8-氯腺苷通过磷酸化修饰提高转录因子CREB的活性  

8-Chloro adenosine Increases the Activity of Transcription Factor CREB by Modification of Phosphorylation

作　　者：畅晓燕[1] 方家椿[1] 王耐勤[1] 刘彤[1] 苏虹[1] 

机构地区：[1]北京大学临床肿瘤学院肿瘤防治研究所细胞生物学研究室,北京100034

出　　处：《癌症》2003年第4期350-353,共4页Chinese Journal of Cancer

基　　金：国家"1035"医药工程项目(96-901-01-03)

摘　　要：背景与目的:8-氯腺苷是国内新近研发的抗肿瘤药物,其分子作用机制尚未完全阐明。本实验研究该药对HL-60细胞转录因子cAMP应答元件结合蛋白(cAMP-response-element-bindingprotein,CREB)活性的影响及其相关机制。方法:应用MTT法检测8-氯腺苷对人早幼粒细胞白血病细胞株HL-60的半数抑制浓度(IC50),用蛋白印迹实验(Westernblotanalysis)检测药物作用不同时间CREB蛋白水平和CREB磷酸化水平,用凝胶阻滞实验(Gelshiftassay)检测药物作用不同时间CREB与DNA的结合水平。结果:(1)8-氯腺苷对HL-60细胞的IC50约为0.42μmol/L。(2)8-氯腺苷快速诱导CREB磷酸化,30min达到最高水平,6h恢复至基础水平,CREB蛋白水平在药物作用期间保持恒定。(3)8-氯腺苷不改变CREB与DNA结合能力。结论:8-氯腺苷诱导CREB转录因子活性的提高依赖于CREB磷酸化水平的提高,而与CREB蛋白水平及CREB与DNA的结合水平无关。BACKGROUND ＆OBJECTIVE:8 Chloro adenosine(8 Cl Ado) is an anti tumor drug,which has been investigated and developed recently in China. The molecular mechanism of 8 Cl Ado is now unclear. This study was conducted to investigate the effect of 8 Cl Ado on the activity of transcription factor cAMP response element binding protein(CREB) and its correlative mechanism in human promyelocytic leukemia cell line HL 60. METHODS:MTT method was used to determine IC50（the concentration showing 50％〓of inhibition rate）of 8 Cl Ado to HL 60. The protein levels and phosphorylated levels of CREB at different time following 8 Cl Ado stimulation were examined using Western blot analysis. Gel shift assay was performed to examine DNA binding levels of CREB at different time following 8 Cl Ado stimulation.RESULTS:(1)The IC50 of 8 Cl Ado to HL 60 was about 0 42 μmol/L. (2)8 Cl Ado increased phosphorylated level of CREB rapidly, which was maximal at 30 minutes, and returned to basal level in 6 hours. The protein level of CREB was constant during 8 Cl Ado stimulation. (3)8 Cl Ado did not influence binding level of DNA and CREB. CONCLUSION:The increase of transcriptional activity of CREB induced by 8 Cl Ado depends on the increased level of phosphorylated CREB, but is not associated with the protein level and DNA binging level of CREB.

关 键 词：8-氯腺苷 肿瘤 转录因子 

分 类 号：R73-36[医药卫生—肿瘤]