Allegra C J, Fine R L, Drake J C, Chabner B A
J Biol Chem. 1986 May 15;261(14):6478-85.
This report details the effects of methotrexate on the intracellular folate pools of the MCF-7 human breast cancer cell line. To achieve this goal, we designed a high-pressure liquid chromatography system capable of separating the physiologic folates. The folate pools were quantitated following growth and equilibration in 2.25 microM radiolabeled folic acid. Each of the intracellular folates was identified by coelution with standard folates and by chemical/biochemical tests unique to each of the various folates. The 10-formyl-H4PteGlu (where H4PteGlu represents dl-tetrahydrofolic acid) pool accounted for 20.5% of the total intracellular folate pool in untreated cells, whereas 5-formyl-H4PteGlu and H4PteGlu accounted for 6.5 and 10.6%, respectively. The levels of these three folates remained stable throughout cell growth. The 5-methyl-H4PteGlu pool accounted for less than 10% in early growth phase cells but assumed greater than 60% of the total pool by the mid- and late-log phases of cell growth. When the MCF-7 cells were exposed to 1 microM methotrexate, de novo purine synthesis and de novo thymidylate synthesis were rapidly inhibited to less than 20% of control within 3 h. During this time period, rapid alterations in the folate pools also occurred such that dihydrofolic acid levels rose from less than 1% in untreated cells to greater than 30% of the total pool. This rise was accompanied by a parallel fall in 5-methyl-H4PteGlu. H4PteGlu and 5-formyl-H4PteGlu were undetectable following 2 h of methotrexate exposure, but 10-formyl-H4PteGlu, the required cosubstrate for de novo purine synthesis, was preserved at greater than 80% of pretreatment values following a 1 microM methotrexate exposure of up to 21 h. The rapid inhibition of de novo purine synthesis in these cells following methotrexate exposure coupled with a relatively preserved 10-formyl-H4PteGlu pool suggests direct inhibition of this synthetic pathway by the temporally coincident accumulation of dihydrofolic acid and/or methotrexate polyglutamates. This inhibition cannot be ascribed to depletion of the folate cofactor 10-formyl-H4PteGlu.
本报告详细阐述了甲氨蝶呤对MCF - 7人乳腺癌细胞系细胞内叶酸池的影响。为实现这一目标，我们设计了一种能够分离生理性叶酸的高压液相色谱系统。在2.25微摩尔放射性标记叶酸中生长并达到平衡后，对叶酸池进行定量分析。通过与标准叶酸共洗脱以及通过各种叶酸特有的化学/生化测试来鉴定每种细胞内叶酸。在未处理的细胞中，10 - 甲酰 - H4PteGlu（其中H4PteGlu代表dl - 四氢叶酸）池占细胞内总叶酸池的20.5%，而5 - 甲酰 - H4PteGlu和H4PteGlu分别占6.5%和10.6%。在细胞生长过程中，这三种叶酸的水平保持稳定。5 - 甲基 - H4PteGlu池在细胞生长早期阶段占比不到10%，但在细胞生长的对数中期和后期占总池的比例超过60%。当MCF - 7细胞暴露于1微摩尔甲氨蝶呤时，在3小时内，嘌呤从头合成和胸苷酸从头合成迅速被抑制至对照的不到20%。在此期间，叶酸池也迅速发生变化，使得二氢叶酸水平从未处理细胞中的不到1%升至总池的30%以上。这种升高伴随着5 - 甲基 - H4PteGlu的平行下降。甲氨蝶呤暴露2小时后，未检测到H4PteGlu和5 - 甲酰 - H4PteGlu，但在高达21小时的1微摩尔甲氨蝶呤暴露后，作为嘌呤从头合成所需辅酶的10 - 甲酰 - H4PteGlu保持在预处理值的80%以上。甲氨蝶呤暴露后这些细胞中嘌呤从头合成的快速抑制以及相对保留的10 - 甲酰 - H4PteGlu池表明，二氢叶酸和/或甲氨蝶呤多聚谷氨酸的同时积累直接抑制了该合成途径。这种抑制不能归因于叶酸辅因子10 - 甲酰 - H4PteGlu的消耗。
