Brigle K E, Spinella M J, Sierra E E, Goldman I D
Department of Medicine, Massey Cancer Center, Virginia Commonwealth University, Medical College of Virginia, Richmond 23298, USA.
J Biol Chem. 1995 Sep 29;270(39):22974-9. doi: 10.1074/jbc.270.39.22974.
This laboratory previously described an L1210 leukemia cell line (MTXrA) selected for resistance to methotrexate by virtue of impaired transport due to a functional defect in the translocation process. We now report on the sequence analysis of cDNAs encoding the reduced folate carrier from this line and identify a single mutation that results in the substitution of a proline for an alanine in a highly conserved transmembrane region of the protein. Transfection of the parental reduced folate carrier into MTXrA cells resulted in a cell line which exhibited a complete restoration of methotrexate uptake and an enhanced sensitivity to methotrexate. Northern analysis and specific [3H]MTX cell surface binding indicated that expression of the reduced folate carrier was elevated approximately 5-fold in the transfectant compared to parental and MTXrA cells. The MTX influx properties of the transfectant cell line were identical to those of the well characterized reduced folate carrier from parental L1210 cells in terms of: 1) patterns of sensitivity to competing folates, 2) sensitivity to the organic anion sulfobromophthalein, 3) lack of energy dependence, and 4) capacity for trans-stimulation. We also provide new data which suggests that the nucleotide sequence 5' of the predicted ATG initiation codon may encode additional protein information in the form of a leader sequence. Finally, we demonstrate that the MTXrA line has both the mutant and the parental reduced folate carrier alleles but that expression appears to be restricted to the mutant allele. Thus, the methotrexate transport phenotype and resultant drug resistance in this cell line result from genetic/regulatory events at both alleles.
本实验室先前描述了一种L1210白血病细胞系（MTXrA），该细胞系因转运过程中的功能缺陷导致转运受损而被选择为对甲氨蝶呤具有抗性。我们现在报告对该细胞系中编码还原型叶酸载体的cDNA进行的序列分析，并鉴定出一个单一突变，该突变导致蛋白质高度保守的跨膜区域中的丙氨酸被脯氨酸取代。将亲本还原型叶酸载体转染到MTXrA细胞中，产生了一个细胞系，该细胞系表现出甲氨蝶呤摄取的完全恢复以及对甲氨蝶呤的敏感性增强。Northern分析和特异性[3H]MTX细胞表面结合表明，与亲本和MTXrA细胞相比，转染细胞中还原型叶酸载体的表达提高了约5倍。转染细胞系的MTX流入特性在以下方面与亲本L1210细胞中特征明确的还原型叶酸载体相同：1）对竞争性叶酸的敏感模式，2）对有机阴离子磺溴酞的敏感性，3）缺乏能量依赖性以及4）反式刺激能力。我们还提供了新的数据，表明预测的ATG起始密码子5'端的核苷酸序列可能以引导序列的形式编码额外的蛋白质信息。最后，我们证明MTXrA细胞系同时具有突变型和亲本还原型叶酸载体等位基因，但表达似乎仅限于突变等位基因。因此，该细胞系中甲氨蝶呤的转运表型和由此产生的耐药性是由两个等位基因的遗传/调控事件导致的。
