Gong M, Yess J, Connolly T, Ivy S P, Ohnuma T, Cowan K H, Moscow J A
Medicine and Pediatric Branches, National Cancer Institute, Bethesda, MD 20892,USA.
Blood. 1997 Apr 1;89(7):2494-9.
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.
大沼等人报道了一系列对甲氨蝶呤耐药的MOLT - 3人T细胞急性淋巴细胞白血病细胞系，随着亚系获得越来越高的甲氨蝶呤（MTX）耐药性，其MTX摄取量逐渐减少（《癌症研究》45:1815，1985年）。在这些细胞中，即中度耐药的MOLT - 3/MTX200和高度耐药的MOLT - 3/MTX10,000细胞系中，MTX摄取动力学的改变归因于甲氨蝶呤转运的最大速率（Vmax）变化，而转运体对MTX的亲和力没有明显变化。我们研究了这些细胞系，以确定最近分离的还原型叶酸载体基因（RFC1）转录或翻译的改变是否是这些细胞系中MTX转运缺陷的原因。通过用鼠RFC逆转录病毒载体转导，在MOLT - 3/MTX10,000细胞中重建RFC活性，逆转了MTX耐药性和三甲曲沙敏感性。尽管耐药细胞系中的RFC1 RNA水平没有变化，但使用多克隆抗RFC1抗体的流式细胞术分析显示，在MOLT - 3/MTX10,000细胞中未检测到RFC1蛋白。对MOLT - 3/MTX10,000细胞的RFC1基因核苷酸序列测定表明，该细胞系包含3个RFC1等位基因：一个野生型等位基因、一个在第40密码子处含有过早终止密码子的等位基因和第三个含有另一个突变的等位基因，该突变导致在第25密码子处出现过早终止密码子。我们通过测定来自MOLT - 3/MTX10,000细胞的24个RFC1 cDNA亚克隆的核苷酸序列，检查了这些等位基因的相对表达，发现这些克隆中只有三分之一包含野生型序列。对MOLT - 3/MTX200细胞中RFC1基因组序列的测定表明，这些细胞在第40密码子处的突变是杂合的，但在第25密码子处的野生型序列是纯合的。因此，MOLT - 3/MTX10,000细胞中MTX转运缺陷的获得是由于RFC1基因等位基因的失活突变。
