Endovascular hypothermia improves post-resuscitation myocardial dysfunction by increasing mitochondrial biogenesis in a pig model of cardiac arrest

The aim of the study was to investigate the effects of endovascular hypothermia on mitochondrial biogenesis in a pig model of prolonged cardiac arrest (CA). Ventricular fibrillation was electrically induced, and animals were left untreated for 10 min; then after 6min of cardiopulmonary resuscitation (CPR), defibrillation was attempted. 25 animals that were successfully resuscitated were randomized into three groups: Sham group (SG, 5, no CA), normal temperature group (NTG, 5 for 12 h observation and 5 for 24 h observation), and endovascular hypothermia group (EHG, 5 for 12 h observation and 5 for 24 h observation). The core temperatures (T_c) in the EHG were maintained at 34 ± 0.5 °C for 6 h by an endovascular hypothermia device (Coolgard 3000), then actively increased at the speed of 0.5 °C per hour during the next 6 h to achieve a normal body temperature, while T_c were maintained at 37.5 ± 0.5 °C in the NTG. Cardiac and mitochondrial functions, the quantification of myocardial mitochondrial DNA (mtDNA), peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), nuclear respiratory factor (NRF)-1, and NRF-2 were examined. Results showed that myocardial and mitochondrial injury and dysfunction increased significantly at 12 h and 24 h after CA. Endovascular hypothermia offered a method to rapidly achieve the target temperature and provide stable target temperature management (TTM). Cardiac outcomes were improved and myocardial injuries were alleviated with endovascular hypothermia. Compared with NTG, endovascular hypothermia significantly increased mitochondrial activity and biogenesis by amplifying mitochondrial biogenesis factors’ expressions, including PGC-1α, NRF-1, and NRF-2. In conclusions, endovascular hypothermia after CA alleviated myocardial and mitochondrial dysfunction, and was associated with increasing mitochondrial biogenesis.     

Beneficial effects of carbon monoxide-releasing molecules on post-ischemic myocardial recovery

There is increasing evidence corroborating a protective role of carbon monoxide releasing molecules (CORMs) in injured tissues. Carbon monoxide (CO) carriers have been recently developed as a pharmacological tool to simulate the effect of heme oxygenase-1-derived CO. The effects of CORM-3, a water-soluble CO releaser, on the incidence of reperfusion-induced ventricular fibrillation (VF) and tachycardia (VT) were studied in isolated rat hearts. Hearts were treated with different doses of CORM-3 before the induction of 30 min global ischemia followed by 120 min reperfusion. We found that at concentrations of 25 μM and 50 μM of CORM-3 promoted a significant reduction in the incidence of VF and VT. Thus, the incidence of VF was reduced by 67% (p < 0.05) and 92% (p < 0.05) with 25 μM and 50 μM of CORM-3, respectively. The protective effect of CORM-3 on the incidence of VT followed the same pattern. The antiarrhythmic protection was associated with a marked attenuation in infarct size, significant decreases in cellular Na⁺ and Ca²⁺ gains and K⁺ loss. Consequently, the recovery of post-ischemic function was significantly improved. In conclusion, CORM-3 exerts beneficial effects against ischemia/reperfusion-induced injury through its abilities to release CO which mediates a cardioprotective action by regulating tissue Na⁺, K⁺, and Ca²⁺ levels.     

Regulation of oogenesis in honey bee workers via programed cell death

Reproductive division of labour characterises eusociality. Currently little is known about the mechanisms that underlie the ‘sterility’ of the worker caste, but queen pheromone plays a major role in regulating the reproductive state. Here we investigate oogenesis in the young adult honey bee worker ovary in the presence of queen pheromone and in its absence. When queen pheromone is absent, workers can activate their ovaries and have well-developed follicles. When queen pheromone is present, even though workers have non-activated ovaries, they continually produce oocytes which are aborted at an early stage. Therefore, irrespective of the presence of the queen, the young adult worker ovary contains oocytes. By this means young workers retain reproductive plasticity. The degeneration of the germ cells in the ovarioles of workers in the presence of queen pheromone has the morphological hallmarks of programmed cell death. Therefore the mechanistic basis of ‘worker sterility’ relies in part on the regulation of oogenesis via programmed cell death. Our results suggest that honey bees have co-opted a highly conserved checkpoint at mid-oogenesis to regulate the fertility of the worker caste.     

Risk of death and recurrent ventricular arrhythmias in survivors of cardiac arrest concurrent with acute myocardial infarction

Aims

Cardiac arrest (CA) is an indication for defibrillator (ICD) implantation unless it occurs in the context of an acute myocardial infarction (AMI). We investigated the ventricular arrhythmia (VA)-free survival of patients resuscitated from CA in the setting of AMI.

Methods

We reviewed a database of 1600 AMI and CA survivors from which 48 patients were identified as having concurrent CA and AMI (CA+AMI group). Those patients were matched by age, gender, race, and left ventricular ejection fraction (LVEF) to 96 patients with AMI but no CA (AMI group) and 48 patients with CA but no AMI (CA group).

Results

Patients and controls were followed for 3.9±3.2 years. Patients in the 3 groups had similar baseline characteristics (age 63±14 yrs, 78% men, 98% white, 53% with CAD, LVEF 33±14%). The 5-year VA-free survival was 67%, 92%, and 80% for the CA+AMI, AMI, and CA groups, respectively, p<0.001.

Conclusion

Patients with concurrent CA and AMI are at high risk of recurrent VA, with VA-free survival rates significantly worse than those of patients with AMI but no CA, and comparable to those of patients with CA outside the context of an AMI. Accordingly, these patients should be considered for ICD implantation.     

Effects of regional body temperature variation during asphyxial cardiac arrest on mortality and brain damage in a rat model

To date, hypothermia has focused on improving rates of resuscitation to increase survival in patients sustaining cardiac arrest (CA). Towards this end, the role of body temperature in neuronal damage or death during CA needs to be determined. However, few studies have investigated the effect of regional temperature variation on survival rate and neurological outcomes. In this study, adult male rats (12 week-old) were used under the following four conditions: (i) whole-body normothermia (37 ± 0.5 °C) plus (+) no asphyxial CA, (ii) whole-body normothermia + CA, (iii) whole-body hypothermia (33 ± 0.5 °C)+CA, (iv) body hypothermia/brain normothermia + CA, and (v) brain hypothermia/body normothermia + CA. The survival rate after resuscitation was significantly elevated in groups exposed to whole-body hypothermia plus CA and body hypothermia/brain normothermia plus CA, but not in groups exposed to whole-body normothermia combined with CA and brain hypothermia/body normothermia plus CA. However, the group exposed to hypothermia/brain normothermia combined with CA exhibited higher neuroprotective effects against asphyxial CA injury, i.e. improved neurological deficit and neuronal death in the hippocampus compared with those involving whole-body normothermia combined with CA. In addition, neurological deficit and neuronal death in the group of rat exposed to brain hypothermia/body normothermia and CA were similar to those in the rats subjected to whole-body normothermia and CA. In brief, only brain hypothermia during CA was not associated with effective survival rate, neurological function or neuronal protection compared with those under body (but not brain) hypothermia during CA. Our present study suggests that regional temperature in patients during CA significantly affects the outcomes associated with survival rate and neurological recovery.     

Serum tau fragments as predictors of death or poor neurological outcome after out-of-hospital cardiac arrest

Background: Anoxic brain injury is the primary cause of death after resuscitation from out-of-hospital cardiac arrest (OHCA) and prognostication is challenging. The aim of this study was to evaluate the potential of two fragments of tau as serum biomarkers for neurological outcome.

Methods: Single-center sub-study of 171 patients included in the Target Temperature Management (TTM) Trial randomly assigned to TTM at 33?°C or TTM at 36?°C for 24?h after OHCA. Fragments (tau-A and tau-C) of the neuronal protein tau were measured in serum 24, 48 and 72?h after OHCA. The primary endpoint was neurological outcome.

Results: Median (quartile 1 – quartile 3) tau-A (ng/ml) values were 58 (43–71) versus 51 (43–67), 72 (57–84) versus 71 (59–82) and 76 (61–92) versus 75 (64–89) for good versus unfavourable outcome at 24, 48 and 72?h, respectively (p_group = 0.95). Median tau C (ng/ml) values were 38 (29–50) versus 36 (29–49), 49 (38–58) versus 48 (33–59) and 48 (39–59) versus 48 (36–62) (p_group = 0.95). Tau-A and tau-C did not predict neurological outcome (area under the receiver-operating curve at 48?h; tau-A: 0.51 and tau-C: 0.51).

Conclusions: Serum levels of tau fragments were unable to predict neurological outcome after OHCA.     

2-methoxyestradiol-induced cell death in osteosarcoma cells is preceded by cell cycle arrest

2-Methoxyestradiol (2-ME), a naturally occurring mammalian metabolite of 17beta-Estradiol (E2), induces cell death in osteosarcoma cells. To further understand the molecular mechanisms of action, we have investigated cell cycle progression in 2-ME-treated human osteosarcoma (MG63, SaOS-2 and LM7 [corrected]) cells. At 5 microM, 2-ME induced growth arrest by inducing a block in cell cycle; 2-ME-treatment resulted in 2-fold increases in G1 phase cells and a decrease in S phase cells in MG63 and SaOS-2 osteosarcoma cell lines, compared to the appropriate vehicle controls. 2-ME-treatment induced a threefold increase in the G2 phase in LM7 [corrected] osteosarcoma cells. The results demonstrated steroid specificity, as the tumorigenic metabolite, 16alpha-hydroxyestradiol (16-OHE), did not have any effect on cell cycle progression in osteosarcoma cells. The cell cycle arrest coincided with an increase in expression of the cell cycle markers p21, p27 and p53 proteins in 2-ME-treated osteosarcoma cells. Also, MG63 cells, transiently transfected with cDNA for a 'loss of function mutant' RNA-dependent protein kinase (PKR) protein, were resistant to 2-ME-induced cell cycle arrest. These results suggest that 2-ME works in concert with factors regulating cell cycle progression, and cell cycle arrest precedes cell death in 2-ME-treated osteosarcoma cells.     

Usefulness of F2-isoprostanes in early prognostication after cardiac arrest: a topical review of the literature and meta-analysis of preclinical data

Abstract

Prognostication after cardiac arrest (CA) represents a challenging issue, and several biomarkers have been proposed in the attempt to predict outcome. Among these, F2-isoprostanes stand out as potential biomarkers for early prognostication, providing information on the magnitude of global oxidative injury after return of spontaneous circulation (ROSC). We performed a topical review searching PubMed and Scopus databases to identify studies evaluating the modifications of F2-isoprostanes in the early period after CA, and a meta-analysis of studies providing curves of F2-isoprostanes plasma levels seeking to describe the biomarker’s kinetics after CA. Evidence suggests that plasma levels of F2-isoprostanes increase in the early post-resuscitation period and seem well correlated with the burden of ischaemia-reperfusion injury. Our meta-analysis shows a possible increase as early as 5?minutes after ROSC, which persists at 2?hours and is attenuated at 4?hours. Clinical studies are warranted to evaluate the utility of this biomarker for prognostication purposes in CA survivors.     

Crosstalk between elicitor-induced cell death and cell cycle regulation in tobacco BY-2 cells

The molecular links between cell cycle control and the regulation of programmed cell death are largely unknown in plants. Here we studied the relationship between the cell cycle and elicitor-induced cell death using synchronized tobacco BY-2 cells. Flow cytometry and fluorescence microscopy of nuclear DNA, and RNA gel-blot analyses of cell cycle-related genes revealed that the proteinaceous elicitor cryptogein induced cell cycle arrest at the G1 or G2 phase before the induction of cell death. Furthermore, the patterns of cell death induction and defence-related genes were different in different phases of the cell cycle. Constitutive treatment with cryptogein induced cell cycle arrest and cell death at the G1 or G2 phase. With transient treatment for 2 h, cell cycle arrest and cell death were only induced by treatment with the elicitor during the S or G1 phase. By contrast, the elicitor-induced production of reactive oxygen species was observed during all phases of the cell cycle. These results indicate that although recognition of the elicitor signal is cell cycle-independent, the induction of cell cycle arrest and cell death depends on the phase of the cell cycle.     

Metaphase arrest and cell death induced by etoposide on HeLa cells

DNA damage, cell cycle and apoptosis form a network with important implications for cancer chemotherapy. Dysfunctions of the cycle checkpoints can allow cancer cells to acquire drug resistance. Etoposide is a well-known inducer of apoptosis, which is widely used in cell biology and in clinical practice. In this work we report that a pulse of 50 μM etoposide (incubation for only 3 h) on HeLa cells causes a sequence of events that leads to abnormal mitotic figures that could be followed either by cell death or, more commonly, by interphase restitution and endocycle. The endocycling polyploid cells enter immediately into mitosis and suffer metaphase blockage with multiple spindle poles, which were generally followed by a direct triggering of apoptosis from metaphase (mitotic catastrophe), or by a new process of endocycling, until surviving cells finally became apoptotic (96 h after the treatment).     

Change in myocardial function after resuscitated sudden cardiac arrest and its impact on long-term mortality and defibrillator implantation

BackgroundThe impact of left ventricular ejection fraction (LVEF) changes after sudden cardiac arrest (SCA) on implantable defibrillator (ICD) utilization and long-term survival is not known. We therefore evaluated the influence of LVEF on these parameters in SCA survivors.MethodsData were collected on consecutive SCA survivors who had ≥1 echocardiogram after SCA and who survived to hospital discharge (n = 655). The median time from baseline to first follow-up echocardiogram was 162 days. LVEF ≥50% was defined as normal. Patients were classified into 4 groups according to baseline (LVEFb) and follow-up (LVEFf) myocardial function: normal LVEFb and LVEFf (group1, n = 261); reduced LVEFb and normal LVEFf (group 2, n = 104); normal LVEFb but reduced LVEFf (group 3, n = 41); and reduced LVEFb and LVEFf (group 4, n = 249). All-cause mortality and time to ICD implantation were examined in all groups.ResultsOver a median follow up of 4.3 years, death occurred in 279 (42%) of patients. Compared with patients in group 1, patients with any reduced LVEF at any time (groups 2–4) had significantly higher mortality, even after adjusting for unbalanced covariates (HR = 1.44, 95.0% CI 1.05–1.95, p = 0.022). ICDs were most commonly implanted in patients with persistently reduced LVEF (group 4: HR = 1.72, 95% CI = 1.26–2.35, p = 0.001).ConclusionWe demonstrate that, in survivors of SCA, a reduced LVEF at or after the index event is associated with higher mortality but that patients with persistently reduced LVEF were most likely to receive ICD therapy. These findings have implications on the management of SCA survivors.     

The effect of lidocaine and bretylium on the defibrillation threshold during cardiac arrest and cardiopulmonary resuscitation

The effect of intravenous lidocaine, 2 mg/kg, and bretylium, 5 mg/kg, on defibrillation threshold (DFT) was investigated in alpha-chloralose anesthetized dogs undergoing conventional closed chest cardiopulmonary resuscitation (CPR) following induced ventricular fibrillation. Ventricular fibrillation was induced electrically and CPR was performed by a pneumatic device set to compress the chest 60 times and inflate the lung 12 times a minute. Defibrillation was achieved using underdamped sinusoidal current shocks from a special defibrillator which allowed determination of delivered energy. The DFT was defined as the peak current which defibrillated, but no more than 20% higher than a current which did not defibrillate. All DFTs were obtained within 5 min of CPR. The mean +/- SD current and energy thresholds required for defibrillation during lidocaine-CPR (seven dogs) were 17.0 +/- 8.9 A and 53.0 +/- 40.7 J as compared to 12.5 +/- 6.2 A and 34.3 +/- 30.7 J, respectively during control-CPR (P less than 0.05). The mean +/- SD current and energy thresholds during bretylium-CPR were 11.0 +/- 3.4 A and 24.1 +/- 1.3 J as compared to 11.8 +/- 1.7 A and 29.4 +/- 9.6 J, respectively, during control-CPR (NS). These results show that lidocaine acutely elevated defibrillation threshold whereas bretylium did not produce such an effect. The effect on DFT along with other pharmacologic properties should be considered when lidocaine or bretylium is used in the setting of cardiac arrest and CPR.     

Improved ROSC rates in out-of-hospital cardiac arrest patients after introduction of a text message alert system for trained volunteers

ObjectiveTo evaluate whether a text message (TM) alert system for trained volunteers contributed to early cardiopulmonary resuscitation, the use of automated external defibrillators (AEDs), return of spontaneous circulation (ROSC) and survival in out-of-hospital cardiac arrest (OHCA) patients in a region with above-average survival rates.DesignData on all OHCA patients in 2012 (non-TM group) were compared with those of all OHCA patients in 2018 (TM group). The association of the presence of a TM alert system with ROSC and survival was assessed with multivariate regression analyses.ResultsTM responders reached 42 OHCA patients (15.9%) earlier than the first responders or ambulance. They connected 31 of these 42 OHCA patients (73.8%) to an AED before the ambulance arrived, leading to a higher percentage of AEDs being attached in 2018 compared to the 2012 non-TM group (55% vs 46%, p = 0.03). ROSC was achieved more often in the TM group (61.0% vs 29.4%, p < 0.01). Three-month and 1‑year survival did not differ significantly between the two groups (29.3% vs 24.3%, p = 0.19, and 25.9% vs 23.5%, p = 0.51). Multivariate regression analyses confirmed the positive association of ROSC with the TM alert system (odds ratio 1.49, 95% confidence interval 1.02‑2.19, p = 0.04).ConclusionA TM alert system seems to improve the chain of survival; because TM responders reached patients early, AEDs were attached more often and more OHCA patients achieved ROSC. However, the introduction of a TM alert system was not associated with improved 3‑month or 1‑year survival in a region with above-average survival rates.     

Inhibition of Ephrin B3-mediated survival signaling contributes to increased cell death response of non-small cell lung carcinoma cells after combined treatment with ionizing radiation and PKC 412

Radiation therapy is frequently used to treat non-small cell lung cancers (NSCLCs). We have previously shown that a combination of ionizing radiation (IR) and the staurosporine analog PKC 412, but not Ro 31–8220, increases cell death in NSCLC cells. To identify genes involved in the enhancement of cell death, a total gene profiling in response to co-administration of (i) PKC 412 with IR, or (ii) Ro 31–8220 with IR was implemented. These combined treatments caused upregulation of 140 and 179 genes and downregulation of 253 and 425 genes, respectively. Certain genes were selected and verified by real-time quantitative PCR and, of these genes, robust suppression of Ephrin B3 expression was suggested as a possible cell death-inducing mechanism of combined treatment with IR and PKC 412. Indeed, silencing of Ephrin B3 using siRNA in NSCLC cells resulted in a major alteration of their morphology with an elongated phenotype, decreased proliferation and increased cell death signaling. Moreover, silencing of Ephrin B3 in combination with IR caused a decrease in IR-mediated G₂-arrest, induced cellular senescence, inhibited MAPK ERK and p38 phosphorylation, and caused an upregulation of p27^kip1 expression. Finally, silencing of Ephrin B3 in combination with IR sensitized U-1810 cells to IR-induced apoptosis. In conclusion, we identify and describe Ephrin B3 as a putative signaling molecule involved in the response of NSCLC cells to combined treatment with PKC 412 and ionizing radiation.     

4-Deoxyraputindole C induces cell death and cell cycle arrest in tumor cell lines

Several molecules extracted from natural products exhibit different biological activities, such as ion channel modulation, activation of signaling pathways, and anti-inflammatory or antitumor activity. In this study, we tested the antitumor ability of natural compounds extracted from the Raputia praetermissa plant. Among the compounds tested, an alkaloid, here called compound S4 (4-Deoxyraputindole C), showed antitumor effects against human tumor lineages. Compound S4 was the most active against Raji, a lymphoma lineage, promoting cell death with characteristics that including membrane permeabilization, dissipation of the mitochondrial potential, increased superoxide production, and lysosomal membrane permeabilization. The use of cell death inhibitors such as Z-VAD-FMK (caspase inhibitor), necrostatin-1 (receptor-interacting serine/threonine-protein kinase 1 inhibitor), E-64 (cysteine peptidases inhibitor), and N-acetyl- L -cysteine (antioxidant) did not decrease compound S4-dependent cell death. Additionally, we tested the effect of cellular activity on adherent human tumor cells. The highest reduction of cellular activity was observed in A549 cells, a lung carcinoma lineage. In this lineage, the effect on the reduction of the cellular activity was due to cell cycle arrest, without plasma membrane permeabilization, loss of the mitochondrial potential or lysosomal membrane permeabilization. Compound S4 was able to inhibit cathepsin B and L by a nonlinear competitive (negative co-operativity) and simple-linear competitive inhibitions, respectively. The potency of inhibition was higher against cathepsin L. Compound S4 promoted cell cycle arrest at G ₀ and G ₂ phase, and increase the expression of p16 and p21 proteins. In conclusion, compound S4 is an interesting molecule against cancer, promoting cell death in the human lymphoma lineage Raji and cell cycle arrest in the human lung carcinoma lineage A549.     

Biochemical and morphological identification of ceramide-induced cell cycle arrest and apoptosis in cultured granulosa cells

We have investigated the effects of ceramide on the progression of cell cycle and on apoptotic cell death in ovarian cultured granulosa cells. Rates of cellular proliferation were measured by immunocytochemical staining for proliferating cell nuclear antigen (PCNA) and flow cytometric cell cycle analysis. We also examined for morphological and biochemical signs of apoptosis. The PCNA expression was downregulated in a dose-dependent manner after treatment with C6-ceramide. Flow cytometric analysis demonstrated that the exposure of granulosa cells to C6-ceramide markedly decreased the population associated with G0/G1 DNA content and the reduction of cell numbers in G0/G1 phase was accompanied by the elevation of the A0 phase. The exposure of granulosa cells to exogenous C6-ceramide induced drastic morphological changes including cytoplasmic- or nuclear condensation and typical apoptotic DNA degradation. We also observed that phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, significantly inhibited the ceramide-induced apoptosis. These results suggested that ceramide might block the progression of cell cycle at G0/G1 phase and as a consequence, granulosa cells would be committed to apoptosis. Our findings also indicated that down-regulation of the PKC activity might be involved in the ceramide-induced apoptosis in cultured granulosa cells.     

Human umbilical cord blood cells improve cardiac function after myocardial infarction

Human umbilical cord blood (UCB) contains an abundance of immature stem/progenitor cells and has been clinically used as an alternative to bone marrow transplantation. In addition, cord blood can be obtained non-invasively, in contrast to invasive bone marrow aspiration. We investigated the potential of human UCB CD34(+) cells to improve cardiac function following myocardial infarction. Myocardial infarction was induced in Wistar rats by ligation of the left coronary artery. Either 2x10(5) human UCB CD34(+) cells or equivalent cell-free medium was injected into the injured myocardium of the rats following induction of myocardial infarction. CD34(+) cell transplantation significantly improved ventricular function as compared to the control group. Immunofluorescence staining for human CD34, CD45, and PECAM-1 revealed surviving cells in the myocardium. Our findings suggest that transplanted human cells survived and improved cardiac function following myocardial infarction. These results may show the usefulness of UCB CD34(+) cells for myocardial infarction.     

Effect of myocardial infarction and ischemia on induction of cardiac reentries and ventricular fibrillation

The present work is aimed at investigating the effects of myocardial infarction and ischemia on induction of ventricular fibrillation. Electrophysiologic effects of global and local ischemia (variation of the dispersion of refractory periods as well as conduction velocity) on initiation of reentry mechanisms was studied by means of computer simulations based on a cellular automata model of propagation of activation wave through a ventricular surface element. A local area of ischemia where effects of the dispersion of refractory periods are investigated is then simulated. This is made using a Gaussian distribution characterized by its mean and standard deviation. These simulations show that ischemia is capable of initiating reentry phenomena which propagate through the whole ventricle; they are responsible for ventricular fibrillation which causes sudden cardiac death, even when ischemia only involves limited parts of the myocardium. Statistical study of the probability of reentries as a function of both of the size of ischemic zones and the rate of dispersion of refractory periods shows that the latter parameter is of primary importance in triggering cardiac reentries.     

PARG dysfunction enhances DNA double strand break formation in S-phase after alkylation DNA damage and augments different cell death pathways

Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for the degradation of poly(ADP-ribose). PARG dysfunction sensitizes cells to alkylating agents and induces cell death; however, the details of this effect have not been fully elucidated. Here, we investigated the mechanism by which PARG deficiency leads to cell death in different cell types using methylmethanesulfonate (MMS), an alkylating agent, and Parg^−/− mouse ES cells and human cancer cell lines. Parg^−/− mouse ES cells showed increased levels of γ-H2AX, a marker of DNA double strand breaks (DSBs), accumulation of poly(ADP-ribose), p53 network activation, and S-phase arrest. Early apoptosis was enhanced in Parg^−/− mouse ES cells. Parg^−/− ES cells predominantly underwent caspase-dependent apoptosis. PARG was then knocked down in a p53-defective cell line, MIAPaCa2 cells, a human pancreatic cancer cell line. MIAPaCa2 cells were sensitized to MMS by PARG knockdown. Enhanced necrotic cell death was induced in MIAPaCa2 cells after augmenting γ-H2AX levels and S-phase arrest. Taken together, these data suggest that DSB repair defect causing S-phase arrest, but p53 status was not important for sensitization to alkylation DNA damage by PARG dysfunction, whereas the cell death pathway is dependent on the cell type. This study demonstrates that functional inhibition of PARG may be useful for sensitizing at least particular cancer cells to alkylating agents.     

Nitric oxide: a signaling molecule against mitochondrial permeability transition- and pH-dependent cell death after reperfusion

Reperfusion of ischemic tissue can precipitate cell death. Much of this cell killing is related to the return of physiological pH after the tissue acidosis of ischemia. The mitochondrial permeability transition (MPT) is a key mechanism contributing to this pH-dependent reperfusion injury in hepatocytes, myocytes, and other cell types. When ATP depletion occurs after the MPT, necrotic cell death ensues. If ATP levels are maintained, at least in part, the MPT initiates apoptosis caused by mitochondrial swelling and release of cytochrome c and other proapoptotic factors. Cyclosporin A and acidotic pH inhibit opening of permeability transition pores and protect cells against oxidative stress and ischemia/reperfusion injury, whereas Ca²⁺, mitochondrial reactive oxygen species, and pH above 7 promote mitochondrial inner membrane permeabilization. Reperfusion with nitric oxide (NO) donors also blocks the MPT via a guanylyl cyclase and protein kinase G-dependent signaling pathway, which in turn prevents reperfusion-induced cell killing. In isolated mitochondria, a combination of cGMP, cytosolic extract, and ATP blocks the Ca²⁺-induced MPT, an effect that is reversed by protein kinase G inhibition. Thus, NO prevents pH-dependent cell killing after ischemia/reperfusion by a guanylyl cyclase/cGMP/protein kinase G signaling cascade that blocks the MPT.