Recent studies have demonstrated that the multidrug resistance-associated protein encoded by the MRP is involved in the transport of substrates containing glutathione (GSH) conjugates. These findings suggest that intracellular GSH levels and activities of gamma- glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme for de novo biosynthesis of GSH, are important in MRP-mediated drug resistance. Indeed, the applicant has demonstrated coordinated expression of MRP and gamma-GCS in many independently established human drug-resistant tumor cell lines, in cultured cells transiently exposed to cytotoxic agents (heavy metals, alkylating agents, phenolic prooxidants and nitric oxide), and in human colorectal tumor biopsies. Because many of these agents are known to generate oxidative stress and his preliminary findings showed that overexpression of antioxidant GSH downregulates MRP, the central theme of this application is that oxidative stress is the underlying mechanism that controls the expression of MRP and gamma-GCS. To test this hypothesis, the applicant proposes in this application: (i) to determine the roles of antioxidant GSH in the coordinated regulation of these genes using inducible expression system to up-regulate the GSH levels and depletors to down-regulate the levels, (ii) to identify and characterize DNA sequences and transcription factors that are involved in the up- and down-regulation of MRP by prooxidants and antioxidants, (iii) to investigate regulation of MRP expression in colon cancer cells under nitrosative stress generated by nitric oxide signaling, and (iv) to establish animal models for the expression of MRP and gamma-GCS in colorectal cancers, because association of oxidative stress with colorectal carcinogenesis has been implicated. The applicant anticipates from these studies to learn the molecular mechanisms that control multidrug resistance gene expression in cancer cells, and thus to improve the efficacy of cancer chemotherapy.
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