Redox Regulation of Multidrug Resistance Gene Expression

描述（由申请人提供）：本更新申请要求支持扩展通过氧化还原条件调节耐药性和肿瘤进展的研究。本申请的长期目标是开发用于治疗人类恶性肿瘤的化疗剂的有效用途，从而提高人类癌症的治愈率。这与国家癌症研究所的使命是一致的。过去几年的研究已经确定，γ-谷氨酰半胱氨酸合成（γ-GCS）是谷胱甘肽（GSH）生物合成的限速酶，是细胞内氧化还原条件的重要调节剂。γ-GCS重亚基（γ-GCSh）的过表达降低了细胞内的氧化还原条件，因此改变了涉及耐药性（MDR 1、MRP 1、MRP 3和HCtr 1）和肿瘤进展（MMP 1、MMP 3和MMP 10）的整个基因宿主的表达谱。因此，了解调节γ-GCS表达的机制在癌症药理学和肿瘤生物学中是重要的。尽管细胞毒性剂对γ-GCSh的转录调控已进行了相当广泛的研究，但转录后调控尚未进行详细研究。我们最近观察到，一种新的转录后调节机制，增强mRNA的稳定性是重要的上调γ-GCSh在各种氧化应激条件下。该更新申请在具体目标1中提出，阐明氧化还原调节的γ-GCSh mRNA稳定性由MAPK途径介导的假设。具体目的2中提出了检验MAPK途径也参与γ-GCSh过表达下调MMP表达的假设的方法。引人注目的是，γ-GCSh的表达升高与铜转运蛋白hCtr 1的上调有关，该转运蛋白将顺铂转运到培养的细胞中。因此，我们在具体目标3中提出研究hCtr 1在各种氧化还原条件下的调节机制。我们希望从这些研究中了解氧化还原条件的分子基础，通过调节各自的耐药基因表达来影响药物敏感性。

DESCRIPTION (provided by applicant): This renewal application requests support to extend studies of the regulation of drug resistance and tumor progression by redox conditions. The long-term objectives of this application are to develop effective use of chemotherapeutic agents for the treatment of human malignancies, thereby improving the cure rates of human cancers. This is consistent with the mission of National Cancer Institute. Studies from the previous funding years have established that gamma-glutamylcysteine synthesis (gamma-GCS), which is the rate-limiting enzyme for the biosynthesis of glutathione (GSH), is an important regulator of intracellular redox conditions. Overexpression of a heavy subunit of gamma-GCS (gamma-GCSh) reduces intracellular redox conditions, as a consequence, changes the expression profile of a whole host of genes involved in drug resistance (MDR1, MRP1, MRP3, and HCtr1) and tumor progression (MMP1, MMP3, and MMP10). Thus, understanding mechanisms that regulate gamma-GCS expression is important in cancer pharmacology and tumor biology. Whereas transcriptional regulation of gamma-GCSh by cytotoxic agents has been studied quite extensively, posttranscriptional regulation has not been investigated in detail. We recently observed that a novel posttranscriptional regulation mechanism of enhancement of mRNA stability is important for the upregulation of gamma-GCSh under various oxidative stress conditions. This renewal application proposes in Specific Aim 1 to elucidate the hypothesis that redox-regulated gamma-GCSh mRNA stability is mediated by the MAPK pathway. Approaches to testing the hypothesis that the MAPK pathway is also involved in the down regulation of MMP expression by overexpression of gamma-GCSh are proposed in Specific Aim 2. Strikingly, elevated expression of gamma-GCSh is associated with upregulation of the copper transporter hCtr1, which transport cisplatin into cultured cells. We therefore propose in Specific Aim 3 to investigate the regulation mechanism of hCtr1 under various redox conditions. We hope from these studies to learn the molecular bases of redox conditions that affect drug sensitivity through the regulation of their respective drug resistance gene expression.