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

本研究旨在通过关注DNA损伤反应（DDR）信号，阐明HTB94软骨肉瘤细胞缺乏长期旁观者反应的分子机制。HTB94细胞用x射线照射（0.5或1 Gy），并与未照射的细胞共培养，使用跨井插入系统，含或不含两种主要DDR信号蛋白共济失调毛细血管扩张和rad3相关蛋白（ATR）或共济失调毛细血管扩张突变（ATM）抑制剂。微核（MN）的形成是通过细胞分裂阻滞MN测定来检测的，这是一种检测持续DNA损伤的强大方法。免疫荧光染色检测DDR蛋白活化情况。MN分析显示，旁观者HTB94细胞在72 h后未形成MN，但ATR而非ATM的存在显著增加了MN的形成，表明ATR介导的DNA损伤反应对旁观者反应细胞是重要的。此外，ATR信号的下游蛋白包括γH2AX、pRPA2 （S33）、BRCA1和RAD51在旁观者HTB94细胞中形成病灶。RAD51的阻滞剂促进了旁观者MN的形成。这些数据表明，atr依赖性信号可能在非靶向HTB94细胞中发挥关键作用，以修复由辐照细胞释放的信号引起的DNA损伤，因此在这些细胞中没有观察到长期的旁观者反应。

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.