辐射诱导的旁观者效应 (RIBE) 通常被视为未受辐射的旁观者细胞中的 DNA 损伤，但针对 DNA 损伤而启动的修复过程尚不清楚。 RIBE 介导的微核 (MN) 形成是持久性 DNA 损伤的生物标志物，之前在旁观者正常成纤维细胞 (AG01522) 细胞中观察到，但在旁观者人软骨肉瘤 (HTB94) 细胞中未观察到。造成这种差异的分子机制尚不清楚。在此，我们研究了 DNA 修复在两种细胞系的旁观者反应中的作用。用 X 射线照射细胞，并立即使用跨孔插入系统与未照射的细胞共培养，其中它们共享相同的培养基。通过免疫荧光染色或蛋白质印迹检测 DNA 损伤反应 (DDR) 蛋白的激活。通过胞质分裂阻断 MN 测定来检查 MN 的形成，这是一种检测持续性 DNA 损伤的可靠方法。γH2AX 和 53BP1（DNA 损伤和修复的生物标志物）的免疫荧光焦点显示，HTB94 细胞的 DNA 修复能力高于 AG01522受辐射群体和旁观者群体中的细胞。与基线时的AG01522细胞相比，HTB94细胞中苏氨酸1989位点的ATR自磷酸化以更高水平表达，并且响应于羟基脲处理或暴露于1Gy的X射线。 ATR 抑制剂（而非 ATM）促进旁观者 HTB94 细胞中 MN 的形成。相比之下，在旁观者 AG01522 细胞中没有观察到任何一种抑制剂的作用，表明 ATR 信号传导可能是防止旁观者 HTB94 细胞中 MN 形成的关键途径。支持这一观点的是，我们发现具有 pRPA2 S33 和 RAD51 焦点的旁观者 HTB94 细胞的分数存在 ATR 依赖性增加。 RAD51 的阻断剂促进旁观者 HTB94 细胞中 MN 的形成。我们的结果表明，HTB94 细胞的 DNA 修复可能比 AG01522 细胞更有效，特别是通过 ATR 信号传导，该信号抑制旁观者信号诱导的 MN 形成。这项研究强调了 DNA 修复效率在旁观者细胞反应中的重要性。

Radiation-induced bystander effect (RIBE) frequently is seen as DNA damage in unirradiated bystander cells, but the repair processes initiated in response to that DNA damage are not well understood. RIBE-mediated formation of micronuclei (MN), a biomarker of persistent DNA damage, was previously observed in bystander normal fibroblast (AG01522) cells, but not in bystander human chondrosarcoma (HTB94) cells. The molecular mechanisms causing this disparity are not clear. Herein, we investigate the role of DNA repair in the bystander responses of the two cell lines.Cells were irradiated with X-rays and immediately co-cultured with un-irradiated cells using a trans-well insert system in which they share the same medium. The activation of DNA damage response (DDR) proteins was detected by immunofluorescence staining or Western blotting. MN formation was examined by the cytokinesis-block MN assay, which is a robust method to detect persistent DNA damage.Immunofluorescent foci of γH2AX and 53BP1, biomarkers of DNA damage and repair, revealed a greater capacity for DNA repair in HTB94 cells than in AG01522 cells in both irradiated and bystander populations. Autophosphorylation of ATR at the threonine 1989 site was expressed at a greater level in HTB94 cells compared to AG01522 cells at the baseline and in response to hydroxyurea treatment or exposure to 1 Gy of X-rays. An inhibitor of ATR, but not of ATM, promoted MN formation in bystander HTB94 cells. In contrast, no effect of either inhibitor was observed in bystander AG01522 cells, indicating that ATR signaling might be a pivotal pathway to preventing the MN formation in bystander HTB94 cells. Supporting this idea, we found an ATR-dependent increase in the fractions of bystander HTB94 cells with pRPA2 S33 and RAD51 foci. A blocker of RAD51 facilitated MN formation in bystander HTB94 cells.Our results indicate that HTB94 cells were likely more efficient in DNA repair than AG01522 cells, specifically via ATR signaling, which inhibited the bystander signal-induced MN formation. This study highlights the significance of DNA repair efficiency in bystander cell responses.