DNA-PKcs subunits in radiosensitization by hyperthermia on hepatocellular carcinoma hepG2 cell line

Abstract

Aim: To investigate the role of DNA-PKcs subunits in radiosensitization by hyperthermia on hepatocellular carcinoma HepG(2) cell lines.

Methods: Hep G(2) cells were exposed to hyperthermia and irradiation. Hyperthermia was given at 45.5 degrees C. Cell survival was determined by an in vitro clonogenic assay for the cells treated with or without hyperthermia at various time points. DNA DSB rejoining was measured using asymmetric field inversion gel electrophoresis (AFIGE). The DNA-PKcs activities were measured using DNA-PKcs enzyme assay system.

Results: Hyperthermia can significantly enhance irradiation-killing cells. Thermal enhancement ratio as calculated at 10 % survival was 2.02. The difference in radiosensitivity between two treatment modes manifested as a difference in the alpha components and the almost same beta components, which alpha value was considerably higher in the cells of combined radiation and hyperthermia as compared with irradiating cells (1.07 Gy(-1) versus 0.44 Gy(-1)). Survival fraction showed 1 logarithm increase after an 8-hour interval between heat and irradiation, whereas DNA-PKcs activity did not show any recovery. The cells were exposed to heat 5 minutes only, DNA-PKcs activity was inhibited at the nadir, even though the exposure time was lengthened. Whereas the ability of DNA DSB rejoining was inhibited with the increase of the length of hyperthermic time. The repair kinetics of DNA DSB rejoining after treatment with Wortmannin is different from the hyperthermic group due to the striking high slow rejoining component.

Conclusion: Determination with the cell extracts and the peptide phosphorylation assay, DNA-PKcs activity was inactivated by heat treatment at 45.5 degrees C, and could not restore. Cell survival is not associated with the DNA-PKcs inactivity after heat. DNA-PKcs is not a unique factor affecting the DNA DSB repair. This suggests that DNA-PKcs do not play a crucial role in the enhancement of cellular radiosensitivity by hyperthermia.

Figure 1

Survival fraction of HepG₂. A: Exposure to X-rays combined with (close circles) or without (open circles) 45.5 °C for 15 min; B: Heat-induced clonogenic cell death as a function of time combined with (close circles) or without (open circles) 3 Gy of X-rays; C: Irradiated with 3 Gy of X-rays at different hours after heat of 15 min at 45.5 °C.

Figure 2

DNA-PKcs activity of HepG2 cells. A: Cells were heated at 45.5 °C for 20 min and then received 40 Gy of X-rays (closed circles) or irradiated with 40 Gy of X-rays only (open circles). At various periods, DNA-PKcs activity in heated cells was inhibited, and kept at low level about 30%. The DNA-PKcs activity in both groups showed slight increase; B: After heated at 45.5 °C for 20 min, DNA-PKcs activity was still inhibited at various periods; C: DNA-PKcs activity was inhibited after exposure to heat at 45.5 °C for 5 min, even though the heat time was prolonged, DNA-PKcs activity levels still remained unchanged.

Figure 3

Dose response curves for HepG2 cells received radiation combined with or without hyperthermia. The upper panel shows a typical gel scanned with ¹⁴C-TdR, while the lower panel shows quantitative data as described in the Methods. The FAR increases almost linearly with dose up to 30 Gy but bends downward at higher doses. Similar increases in FAR as a function of dose are observed in radiation alone or combined with hyperthermia, suggesting similar yields of DNA DSBs.

Figure 4

DNA DSBs repair kinetics in HepG2 cells. Cells were irradiated with 40 Gy and prepared for AFIGE after various periods of incubation at 37 °C to allow for repair. The upper panel in the Figure shows a typical AFIGE gel, while the lower panel shows its quantification as described in the materials and Methods. The rejoining of DNA DSBs was decreasing with the increase of the lengths of hyperthermic time at 45.5 °C.

Figure 5

The kinetics of DNA DSBs rejoining after treatment with Wortmannin. The slow rejoining component is strikingly high. The deficiency of DNA DSBs rejoining was found in Wortmannin treatment cells after 2 h.