GLUTATHIONE BIOSYSTHESIS AND DRUG RESISTANCE

细胞内谷胱甘肽(GSH)水平的升高是最早的 以及与囚犯或后天获得性疾病相关的最常见的生化变化 对烷化剂、顺铂和阿霉素类似物的抵抗力。 尽管长期以来，GSH升高与药物之间存在联系 抗性，导致变化的分子机制的细节 GSH在耐药肿瘤细胞中的动态平衡尚不清楚。我们 已经证明，耐药细胞中GSH浓度的增加 通常与伽马活动的增加有关- 谷氨酰半胱氨酸合成酶(GCS)，催化限速 谷胱甘肽从头合成中的反应。我们还确定， GCS表达的增加涉及到稳定状态的mRNA水平的增加 重(催化，GCSh)和轻(调节，GCS1)亚基 由GCS全酶组成。在重亚单位基因的情况下 (尚未检测到轻亚基)这种过度表达可归因于 基因转录速度的提高。此外，我们还克隆了 人GCS催化调节亚基基因及其序列分析 5‘侧翼区域。我们已经进一步证明了与 这两个亚基的cDNA导致谷胱甘肽升高和抵抗力增强 烷基化剂L-PAM。 我们研究的长期目标是定义分子 细胞中常见的与谷胱甘肽升高相关的途径(S) 耐烷基化剂和铂类似物。我们假设 在耐药细胞中观察到的GSH水平经常增加是 谷胱甘肽限速酶(GCS)上调的结果 对药物暴露作出反应的合成途径，并涉及特定的顺式- 以及调节对挑战的反应的反式作用元件 细胞毒剂。拟议调查的具体目标是 定义那些调节上调的顺式和反式作用因子 耐药肿瘤细胞中GCSh和GCSI亚单位基因的 表示谷胱甘肽水平升高。为此，我们建议：1.确定 共转染GCSh和GCS1 cDNAs是否导致GSH升高 用诱导剂测定烷化剂和顺铂的水平和抗药性 基于四环素抗性操纵子的大肠杆菌TO基因表达系统 建立GCS活性升高与药物之间的直接关系 并建立两个亚基之间的关系 抗性表达所必需的；2.确定是否表达 GCS1在耐药肿瘤细胞中转录上调， 重组病毒启动子区域的全序列测定及特性分析 人类GCS1和GCSh亚单位基因；3.识别顺式作用元件 GCSh和GCS1亚单位基因在细胞内的表达调控 耐烷基化剂或顺铂；以及4.识别特定的 在耐药细胞中表达的反式作用因子 GCS亚单位基因转录增加。 对致病分子机制的全面研究 GSH水平的升高将大大有助于理解 这种重要的硫醇在反应中的分子和细胞药理学 细胞对抗肿瘤药物的作用及其在药物进化中的作用 抵抗。

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.