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


CONCLUSIONS
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.
Zeng
Zhao-Chong
ZC
Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai, 200032, China. zeng@guomai.sh.cn
Jiang
Guo-Liang
GL
Wang
Guo-Min
GM
Tang
Zhao-You
ZY
Curran
Walter J
WJ
Iliakis
George
G
D006528:Carcinoma, Hepatocellular; D002470:Cell Survival / Q000528:radiation effects; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D006801:Humans; D006979:Hyperthermia, Induced; D008113:Liver Neoplasms; D009687:Nuclear Proteins; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011836:Radiation Tolerance; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases