title: ABCG2 harboring the Gly482 mutation confers high-level resistance to various hydrophilic antifolates.
abstract: ABCG2 is an ATP-binding cassette transporter that confers resistance to various chemotherapeutic agents. Recent studies have established that an Arg (wild-type) to Gly mutation at amino acid 482 in ABCG2 alters substrate specificity. Here, we explored the role of this G482 mutation in antifolate resistance using a clinically relevant 4-hour drug exposure. Stable transfectants overexpressing the mutant G482 transporter displayed 120-, 1,000-, and >6,250-fold resistance to the antifolates methotrexate, GW1843, and Tomudex, respectively, relative to parental human embryonic kidney cells. Moreover, although overexpressing equal transporter levels at the plasma membrane, G482-ABCG2 cells were 6-, 23-, and >521-fold more resistant to methotrexate, GW1843, and Tomudex, respectively, than R482-ABCG2 cells. In contrast, upon a continuous (72-hour) drug exposure, both the G482- and R482-ABCG2 cells lost almost all their antifolate resistance; this result was consistent with the inability of ABCG2 to extrude long-chain antifolate polyglutamates. Ko143, a specific and potent ABCG2 inhibitor reversed methotrexate resistance in both G482- and R482-ABCG2 cells. Consistently, whereas the pool of free methotrexate in parental human embryonic kidney cells was prominent after 4 hours of transport with 1 micromol/L [3H]methotrexate, in R482- and G482-ABCG2 cells, it was minimal. Furthermore, G482-ABCG2 cells contained marked decreases in the di- and triglutamate species of [3H]methotrexate at 4 hours of incubation with methotrexate and in the tetra- and pentaglutamates at 24 hours. These changes were not associated with any significant decrease in folylypoly-gamma-glutamate synthetase activity. These results provide the first evidence that the G482-ABCG2 mutation confers high-level resistance to various hydrophilic antifolates.
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Assaf
authors.initials: A
authors.lastname: Shafran
authors.forename: Ilan
authors.initials: I
authors.lastname: Ifergan
authors.forename: Eran
authors.initials: E
authors.lastname: Bram
authors.forename: Gerrit
authors.initials: G
authors.lastname: Jansen
authors.forename: Ietje
authors.initials: I
authors.lastname: Kathmann
authors.forename: Godefridus J
authors.initials: GJ
authors.lastname: Peters
authors.forename: Robert W
authors.initials: RW
authors.lastname: Robey
authors.forename: Susan E
authors.initials: SE
authors.lastname: Bates
authors.forename: Yehuda G
authors.initials: YG
authors.lastname: Assaraf
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D005493:Folic Acid Antagonists / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D008727:Methotrexate / Q000031:analogs & derivatives / Q000493:pharmacokinetics / Q000494:pharmacology; D009154:Mutation; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000493:pharmacokinetics / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D014316:Tritium
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D005493:Folic Acid Antagonists; D009363:Neoplasm Proteins; D014316:Tritium; D011099:Polyglutamic Acid; D004317:Doxorubicin; C014085:methotrexate polyglutamate; D008727:Methotrexate