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Originally published In Press as doi:10.1074/jbc.M003988200 on July 17, 2000

J. Biol. Chem., Vol. 275, Issue 40, 30855-30863, October 6, 2000
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Clustering of Mutations in the First Transmembrane Domain of the Human Reduced Folate Carrier in GW1843U89-resistant Leukemia Cells with Impaired Antifolate Transport and Augmented Folate Uptake*

Stavit DroriDagger §, Gerrit Jansen, Robert Mauritz, Godefridus J. Peters, and Yehuda G. AssarafDagger ||

From the Dagger  Department of Biology, The Technion, Haifa 32000, Israel and the  Department of Oncology, University Hospital Vrije Universteit, 1081 HV Amsterdam, The Netherlands

We have studied the molecular basis for the resistance of human CEM leukemia cells to GW1843, a thymidylate synthase inhibitor. GW1843-resistant cells displayed a ~100-fold resistance to GW1843 and methotrexate but were collaterally sensitive to the lipophilic antifolates trimetrexate and AG337, which enter cells by diffusion. These cells exhibited a 12-fold decreased methotrexate influx but surprisingly had a 2-fold decreased folic acid growth requirement. This was associated with a 4-fold increased influx of folic acid, a 3.5-fold increased steady-state level of folic acid, and a 2.3-fold expansion of the cellular folate pool. Characterization of the transport kinetic properties revealed that GW1843-resistant cells had the following alterations: (a) 11-fold decreased transport Km for folic acid; (b) 6-fold increased transport Km for GW1843; and (c) a slightly increased transport Vmax for folic acid. Sequence analysis showed that GW1843-resistant cells contained the mutations Val-29 right-arrow Leu, Glu-45 right-arrow Lys, and Ser-46 right-arrow Ile in the first transmembrane domain of the reduced folate carrier. Transfection of the mutant-reduced folate carrier cDNA into methotrexate transport null cells conferred resistance to GW1843. This is the first demonstration of multiple mutations in a confined region of the human reduced folate carrier in an antifolate-resistant mutant. We conclude that certain amino acid residues in the first transmembrane domain play a key role in (anti)folate binding and in the conferring of drug resistance.

* This study was supported by research grants from the Zimmer Cancer Research Fund (to Y. G. A.), the Hedson Fund for Medical Research (to Y. G. A.), and by the Dutch Cancer Society Grant NKB-VU-96-1260 (to G. J).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of a Young Investigator Exchange Fellowship of the EORTC-Pharmacology and Molecular Mechanisms Group.

|| To whom correspondence should be addressed: Dept. of Biology, the Technion-Israel Institute of Technology, Haifa 32 000, Israel. Tel.: 972-4-8293744; Fax: 972-4-8225153; E-mail: assaraf@tx.technion.ac.il.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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