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Molecular mechanism of antifolate transport-deficiency in a
methotrexate-resistant MOLT-3 human leukemia cell line
M Gong, J Yess, T Connolly, SP Ivy, T Ohnuma, KH Cowan and JA Moscow
Medicine and Pediatric Branches, National Cancer Institute, Bethesda, MD
20892,USA.
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.
Volume 89,
Issue 7,
pp. 2494-2499,
04/01/1997
Copyright © 1997 by The American Society of Hematology
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