Mitochondrial dynamics has been suggested to be indispensable for the maintenance of cellular quality and function in response to various stresses. While ionizing radiation (IR) elicits mitochondrial fission mediated by the mitochondrial fission protein dynamin-related protein 1 (Drp1), it remains unclear how IR promotes Drp1 activation and subsequent mitochondrial fission. Therefore, we conducted this study to answer these questions. First, we found that X-irradiation triggered Drp1 phosphorylation at serine 616 (S616), but not at serine 637 (S637). Reconstitution analysis revealed that introduction of WT Drp1 recovered radiation-induced mitochondrial fission, which was lost in Drp1-deficient cells. Compared with the cells transfected with WT or S637A Drp1, the mitochondrial shape change after irradiation was mitigated in S616A Drp1-introduced cells. Furthermore, inhibition of CaMKII significantly suppressed Drp1 S616 phosphorylation and mitochondria fission induced by IR. These results suggest that Drp1 phosphorylation at S616, but not at S637, is prerequisite for radiation-induced mitochondrial fission and that CaMKII regulates Drp1 phosphorylation at S616 after irradiation.

Excessive DNA damage induced by ionising radiation (IR) to normal tissue cells is known to trigger cellular senescence, a process termed stress-induced premature senescence (SIPS). SIPS is often accompanied by the production of reactive oxygen species (ROS), and this is reported to be important for the initiation and maintenance of SIPS. However, the source of ROS during SIPS after IR and their significance in radiation-induced normal tissue damage remain elusive. In the present study, we tested the hypothesis that the NADPH oxidase (NOX) family of proteins mediates ROS production in SIPS-induced cells after IR and plays a role in SIPS-associated biological events. X-irradiation of primary mouse embryonic fibroblasts (MEFs) resulted in cellular senescence and the concomitant increase of intracellular ROS. Among all six murine NOX isoforms (NOX1-4 and DUOX1/2), only NOX4 was detectable under basal conditions and was upregulated following IR. In addition, radiation-induced ROS production was diminished by genetic or pharmacological inhibition of NOX4. Meanwhile, NOX4 deficiency did not affect the induction of cellular senescence after IR. Furthermore, the migration of human monocytic U937 cells to the culture medium collected from irradiated MEFs was significantly reduced by NOX4 inhibition, suggesting that NOX4 promotes the recruitment of inflammatory cells. Collectively, our findings imply that NOX4 mediates ROS production in radiation-induced senescent cells and contributes to normal tissue damage after IR via the recruitment of inflammatory cells and the exacerbation of tissue inflammation.

The role of mitochondrial dynamics in cellular responses to ionizing radiation (IR) is still largely unknown. This study demonstrates that IR triggers Drp1-dependent mitochondrial fission and that Drp1 inhibition attenuates radiation-induced mitotic catastrophe, suggesting that Drp1 is involved in determining the fate of cells after irradiation.

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Checkpoint kinase 1 (Chk1) is an evolutionarily conserved serine/threonine kinase that plays an important role in G2/M checkpoint signaling. Here, we evaluate the radiosensitizing effects of a novel selective Chk1 inhibitor MK-8776, comparing its efficacy with a first-generation Chk1 inhibitor UCN-01, and attempt to elucidate the mechanism of radiosensitization. In a clonogenic survival assay, MK-8776 demonstrated a more pronounced radiosensitizing effect than UCN-01, with lower cytotoxicity. Importantly, radiosensitization by MK-8776 can be achieved at doses as low as 2.5 Gy, which is a clinically applicable irradiation dose. MK-8776, but not UCN-01, exacerbated mitotic catastrophe (MC) and centrosome abnormalities, without affecting repair kinetics of DNA double strand breaks. Furthermore, live-cell imaging revealed that MK-8776 significantly abrogated the radiation-induced G2/M checkpoint, prolonged the mitotic phase, and enhanced aberrant mitosis. This suggests that Chk1 inhibition by MK-8776 activates a spindle assembly checkpoint and increases mitotic defects in irradiated EMT6 cells. In conclusion, we have shown that, at minimally toxic concentrations, MK-8776 enhances radiation-induced cell death through the enhancement of aberrant mitosis and MC, without affecting DNA damage repair.