Posttreatment with the Ca(2+)-Mg(2+)-dependent endonuclease inhibitor aurintricarboxylic acid abolishes genotoxic agent-induced nuclear condensation and DNA fragmentation and decreases death of astrocytes

DNA fragmentation and nuclear condensation are important nuclear changes in apoptosis. In this study we determined whether DNA fragmentation and nuclear condensation occur in astrocytes treated with 100-200 microM of the genotoxic agent M-nitroso-N-nitroguanidine (MNNG). Our study also investigated the roles of Ca(2+)-Mg(2+)-dependent endonuclease (CME) in the MNNG-induced nuclear changes. We found that MNNG induced profound ATP depletion as well as marked nuclear condensation and DNA fragmentation in the cells. Both the nuclear condensation and the DNA fragmentation were abolished by posttreatment of the cells with the CME inhibitor aurintricarboxylic acid (ATA). The ATA posttreatment also significantly, but only partially, decreased MNNG-induced cell death. In contrast, pretreatment plus cotreatment with ATA did not affect either MNNG-induced nuclear condensation or cell death. Our study further suggests that ATA does not decrease the cytotoxicity of MNNG by directly inhibiting poly(ADP-ribose) polymerases. Collectively, our observations suggest that MNNG can induce both DNA fragmentation and nuclear condensation in astrocytes by a CME-dependent mechanism, which partially contributes to the genotoxic agent-induced cell death. Published 2008 Wiley-Liss, Inc.     

Immunohistochemical study of p53-associated proteins in rat brain following lithium-pilocarpine status epilepticus

Activation of the p53-stress response pathway has been implicated in excitotoxic neuronal cell death. Recent studies have demonstrated an age-dependent induction of both p53 mRNA and protein in the rat brain following lithium-pilocarpine-mediated status epilepticus (LPSE). We investigated whether other proteins that have been shown to participate in the p53 cascade are induced by LPSE. We used immunohistochemistry to examine the expression of Mdm2, Bax, CD95/Fas/APO-1, ATM, Ref-1 and ubiquitin. A significant increase in nuclear Mdm2 immunoreactivity, which colocalized with p53, was observed in cells within hippocampal pyramidal cell layers, dentate gyrus, piriform cortex, amygdala and thalamus. Dual immunofluorescence microscopy revealed a reduction in free ubiquitin expression in cells with p53 and Mdm2 accumulation. Increased immunoreactivity for CD95/Fas/APO-1 and Bax was also detected in the same p53-positive cells. Moreover, expression of Ref-1 and ATM, which are involved in the response to oxidative stress-induced DNA damage and regulation of p53 function, were increased. Colocalization of Ref-1 and p53 suggests that Ref-1 might activate p53 function in LPSE-induced neurodegeneration. In contrast, ATM immunoreactivity was predominantly cytoplasmic suggesting that ATM may not directly modulate p53 activity in injured neurons. These results extend our previous observations with regard to activation and stabilization of p53 in injured central nervous system neurons. The data indicate that p53 induction following LPSE may activate downstream pro-apoptotic genes leading to neurodegeneration.     

DNA damage induces cdk2 protein levels and histone H2B phosphorylation in SH-SY5Y neuroblastoma cells

DNA damage and activation of the cell cycle have been implicated in numerous neurodegenerative diseases, including Alzheimer disease, Parkinson's disease, and amyotrophic lateral sclerosis. To better understand the role of cell cycle proteins in DNA-damage induced neuronal cell death, we examined various cell cycle proteins during camptothecin-induced death of human neuroblastoma cells. We report a rapid induction of p53 and increased expression of p21, concurrent with reduced levels of many cell cycle proteins that regulate G1 to S phase cell cycle progression. However, we found increased levels of cdk2 and cyclin E, and formation of a cyclin E-cdk2-p21 protein complex. DNA damage failed to induce activation and progression of the cell cycle. Finally, camptothecin-induced neuronal cell death occurred concurrent with phosphorylation of histone H2B. Pretreatment of cells with cdk inhibitor olomoucine impeded cdk2-cyclin E accumulation, but not the induction of p53. Olomucine concurrently delayed histone H2B phosphorylation, caspase-3 activation and cell death. These findings suggest that DNA-damage of differentiated neuroblastoma cells induces a rapid p53-mediated inhibition of cell cycle progression and induction of cdk2-cyclin E, followed by caspase-3 activation, phosphorylation of histone and cell death.     

Regulation of the hypoxia-dependent plasminogen activator inhibitor 1 expression by MAP kinases

Mitogen-activated protein kinases (MAPKs) and protein kinase B (PKB) mediate growth and stress signals and have been implicated in the hypoxic response. Under hypoxic conditions, the expression of plasminogen activator inhibitor-1 (PAI-1) is mainly controlled by the hypoxia-inducible factor HIF-1. However, the role of MAPKs and PKB in HIF-1-mediated PAI-1 regulation is not clear. Treatment with the p38 inhibitor SB203580 and the PI3K inhibitor LY294002, but not with the MEK1 inhibitor PD98059, abrogated hypoxia-dependent PAI-1 induction in HepG2 cells. Consistently, overexpression of PKB or of the p38 upstream kinases MKK6 and MKK3 and of JNK, but not of ERK, enhanced PAI-1 mRNA levels. In MKK3-, MKK6- and PKB-expressing cells luciferase (Luc) activities from a hypoxia-inducible PAI-1-Luc construct or from a HIF-dependent Luc construct and, concomitantly, HIF-1alpha protein levels were enhanced. These findings indicate that p38- and PKB-dependent signalling pathways contribute to enhanced PAI-1 levels in the hypoxic response.     

Distal spinal muscular atrophy as a major feature in adult-onset ataxia telangiectasia

The authors report four adult-onset ataxia telangiectasia (AT) patients belonging to two families lacking pronounced cerebellar ataxia but displaying distal spinal muscular atrophy. AT was proven by genetic studies showing ATM mutations and a reduced level of ATM. ATM activity, as measured by phosphorylation of p53, was close to normal, indicating that the p53 response is not the only factor in preventing neural damage in anterior horn cells in AT.     

Dichlorvos‐induced cell cycle arrest and DNA damage repair activation in primary rat microglial cells

Dichlorvos, an organophosphate (OP), is known to cause oxidative stress in the central nervous system (CNS). Previously we have shown that dichlorvos treatment promoted the levels of proinflammatory molecules and ultimately induced apoptotic cell death in primary microglial cells. Here we studied the effect of dichlorvos on crucial cell cycle regulatory proteins and the DNA damage sensor ataxia‐telangiectasia mutated (ATM). We found a significant increase in p53 and its downstream target, p21, levels in dichlorvos‐treated microglial cells compared with control cells. Moreover, dichlorvos exposure promoted the levels of different cell cycle regulatory proteins. These results along with flow cytometry results suggested that primary microglial cells were arrested at G1 and G2/M phase after dichlorvos exposure. We have shown in a previous study that dichlorvos can induce DNA damage in microglia; here we found that microglial cells also tried to repair this damage by inducing a DNA repair enzyme, i.e., ATM. We observed a significant increase in the levels of ATM after dichlorvos treatment compared with control. © 2012 Wiley Periodicals, Inc.     

Sodium tungstate decreases the phosphorylation of tau through GSK3 inactivation

Tungstate treatment increases the phosphorylation of glycogen synthase kinase-3beta (GSK3beta) at serine 9, which triggers its inactivation both in cultured neural cells and in vivo. GSK3 phosphorylation is dependent on the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) induced by tungstate. As a consequence of GSK3 inactivation, the phosphorylation of several GSK3-dependent sites of the microtubule-associated protein tau decreases. Tungstate reduces tau phosphorylation only in primed sequences, namely, those prephosphorylated by other kinases before GSK3beta modification, which are serines 198, 199, or 202 and threonine 231. The phosphorylation at these sites is involved in reduction of the interaction of tau with microtubules that occurs in Alzheimer's disease.     

Changes in c-Jun but not Bcl-2 family proteins in p53-dependent apoptosis of mouse cerebellar granule neurons induced by DNA damaging agent bleomycin

Tumor suppressor gene p53 is a critical regulator of the cellular response to DNA damage. To examine the function of p53 in postmitotic CNS neurons, we cultured cerebellar granule cells from 15-day-old wild type and p53-deficient mice, and analyzed changes of protein expression in apoptosis elicited by DNA damage. When cerebellar granule cells from wild type mice were treated with bleomycin, a DNA strand-break inducing agent, neuronal death occurred. In contrast, cells from p53-deficient mice were resistant to bleomycin-induced neuronal death. Furthermore, cells from p53 heterozygous mice showed an intermediate resistance between wild type and p53-deficient mice. These results show that p53 is required for the bleomycin-induced cerebellar granule cell death. To examine which proteins are involved in this apoptosis, we examined changes in protein levels of the Bcl-2 family, including Bcl-2, Bcl-X and Bax. The relative amounts of these proteins did not change after bleomycin treatment, suggesting that the changes in the levels of these Bcl-2 family proteins are not necessary for apoptosis in this system. In contrast, the levels of c-Jun protein significantly increased 6 h after treatment with bleomycin in wild type but not in p53-deficient cerebellar granule cells. These results raise the possibility that c-Jun is required for p53-dependent neuronal apoptosis induced by bleomycin.     

Photoreceptor cell apoptosis induced by the 2-nitroimidazole radiosensitizer, CI-1010, is mediated by p53-linked activation of caspase-3

The nitroimidazole radiosensitizer CI-1010 ((R)-alpha-[[(2-bromoethyl)-amino]methyl]-2-nitro-1H-imidazole-1-ethanol monohydrobromide) causes selective, irreversible, retinal photoreceptor apoptosis in vivo. The mouse 661 W photoreceptor cell line was used as a neuronotypic model of CI-1010-mediated retinal degeneration. Exposure to CI-1010 for 24 h induced apoptosis in 661 W cells, as determined by ultrastructural analysis, agarose electrophoresis and analysis of TUNEL-positive nuclei. CI-1010 caused a loss of viability in 661 W cells, as measured by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). A clear link was established between the onset of apoptosis and activity of caspase-3 and caspase-8, prior to poly[ADP-ribose]polymerase (PARP) cleavage. Pretreatment with caspase inhibitors, ZVAD.fmk or DEVD-CHO, prevented morphological changes in most CI-1010-treated cells. Evaluation of mitochondrial inner membrane potential (Deltapsi(m)) in live 661 W cells using the fluorescent dye, tetramethylrhodamine methyl ester revealed retention of (Deltapsi(m)) until after caspase activation. Absence of cytochrome c in the cytoplasm in treated cells further supports the hypothesis of a mitochondrial-independent mechanism of cell death. Significant increase in DNA crosslinks observed in 661 W cells correlates with induction and phosphorylation of p53 at multiple serine sites. Cell cycle analysis of 661 W cells reveals a G(2) arrest in response to CI-1010-induced DNA damage and neuronal cell death. Increased protein expression of Bax, Fas, and FasL, concomitant to the loss of Bcl-xL in treated 661 W cells may be modulated by p53. This study evaluates in vitro mechanisms of CI-1010-induced cell death in photoreceptors and provides evidence in support of a p53-linked activation of caspase-3 in response to DNA damage caused by CI-1010.     

Radiation-induced apoptosis in retinal progenitor cells is p53-dependent with caspase-independent DNA fragmentation

Caspases are important executioners of the endogenous cell death program. However, their function is not restricted to the induction of cell death. Caspases may process cytokines and contribute to cell differentiation or lymphocyte proliferation. In addition to their pleiotropic functions we show evidence that, under certain conditions, caspases are activated during apoptosis without executing the cell death program. Following whole body irradiation, p53 and caspases were activated in both the cerebellum and eye of postnatal day 5 mice. Although p53 activation and cell death kinetics were similar in both the cerebellum and eye, the processing of caspases was protracted and reduced in the eye. In particular, retinal caspase activation appeared not to be the executioner of cell death; incubation of retinal and cerebellar explants in the presence of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone prevented DNA fragmentation, a hallmark of apoptosis, only in cerebellar granule cells. In contrast, in retinal cells no impairment of DNA fragmentation was observed in the presence of N-benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone, indicating p53-dependent but caspase-independent cell death pathways despite caspase activation.     

Neurotensin and the neurotensin receptor-3 in microglial cells

Microglia motility plays a crucial role in response to lesion or exocytotoxic damage of the cerebral tissue. The neuropeptide neurotensin elicited the migration of the human microglial cell line C13NJ by a mechanism dependent on both phosphatidylinositol-3 kinase (PI3 kinase) and mitogen-activated protein (MAP) kinases pathways. The effect of neurotensin on cell migration was blocked by the neurotensin receptor-3 propeptide, a selective ligand of this receptor. The type I neurotensin receptor-3 was the only known neurotensin receptor expressed in these microglial cells, and its activation led to the phosphorylation of both extracellular signaling-regulated kinases Erk1/2 and Akt. Furthermore, the effect of neurotensin on cell migration was preceded by a profound modification of the F-actin cytoskeleton, particularly by the rapid formation of numerous cell filopodia. Both the motility and the filopodia appearance induced by neurotensin were totally blocked by selective inhibitors of MAP kinases or PI3 kinase pathways. In the murine microglial cell line N11, the neurotensin receptor-3 is also the only neurotensin receptor expressed, and its activation by neurotensin leads to the phosphorylation of both Erk1/2 and Akt. In these cells, neurotensin induces the gene expression of several cytokines/chemokines, including MIP-2, MCP-1, interleukin-1beta and tumor necrosis factor-alpha. This induction is dependent on both protein kinases pathways. We observed that the effect of neurotensin on the cytokine/chemokine expression is also inhibited by the neurotensin receptor-3 propeptide. This is the demonstration that the neurotensin receptor-3 is functional and mediates both the migratory action of neurotensin and its induction of chemokines/cytokines expression.     

Modifications of p53 and the DNA damage response in cells expressing mutant form of the protein huntingtin

Huntington’s disease (HD) occurs through an expansion of the trinucleotide repeat in the HD gene resulting in the lengthening of the polyglutamine stretch within the N terminus of the protein, huntingtin (Htt). While the function of the protein is still being fully elucidated, we have shown that genomic DNA damage is associated with the expression of mutant Htt (mHtt) in a time-dependent fashion. With the accumulation of mHtt and its development into a micro-aggregated complex, the initiation of genomic damage engages a cellular stress signal that activates the DNA damage and stress response pathway. Here we explore the modifications and activation of p53 and keystone regulators of the cell stress response pathway using expression of a fragment of mHtt in HEK293T cells. We find an increase in phosphorylated p53 at serine 15 (S15), diminished acetylation at lysine 382 (K382), altered ubiquitination pattern, and oligomerization activity as a function of mHtt expression. As one might predict, upstream regulators of p53, such as CREB-binding protein/p300 and MDM2, are also seen to be affected by the expression of mHtt, albeit in different ways. These data suggest a possible relationship between p53 and the slow accumulation of DNA damage resulting from the expression of mHtt. The lack of a proper p53-mediated signaling cascade or its alteration in the presence of DNA damage may contribute to the slow progression of cellular dysfunction which is a hallmark of HD pathology.     

Molecular mechanisms of neuronal cell death: implications for nuclear factors responding to cAMP and phorbol esters

Chronic treatment with calcium ionophore A23187 in NGF-differentiated cells results in cell death that is time- and concentration-dependent. Additionally, PC12 cells codifferentiated with NGF and dBcAMP become dependent on these factors for survival and undergo apoptosis when both factors are withdrawn. We show that in both cases there is a prolonged induction of c-Fos which correlates with cell death. Its continual activation in PC12 cells overexpressing c-FosER results in caspase-3 cleavage and rapid cell death. Specific phosphorylation of CREB/CREM(tau) transactivators or their binding to CRE of c-fos was observed. Our results indicate that prolonged c-Fos induction activates p53. There is increased nuclear localization of p53, p21 and Bax levels are induced in NGF/dBcAMP-deprived c-FosER cells, and dominant negative p53 inhibits cell death induced either by serum deprivation or by c-Fos. Overall these data implicate AP-1 as a nuclear target of signal transduction pathways which plays a role in the activation of apoptosis.     

C-reactive protein increases plasminogen activator inhibitor-1 expression in human endothelial cells

C-reactive protein (CRP) is an inflammatory marker which predicts cardiovascular disease. However, it is not fully understood whether CRP has direct effects on endothelial functions and gene expression. The purpose of current study was to determine the effects and molecular mechanisms of CRP on the expression of plasminogen activator inhibitor-1 (PAI-1) in human endothelial cells. Human coronary artery endothelial cells (HCAEC) were treated with CRP at clinically relevant concentrations for different durations. PAI-1 mRNA, protein and enzyme activities were studied. The effects of CRP on MAPK p38 phosphorylation was also studied by Bio-Plex luminex immunoassay. In addition, other types of human endothelial cells isolated from umbilical vein, skin, and lung microvessels were tested. CRP significantly increased PAI-1 mRNA levels in a time- and concentration-dependent manner. The protein level and enzyme activity of PAI-1 in the supernatant of CRP-treated HCAEC cultures were significantly increased. Anti-CD32 antibody effectively blocked CRP-induced PAI-1 mRNA expression. In addition, CRP significantly increased CD32 mRNA levels and enhanced phosphorylation of MAPK p38. Furthermore, antioxidant curcumin dramatically inhibited CRP-induced PAI-1 mRNA expression. The effect of CRP on PAI-1 expression was also confirmed in other types of human endothelial cells. In conclusion, CRP significantly increased the expression of PAI-1 in HCAEC and other human endothelial cells. CRP also increased its receptor CD32 expression which may further enhance its action. CRP-induced PAI-1 expression may be mediated by oxidative stress and p38 signal pathway as antioxidant effectively blocks the effect of CRP on HCAEC.