P53蛋白二阶段磷酸化修饰在砷"致癌与治癌"双重作用中的机制研究

Cell fate decision-proliferation or apoptosis-decides arsenic-exposure outcomes-carcinogenicity or chemotherapeutic effects. As the key cell fate decision maker, P53 governs cell survival/proliferation and apoptosis though a two-phase phosphorylation when responses to DNA damage, and WIP1, a dephosphorylation enzyme, is crucial in that P53 dynamical phosphorylation. Arsenic was reported to active P53 by DNA oxidative damage, suggesting that P53 may involved in arsenic-induced DNA damage response. Thus, we hypnotize that P53 may control cellular outcome by two-phase phosphorylation in arsenic-induced DNA damage response that leads to carcinogenicity or chemotherapeutic effects. To test this hypothesis, arsenic trioxide and sodium arsenite will be utilized, and tumor cells derived from arsenical target organs and their corresponding normal cells will be chosen as model cells. WIP1-specific siRNA/shRNA or plasmid DNA will be transfected to model cells to interfere the process of P53 two-phase phosphorylation. Then, the link between P53 phosphorylation levels and their following signal protein, and cellular outcome-cell cycle arrest and apoptosis will be examined to determine whether P53 two-phase phosphorylation is involved in low-level arsenic induced tumorigenesis and high-level arsenic induced chemotherapeutic effects. Our proposed research will significantly advance our understanding of the paradox of arsenic, and will further facilitate identification of WIP1 as a susceptive biomarker in arsenic carcinogenesis or new molecular target for improvement of arsenic treatment efficiency.

细胞在砷诱导DNA损伤反应中的命运抉择（增殖或凋亡）决定了砷是"致癌"还是"治癌"，而命运抉择的机制与P53蛋白二阶段磷酸化修饰及调控其转换的去磷酸化酶WIP1密切相关。已有研究显示砷诱导的DNA损伤可以激活P53，提示砷可能通过P53信号通路以P53蛋白二阶段磷酸化修饰机制决定细胞暴露的结局。然而，P53蛋白二阶段磷酸化修饰是否参与砷诱导的DNA损伤反应？不同的磷酸化修饰是否会导致不同的结局？调控WIP1是否可以成为砷治癌增敏的有效手段？课题以砷主要靶器官的正常细胞和癌细胞作为研究对象，分析砷作用后P53蛋白磷酸化水平及其下游因子的变化规律；同时构建WIP1低/高表达细胞模型以干预P53蛋白二阶段磷酸化的进程，检测细胞周期阻滞/DNA修复和凋亡，以阐明P53蛋白二阶段磷酸化修饰在低剂量砷暴露引起细胞增殖和治疗剂量砷处理诱导细胞凋亡中的关键作用，从而揭示砷"致癌与治癌"双重作用的分子机制。

课题主要以人支气管上皮细胞HBE和人肺腺癌A549细胞为研究对象，探讨了P53蛋白二阶段磷酸化修饰在砷“致癌”与“治癌”中的作用，主要结果如下：(1) 以不同浓度三氧化二砷（ATO）处理A549和HBE细胞，结果显示：正常细胞会在损伤达到一定程度时发生凋亡，而癌细胞则会通过延长周期阻滞帮助细胞修复以躲避凋亡；P53 ser15向ser46转换受阻可能参与了肺癌细胞延长周期阻滞躲避凋亡的机制；促磷酸化蛋白的缺乏及去磷酸化蛋白高表达是肺癌细胞中P53 ser15向ser46转换受阻的原因。(2) 以亚砷酸钠低剂量（2.5 µM）、长期间（13周）诱导HBE细胞恶性转化，观察转化过程中TP53INP1和WIP1的表达变化。结果显示：随着转化代数增加，TP53INP1蛋白表达逐渐降低，而WIP1蛋白则显著升高。提示TP53INP1降低及WIP1的增高可能在砷诱导细胞恶性转化过程中扮演重要角色，参与了砷的致癌过程。(3) 探究干预TP53及WIP1表达以促进或削弱P53磷酸化进程，从而增敏砷剂的抗癌效应：（1）通过siRNA技术靶向沉默A549细胞中WIP1蛋白的编码基因PPM1D，抑制WIP1蛋白表达，加快了ATO处理后P53 ser15向P53 ser46的转换，降低P53 ser15下游效应因子P21的表达，提高P53 ser46下游效应因子—凋亡诱导蛋白PUMA表达；从而减少细胞保护性G2/M期，提高细胞凋亡率。提示抑制WIP1可显著提高肺腺癌细胞对砷剂的敏感性。（2）构建TP53INP1重组真核表达质粒，以慢病毒包装后转染A549细胞，使细胞中TP53INP1蛋白表达水平提高6倍，显著加快ATO处理后P53 ser15向P53 ser46的转换，降低了P21的表达，提高了PUMA表达和细胞凋亡。提示提高TP53INP1表达可显著增加砷剂对A549细胞的杀灭作用。（3）同时调控WIP1和TP53INP1的表达比单独下调WIP1或单独上调TP53INP1促进P53磷酸化进程的效率更高，因而对砷剂的增敏效果更为显著。.以上部分结果在人正常肝细胞L02和肝癌细胞HepG2进行了验证，课题组还增加了TP53(促磷酸化)蛋白的研究，与WIP1(去磷酸化)从正反两方面验证学术假说。另外，我们研究了项目中没有计划的miRNA-155在砷诱导细胞转化中的作用。研究达到了预期目标!