Determination of Clonogenic Radiosensitivity of HeLa Cells in Early and Late G Phases.

BACKGROUND/AIM: Using a fluorescent ubiquitination-based cell cycle indicator (Fucci), we recently reported that post irradiation of HeLa cells, micronuclei frequency increased in the early G phase in comparison with that in the late G phase. This is inconsistent with the results of well-recognized studies that used clonogenic assays. In this study, we determined radiosensitivity of the cells using a clonogenic assay by making the best use of the Fucci property, while simultaneously characterizing cell cycle kinetics and DNA damage responses.

MATERIALS AND METHODS

Early and late G phase cell fractions were isolated using a cell sorter by exploiting the different red fluorescence intensities of Fucci. Radiosensitivity was determined by the colony formation assay. Time-lapse imaging and immunostaining were performed to analyze cell cycle kinetics and DNA damage.

RESULTS

Late G cells were more radioresistant than early G cells. Cells irradiated in the early and late G phases induced G arrest, while the latter demonstrated a significantly longer duration of G arrest. This difference became more evident as the radiation dose increased. Furthermore, 16 h after irradiation, a greater number of γH2AX foci remained in cells irradiated in the early G phase than in those irradiated in the late G phase.

CONCLUSION

HeLa cells in the late G phase are more radioresistant than those in the early G phase, presumably because DNA damage is efficiently repaired during a longer G arrest in late G cells.