The role of ATR signaling in chondrosarcoma cells responding to radiation-induced bystander effects

本研究旨在从DNA损伤反应（DDR）信号的角度阐明软骨肉瘤HTB 94细胞缺乏长期旁观者反应的分子机制（0.5或1戈伊），并使用trans-well insert系统与未照射的细胞共培养，所述trans-well insert系统具有和不具有共济失调毛细血管扩张和Rad 3相关蛋白（ATR）的抑制剂或共济失调毛细血管扩张突变（ATM），其是两种主要的DDR信号传导蛋白。微核（MN）的形成进行了检查，通过分裂阻断MN测定，这是一个强大的方法来检测持续的DNA损伤。免疫荧光染色检测到DDR蛋白的激活，MN分析显示，在旁观者HTB 94细胞中，72 h后没有MN形成，而ATR抑制剂的存在显著增加了MN的形成，而ATM抑制剂则没有，表明ATR介导的DNA损伤反应对于旁观者应答细胞是重要的。此外，ATR信号的下游蛋白包括γ H2 AX、pRPA 2（S33）、BRCA 1和RAD 51在旁观者HTB 94细胞中形成病灶。RAD 51的阻断剂促进旁观者MN形成。这些数据表明，ATR依赖性信号传导可能在非靶向HTB 94细胞中起关键作用，以修复由辐射细胞释放的信号引起的DNA损伤，使得在这些细胞中没有观察到长期旁观者应答。

This study aims to elucidate the molecular mechanisms underlying the lack of long-term bystander responses in chondrosarcoma HTB94 cells by focusing on DNA damage response (DDR) signaling.HTB94 cells were irradiated with X-rays (0.5 or 1 Gy) and co-cultured with un-irradiated cells using a trans-well insert system with and without inhibitors of Ataxia telangiectasia and Rad3-related protein (ATR) or Ataxia telangiectasia mutated (ATM) that are two-main DDR signaling proteins. Micronucleus (MN) formation was examined by the cytokinesis-block MN assay, which is a robust method to detect persistent DNA damage. The activation of DDR proteins was detected by immunofluorescence staining.MN analysis showed that MN were not formed after 72 h in the bystander HTB94 cells, however, the presence of ATR, but not ATM, inhibitor significantly increased the MN formation, indicating that ATR-mediated DNA damage response is important for the bystander responsive cells. Furthermore, downstream proteins of ATR signaling including γH2AX, pRPA2 (S33), BRCA1, and RAD51 were formed as foci in the bystander HTB94 cells. A blocker of RAD51 facilitated bystander MN formation. These data suggest that ATR-dependent signaling may play a critical role in the non-targeted HTB94 cells to repair DNA damage caused by the signals released from the irradiated cells so that no long-term bystander responses are observed in these cells.