Molecular mechanism of antifolate transport-deficiency in a methotrexate-resistant MOLT-3 human leukemia cell line

被引:53
作者
Gong, MK
Yess, J
Connolly, T
Ivy, SP
Ohnuma, T
Cowan, KH
Moscow, JA
机构
[1] NCI,MED BRANCH,NIH,BETHESDA,MD 20892
[2] NCI,PEDIAT BRANCH,BETHESDA,MD 20892
[3] CHILDRENS NATL MED CTR,WASHINGTON,DC 20010
[4] MT SINAI SCH MED,DEPT MED,NEW YORK,NY
关键词
D O I
10.1182/blood.V89.7.2494
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
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/MTX(200) and the highly resistant MOLT-3/MTX(10,000) 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/MTX(10,000) 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/MTX(10,000) cells. Determination of the nucleotide sequence of RFC1 genes from MOLT-3/MTX(10,000) 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/MTX(10,000) 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/MTX(200) 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/MTX(10,000) cells results from inactivating mutations of RFC1 gene alleles.
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页码:2494 / 2499
页数:6
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