AMPK参与电离辐射DNA损伤修复及其作为辐射防护靶点的研究

Genetic instability is one of the hallmarks of cancer. It often arises from DNA damage and DNA replication errors. Recent findings demonstrated that AMP-activated protein kinase (AMPK), an established cell metabolic sensor, plays an important role in the detection of genomic stresses. However, it remains unknown how AMPK responds to those genomic stresses, in particular ionizing radiation (IR)-induced DNA damage, as yet no identifying hypothesis has convincingly explained how activation of AMPK contributes to DNA damage response (DDR), a critical mechanism that guards against genome instability.. The stimulus to propose this grant application is our preliminary findings that AMPK was recruited to DNA double strand break sites and forms nuclear foci after IR. AMPK foci co-localized with gama-H2AX and were predominantly dependent on the activity of the ATM kinase. Depletion of AMPK resulted in delayed DDR and reduced DNA repair capability after IR. More importantly, our protein profiling identified several clusters of DDR molecules, which were down-regulated by the depletion of AMPK in response to IR. Thus, we hypothesize that AMPK safeguards genomic stability through regulating DNA damage/repair response. As a corollary, activation of AMPK may possess a protective effect against IR. To address our hypothesis, we designed 3 specific aims: (1) to determine the role of AMPK in DDR and in the maintenance of genomic stability. (2) To explore the possible molecular mechanisms by which AMPK are activated under IR and identify downstream targets of AMPK in DDR. (3) To validate the effect of metformin, an AMPK activator, on the protection against ionizing radiation. . Results obtained from the proposed studies will yield novel findings that will suggest how AMPK integrates different signals in DDR and provide valuable new information to further our current knowledge in IR-induced DDR. These studies will also provide rationale for pharmacologic activation of AMPK in the protection against ionizing radiation.

DNA损伤是诱发基因组不稳定的重要原因。研究表明AMP激活的蛋白激酶(AMPK)不仅是调节细胞能量代谢的靶基因，而且还是基因组应激损伤时的感受分子。但AMPK在电离辐射（IR）DNA损伤中的作用至今不明。我们发现IR后AMPK形成核灶，募集至DNA双链断裂处。AMPK缺失细胞DNA损伤修复能力降低。蛋白组分析显示，AMPK缺乏致多个DNA损伤应答(DDR)蛋白表达降低。据此，我们认为AMPK在IR引起的DNA损伤修复和保持基因组稳定性中起重要作用，应用AMPK激活剂可起到辐射防护作用（假说）。本研究将明确IR后，AMPK表达、分布、磷酸化及其与 DDR 蛋白的相互作用；研究IR后AMPK在调控细胞周期检查点、凋亡、DNA损伤修复及保持基因组稳定中的作用；探讨IR后AMPK激活及调控DDR的分子机制；评估AMPK作为辐射防护靶点的可行性。研究结果对辐射损伤个体的防护具有高度的指导和应用价值。

DNA损伤是诱发基因组不稳定的重要原因。有研究表明AMP激活的蛋白激酶(AMPK)不仅是调节细胞能量代谢的靶基因，而且还是基因组应激损伤时的感受分子。但AMPK在电离辐射（IR）DNA损伤中的作用之前一直不明。基于我们的前期研究结果，我们初始的研究假设是＂AMPK在IR引起的DNA损伤修复和保持基因组稳定性中起重要作用，应用AMPK激活剂可起到辐射防护作用＂。..为明确AMPK在IR引起的DNA损伤应答（DDR）中的作用，我们观察了放射损伤对AMPK表达、分布、磷酸化的影响，探讨AMPK与DDR蛋白的相互作用；研究AMPK在放射损伤后调控细胞周期检查点、细胞凋亡、DNA损伤修复、及保持基因组稳定性中的作用；探讨放射损伤后，AMPK激活及调控DDR的上下游分子机制；评估AMPK激活剂二甲双胍在受辐照小鼠辐射防护中的作用。..我们的研究发现-射线照射可致AMPK激活。激活的AMPK在DNA损伤后形成核灶，并与-H2AX共定位，该作用依赖于ATM。AMPK作为DNA损伤反应蛋白，其激活亦受到UV和ROS等应激处理的调节。AMPK参与了IR引起的DNA损伤修复。用AMPK基因敲除及沉默细胞行IR照射，对照射后的细胞裂解成分行蛋白组分析，及其后的一些列验证试验，证实FKHRL1 (FOXO3a)在介导AMPK相关的DNA损伤修复中起了重要作用。FOXO3a还在辐射损伤后，AMPK介导的细胞周期阻滞、细胞老化和凋亡等一系列反应中起重要作用。AMPK激活剂二甲双胍对辐射损伤小鼠免疫及骨髓具有显著的保护作用。二甲双胍促进受X射线照射小鼠白细胞、中性粒细胞和血小板的恢复，明显降低X射线照射诱导的小鼠骨髓细胞染色体畸变率，降低受照射小鼠骨髓嗜多染红细胞中的微核率，维持受照射小鼠的脾脏指数，改善小鼠肠道菌群，对辐射引起的多脏器具有明显的保护作用。..总之，我们成功地完成了本项目研究计划的实验设计内容，验证了我们的科学假说，为最终阐明AMPK在DNA损伤修复中的作用奠定了坚实基础。研究结果提示AMPK激活可以作为辐射防护的靶点。AMPK激活剂二甲双胍对受照射小鼠具有明显的保护作用。研究结果对辐射损伤个体的防护具有高度的指导和应用价值。