Shi Xin-li, Yang Jing, Mao Nan, Wu Jing-hua, Ren Lai-feng, Yang Yuan, Yin Xiao-lin, Wei Lin, Li Ming-yuan, Wang Bao-ning
1] Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China [2] Department of Pathobiology and Immunology, Hebei University of Chinese Medicine, Shijiazhuang 050200, China [3] Key Laboratory of Immune Mechanism and Intervention on Serious Disease in Hebei Province, Hebei Medical University, Shijiazhuang 050017, China.
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Acta Pharmacol Sin. 2015 Feb;36(2):252-8. doi: 10.1038/aps.2014.106. Epub 2014 Dec 29.
Interferon-γ inducible protein 16 (IFI16), a DNA sensor for DNA double-strand break (DSB), is expressed in most human hepatocellular carcinoma cell (HCC) lines. In this study we investigated the re-localization of chromatin-bound IFI16 by Nutlin-3, a DNA damage agent, in HCC cells in vitro, and the potential mechanisms.
Human HCC SMMC-7721 (wild-type TP53), Huh-7 (mutant TP53), Hep3B (null TP53) and normal fetal liver L02 cell lines were examined. DSB damage in HCC cells was detected via γH2AX expression and foci formation assay. The expression of IFI16 and IFNB mRNA was measured using RT-PCR, and subcellular localization and expression of the IFI16 protein were detected using chromatin fractionation, Western blot analysis, and fluorescence microscopy.
Treatment of SMMC-7721 cells with Nutlin-3 (10 μmol/L) or etoposide (40 μmol/L) induced significant DSB damage. In SMMC-7721 cells, Nutlin-3 significantly increased the expression levels of IFI16 and IFNB mRNA, and partially redistributed chromatin-bound IFI16 protein to the cytoplasm. These effects were blocked by pretreatment with pifithrin-α, a p53 inhibitor. Furthermore, Nutlin-3 did not induce ectopic expression of IFI16 protein in Huh-7 and Hep3B cells. Moreover, the association of IFI16 with chromatin and Nutlin-3-induced changes in localization were not detected in L02 cells.
Nutlin-3 regulates the subcellular localization of IFI16 in HCC cells in vitro in a p53-dependent manner.
干扰素γ诱导蛋白16（IFI16）是一种用于检测DNA双链断裂（DSB）的DNA传感器，在大多数人肝癌细胞（HCC）系中均有表达。在本研究中，我们在体外研究了DNA损伤剂Nutlin-3对HCC细胞中与染色质结合的IFI16重新定位的影响及其潜在机制。
检测人肝癌SMMC-7721（野生型TP53）、Huh-7（突变型TP53）、Hep3B（TP53缺失）细胞系以及正常胎儿肝L02细胞系。通过γH2AX表达和灶点形成试验检测HCC细胞中的DSB损伤。采用逆转录聚合酶链反应（RT-PCR）检测IFI16和IFNB mRNA的表达，并通过染色质分级分离、蛋白质免疫印迹分析和荧光显微镜检测IFI16蛋白的亚细胞定位和表达。
用Nutlin-3（10 μmol/L）或依托泊苷（40 μmol/L）处理SMMC-7721细胞可诱导显著的DSB损伤。在SMMC-7721细胞中，Nutlin-3显著增加了IFI16和IFNB mRNA的表达水平，并使与染色质结合的IFI16蛋白部分重新分布到细胞质中。这些作用被p53抑制剂pifithrin-α预处理所阻断。此外，Nutlin-3未诱导Huh-7和Hep3B细胞中IFI16蛋白的异位表达。而且，在L02细胞中未检测到IFI16与染色质的结合以及Nutlin-3诱导的定位变化。
Nutlin-3在体外以p53依赖的方式调节HCC细胞中IFI16的亚细胞定位。
