泛素连接酶β-TrCP降角翠BCR-ABL融合蛋白对白血病K562细胞增殖的抑制  

Antiproliferation Effect of Beta-TrCP Ubiquitin Ligase Mediated BCR-ABL Protein Degradation in Leukemia K562 Cells

作　　者：田文君[1] 罗红伟[1] 袁颖[2] 黄世峰[1] 刘钉宾[1] 陶昆[1] 冯文莉[1] 

机构地区：[1]重庆医科大学临床血液学教研室临床检验诊断学教育部重点实验室,重庆市400016 [2]重庆医科大学临床学院

出　　处：《中国肿瘤临床》2010年第2期66-70,共5页Chinese Journal of Clinical Oncology

基　　金：国家自然科学基金资助(编号:30670901)~~

摘　　要：目的:t(9;22)(q34;q11)突变导致的BCR-ABL融合基因及其编码的融合蛋白在慢性粒细胞白血病(chronic myeloid leukemia,CML)发病中发挥重要作用。本研究观察可与BCR-ABL融合蛋白N端寡聚化区域(Oligomerization domain,OD)特异性结合的嵌合泛素连接酶E3 β-TrCP的β-TrCP-OD-HA的重组腺病毒载体,经泛素-蛋白酶体途径降解BCR-ABL融合蛋白对白血病K562细胞增殖的影响。方法:HEK293细胞扩增野生型Ad5β-TrCP-OD-HA、突变型Ad5ΔF-TrCP-OD-HA及空载Ad5GFP重组腺病毒载体,感染K562细胞。以未感染K562细胞作空白对照,流式细胞术检测重组腺病毒载体感染效率,Western blot检测外源性重组蛋白和BCR-ABL融合蛋白的表达,通过细胞增殖曲线、甲基纤维素集落形成试验、细胞周期检测β-TrCP-OD-HA对K562细胞增殖的影响。结果:成功建立携β-TrCP-OD-HA重组腺病毒载体的K562细胞株,重组腺病毒载体感染效率达66.4%以上,β-TrCP-OD-HA、ΔF-TrCP-OD-HA可在K562细胞中表达。三组重组腺病毒载体感染K562细胞后,Ad5β-TrCP-OD-HA组Western blot检测BCR-ABL融合蛋白含量下降;K562细胞生长和集落形成能力均受抑制;细胞周期显示S期细胞数下降至10.88%±2.42%、G_0/G_1期升高至85.6%±5.61%,与Ad5β-TrCP-OD-HA组、Ad5GFP组及对照组相比,差异具有统计学意义(P均<0.05)。结论:重组腺病毒介导的β-TrCP-OD-HA、ΔF-TrCP-OD-HA能够在白血病K562细胞中表达;β-TrCp-OD-HA可以抑制K562细胞的生长增殖,其机制可能与其降解BCR-ABL融合蛋白、阻滞细胞周期而实现。Objective： The BCR-ABL fusion gene induced by reciprocal translocation of t （9; 22） （q34; q11） plays an important role in the pathogenesis of chronic myeloid leukemia （CML）. Using recombinant adenoviruses carrying the N-terminal oligomerizaton domain （OD） of the BCR/ABL and chimeric ubiquitin ligase β-TrCP, this study was to investigate the effect of the targeted degradation of oncoprotein BCR-ABL by Ubiquitin-Proteasome System on the proliferation of leukemia cell line K562. Methods： The recombinant adenoviruses carrying wild-type β-TrCP gene （Ad5β-TrCP-OD-HA）, mutational β-TrCP gene （Ad5 Δ F-TrCP-OD-HA）and green fluorescent protein gene （Δd5GFP）were amplified in 293 cells and co-infected into K562 cells respectively. The rates of infection were analyzed by flow cytometry （FCM）. Recombinant protein and BCR-ABL expression was detected by Western blot. Cell proliferation was determined by cell counting and methylcellu- cose clonal cell culture. Cell cycle was observed through FCM. Untreated K562 cells were used as blank controls. Result： The leukemia K562 cell lines with exogenous recombinant β-TrCP-OD-HA and Δ F-TrCP-OD-HA gene were established. The infection rates in the three groups were over 66.4% and recombinant protein sus- tained to be expressed. Ad5β-TrCP-OD-HA down-regulated the expression of BCR-ABL and inhibited prolifer-ation of K562 cells. FCM showed that the percentage of cells at S phase was decreased to 10.88%±2.42%, while that of cells at G0/G1 was increased to 85.6%±5.61%, with a significant difference （P〈0.05）. No changes were found in the cell cycle in groups of Ad5ΔF-TrCP-OD-HA and Ad5GFP. Conclusion： There is sustained expression of recombinant β-TrCP-OD-HA protein in K562 cells infected by recombinant adenovirus. β-TrCP-OD-HA could inhibit the proliferation and clonogenicity of K562 cells through targeted degradation of oncoprotein BCR-ABL and arresting the progression of cell cycle.

关 键 词：BCR—ABL融合蛋白 泛素连接酶β—TrCP K562细胞细胞增殖 

分 类 号：R733.7[医药卫生—肿瘤]