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 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, whi...

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Bibliographic Details
Published in:	The Journal of biological chemistry 2000-10, Vol.275 (40), p.30855-30863
Main Authors:	Drori, Stavit, Jansen, Gerrit, Mauritz, Robert, Peters, Godefridus J., Assaraf, Yehuda G.
Format:	Article
Language:	eng
Subjects:	Antimetabolites, Antineoplastic - pharmacology Biological Transport Blotting, Northern Blotting, Southern Blotting, Western Carrier Proteins - chemistry Carrier Proteins - genetics Cell Division - drug effects Cell Membrane - metabolism Chlorides - pharmacology DNA Mutational Analysis DNA, Complementary - metabolism Dose-Response Relationship, Drug drug resistance Drug Resistance, Neoplasm - genetics Enzyme Inhibitors - pharmacology Exons folate carrier folic acid Folic Acid - analogs & derivatives Folic Acid - chemistry Folic Acid - metabolism Folic Acid - pharmacokinetics Folic Acid - pharmacology Folic Acid Antagonists - metabolism Folic Acid Antagonists - pharmacology GW1843 Humans Indoles - chemistry Indoles - pharmacology Inhibitory Concentration 50 Isoindoles Kinetics Leucovorin - pharmacology Leukemia - genetics Leukemia - metabolism Membrane Proteins Membrane Transport Proteins Methotrexate - chemistry Methotrexate - pharmacology methotrexate transport Mutagenesis, Site-Directed Mutation Polymorphism, Single-Stranded Conformational Protein Structure, Secondary Protein Structure, Tertiary Quinazolines - chemistry Quinazolines - pharmacology Recombinant Proteins - chemistry Recombinant Proteins - metabolism Reduced Folate Carrier Protein Thymidylate Synthase - antagonists & inhibitors Time Factors Transfection Trimetrexate - pharmacology Tumor Cells, Cultured
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Summary:	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 transportVmax 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. 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.
ISSN:	0021-9258 1083-351X