expression in cells resistant to alkylating agents or cisplatinum; and 4. identify specific trans-acting factors expressed in drug-resistant cells which regulate increased transcription of GCS subunit genes. A comprehensive investigation of the molecular mechanisms responsible for the elevation in GSH will contribute significantly to understanding the molecular and cellular pharmacology of this important thiol in the response of cells to anti-neoplastic agents and its role in the evolution of drug- resistance.