Redox Regulation of Multidrug Resistance Gene Expression

描述(由申请人提供)：此续签申请请求支持延长氧化还原条件调节耐药和肿瘤进展的研究。这一应用的长期目标是开发有效使用化疗药物治疗人类恶性肿瘤，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。过去几年的研究表明，谷氨酰半胱氨酸合成(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因素。过表达一个重亚单位的Gamma-GCS(Gamma-GCSh)可以减少细胞内的氧化还原条件，结果是改变了一系列参与耐药(MDR1、MRP1、MRP3和HCtr1)和肿瘤进展(MMP1、MMP3和MMP10)的基因的表达谱。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具有重要意义。虽然细胞毒剂对γ-GCSh的转录调控已有相当广泛的研究，但对转录后调控的研究还不是很详细。我们最近观察到，在不同的氧化应激条件下，一种新的增强mRNA稳定性的转录后调控机制对于上调Gamma-GCSh是重要的。这一新的应用在特定目的1中提出，以阐明氧化还原调节的Gamma-GCSh mRNA稳定性是由MAPK途径介导的假说。在《特定目的2》中提出了一种方法来验证这一假说，即MAPK通路也参与了通过过表达Gamma-GCSh而下调MMP表达的假设。值得注意的是，Gamma-GCSh的表达上调与铜转运蛋白hCtr1的上调有关，hCtr1负责将顺铂输送到培养细胞中。因此，我们建议在特定的目标3中研究hCtr1在不同氧化还原条件下的调控机制。我们希望从这些研究中了解氧化还原条件通过调节它们各自的耐药基因表达来影响药物敏感性的分子基础。

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.