Zhao R, Seither R, Brigle K E, Sharina I G, Wang P J, Goldman I D
Departments of Medicine, Molecular Pharmacology, and the Albert Einstein College of Medicine Cancer Center, Bronx, New York 10461, USA.
J Biol Chem. 1997 Aug 22;272(34):21207-12. doi: 10.1074/jbc.272.34.21207.
Transport of reduced folates in murine leukemia cells is mediated by the bidirectional reduced folate carrier (RFC1) and independent unidirectional exit pumps. RFC1 has been proposed to be intrinsically equilibrating, generating transmembrane gradients by exchange with inorganic and organic anions. This paper defines the role of high level carrier expression, through transfection with RFC1 cDNA, on concentrative transport of the folate analog, methotrexate (MTX) in murine L1210 leukemia cells. RFC1 was expressed in the MTXrA line, which lacks a functional endogenous carrier to obtain the MTXrA-R16 clonal derivative. Influx was increased approximately 9-fold in MTXrA-R16 cells without a change in Km. The efflux rate constant was increased by a factor of 5.1 relative to L1210 cells, and this resulted in only a 2.1-fold increase in the steady-state level of free intracellular MTX, [MTX]i, when [MTX]e was 1 microM. The concentrative advantage for RFC1 (the ratio of [MTX]i in MTXrA-R16 to L1210 cells) increased from 1.8 at 0.1 microM MTX to 3.8 at an [MTX]e level of 30 microM. Augmented transport in MTXrA-R16 cells was accompanied by a 2-fold increase in accumulation of MTX polyglutamate derivatives and a approximately 50% decrease in the EC50 for 5-formyltetrahydrofolate and folic acid and the MTX IC50 relative to L1210 cells. These alterations paralleled changes in [MTX]i and not the much larger change in influx at low [MTX]e levels, consistent with the critical role that free intracellular folates and drug play in meeting cellular needs for folates and as a determinant of antifolate activity, respectively. The data indicate that RFC1 produces a large and near symmetrical increase in the bidirectional fluxes of MTX resulting in only a small increase in the transmembrane chemical gradient at low extracellular folate levels. Hence, increased expression of RFC1, alone, may not be an efficient adaptive response to folate deprivation, and other factors may come into play to account for the marked increases in concentrative folate transport which occur when cells are subjected to low folate-selective pressure.
还原型叶酸在小鼠白血病细胞中的转运由双向还原型叶酸载体（RFC1）和独立的单向外排泵介导。有人提出RFC1本质上是平衡的，通过与无机和有机阴离子交换产生跨膜梯度。本文通过用RFC1 cDNA转染来定义高水平载体表达对叶酸类似物甲氨蝶呤（MTX）在小鼠L1210白血病细胞中浓缩转运的作用。RFC1在MTXrA细胞系中表达，该细胞系缺乏功能性内源性载体，从而获得MTXrA-R16克隆衍生物。在MTXrA-R16细胞中，内流增加了约9倍，而Km没有变化。相对于L1210细胞，外排速率常数增加了5.1倍，当[MTX]e为1 microM时，这仅导致细胞内游离MTX的稳态水平[MTX]i增加了2.1倍。RFC1的浓缩优势（MTXrA-R16中[MTX]i与L1210细胞的比值）从0.1 microM MTX时的1.8增加到30 microM [MTX]e水平时的3.8。MTXrA-R16细胞中转运的增强伴随着MTX多聚谷氨酸衍生物积累增加2倍，相对于L1210细胞，5-甲酰四氢叶酸和叶酸的EC50以及MTX的IC50降低了约50%。这些变化与[MTX]i的变化平行，而不是在低[MTX]e水平时内流的更大变化，这与游离细胞内叶酸和药物分别在满足细胞对叶酸的需求以及作为抗叶酸活性的决定因素中所起的关键作用一致。数据表明，RFC1使MTX的双向通量大幅且近乎对称地增加，导致在低细胞外叶酸水平时跨膜化学梯度仅小幅增加。因此，单独增加RFC1的表达可能不是对叶酸缺乏的有效适应性反应，当细胞受到低叶酸选择压力时，可能还有其他因素起作用来解释浓缩型叶酸转运的显著增加。
