A structurally altered human reduced folate carrier with increased folic acid transport mediates a novel mechanism of antifolate resistance

G Jansen, R Mauritz, S Drori, H Sprecher, I Kathmann, M Bunni, D G Priest, P Noordhuis, J H Schornagel, H M Pinedo, G J Peters, Y G Assaraf

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CEM/MTX is a subline of human CCRF-CEM leukemia cells which displays >200-fold resistance to methotrexate (MTX) due to defective transport via the reduced folate carrier (RFC). CEM/MTX-low folate (LF) cells, derived by a gradual deprivation of folic acid from 2.3 microM to 2 nM (LF) in the cell culture medium of CEM/MTX cells, resulted in a >20-fold overexpression of a structurally altered RFC featuring; 1) a wild type Km value for MTX transport but a 31-fold and 9-fold lower Km values for folic acid and leucovorin, respectively, relative to wild type RFC; 2) a 10-fold RFC1 gene amplification along with a >20-fold increased expression of the main 3.1-kilobase RFC1 mRNA; 3) a marked stimulation of MTX transport by anions (i.e. chloride); and 4) a G --> A mutation at nucleotide 227 of the RFC cDNA in both CEM/MTX-LF and CEM/MTX, resulting in a lysine for glutamate substitution at amino acid residue 45 predicted to reside within the first transmembrane domain of the human RFC. Upon transfer of CEM/MTX-LF cells to folate-replete medium (2.3 microM folic acid), the more efficient folic acid uptake in CEM/MTX-LF cells resulted in a 7- and 24-fold elevated total folate pool compared with CEM and CEM/MTX cells, respectively (500 versus 69 and 21 pmol/mg of protein, respectively). This markedly elevated intracellular folate pool conferred a novel mechanism of resistance to polyglutamatable (e.g. ZD1694, DDATHF, and AG2034) and lipophilic antifolates (e.g. trimetrexate and pyrimethamine) by abolishing their polyglutamylation and circumventing target enzyme inhibition.

Original languageEnglish
Pages (from-to)30189-98
Number of pages10
JournalJournal of Biological Chemistry
Issue number46
Publication statusPublished - 13 Nov 1998


  • Affinity Labels/metabolism
  • Biological Transport
  • Blotting, Northern
  • Carrier Proteins/chemistry
  • Drug Resistance, Neoplasm/genetics
  • Folic Acid Antagonists/pharmacology
  • Folic Acid/metabolism
  • Glutamates/pharmacology
  • Humans
  • Kinetics
  • Leukemia/metabolism
  • Membrane Proteins
  • Membrane Transport Proteins
  • Methotrexate/metabolism
  • Pyrimethamine/pharmacology
  • Pyrimidines/pharmacology
  • Reduced Folate Carrier Protein
  • Structure-Activity Relationship
  • Tetrahydrofolates/pharmacology
  • Trimetrexate/pharmacology
  • Tumor Cells, Cultured

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