title: H2O2 in the induction of NF-κB-dependent selective gene expression.
abstract: NF-κB is a transcription factor that plays key roles in health and disease. Learning how this transcription factor is regulated by hydrogen peroxide (H2O2) has been slowed down by the lack of methodologies suitable to obtain quantitative data. Literature is abundant with apparently contradictory information on whether H2O2 activates or inhibits NF-κB. There is increasing evidence that H2O2 is not just a generic modulator of transcription factors and signaling molecules but becomes a specific regulator of individual genes. Here, we describe a detailed protocol to obtain rigorous quantitative data on the effect of H2O2 on members of the NF-κB/Rel and IκB families, in which H2O2 is delivered as a steady-state addition instead of the usual bolus addition. Solutions, pilot experiments, and experimental set-ups are fully described. In addition, we outline a protocol to measure the impact of alterations in the promoter κB regions on the H2O2 regulation of the expression of individual genes. As important as evaluating the effects of H2O2 alone is the evaluation of the modulation elicited by this oxidant on cytokine regulation of NF-κB. We illustrate this for the cytokine tumor necrosis factor alpha.
authors.affiliation: Departamento de Química e Bioquímica and Centro de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Luísa
authors.initials: L
authors.lastname: Cyrne
authors.forename: Virgínia
authors.initials: V
authors.lastname: Oliveira-Marques
authors.forename: H Susana
authors.initials: HS
authors.lastname: Marinho
authors.forename: Fernando
authors.initials: F
authors.lastname: Antunes
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation / Q000187:drug effects*; D005947:Glucose / Q000378:metabolism; D005949:Glucose Oxidase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020569:Proto-Oncogene Proteins c-rel / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D016328:NF-kappa B; D020569:Proto-Oncogene Proteins c-rel; D014409:Tumor Necrosis Factor-alpha; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D051550:I-kappa B Kinase; D005947:Glucose
keywords: IκB; Single-nucleotide polymorphisms; Steady state; TNF-α; κB sites