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J. Biol. Chem., Vol. 279, Issue 17, 17134-17141, April 23, 2004

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RNA Helicase A in the MEF1 Transcription Factor Complex Up-regulates the MDR1 Gene in Multidrug-resistant Cancer Cells^*

Xiaoling Zhong and Ahmad R. Safa

From the Department of Pharmacology and Toxicology and Indiana University Cancer Center, Indianapolis, Indiana 46202

RNA helicase A (RHA) is a member of the DEAD/H family of RNA helicases and unwinds duplex RNA and DNA. Recent studies have shown that RHA regulates the activity of gene promoters. However, little information is available about the in vivo relevance of RHA in the regulation of natural genes. We previously characterized a nuclear protein (MEF1) that binds to the proximal promoter of the multidrug resistance gene (MDR1) and up-regulates the promoter activity. In the present study, we isolated and identified RHA as a component of the MEF1 complex by using DNA-affinity chromatography and mass spectrometry. The antibody against RHA specifically disrupted the complex formation in electrophoretic mobility shift assay, confirming the identity of RHA. Western blotting showed that RHA in drug-resistant cells had a higher molecular weight than that in drug-sensitive cells. Similar results were obtained when FLAG-tagged RHA was overexpressed in these cells. This size difference probably reflects posttranslational modification(s) of RHA in drug-resistant cells. Chromatin immunoprecipitation revealed that RHA occupies the MDR1 promoter in vivo. Overexpression of RHA enhanced expression of the MDR1 promoter/reporter construct and endogenous P-glycoprotein (P-gp), the MDR1 gene product, and increased drug resistance of drug-resistant cells but not the drug-sensitive counterpart. Introduction of short interfering RNA targeting the RHA gene sequence selectively knocked-down RHA expression and concomitantly reduced P-gp level. Thus, our study demonstrates, for the first time, the involvement of RHA in up-regulation of the MDR1 gene. Interactions of RHA with other protein factors in the MEF1 complex bound to the promoter element may contribute to P-gp overexpression and multidrug resistance phenotype in drug-resistant cancer cells.

Received for publication, October 8, 2003 , and in revised form, February 3, 2004.

^* This work was supported by National Cancer Institute Grants CA 90878, CA 080734, and CA 101743 (to A. R. S.). 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.

To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology and Indiana University Cancer Center, 1044 West Walnut R4-119, Indianapolis, IN 46202. Tel.: 317-278-4952; Fax: 317-274-8046; E-mail: asafa{at}iupui.edu.

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