Spinella M J, Brigle K E, Sierra E E, Goldman I D
Department of Medicine, Virginia Commonwealth University, Medical College of Virginia, Richmond 23298, USA.
J Biol Chem. 1995 Apr 7;270(14):7842-9. doi: 10.1074/jbc.270.14.7842.
L1210 leukemia cells transport reduced folates and methotrexate via a well defined reduced folate carrier system and, in the absence of low folate selective pressure, do not express an alternate endocytotic route mediated by cell surface folate receptors. This laboratory previously described an L1210 leukemia cell line, MTXrA, with acquired resistance to methotrexate (MTX) due to the loss of mobility of the reduced folate carrier. We now report on the transfection of MTXrA with a cDNA encoding the murine homolog of the human folate receptor isoform of KB cells to produce MTXrA-TF1, which constitutively expresses high levels of FR-alpha. MTXrA-TF1 and L1210 cells were utilized to compare transport of methotrexate mediated by FR-alpha and the reduced folate carrier, respectively. Methotrexate influx in the two lines was similar when the extracellular level was 0.1 microM, but as the methotrexate concentration increased, influx via the reduced folate carrier increased in comparison to influx mediated by FR-alpha. Transport kinetics indicated both a approximately 20-fold lower influx Kb and Vmax for MTXrA-TF1 as compared to L1210 cells. The two cell lines exhibited distinct influx properties. Methotrexate influx in MTXrA-TF1 was markedly inhibited by 50 nM folic acid and metabolic poisons. In L1210 cells, 1.0 microM folic acid did not affect MTX influx, and metabolic poisons either had no effect on or increased methotrexate influx. Removal of extracellular chloride markedly inhibited transport in MTXrA-TF1 but stimulated influx in L1210 cells. When the pH was decreased to 6.2, methotrexate influx was not altered in MTXrA-TF1 but was reduced in L1210 cells. Probenecid and sulfobromophthalein inhibit methotrexate influx in both L1210 and MTXrA-TF1 cell lines; however, inhibition in MTXrA-TF1 could be accounted for on the basis of inhibition of methotrexate binding to FR-alpha. The data indicate that the reduced folate carrier and FR-alpha function independently and exhibit distinct properties. FR-alpha expressed at sufficient levels can mediate influx of MTX and folates into cells at rates comparable to the reduced folate carrier and hence has pharmacologic and physiologic importance.
L1210白血病细胞通过一个明确的还原型叶酸载体系统转运还原型叶酸和甲氨蝶呤，并且在缺乏低叶酸选择压力的情况下，不表达由细胞表面叶酸受体介导的另一种内吞途径。本实验室先前描述了一种L1210白血病细胞系MTXrA，由于还原型叶酸载体的流动性丧失而获得了对甲氨蝶呤（MTX）的抗性。我们现在报告用编码KB细胞人叶酸受体亚型的鼠同源物的cDNA转染MTXrA，以产生MTXrA-TF1，其组成性表达高水平的FR-α。利用MTXrA-TF1和L1210细胞分别比较由FR-α和还原型叶酸载体介导的甲氨蝶呤转运。当细胞外水平为0.1微摩尔时，两种细胞系中甲氨蝶呤的流入相似，但随着甲氨蝶呤浓度的增加，与由FR-α介导的流入相比，通过还原型叶酸载体的流入增加。转运动力学表明，与L1210细胞相比，MTXrA-TF1的流入Kb和Vmax均降低了约20倍。这两种细胞系表现出不同的流入特性。MTXrA-TF1中甲氨蝶呤的流入受到50纳摩尔叶酸和代谢毒物的显著抑制。在L1210细胞中，1.µM叶酸不影响MTX的流入，代谢毒物对甲氨蝶呤的流入要么没有影响，要么增加甲氨蝶呤的流入。去除细胞外氯离子显著抑制MTXrA-TF1中的转运，但刺激L1210细胞中的流入。当pH值降至6.2时，MTXrA-TF1中甲氨蝶呤的流入没有改变，但在L1210细胞中减少。丙磺舒和磺溴酞抑制L1210和MTXrA-TF1细胞系中甲氨蝶呤的流入；然而，MTXrA-TF1中的抑制作用可以基于甲氨蝶呤与FR-α结合的抑制来解释。数据表明，还原型叶酸载体和FR-α独立发挥作用并表现出不同的特性。以足够水平表达的FR-α可以介导MTX和叶酸以与还原型叶酸载体相当的速率流入细胞，因此具有药理学和生理学重要性。
