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Research Articles: Therapeutics

A T3587G germ-line mutation of the MDR1 gene encodes a nonfunctional P-glycoprotein

¹ Department of Chemotherapy, Kyoritsu University of Pharmacy; ² Division of Gene Therapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research; ³ Project Team for Pharmacogenetics and Divisions of ⁴ Xenobiotic Metabolism and Disposition, ⁵ Pharmacology, and ⁶ Biochemistry and Immunochemistry, National Institute of Health Sciences, Tokyo, Japan and ⁷ Laboratory of Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

Requests for reprints: Yoshikazu Sugimoto, Department of Chemotherapy, Kyoritsu University of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan. Phone: 81-3-5400-2670; Fax: 81-3-5400-2669. E-mail: sugimoto-ys{at}kyoritsu-ph.ac.jp

Abstract

The human multidrug resistance gene 1 (MDR1) encodes a plasma membrane P-glycoprotein (P-gp) that functions as an efflux pump for various structurally unrelated anticancer agents. We have identified two nonsynonymous germ-line mutations of the MDR1 gene, C3583T MDR1 and T3587G MDR1, in peripheral blood cell samples from Japanese cancer patients. Two patients carried the C3583T MDR1 allele that encodes H1195Y P-gp, whereas a further two carried T3587G MDR1 that encodes I1196S P-gp. Murine NIH3T3 cells were transfected with pCAL-MDR-IRES-ZEO constructs carrying either wild-type (WT), C3583T, or T3587G MDR1 cDNA and selected with zeocin. The resulting zeocin-resistant mixed populations of transfected cells were designated as 3T3/WT, 3T3/H1195Y, and 3T3/I1196S, respectively. The cell surface expression of I1196S P-gp in 3T3/I1196S cells could not be detected by fluorescence-activated cell sorting, although low expression of I1196S P-gp was found by Western blotting. H1195Y P-gp expression levels in 3T3/H1195Y cells were slightly lower than the corresponding WT P-gp levels in 3T3/WT cells. By immunoblotting analysis, both WT P-gp and H1195Y P-gp were detectable as a 145-kDa protein, whereas I1196S P-gp was visualized as a 140-kDa protein. 3T3/I1196S cells did not show any drug resistance unlike 3T3/H1195Y cells. Moreover, a vanadate-trap assay showed that the I1196S P-gp species lacks ATP-binding activity. Taken together, we conclude from these data that T3587G MDR1 expresses a nonfunctional P-gp and this is therefore the first description of such a germ-line mutation. We contend that the T3587G MDR1 mutation may affect the pharmacokinetics of MDR1-related anticancer agents in patients carrying this allele. [Mol Cancer Ther 2006;5(4):877–84]

Grant support: Ministry of Education, Culture, Sports, Science and Technology and Ministry of Health, Labor and Welfare, Japan.

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 7/12/05; revised 12/25/05; accepted 1/25/06.