Molecular mechanism of antifolate transport-deficiency in a methotrexate-resistant MOLT-3 human leukemia cell line.
Ohnuma et al reported a series of methotrexate-resistant MOLT-3 human T-cell acute lymphoblastic leukemia cell lines that showed decreasing methotrexate (MTX) uptake as the sublines acquired increasing MTX resistance (Cancer Res 45:1815, 1985). The alteration of MTX uptake kinetics in these cells, the intermediately resistant MOLT-3/MTX200 and the highly resistant MOLT-3/MTX10,000 cell lines, was attributed to a change in Vmax for methotrexate transport, without an apparent change in affinity of the transporter for MTX. We studied these cell lines to determine whether alteration of transcription or translation of the recently isolated reduced folate carrier gene (RFC1) was the cause of MTX transport deficiency in these cell lines. Reconstitution of RFC activity in MOLT-3/MTX10,000 cells by transduction with a murine RFC retroviral vector reversed MTX resistance and trimetrexate sensitivity. Although RFC1 RNA levels were unchanged in the resistant cell lines, FACS analysis using a polyclonal anti-RFC1 antibody showed no detectable RFC1 protein in the MOLT-3/MTX10,000 cells. Determination of the nucleotide sequence of RFC1 genes from MOLT-3/MTX10,000 cells revealed that this cell line contained 3 RFC1 alleles: a wild-type allele, an allele containing the premature stop codon at codon 40 and a third allele containing another mutation, which resulted in a premature stop codon at codon 25. We examined the relative expression of these alleles by determining the nucleotide sequence of 24 RFC1 cDNA subclones from MOLT-3/MTX10,000 cells and found that only one-third of these clones contained the wild-type sequence. Determination of the genomic sequence of RFC1 in MOLT-3/ MTX200 cells demonstrated that these cells were heterozygous for a mutation at codon 40, but were homozygous for the wild-type sequence at codon 25. Thus, the acquisition of MTX transport-deficiency in MOLT-3/MTX10,000 cells results from inactivating mutations of RFC1 gene alleles.
Gong
M
M
Medicine and Pediatric Branches, National Cancer Institute, Bethesda, MD 20892,USA.
Yess
J
J
Connolly
T
T
Ivy
S P
SP
Ohnuma
T
T
Cowan
K H
KH
Moscow
J A
JA
D000483:Alleles; D000964:Antimetabolites, Antineoplastic / Q000378:metabolism / Q000494:pharmacology*; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics* / Q000502:physiology; D003062:Codon / Q000235:genetics; D019008:Drug Resistance, Neoplasm; D015973:Gene Expression Regulation, Leukemic; D006801:Humans; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000235:genetics / Q000473:pathology*; D008565:Membrane Proteins / Q000235:genetics* / Q000502:physiology; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000378:metabolism / Q000494:pharmacology*; D009363:Neoplasm Proteins / Q000172:deficiency* / Q000235:genetics / Q000502:physiology; D013728:Terminator Regions, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000964:Antimetabolites, Antineoplastic; D002352:Carrier Proteins; D003062:Codon; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009363:Neoplasm Proteins; C120043:SLC19A2 protein, human; D008727:Methotrexate