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A project assigned by my research mentor to review specific publications on cell death by a renowned professional in the field.
John A. Corbett: DNA damage 1.) Am J Physiol Endocrinol Metab. 2012 Jul 15;303(2):E172-9. Epub 2012 Apr 24. Cytokine-mediated β-cell damage in PARP-1-deficient islets. Andreone T, Meares GP, Hughes KJ, Hansen PA, Corbett JA. Department of Pediatrics, Saint Louis University, St. Louis, MO, USA. email@example.com Poly(ADP)-ribose polymerase (PARP) is an abundant nuclear protein that is activated by DNA damage; once active, it modifies nuclear proteins through attachment of poly(ADP)-ribose units derived from β-nicotinamide adenine dinucleotide (NAD(+)). In mice, the deletion of PARP-1 attenuates tissue injury in a number of animal models of human disease, including streptozotocin-induced diabetes. Also, inflammatory cell signaling and inflammatory gene expression are attenuated in macrophages isolated from endotoxin-treated PARP-1-deficient mice. In this study, the effects of PARP-1 deletion on cytokine-mediated β-cell damage and macrophage activation were evaluated. There are no defects in inflammatory mediator signaling or inflammatory gene expression in macrophages and islets isolated from PARP-1-deficient mice. While PARP-1 deficiency protects islets against cytokine-induced islet cell death as measured by biochemical assays of membrane polarization, the genetic absence of PARP-1 does not effect cytokine-induced inhibition of insulin secretion or cytokine-induced DNA damage in islets. While PARP-1 deficiency appears to provide protection from cell death, it fails to provide protection against the inhibitory actions of cytokines on insulin secretion or the damaging actions on islet DNA integrity. Techniques: Treatment of rat islets with the macrophage-derived cytokine IL-1 or mouse and human islets with IL-1 and IFN-γ Griess assay GSIS (radioimmuno assay) TUNEL staining Neutral red dye assay Inducer: Cytokines (IL-1, IFN-γ) Nitric oxide 2.) Am J Physiol Endocrinol Metab. 2012 Jun 1;302(11):E1390-8. Epub 2012 Mar 20. A role for aberrant protein palmitoylation in FFA-induced ER stress and β-cell death. Baldwin AC, Green CD, Olson LK, Moxley MA, Corbett JA. Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA. Exposure of insulin-producing cells to elevated levels of the free fatty acid (FFA) palmitate results in the loss of β-cell function and induction of apoptosis. The induction of endoplasmic reticulum (ER) stress is one mechanism proposed to be responsible for the loss of β-cell viability in response to palmitate treatment; however, the pathways responsible for the induction of ER stress by palmitate have yet to be determined. Protein palmitoylation is a major posttranslational modification that regulates protein localization, stability, and activity. Defects in, or dysregulation of, protein palmitoylation could be one mechanism by which palmitate may induce ER stress in β-cells. The purpose of this study was to evaluate the hypothesis that palmitate-induced ER stress and β-cell toxicity are mediated by excess or aberrant protein palmitoylation. In a concentration-dependent fashion, palmitate treatment of RINm5F cells results in a loss of viability. Similar to palmitate, stearate also induces a concentration-related loss of RINm5F cell viability, while the monounsaturated fatty acids, such as palmoleate and oleate, are not toxic to RINm5F cells. 2-Bromopalmitate (2BrP), a classical inhibitor of protein palmitoylation that has been extensively used as an inhibitor of G protein-coupled receptor signaling, attenuates palmitate-induced RINm5F cell death in a concentration-dependent manner. The protective effects of 2BrP are associated with the inhibition of [(3)H]palmitate incorporation into RINm5F cell protein. Furthermore, 2BrP does not inhibit, but appears to enhance, the oxidation of palmitate. The induction of ER stress in response to palmitate treatment and the activation of caspase activity are attenuated by 2BrP. Consistent with protective effects on insulinoma cells, 2BrP also attenuates the inhibitory actions of prolonged palmitate treatment on insulin secretion by isolated rat islets. These studies support a role for aberrant protein palmitoylation as a mechanism by which palmitate enhances ER stress activation and causes the loss of insulinoma cell viability. Techniques: RINm5F cells; islets were isolated from male Sprague-Dawley rats Cell viability: neutral red assay Caspase-3/7 activity: spectroscopy PCR Metabolic labeling of palmitoylated proteins with 2BrP and fluorography Palmitate oxidation and esterification measured through [C14]CO2 release Inducer: FFA palmitate-induced ER stress and B-cell apoptosis (suggested through abberant protein palmitoylation) Stearate also reduces cell viability to similar levels Cells treated with RPMI Palmitate analog 2-bromopalmitate (2BrP) (to examine whether changes in posttranslational modification of β-cell proteins may mediate palmitate-induced ER stress induction and β-cell death) 3.) J Biol Chem. 2011 Mar 11;286(10):8338-48. Epub 2011 Jan 1. FoxO1 and SIRT1 regulate beta-cell responses to nitric oxide. Hughes KJ, Meares GP, Hansen PA, Corbett JA. Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA. For many cell types, including pancreatic β-cells, nitric oxide is a mediator of cell death; paradoxically, nitric oxide can also activate pathways that promote the repair of cellular damage. In this report, a role for FoxO1-dependent transcriptional activation and its regulation by SIRT1 in determining the cellular response to nitric oxide is provided. In response to nitric oxide, FoxO1 translocates from the cytoplasm to the nucleus and stimulates the expression of the DNA repair gene GADD45α, resulting in FoxO1-dependent DNA repair. FoxO1-dependent gene expression appears to be regulated by the NAD(+)-dependent deacetylase SIRT1. In response to SIRT1 inhibitors, the FoxO1-dependent protective actions of nitric oxide (GADD45α expression and DNA repair) are attenuated, and FoxO1 activates a proapoptotic program that includes PUMA (p53-up-regulated mediator of apoptosis) mRNA accumulation and caspase-3 cleavage. These findings support primary roles for FoxO1 and SIRT1 in regulating the cellular responses of β-cells to nitric oxide. Techniques: Western blot DAPI staining for FoxO1 translocation Real Time PCR Comet Assay (DNA damage) Immunoprecipitation of Acetylated Lysine Fluor-de-lys Sirt1 fluorometric drug discovery assay kit (Sirt1 activity) Inducer: NO Sirt1 inhibitors (to activate FoxO1 proapoptotic mechanism) PUMA mRNA accumulation (caspase-3 cleavage) 4.) J Biol Chem. 2010 Jan 29;285(5):3191-200. Epub 2009 Nov 20. AMP-activated protein kinase attenuates nitric oxide-induced beta-cell death. Meares GP, Hughes KJ, Jaimes KF, Salvatori AS, Rhodes CJ, Corbett JA. Department of Medicine, Division of Endocrinology, Comprehensive Diabetes Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. During the initial autoimmune response in type 1 diabetes, islets are exposed to a damaging mix of pro-inflammatory molecules that stimulate the production of nitric oxide by beta-cells. Nitric oxide causes extensive but reversible cellular damage. In response to nitric oxide, the cell activates pathways for functional recovery and adaptation as well as pathways that direct beta-cell death. The molecular events that dictate cellular fate following nitric oxide-induced damage are currently unknown. In this study, we provide evidence that AMPK plays a primary role controlling the response of beta-cells to nitric oxide-induced damage. AMPK is transiently activated by nitric oxide in insulinoma cells and rat islets following IL-1 treatment or by the exogenous addition of nitric oxide. Active AMPK promotes the functional recovery of beta-cell oxidative metabolism and abrogates the induction of pathways that mediate cell death such as caspase-3 activation following exposure to nitric oxide. Overall, these data show that nitric oxide activates AMPK and that active AMPK suppresses apoptotic signaling allowing the beta-cell to recover from nitric oxide-mediated cellular stress. Techniques: Griess Assay Aconitase assay TUNEL assay Comet assay Caspasae-3 assay Inducer: Cytokine-induced NO production NO-induced activation of AMPK (β -cell recovery) 5.) Am J Physiol Endocrinol Metab. 2009 Nov;297(5):E1187-96. Epub 2009 Sep 8. Nitric oxides mediates a shift from early necrosis to late apoptosis in cytokine-treated β-cells that is associated with irreversible DNA damage. Hughes KJ, Chambers KT, Meares GP, Corbett JA. The Comprehensive Diabetes Center, Univ. of Alabama Birmingham, 12th Floor Shelby, 1530 3rd Ave. South, Birmingham, AL 35294, USA. For many cell types, including pancreatic β-cells, nitric oxide is a mediator of cell death; however, it is paradoxical that for a given cell type nitric oxide can induce both necrosis and apoptosis. This report tests the hypothesis that cell death mediated by nitric oxide shifts from an early necrotic to a late apoptotic event. Central to this transition is the ability of β-cells to respond and repair nitric oxide-mediated damage. β-Cells have the ability to repair DNA that is damaged following 24-h incubation with IL-1; however, cytokine-induced DNA damage becomes irreversible following 36-h incubation. This irreversible DNA damage following 36-h incubation with IL-1 correlates with the activation of caspase-3 (cleavage and activity). The increase in caspase activity correlates with reductions in endogenous nitric oxide production, as nitric oxide is an inhibitor of caspase activity. In contrast, caspase cleavage or activation is not observed under conditions in which β-cells are capable of repairing damaged DNA (24-h incubation with cytokines). These findings provide evidence that β-cell death in response to cytokines shifts from an early necrotic process to apoptosis and that this shift is associated with irreversible DNA damage and caspase-3 activation. Techniques: Neutral red assay Griess Assay MTT assay (cell viability) Comet assay Caspase-3 Fluorometric Assay Kit RINm5F cells were transiently transfected using the Amaxa Nucleofect electroporator RT PCR Inducer: Cytokine-induced NO production 6.) J Biol Chem. 2009 Oct 2;284(40):27402-8. Epub 2009 Aug 2. Repair of nitric oxide-damaged DNA in beta-cells requires JNK-dependent GADD45alpha expression. Hughes KJ, Meares GP, Chambers KT, Corbett JA. Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA. Proinflammatory cytokines induce nitric oxide-dependent DNA damage and ultimately beta-cell death. Not only does nitric oxide cause beta-cell damage, it also activates a functional repair process. In this study, the mechanisms activated by nitric oxide that facilitate the repair of damaged beta-cell DNA are examined. JNK plays a central regulatory role because inhibition of this kinase attenuates the repair of nitric oxide-induced DNA damage. p53 is a logical target of JNK-dependent DNA repair; however, nitric oxide does not stimulate p53 activation or accumulation in beta-cells. Further, knockdown of basal p53 levels does not affect DNA repair. In contrast, expression of growth arrest and DNA damage (GADD) 45alpha, a DNA repair gene that can be regulated by p53-dependent and p53-independent pathways, is stimulated by nitric oxide in a JNK-dependent manner, and knockdown of GADD45alpha expression attenuates the repair of nitric oxide-induced beta-cell DNA damage. These findings show that beta-cells have the ability to repair nitric oxide-damaged DNA and that JNK and GADD45alpha mediate the p53-independent repair of this DNA damage. Techniques: Commet assay Western blot Griess assay siRNA transfection using NeoFX transfection reagent (Ambion) RT PCR Inducer: NO activation of the base-excision pathway NO activation of p53 and JNK pathway GADD45α 7.) Diabetes. 2008 Jan;57(1):124-32. Epub 2007 Oct 10. The role of nitric oxide and the unfolded protein response in cytokine-induced beta-cell death. Chambers KT, Unverferth JA, Weber SM, Wek RC, Urano F, Corbett JA. Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri, USA. OBJECTIVE: The unfolded protein response (UPR) is a conserved cellular response designed to alleviate damage and promote survival of cells experiencing stress; however, prolonged UPR activation can result in apoptotic cell death. The UPR, activated by cytokine-induced nitric oxide (NO) production, has been proposed to mediate beta-cell death in response to cytokines. In this study, the role of UPR activation in cytokine-induced beta-cell death was examined. RESEARCH DESIGN AND METHODS: The effects of cytokine treatment of rat and human islets and RINm5F cells on UPR activation, NO production, and cell viability were examined using molecular and biochemical methodologies. RESULTS: UPR activation correlates with beta-cell death in interleukin (IL)-1-treated rat islets. NO mediates both cytokine-induced UPR activation and beta-cell death as NO synthase inhibitors attenuate each of these IL-1-stimulated events. Importantly, cytokines and tunicamycin, a classical UPR activator, induce beta-cell death by different mechanisms. Cell death in response to the classical UPR activator is associated with a 2.5-fold increase in caspase-3 activity, while IL-1 fails to stimulate caspase-3 activity. In addition, cell death is enhanced by approximately 35% in tunicamycin-treated cells expressing an S51A eIF2 alpha mutant that cannot be phosphorylated or in cells lacking PERK (protein kinase regulated by RNA/endoplasmic reticulum-like kinase). In contrast, neither the absence of PERK nor the expression of the S51A eIF2 alpha mutant affects the levels of cytokine-induced death. CONCLUSIONS: While cytokine-induced beta-cell death temporally correlates with UPR activation, the lack of caspase activity and the ability of NO to attenuate caspase activity suggest that prolonged UPR activation does not mediate cytokine-induced beta-cell death. Techniques: Western blot Griess assay RNeasy kit (Xbp1 splicing assay) Neutral Red Assay Caspase-3 Fluorometric Assay Kit Inducer: IL-1 Nitric Oxide Prolonged unfolded protein response (UPR) UPR is activated by protein overload in the ER, nutrient deprivation, metabolic changes, viral infection, and the generation of free radicals. Also chemical activators: N-linked glycosylation inhibitor tunicamycin, the SERCA (sarcoplasmic ER calcium inhibitor) thapsigargin, and the thiol-reducing reagent dithiothreitol 8.) PLoS Med. 2006 Feb;3(2):e17. Epub 2005 Dec 20. Interleukin-1 stimulates beta-cell necrosis and release of the immunological adjuvant HMGB1. Steer SA, Scarim AL, Chambers KT, Corbett JA. The Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri, USA. BACKGROUND: There are at least two phases of beta-cell death during the development of autoimmune diabetes: an initiation event that results in the release of beta-cell-specific antigens, and a second, antigen-driven event in which beta-cell death is mediated by the actions of T lymphocytes. In this report, the mechanisms by which the macrophage-derived cytokine interleukin (IL)-1 induces beta-cell death are examined. IL-1, known to inhibit glucose-induced insulin secretion by stimulating inducible nitric oxide synthase expression and increased production of nitric oxide by beta-cells, also induces beta-cell death. METHODS AND FINDINGS: To ascertain the mechanisms of cell death, the effects of IL-1 and known activators of apoptosis on beta-cell viability were examined. While IL-1 stimulates beta-cell DNA damage, this cytokine fails to activate caspase-3 or to induce phosphatidylserine (PS) externalization; however, apoptosis inducers activate caspase-3 and the externalization of PS on beta-cells. In contrast, IL-1 stimulates the release of the immunological adjuvant high mobility group box 1 protein (HMGB1; a biochemical maker of necrosis) in a nitric oxide-dependent manner, while apoptosis inducers fail to stimulate HMGB1 release. The release of HMGB1 by beta-cells treated with IL-1 is not sensitive to caspase-3 inhibition, while inhibition of this caspase attenuates beta-cell death in response to known inducers of apoptosis. CONCLUSIONS: These findings indicate that IL-1 induces beta-cell necrosis and support the hypothesis that macrophage-derived cytokines may participate in the initial stages of diabetes development by inducing beta-cell death by a mechanism that promotes antigen release (necrosis) and islet inflammation (HMGB1 release). Techniques: MTT assay Neutral red assay Annexin V-FITC Staining In Situ Cell Death Detection Kit, Fluorescein (TUNEL staining) Caspase-3 Fluorometric Assay Kit Western Blot Griess assay Inducer: IL-1 Nitric Oxide Caspase-3 HMGB1
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