Functional Characterization of Coding Polymorphisms in the HumanMDR1 Gene Using a Vaccinia Virus Expression System

Abstract

The human MDR1-encoded transporter is a 170-kDa plasma membrane glycoprotein [P-glycoprotein (P-gp)] capable of binding and energy-dependent extrusion of structurally diverse organic compounds and drugs. P-gp seems to play a significant role in uptake, distribution, and excretion of many different drugs. 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. Cell surface expression of wild-type P-gp was time-dependent over a time course of 5.5 to 34.5 h; highest expression was obtained by 22 to 26.5 h after infection/transfection, indicating that a semiquantitative assay for P-gp expression levels was possible. HeLa cells stained with the P-gp specific monoclonal antibodies MRK-16 and Western blots probed with C219 revealed similar cell surface expression for the polymorphisms and for wild-type protein. Time-dependent P-gp pump function maximal at 22 h after infection/transfection was demonstrated for the following MDR1 fluorescence substrates: 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid, succinimidyl ester (bodipy-FL)-verapamil, bodipy-FL-vinblastine, calcein-AM, bodipy-FL-prazosin, bisantrene, and bodipy-FL-forskolin, but not for daunorubicin. Transport studies of all tested substrates indicated that the substrate specificity of the pump was not substantially affected by any of the tested polymorphisms. 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. These results demonstrate that the common MDR1 coding polymorphisms result in P-gps with a cell surface distribution and function similar to wild-type P-gp.

Association of ABCB1 polymorphisms with survival and in vitro cytotoxicty in de novo acute myeloid leukemia with normal karyotype

“…41 This would indicate a higher resistance for the 2677T variant than for 2677G, which is in contradiction to the lower survival we observed in isolated leukemic cells from patients homozygous for T in this position. In another study using HeLa cells the wild-type showed a slightly higher efflux of paclitaxel than the Ser893 variant (2677T) 42 , in agreement with our results. In contrast, transport of other substrates such as verapamil, vinblastine, calcein-AM, prazosin, bisantrene, forskolin, digoxin and cyclosporin A was not affected by G2677T/A or C3435T variants of P-glycoprotein; however, for each substrate only one concentration was tested.…”

“…In contrast, transport of other substrates such as verapamil, vinblastine, calcein-AM, prazosin, bisantrene, forskolin, digoxin and cyclosporin A was not affected by G2677T/A or C3435T variants of P-glycoprotein; however, for each substrate only one concentration was tested. 31,[42][43] In conclusion our findings suggest that certain ABCB1 SNPs (e.g. C1236T and G2677T) affect the survival of AML patients with normal karyotype after chemotherapy and might provide useful information for treatment strategies and individualized chemotherapy.…”

Functional Implications of Genetic Polymorphisms in the Multidrug Resistance Gene MDR1 (ABCB1)

“…A vaccinia virus expression system was used to examine the Asn21Asp, Phe103Leu, Ser400Ala, Ala893Ser, and Ala893Thr P-glycoprotein variants. In all cases, the cell surface distribution and substrate specificity of these variant transporters were similar to reference P-glycoprotein, suggesting no functional impact of these variations (34). However, most substrates used in this study were labeled with bulky fluorescent bodipy groups, which might affect the substrate specificity for P-glycoprotein.…”

ABCB1 genotype and PGP expression, function and therapeutic drug response: a critical review and recommendations for future research

“…61 One study examined the efflux of verapamil, daunorubicin, vinblastine, calcein, prazosin, bisantrene, paclitaxel and forskolin in HeLa cells transiently transfected with 61A4G, 307T4C, 1199G4A, 2677G4T and 3435C4T variants, and demonstrated no substantial difference in transport for all drugs compared with wild-type PGP. 28 By contrast, positive associations have been shown for the following:…”

“…28 A positive association has however been described with the À129T4C polymorphism in exon 1 and PGP expression in placenta tissue Japanese patients, with a P-value of 0.002. 26 …”