脱氧胞苷激酶Ser-74位点在辐射诱导的乳腺癌细胞死亡中的作用

Roles of dCK Ser-74 in radiation-induced cell death in breast cancer cells

目的研究脱氧胞苷激酶（dCK）S74位点在辐射诱导乳腺癌细胞MCF-7死亡中的作用。方法采用脂质体转染方法在MCF-7中分别构建dCK—Vector（空载体）、dCK—WT（野生型）、dCK-S74A（非磷酸化型）及dCK-S74E（过磷酸化型）4种细胞模型，均分别予以0，2，4，6和8GyX射线照射。实时定量PCR（QPCR）及Westernblot验证细胞模型，CCK-8法和集落形成实验检测细胞存活率，单丹磺酰尸胺（MDC）检测自噬发生率，流式细胞术检测细胞凋亡率。结果成功构建4种细胞模型；CCK_8结果显示，与0Gy组相比，MCF-7和dCK—Vector经8Gy照射后存活率下降（t=14．469和9．357，P〈0．05），dCK-WT、dCK-S74E和dCK-S74A没有改变（P〉0．05）；与dCK-Vector比较，dCK-wT和dCK．$74E照射后的总死亡率降低（X2=3．857～3．971和3．857—3．859，P〈0．05），dCK-S74A则没有变化（P〉0．05）；与各自0Gy组比较，MCF7、dCK—Vector和dCK-S74A照射后凋亡发生率升高（t=-4．531、-3．688和-7．076，P〈0．05），而dCK—wT和dCK-S74E使照射诱导的凋亡率逆转了66％和68％；dCK—wT和dCK-S74E照射后自噬率分别增加22％和26％（f=-9．051和-8．411，P〈0．01），dCK-S74A、MCF-7和dCK—Vector照射后的自噬率没有明显变化（P〉0．05）。结论riCK-WT和dCK-S74E使电离辐射诱导的凋亡率增加发生逆转，同时使自噬率增加，总死亡率降低，提示dCK在Ser．74的磷酸化与乳腺癌细胞MCF-7的辐射敏感性有关。

Objective To investigate the roles of dCK Ser-74 in radiation-induced eel1 death in breast cancer cells. Methods Different phenotypes of dCK plasmids were transfeeted into MCF-7 cells by liposome transfection, including dCK-Vector, dCK-WT（ wild type）, dCK-S74A （non-phosphorylation） and dCK-S74E （hyper-phosphorylation）. All these cells were irradiated by 0, 2,4, 6, 8 Gy X-rays, respectively. The transcriptional and translational level of dCK were detected with real time-PCR and Western blot, respectively. Radiosensitivity was analyzed using cell counting kit （CCK-8） and colony formation assays. Monodansyleadaverine staining （MDC） and flow cytometry were used to detect autophagy and apoptosis, respectively. Results Four phenotypes of dCK cell models were established successfully. After irradiation, the cell viabilities of MCF-7 and dCK-Vector decreased significantly and compared with mock group （t = 14. 469 and 9. 357,P 〈 0. 05 ）, the cell viabilities of dCK-WT, dCK-S74A and dCK-S74E showed no changes （P 〉 0. 05 ）. The total mortalities of dCK-WT and dCK-S74E decreased significantly as compared with dCK-Vector （X2 = 3. 857 - 3. 971, P 〈 0. 05 ）, but no changes in dCK-S74A ceils （ P 〉0. 05 ）. The apoptosis rates in dCK-S74A, dCK-Vector and control group were up-regulated after irradiation （t = - 4. 531, - 3. 688 and - 7.076, P 〈 0.05 ）, and the irradiation-induced apoptosis was reversed in dCK-WT and dCK-S74E （66% and 68% of the increase level in dCK-Vector group）. The autophagy in dCK-WTanddCK-S74Eincreased by 22% and 26% （t = -9.051 and -8.411,P 〈0.01）, but no changes were observed in dCK-S74A, dCK-Vector and control groups （ P 〉 0. 05 ）. Conclusions The dCK-WT and dCK-S74E could reverse the irradiation-induced apoptosis, increase the autophagy occurence,and decrease the total mortality, indicating that the phosphorylation of dCK at Ser-74 sites is related to the radiosensitivity of MCF-7 cells.