[Potentials of DNA-PKcs, Ku80, and ATM in enhancing radiosensitivity of cervical carcinoma cells].
OBJECTIVE
DNA double strand break (DSB) is the lethal damage of cells after irradiation. DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and ataxia-telangiectasia mutated (ATM) are the most important repair proteins of DSB. Cervical carcinoma is mainly treated by radiotherapy; however, the tumor cells display different radiosensitivity. This study tried to explore the correlations of DNA-PKcs, Ku80, and ATM expression to radiosensitivity of cervical cancer cells, and to probe their potentials in enhancing the radiosensitivity of cervical carcinoma.


METHODS
The expression of DNA-PKcs, Ku80, and ATM in 41 specimens of cervical carcinoma was detected by immunohistochemistry. Their expression in 8 tumor cell lines (including 4 cervical carcinoma cell lines) was measured by Western blot; the survival fraction at 2 Gy (SF2) and alpha value were measured by colony formation test; the correlations of protein expression to SF2 and alpha values were analyzed by Pearson linear correlation analysis. The small hairpin RNA (shRNA) targeting DNA-PKcs and a competitive DNA-PKcs inhibitor LY294002 were used to inhibit DNA-PKcs expression and activity in cervical carcinoma cell line HeLa. The SF2 and alpha values of HeLa cells after X-ray irradiation were measured by colony formation test; cell apoptosis was analyzed by flow cytometry.


RESULTS
The positive rates of Ku80, DNA-PKcs, and ATM in the 41 specimens of cervical carcinoma were 70.73%, 68.29%, and 19.51%, respectively. The expression of DNA-PKcs was positively related to the SF2 values of the 8 tumor cell lines (r = 0.72, P = 0.04); the expression of Ku80 and ATM had no correlation to SF2 and alpha values. The SF2 value was lower in DNA-PKcs shRNA-transfected HeLa cells than in control HeLa cells (0.37 vs. 0.53, P < 0.05). When treated with 50 mumol/L LY294002 for 1 h, the apoptosis rate of HeLa cells had no significant change ( P > 0.05); when irradiated by 6 Gy X-ray for 48 h and 72 h, the apoptosis rate was significantly higher in LY294002-pretreated HeLa cells than in control HeLa cells (t = 3.25, P = 0.03; t = 3.01, P = 0.04).


CONCLUSIONS
DNA-PKcs protein is highly expressed in cervical carcinoma, and its expression level could prognosticate the radiosensitivity of tumor cells. Inhibiting DNA-PKcs expression or activity may sensitize HeLa cells to X-ray.
Zhuang
Liang
L
Cancer Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, P. R. China.
Yu
Shi-Ying
SY
Huang
Xiao-Yuan
XY
Cao
Yang
Y
Xiong
Hui-Hua
HH
D000328:Adult; D000368:Aged; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002867:Chromones / Q000494:pharmacology; D004265:DNA Helicases / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D006367:HeLa Cells / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D000072200:Ku Autoantigen; D008875:Middle Aged; D009025:Morpholines / Q000494:pharmacology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D011836:Radiation Tolerance; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D002583:Uterine Cervical Neoplasms / Q000378:metabolism* / Q000473:pathology; D055815:Young Adult
D018797:Cell Cycle Proteins; D002867:Chromones; D004268:DNA-Binding Proteins; D009025:Morpholines; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen