ABCMdb

PMID: 12874005

Chen ZS, Robey RW, Belinsky MG, Shchaveleva I, Ren XQ, Sugimoto Y, Ross DD, Bates SE, Kruh GD
Transport of methotrexate, methotrexate polyglutamates, and 17beta-estradiol 17-(beta-D-glucuronide) by ABCG2: effects of acquired mutations at R482 on methotrexate transport.
Cancer Res. 2003 Jul 15;63(14):4048-54., 2003-07-15 [PubMed]

Sentences

No. Mutations Sentence Comment

12 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly By contrast with the wild-type protein (ABCG2-R482), two ABCG2 variants that have been identified in drug selected cell lines, R482T and R482G, were unable to transport MTX to any extent. Login to comment 25 ABCG2 p.Arg482Thr However, a puzzling feature of this study was that MCF7 cells stably transfected with an ABCG2 cDNA, which has since been determined to have an R482T mutation, did not recapitulate MTX resistance. Login to comment 29 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The results of these experiments show that wild-type ABCG2 is indeed capable of mediating the transport of MTX, but that neither the R482G nor the R482T mutants are able to transport this agent to any extent. Login to comment 49 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly HEK293 cells transfected with ABCG2-R482, ABCG2-R482G, and ABCG2-R482T were generated using the respective cDNAs cloned into pcDNA3.4 LLC/PK1 cells transfected with MRP2 expression vector, and HEK293 cells transfected with MRP3 expression vector were described previously (19, 20). Login to comment 61 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly RESULTS Analysis of MTX Transport by Wild-Type ABCG2, ABCG2R482G, and ABCG2-R482T. Login to comment 68 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Membrane vesicles were prepared from HEK293 cells transfected with ABCG2-R482G and ABCG2-R482T, so as to analyze both types of R482 mutations that have been identified in drug-resistant cell lines (4). Login to comment 70 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly However, by contrast with ABCG2-R482, neither of the mutants were able to transport [3 H]MTX to any extent, in that MgATP-dependent uptake of this agent by ABCG2-R482G and ABCG2-R482T-enriched vesicles was indistinguishable from uptake by the same vesicles in the presence of MgAMP, or uptake by negative control vesicles in the presence of either MgATP or MgAMP (Fig. 2, B and C). Login to comment 82 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Membrane vesicles were prepared from HEK293 cells transfected with parental vector (Lane 1), ABCG2-R482 (Lane 2), ABCG2-R482G (Lane 3), and ABCG2-R482T (Lane 4). Login to comment 99 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly We reported previously that several members of the MRP family that are capable of transporting MTX are also competent in mediating the Fig. 2. Time course of ATP-dependent uptake of [3 H]MTX by ABCG2-R482, ABCG2-R482G, and ABCG2-R482T. Login to comment 100 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Membrane vesicles (10 ␮g) prepared from HEK293 cells transfected with ABCG2-R482 (A), ABCG2-R482G (B), and ABCG2-R482T (C), and from parental plasmid-transfected control cells, were incubated at 37°C in uptake medium containing 100 ␮M [3 H]MTX and 4 mM MgATP (solid symbols) or 4 mM MgAMP (open symbols). Login to comment 107 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The results of experiments in which uptake of FA was examined were in complete accord with the properties of ABCG2 as determined from the MTX transport experiments, in that whereas membrane vesicles prepared from wild-type ABCG2-transfected cells were able to catalyze the uptake of 100 ␮M [3 H]FA, uptake was not detected for R482G or R482T (Fig. 6, A-C). Login to comment 143 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly Membrane vesicles (10 ␮g) prepared from HEK293 cells transfected with ABCG2-R482 (A and D), ABCG2-R482G (B), ABCG2-R482T (C), and MRP3 (E), from LLC/PK1 cells transfected with MRP2 (F), and from the respective parental plasmid-transfected counterparts, were incubated at 37°C in uptake medium containing 100 ␮M [3 H]FA (A-C) or 100 ␮M [3 H]leucovorin (D-F), and 4 mM MgATP (solid symbols) or 4 mM MgAMP (open symbols). Login to comment