DNA-PKcs-HDAC6复合物调控放射损伤细胞有丝分裂进程机制研究

Mitosis regulation and spindle formation stability are as important as other DNA damage checkpoints and repair mechanisms in maintaining genomic stability/chromosomal stability and preventing carcinogenesis after irradiation. How DNA damage response coupled mitotic progression regulation has not been fully clarified. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is well known as a critical component involving the nonhomologous end joining pathway of DNA double-strand breaks repair. Our preliminary data showed another important role of DNA-PKcs in stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage. Inactivation of DNA-PKcs by specific inhibitor NU7441 resulted in an increased outcome of multipolar spindles and misalignment chromosome cells. Further, DNA-PKcs deficient cells are highly sensitive to HDACs inhibitor TSA and SAHA, and exhibit high incidence of mitotic cells with abnormal spindle structure, suggesting that the coordination between DNA-PKcs and HDACs is crucial to ensure mitosis progression. HDAC6 belongs to class II of the histone deacetylase family and participates in mitosis regulation through mediating deacetylation of several non-histone proteins, including HSP90 and a-tubulin. Loss of HDAC6 stabilizes acetylation of HSP90, thus inhibiting HSP90 chaperone function. The inhibition of HSP90 leads to mitotic defects which appear to be a consequence of the destabilization of mitosis-specific HSP90 clients: Aurora A and PLK1. Our preliminary data also proved that DNA-PKcs protect Aurora A and PLK1 protein stability after HDACi treatment. And DNA-PKcs interacts with HDAC6 directly. Here, we hypothesize that DNA-PKcs-HDAC6 signal pathway might be an important regulator coupled irradiation induced DNA damage response and mitosis progression regulation. In this project, we will further determine: 1) whether DNA-PKcs coordinates enzyme activity of HDAC6 through DNA-PKcs mediated phosphorylation; 2) whether the DNA-PKcs-mediated HDAC6 phosphorylation contributes to mitosis regulation in response to irradiation; 3) if the DNA-PKcs-mediated HDAC6 phosphorylation contributes to genomic stability/chromosomal stability maintenance after irradiation; HDAC6 also participate in primary cilia organization, which was reported to coordinate DNA damage response and mitosis progression, therefore 4) we will also test whether DNA-PKcs-HDAC6 complex involves in cilia assembly.

有丝分裂调控与纺锤体稳定性同细胞周期监测点一样，在电离辐射后维持细胞基因组/染色体稳定性、抑制肿瘤发生过程中发挥功能。DNA-PKcs是DNA双链断裂损伤非同源末端连接信号通路重要分子。DNA-PKcs在DNA损伤后稳定纺锤体结构，阻止有丝分裂灾变。我们发现DNA-PKcs与去乙酰化酶 HDAC6相互作用，DNA-PKcs缺失细胞HDAC抑制剂的敏感性增加。本项目将探索：1）DNA-PKcs是否通过磷酸化促进HDAC6去乙酰化酶活性；2）DNA-PKcs介导的HDAC6磷酸化是否参与调控电离辐射后有丝分裂进程；3）DNA-PKcs介导的HDAC6磷酸化是否有助于维持电离辐射后基因组和染色体稳定性； HDAC6调控纤毛装配，最新报道纤毛在有丝分裂和DNA损伤反应中发挥功能，因此4）探讨DNA-PKcs-HDAC6在纤毛装配中的作用。

本研究探讨了 DNA-PKcs 与 HDAC6 之间的关系, 并确定了该复合物在DNA 损伤耦合有丝分裂调控中的新作用。.(1) 在这里, 我们发现DNA-PKcs 缺失或 DNA-PKcs 激酶活性的抑制使细胞对 HDAC抑制剂更敏感, 包括TSA和SAHA。与野生型细胞相比，DNA-PKcs缺失HCT116细胞在HDAC抑制剂处理后呈现更高的细胞凋亡水平。由于 HDAC6 在有丝分裂期微管组装中的重要作用, 我们分析了 HDAC 抑制剂诱导的有丝分裂突变, 发现DNA-PKcs缺失细胞与对照细胞相比表现出更高水平的异常有丝分裂。与野生类型细胞相比, Aurora A 的蛋白质稳定性显著下降, 这是由于DNA-PKcs敲除HCT116细胞在TSA处理后HSP90的乙酰化增加更显著。.（2）一种新型的香兰素衍生物, 6-溴-5-羟基-4-甲氧基苯甲醛 (BVAN08) 最近被报道激活了癌细胞中不同的细胞死亡途径。在本研究中, 我们证明 BVAN08通过抑制重要的有丝分裂激酶 PLK1 的表达, 对ESCC细胞（TE-1和ECA-109）具有强大的抗增殖作用。与此一致的是, BVAN08会诱导ESCC细胞中的有丝分裂抑制和染色体错位。在BVAN08处理的ESCC细胞中也观察到微管核在质心周围的破坏。此外, BVAN08通过延长DNA损伤修复时间, 提高了ESCC细胞的放射敏感性。