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

Kazuyoshi Mutoh, Junko Mitsuhashi, Yasuhisa Kimura, Satomi Tsukahara, Etsuko Ishikawa, Kimie Sai, Shogo Ozawa, Jun Ichi Sawada, Kazumitsu Ueda, Kazuhiro Katayama, Yoshikazu Sugimoto

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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 11196S 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 11196S 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.

Original languageEnglish
Pages (from-to)877-884
Number of pages8
JournalMolecular Cancer Therapeutics
Volume5
Issue number4
DOIs
Publication statusPublished - 2006 Apr

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MDR Genes
Germ-Line Mutation
P-Glycoprotein
3T3 Cells
Genes
Antineoplastic Agents
Alleles
Vanadates
Immunoblotting
Drug Resistance
Blood Cells
Flow Cytometry
Proteins

ASJC Scopus subject areas

  • Oncology
  • Drug Discovery
  • Pharmacology

Cite this

A T3587G germ-line mutation of the MDR1 gene encodes a nonfunctional P-glycoprotein. / Mutoh, Kazuyoshi; Mitsuhashi, Junko; Kimura, Yasuhisa; Tsukahara, Satomi; Ishikawa, Etsuko; Sai, Kimie; Ozawa, Shogo; Sawada, Jun Ichi; Ueda, Kazumitsu; Katayama, Kazuhiro; Sugimoto, Yoshikazu.

In: Molecular Cancer Therapeutics, Vol. 5, No. 4, 04.2006, p. 877-884.

Research output: Contribution to journalArticle

Mutoh, K, Mitsuhashi, J, Kimura, Y, Tsukahara, S, Ishikawa, E, Sai, K, Ozawa, S, Sawada, JI, Ueda, K, Katayama, K & Sugimoto, Y 2006, 'A T3587G germ-line mutation of the MDR1 gene encodes a nonfunctional P-glycoprotein', Molecular Cancer Therapeutics, vol. 5, no. 4, pp. 877-884. https://doi.org/10.1158/1535-7163.MCT-05-0240
Mutoh, Kazuyoshi ; Mitsuhashi, Junko ; Kimura, Yasuhisa ; Tsukahara, Satomi ; Ishikawa, Etsuko ; Sai, Kimie ; Ozawa, Shogo ; Sawada, Jun Ichi ; Ueda, Kazumitsu ; Katayama, Kazuhiro ; Sugimoto, Yoshikazu. / A T3587G germ-line mutation of the MDR1 gene encodes a nonfunctional P-glycoprotein. In: Molecular Cancer Therapeutics. 2006 ; Vol. 5, No. 4. pp. 877-884.
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AU - Ishikawa, Etsuko

AU - Sai, Kimie

AU - Ozawa, Shogo

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N2 - 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 11196S 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 11196S 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.

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