Literature Review- Cell Death by elazek


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									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.

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.

    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

    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

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.

    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

    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.

    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)

    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.

    Griess Assay
    Aconitase assay
    TUNEL assay
    Comet assay
    Caspasae-3 assay

    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

    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

    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.

    Commet assay
    Western blot
    Griess assay
    siRNA transfection using NeoFX transfection reagent (Ambion)
    RT PCR

    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.

    Western blot
    Griess assay
    RNeasy kit (Xbp1 splicing assay)
    Neutral Red Assay
    Caspase-3 Fluorometric Assay Kit

    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).

    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

    IL-1
    Nitric Oxide
    Caspase-3
    HMGB1

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