文献类型：期刊文献

中文题名：癌蛋白NPM-ALK关键磷酸化位点对细胞周期的影响及其机制研究

英文题名：Effect of Tyrosine Phosphorylation Sites of Oncogenic Protein NPM-ALK on Cell Cycle and Its Related Mechanisms

作者：胡林林[1];甄红[1];张晓楠[1];周利[1];Hesham M.Amin[2];史萍[1]

机构：[1]华东理工大学生物反应器国家重点实验室,上海200237;[2]Department of Hematopathology,The University of Texas M.D Anderson Cancer Center,houstontx77030

年份：2016

卷号：24

期号：4

起止页码：1201

中文期刊名：中国实验血液学杂志

外文期刊名：Journal of Experimental Hematology

收录：MEDLINE(收录号：27531800);CSTPCD;;Scopus;北大核心:【北大核心2014】；CSCD:【CSCD2015_2016】；PubMed;

基金：国家自然科学基金青年项目(31100549);上海市科学技术委员会国际学术合作交流项目(14520720700);中央高校基本科研业务费(222201313010);美国国家癌症研究所项目(R01CA151533)

语种：中文

中文关键词：癌蛋白NPM-ALK;磷酸化位点;细胞周期;双点突变

外文关键词：oncogenic protein NPM-ALK;;phosphorylation site;;cell cycle;;double mutation

摘要：目的:探讨NPM-ALK蛋白关键磷酸化位点Tyr644和Tyr664对细胞周期的影响及相关的分子机制。方法:建立NPM-ALK和NPM-ALK^(644,664)稳定表达的Jurkat细胞以及瞬时转染的293T细胞,检测各个细胞系的周期分布;应用软琼脂集落形成实验检验野生型NPM-ALK与双点突变型NPM-ALK致瘤能力的差异;利用蛋白质印迹法检测NPM-ALK关键磷酸化位点对下游相关蛋白表达、修饰的调控作用。结果:野生和突变NPM-ALK在瞬时转染的293T细胞和稳定转染的Jurkat细胞中成功表达,转入双点突变型NPM-ALK的Jurkat细胞与转入野生型NPM-ALK的细胞相比呈现出明显的细胞S期阻滞;酪氨酸激酶抑制剂PPP对野生型NPM-ALK转染的Jurkat细胞杀伤效果最强,对双点突变型NPM-ALK转染的细胞的杀伤作用与阴性对照无明显差异;随着NPM-ALK的转入,下游与细胞生长相关分子STAT3、AKT和ERK的磷酸化水平有所升高(P<0.05),双点突变型NPM-ALK转入后,相关蛋白的磷酸化水平与野生型相比显著降低(P<0.05)。结论:癌蛋白NPM-ALK的Tyr644和Tyr664两个关键磷酸化位点的突变会引起细胞S期阻滞和对药物PPP敏感性的降低,其机制可能与NPM-ALK下游的细胞生长相关分子的磷酸化表达变化相关。
Objective:To explore the effect of tyrosine phosphorylation sites Tyr644 and Tyr664 in oncogenic protein NPM-ALK on cell cycle and its related mechanisms.Methods:Transiently transfected 293 T cells and stably transfected Jurkat cells were used for analysis of cell cycle and protein after the transfection with the constructed recombinant plasmid pEGFP-N1,pEGFP-N1-NPM-ALK and pEGFP-N1-NPM-ALK644,664;soft agar assay for colony formation was performed to examine the different carcinogenicity of stable cell lines;cell viability of stable cell lines was examined by CCK-8 after the treatment with PPP.Results:The S arrest occurred in both NPM-ALK644,664 transfected 293 T and Jurkat cells;the susceptibility of NPM-ALK transfected Jurkat cells to PPP was highest among the 3 stable cell lines;the phosphorylated levels of AKT,ERK and STAT3 were decreased in NPM-ALK644,664 cells compared with the NPM-ALK ones.Additionally,the double mutation induced the increase of CDK2 and the decrease of P27（P < 0.05）.Conclusion:The mutation of Tyr644 and 664 sites in NPM-ALK can induce cell cycle arrest in S phase and lower susceptibility to PPP that may be related with the phosphorylation change of cell growth related molecules in the downstream of NPM-ALK.