Seither R L, Trent D F, Mikulecky D C, Rape T J, Goldman I D
Department of Medicine, Virginia Commonwealth University, Medical College of Virginia, Richmond 23298.
J Biol Chem. 1989 Oct 15;264(29):17016-23.
Folate analogs that inhibit dihydrofolate reductase result in only partial interconversion of tetrahydrofolate cofactors to dihydrofolate with preservation of the major portion of reduced cellular folate cofactors in L1210 leukemia cells. One possible explanation for this phenomenon is that low levels of dihydrofolate polyglutamates that accumulate in the presence of antifolates block thymidylate synthase to prevent depletion of reduced folate pools. This paper correlates biochemical analyses of rapid interconversions of radiolabeled folates and changes in purine and pyrimidine biosynthesis in L1210 murine leukemia cells exposed to antifolates with network thermodynamic computer modeling to assess this hypothesis. When cells are exposed to 1 microM trimetrexate there is an almost instantaneous inhibition of [3H] deoxyuridine or [14C]formate incorporation into nucleotides which is maximal within 5 min. This is associated with a rapid rise in cellular dihydrofolate (t1/2 approximately 1.5 min), which reaches a steady state that represents only 27.9% of the total folate pool. Pretreatment of cells with fluorodeoxyuridine, to inhibit thymidylate synthase by about 95% followed by trimetrexate only slows the rate of folate interconversion (t1/2 approximately 25 min) but not the final dihydrofolate level achieved. This is consistent with computer simulations which predict that direct inhibition of thymidylate synthase by 97, 98, and 99% should increase the half-time of dihydrofolate rise after trimetrexate to 40, 60, and 124 min, respectively, but the final level achieved is always the same as in cells with normal thymidylate synthase activity. The data reflect the high degree of catalytic activity of thymidylate synthase relative to tetrahydrofolate cofactor pools in the cells and the enormous extent of inhibition of this enzyme that is necessary to slow the rate of folate interconversions after addition of antifolates. The model predicts, and the data demonstrate, that virtually any residual thymidylate synthase activity will permit the interconversion of all tetrahydrofolate cofactors available for oxidation to dihydrofolate when dihydrofolate reductase activity is abolished, but the rate of interconversion will be slowed. Additional simulations indicate that the time course of cessation of tetrahydrofolate-dependent purine and pyrimidine biosynthesis after antifolates in these cells can be accounted for solely on the basis of tetrahydrofolate cofactor depletion alone. These data exclude the possibility that direct inhibition of thymidylate synthase by dihydrofolate polyglutamates, or any other intracellular folates that accumulate in cells after antifolates, can account for the rapid but partial interconversion of reduced folate cofactors to dihydrofolate.(ABSTRACT TRUNCATED AT 400 WORDS)
抑制二氢叶酸还原酶的叶酸类似物只会导致四氢叶酸辅因子部分转化为二氢叶酸，L1210白血病细胞中大部分还原型细胞叶酸辅因子得以保留。对此现象的一种可能解释是，在抗叶酸药物存在的情况下积累的低水平二氢叶酸多聚谷氨酸会阻断胸苷酸合酶，以防止还原型叶酸池的耗尽。本文将对暴露于抗叶酸药物的L1210小鼠白血病细胞中放射性标记叶酸的快速相互转化的生化分析以及嘌呤和嘧啶生物合成的变化与网络热力学计算机模型相关联，以评估这一假设。当细胞暴露于1微摩尔三甲氧苄氨嘧啶时，[3H]脱氧尿苷或[14C]甲酸掺入核苷酸的过程几乎会立即受到抑制，5分钟内达到最大值。这与细胞中二氢叶酸的快速增加（半衰期约1.5分钟）相关，其达到的稳态仅占总叶酸池的27.9%。先用氟脱氧尿苷预处理细胞，以抑制胸苷酸合酶约95%，然后再加入三甲氧苄氨嘧啶，只会减慢叶酸相互转化的速度（半衰期约25分钟），但不会改变最终达到的二氢叶酸水平。这与计算机模拟结果一致，模拟结果预测，将胸苷酸合酶直接抑制97%、98%和99%，应分别使三甲氧苄氨嘧啶处理后二氢叶酸增加的半衰期延长至40分钟、60分钟和124分钟，但最终达到的水平始终与胸苷酸合酶活性正常的细胞相同。这些数据反映了细胞中胸苷酸合酶相对于四氢叶酸辅因子池的高度催化活性，以及添加抗叶酸药物后减缓叶酸相互转化速度所需的该酶的巨大抑制程度。该模型预测并经数据证明，当二氢叶酸还原酶活性被消除时，几乎任何残留的胸苷酸合酶活性都将使所有可用于氧化为二氢叶酸的四氢叶酸辅因子相互转化，但相互转化的速度会减慢。额外的模拟表明，这些细胞在抗叶酸药物作用后，四氢叶酸依赖性嘌呤和嘧啶生物合成停止的时间进程仅可基于四氢叶酸辅因子的耗尽来解释。这些数据排除了二氢叶酸多聚谷氨酸或抗叶酸药物作用后细胞中积累的任何其他细胞内叶酸直接抑制胸苷酸合酶，能够解释还原型叶酸辅因子快速但部分转化为二氢叶酸的可能性。（摘要截短至400字）
