Novel Stably Transfected Gene Reporter Human Hepatoma Cell Line for Assessment of Aryl Hydrocarbon Receptor Transcriptional Activity: Construction and Characterization
Novel Stably Transfected Gene Reporter Human Hepatoma Cell Line for Assessment of Aryl Hydrocarbon Receptor Transcriptional Activity: Construction and Characterization
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DOI：
10.1021/es2029334
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发表时间：
2011-12-01
影响因子：
11.4
通讯作者：
Dvorak, Zdenek
中科院分区：
文献类型：
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作者：
Novotna, Arleta;Petr, Pavek;Dvorak, Zdenek
我们构建了稳定转染的基因报告细胞系AZ-AHR，可以测量芳烃受体（AhR）的转录活性。用荧光素酶报告基因上游含有多个AhR结合位点的构建体转染人肝癌HepG2细胞。我们准备了12个克隆，并鉴定了对TCDD响应性最好的5个。对不同的AhR配体，包括TCDD、3-甲基胆蒽、靛玉红、白藜芦醇、奥美拉唑和SP600125进行剂量效应分析。所有克隆的EC（50）值相似。荧光素酶的诱导具有时间依赖性，用5 nM TCDD处理6小时足以评估96孔板格式下AhR的转录活性（8-24倍诱导）。冻存细胞解冻后对AhR配体的反应与新鲜细胞无显著差异。在15代和30天的培养中，细胞系对AhR配体保持完全反应，无明显变化。总之，我们开发了新的人类荧光素酶报告细胞系AZ-AHR，用于监测AhR的转录活性。该检测的灵敏度允许高通量格式（96孔板），并在孵育6小时后立即评估荧光素酶活性，这对细胞毒性化合物的研究具有潜在的意义。
We constructed stably transfected gene reporter cell line AZ-AHR, allowing measurement of aryl hydrocarbon receptor (AhR) transcriptional activity. Human hepatoma HepG2 cells were transfected with a construct containing several AhR binding sites upstream of luciferase reporter gene. We prepared 12 clones and we characterized the best five in responsiveness to TCDD. Dose response analyses were performed for various AhR ligands, including TCDD, 3-methylcholanthrene, indirubin, resveratrol, omeprazole, and SP600125. The EC(50) values were similar in all tested clones. Induction of luciferase was time-dependent, and treatment for 6 h with 5 nM TCDD was sufficient to evaluate AhR transcriptional activity in 96-well plate format (8-24 fold induction). Response to AhR ligands of cryopreserved cells after thawing was not significantly different from that of fresh cells. Cell line remained fully responsive to AhR ligands over 15 passages and 30 days in culture without significant alterations. Overall, we have developed novel human luciferase reporter cell line AZ-AHR for monitoring AhR transcriptional activity. The sensitivity of the assay allows high throughput format (96-well plate) and evaluation of luciferase activity as soon as after 6 h of incubation, which has potential implication for studies of cytotoxic compounds.