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

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...

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Published in:Molecular cancer therapeutics 2006-04, Vol.5 (4), p.877-884
Main Authors: Mutoh, Kazuyoshi, Mitsuhashi, Junko, Kimura, Yasuhisa, Tsukahara, Satomi, Ishikawa, Etsuko, Sai, Kimie, Ozawa, Shogo, Sawada, Jun-ichi, Ueda, Kazumitsu, Katayama, Kazuhiro, Sugimoto, Yoshikazu
Format: Article
Language:English
Subjects:
3T3 Cells
ABCB1
Adenosine Triphosphate - metabolism
Amino Acid Sequence
Animals
Antineoplastic Agents - pharmacology
ATP-Binding Cassette, Sub-Family B, Member 1 - blood
ATP-Binding Cassette, Sub-Family B, Member 1 - genetics
ATP-Binding Cassette, Sub-Family B, Member 1 - metabolism
Base Sequence
DNA Primers
drug resistance
Drug Resistance, Neoplasm
Gene Transfer Techniques
Germ-Line Mutation
Humans
Japan
Mice
Molecular Sequence Data
Neoplasms - blood
Neoplasms - genetics
P-gp
Polymerase Chain Reaction
Polymorphism, Single Nucleotide
Retroviridae - genetics
Reverse Transcriptase Polymerase Chain Reaction
SNP
vincristine
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Summary: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]
ISSN:1535-7163
1538-8514
DOI:10.1158/1535-7163.MCT-05-0240