DNA-PKcs、Ku80及ATM备选宫颈癌放疗增敏靶点的体外研究

背景与目的:DNA双链断裂(DNA double strand break,DSB)是细胞受辐射后最致命的损伤,而DNA依赖蛋白激酶催化亚单位(DNA-dependent protein kinase catalytic subunit,DNA-PKcs)、Ku80和ATM(ataxia-telangiectasia mutated)为DSB的主要修复蛋白.宫颈癌是以放疗为主要治疗手段的肿瘤.但其肿瘤细胞对放射线的敏感性不同.本实验拟研究3种DSB修复蛋白的表达与宫颈癌细胞放射敏感性的关系.并探讨DSB修复蛋白成为宫颈癌放疗增敏靶点的可能性.方法:免疫组化法检测41例宫颈癌患者组织中DNA-PKcs、Ku80和ATM蛋白的表达情况;Western blot检测8株肿瘤细胞(包括4株宫颈癌细胞)中3种蛋白的表达,克隆形成实验检测SF2 (suivival fraction at 2 Gv)、α值,分析蛋白表达水平和SF2、α值的关系;利用靶向抑制DNA-PKcs的shRNA表达质粒和小分子抑制剂LY294002,分别抑制HeLa细胞DNA-PKcs蛋白表达和活性后,克隆形成实验和流式细胞仪检测HeLa细胞受6 MVX线照射后的SF2、α值和凋亡率变化.结果:在41例宫颈癌组织中,Ku80、DNA-PKcs和ATM的阳性率分别为70.73%、68.29%和19.51%:8株肿瘤细胞中Ku80、DNA.PKcs和ATM蛋白的相对表达量与各细胞SF2、α值各不相同,作Pearson线性相关分析后得出DNA-PKcs的表达水平与SF2之间有明显的正相关关系(r=0.72,P=0.04);靶向抑制DNA-PKcs的shRNA可以促进HeLa细胞的放射敏感性,其SF2值为0.37,显著低于对照HeLa细胞的0.53(P＜0.05);单独接受50 μmol/L LY294002作用1 h HeIa细胞的凋亡率未见明显增加,但先经LY294002处理再照射6 Gy的HeLa细胞在48 h和72 h的凋亡率比单独照射6 Gy的HeLa细胞凋亡率显著增加(48 h点:t=3.25,P=0.03;72h点:t=3.01,P=0.04).结论:DNA-PKcs在宫颈癌组织中表达较高,且其表达水平可以预示肿瘤细胞的放射敏感性:抑制DNA-PKcs的表达或活性可以促进HeLa细胞的放射敏感性.

Potentials of DNA-PKcs, Ku80, and ATM in enhancing radiosensitivity of cervical carcinoma cells

BACKGROUND & 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.