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 resi stance (Cancer Res 45:1815, 1985). The alteration of MTX uptake kineti cs in these cells, the intermediately resistant MOLT-3/MTX(200) and th e 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 r ecently isolated reduced folate carrier gene (RFC1) was the cause of M TX transport deficiency in these cell lines. Reconstitution of RFC act ivity in MOLT-3/MTX(10,000) cells by transduction with a murine RFC re troviral vector reversed MTX resistance and trimetrexate sensitivity. Although RFC1 RNA levels were unchanged in the resistant cell lines, F ACS analysis using a polyclonal anti-RFC1 antibody showed no detectabl e RFC1 protein in the MOLT-3/MTX(10,000) cells. Determination of the n ucleotide sequence of RFC1 genes from MOLT-3/MTX(10,000) cells reveale d 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 al lele containing another mutation, which resulted in a premature stop c odon 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 clone s contained the wild-type sequence. Determination of the genomic seque nce of RFC1 in MOLT-3/MTX(200) cells demonstrated that these cells wer e heterozygous for a mutation at codon 40, but were homozygous for the wild-type sequence at codon 25. Thus, the acquisition of MTX transpor t-deficiency in MOLT-3/MTX(10,000) cells results from inactivating mut ations of RFC1 gene alleles.