脱氧胞苷激酶dCK在电离辐射诱导自噬中的调控作用

Ionozing radiation could induce DNA damage and autophagy. Deoxycytidine kinase (dCK) is a rate-limiting enzyme in deoxyribonucleoside salvage and is required for the phosphorylation of several deoxyribonucleosides and their nucleoside analogs. The roles of dCK in drugs metabolism is a wide concern, while its application in radiobiological regulation is still unclear. We have found that dCK functions in the processes of DNA damage and radiation-induced autophagy. In this study we are aiming to elucidate the roles of dCK in radiation-induced autophagy and the underlying mechanisms. Five parts will be included: (1) to validate the fact that dCK participates in the radiation-induced autophagy; (2) to verify that the phosphorylation of dCK-Ser 74 by ATM is required for the radiation-induced autophagy;(3) to confirm the regulatory roles of dCK-AKT-mTOR pathway; (4) to confirm the regulatory roles of HSP90-dCK-AKT-IKK-NFkB- BECLIN pathway; (5) to elucidate if dCK interacts with MAPLC3 via its LIR （LC3-interacting region）domain and consequently regulate the process of autophagy. This study will provide a new insight and molecular mechanisms for radiobiology theory. In addition, based on the facts that dCK could change the radiosensitivity and DNA damage could alter dCK expression and activity, dCK could be used as a biological dosimeter and a target for both radiotherapy and radiation protection.

电离辐射诱导DNA损伤进而引起自噬的发生。脱氧胞苷激酶（dCK）是磷酸化天然脱氧核糖核苷酸以及外源性抗肿瘤、抗病毒的核苷类似物药物的关键酶，在DNA合成及药物活化方面起关键作用。dCK在放射生物学领域少有报道。我们发现，dCK参与电离辐射诱导DNA损伤和自噬。本项目拟就dCK如何调控电离辐射诱导的自噬开展探索：（1）验证dCK参与辐射诱导自噬的调控；（2）确定ATM磷酸化dCK Ser74为辐射诱导自噬所必需；（3）确定dCK-AKT-mTOR通路的调控作用；（4） 确定HSP90-dCK-AKT-NFkB- BECLIN通路的调控作用；（5）确定dCK的LIR（LC3 interacting region）结构域结合LC3调控自噬。本研究将为辐射诱导自噬的调控机制提供新的理论基础，鉴于dCK在辐射诱导的DNA损伤中变化显著，可作辐射剂量计，以dCK为靶点对于辐射防护有重要的意义。

脱氧胞苷激酶（dCK）是磷酸化天然脱氧核糖核苷酸和外源性核苷类抗肿瘤药物的关键酶，在DNA合成及药物活化方面起重要作用，本研究主要探讨dCK参与调控电离辐射诱导的自噬的可能机制。.研究发现：1）dCK与辐射敏感性：沉默dCK基因增加Hela细胞和乳腺癌SKBR3和MDAMB231细胞的辐射敏感性，过表达dCK野生型和过磷酸化型S74E能够挽救dCK基因沉默引起的细胞死亡，但是空载体和突变子S74A无挽救作用，表明S74位点对辐射敏感性是必须的；2）ATM介导dCK发生磷酸化：辐射诱导ATM+/+细胞GM0536发生dCK激活，而ATM-/-细胞GM1526则无此反应，沉默ATM后细胞对Ara-C抗性增加，提示dCK活性下降，激酶活性分析实验发现dCK Ser74被ATM直接磷酸化；3）dCK Ser-74磷酸化是辐射诱导自噬必需位点：将野生型wt、S74A、S74E表达载体分别转染到dCK或ATM沉默细胞中，发现野生型dCK和S74E可恢复dCK沉默所致的自噬降低，增加细胞自噬水平；4）确定了dCK-mTOR-AKT通路参与了辐射诱导的自噬；5）确定了HSP90-dCK-AKT-NFkB- BECLIN通路参与了辐射诱导自噬的调控。6） 确定dCK通过增加辐射诱导的ROS的产生，进一步增加自噬；7）确定了dCK与铁依赖的ROS产生基因的调控关系。本研究揭示了dCK参与调控电离辐射诱导宫颈癌和乳腺癌细胞自噬的可能机制，对丰富肿瘤细胞死亡机制有重要的意义，并对协同放化疗敏感性起指导作用。