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Research Articles: Therapeutics, Targets, and Development

Thioredoxin-like domains required for glucose regulatory protein 58–mediated reductive activation of mitomycin C leading to DNA cross-linking

Department of Pharmacology, Baylor College of Medicine, Houston, Texas

Requests for reprints: Anil K. Jaiswal, Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201. Phone: 410-706-7333. E-mail: ajaiswal{at}umaryland.edu

Abstract

Glucose regulatory protein (GRP58) is known to mediate mitomycin C (MMC)–induced DNA cross-linking. However, the mechanism remains elusive. We hypothesized that thioredoxin-like domains, one at NH₂ terminus and another at COOH terminus, are required for GRP58-mediated MMC reductive activation leading to DNA cross-linking. Site-directed mutagenesis mutated cysteines in thioredoxin domains to serines. Wild-type (WT) and mutant GRP58 were cloned in pcDNA to produce GRP58 V5-tagged WT and mutant proteins on transfection in mammalian cells. Human colon carcinoma (HCT116) cells transiently expressing and Chinese hamster ovary cells stably expressing WT and mutant GRP58 were analyzed for MMC-induced DNA cross-linking. WT GRP58 was highly efficient in MMC-induced DNA cross-linking. However, both NH₂- and COOH-terminal thioredoxin mutants showed significant reduction in MMC-induced DNA cross-linking. The coexpression of GRP58 with thioredoxin reductase 1 and/or treatment of cells with NADPH increased MMC-induced DNA cross-linking from the WT GRP58. In similar experiments, siRNA inhibition of thioredoxin reductase 1 led to decreased MMC-induced DNA cross-linking. Further experiments revealed that mutations in thioredoxin domains led to significant decrease in metabolic reductive activation of MMC. These results led to conclusion that GRP58, through its two thioredoxin-like domains, functions as a reductase leading to bioreductive drug MMC activation and DNA cross-linking. [Mol Cancer Ther 2007;6(10):2719–27]

Grant support: NIH grant RO1 CA81057.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3/ 7/07; revised 6/15/07; accepted 8/17/07.