Naito Tomoharu, Yokogawa Tatsushi, Takatori Satoshi, Goda Kazato, Hiramoto Akiko, Sato Akira, Kitade Yukio, Sasaki Takuma, Matsuda Akira, Fukushima Masakazu, Wataya Yusuke, Kim Hye-Sook
Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushimanaka, Okayama, 700-8530, Japan.
Cancer Chemother Pharmacol. 2009 Apr;63(5):837-50. doi: 10.1007/s00280-008-0810-y. Epub 2008 Jul 31.
1-(3-C-Ethynyl-beta-D: -ribo-pentofuranosyl)cytosine (ECyd), a ribonucleoside analog, has a potent cytotoxic activity against cancer cells. The present studies have been performed to elucidate the overall mechanisms of ECyd-induced apoptotic cell death.
Cultured cells of mouse mammary carcinoma FM3A and human fibrosarcoma HT 1080 lines were used. The efficacy of RNA synthesis inhibition by ECyd was assessed by kinetic analysis using nuclei isolated from FM3A cells. RNA status in ECyd-treated cells was investigated by Northern blots, and the cleavage sites of RNA were identified by rapid amplification of 5' cDNA ends (5'-RACE). The effect of protein functions on the ECyd-induced apoptotic pathway was analyzed by siRNA and immunohistochemical techniques. Apoptotic cells were detected by TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) assay.
ECyd induces inhibition of RNA synthesis in vitro and in vivo, which appears to be a major cause for the apoptosis. It is known that ECyd is converted inside the cell into its 5'-triphosphate (ECTP). We have now found in test-tube experiments that ECTP strongly inhibits the activity of RNA polymerase I by competing with CTP. In the absence of robust RNA synthesis, the cellular RNAs would be destined to break down. RNase L was found to be playing a role in the breakdown: thus, the 28S rRNA-fragmentation pattern observed for the ECyd-treated cells was very similar to that observable in an in vitro treatment of the 28S ribosomes with RNase L. Association of RNase L with the cytotoxic action of ECyd was confirmed by use of the siRNA-mediated suppression of the cellular RNase L. Thus, the cells in which the RNase L was knocked-down were highly resistant to the cytotoxic action of ECyd. Further events, downstream of the RNase L action that can lead to the eventual apoptosis, would conceivably involve the phosphorylation of c-jun N-terminal kinase and subsequent decrease in mitochondrial membrane-potential. Evidence to support this flow of events was obtained by siRNA-experiments.
The results from this study demonstrated that RNase L is activated after the inhibition of RNA polymerase, and induces mitochondria-dependent apoptotic pathway. We propose this new role for RNase L in the apoptotic mechanism. These findings may open up the possibility of finding new targets for anticancer agents.
1-(3-C-乙炔基-β-D-核糖-戊呋喃糖基)胞嘧啶（ECyd），一种核糖核苷类似物，对癌细胞具有强大的细胞毒活性。进行本研究以阐明ECyd诱导凋亡性细胞死亡的整体机制。
使用小鼠乳腺癌FM3A细胞系和人纤维肉瘤HT 1080细胞系的培养细胞。通过对从FM3A细胞分离的细胞核进行动力学分析来评估ECyd对RNA合成的抑制效果。通过Northern印迹法研究经ECyd处理的细胞中的RNA状态，并通过5' cDNA末端快速扩增（5'-RACE）鉴定RNA的切割位点。通过小干扰RNA（siRNA）和免疫组织化学技术分析蛋白质功能对ECyd诱导的凋亡途径的影响。通过TdT介导的dUTP-生物素缺口末端标记（TUNEL）测定法检测凋亡细胞。
ECyd在体外和体内均诱导RNA合成的抑制，这似乎是凋亡的主要原因。已知ECyd在细胞内转化为其5'-三磷酸酯（ECTP）。我们现在在试管实验中发现ECTP通过与CTP竞争而强烈抑制RNA聚合酶I的活性。在缺乏强劲RNA合成的情况下，细胞RNA将注定会分解。发现核糖核酸酶L（RNase L）在这种分解过程中起作用：因此，在经ECyd处理的细胞中观察到的28S核糖体RNA片段化模式与用RNase L对28S核糖体进行体外处理时可观察到的模式非常相似。通过使用siRNA介导的细胞RNase L抑制，证实了RNase L与ECyd的细胞毒作用相关。因此，RNase L被敲低的细胞对ECyd的细胞毒作用具有高度抗性。在RNase L作用下游可能导致最终凋亡的进一步事件可能涉及c-jun N末端激酶的磷酸化以及随后线粒体膜电位的降低。通过siRNA实验获得了支持这一系列事件的证据。
本研究结果表明，RNase L在RNA聚合酶受到抑制后被激活，并诱导线粒体依赖性凋亡途径。我们提出了RNase L在凋亡机制中的这一新作用。这些发现可能为寻找抗癌药物的新靶点开辟可能性。
