Oleinik Natalia V, Krupenko Natalia I, Priest David G, Krupenko Sergey A
Department of Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.
Biochem J. 2005 Nov 1;391(Pt 3):503-11. doi: 10.1042/BJ20050533.
A folate enzyme, FDH (10-formyltetrahydrofolate dehydrogenase; EC 1.5.1.6), is not a typical tumour suppressor, but it has two basic characteristics of one, i.e. it is down-regulated in tumours and its expression is selectively cytotoxic to cancer cells. We have recently shown that ectopic expression of FDH in A549 lung cancer cells induces G1 arrest and apoptosis that was accompanied by elevation of p53 and its downstream target, p21. It was not known, however, whether FDH-induced apoptosis is p53-dependent or not. In the present study, we report that FDH-induced suppressor effects are strictly p53-dependent in A549 cells. Both knockdown of p53 using an RNAi (RNA interference) approach and disabling of p53 function by dominant-negative inhibition with R175H mutant p53 prevented FDH-induced cytotoxicity in these cells. Ablation of the FDH-suppressor effect is associated with an inability to activate apoptosis in the absence of functional p53. We have also shown that FDH elevation results in p53 phosphorylation at Ser-6 and Ser-20 in the p53 transactivation domain, and Ser-392 in the C-terminal domain, but only Ser-6 is strictly required to mediate FDH effects. Also, translocation of p53 to the nuclei and expression of the pro-apoptotic protein PUMA (Bcl2 binding component 3) was observed after induction of FDH expression. Elevation of FDH in p53 functional HCT116 cells induced strong growth inhibition, while growth of p53-deficient HCT116 cells was unaffected. This implies that activation of p53-dependent pathways is a general downstream mechanism in response to induction of FDH expression in p53 functional cancer cells.
一种叶酸酶，FDH（10-甲酰四氢叶酸脱氢酶；EC 1.5.1.6），并非典型的肿瘤抑制因子，但它具有肿瘤抑制因子的两个基本特征，即它在肿瘤中表达下调，且其表达对癌细胞具有选择性细胞毒性。我们最近发现，在A549肺癌细胞中异位表达FDH会诱导G1期阻滞和凋亡，同时伴有p53及其下游靶点p21的升高。然而，FDH诱导的凋亡是否依赖p53尚不清楚。在本研究中，我们报告FDH诱导的抑制作用在A549细胞中严格依赖p53。使用RNAi（RNA干扰）方法敲低p53以及用R175H突变型p53通过显性负抑制使p53功能失活，均可阻止这些细胞中FDH诱导的细胞毒性。在缺乏功能性p53的情况下，FDH抑制作用的消除与无法激活凋亡相关。我们还发现，FDH水平升高会导致p53转录激活结构域的Ser-6和Ser-20以及C末端结构域的Ser-392发生磷酸化，但只有Ser-6是介导FDH效应所必需的。此外，在诱导FDH表达后，观察到p53转位至细胞核以及促凋亡蛋白PUMA（Bcl2结合成分3）的表达。在p53功能正常的HCT116细胞中，FDH水平升高会诱导强烈的生长抑制，而p53缺陷的HCT116细胞的生长则不受影响。这意味着激活p53依赖的信号通路是p53功能正常的癌细胞中响应FDH表达诱导的一般下游机制。
