EBV-LMP1-targeted DNAzyme induces DNA damage and causes cell cycle arrest in LMP1-positive nasopharyngeal carcinoma cells
EBV-LMP1-targeted DNAzyme induces DNA damage and causes cell cycle arrest in LMP1-positive nasopharyngeal carcinoma cells
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EBV-LMP1 靶向 DNAzyme 诱导 DNA 损伤并导致 LMP1 阳性鼻咽癌细胞细胞周期停滞
DOI：
10.3892/ijo.2013.2098
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发表时间：
2013-11-01
影响因子：
5.2
通讯作者：
Cao, Ya
中科院分区：
文献类型：
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作者：
Ma, Xiaoqian;Xu, Zhijie;Cao, Ya
本研究旨在探讨靶向LMP 1的DNAzyme 1（DZ 1）对鼻咽癌细胞周期进程影响的分子机制。我们发现，活性DZ 1抑制潜伏膜蛋白1（LMP 1）的表达，并诱导G1期阻滞。此外，这种细胞周期失调显示伴随着DNA损伤标记物γ-H2 AX的上调、DNA损伤反应因子p-p53-Ser 15的下调和细胞增殖抑制。为了研究是什么影响了细胞周期进程，我们检测了两个检查点相关的细胞周期蛋白和细胞周期蛋白依赖性激酶（CDKs）的表达。我们发现DZ 1处理24 h后细胞周期蛋白D1和细胞周期蛋白E蛋白水平下降。此外，我们观察到CDK 4抗体免疫沉淀复合物中CDK 4活性的抑制和细胞周期蛋白D1的表达减少。我们还发现，在CDC 2磷酸化的Thr 161，这部分代表在DZ 1处理的CNE 1-LMP 1细胞中的CDC 2激酶活性的减少，虽然LMP 1表达的下调对细胞周期蛋白B1和CDC 2的表达没有影响。此外，我们分析了DZ 1诱导的cdc 2激酶活性的变化，发现LMP 1表达的下调导致CNE 1-LMP 1中cdc 2激酶活性降低5倍。结果表明，DZ 1下调LMP 1的表达，导致细胞DNA损伤，进而抑制细胞增殖，导致细胞周期检查点功能障碍，使G1期阻滞，G2/M期细胞减少。
This study aimed to determine the molecular mechanisms underlying the effect of the LMP1-targeted DNAzyme 1 (DZ1) on cell cycle progression in nasopharyngeal carcinoma (NPC) cells. We showed that the active DZ1 inhibited the expression of latent membrane protein 1 (LMP1) and induced a G1 phase arrest. In addition, this cell cycle deregulation was shown to be accompanied by upregulation of the DNA damage marker gamma-H2AX, downregulation of the DNA damage response factor p-p53-Ser15 and cell proliferation inhibition. To investigate what affected the cell cycle progression, we examined the expression of two checkpoint-related cyclins and cyclin-dependent kinases (CDKs). We found a decrease of cyclin D1 and cyclin E protein levels at 24 h from the DZ1 treatment. Moreover, we observed inhibition of CDK4 activity and decreased cyclin D1 expression in the complexes immunoprecipitated with CDK4 antibody. We also found a reduction in cdc2 phosphorylation at Thr161 which partially stands for the cdc2 kinase activity in DZ1-treated CNE1-LMP1 cells, although the downregulation of LMP1 expression had no effect on the cyclin B1 and cdc2 expression. Further, we analyzed changes in cdc2 kinase activity induced by DZ1 and found that the downregulation of the LMP1 expression resulted in a 5-fold reduction in cdc2 kinase activity in CNE1-LMP1. The data suggest that the downregulation of the LMP1 expression by DZ1 was able to induce DNA damage, which then further inhibited the cell proliferation and resulted in malfunction of cell cycle checkpoints that led to G1 phase arrest and the decrease in number of cells in G2/M phase.