Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest.

Besides the well-understood DNA damage response via establishment of G(2) checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re-entry. The aim of this study was to investigate the role of Chk1 in the recovery from G(2) checkpoint arrest in HCT116 (human colorectal cancer) wt, p53(-/-) and p21(-/-) cell lines following H(2) O(2) treatment. Firstly, DNA damage caused G(2) checkpoint activation via Chk1. Secondly, overriding G(2) checkpoint led to (i) mitotic slippage, cell cycle re-entry in G(1) and subsequent G(1) arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21(WAF1) causing mitotic catastrophe. We revealed subtle differences in the initial Chk1-involved G(2) arrest with respect to p53/p21(WAF1) : absence of either protein led to late G(2) arrest instead of the classic G(2) arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G(2) arrest correlated with downstream senescence, but late G(2) arrest led to mitotic catastrophe, although both cell cycle re-entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long-term DNA damage responses causing cell cycle re-entry. We propose that recovery from oxidative DNA damage-induced G(2) arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21(WAF1) . The general relevance of Chk1 as an important determinant of recovery from G(2) checkpoint arrest was verified in HT29 colorectal cancer cells.

Hypothermia protects H9c2 cardiomyocytes from H2O2 induced apoptosis.

The purpose of our study was to investigate underlying basic mechanisms of hypothermia-induced cardioprotection during oxidative stress in a cardiomyocyte cell culture model. For hypothermic treatment we cooled H9c2 cardiomyocytes to 20°C, maintained 20min at 20°C during which short-term oxidative damage was inflicted with 2mM H(2)O(2,) followed by rewarming to 37°C. Later on, we analyzed lactate dehydrogenase (LDH), caspase-3 cleavage, reactive oxygen species (ROS), mitochondrial activity, intracellular ATP production, cytoprotective signal molecules as well as DNA damage. Hypothermia decreased H(2)O(2) damage in cardiomyocytes as demonstrated in a lower LDH release, less caspase-3 cleavage and less M30 CytoDeath staining. After rewarming H(2)O(2) damaged cells demonstrated a significantly higher reduction rate of intracellular ROS compared to normothermic H(2)O(2) damaged cardiomyocytes(.) This was in line with a significantly greater mitochondrial dehydrogenase activity and higher intracellular ATP content in cooled and rewarmed cells. Moreover, hypothermia preserved cell viability by up-regulation of the anti-apoptotic protein Bcl-2 and a reduction of p53 phosphorylation. DNA damage, proven by PARP-1 cleavage and H2AX phosphorylation, was significantly reduced by hypothermia. In conclusion, we could demonstrate that hypothermia protects cardiomyocytes during oxidative stress by preventing apoptosis via inhibiting mitochondrial dysfunction and DNA damage.

Activation of microglial poly(ADP-ribose)-polymerase-1 by cholesterol breakdown products during neuroinflammation: a link between demyelination and neuronal damage.

Multiple sclerosis (MS) is a chronic demyelinating disease in which it has only recently been suggested that damage to neuronal structures plays a key role. Here, we uncovered a link between the release of lipid breakdown products, found in the brain and cerebrospinal fluid (CSF) of MS patients as well as in experimental autoimmune encephalomyelitis, and neuronal damage mediated by microglial activation. The concentrations of the breakdown product 7-ketocholesterol detected in the CSF of MS patients were capable of inducing neuronal damage via the activation and migration of microglial cells in living brain tissue. 7-ketocholesterol rapidly entered the nucleus and activated poly(ADP-ribose)-polymerase (PARP)-1, followed by the expression of migration-regulating integrins CD11a and intercellular adhesion molecule 1. These findings reveal a novel mechanism linking demyelination and progressive neuronal damage, which might represent an underlying insidious process driving disease beyond a primary white matter phenomenon and rendering the microglial PARP-1 a possible antiinflammatory therapeutic target.

Mechanisms of hypothermia-induced cell protection mediated by microglial cells in vitro.

Despite the widespread interest in the clinical applications of hypothermia, the cellular mechanisms of hypothermia-induced neuroprotection have not yet been clearly understood. Therefore, the aim of this study was to elucidate the cellular effects of clinically relevant hypothermia and rewarming on the morphological and functional characteristics of microglia. Microglial cells were exposed to a dynamic cooling and rewarming protocol. For stimulation, microglial cells were treated with 1 microg/mL lipopolysaccharide (LPS). We found that hypothermia led to morphological changes from ramified to ameboid cell shapes. At 2 h after hypothermia and rewarming, microglial cells were again ramified with extended branches. Moreover, we found enhanced cell activation after rewarming, accompanied by increased phagocytosis and adenosine triphosphate consumption. Interestingly, hypothermia and rewarming led to a time-dependent significant up-regulation of the anti-inflammatory cytokines interleukin-10 and interleukin-1 receptor antagonist in stimulated microglial cells. This is in line with the reduced proliferation and time-dependent down-regulation of the pro-inflammatory cytokines tumor necrosis factor-alpha and monocyte chemotactic protein-1 in comparison to normothermic control cells after LPS stimulation. Furthermore, degradation of the inhibitor of the nuclear transcription factor-kappaB (IkappaB-alpha) was diminished and delayed under conditions of cooling and rewarming in LPS-stimulated microglial cells. Thus, our results show that hypothermia and rewarming activate microglial cells, increase phagocytosis and shift the balance of cytokine release in stimulated microglial cells towards the anti-inflammatory cytokines. This could be a new cellular mechanism of hypothermia-induced neuroprotection mediated by activated microglial cells.

Helicobacter pylori regulates p21(WAF1) by histone H4 acetylation.

Helicobacter pylori are bacteria that colonize the stomach persistently, which confers risk of serious diseases, including peptic ulceration and gastric neoplasia. Aberrant expression of cell cycle control proteins has been demonstrated in H. pylori infected gastric epithelial cells, suggesting that perturbation of the cell cycle plays a role in the pathogenesis of various H. pylori associated diseases. In this study, we investigate the modulation of the cell cycle control protein p21(WAF1) by H. pylori in the gastric carcinoma cell line NCI-N87 and in primary gastric cells derived from healthy tissue. We observed an up-regulation of p21(WAF1) in both NCI-N87 and primary cells. Chromatin immunoprecipitation analysis revealed that the increased expression of p21(WAF1) induced by H. pylori is associated with the release of HDAC-1 from the p21(WAF1) promoter and hyper-acetylation of histone H4. The elucidation of the epigenetic regulation of p21(WAF1) by H. pylori may help to dissect the pathogenetic mechanisms underlying the development and progression of H. pylori associated diseases.

Hypothermia downregulates inflammation but enhances IL-6 secretion by stimulated endothelial cells.

Hypothermia is a standard method for organ protection during cardiac surgery in children. However, the mechanisms of hypothermia-induced cell protection have not yet been clearly established. Therefore, the aim of our studies was to elucidate molecular effects of clinically relevant mild and deep hypothermia on endothelial cells. The endothelium plays a pivotal role in the interaction between blood cells and actively participates in complex inflammatory events. We isolated primary human umbilical vein endothelial cells (HUVEC) and investigated cell viability, proliferation and inflammatory characteristics after TNF-alpha stimulation under mild (32 degrees C) and deep (17 degrees C) hypothermia in comparison to normothermia (37 degrees C). As a protective mechanism of endothelial cells kept under hypothermic conditions we found a significant upregulation of the antiapoptotic protein Bcl-2, resulting in the same cell viability under hypothermic conditions. Unexpectedly we demonstrated significantly higher IL-6 release after 6h of mild hypothermia. In contrast, hypothermia diminished inflammatory chemokines such as IL-8, MCP-1 and COX-2 protein expression which could lead to reduced leukocyte recruitment under hypothermia. Underlying mechanisms of this downregulation were found to be reduced ERK 1/2 phosphorylation and incomplete IkappaB-alpha degradation resulting in reduced NFkappaB-dependent proinflammatory gene expression. The upregulation of Bcl-2 protein and the higher IL-6 release after 6h of mild hypothermia are new and interesting cellular mechanisms of hypothermia in endothelial cell biology. Both factors may play a major role as cell protective mechanisms in hypothermia.

Lack of p53 augments thymoquinone-induced apoptosis and caspase activation in human osteosarcoma cells.

We have recently shown that thymoquinone (TQ) is an antineoplastic drug that induces p53-dependent apoptosis in human colon cancer cells. This study evaluated the antiproliferative and pro-apoptotic effects of TQ in two human osteosarcoma cell lines with different p53 mutation status. TQ decreased cell survival dose-dependently and, more significantly, in p53-null MG63 cells (IC(50) = 17 muM) than in p53-mutant MNNG/HOS cells (IC(50) = 38 muM). Cell viability was reduced more selectively in MG63 tumor cells than in normal human osteoblasts. Flow cytometric analysis showed that TQ induced a much greater increase in the PreG(1) (apoptotic) cell population, but no cell cycle arrest in MG63. G(2)/M arrest in MNNG/HOS cells was associated with p21(WAF1) upregulation. Using three DNA damage assays, TQ was confirmed to result in a significantly greater extent of apoptosis in p53 null MG63 cells. Although the Bax/Bcl-2 ratios were not differentially modulated in both cell lines, the mitochondrial pathway appeared to be involved in TQ-induced apoptosis in MG63 by showing the cleavage of caspases-9 and -3. Oxidative stress and mitochondrial O(2)(*-) generation in isolated rat mitochondria were enhanced by TQ as measured by the dose-dependent reduction in aconitase enzyme activity and Amplex Red oxidation respectively. TQ-induced oxidative damage, reflected by an increase in gamma-H2AX foci and increased protein expression levels of gamma-H2AX and the DNA repair enzyme, NBS1, was more pronounced in MNNG/HOS than in MG63. We suggest that the resistance of MNNG/HOS cells to drug-induced apoptosis is caused by the up-regulation of p21(WAF1) by the mutant p53 (transcriptional activity was shown by p53 siRNA treatment) which induces cell cycle arrest and allows to repair DNA damage. Collectively, these findings show that TQ induces p53-independent apoptosis in human osteosarcoma cells. As the loss of p53 function is frequently observed in osteosarcoma patients, our data suggest the potential clinical usefulness of TQ for the treatment of these malignancies.

Hypothermia suppresses inflammation via ERK signaling pathway in stimulated microglial cells.

Hypothermic perfusion is a standard method for neuroprotection during cardiac surgery in children. However, the cellular responses underlying these mechanisms have not been clearly elucidated. In the present study we demonstrated that the inflammatory response of stimulated microglial cells is significantly reduced after moderate hypothermia. Continuous hypothermia caused a diminished NO release. Moderate hypothermia and rewarming caused a downregulation of phosphorylated MEK, ERK and iNOS-expression, diminished cytokine release and reduced CD-11a and ICAM-1 expression. Thus, neuroprotection offered by hypothermia could be attributed to reduced cytotoxic products released from stimulated microglial cells mediated by the MEK/ERK signal transduction pathway.

How does hypothermia protect cardiomyocytes during cardioplegic ischemia?

OBJECTIVE: Insufficient myocardial protection is still a considerable cause for in-hospital mortality in children. The purpose of our study was to investigate underlying the basic mechanisms of cardioplegic cardioprotection during hypothermic and normothermic ischemia in a cardiomyocyte cell culture model. METHODS: We cooled cardiomyocytes to 20°C for 20min; during this time, cardiac arrest was simulated by oxidative damage with 2mM H2O2 and cardioplegic solution, followed by rewarming to 37°C. Later on, we analyzed cardiomyocyte cell morphology (phase-contrast-microscopy), viability (trypan blue staining), inflammation (cyclooxygenase-2 (Cox-2) and phosphorylated-extracellular signal-regulated kinase (pERK) 1/2 expression in Western blot analysis), and expression of Akt survival protein (Western blot technique). RESULTS: Hypothermia increases cell survival of cardiomyocytes after cardioplegic ischemia, as demonstrated in significantly higher cell viability and less cell death in these cells compared with normothermic H2O2-damaged cardiomyocytes. As a possible underlying cellular mechanism, we found that, during cold cardioplegic ischemia, ERK 1/2 enzyme is less phosphorylated than under conditions of normothermic cardioplegic ischemia. This is in line with significantly diminished Cox-2 expression during cold cardioplegic ischemia. Moreover, hypothermic cardioplegia preserved cell survival by upregulation of Akt transcription factor in cardiomyocytes. CONCLUSION: In the present cell culture study, we clearly demonstrated that hypothermia exerts additional protection for cardiomyocytes during cardioplegic ischemia. The understanding of underlying basic mechanisms is evident to improve current techniques of myocardial protection.

Hypothermia-induced neurite outgrowth is mediated by tumor necrosis factor-alpha.

Systemic or brain-selective hypothermia is a well-established method for neuroprotection after brain trauma. There is increasing evidence that hypothermia exerts beneficial effects on the brain and may also support regenerative responses after brain damage. Here, we have investigated whether hypothermia influences neurite outgrowth in vitro via modulation of the post-injury cytokine milieu. Organotypic brain slices were incubated: deep hypothermia (2 h at 17 degrees C), rewarming (2 h up to 37 degrees C), normothermia (20 h at 37 degrees C). Neurite density and cytokine release (IL 1beta, IL-6, IL-10, and TNF-alpha) were investigated after 24 h. For functional analysis mice deficient in NT-3/NT-4 and TNF-alpha as well as the TNF-alpha inhibitor etanercept were used. Hypothermia led to a significant increase of neurite outgrowth, which was independent of neurotrophin signaling. In contrast to other cytokines investigated, TNF-alpha secretion by organotypic brain slices was significantly increased after deep hypothermia. Moreover, hypothermia-induced neurite extension was abolished after administration of the TNF-alpha inhibitor and in TNF-alpha knockout mice. We demonstrate that TNF-alpha is responsible for inducing neurite outgrowth in the context of deep hypothermia and rewarming. These data suggest that hypothermia not only exerts protective effects in the CNS but may also support neurite outgrowth as a potential mechanism of regeneration.