Studies on Molecular Mechanism of DNA Fragmentation in thymocytes undergoing Apoptosis

1)细胞凋亡前磷酸化组蛋白的鉴定据报道，细胞凋亡是由calyculin A和冈田酸诱导的，它们是多种细胞系中的蛋白磷酸酶抑制剂。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。本研究尝试了磷酸化组蛋白的鉴定。组蛋白H1、H2 A和H3的磷酸化在calyculin A诱导的胸腺细胞DNA片段化之前增加。另一方面，用calyculin A处理细胞后，各组蛋白含量没有变化。为了明确组蛋白的化学修饰作为细胞凋亡的常见机制是否重要，还检查了经历人星形胶质细胞细胞凋亡的组蛋白的化学修饰。将人星形胶质细胞暴露于生理盐水后，用生长培养基培养，细胞受到损伤。这种细胞损伤具有凋亡的特征。磷光体 关于我们 2）组蛋白的化学修饰对染色质结构的影响DNA断裂是细胞凋亡的生化标志，与凋亡DNA断裂有关的DNA酶也已阐明。然而，DNA片段化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通过改变细胞核中染色质DNA的结构而与转录调控有关。我们试图验证细胞凋亡过程中染色质结构的变化。用圆二色谱法测定了经calyculin A处理的细胞核可溶性染色质的圆二色谱。虽然calyculin A处理不影响DNA的结构，但calyculin A处理的染色质中的α-螺旋含量减少。在地塞米松诱导的胸腺细胞凋亡中也观察到了染色质结构的改变。对DNA酶的敏感性增加了来自对照的calyculin A处理的细胞的制备的染色质。这些结果表明，组蛋白H2 A的磷酸化是重要的凋亡过程中的一个共同的机制。此外，组蛋白磷酸化通过减少染色质的螺旋含量使染色质结构松弛，并通过改变染色质结构增加对DNA酶的敏感性。3）寻找新的凋亡诱导剂。寻找一种新的凋亡诱导剂，以明确染色质结构变化是否是细胞凋亡的共同机制。由Okada合成的YO-et.al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，这表明了染色质结构变化在凋亡性细胞死亡中的重要性。4）培养的大鼠星形胶质细胞凋亡性细胞死亡机制的研究。将培养的大鼠星形胶质细胞暴露于无Ca^<2 +>的培养基后，再灌注含Ca ^<2 +>的培养基，导致细胞内Ca^<2+>浓度增加，随后出现延迟的细胞死亡，包括凋亡。一些化合物如T-588和CV-2619保护培养的大鼠星形胶质细胞免受再灌注损伤。此外，CGMP通过激活PKG抑制线粒体通透性转换孔，防止凋亡细胞死亡。少

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