Role of the E45K-reduced folate carrier gene mutation in methotrexate resistance in human leukemia cells.
Resistance to the antifolate methotrexate (MTX) can cause treatment failure in childhood acute lymphoblastic leukemia (ALL). This may result from defective MTX accumulation due to alterations in the human reduced folate carrier (hRFC) gene. We have identified an hRFC gene point mutation in a transport-defective CCRF-CEM human T-ALL cell line resulting in a lysine to glutamic acid substitution at codon 45 (E45K), which has been identified in other antifolate-resistant sublines (JBC 273:30 189, 1998; JBC 275:30 855, 2000). To characterize the role of this mutation in MTX resistance, transfection experiments were performed using hRFC-null CCRF-CEM cells. E45K transfectants demonstrated an initial rate of MTX influx that was approximately 0.5-fold that of CCRF-CEM cells, despite marked protein overexpression. Cytotoxicity studies revealed partial reversal of MTX and raltitrexed resistance in E45K transfectants, while trimetrexate resistance was significantly increased. Kinetic analysis indicated only minor differences in MTX kinetics between wild-type and E45K hRFCs, however, K(i)s for folic acid and 5-formyltetrahydrofolate were markedly reduced for E45K hRFC. This was paralleled by increased folic acid transport and reduced synthesis of MTX polyglutamates. Collectively, the results demonstrate that expression of E45K hRFC leads to increased MTX resistance due to decreased membrane transport and, secondarily, from alterations in binding affinities and transport of folate substrates. However, despite these findings, we could find no evidence of this mutation in 121 childhood ALL samples, suggesting that it does not contribute to clinical MTX resistance in this disease.
Gifford
A J
AJ
Children's Cancer Institute Australia for Medical Research, Sydney, Australia.
Haber
M
M
Witt
T L
TL
Whetstine
J R
JR
Taub
J W
JW
Matherly
L H
LH
Norris
M D
MD
D019943:Amino Acid Substitution; D000964:Antimetabolites, Antineoplastic / Q000493:pharmacokinetics*; D001854:Bone Marrow Cells / Q000473:pathology; D002352:Carrier Proteins / Q000235:genetics* / Q000502:physiology; D002648:Child; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005492:Folic Acid / Q000493:pharmacokinetics; D006801:Humans; D007700:Kinetics; D007938:Leukemia / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000493:pharmacokinetics*; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D058978:Reduced Folate Carrier Protein; D014162:Transfection; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D005492:Folic Acid; D008727:Methotrexate