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

We have studied the molecular basis for the resistance of human CEM leukemi a cells to GW1843, a thymidylate synthase inhibitor. GW1843-resistant cells displayed a similar to 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 decr eased methotrexate influx but surprisingly had a a-fold decreased folic aci d growth requirement. This was associated with a I-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 tran sport kinetic properties revealed that GW1843 resistant cells had the follo wing 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 tran sport V-max for folic acid. Sequence analysis showed that GW1843-resistant cells contained the mutations Val-29 --> Leu, Glu-45 --> Lys, and Ser-46 -- > Ile in the first transmembrane domain of the reduced folate carrier, Tran sfection of the mutant-reduced folate carrier cDNA into methotrexate transp ort null cells conferred resistance to GW1843, This is the first demonstrat ion of multiple mutations in a confined region of the human reduced folate carrier in an antifolate-resistant mutant. We conclude that certain amino a cid residues in the first transmembrane domain play a key role in (anti)fol ate binding and in the conferring of drug resistance.