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 approximately 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 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 --> 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.
Drori
S
S
Department of Biology, The Technion, Haifa 32000, Israel.
Jansen
G
G
Mauritz
R
R
Peters
G J
GJ
Assaraf
Y G
YG
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001692:Biological Transport; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics*; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000494:pharmacology; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005091:Exons; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D054833:Isoindoles; D007700:Kinetics; D002955:Leucovorin / Q000494:pharmacology; D007938:Leukemia / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000737:chemistry / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018807:Polymorphism, Single-Stranded Conformational; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011799:Quinazolines / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D058978:Reduced Folate Carrier Protein; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors; D013997:Time Factors; D014162:Transfection; D016597:Trimetrexate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D002352:Carrier Proteins; D002712:Chlorides; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; D007211:Indoles; D054833:Isoindoles; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011799:Quinazolines; D011994:Recombinant Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; C120043:SLC19A2 protein, human; C079601:1843U89; C010920:dihydrofolate; D005492:Folic Acid; D013940:Thymidylate Synthase; C099178:nolatrexed; D002955:Leucovorin; D016597:Trimetrexate; D008727:Methotrexate