ACCELERATED COMMUNICATION Functional Characterization of Coding Polymorphisms in the Human MDR1 Gene Using a Vaccinia Virus Expression System

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 S400NA893S) 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. The pharmacokinetics of commonly used drugs varies from person to person, reflecting differences in absorption, distribution, metabolism, and excretion. A family of ATP-dependent ABC transporters has been described, several members of which seem to be involved in drug absorption, distribution, and excretion (Gottesman, 2002). One of the best-characterized members of this superfamily is P-glycoprotein (P-gp), the product of the human MDR1 gene (Ambudkar et al., 1999). P-gp was first described as an energy-dependent efflux pump for diverse hydrophobic natural product anticancer drugs such as doxorubicin, vinblastine, and paclitaxel (Taxol) but has since been shown to transport dozens of different commonly used drugs including HIV protease inhibitors (Kim et al., 1998; Lee et al., 1998), cholesterol-lowering statins (Bogman et al., 2001), antihistamines (Chiou et al., 2001), and digoxin (Mayer et al., 1996). The localization of P-gp in the mucosa of the small and large intestine, at bloodbrain barrier sites, in biliary hepatocytes, and in proximal tubules of the kidney (Thiebaut et al., 1987; Cordon-Cardo et al., 1989; Thiebaut et al., 1989) together with evidence from mdr knockout transgenic mice, indicates a significant role for P-gp in drug pharmacokinetics (Schinkel et al., 1997; Borst et al., 1999, 2000). Several recent reports indicate that polymorphisms are relatively common in the human MDR1 gene (Yoshimoto et al., 1988; Mickley et al., 1998; Decleves et al., 2000; Hoffmeyer et al., 2000; Liu and Hu 2000; Ameyaw et al., 2001; Brinkmann et al., 2001; Cascorbi et al., 2001; Hitzl et al., C.K.-S. and J.J.G. contributed equally to this work. 1 Present Address: Jefferson Medical College, 1025 Walnut Street, Philadelphia, PA 19107-5083 ABBREVIATIONS: ABC, ATP binding cassette; P-gp, P-glycoprotein; MDR, multidrug resistance; FACS, fluorescence-activated cell sorting; IMDM, Iscove’s modified Dulbecco’s medium; bodipy-FL, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid, succinimidyl ester. 0026-895X/02/6201-1–6 MOLECULAR PHARMACOLOGY Vol. 62, No. 1 U.S. Government work not protected by U.S. copyright 1617/989398 Mol Pharmacol 62:1–6, 2002 Printed in U.S.A. 1 at A PE T Jornals on Jne 5, 2017 m oharm .aspeurnals.org D ow nladed from 2001; Ito et al., 2001; Kerb et al., 2001; Kim et al., 2001; Schaeffeler et al., 2001). This finding has stimulated interest in whether common coding polymorphisms affect function of P-gp and/or whether polymorphic variants are linked to altered drug pharmacokinetics. In this study, we examined the five most common P-gp coding polymorphisms previously reported in the literature (N21D, F103L, S400N, A893S, and A998T). We show, using a transient vaccinia expression system to avoid bias resulting from selecting for P-gp expression and optimizing this system to allow for semiquantitative interpretation of results, that none of the common coding polymorphisms alter cell surface localization or transport function of P-gp, as measured using monoclonal antibodies to P-gp and six diverse fluorescent substrates. Materials and Methods Cell Line, Cell Culture, and Propagation of the Vaccinia Virus. HeLa cells (cervical epidermoid carcinoma) were maintained as described previously (Ramachandra et al., 1998). Recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase (vTF7-3) was propagated in HeLa cells and purified as described previously (Earl et al., 1991). Titration of the virus was performed on HeLa infected/transfected cells using pTM1-MDR1 vector (Hrycyna et al.,