title: A structurally altered human reduced folate carrier with increased folic acid transport mediates a novel mechanism of antifolate resistance.
abstract: 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.
authors.affiliation: Department of Oncology, University Hospital Vrije Universiteit, 1081 HV Amsterdam, The Netherlands. g.jansen@azvu.nl
authors.forename: G
authors.initials: G
authors.lastname: Jansen
authors.forename: R
authors.initials: R
authors.lastname: Mauritz
authors.forename: S
authors.initials: S
authors.lastname: Drori
authors.forename: H
authors.initials: H
authors.lastname: Sprecher
authors.forename: I
authors.initials: I
authors.lastname: Kathmann
authors.forename: M
authors.initials: M
authors.lastname: Bunni
authors.forename: D G
authors.initials: DG
authors.lastname: Priest
authors.forename: P
authors.initials: P
authors.lastname: Noordhuis
authors.forename: J H
authors.initials: JH
authors.lastname: Schornagel
authors.forename: H M
authors.initials: HM
authors.lastname: Pinedo
authors.forename: G J
authors.initials: GJ
authors.lastname: Peters
authors.forename: Y G
authors.initials: YG
authors.lastname: Assaraf
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D001692:Biological Transport; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005492:Folic Acid / Q000378:metabolism*; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D005971:Glutamates / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D007938:Leukemia / Q000378:metabolism; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000378:metabolism; D011739:Pyrimethamine / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D058978:Reduced Folate Carrier Protein; D013329:Structure-Activity Relationship; D013763:Tetrahydrofolates / Q000494:pharmacology; D016597:Trimetrexate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D000345:Affinity Labels; D002352:Carrier Proteins; D005493:Folic Acid Antagonists; D005971:Glutamates; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011743:Pyrimidines; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; C120043:SLC19A2 protein, human; D013763:Tetrahydrofolates; C045894:lometrexol; D005492:Folic Acid; C105066:AG 2034; D016597:Trimetrexate; D008727:Methotrexate; D011739:Pyrimethamine