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

S Drori, G Jansen, R Mauritz, G J Peters, Y G Assaraf

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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.

Original languageEnglish
Pages (from-to)30855-63
Number of pages9
JournalJournal of Biological Chemistry
Issue number40
Publication statusPublished - 6 Oct 2000


  • Antimetabolites, Antineoplastic/pharmacology
  • Biological Transport
  • Blotting, Northern
  • Blotting, Southern
  • Blotting, Western
  • Carrier Proteins/chemistry
  • Cell Division/drug effects
  • Cell Membrane/metabolism
  • Chlorides/pharmacology
  • DNA Mutational Analysis
  • DNA, Complementary/metabolism
  • Dose-Response Relationship, Drug
  • Drug Resistance, Neoplasm/genetics
  • Enzyme Inhibitors/pharmacology
  • Exons
  • Folic Acid Antagonists/metabolism
  • Folic Acid/analogs & derivatives
  • Humans
  • Indoles/chemistry
  • Inhibitory Concentration 50
  • Isoindoles
  • Kinetics
  • Leucovorin/pharmacology
  • Leukemia/genetics
  • Membrane Proteins
  • Membrane Transport Proteins
  • Methotrexate/chemistry
  • Mutagenesis, Site-Directed
  • Mutation
  • Polymorphism, Single-Stranded Conformational
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Quinazolines/chemistry
  • Recombinant Proteins/chemistry
  • Reduced Folate Carrier Protein
  • Thymidylate Synthase/antagonists & inhibitors
  • Time Factors
  • Transfection
  • Trimetrexate/pharmacology
  • Tumor Cells, Cultured

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