Zhang Xiuwu, Hadley Caroline, Jackson Isabel L, Zhang Yi, Zhang Angel, Spasojevic Ivan, Haberle Ines Batinic, Vujaskovic Zeljko
a Division of Translational Radiation Sciences, Department of Radiation Oncology , University of Maryland, School of Medicine , Baltimore , MD , USA ;
b Baylor University School of Medicine , Houston , TX , USA ;
Free Radic Res. 2016 Aug;50(8):875-86. doi: 10.1080/10715762.2016.1189078. Epub 2016 Jul 1.
The current study was designed to address our hypothesis that oxidative stress secondary to the ionizing event upregulates phosphatase and tensin homolog (PTEN) mRNA and protein in the lungs of C57BL/6J mice through oxidative DNA damage resulting in CpG hypomethylation in the PTEN promoter.
Fibrosis-prone C57BL/6J mice were exposed to 0 or 15 Gy of 320 kVp X-rays to the whole thorax. Lung tissue was serially harvested at time points between one day and six months postirradiation. Tissue levels of PTEN mRNA, total protein, and phosphorylated PTEN, as well as CpG methylation of the PTEN promoter, expression of DNA methyltransferases 1 (Dnmt1) and 3a (Dnmt3a), NADPH oxidase 4 (Nox4) protein expression, and DNA damage levels were measured. The induction of DNA damage and global methylation changes were also examined in hydrogen peroxide (H2O2)-treated human umbilical vein endothelial cells (HUVECs) and human bronchial epithelial cells in vitro.
These experiments demonstrate that PTEN mRNA and protein, Nox4 protein, and DNA damage levels increase continuously from one day to six months following radiation exposure. Elevated PTEN transcription and translation are likely the result of the observed decrease in CpG methylation of the PTEN promoter region. This finding is not consistent with the observed increase in Dnmt1 and Dnmt3a protein expression, implicating an alternative mechanism as the driving force behind hypomethylation. In vitro results provide evidence that H2O2 can induce DNA damage and affect DNA methylation status. The Mn porphyrin-based superoxide dismutase (SOD) mimic MnTnHEx-2-PyP(5+ )exhibited partial rescue from radiation-induced hypomethylation.
Taken together, these data suggest that reactive oxygen species (ROS)-induced DNA damage results in hypomethylation of the PTEN promoter, upregulation of PTEN mRNA and protein, and a subsequent increase in apoptosis in irradiated lung tissue.
本研究旨在验证我们的假设，即电离事件继发的氧化应激通过导致PTEN启动子区域CpG低甲基化的氧化性DNA损伤，上调C57BL/6J小鼠肺组织中磷酸酶和张力蛋白同源物（PTEN）的mRNA和蛋白质水平。
将易发生纤维化的C57BL/6J小鼠全胸暴露于0或15 Gy的320 kVp X射线下。在照射后1天至6个月的时间点连续收集肺组织。检测PTEN mRNA、总蛋白和磷酸化PTEN的组织水平，以及PTEN启动子的CpG甲基化、DNA甲基转移酶1（Dnmt1）和3a（Dnmt3a）的表达、NADPH氧化酶4（Nox4）蛋白表达和DNA损伤水平。还在体外检测了过氧化氢（H2O2）处理的人脐静脉内皮细胞（HUVECs）和人支气管上皮细胞中DNA损伤的诱导和整体甲基化变化。
这些实验表明，辐射暴露后1天至6个月，PTEN mRNA和蛋白、Nox4蛋白以及DNA损伤水平持续增加。PTEN转录和翻译的升高可能是由于观察到的PTEN启动子区域CpG甲基化减少所致。这一发现与观察到的Dnmt1和Dnmt3a蛋白表达增加不一致，提示存在另一种机制作为低甲基化的驱动力。体外实验结果表明，H2O2可诱导DNA损伤并影响DNA甲基化状态。基于锰卟啉的超氧化物歧化酶（SOD）模拟物MnTnHEx-2-PyP(5+ )可部分挽救辐射诱导的低甲基化。
综上所述，这些数据表明活性氧（ROS）诱导的DNA损伤导致PTEN启动子低甲基化、PTEN mRNA和蛋白上调，以及照射后肺组织中凋亡的随后增加。
