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

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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.