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 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.
Mutoh
Kazuyoshi
K
Department of Chemotherapy, Kyoritsu University of Pharmacy, 1-5-30 Shibakoen, Tokyo 105-8512, Japan.
Mitsuhashi
Junko
J
Kimura
Yasuhisa
Y
Tsukahara
Satomi
S
Ishikawa
Etsuko
E
Sai
Kimie
K
Ozawa
Shogo
S
Sawada
Jun-ichi
J
Ueda
Kazumitsu
K
Katayama
Kazuhiro
K
Sugimoto
Yoshikazu
Y
D016475:3T3 Cells; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001483:Base Sequence; D017931:DNA Primers; D019008:Drug Resistance, Neoplasm; D018014:Gene Transfer Techniques; D018095:Germ-Line Mutation; D006801:Humans; D007564:Japan; D051379:Mice; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000097:blood / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D017931:DNA Primers; D000255:Adenosine Triphosphate