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Molecular analysis of the role of DNases in radiation-induced apoptosis.

DNA酶在辐射诱导细胞凋亡中作用的分子分析。

基本信息

批准号：
11680547
负责人：
NODA Asao
金额：
$ 1.47万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680547/
关键词：
p53 apoptosis DNase γ Bax Bcl-2 Caspase VDAC Cytochrome C 紫外線 X線アポトーシス DNase CAD

项目摘要

Ionizing radiation (IR) induced apoptosis is a major causative mechanism underlying cell death by radiation therapy, and is dependent on the function of tumor suppressor p53 protein. We have analyzed the molecular mechanisms of IR-apoptosis in p53 (+) and p53 (-) cells, and found that (1) the final effector molecule of the IR-apoptosis is a well-known CAD (Caspase activated DNase) as well as unknown, Ca^<2+> and Mg^<2+> activated DNase. We also determined that (2) activated p53 induced Bax and Bcl-2 translocation in the miotchondrial (MT) outer menbrane, followed by cytochrome C (CytC) release.With regards to apoptotic DNase (1), treatments of Caspase 3 inhibitors blocked about 50% of DNA ladder formation, while remaining half of the activity was completely suppressed by the addition of Ca^<2+>/Mg^<2+> chelator. We isolated one of the candidate DNase genes, human DNase γ, and analyzed its possible involvement in IR-apoptosis. However, human DNase γ gene and protein were scarcely expressed in the typical IR-apoptotic cells, indicating that another DNase or unknown regulation of the DNase γ protein would be responsible for the Ca^<2+>/Mg^<2+> dependent DNase activity.With regards to MT regulation by IR (2), activation of p53 by IR was attributed to Ser 15 phosphorylation in its polypeptide, and then the activated form induced cytosolic Bax translocation to MT outermenbrane. We have found that Bcl-2 was sequestered from MT, and instead Bax traslocation accumulated by a VDAC binding from, indicating that VDAC pore opening was regulated by Bcl-2 negatively but by Bax positively. Our results suggestes that VDAC opening induces CytC release, followed by Caspase activation, which is the major mechanism of IR-apoptosis.

电离辐射（IR）诱导的细胞凋亡是放射治疗引起细胞死亡的主要机制，并依赖于抑癌基因p53蛋白的功能。我们分析了p53（+）和p53（-）细胞IR-凋亡的分子机制，发现（1）IR-凋亡的最终效应分子是已知的CAD（Caspase激活的DNA酶）以及未知的Ca^2+和Mg^2+激活的DNA酶。我们还确定：（2）激活p53可诱导线粒体外膜Bax和Bcl-2易位，随后细胞色素C（CytC）释放。对于凋亡DNA酶（1），Caspase 3抑制剂可阻断约50%的DNA梯状条带形成，而其余一半的活性可被Ca^2+/Mg^2+螯合剂完全抑制。我们分离了候选DNA酶基因之一，人DNA酶γ，并分析了其可能参与IR-凋亡。然而，在典型的IR-凋亡细胞中，人DNase γ基因和蛋白几乎不表达，这表明另一种DNase或未知的DNase γ蛋白调节可能是Ca^2+/Mg^2+依赖的DNase活性的原因。关于IR对MT的调节（2），IR激活p53是由于其多肽中Ser 15磷酸化，激活后的Bax可转位到MT外膜。我们已经发现Bcl-2与MT隔离，而Bax易位通过VDAC结合积累，表明VDAC孔开放由Bcl-2负调控，但由Bax正调控。我们的研究结果表明，VDAC开放诱导CytC释放，然后激活Caspase，这是IR凋亡的主要机制。