Yang C H, Pain J, Sirotnak F M
Program in Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
J Biol Chem. 1992 Apr 5;267(10):6628-34.
Studies are described examining further the decline in folate analogue influx mediated by the one-carbon reduced-folate transport system in HL-60 cells following induction of maturation by cytodifferentiation agents. To facilitate the investigation of the underlying basis of this phenomenon, we derived a variant (HL-60/LCV) with 4-5-fold elevated influx capacity (Vmax) for folate analogues. A commensurate increase in the putative transporter for this system was documented by affinity labeling of these cells with N-hydroxysuccinimide-[3H]aminopterin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the affinity labeled plasma membrane in HL-60/LCV cells delineated a protein peak at Mr = 75,000-80,000. This was substantially greater than the analogous transporter (Mr = 45,000-47,000) we had delineated (Yang, C.-H., Sirotnak, F.M., and Mines, L.S. (1988) J. Biol. Chem. 263, 9703-9709) with the same methodology in the L1210 cell plasma membrane. In addition, the rate of translocation of the Mr = 75,000-80,000 transporter in HL-60 and HL-60/LCV cells was 2-fold lower than the rate of translocation determined for the Mr = 45,000-47,000 transporter in L1210 cells. During induced maturation of HL-60/LCV cells toward the granulocyte pathway, [3H]methotrexate (MTX) influx capacity and the amount of the affinity labeled transporter decreased rapidly in a parallel fashion. The decrease in [3H]MTX influx and in affinity labeling and in the amount of the Mr = 75,000-80,000 transporter was 5-fold following exposure to 210 mM dimethyl sulfoxide (Me2SO) for 5 days during growth in culture. Moreover, during cycloheximide treatment, the decay in [3H]MTX influx at 37 degrees C and in amount of affinity labeled transporter was the same (t1/2 = 144-155 min) for both control and Me2SO-treated HL-60/LCV cells. These results, which reveal no difference in metabolic turnover for control and Me2SO-treated cells, suggest that the decline in folate analogue influx in HL-60/LCV influx cells is a very early event in the program of differentiation and probably occurs by down-regulation of synthesis of the transporter for the one-carbon reduced-folate transport system.
本研究描述了在细胞分化剂诱导HL-60细胞成熟后，进一步检测一碳还原叶酸转运系统介导的叶酸类似物流入量的下降情况。为便于研究这一现象的潜在基础，我们获得了一种变体（HL-60/LCV），其叶酸类似物的流入能力（Vmax）提高了4 - 5倍。用N-羟基琥珀酰亚胺-[3H]氨甲蝶呤对这些细胞进行亲和标记，记录了该系统推定转运蛋白的相应增加。对HL-60/LCV细胞中经亲和标记的质膜进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，在Mr = 75,000 - 80,000处勾勒出一个蛋白峰。这比我们用相同方法在L1210细胞质膜中勾勒出的类似转运蛋白（Mr = 45,000 - 47,000）大得多。此外，HL-60和HL-60/LCV细胞中Mr = 75,000 - 80,000转运蛋白的转运速率比L1210细胞中Mr = 45,000 - 47,000转运蛋白的转运速率低2倍。在HL-60/LCV细胞向粒细胞途径的诱导成熟过程中，[3H]甲氨蝶呤（MTX）流入量和亲和标记转运蛋白的量以平行方式迅速下降。在培养生长期间，用210 mM二甲基亚砜（Me2SO）处理5天后，[3H]MTX流入量、亲和标记以及Mr = 75,000 - 80,000转运蛋白的量下降了5倍。此外，在环己酰亚胺处理期间，对照和Me2SO处理的HL-60/LCV细胞在37℃时[3H]MTX流入量的衰减以及亲和标记转运蛋白的量的衰减是相同的（t1/2 = 144 - 155分钟）。这些结果表明对照细胞和Me2SO处理细胞在代谢周转方面没有差异，提示HL-60/LCV流入细胞中叶酸类似物流入量的下降是分化程序中的一个非常早期的事件，可能是通过下调一碳还原叶酸转运系统转运蛋白的合成而发生的。
