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Molecular mechanism of antifolate transport-deficiency in a methotrexate-resistant MOLT-3 human leukemia cell line
1Medicine and Pediatric Branches, National Cancer Institute, Bethesda, MD 20892,USA.
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.
Insights
Methotrexate resistance in leukemia cells is linked to mutations in the reduced folate carrier 1 (RFC1) gene, affecting methotrexate transport. These RFC1 gene mutations inactivate the transporter, causing drug resistance.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- Methotrexate (MTX) resistance in cancer cells can arise from altered drug uptake.
- Previous studies indicated changes in MTX transport kinetics in resistant cell lines.
- The reduced folate carrier 1 (RFC1) gene is crucial for MTX transport.
Purpose of the Study:
- To investigate if alterations in RFC1 gene transcription or translation cause MTX transport deficiency.
- To determine the role of RFC1 gene mutations in MTX resistance in human T-cell leukemia cell lines.
Main Methods:
- Studied methotrexate-resistant MOLT-3 cell lines (MOLT-3/MTX200 and MOLT-3/MTX10,000).
- Analyzed RFC1 gene transcription and protein expression using RNA and FACS analysis.
- Determined RFC1 gene nucleotide and genomic sequences to identify mutations.
- Reconstituted RFC activity via retroviral vector transduction.
Main Results:
- RFC1 RNA levels were unchanged in resistant cell lines.
- No detectable RFC1 protein was found in highly resistant MOLT-3/MTX10,000 cells.
- MOLT-3/MTX10,000 cells possessed three RFC1 alleles: wild-type, premature stop codon at codon 40, and premature stop codon at codon 25.
- Only one-third of RFC1 cDNA subclones from MOLT-3/MTX10,000 cells contained the wild-type sequence.
- MOLT-3/MTX200 cells were heterozygous for a codon 40 mutation and homozygous for wild-type at codon 25.
- Reconstitution of RFC activity reversed MTX resistance.
Conclusions:
- Acquisition of MTX transport deficiency in MOLT-3/MTX10,000 cells results from inactivating mutations in RFC1 gene alleles.
- Mutations leading to premature stop codons in RFC1 are responsible for MTX resistance.
- RFC1 gene mutations play a critical role in methotrexate resistance mechanisms.