Inhibition of p53 by lentiviral mediated shRNA abrogates G1 arrest and apoptosis in retinal pigmented epithelial cell line
Inhibition of p53 by lentiviral mediated shRNA abrogates G1 arrest and apoptosis in retinal pigmented epithelial cell line
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DOI：
10.4161/cc.4.5.1672
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发表时间：
2005-05-01
期刊：
影响因子：
4.3
通讯作者：
Ramabhadran, R
中科院分区：
文献类型：
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作者：
Nair, AR;Schliekelman, M;Ramabhadran, R
我们使用 RNA 干扰沉默了 ARPE-19（一种人视网膜色素上皮细胞系）中的 p53 基因表达。研究了沉默 p53 基因对 ARPE-19 细胞增殖的影响。使用慢病毒载体将四种针对人 p53 mRNA 不同区域的短发夹 RNA (shRNA) 分别递送至 ARPE-19 细胞中，以产生稳定的细胞系。在四种 shRNA 转导的 ARPE-19 细胞系中，p53 mRNA 和蛋白质水平都有不同程度的降低。 p53 表达显着降低 (85-90%) 的细胞系在用喜树碱、依托泊苷和 MMS 损伤 DNA 后，p21 启动子激活降低。用喜树碱处理野生型 ARPE-19 细胞会导致细胞凋亡，而沉默 p53 表达则会增加其存活率。细胞周期分析表明，辐射导致 ARPE-19 细胞中 G(1) 停滞，并且 p53 沉默细胞中的停滞显着减少。因此，p53 在 ARPE-19 细胞对通过不同机制起作用的 DNA 损伤剂的反应中发挥着核心作用。此外，p53 表达减少的 ARPE-19 细胞的行为与 p53 突变或无功能的肿瘤细胞系相似。目前的数据证明了慢病毒载体可用于创建特定基因表达减少的稳定的同基因细胞系，从而允许以成对方式研究基因的功能、其控制的途径以及治疗对基因组成改变的细胞的影响。
We silenced p53 gene expression in ARPE-19, a human retinal pigmented epithelial cell line using RNA interference. The effect of silencing the p53 gene in proliferating ARPE-19 cells was studied. Four short hairpin RNAs (shRNAs) targeting different regions of human p53 mRNA were delivered individually into ARPE-19 cells using lentiviral vector to produce stable cell lines. p53 mRNA and protein levels were reduced to varying extents in the four shRNA-transduced ARPE-19 cell lines. The cell line that showed greatest reduction (85-90%) of p53 expression showed decreased p21 promoter activation after DNA damage with camptothecin, etoposide and MMS. Whereas treatment of wild type ARPE-19 cells with camptothecin resulted in apoptosis, silencing p53 expression increased their survival. Cell cycle analyses indicated that irradiation resulted in a G(1) arrest in ARPE-19 cells, and that the arrest was significantly reduced in p53-silenced cells. Thus, p53 plays a central role in the response of ARPE-19 cells to DNA damaging agents that act via different mechanisms. Additionally, ARPE-19 cells with reduced p53 expression behave similar to tumor cell lines with mutated or non-functional p53. The present data demonstrate the utility of lentiviral vectors to create stable isogenic cell lines with reduced expression of a specific gene, thereby permitting the study of the function of a gene, the pathways controlled by it, and the effect of therapeutics on a cell with altered genetic makeup in a pair-wise fashion.