ABCMdb

PMID: 15598974

Ejendal KF, Hrycyna CA
Differential sensitivities of the human ATP-binding cassette transporters ABCG2 and P-glycoprotein to cyclosporin A.
Mol Pharmacol. 2005 Mar;67(3):902-11. Epub 2004 Dec 14., [PubMed]

Sentences

No. Mutations Sentence Comment

31 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly A selection of substrates transported by wild-type ABCG2 or the R482G or R482T variants, include the fluorescent dye rhodamine 123, the anthracenes bisantrene and mitoxantrone, the anthracyclines doxorubicin and daunorubicin, and the camptothecins topotecan and SN-38 (Litman et al., 2001; Gottesman et al., 2002). Login to comment 85 ABCG2 p.Arg482Thr Microsomal membranes were isolated from transiently cotransfected-infected HeLa cells, drug-sensitive MCF-7 cells, drug-resistant MCF-7/AdVp3000, which overexpress the R482T variant of human ABCG2, and drug resistant MCF-7/DX1 cells, which overexpress human P-gp. Login to comment 97 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Our data show that ABCG2 is localized at the surface in both HeLa cells transfected with any of the three ABCG2 variants (R482G, R482, or R482T) and in MCF-7/AdVp3000 cells (Fig. 2, A and B). Login to comment 98 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The R482 (wild type), R482G, and R482T ABCG2 variants are expressed equivalently in HeLa cells (Fig. 2A). Login to comment 113 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Rhodamine 123 is a well established substrate of the R482G and R482T variants of ABCG2 (Honjo et al., 2001) and of P-gp (Hrycyna et al., 1998), but it is not a substrate of wild-type R482 variant of ABCG2 (Honjo et al., 2001). Login to comment 117 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly As can be seen in Fig. 3A, cells expressing ABCG2 (R482G or R482T) or P-gp exclude rhodamine 123 equivalently in the absence of inhibitor. Login to comment 123 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Transport of mitoxantrone by the R482T ABCG2 variant seems to be the least sensitive to addition of 5 ␮M cyclosporin A, followed by the R482G and the wild-type R482 ABCG2 proteins. Login to comment 130 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly ABCG2 p.Arg482Gly A, transiently transfected HeLa cells expressing ABCG2 labeled with 5D3; R482G (-), R482 (⅐ ⅐ ⅐), R482T (-), R482G cells labeled with IgG2b (- ⅐ - ⅐ -). Login to comment 143 ABCG2 p.Arg482Gly In contrast, it has been reported that 5 ␮M cyclosporin A increased the accumulation of 99m Tc-tetrofosmin 1.8-fold and 99m Tc-sestamibi 1.3-fold in S1-M1/S1-M1-80 cells that express the R482G variant of ABCG2 (Chen et al., 2000b). Login to comment 156 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly A to F, histograms show HeLa cells transfected with the empty pTM1 vector (shaded peak), R482G (-), R482 (- ⅐ - ⅐ -), R482T (- - -), or P-gp (-). Login to comment 168 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly These experiments show that cells expressing ABCG2 (R482T or R482G) or P-gp extrude daunomycin in the absence of the inhibitor and that this transport is inhibited in the presence of GF120918. Login to comment 170 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Together, our data demonstrate that cyclosporin A is only transported by P-gp, whereas daunomycin is transported by P-gp and the R482G and R482T variants of ABCG2 but not the wild-type ABCG2 (R482). Login to comment 175 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly To assess whether different variants of ABCG2 can be labeled with [125 I]IAAP, we performed the experiment with membrane isolates from S1-M1-80, MCF-7/FLV1000, and MCF-7/AdVp3000 cells that express the R482G, R482, and R482T variants of ABCG2, respectively. Login to comment 197 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The ATPase activity of the R482G and R482T variants of ABCG2 are stimulated 2.2- and 1.4-fold, respectively, by the addition of 40 ␮M prazosin (Fig. 6, A and C). Login to comment 201 ABCG2 p.Arg482Thr A, 50 ␮g of membrane protein from MCF-7 or MCF-7/AdVp3000 cells expressing the R482T variant of ABCG2. Login to comment 203 ABCG2 p.Arg482Gly C, 60 ␮g of membrane protein from S1 or S1-M1-80 expressing the R482G variant of ABCG2. Login to comment 211 ABCG2 p.Arg482Gly A previous study reported that cyclosporin A inhibits both basal and prazosin-stimulated ATPase activity of ABCG2 (R482G variant) expressed in Sf9 insect cells (O¨ zvegy et al., 2001). Login to comment 224 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The assays were performed both in the absence (᭜) and presence (E) of 40 ␮M prazosin (for ABCG2) or 30 ␮M verapamil (for P-gp), at increasing concentrations of cyclosporin A. ATPase activity of ABCG2 proteins transiently expressed in HeLa cells R482G (A), R482 (B), and R482T (C), and in MCF-7/AdVp3000 cells (D). Login to comment 228 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly In this study, we used both the drug-resistant cell lines MCF-7/AdVp3000 that overexpress ABCG2 with the R482T gain of function mutation (Miyake et al., 1999) and MCF-7/ DX1 that overexpress P-gp, as well as HeLa cells transiently expressing ABCG2 (R482G, R482, or R482T variants) or P-gp to explore, in detail, the molecular effects that the immunomodulator cyclosporin A has on these transporters. Login to comment 233 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly In addition, we observe subtle differences in the transport of mitoxantrone in the absence and presence of cyclosporin A between the three ABCG2 variants (R482G, Arg482, and R482T) (Fig. 3, D-F), corroborating previous data suggesting amino acid 482 is an important determinant of substrate specificity. Login to comment 254 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly In conclusion, our findings demonstrate that the immunomodulator cyclosporin A is a substrate and an inhibitor of human P-glycoprotein, but that under the conditions examined, it is neither a substrate nor an inhibitor of any of the variants (R482G, R482, and R482T) of the related ABC transporter ABCG2. Login to comment