Xue Lian, Furusawa Yoshiya, Okayasu Ryuichi, Miura Masahiko, Cui Xing, Liu Cuihua, Hirayama Ryoichi, Matsumoto Yoshitaka, Yajima Hirohiko, Yu Dong
School of Public Health, Medical College of Soochow University, Suzhou, China; Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, School of Public Health, Soochow University, Suzhou, China.
Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba, Japan.
DNA Repair (Amst). 2015 Jan;25:72-83. doi: 10.1016/j.dnarep.2014.11.004. Epub 2014 Nov 24.
DNA double strand break (DSB) repair pathway choice following ionizing radiation (IR) is currently an appealing research topic, which is still largely unclear. Our recent paper indicated that the complexity of DSBs is a critical factor that enhances DNA end resection. It has been well accepted that the RPA-coated single strand DNA produced by resection is a signaling structure for ATR activation. Therefore, taking advantage of high linear energy transfer (LET) radiation to effectively produce complex DSBs, we investigated how the complexity of DSB influences the function of ATR pathway on the G2/M checkpoint regulation. Human skin fibroblast cells with or without ATM were irradiated with X rays or heavy ion particles, and dual-parameter flow cytometry was used to quantitatively assess the mitotic entry at early period post radiation by detecting the cells positive for phosphor histone H3. In ATM-deficient cells, ATR pathway played a pivotal role and functioned in a dose- and LET-dependent way to regulate the early G2/M arrest even as low as 0.2Gy for heavy ion radiation, which indicated that ATR pathway could be rapidly activated and functioned in an ATM-independent, but DSB complexity-dependent manner following exposure to IR. Furthermore, ATR pathway also functioned more efficiently in ATM-proficient cells to block G2 to M transition at early period of particle radiation exposure. Accordingly, in contrast to ATM inhibitor, ATR inhibitor had a more effective radiosensitizing effect on survival fraction following heavy ion beams as compared with X ray radiation. Taken together, our results reveal that the complexity of DSBs is a crucial factor for the activation of ATR pathway for G2/M checkpoint regulation, and ATM-dependent end resection is not essential for the activation.
电离辐射（IR）后DNA双链断裂（DSB）修复途径的选择是当前一个引人关注的研究课题，目前仍在很大程度上不清楚。我们最近的论文表明，DSB的复杂性是增强DNA末端切除的关键因素。人们已经普遍接受，由切除产生的RPA包被的单链DNA是ATR激活的信号结构。因此，利用高线性能量传递（LET）辐射有效地产生复杂的DSB，我们研究了DSB的复杂性如何影响ATR途径在G2/M期检查点调控中的功能。对有或没有ATM的人皮肤成纤维细胞进行X射线或重离子粒子照射，并使用双参数流式细胞术通过检测磷酸化组蛋白H3阳性细胞来定量评估辐射后早期的有丝分裂进入。在ATM缺陷细胞中，ATR途径发挥了关键作用，并以剂量和LET依赖性方式发挥作用，以调节早期G2/M期阻滞，即使对于重离子辐射低至0.2Gy，这表明ATR途径在暴露于IR后可以快速激活，并以不依赖ATM但依赖DSB复杂性的方式发挥作用。此外，ATR途径在ATM功能正常的细胞中也更有效地发挥作用，以在粒子辐射暴露早期阻断G2向M期的转变。因此，与ATM抑制剂相比，ATR抑制剂对重离子束照射后的存活分数具有比X射线辐射更有效的放射增敏作用。综上所述，我们的结果表明，DSB的复杂性是激活ATR途径进行G2/M期检查点调控的关键因素，并且依赖ATM的末端切除对于激活不是必需的。
