腺病毒介导的SD—HA对K562细胞增殖抑制和凋亡诱导的作用机制  

Mechanisms of recombinant adenovirus-mediated SD-HA fusion protein proliferation inhibition and induced apoptosis ofK562 cells

作　　者：黄勇[1] 张萍[2] 杜莉[3] 桂敏[3] 冯文莉[5] 彭智[3,4] Huang Yong;Zhang Ping;Du Li;Gui Min;Feng Wenli;Peng Zhi(Department of Infectious Diseases, Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China)

机构地区：[1]重庆医科大学第二附属医院检验科,400010 [2]重庆医科大学第二附属医院血液科,400010 [3]重庆医科大学第二附属医院感染科,400010 [4]中华肝脏病杂志编辑部 [5]重庆医科大学检验医学部血液学教研室

出　　处：《中华血液学杂志》2018年第4期314-319,共6页Chinese Journal of Hematology

基　　金：国家自然科学基金（81501811）

摘　　要：目的探讨融合蛋白SD—HA能否通过竞争结合BCR—ABL的第177位酪氨酸磷酸化位点（Y177p），调控与其相关的下游信号分子活性，进而抑制K562细胞增殖并诱导凋亡。方法Westernblot结合免疫共沉淀技术分析融合蛋白SD—HA与BCR．ABL的Y177p的相互作用，及其对下游信号途径Ras—MAPK和磷脂酰肌醇3-激酶（P13K）．Akt的影响；Westernblot分析融合蛋白SD—HA对细胞膜受体死亡途径级联反应caspase-8、caspase-3和PRAP的影响。结果双向免疫共沉淀实验可检测到融合蛋白SD—HA能与Grb2竞争性结合BCR—ABLY177p，形成复合物。融合蛋白SD—HA可降低活化Ras、磷酸化MAPK（p—MAPK）、p-ELK的表达水平，抑制Ras—MAPK信号途径；融合蛋白SD—HA还可降低P—Akt及Akt的底物P—GSK的表达水平，抑制P13K—Akt信号途径，从而抑制K562细胞增殖；通过死亡效应结构域（DED）与caspases-8前体DED结合而寡聚化，激活caspases-8、caspase-3和PRAP，诱导K562细胞凋亡。结论融合蛋白SD—HA抑制BCR-ABL—Y177与Grb2结合的策略，可作为慢性髓性白血病治疗新的切入点。Objective To investigate whether fusion protein SD-HA could regulate its downstream signaling molecule activity by competing with the phospho-BCR-ABL Y177 site, and its mechanisms to inhibit proliferation and induce apoptosis of K562 cells. Methods Co-immunoprecipitation interaction technology analysis of fusion protein SD-HA functioned by potently binding to the phospho-BCR-ABL Y177 site, Ras, MAPK and Akt activities were observed in the Ad5F35-SD-HA-treated cells. Western blot analyses of SD-HA fusion protein on cell membrane receptor pathway to death cascade caspase-8, caspase-3 and PRAP were performed. Results Exploration into the underlying mechanisms revealed that Ad5F35- SD-HA infection functioned by binding to the phospho-BCR-ABL Y177 site, which lead to a complex with Grb2. competitively disrupted the Grb2 SH2-phospho-BCR-ABL Y177 formation. The fusion protein SD-HA could reduce the activation of Ras and phosphorylation of MAPK （p-MAPK） and the expression level of p-ELK, inhibition of Ras-MAPK signaling pathway; SD-HA fusion protein could reduce p-Akt and Akt substrate p-GSK with inhibition of PI3K-Akt signaling pathway, thereby inhibiting the proliferation of K562 cells. Caspases- 8- induced apoptosis signal could be activated by DED protein binding to DED domain of precursor caspases-8. Conclusions The strategy of fusion protein SD-HA inhibiting- Y177 BCR-ABL and Grb2 binding could be used as a novel entry point for the treatment of chronic myeloid leukemia.

关 键 词：白血病 髓系 慢性 BCR—ABL融合蛋白 Src同源结构域2 死亡效应结构域 细胞增殖 细胞凋亡 

分 类 号：R733.72[医药卫生—肿瘤]