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Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity

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Author: Boerboom, A.M.J.F. · Vermeulen, M. · Woude, H. van der · Bremer, B.I. · Lee-Hilz, Y.Y. · Kampman, E. · Bladeren, P.J. van · Rietjens, I.M.C.M. · Aarts, J.M.M.J.G.
Type:article
Date:2006
Institution: TNO Kwaliteit van Leven
Source:Biochemical Pharmacology, 2, 72, 217-226
Identifier: 280129
doi: doi:10.1016/j.bcp.2006.04.002
Keywords: Biology · Benzyl isothiocyanate · Fisetin · Glutathione transferase · Kaempferol · Luteolin · Myricetin · Quercetin · Reduced nicotinamide adenine dinucleotide (phosphate) dehydrogenase (quinone) · Taxifolin · Tert butylhydroquinone · Animal cell · Antioxidant activity · Cancer cell culture · Cancer screening · Chemoprophylaxis · Controlled study · Drug potency · Drug structure · Electrophile responsive element mediated gene · Enzyme induction · Gene · Gene activation · Gene expression · Gene induction · Genetic transcription · Human cell · Molecular interaction · Mouse · Nonhuman · Reporter gene · Screening test · Stereospecificity · Structure activity relation · Tandem repeat · Transcription initiation · Validation study · Animals · Base Sequence · Cell Line · DNA Primers · Enzyme Induction · Flavonoids · Gene Expression · Genes, Reporter · Humans · Luciferases · Mice · Plasmids · Structure-Activity Relationship · Transcription, Genetic · Benzyl isothiocyanate · Electrophile-responsive element (EpRE) · Hepa-1c1c7 cells · Stable luciferase reporter cell lines · Transient transfection

Abstract

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. © 2006 Elsevier Inc. All rights reserved. Chemicals/CAS: benzyl isothiocyanate, 622-78-6; fisetin, 528-48-3; glutathione transferase, 50812-37-8; kaempferol, 520-18-3; luteolin, 491-70-3; myricetin, 529-44-2; quercetin, 117-39-5; reduced nicotinamide adenine dinucleotide (phosphate) dehydrogenase (quinone), 9032-20-6; taxifolin, 480-18-2; tert butylhydroquinone, 1948-33-0; DNA Primers; Flavonoids; Luciferases, EC 1.13.12.-