Chk1在K562/A02细胞耐药中的作用机制

The Role of Chk1 in Regulation of Drug Resistance in K562/A02 Cell Line

目的研究检测点激酶1(Chk1)对K562/A02细胞周期及凋亡的影响,探讨Chk1在肿瘤细胞耐药中的作用机制。方法以人红白血病细胞系K562及其耐药细胞株K562/A02为研究对象,与阿霉素共同孵育后,RT-PCR检测Chk1mRNA表达水平,Western blot检测其蛋白质的表达及磷酸化水平,MTT法检测药物敏感性,流式细胞术检测细胞周期分布和细胞凋亡。结果Chk1 mRNA及蛋白表达水平在两种细胞间无显著差异(均P>0.05);Chk1磷酸化水平在K562/A02细胞为(0.79±0.56),K562细胞为(0.27±1.47),其差异有显著性意义(P<0.05)。阿霉素作用6h后,致K562/A02细胞阻滞在G2/M期的细胞百分率为(54.12±0.57)%,显著高于K562细胞的(36.99±1.28)%(P<0.05),K562/A02细胞凋亡率为(6.25±0.81)%,显著低于K562细胞的(66.47±1.26)%(P<0.05)。阿霉素对K562/A02和K562细胞的IC50值分别为(109.65±0.26)mg/L、(1.08±0.74)mg/L,耐药倍数为102倍。结论由于G2/M期阻滞是导致白血病细胞耐药的机制之一,Chk1磷酸化水平与G2/M期阻滞和细胞凋亡密切相关,提示Chk1的活化状态可能参与K562/A02细胞的耐药机制。

Objective To investigate the effect of phosphorylated Chk1 on cell cycle and apoptosis of two kinds of human erythroleukemic cell lines, K562 and K562/A02, and then explore the role of Chkl in drug resistance of two leukemia cell lines. Methods After treatment with adriamycin for 6 h, cell cycle and apoptosis of two cell lines were detected by flow cytometry, and the sensitivity to adriamycin was analyzed by using MTT assay. Chk1 mRNA expression of these two cell lines was detected by RT-PCR, and the expression and phosphorylation level of Chkl protein were analyzed by Western blot. Results The proportion of K562/A02 cells arrested in G2/M phase was （54.12±0.57） % after adriamycin treatment, which was significantly higher than in K562 cells （P〈0.05）. There was no significant difference in the Chkl mRNA and protein expression levels between K562 and K562/A02 cells （P〉0.05）. But elevated Chkl phosphorylation was observed after treatment with adriamycin in K562/A02 cells （0.79±0.56）, which was obviously higher than that in K562 cells （0.27± 1.47） （P〈0. 05）. Conclusion Because G2/M arrest is related with development of leukemia, the phosphorylation level of Chkl is associated with G2/M arrest and apoptosis, which may be involved in adriamycin resistance in K562/A02 cells.