Assaraf Yehuda G, Rothem Lilah, Hooijberg Jan Hendrik, Stark Michal, Ifergan Ilan, Kathmann Ietje, Dijkmans Ben A C, Peters Godefridus J, Jansen Gerrit
Department of Biology, The Technion, Haifa 32000, Israel.
J Biol Chem. 2003 Feb 28;278(9):6680-6. doi: 10.1074/jbc.M209186200. Epub 2002 Dec 16.
We studied the molecular basis of the up to 46-fold increased accumulation of folates and methotrexate (MTX) in human leukemia CEM-7A cells established by gradual deprivation of leucovorin (LCV). CEM-7A cells consequently exhibited 10- and 68-fold decreased LCV and folic acid growth requirements and 23-25-fold hypersensitivity to MTX and edatrexate. Although CEM-7A cells displayed a 74-86-fold increase in the reduced folate carrier (RFC)-mediated influx of LCV and MTX, RFC overexpression per se cannot induce a prominently increased folate/MTX accumulation because RFC functions as a nonconcentrative anion exchanger. We therefore explored the possibility that folate efflux activity mediated by members of the multidrug resistance protein (MRP) family was impaired in CEM-7A cells. Parental CEM cells expressed substantial levels of MRP1, MRP4, poor MRP5 levels, whereas MRP2, MRP3 and breast cancer resistance protein were undetectable. In contrast, CEM-7A cells lost 95% of MRP1 levels while retaining parental expression of MRP4 and MRP5. Consequently, CEM-7A cells displayed a 5-fold decrease in the [(3)H]folic acid efflux rate constant, which was identical to that obtained with parental CEM cells, when their folic acid efflux was blocked (78%) with probenecid. Furthermore, when compared with parental CEM, CEM-7A cells accumulated 2-fold more calcein fluorescence. Treatment of parental cells with the MRP1 efflux inhibitors MK571 and probenecid resulted in a 60-100% increase in calcein fluorescence. In contrast, these inhibitors failed to alter the calcein fluorescence in CEM-7A cells, which markedly lost MRP1 expression. Replenishment of LCV in the growth medium of CEM-7A cells resulted in resumption of normal MRP1 expression. These results establish for the first time that MRP1 is the primary folate efflux route in CEM leukemia cells and that the loss of folate efflux activity is an efficient means of markedly augmenting cellular folate pools. These findings suggest a functional role for MRP1 in the maintenance of cellular folate homeostasis.
我们研究了通过逐渐剥夺亚叶酸（LCV）建立的人白血病CEM - 7A细胞中叶酸和甲氨蝶呤（MTX）积累增加高达46倍的分子基础。因此，CEM - 7A细胞对LCV和叶酸的生长需求分别降低了10倍和68倍，对MTX和依达曲沙的敏感性增加了23 - 25倍。尽管CEM - 7A细胞中还原型叶酸载体（RFC）介导的LCV和MTX内流增加了74 - 86倍，但RFC的过表达本身并不能显著增加叶酸/MTX的积累，因为RFC作为一种非浓缩性阴离子交换器发挥作用。因此，我们探讨了多药耐药蛋白（MRP）家族成员介导的叶酸外排活性在CEM - 7A细胞中受损的可能性。亲代CEM细胞表达大量的MRP1、MRP4，MRP5水平较低，而未检测到MRP2、MRP3和乳腺癌耐药蛋白。相比之下，CEM - 7A细胞失去了95%的MRP1水平，同时保留了MRP4和MRP5的亲代表达。因此，当用丙磺舒阻断其叶酸外排（78%）时，CEM - 7A细胞的[³H]叶酸外排速率常数降低了5倍，这与亲代CEM细胞的情况相同。此外，与亲代CEM相比，CEM - 7A细胞积累的钙黄绿素荧光增加了2倍。用MRP1外排抑制剂MK571和丙磺舒处理亲代细胞导致钙黄绿素荧光增加60 - 100%。相比之下，这些抑制剂未能改变CEM - 7A细胞中钙黄绿素荧光，CEM - 7A细胞明显失去了MRP1表达。在CEM - 7A细胞的生长培养基中补充LCV导致MRP1表达恢复正常。这些结果首次证实MRP1是CEM白血病细胞中主要的叶酸外排途径，并且叶酸外排活性的丧失是显著增加细胞叶酸池的有效手段。这些发现表明MRP1在维持细胞叶酸稳态中具有功能性作用。
