三氧化二砷抑制cdc2基因表达诱导K562白血病细胞G_2/M周期停滞

Arsenic Trioxide Inducing G_2/M Arrest of K562 Cell Line via Down-regulating Expression of cdc2 Gene in vitro

目的检测三氧化二砷(arsenic trioxide,As2O3)诱导慢性髓系白血病急变细胞K562周期停滞中细胞周期相关调节基因的转录、蛋白表达及其磷酸化改变,以期阐明As2O3诱导其周期停滞的分子生物学机制,为克服K562细胞的As2O3抵抗性提供实验基础。方法体外培养K562细胞,采用PI染色、流式细胞仪检测As2O3作用后细胞周期分布的改变;逆转录聚合酶链式反应(RT-PCR)检测细胞周期相关调节基因mRNA表达变化;Westernblot检测相关蛋白及磷酸化改变。结果As2O3作用K562细胞24h后,G2/M期细胞比例明显增加,浓度为0、2、5、10μmol/L时G2/M期细胞比例分别为(22.6±3.4)%、(27.2±2.3)%、(43.8±4.5)%、(36.7±4.1)%;K562细胞在As2O3处理前有Sur-vivin、cdc2、chk1基因表达,As2O3处理后Survivin、chk1的表达无明显变化,但cdc2表达呈浓度依赖性下调;As2O3作用前可见Survivin、cdc2、cdc2-p、chk1等4种蛋白的表达,作用24h后,Survivin的表达条带增强,cdc2蛋白表达水平呈浓度依赖性下调,cdc2-p水平在2～5μmol/L时无明显变化,在10μmol/L时受抑明显,chk1蛋白表达水平在As2O3作用前后无明显改变。结论As2O3显著诱导K562白血病细胞G2/M周期停滞,其机制与抑制cdc2转录水平,下调活性化cdc2蛋白表达有关。不能有效抑制抗凋亡蛋白Survivin的表达可能是K562白血病细胞对As2O3诱导凋亡抵抗的一个重要机制。

Objective To elucidate the possible molecular biologic role of arsenic trioxide （As2O3） inducing G2/M arrest of K562 cell. Methods K562 cells were cultured in vitro and treated with various concentrations of As2O3. Flow cytometry was used to measure the changes of cell cycle distributions. Reverse-transcription PCR （RT-PCR） assays were used to detect the mRNA expression level and Western blot assay to detect the level of protein expression and phosphated change. Results K562 cells arrested in G2/M phase were obviously increased after co-cultured with 2 to 10 μmol/L As2 03 for 24 h. The ratio of G2/ M cells in control, 2 μmol/L As203 , 5μmol/L As203 and 10μmol/L As203 groups were （22.6±3.4）0/6, （27.2±2.3）0/60, （43.8±4.5） 0/6 and （36.7±4.1）%, respectively. The mRNA expression level of Survivin, cdc2 and chkl could be detected in the absence of As2O3 , in the presence of As2O3 the expression level of Survivin and chkl had no significant change, but that of cdc2 was down-regulated in a concentration-dependent manner. In the absence of As2O3 the protein expression of Survivin, cdc2, cdc2-p and chkl could be detected, after treatment with As2 03 for 24 h, the expression level of Survivin was not inhibited, cdc2 down-regulated in a concentration-dependent manner, and cdc2-p and chkl were maintained at the original levels. Conclusion As2O3could obviously induce G2/M arrest of K562 cells by inhibiting transcriptional level of cdc2 and down-regulating activated cdc2 protein expression. Not effectively inhibiting the expression of Survivin protein might be an important mechanism by which K562 cells are resistant to As2O3.