Superinduction of estrogen receptor mediated gene expression in luciferase based reporter gene assays is mediated by a post-transcriptional mechanism

A.M. Sotoca Covaleda, T.F.H. Bovee, W. Brand, N.R. Velikova, A.J. Murk, J.J.M. Vervoort, I.M.C.M. Rietjens

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Abstract

Several estrogenic compounds including the isoflavonoid genistein have been reported to induce a higher maximal response than the natural estrogen 17ß-estradiol in in vitro luciferase based reporter gene bioassays for testing estrogenicity. The phenomenon has been referred to as superinduction. The mechanism underlying this effect and thus also its biological relevance remain to be elucidated. In the present study several hypotheses for the possible mechanisms underlying this superinduction were investigated using genistein as the model compound. These hypotheses included (i) a non-estrogen receptor (ER)-mediated mechanism, (ii) a role for an ER activating genistein metabolite with higher ER inducing activity than genistein itself, and (iii) a post-transcriptional mechanism that is not biologically relevant but specific for the luciferase based reporter gene assays. The data presented in this study indicate that induction and also superinduction of the reporter gene is ER-mediated, and that superinduction by genistein could be ascribed to stabilization of the firefly luciferase reporter enzyme increasing the bioluminescent signal during the cell-based assay. This indicates that the phenomenon of superinduction may not be biologically relevant but may rather represent a post-transcriptional effect on enzyme stability.
Original languageEnglish
Pages (from-to)204-211
JournalJournal of Steroid Biochemistry and Molecular Biology
Volume122
Issue number4
DOIs
Publication statusPublished - 2010

Fingerprint

Genistein
Luciferases
Reporter Genes
Gene expression
Estrogen Receptors
Assays
Genes
Gene Expression
Estrogens
Firefly Luciferases
Enzyme Stability
Bioassay
Enzymes
Metabolites
Biological Assay
Estradiol
Stabilization
Testing

Keywords

  • human breast-cancer
  • in-vitro
  • er-beta
  • firefly luciferase
  • cell-proliferation
  • genistein
  • phytoestrogens
  • metabolites
  • daidzein
  • alpha

Cite this

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title = "Superinduction of estrogen receptor mediated gene expression in luciferase based reporter gene assays is mediated by a post-transcriptional mechanism",
abstract = "Several estrogenic compounds including the isoflavonoid genistein have been reported to induce a higher maximal response than the natural estrogen 17{\ss}-estradiol in in vitro luciferase based reporter gene bioassays for testing estrogenicity. The phenomenon has been referred to as superinduction. The mechanism underlying this effect and thus also its biological relevance remain to be elucidated. In the present study several hypotheses for the possible mechanisms underlying this superinduction were investigated using genistein as the model compound. These hypotheses included (i) a non-estrogen receptor (ER)-mediated mechanism, (ii) a role for an ER activating genistein metabolite with higher ER inducing activity than genistein itself, and (iii) a post-transcriptional mechanism that is not biologically relevant but specific for the luciferase based reporter gene assays. The data presented in this study indicate that induction and also superinduction of the reporter gene is ER-mediated, and that superinduction by genistein could be ascribed to stabilization of the firefly luciferase reporter enzyme increasing the bioluminescent signal during the cell-based assay. This indicates that the phenomenon of superinduction may not be biologically relevant but may rather represent a post-transcriptional effect on enzyme stability.",
keywords = "human breast-cancer, in-vitro, er-beta, firefly luciferase, cell-proliferation, genistein, phytoestrogens, metabolites, daidzein, alpha",
author = "{Sotoca Covaleda}, A.M. and T.F.H. Bovee and W. Brand and N.R. Velikova and A.J. Murk and J.J.M. Vervoort and I.M.C.M. Rietjens",
year = "2010",
doi = "10.1016/j.jsbmb.2010.06.010",
language = "English",
volume = "122",
pages = "204--211",
journal = "Journal of Steroid Biochemistry and Molecular Biology",
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}

TY - JOUR

T1 - Superinduction of estrogen receptor mediated gene expression in luciferase based reporter gene assays is mediated by a post-transcriptional mechanism

AU - Sotoca Covaleda, A.M.

AU - Bovee, T.F.H.

AU - Brand, W.

AU - Velikova, N.R.

AU - Murk, A.J.

AU - Vervoort, J.J.M.

AU - Rietjens, I.M.C.M.

PY - 2010

Y1 - 2010

N2 - Several estrogenic compounds including the isoflavonoid genistein have been reported to induce a higher maximal response than the natural estrogen 17ß-estradiol in in vitro luciferase based reporter gene bioassays for testing estrogenicity. The phenomenon has been referred to as superinduction. The mechanism underlying this effect and thus also its biological relevance remain to be elucidated. In the present study several hypotheses for the possible mechanisms underlying this superinduction were investigated using genistein as the model compound. These hypotheses included (i) a non-estrogen receptor (ER)-mediated mechanism, (ii) a role for an ER activating genistein metabolite with higher ER inducing activity than genistein itself, and (iii) a post-transcriptional mechanism that is not biologically relevant but specific for the luciferase based reporter gene assays. The data presented in this study indicate that induction and also superinduction of the reporter gene is ER-mediated, and that superinduction by genistein could be ascribed to stabilization of the firefly luciferase reporter enzyme increasing the bioluminescent signal during the cell-based assay. This indicates that the phenomenon of superinduction may not be biologically relevant but may rather represent a post-transcriptional effect on enzyme stability.

AB - Several estrogenic compounds including the isoflavonoid genistein have been reported to induce a higher maximal response than the natural estrogen 17ß-estradiol in in vitro luciferase based reporter gene bioassays for testing estrogenicity. The phenomenon has been referred to as superinduction. The mechanism underlying this effect and thus also its biological relevance remain to be elucidated. In the present study several hypotheses for the possible mechanisms underlying this superinduction were investigated using genistein as the model compound. These hypotheses included (i) a non-estrogen receptor (ER)-mediated mechanism, (ii) a role for an ER activating genistein metabolite with higher ER inducing activity than genistein itself, and (iii) a post-transcriptional mechanism that is not biologically relevant but specific for the luciferase based reporter gene assays. The data presented in this study indicate that induction and also superinduction of the reporter gene is ER-mediated, and that superinduction by genistein could be ascribed to stabilization of the firefly luciferase reporter enzyme increasing the bioluminescent signal during the cell-based assay. This indicates that the phenomenon of superinduction may not be biologically relevant but may rather represent a post-transcriptional effect on enzyme stability.

KW - human breast-cancer

KW - in-vitro

KW - er-beta

KW - firefly luciferase

KW - cell-proliferation

KW - genistein

KW - phytoestrogens

KW - metabolites

KW - daidzein

KW - alpha

U2 - 10.1016/j.jsbmb.2010.06.010

DO - 10.1016/j.jsbmb.2010.06.010

M3 - Article

VL - 122

SP - 204

EP - 211

JO - Journal of Steroid Biochemistry and Molecular Biology

JF - Journal of Steroid Biochemistry and Molecular Biology

SN - 0960-0760

IS - 4

ER -