title: Loss of C/EBPδ enhances IR-induced cell death by promoting oxidative stress and mitochondrial dysfunction.
abstract: Exposure of cells to ionizing radiation (IR) generates reactive oxygen species (ROS). This results in increased oxidative stress and DNA double strand breaks (DSBs) which are the two underlying mechanisms by which IR causes cell/tissue injury. Cells that are deficient or impaired in the cellular antioxidant response are susceptible to IR-induced apoptosis. The transcription factor CCAAT enhancer binding protein delta (Cebpd, C/EBPδ) has been implicated in the regulation of oxidative stress, DNA damage response, genomic stability and inflammation. We previously reported that Cebpd-deficient mice are sensitive to IR and display intestinal and hematopoietic injury, however the underlying mechanism is not known. In this study, we investigated whether an impaired ability to detoxify IR-induced ROS was the underlying cause of the increased radiosensitivity of Cebpd-deficient cells. We found that Cebpd-knockout (KO) mouse embryonic fibroblasts (MEFs) expressed elevated levels of ROS, both at basal levels and after exposure to gamma radiation which correlated with increased apoptosis, and decreased clonogenic survival. Pre-treatment of wild type (WT) and KO MEFs with polyethylene glycol-conjugated Cu-Zn superoxide dismutase (PEG-SOD) and catalase (PEG-CAT) combination prior to irradiation showed a partial rescue of clonogenic survival, thus demonstrating a role for increased intracellular oxidants in promoting IR-induced cell death. Analysis of mitochondrial bioenergetics revealed that irradiated KO MEFs showed significant reductions in basal, adenosine triphosphate (ATP)-linked, maximal respiration and reserved respiratory capacity and decrease in intracellular ATP levels compared to WT MEFs indicating they display mitochondrial dysfunction. KO MEFs expressed significantly lower levels of the cellular antioxidant glutathione (GSH) and its precursor- cysteine as well as methionine. In addition to its antioxidant function, GSH plays an important role in detoxification of lipid peroxidation products such as 4-hydroxynonenal (4-HNE). The reduced GSH levels observed in KO MEFs correlated with elevated levels of 4-HNE protein adducts in irradiated KO MEFs compared to respective WT MEFs. We further showed that pre-treatment with the GSH precursor, N-acetyl L-cysteine (NAC) prior to irradiation showed a significant reduction of IR-induced cell death and increases in GSH levels, which contributed to the overall increase in clonogenic survival of KO MEFs. In contrast, pre-treatment with the GSH synthesis inhibitor- buthionine sulfoximine (BSO) further reduced the clonogenic survival of irradiated KO MEFs. This study demonstrates a novel role for C/EBPδ in protection from basal as well as IR-induced oxidative stress and mitochondrial dysfunction thus promoting post-radiation survival.
authors.affiliation: Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States.
authors.forename: Sudip
authors.initials: S
authors.lastname: Banerjee
authors.affiliation: Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States.
authors.forename: Nukhet
authors.initials: N
authors.lastname: Aykin-Burns
authors.affiliation: Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States.
authors.forename: Kimberly J
authors.initials: KJ
authors.lastname: Krager
authors.affiliation: Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States.
authors.forename: Sumit K
authors.initials: SK
authors.lastname: Shah
authors.affiliation: Arkansas Children's Hospital Research Institute, Little Rock, AR 72205, United States.
authors.forename: Stepan B
authors.initials: SB
authors.lastname: Melnyk
authors.affiliation: Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States; Surgical Services, Central Arkansas Veterans Healthcare System, Little Rock, AR 72205, United States.
authors.forename: Martin
authors.initials: M
authors.lastname: Hauer-Jensen
authors.affiliation: Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States. Electronic address: SAPawar@uams.edu.
authors.forename: Snehalata A
authors.initials: SA
authors.lastname: Pawar
mesh_terms: D000111:Acetylcysteine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D000447:Aldehydes / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D051743:CCAAT-Enhancer-Binding Protein-delta / Q000172:deficiency / Q000235:genetics*; D002374:Catalase / Q000494:pharmacology; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects*; D005720:Gamma Rays; D005786:Gene Expression Regulation; D005978:Glutathione / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects*; D018384:Oxidative Stress; D011092:Polyethylene Glycols / Q000494:pharmacology; D061251:Primary Cell Culture; D017382:Reactive Oxygen Species / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction; D013482:Superoxide Dismutase / Q000494:pharmacology
chemical_list: D000447:Aldehydes; C497253:Cebpd protein, mouse; D017382:Reactive Oxygen Species; C070295:catalase-polyethylene glycol; D051743:CCAAT-Enhancer-Binding Protein-delta; D011092:Polyethylene Glycols; D019328:Buthionine Sulfoximine; D000255:Adenosine Triphosphate; D004247:DNA; D002374:Catalase; D013482:Superoxide Dismutase; C063471:polyethylene glycol-superoxide dismutase; D005978:Glutathione; C027576:4-hydroxy-2-nonenal; D000111:Acetylcysteine
keywords: CCAAT enhancer binding protein delta; Glutathione; Ionizing radiation; Mitochondrial dysfunction; Oxidative stress; Reactive oxygen species