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Role of the E45K-reduced folate carrier gene mutation in methotrexate resistance in human leukemia cells

Leukemia volume 16, pages 2379–2387 (2002)Cite this article

Abstract

Resistance to the antifolate methotrexate (MTX) can cause treatment failure in childhood acute lymphoblastic leukemia (ALL). This may result from defective MTX accumulation due to alterations in the human reduced folate carrier (hRFC) gene. We have identified an hRFC gene point mutation in a transport-defective CCRF-CEM human T-ALL cell line resulting in a lysine to glutamic acid substitution at codon 45 (E45K), which has been identified in other antifolate-resistant sublines (JBC 273:30 189, 1998; JBC 275:30 855, 2000). To characterize the role of this mutation in MTX resistance, transfection experiments were performed using hRFC-null CCRF-CEM cells. E45K transfectants demonstrated an initial rate of MTX influx that was approximately 0.5-fold that of CCRF-CEM cells, despite marked protein overexpression. Cytotoxicity studies revealed partial reversal of MTX and raltitrexed resistance in E45K transfectants, while trimetrexate resistance was significantly increased. Kinetic analysis indicated only minor differences in MTX kinetics between wild-type and E45K hRFCs, however, Kis for folic acid and 5-formyltetrahydrofolate were markedly reduced for E45K hRFC. This was paralleled by increased folic acid transport and reduced synthesis of MTX polyglutamates. Collectively, the results demonstrate that expression of E45K hRFC leads to increased MTX resistance due to decreased membrane transport and, secondarily, from alterations in binding affinities and transport of folate substrates. However, despite these findings, we could find no evidence of this mutation in 121 childhood ALL samples, suggesting that it does not contribute to clinical MTX resistance in this disease.

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Acknowledgements

The Children's Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and Sydney Children's Hospital. This work was supported by grants from the National Health and Medical Research Council (Australia) (MDN, MH), the New South Wales Cancer Council (Australia) (MDN, MH), and grants CA53535 and CA77641 from the National Cancer Institute, National Institutes of Health (LHM, JWT). Mr. Gifford is the recipient of an Australian Postgraduate Award, and an International Cancer Technology Transfer Fellowship (ICRETT) from the International Union Against Cancer.

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  1. Children's Cancer Institute Australia for Medical Research, Sydney, Australia

    AJ Gifford, M Haber & MD Norris

  2. Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USA

    TL Witt, JR Whetstine, JW Taub & LH Matherly

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Gifford, A., Haber, M., Witt, T. et al. Role of the E45K-reduced folate carrier gene mutation in methotrexate resistance in human leukemia cells. Leukemia 16, 2379–2387 (2002). https://doi.org/10.1038/sj.leu.2402655

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