Boerboom Anne-Marie J F, Vermeulen Martijn, van der Woude Hester, Bremer Birgit I, Lee-Hilz Yee Y, Kampman Ellen, van Bladeren Peter J, Rietjens Ivonne M C M, Aarts Jac M M J G
Division of Toxicology, Wageningen University, P.O. Box 8000, 6700 EA Wageningen, The Netherlands.
Biochem Pharmacol. 2006 Jul 14;72(2):217-26. doi: 10.1016/j.bcp.2006.04.002. Epub 2006 Apr 25.
The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from the human NAD(P)H:quinone oxidoreductase (hNQO1) and the mouse glutathione-S-transferase Ya (mGST-Ya) gene, containing one and two tandem EpRE core sequences, respectively. The standard inducer tert-butylhydroquinone (tBHQ), the electrophile benzyl isothiocyanate (BITC), and the antioxidant flavonoid quercetin were found to induce luciferase expression, thereby validating these newly developed reporter cell lines. For tBHQ and BITC, but not for quercetin, higher maximum luciferase induction was found under control of the mGST-Ya EpRE as compared to the hNQO1 EpRE, pointing at different induction mechanisms. Furthermore, we investigated the structure-activity relationship for induction of luciferase expression by flavonoids in EpRE(mGST-Ya)-LUX cells, and also the relation between luciferase induction and flavonoid antioxidant potency. Five different flavonoids with a planar molecular structure were found to induce various levels of luciferase activity, whereas taxifolin, a non-planar flavonoid, did not induce luciferase activity. This suggests that a stereospecific molecular interaction may be important for EpRE-mediated gene activation, possibly with Keap1, a regulator of EpRE-controlled transcription, or with another effector or receptor protein. No consistent relation between luciferase induction level and flavonoid antioxidant potential was observed. Altogether, these results point to differences in induction mechanism between the various chemoprotective compounds tested. The newly developed stably transfected reporter cell lines provide a validated tool for future screening and mechanistic studies of EpRE-mediated gene transcription.
亲电反应元件（EpRE）是一种转录增强子，参与某些食物成分对癌症化学保护基因表达的调控。基于人NAD(P)H:醌氧化还原酶（hNQO1）和小鼠谷胱甘肽-S-转移酶Ya（mGST-Ya）基因的EpRE序列，构建了两种稳定转染荧光素酶报告基因的细胞系，即EpRE(hNQO1)-LUX和EpRE(mGST-Ya)-LUX，分别含有一个和两个串联的EpRE核心序列。研究发现，标准诱导剂叔丁基对苯二酚（tBHQ）、亲电试剂苄基异硫氰酸酯（BITC）和抗氧化剂类黄酮槲皮素均可诱导荧光素酶表达，从而验证了这些新构建的报告基因细胞系。对于tBHQ和BITC，而非槲皮素，与hNQO1 EpRE相比，在mGST-Ya EpRE的调控下，荧光素酶诱导的最大值更高，这表明诱导机制不同。此外，我们研究了EpRE(mGST-Ya)-LUX细胞中类黄酮诱导荧光素酶表达的构效关系，以及荧光素酶诱导与类黄酮抗氧化能力之间的关系。发现五种具有平面分子结构的不同类黄酮可诱导不同水平的荧光素酶活性，而非平面类黄酮紫杉叶素则不诱导荧光素酶活性。这表明立体特异性分子相互作用可能对EpRE介导的基因激活很重要，可能与EpRE控制转录的调节因子Keap1或其他效应器或受体蛋白有关。未观察到荧光素酶诱导水平与类黄酮抗氧化潜力之间的一致关系。总之，这些结果表明所测试的各种化学保护化合物之间的诱导机制存在差异。新构建的稳定转染报告基因细胞系为未来EpRE介导的基因转录的筛选和机制研究提供了一个经过验证的工具。
