p21剪切的发生和调控机制及其对肺癌细胞化疗增敏作用的研究

Resistance to apoptosis is the main reason for failure of chemotherapy in lung cancer. p21 is the important regulator of chemotherapy-induced DNA damage response (DDR) and thus becomes a promising target for cancer therapy.It has been shown that p21 could induce cell cycle arrest and allow time for DNA repair, while inhibit apoptosis. Our previous work showed that some anti-cancer drugs could induce p21 expression, cell cycle arrest, and p21 cleavage which was correlated with apoptosis. Inhibition of p21 cleavage decreased apoptosis, indicating that p21 cleavage is the crucial step in chemotherapy-induced apoptosis in lung cancer, and exploring the mechanisms of p21 cleavage would be important to increase chemosensitivity. Based on these results, for the first time we addressed the p21 cleavage-targeted chemosensitizing strategy. In this study, we re going to use methods such as co-immunoprecipitation, mutagenesis, gene transfection and so on, to investigate the time and cellular distriution of p21 cleavage upon drug treatment, function of p21 cleavage fragments, and the influence of p21 cleavage on cell fate and chemosensitivity after DNA damage. Since the function of p21 is closely regulated by post-translational modification (PTM) and cellular location, we also would investigate the regulatory mechanisms by p21 PTM and location of drug-induced p21 cleavage. Our study would help to add to new function of p21, and provide evidence for the p21 cleavage-targeted therapy to increase chemosensitivity of lung cancer.

肺癌细胞凋亡抵抗是导致化疗耐受的重要原因。p21对化疗引起的DNA损伤应答具有重要调控作用而成为肿瘤治疗靶点。研究表明化疗致细胞损伤时p21促使细胞周期阻滞并进行DNA修复，对凋亡却有抑制作用。我们前期工作发现多种抗癌药物致肺癌细胞损伤时，p21表达增高、细胞周期阻滞并伴p21剪切，剪切程度与凋亡呈正相关，抑制其剪切则抑制细胞凋亡。提示p21剪切是化疗引起肺癌细胞凋亡的关键，揭示其性质和调控机制有助于提高肺癌化疗敏感性。因此本课题首次提出靶向p21剪切的化疗增敏策略，拟采用免疫共沉淀、点突变、基因转染等研究化疗药物引起p21剪切发生的时间、地点、执行者、剪切片段的作用以及p21剪切对肺癌细胞化疗敏感性的影响；由于p21功能受翻译后修饰和细胞内分布的密切调节，我们还将从此入手研究p21剪切的调控机制。本研究将对p21的作用做新的补充，并为以p21剪切调控为靶点的肺癌化疗增敏治疗提供依据。

DNA损伤应答与肿瘤治疗密切相关，P21对DNA损伤修复和细胞凋亡过程具有调控作用，化疗药物如何影响P21剪切以及P21自身表达调控机制如何尚不明确。本课题中，我们研究了化疗药物诱导P21剪切作用、P21对化疗药物敏感性的影响及P21的表达调控机制，有如下发现：1. P21剪切与化疗药物诱导的肿瘤细胞凋亡密切相关，且与凋亡呈正相关，包括多西紫杉醇、CDK抑制剂Roscovitine（Ros）、顺铂（cDDP）以及组蛋白去乙酰化酶抑制剂TSA；2. P21表达水平影响药物诱导肺癌细胞凋亡， P21高表达显著降低TSA和cDDP引起的肺癌细胞凋亡，而P21基因敲减则促进TSA和cDDP诱导的肺癌细胞凋亡；3.P21表达受到P300的调控，包括3方面的机制：①肺癌细胞过表达生长抑制因子5（ING5）可促进乙酰化酶P300自乙酰化，进而激活其HAT酶活性，通过乙酰化P53K382促进P53对靶基因P21的转录激活；②P300作为转录co-activator直接结合于P21启动子区，促进P21转录，ING5过表达显著促进P300在P21启动子区的富集；③P300可乙酰化组蛋白H3K18，ING5高表达可显著促进H3K18乙酰化，P21启动子区组蛋白H3乙酰化增加促进P21基因转录表达；4. 阻断P300活性可通过抑制P21提高肺癌细胞化疗敏感性，P300HAT酶特异性抑制剂C646抑制H3K18和P53K382乙酰化，并降低P21水平而显著提高cDDP诱导的肺癌细胞凋亡，并增强cDDP对肺癌细胞克隆形成能力的抑制作用。以上研究结果提示，通过调控P21转录表达水平可提高肺癌细胞对药物的敏感性。该课题的研究进一步拓展了我们对P21剪切作用及P21表达调控机制的认识，为后续的研究工作提供了线索和方向。