Zhao Peng, Zhong Weijian, Ying Xianping, Yuan Zhun, Fu Juanling, Zhou Zongcan
Department of Toxicology, Peking University Health Science Center, Beijing 100083, China.
Toxicology. 2008 Aug 19;250(1):39-46. doi: 10.1016/j.tox.2008.05.016. Epub 2008 Jun 3.
In the present study, we investigated the effects of manganese chloride (MnCl2) on cell cycle progression in A549 cells used as a model of Mn-induced lung toxicity. Cells were treated with various concentrations of MnCl2 (0, 0.01, 0.1, 0.5, 1.0 or 2.0 mM) for 24, 48 or 72 h. Cell proliferation was determined with MTT assay and mitotic index measurement and apoptosis was measured by flow cytometer. The results showed that MnCl2 inhibited A549 cells proliferation in a dose- and time-dependent manner, and induced apoptosis in A549 cells. When G0/G1 cells obtained by serum starvation were incubated with 0.5 mM of MnCl2 in the presence of 10% serum for several time intervals, the disruption of cell cycle progression was observed. The G0/G1 arrest was induced by MnCl2 treatment at 16 h and the arrest maintained for 8 h. Following the G0/G1 arrest, MnCl2 blocked the cells at S phase at 28 h and the S phase arrest maintained for at least 4 h. And moreover, proteasome inhibitor MG132 was able to prolong the duration of G0/G1 arrest induced by MnCl2 treatment. Results of western blotting assay revealed that cellular Cdk4, Cdk2 and phospho-Cdk2 (Thr160) levels decreased in manganese-treated cells at both 20 and 28 h. In addition, the decreasing of Cyclin A level and the increasing of p53 and WAF1/p21 were also induced by MnCl2 treatment at 20 h. The expression of Cyclin D1, Cyclin E and Cdc25A proteins was not altered in manganese-treated cells at both 20 and 28 h. Our results indicate that MnCl2 orderly induces G0/G1 and S phase arrest in A549 cells, the decreasing of Cdk4, Cdk2 and Cyclin A, and the increasing of p53 and Cdks inhibitor WAF1/p21 might be responsible for the G0/G1 arrest, and the decreasing of Cdk4 and Cdk2 levels for the S phase arrest.
在本研究中，我们以A549细胞作为锰诱导肺毒性的模型，研究了氯化锰（MnCl₂）对细胞周期进程的影响。用不同浓度的MnCl₂（0、0.01、0.1、0.5、1.0或2.0 mM）处理细胞24、48或72小时。通过MTT法和有丝分裂指数测定来确定细胞增殖，并通过流式细胞仪检测细胞凋亡。结果表明，MnCl₂以剂量和时间依赖性方式抑制A549细胞增殖，并诱导A549细胞凋亡。当通过血清饥饿获得的G0/G1期细胞在10%血清存在的情况下与0.5 mM的MnCl₂孵育几个时间间隔时，观察到细胞周期进程的破坏。MnCl₂处理在16小时诱导G0/G1期阻滞，并持续8小时。在G0/G1期阻滞之后，MnCl₂在28小时将细胞阻滞在S期，且S期阻滞至少持续4小时。此外，蛋白酶体抑制剂MG132能够延长MnCl₂处理诱导的G0/G1期阻滞的持续时间。蛋白质印迹分析结果显示，在20小时和28小时时，锰处理的细胞中细胞周期蛋白依赖性激酶4（Cdk4）、细胞周期蛋白依赖性激酶2（Cdk2）和磷酸化Cdk2（Thr160）水平均下降。此外，在20小时时，MnCl₂处理还诱导细胞周期蛋白A（Cyclin A）水平降低以及p53和WAF1/p21水平升高。在20小时和28小时时，锰处理的细胞中细胞周期蛋白D1（Cyclin D1）、细胞周期蛋白E（Cyclin E）和细胞分裂周期蛋白25A（Cdc25A）的表达均未改变。我们的结果表明，MnCl₂在A549细胞中依次诱导G0/G1期和S期阻滞，Cdk4、Cdk2和Cyclin A的减少以及p53和细胞周期蛋白依赖性激酶抑制剂WAF1/p21的增加可能是G0/G1期阻滞的原因，而Cdk4和Cdk2水平的降低是S期阻滞的原因。
