Spinella M J, Brigle K E, Freemantle S J, Sierra E E, Goldman I D
Department of Medicine, Virginia Commonwealth University, Medical College of Virginia, Richmond 23298, USA.
Biochem Pharmacol. 1996 Sep 13;52(5):703-12. doi: 10.1016/0006-2952(96)00347-4.
We previously described a methotrexate-resistant L1210 cell line (MTXrA) that lacks a functional reduced folate carrier and does not appreciably express the folate receptor. In the present study, we utilized MTXrA cell lines stably transfected with cDNAs encoding either the folate receptor or the reduced folate carrier to investigate the influence of the route of folate influx on the rate and extent of methotrexate polyglutamylation. At an extracellular methotrexate concentration of 0.1 microM, influx in the folate receptor transfectant (MTXrA-TF1) and in the reduced folate carrier transfectant (MTXrA-R1) was equal and methotrexate polyglutamates accumulated at an identical rate, but the onset was delayed until dihydrofolate reductase was saturated with the monoglutamate (approxmately 3 hr). The onset of polyglutamate formation was immediate and identical among the lines in cells pretreated with the lipophilic dihydrofolate reductase inhibitor trimetrexate to block methotrexate binding to dihydrofolate reductase. The spectra of individual methotrexate polyglutamates that accumulated were similar, with the tetraglutamate present as the predominant form. A 100-fold higher methotrexate concentration was required to detect methotrexate uptake and polyglutamylation in the transport defective parent MTXrA line, demonstrating that diffusion or an unidentified low affinity route also supports polyglutamylation. Since the folate receptor and the reduced folate carrier achieve nearly identical rates of polyglutamylation despite very different mechanisms of methotrexate delivery, the data suggest that transport-mediated substrate channeling to folylpolyglutamate synthetase is unlikely to play a role in tetrahydrofolate metabolism. This study supports the notion that it is the intracellular concentration of methotrexate achieved within the cell that drives polyglutamylation irrespective of its route of entry.
我们之前描述过一种对甲氨蝶呤耐药的L1210细胞系（MTXrA），该细胞系缺乏功能性的还原型叶酸载体，且叶酸受体表达不明显。在本研究中，我们利用稳定转染了编码叶酸受体或还原型叶酸载体的cDNA的MTXrA细胞系，来研究叶酸流入途径对甲氨蝶呤多聚谷氨酸化的速率和程度的影响。在细胞外甲氨蝶呤浓度为0.1微摩尔时，叶酸受体转染细胞系（MTXrA-TF1）和还原型叶酸载体转染细胞系（MTXrA-R1）中的流入量相等，甲氨蝶呤多聚谷氨酸以相同的速率积累，但在二氢叶酸还原酶被单谷氨酸饱和（约3小时）之前，其起始过程会延迟。在用亲脂性二氢叶酸还原酶抑制剂三甲曲沙预处理以阻断甲氨蝶呤与二氢叶酸还原酶结合的细胞中，各细胞系中多聚谷氨酸形成的起始过程是即时且相同的。积累的单个甲氨蝶呤多聚谷氨酸的谱图相似，以四聚谷氨酸为主要形式。在转运缺陷的亲本MTXrA细胞系中，检测甲氨蝶呤摄取和多聚谷氨酸化需要高一百倍的甲氨蝶呤浓度，这表明扩散或一种未确定的低亲和力途径也支持多聚谷氨酸化。由于尽管甲氨蝶呤递送机制非常不同，但叶酸受体和还原型叶酸载体实现了几乎相同的多聚谷氨酸化速率，因此数据表明，转运介导的底物向叶酰多聚谷氨酸合成酶的通道化不太可能在四氢叶酸代谢中起作用。这项研究支持这样一种观点，即驱动多聚谷氨酸化的是细胞内达到的甲氨蝶呤细胞内浓度，而与其进入途径无关。
