Molecular analysis of the role of DNases in radiation-induced 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之一，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^&lt；2+&gt；和Mg^&lt；2+&gt；我们还发现，(2)激活的P53诱导线粒体(MT)外膜Bax和Bcl2易位，随后细胞色素C(CytC)释放。对于凋亡DNA酶(1)，Caspase3抑制剂处理阻止了约50%的DNA梯带形成，而其余一半的活性完全被添加Ca^&lt；2+&gt；/mg^&lt；2+&gt；我们分离了候选脱氧核糖核酸酶基因之一，人脱氧核糖核酸酶γ，并分析了它可能参与IR细胞凋亡。然而，人脱氧核糖核酸酶γ基因和蛋白在典型的IR凋亡细胞中几乎没有表达，这表明另一种脱氧核糖核酸酶γ蛋白的未知调控可能是依赖于钙依赖的脱氧核糖核酸酶活性的原因。关于IR(2)对MT的调节，IR对P53的激活归因于其多肽中的Ser15磷酸化，然后激活的形式诱导胞浆Bax转位到MT外膜。我们发现，Bcl2是从MT中分离出来的，而Bax转位是通过VDAC结合的形式积累起来的，这表明VDAC的孔道开放受Bcl2负向调节，而Bax正向调节。我们的结果提示，VDAC开放诱导细胞释放CytC，继而激活Caspase，这是IR细胞凋亡的主要机制。

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.