Lam R K K, Han Wei, Yu K N
Department of Physics and Materials Science, City University of Hong Kong, Hong Kong.
Center of Medical Physics and Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, PR China.
Mutat Res. 2015 Dec;782:23-33. doi: 10.1016/j.mrfmmm.2015.10.004. Epub 2015 Oct 23.
We studied the involvement of NF-κB pathway activation in the rescue effect in HeLa and NIH/3T3 cells irradiated by α particles. Firstly, upon irradiation by 5 cGy of α particles, for both cell lines, the numbers of 53BP1 foci/cell at 12 h post-irradiation were significantly smaller when only 2.5% of the cell population was irradiated as compared to 100% irradiation, which demonstrated the rescue effect. Secondly, we studied the effect of NF-κB on the rescue effect through the use of the NF-κB activation inhibitor BAY-11-7082. Novel experimental setup and procedures were designed to prepare the medium (CM) which had conditioned the bystander cells previously partnered with irradiated cells, to ensure physical separation between rescue and bystander signals. BAY-11-7082 itself did not inflict DNA damages in the cells or have effects on activation of the NF-κB response pathway in the irradiated cells through direct irradiation. The rescue effect was induced in both cell lines by the CM, which was abrogated if BAY-11-7082 was added to the CM. Thirdly, we studied the effect of NF-κB on the rescue effect through staining for phosphorylated NF-κB (p-NF-κB) expression using the anti-NF-κB p65 (phospho S536) antibody. When the fraction of irradiated cells dropped from 100% to 2.5%, the p-NF-κB expression in the cell nuclei of irradiated NIH/3T3 cells increased significantly, while that in the cell nuclei of irradiated HeLa cells also increased although not significantly. Moreover, the p-NF-κB expression in the cell nuclei of irradiated HeLa cells and NIH/3T3 cells treated with CM also increased significantly.
我们研究了NF-κB信号通路激活在α粒子辐照的HeLa和NIH/3T3细胞中的挽救效应。首先，用5 cGy的α粒子进行辐照，对于这两种细胞系，与100%辐照相比，仅2.5%的细胞群体受到辐照时，辐照后12小时每个细胞中53BP1病灶的数量显著减少，这证明了挽救效应。其次，我们通过使用NF-κB激活抑制剂BAY-11-7082研究了NF-κB对挽救效应的影响。设计了新的实验装置和程序来制备之前与辐照细胞配对的旁观者细胞所条件化的培养基（CM），以确保挽救信号和旁观者信号在物理上分离。BAY-11-7082本身不会对细胞造成DNA损伤，也不会通过直接辐照对辐照细胞中的NF-κB反应通路激活产生影响。CM在两种细胞系中均诱导出挽救效应，如果向CM中添加BAY-11-7082，该效应则被消除。第三，我们通过使用抗NF-κB p65（磷酸化S536）抗体对磷酸化NF-κB（p-NF-κB）表达进行染色，研究了NF-κB对挽救效应的影响。当辐照细胞的比例从100%降至2.5%时，辐照的NIH/3T3细胞核中p-NF-κB表达显著增加，而辐照的HeLa细胞核中p-NF-κB表达也有所增加，尽管增幅不显著。此外，用CM处理的辐照HeLa细胞和NIH/3T3细胞核中p-NF-κB表达也显著增加。
