Hellweg Christine E, Baumstark-Khan Christa, Horneck Gerda
Radiation Biology, Institute of Aerospace Medicine, DLR, Linder Höhe, D-51170 Köln, Germany.
J Biomol Screen. 2003 Oct;8(5):511-21. doi: 10.1177/1087057103257204.
Cellular stress protection responses lead to increased transcription of several genes via modulation of transcription factors. Activation of the Nuclear Factor kappaB (NF-kappaB) pathway as a possible antiapoptotic route represents one important cellular stress response. To identify conditions that are capable of modifying this pathway, a screening assay for detection of NF-kappaB-dependent gene activation using the reporter protein Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) was developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing 4 copies of the NF-kappaB response element. Treatment with tumor necrosis factor alpha (TNF-alpha) gave rise to substantial EGFP/d2EGFP expression in up to 90% of the cells and was therefore used to screen different stably transfected clones for induction of NF-kappaB-dependent gene expression. The time course of NF-kappaB activation leading to d2EGFP expression was measured in an oligonucleotide-based NF-kappaB-ELISA. NF-kappaB binding in-creased after 15-min incubation with TNF-alpha. In parallel, d2EGFP increased after 3 h and reached its maximum at 24 h. These results show (1) the time lag between NF-kappaB activation and d2EGFP transcription, translation, and protein folding and (2) the increased reporter gene expression after treatment with TNF-alpha to be caused by the activation of NF-kappaB. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening.
细胞应激保护反应通过转录因子的调控导致多个基因的转录增加。核因子κB（NF-κB）信号通路的激活作为一种可能的抗凋亡途径，是一种重要的细胞应激反应。为了确定能够调节该信号通路的条件，开发了一种使用报告蛋白增强型绿色荧光蛋白（EGFP）及其不稳定变体（d2EGFP）检测NF-κB依赖性基因激活的筛选试验。人胚肾（HEK/293）细胞用携带EGFP或d2EGFP的载体进行稳定转染，该载体受含有4个拷贝NF-κB反应元件的合成启动子控制。用肿瘤坏死因子α（TNF-α）处理后，高达90%的细胞中出现大量EGFP/d2EGFP表达，因此用于筛选不同的稳定转染克隆以诱导NF-κB依赖性基因表达。在基于寡核苷酸的NF-κB-ELISA中测量导致d2EGFP表达的NF-κB激活的时间进程。与TNF-α孵育15分钟后，NF-κB结合增加。同时，d2EGFP在3小时后增加，并在24小时达到最大值。这些结果表明：（1）NF-κB激活与d2EGFP转录、翻译和蛋白质折叠之间存在时间滞后；（2）TNF-α处理后报告基因表达增加是由NF-κB激活引起的。d2EGFP表达的检测需要流式细胞术分析或荧光显微镜检查，而EGFP也可以在酶标仪中测量，使得该试验可用于高通量筛选。
