ABCG2 harboring the Gly482 mutation confers high-level resistance to various hydrophilic antifolates

Assaf Shafran, Ilan Ifergan, Eran Bram, Gerrit Jansen, Ietje Kathmann, Godefridus J Peters, Robert W Robey, Susan E Bates, Yehuda G Assaraf

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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.

Original languageEnglish
Pages (from-to)8414-8422
Number of pages9
JournalCancer research
Issue number18
Publication statusPublished - 15 Sept 2005


  • ATP Binding Cassette Transporter, Subfamily G, Member 2
  • ATP-Binding Cassette Transporters/biosynthesis
  • Cell Line
  • Cell Membrane/metabolism
  • Doxorubicin/pharmacology
  • Drug Resistance, Neoplasm
  • Folic Acid Antagonists/pharmacokinetics
  • Humans
  • Methotrexate/analogs & derivatives
  • Mutation
  • Neoplasm Proteins/biosynthesis
  • Polyglutamic Acid/analogs & derivatives
  • Subcellular Fractions/metabolism
  • Transfection
  • Tritium

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