Liu Jing, Wang Qian, Yu Shi-zhu, Zhao Wen-juan, Sun Cui-yun, An Tong-ling, Wang Li-li, Chen Xiu-ju
Department of Neuropathology, Neurological Institute of Tianjin Medical University General Hospital, Tianjin, China.
Zhonghua Bing Li Xue Za Zhi. 2009 Mar;38(3):183-8.
To investigate the pharmacological effects and underlying mechanism of azidothymidine (AZT) on human glioblastoma cells in vitro.
The telomerase activity of human glioblastoma TJ905 cells was determined by TRAP assay after 24 hrs' incubation with 50, 100, 200 micromol/L AZT and control vehicle solution. Colony formation efficiencies of the cells were recorded. Cells of the 1st, 3rd and 6th generations were harvested, followed by evaluations of cyclin A protein expression by Western blot, cell cycle distribution by flow cytometry, apoptotic level by single cell gel electrophoresis and proliferation index by Ki-67 immunocytochemical staining.
AZT inhibited telomerase activity of TJ905 cells. Cyclin A expression levels in the cells treated with 50 and 100 micromol/L AZT were significantly lower than controls (P < 0.01), and down-regulation of the expression was in a dose- and time-dependent manner. Compared with controls, G(0)/G(1) phase cells were obviously decreased (P < 0.05 approximately 0.01) and S phase cells significantly increased (P < 0.05 approximately 0.01) after treatment with 50, 100 and 200 micromol/L AZT. The cell numbers of G(0)/G(1) and S phases at the 1st generation of above three treated groups changed in a dose-dependent manner, whereas S phase cells increases in all AZT treatment groups and G(0)/G(1) phase cell decrease in group treated with 50 micromol/L AZT were also in a time-dependent manner. Both the apoptotic cells of the 1st and 6th generations of all AZT treatment groups were significantly more than controls (P < 0.05 approximately 0.01), their numbers of the 6th generations of the three groups increased with AZT concentration (P < 0.05 approximately 0.01), and all of them were more than the 1st and 3rd generations of the same dosage group (P < 0.05 approximately 0.01). Colony formation efficiencies and Ki-67 labeling indexes of the three AZT treatment groups were distinctly lower than controls (P < 0.01), and they were also decreased with the elevation of AZT concentration and/or the elongation of the incubating time. The difference of any above parameter had no significance among the 1st, 3rd and 6th generations of control group (P > 0.05).
AZT blocks S/G(2) conversion of TJ905 cells by inhibition of telomerase activity and cyclin A expression, leading to an enhancement of apoptosis and suppression of cell proliferation.
研究叠氮胸苷（AZT）对人胶质母细胞瘤细胞的药理作用及其潜在机制。
用50、100、200 μmol/L AZT及对照溶剂溶液孵育人胶质母细胞瘤TJ905细胞24小时后，通过端粒重复扩增法（TRAP）检测细胞端粒酶活性。记录细胞集落形成效率。收集第1、3、6代细胞，通过蛋白质免疫印迹法评估细胞周期蛋白A的表达，通过流式细胞术检测细胞周期分布，通过单细胞凝胶电泳检测凋亡水平，通过Ki-67免疫细胞化学染色检测增殖指数。
AZT抑制TJ905细胞的端粒酶活性。50和100 μmol/L AZT处理的细胞中细胞周期蛋白A的表达水平明显低于对照组（P < 0.01），且表达下调呈剂量和时间依赖性。与对照组相比，50、100和200 μmol/L AZT处理后，G(0)/G(1)期细胞明显减少（P < 0.05至0.01），S期细胞显著增加（P < 0.05至0.01）。上述三个处理组第1代细胞中G(0)/G(1)期和S期的细胞数量呈剂量依赖性变化，而所有AZT处理组中S期细胞增加以及50 μmol/L AZT处理组中G(0)/G(1)期细胞减少也呈时间依赖性。所有AZT处理组第1代和第6代的凋亡细胞均明显多于对照组（P < 0.05至0.01），三组第6代细胞的凋亡细胞数量随AZT浓度增加而增加（P < 0.05至0.01），且均多于相同剂量组的第1代和第3代（P < 0.05至0.01）。三个AZT处理组的集落形成效率和Ki-67标记指数均明显低于对照组（P < 0.01），且随AZT浓度升高和/或孵育时间延长而降低。对照组第1、3、6代之间上述任何参数的差异均无统计学意义（P > 0.05）。
AZT通过抑制端粒酶活性和细胞周期蛋白A的表达来阻断TJ905细胞的S/G(2)转换，从而导致细胞凋亡增加和细胞增殖受到抑制。
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