Leucovorin-induced resistance against FDH growth suppressor effects occurs through DHFR up-regulation
Leucovorin-induced resistance against FDH growth suppressor effects occurs through DHFR up-regulation
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DOI：
10.1016/j.bcp.2006.04.005
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发表时间：
2006-07-14
影响因子：
5.8
通讯作者：
Krupenko, Sergey A.
中科院分区：
文献类型：
--
作者：
Oleinik, Natalia V.;Krupenko, Natalia I.;Krupenko, Sergey A.
10-甲酰四氢叶酸脱氢酶（FDH）将10-甲酰四氢叶酸转化为四氢叶酸（THF）。该酶在FDH缺陷型癌细胞中的表达诱导细胞毒性，该细胞毒性可以通过补充高浓度的还原叶酸5-甲酰-THF（亚叶酸）来逆转。相反，非肿瘤细胞对FDH具有抗性。本研究旨在研究保护细胞免受FDH抑制剂影响的机制。使用10 μ M leucovorin补充的FDH-敏感的A549细胞转染FDH表达，我们选择克隆，已获得对FDH的抗性。耐药细胞表达高水平的FDH，并能够生长后，撤出亚叶酸。然而，这些细胞由于S期延长而具有增加的倍增时间。它们还具有显著增加的总叶酸池和THF/5，10-亚甲基-THF池的水平，而10-甲酰基-THF的水平比亲本FDH敏感性细胞低两倍。我们已经表明，由于产物抑制（K-I为2.35 μ M），在对应于抗性细胞的10-甲酰基-THF/THF的比率下，FDH催化的反应进行的速率比对应于亲本敏感细胞的比率下慢约三倍。FDH抗性细胞具有强烈上调的二氢叶酸还原酶（DHFR），其被认为是改变叶酸库的机制和获得性抗性的关键组分。通过瞬时转染在A549细胞中升高DHFR降低了对FDH毒性的敏感性，并允许选择FDH抗性克隆。DHFR诱导的FDH催化抑制可能是一种S期相关的代谢调节，可提供针对FDH抑制剂效应的保护。(c)2006年爱思唯尔公司All rights reserved.
10-Formyltetrahydrofolate dehydrogenase (FDH) converts 10-formyltetrahydrofolate to tetrahydrofolate (THF). Expression of the enzyme in FDH-deficient cancer cells induces cytotoxicity that can be reversed by supplementation with high concentrations of a reduced folate, 5-formyl-THF (leucovorin). In contrast, non-tumor cells are resistant to FDH. The present study was undertaken to investigate mechanisms that could protect cells against FDH suppressor effects. Using 10 mu M leucovorin supplementation of FDH-sensitive A549 cells transfected for FDH expression, we selected clones that have acquired resistance against FDH. Resistant cells expressed high levels of FDH and were capable of growing after withdrawal of leucovorin. These cells, however, have increased doubling time due to prolonged S phase. They also have significantly increased levels of total folate pool and THF/5,10-methylene-THF pool while the level of 10-formyl-THF was two-fold lower than in parental FDH-sensitive cells. We have shown that the FDH-catalyzed reaction proceeds at about a three-fold slower rate at the ratio of 10-formyl-THF/THF corresponding to the resistant cells than at the ratio corresponding to parental sensitive cells, due to product inhibition (K-I is 2.35 mu M). FDH-resistant cells have strongly up-regulated dihydrofolate reductase (DHFR) that is proposed to be a mechanism for the alteration of folate pools and a key component of the acquired resistance. Elevation of DHFR in A549 cells by transient transfection decreased sensitivity to FDH toxicity and allowed selection of FDH-resistant clones. DHFR-induced repression of FDH catalysis could be an S phase-related metabolic adjustment that provides protection against FDH suppressor effects. (c) 2006 Elsevier Inc. All rights reserved.