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
Journal of Biological Chemistry
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 ϳ100-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
... d with a 4-fold increased influx of folic acid, a 3.5-fold increased steadystate 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 K m for folic acid; (b) 6-fold increased transport K m for GW1843; and (c) a slightly increased transport V max for folic acid. Sequence analysis showed that GW1843-resistant cells contained the mutations Val-29 3 Leu, Glu-45 3 Lys, and Ser-46 3 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.