三氧化二砷对T315I突变细胞株KBM5R细胞周期的影响

J4 ›› 2011, Vol. 37 ›› Issue (2): 245-249.

三氧化二砷对T315I突变细胞株KBM5R细胞周期的影响

李小丰¹|刘秋菊¹|李景贺¹|王秀丽¹|沈卫章¹|卢爱萍¹|郏博^*|陈鹏²

1. 吉林大学第二医院肿瘤血液科|吉林长春 130041； 2. 吉林大学第二医院儿科|吉林长春 130041

收稿日期:2010-12-09 出版日期:2011-03-28 发布日期:2011-03-28
基金资助:
吉林省科技发展计划项目资助课题（200705206）

Effect of arsenic trioxide on cell cycle progression of cell line KBM5R with T315I point mutation

LI Xiao-feng¹,LIU Qiu-ju¹,LI Jing-he¹,WANG Xiu-li¹,SHEN Wei-zhang¹,LU Ai-ping¹,JIA Bo*,CHEN Peng²

1. Department of Oncology and Hematology,Second Hospital,Jilin University,Changchun 130041,China;2. Department of Pediatrics,Second Hospital,Jilin University,Changchun 130041,China

Received:2010-12-09 Online:2011-03-28 Published:2011-03-28
Contact: 王秀丽（Tel：0431-88796994，E-mail：xlwang59@sina.com ） E-mail:xlwang59@sina.com
About author:李小丰（1979-）|男|吉林省榆树市人|医师|医学硕士|主要从事白血病发病机制方面的研究。

摘要/Abstract

摘要：

目的：探讨三氧化二砷（ATO）对T315I突变的伊马替尼（IM）耐药慢性粒细胞白血病（CML）细胞株KBM5R细胞周期的影响，为对抗CML患者的IM耐药性提供理论依据。方法：选择野生型KBM5细胞作为T315I点突变KBM5R细胞的阴性对照组，根据是否经过ATO处理将实验分为KBM5R细胞空白对照组、KBM5R细胞ATO处理组、KBM5细胞空白对照组和KBM5细胞ATO处理组。应用MTT法检测IM和ATO作用后KBM5和KBM5R细胞的增殖活性；流式细胞术检测ATO作用后KBM5和KBM5R细胞周期的变化；Western blotting法检测ATO作用后KBM5和KBM5R细胞周期调节相关蛋白P21和P27表达的变化。结果：IM作用后耐药组KBM5R细胞IC50与野生型KBM5细胞比较明显增高（P<0.01）；不同浓度（0.5、1.0、2.0、4.0和8.0 μmol·L-1）ATO于不同时间点（24、48、72和96 h）作用于KBM5和KBM5R细胞，与空白对照组比较，ATO处理组细胞均表现为显著的增殖抑制，且随药物浓度增加或作用时间延长细胞的增殖抑制率显著增加（P<0.05）；在相同的药物浓度和时间点，ATO对KBM5R细胞的增殖抑制作用强于野生型KBM5细胞（P<0.05）；2.0、4.0和8.0 μmol· L-1 ATO作用细胞48 h后，KBM5和KBM5R细胞周期中G2/M期细胞所占比例均随药物剂量的增加而增加，且相同药物浓度时KBM5R细胞周期中G2/M期细胞所占比例与KBM5细胞比较差异无统计学意义(P>0.05)； ATO作用KBM5和KBM5R细胞48 h后，2.0、4.0和8.0 μmol·L-1 ATO处理组与空白对照组比较细胞内P21和P27蛋白表达均增加，且随药物剂量增加而增加。结论：ATO通过上调P21和P27蛋白水平使KBM5R细胞周期阻滞于G2/M期，细胞周期阻滞是ATO抑制T315I点突变细胞株KBM5R增殖的原因之一。

关键词: 三氧化二砷；伊马替尼耐药；T315I点突变；细胞周期

Abstract:

Abstract:Objective To investigate the effect of arsenic trioxide (ATO) on cellcycle progression of imatinib(IM)-resistant chronic myeloid leukemia (CML) cellline KBM5R with T315I point mutation,and provide theoretical foundation for overcoming resistance of CML patients to IM. Methods The wild-type cell line KBM5was selected as negative control of KBM5R with T315I point mutation，and the cells were divided into four groups according to ATO treatment：control group of KBM5R cells,ATO group of KBM5R cells,control group of KBM5 cells and ATO group of KBM5 cells. The proliferation of KBM5 and KBM5R cells after treated with IM or ATO were detected by MTT. The changes of cell cycles of KBM5 and KBM5R cells were quantified by flow cytometry. The expressions of P21 and P27 proteins in KBM5 and KBM5R cells after treated with ATO were determined by Western blotting. Results The IC50 of KBM5R cells treated with IM was higher than that of wild-type cell line KBM5 (P<0.01). The inhibitory rates of proliferation of KBM5 and KBM5R cells after treated with 0.5,1.0,2.0,4.0 and 8.0 μmol·L^-1 of ATO for 24,48,72 and 96 h were higher than that of control group,which were increased according to the increase of concentration or the treatment time of ATO （P<0.05）. The inhibitory effect of KBM5R cell oliferation was stronger than that of KBM5 at the same drug concentration and time （P<0.05）. The cell cycle detection of KBM5 and KBM5R cells after treated with 2.0,4.0 and 8.0 μmol·L^-1 ATO for 48 h showed that the G2/M arrest rate was increased according to the increase of concentration of ATO,and the G2/M arrest rate of KBM5R had no signficant difference compared with KBM5 at the same drug concentration. The expressions of P21 and P27 proteins in KBM5 and KBM5R cells after treated with 2.0,4.0 and 8.0 μmol·L^-1 ATO for 48 h were significantly increased in a concentration-depenent manner. Conclusion ATO can arrest cell cycle of KBM5R cell line in G2/M phase through up-regulation of P21 and P27 protein levels,and it is one of thereasons that ATO can inhibit the proliferation of KBM5R cells with T315I point mutation.

Key words: arsenic trioxide；imatinib-resistance；T315I point mutation；cell cycle

中图分类号:

R557.3

李小丰|刘秋菊|李景贺|王秀丽|沈卫章|卢爱萍|郏博|陈鹏. 三氧化二砷对T315I突变细胞株KBM5R细胞周期的影响[J]. J4, 2011, 37(2): 245-249.