Abstract
We previously reported that the delayed production of intracellular ROS induced by X-irradiation was derived from mitochondrial electron transport chain in human lung carcinoma A549 cells. On the other hand, X-irradiation is known to cause cell cycle arrest in G2 phase under the control of G2 check point. Recent studies indicated that cell cycle took part in the regulation of mitochondrial fission and fusion. We therefore hypothesized that cell cycle was involved in the regulation of intracellular ROS level in X-irradiated cells. To test this hypothesis, we performed a simultaneous analysis of intercellular ROS level and cell cycle.
A549 cells were irradiated with 10 Gy X-rays. Propidium iodide (PI) or Nuclear-IDTM Red DNA Stain was used for the assessment of cell cycle. Intracellular ROS level and mitochondrial membrane potential were evaluated with DCFDA and DiOC6, respectively. Fluorescent intensities of these dyes were analyzed by flow cytometry. Nuclear-IDTM Red DNA Stain was used for double fluorescent staining with DCFDA or DiOC6.
Intracellular ROS level was significantly increased at 12 hours after irradiation. When intracellular ROS level in each cell cycle fraction (G1, S, G2/M) was analyzed, similar degree of increase in ROS level was observed in each cell cycle fraction after X-irradiation. In addition, intracellular ROS level and mitochondrial membrane potential in G2 phase were higher than in G1 and S phase without X-irradiation. These results suggested that both mitochondrial activation and the accumulation of cell population in G2 phase contributed to the increase of intracellular ROS level after X-irradiation.
