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Molecular mechanism of antifolate transport-deficiency in a methotrexate-resistant MOLT-3 human leukemia cell line.
- Source :
-
Blood [Blood] 1997 Apr 01; Vol. 89 (7), pp. 2494-9. - Publication Year :
- 1997
-
Abstract
- 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.
- Subjects :
- Alleles
Antimetabolites, Antineoplastic metabolism
Biological Transport
Carrier Proteins physiology
Codon genetics
Drug Resistance, Neoplasm
Gene Expression Regulation, Leukemic
Humans
Leukemia-Lymphoma, Adult T-Cell genetics
Membrane Proteins physiology
Methotrexate metabolism
Neoplasm Proteins genetics
Neoplasm Proteins physiology
Terminator Regions, Genetic
Transfection
Tumor Cells, Cultured drug effects
Antimetabolites, Antineoplastic pharmacology
Carrier Proteins genetics
Leukemia-Lymphoma, Adult T-Cell pathology
Membrane Proteins genetics
Membrane Transport Proteins
Methotrexate pharmacology
Neoplasm Proteins deficiency
Subjects
Details
- Language :
- English
- ISSN :
- 0006-4971
- Volume :
- 89
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- Blood
- Publication Type :
- Academic Journal
- Accession number :
- 9116294