title: Comprehensive Landscape of Nrf2 and p53 Pathway Activation Dynamics by Oxidative Stress and DNA Damage.
abstract: A quantitative dynamics pathway map of the Nrf2-mediated oxidative stress response and p53-related DNA damage response pathways as well as the cross-talk between these pathways has not systematically been defined. To allow the dynamic single cell evaluation of these pathways, we have used BAC-GFP recombineering to tag for each pathway's three key components: for the oxidative stress response, Keap1-GFP, Nrf2-GFP, and Srxn1-GFP; for the DNA damage response, 53bp1-GFP, p53-GFP, and p21-GFP. The dynamic activation of these individual components was assessed using quantitative high throughput confocal microscopy after treatment with a broad concentration range of diethyl maleate (DEM; to induce oxidative stress) and etoposide (to induce DNA damage). DEM caused a rapid activation of Nrf2, which returned to baseline levels at low concentrations but remained sustained at high concentrations. Srxn1-GFP induction and Keap1-GFP translocation to autophagosomes followed later, with upper boundaries reached at high concentrations, close to the onset of cell death. Etoposide caused rapid accumulation of 53bp1-GFP in DNA damage foci, which was later followed by the concentration dependent nuclear accumulation of p53-GFP and subsequent induction of p21-GFP. While etoposide caused activation of Srxn1-GFP, a modest activation of DNA damage reporters was observed for DEM at high concentrations. Interestingly, Nrf2 knockdown caused an inhibition of the DNA damage response at high concentrations of etoposide, while Keap1 knockdown caused an enhancement of the DNA damage response already at low concentrations of etoposide. Knockdown of p53 did not affect the oxidative stress response. Altogether, the current stress response landscapes provide insight in the time course responses of and cross-talk between oxidative stress and DNA-damage and defines the tipping points where cell injury may switch from adaptation to injury.
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Steven
authors.initials: S
authors.lastname: Hiemstra
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Marije
authors.initials: M
authors.lastname: Niemeijer
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Esmee
authors.initials: E
authors.lastname: Koedoot
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Steven
authors.initials: S
authors.lastname: Wink
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Janna E
authors.initials: JE
authors.lastname: Klip
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Matthijs
authors.initials: M
authors.lastname: Vlasveld
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: de Zeeuw
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Bram
authors.initials: B
authors.lastname: van Os
authors.affiliation: SEAC, Unilever, Colworth, United Kingdom.
authors.forename: Andrew
authors.initials: A
authors.lastname: White
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University , Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Bob van de
authors.identifier: 0000-0002-5839-2380
authors.initials: BV
authors.lastname: Water
mesh_terms: D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects*; D005047:Etoposide / Q000633:toxicity*; D017930:Genes, Reporter; D056945:Hep G2 Cells; D006801:Humans; D000072019:Kelch-Like ECH-Associated Protein 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008298:Maleates / Q000633:toxicity*; D051267:NF-E2-Related Factor 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D050759:Cyclin-Dependent Kinase Inhibitor p21; D000072019:Kelch-Like ECH-Associated Protein 1; D008298:Maleates; D051267:NF-E2-Related Factor 2; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D005047:Etoposide; D050862:Oxidoreductases Acting on Sulfur Group Donors; C504024:SRXN1 protein, human; C014476:diethyl maleate