ABCMdb

PMID: 12065748

Kimchi-Sarfaty C, Gribar JJ, Gottesman MM
Functional characterization of coding polymorphisms in the human MDR1 gene using a vaccinia virus expression system.
Mol Pharmacol. 2002 Jul;62(1):1-6., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Phe103Leu To determine whether common polymorphic forms of P-gp are likely to alter function of P-gp, we characterized five known MDR1 coding polymorphisms (N21D, F103L, S400N, A893S, and A998T) using a vaccinia virus-based transient expression system. Login to comment 7 ABCB1 p.Ser400Asn ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Asn21Asp Cell surface expression and function of double mutants including the more common polymorphisms (N21D-S400N, N21D-A893S, and S400N-A893S) showed no differences from wild-type. Login to comment 20 ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Phe103Leu In this study, we examined the five most common P-gp coding polymorphisms previously reported in the literature (N21D, F103L, S400N, A893S, and A998T). Login to comment 30 ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Phe103Leu Using the technique described by Kunkel et al. (1987), five different mutated sites were introduced into the MDR1 gene with the following primers: for the N21D (A3G) polymorphism, 5Ј TTT TTC ACT TTT ATC GTT CAG TTT AA 3Ј; for the F103L (C3T) polymorphism, 5Ј CAG ATT CAT GAA GAG CCC TGT ATC A 3Ј; for the S400N (G3T) polymorphism, 5Ј TCG AGA TGG GTA ATT GAA GTG AAC AT 3Ј; for the A893S (G3T) polymorphism, 5Ј AGC GAT CTT CCC AGA ACC TTC TAG TT 3Ј; and for the A998T (G3A) polymorphism, 5Ј TAT TTT GGC TTT GGT ATA GTC AGG AGC 3Ј. Login to comment 31 ABCB1 p.Ser400Asn ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Asn21Asp Double mutant MDR1s were generated using the NdeI and XhoI restriction enzymes on single mutant templates (N21D-S400N, N21D-A893S, and S400N-A893S). Login to comment 48 ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Phe103Leu We have chosen HeLa cells for these studies because of their low level of endogenous P-gp expression, their ability to express high TABLE 1 Common MDR1 polymorphisms that change amino acids Location Polymorphic Variant Heterozygous Frequency Reference Exon Nucleotide % 2 61 N21D 17.6 Hoffmeyer et al. (2000) 11.2 Cascorbi et al. (2001) 5.7 Decleves et al. (2000) 5 307 F103L 1.2 Hoffmeyer et al. (2000) 10 1107 G369P 0.2 Cascorbi et al. (2001) 11 1199 S400N 12.9 Hoffmeyer et al. (2000) 5.5 Cascorbi et al. (2001) A893S 43.0 Mickley et al. (1998) A893T 41.6 Cascorbi et al. (2001) 21 2677 A893S 62.0a , 13.0b Kim et al. (2001) A893G 56.4 Cascorbi et al. (2001) 24 2995 A998T 11.0 Mickley et al. (1998) a European Americans. b African Americans. levels of wild-type and mutant P-gp after vaccinia infection/ transfection, and their relative ease of transfection (Hrycyna et al., 1998; Ramachandra et al., 1998; Gribar et al., 2000). Login to comment 63 ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Phe103Leu HeLa cells were infected/transfected with the pTM1-MDR1 vector harboring the MDR1 polymorphisms N21D, F103L, S400N, A893S, and A998T (described in Table 1). Login to comment 70 ABCB1 p.Ser400Asn ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Asn21Asp All cells infected/ transfected with double mutants (N21D-S400N, N21D-A893S, and S400N-A893S) revealed results similar to those of the single mutants (data not shown). Login to comment 71 ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Phe103Leu Discussion In this study a transient vaccinia expression system was used to determine the effect of five known coding human MDR1 polymorphisms on P-gp function: N21D, F103L, S400N, A893S, and A998T. Login to comment 113 ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Phe103Leu Infected/transfected HeLa cells with wild-type pTM1-MDR1 (--), pTM1-MDR1-N21D (- -⅐- -⅐), pTM1-MDR1-F103L (⅐⅐⅐⅐), pTM1-MDR1-S400N (-⅐-⅐-), pTM1-MDR1-A998T (- - - -), and pTM1-MDR1-A893S (⅐⅐⅐-⅐⅐⅐) were incubated and analyzed by FACS as described under Materials and Methods, with MRK-16 or control IGg2a␬ monoclonal antibodies (-⅐-⅐-⅐) 13.5 h after infection/transfection. Login to comment 117 ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Phe103Leu Cells were transfected with pTM1 (control), pTM1-MDR1, (wild-type P-gp), pTM1-MDR1- N21D, pTM1-MDR1-F103L, pTM1-MDR1-S400N, pTM1-MDR1-A893S, and pTM1-MDR1-A998T. Login to comment 119 ABCB1 p.Ser400Asn ABCB1 p.Ser400Asn ABCB1 p.Ser400Asn ABCB1 p.Asn21Asp ABCB1 p.Asn21Asp ABCB1 p.Asn21Asp ABCB1 p.Phe103Leu ABCB1 p.Phe103Leu with A, 0.5 ␮M Calcein-AM: wild-type (--), N21D (- -⅐- -⅐), and S400N (-⅐-⅐-), in the presence of an inhibitor, 5 ␮M cyclosporin A (- - -); B, 0.5 ␮M bodipy-FL-forskolin: wild-type (--), N21D (- -⅐- -⅐), F103L (⅐⅐⅐⅐), and S400N (-⅐-⅐-), in the presence of an inhibitor, 5 ␮M cyclosporin A (- - -); C, 0.5 ␮M bodipy-FL-verapamil: wild-type (--), N21D (- -⅐- -⅐), F103L (⅐⅐⅐⅐), and S400N (-⅐- ⅐-), in the presence of an inhibitor, 5 ␮M cyclosporin A (- - -); D, 0.1 ␮M bodipy-FL-paclitaxel: wild-type (--), A893S (- -⅐- -⅐), in the presence of an inhibitor, 5 ␮M cyclosporin A for the wild-type (- -), and for A893S (- - -). Login to comment