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Mutant Gly482 and Thr482 ABCG2 mediate high-level resistance to lipophilic antifolates

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Abstract

Cellular uptake of hydrophilic antifolates proceeds via the reduced folate carrier whereas lipophilic antifolates enter cells by diffusion. Recently we have shown that transfectant cells overexpressing the mutant G482 ABCG2 displayed 120–6,250-fold resistance to hydrophilic antifolates than untransfected cells upon 4 h drug exposure, but lost almost all their antifolate resistance upon 72 h drug exposure (Shafran et al. in Cancer Res 65:8414–8422, 2005). Here we explored the ability of the wild type (WT) R482—as well as the mutant G482—and T482 ABCG2 to confer resistance to lipophilic antifolate inhibitors of dihydrofolate reductase (trimetrexate, piritrexim, metoprine and pyrimethamine) and thymidylate synthase (AG337, AG377 and AG331). Lipophilic antifolate resistance was determined using growth inhibition assays upon 72 h drug exposure. Cells overexpressing these mutant efflux transporters displayed up to 106-fold resistance to lipophilic antifolates relative to untransfected cells; this resistance was reversed by the specific and potent ABCG2 efflux inhibitor Ko143. In contrast, cells overexpressing the WT R482 ABCG2 exhibited either no or only a low-level of lipophilic antifolate resistance. These results provide the first evidence that overexpression of the mutant G482- and T482 but not the WT R482 ABCG2 confers a high-level of resistance to lipophilic antifolates. The high membrane partitioning of lipophilic antifolates along with the large confinement of ABCG2 to the plasma membrane suggest that these mutant ABCG2 transporters may possibly recognize and extrude lipophilic antifolates from the lipid bilayer. The potential implications to cancer chemotherapy as well as the mechanism of anticancer drug extrusion by these mutant exporters are discussed.

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Abbreviations

ABC:

ATP-binding cassette protein

BCRP:

Breast cancer resistance protein

MRP:

Multidrug resistance protein

Pgp:

P-glycoprotein

DHFR:

Dihydrofolate reductase

TS:

Thymidylate synthase

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Acknowledgments

Grant support: The Fred Wyszkowski Cancer Research Fund, Israel Cancer Association and the Star Foundation (to YGA) as well as the Dutch Arthritis Association (NRF 03-40I to GJ). We thank Dr S.E. Bates (Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD) for the HEK293 transfectants overexpressing the R482-, G482- and T482 ABCG2. We extend our gratitude to Dr G.L. Scheffer and Prof. R.J. Scheper (Department of Pathology, VU University Medical Centre, Amsterdam, The Netherlands) for the BXP-53 monoclonal antibody, Prof. A.H. Schinkel for the kind gift of Ko143 as well as Prof. S.J. Karlish (Department of Biological Chemistry, The Weizmann Institute of Science, Rehovoth, Israel) for the polyclonal antiserum against Na+–K+-ATPase.

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Correspondence to Yehuda G. Assaraf.

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Bram, E., Ifergan, I., Shafran, A. et al. Mutant Gly482 and Thr482 ABCG2 mediate high-level resistance to lipophilic antifolates. Cancer Chemother Pharmacol 58, 826–834 (2006). https://doi.org/10.1007/s00280-006-0230-9

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  • DOI: https://doi.org/10.1007/s00280-006-0230-9

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