Abrogation of Chk1-mediated S/G2 checkpoint by UCN-01 enhances ara-C-induced cytotoxicity in human colon cancer cells.
Abrogation of Chk1-mediated S/G2 checkpoint by UCN-01 enhances ara-C-induced cytotoxicity in human colon cancer cells.
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发表时间：
2004-06
影响因子：
8.2
通讯作者：
R. Shao;C. Cao;Y. Pommier
中科院分区：
文献类型：
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作者：
R. Shao;C. Cao;Y. Pommier
目的探讨7-羟基星形孢菌素（UCN-01）对阿糖胞苷（ara-C）诱导的人结肠癌HT-29细胞周期进程的影响。方法采用克隆形成实验、流式细胞术、DNA合成实验、免疫印迹和激酶实验分别测定细胞毒性、DNA合成、细胞周期分布、蛋白水平和激酶活性。结果UCN-01可阻断阿糖胞苷诱导的HT-29细胞S/G2期关卡。在用阿糖胞苷处理后再加入UCN-01，DNA合成的恢复速度加快，而集落形成能力减弱。因此，DNA合成的过早恢复与细胞毒性增加有关。细胞周期蛋白A和B蛋白水平、Cdk 2和Cdc 2激酶活性、Cdc 25 C磷酸化和Chk 1激酶活性的测量结果与UCN-01诱导的阿糖胞苷处理细胞中S/G2期检查点的消除一致。结论UCN-01可通过抑制Chk 1激酶而使S/G2检查点消失。当UCN-01与ara-C组合时产生的增强的细胞毒性表明了将这种药物组合用于可能对细胞周期检查点废除敏感的肿瘤的基本原理。
AIM To investigate whether 7-hydroxystaurosporine (UCN-01) affects cell cycle progression in arabinosylcytosine (ara-C) treated human colon carcinoma HT-29 cells. METHODS Cytotoxicity, DNA synthesis, cell cycle distribution, protein level, and kinase activity were determined by clonogenic assay, flow cytometry, DNA synthesis assay, immunoblotting, and kinase assays, respectively. RESULTS UCN-01 abrogated an S/G2-phase checkpoint in HT-29 cells treated with ara-C. When UCN-01 was added after treatment with ara-C, the rate of recovery of DNA synthesis was enhanced and colony-forming ability diminished. Thus, premature recovery of DNA synthesis was associated with increased cytotoxicity. Measurements of cyclin A and B protein levels, Cdk2 and Cdc2 kinase activities, Cdc25C phosphorylation, and Chk1 kinase activity were consistent with UCN-01-induced abrogation of the S/G2-phase checkpoint in ara-C treated cells. CONCLUSION The abrogation of the S/G2 checkpoint may be due to inhibition of Chk1 kinase by UCN-01. The enhanced cytotoxicity produced when UCN-01 was combined with ara-C suggested a rationale for the use of this drug combination for tumors that might be susceptible to cell cycle checkpoint abrogation.