Studies on Molecular Mechanism of DNA Fragmentation in thymocytes undergoing Apoptosis

胸腺细胞凋亡过程中DNA断裂的分子机制研究

• 批准号: 12672234
• 负责人: LEE Eibai
• 金额: $ 0.32万
• 依托单位: Kobe Gakuin University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12672234/
• 关键词: Apoptosis; Histone; Phosphorylation; Acetylation; Chromatin; DNA fragmentation; α-Helix; Calyculin A

1) The identification of phosphorylated histones preceding apoptotic cell death It is reported that the apoptosis is induced by calyculin A and okadaic acid, which are the protein phosphatase inhibitors in various cell lines. We had already found that the histone phosphorylation precedes thymocyte apoptosis induced by these drugs. In this study, the identification of phosphorylated histones was tried. The phosphorylation of histone HI, H2A and H3 increased prior to calyculin A-induced DNA fragmentation in thymocytes. On the other hand, each histone contents did not change by the treatment of the cells with calyculin A. To clear whether the chemical modification of histones is important as a common mechanism of apoptosis, the chemical modification of histones undergoing apoptosis of human astrocytes was also examined. When the human astrocytes were cultured by growth medium after exposure to saline, the cells were injured. This cell damage showed features of the apoptosis. The phosphory … More lation of histones H2A preceded in apoptotic cell deatn.2) The effect of chemical modification of histones for the chromatin structure The DNA fragmentation is the biochemical hallmark of the apoptosis, and the DNase concerning apoptotic DNA fragmentation has also been clarified. However, the detailed molecular mechanism of DNA fragmentation has not sufficiently been clarified. It is known that chemical modification of histones is related to the transcriptional control by the change of the structure of chromatin DNA in the nucleus. We tried to verify the chromatin structural change in apoptotic process. CD spectrum of the soluble chromatin prepared from the calyculin A treated cell nucleus was measured. Though the calyculin A treatment did not affect for the structure of DNA, cc-helical content in the calyculin A treated chromatin was decreased. The chromatin structural change was also observed in dexamethasone induced thymocyte apoptosis. The sensitivity for the DNase increased prepared chromatin from the calyculin A treated cell from control. These results suggest that the phosphorylation of histone H2A was important as a common mechanism of the apoptotic process. In addition, the histone phosphorylation made the chromatin structure relax by decreasing ahelical content of the chromatin and the sensitivity for DNase increased by change of chromatin structure.3) The search of a novel apoptosis inducer. A novel apoptosis inducer was searched in order to clear whether the chromatin structural change is a common mechanism of apoptosis. YO-2 synthesized by Okada et.al induced apoptosis in rat thymocytes. Therefore, this compound seemed to be useful as an inducer of apoptosis, which examined the importance of the chromatin structural change in the apoptotic cell death.4) The study on the mechanism of apoptotic cell death in cultured rat astrocytes. Reperfusion of cultured rat astrocytes with Ca^<2+>-containing medium after exposure to Ca^<2+>-free medium caused an increase in intracellular Ca^<2+> concentration followed by delayed cell death, including apoptosis. Some compounds such as T-588 and CV-2619 protected cultured rat astrocytes against reperfusion injury. In addition, CGMP inhibited the mitochondrial permeable transition pore via the activation of PKG and prevented apoptotic cell death. Less

1)凋亡细胞死亡前磷酸化组蛋白的鉴定据报道，calyculin A和冈田酸是多种细胞系蛋白磷酸酶抑制剂，可诱导细胞凋亡。我们已经发现组蛋白磷酸化先于这些药物诱导的胸腺细胞凋亡。本研究尝试鉴定磷酸化组蛋白。在花青素a诱导的胸腺细胞DNA断裂之前，组蛋白HI、H2A和H3的磷酸化水平升高。另一方面，各组蛋白含量未因calyculin a处理而改变。为了明确组蛋白的化学修饰是否作为细胞凋亡的共同机制，我们还检测了人星形胶质细胞凋亡过程中组蛋白的化学修饰。人星形胶质细胞经生理盐水培养后，用生长培养基培养，细胞受到损伤。细胞损伤表现为凋亡特征。在凋亡细胞死亡之前，磷酸化组蛋白H2A的表达增加。2)组蛋白的化学修饰对染色质结构的影响DNA片段化是细胞凋亡的生化标志，与细胞凋亡DNA片段化有关的DNA酶也已被阐明。然而，DNA断裂的详细分子机制尚未得到充分阐明。已知组蛋白的化学修饰与细胞核内染色质DNA结构的改变对转录的控制有关。我们试图验证凋亡过程中染色质结构的变化。测定了calyculin A处理后的细胞核制备的可溶性染色质的CD谱。虽然花青素A处理对DNA的结构没有影响，但处理后的染色质中cc-螺旋含量降低。地塞米松诱导胸腺细胞凋亡时染色质结构也发生改变。对dna酶的敏感性增加了从花青素A处理的细胞制备的染色质。这些结果表明，组蛋白H2A的磷酸化是凋亡过程的一个重要的共同机制。此外，组蛋白磷酸化使染色质的非螺旋含量降低，使染色质结构松弛，染色质结构的改变使dna酶的敏感性增加。3)寻找一种新的细胞凋亡诱导剂。寻找一种新的细胞凋亡诱导剂，以明确染色质结构变化是否为细胞凋亡的共同机制。Okada等合成的YO-2诱导大鼠胸腺细胞凋亡。因此，该化合物似乎是一种有用的凋亡诱导剂，这就检验了染色质结构改变在凋亡细胞死亡中的重要性。4)培养大鼠星形胶质细胞凋亡机制的研究。培养的大鼠星形胶质细胞暴露于不含Ca^<2+>的培养基后，用含Ca^<2+>的培养基再灌注，导致细胞内Ca^<2+>浓度升高，随后细胞死亡，包括凋亡。T-588、CV-2619等化合物对培养大鼠星形胶质细胞的再灌注损伤具有保护作用。CGMP通过激活PKG抑制线粒体可渗透过渡孔，防止凋亡细胞死亡。少

项目成果

K.Takuma, T.Fujita, Y.Kimura, M.Tanabe A.Yamamuro, E.Lee: "T-588 inhibits astrocyte apoptosis via mitogen-activated protein kinase signal pathway."Eur. J. Pharmacol. 399. 1-8 (2000)

K.Takuma、T.Fujita、Y.Kimura、M.Tanabe A.Yamamuro、E.Lee：“T-588 通过丝裂原激活蛋白激酶信号通路抑制星形胶质细胞凋亡。”Eur。

K.Takuma, E.Lee, R.Enomoto, K.Mori, A.Baba, T.Matsuda: "Ibudilast attenuates astrocyte apoptosis via cyclic GMP signalling pathway in an in vitro reperfusion model."Br. J. Pharmacol. 133(6). 841-8 (2001)

K.Takuma、E.Lee、R.Enomoto、K.Mori、A.Baba、T.Matsuda：“在体外再灌注模型中，异丁司特通过环 GMP 信号通路减弱星形胶质细胞凋亡。”Br。

Takuma, K.et al.: "Anti-apoptotic effect of cGMP in cultured astrocytes : Inhibition by cGMP-dependent protein kinase of mitochondrial permeable transition pore"J.Biol.Chem.. 276(51). 48093-48099 (2001)

Takuma, K.等人：“培养星形胶质细胞中 cGMP 的抗凋亡作用：线粒体通透性转换孔的 cGMP 依赖性蛋白激酶的抑制”J.Biol.Chem.. 276(51)。

Lee, E.et al.: "A Selective Plasmin Inhibitor, trans-aminomethylcyclohexanecarbonyl-L-(O-picolyl)tyrosine-octylamide (YO-2), Induces Thymocyte Apoptosis"Biochem.Pharmacol.. (in press). (2002)

Lee, E.等人：“选择性纤溶酶抑制剂，反式氨基甲基环己烷羰基-L-(O-吡啶甲基)酪氨酸-辛酰胺 (YO-2)，诱导胸腺细胞凋亡”Biochem.Pharmacol..（出版中）。

Enomoto, R.et al.: "Phosphorylation of histones triggers DNA fragmentation in thymocytes undergoing apoptosis induced by protein phosphatase inhibitors"Mol.Cell Biol.Res.Commun.. 4. 276-281 (2001)

Enomoto, R.等人：“组蛋白的磷酸化会触发胸腺细胞中的 DNA 片段化，从而经历蛋白磷酸酶抑制剂诱导的细胞凋亡”Mol.Cell Biol.Res.Commun.. 4. 276-281 (2001)