Reduction Of Apoptotic Cell Death Research Articles

GLP-1 receptor agonist attenuates endoplasmic reticulum stress-mediated β-cell damage in Akita mice.

Aims/Introduction: Endoplasmic reticulum (ER) stress is one of the contributing factors in the development of type 2 diabetes. To investigate the cytoprotective effect of glucagon-like peptide 1 receptor (GLP-1R) signaling in vivo, we examined the action of exendin-4 (Ex-4), a potent GLP-1R agonist, on β-cell apoptosis in Akita mice, an animal model of ER stress-mediated diabetes. Ex-4, phosphate-buffered saline (PBS) or phlorizin were injected intraperitoneally twice a day from 3 to 5 weeks-of-age. We evaluated the changes in blood glucose levels, bodyweights, and pancreatic insulin-positive area and number of islets. The effect of Ex-4 on the numbers of C/EBP-homologous protein (CHOP)-, TdT-mediated dUTP-biotin nick-end labeling (TUNEL)- or proliferating cell nuclear antigen-positive β-cells were also evaluated. Ex-4 significantly reduced blood glucose levels and increased both the insulin-positive area and the number of islets compared with PBS-treated mice. In contrast, there was no significant difference in the insulin-positive area between PBS-treated mice and phlorizin-treated mice, in which blood glucose levels were controlled similarly to those in Ex-4-treated mice. Furthermore, treatment of Akita mice with Ex-4 resulted in a significant decrease in the number of CHOP-positive β-cells and TUNEL-positive β-cells, and in CHOP mRNA levels in β-cells, but there was no significant difference between the PBS-treated group and the phlorizin-treated group. Proliferating cell nuclear antigen staining showed no significant difference among the three groups in proliferation of β-cells. These data suggest that Ex-4 treatment can attenuate ER stress-mediated β-cell damage, mainly through a reduction of apoptotic cell death that is independent of lowered blood glucose levels. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00075.x, 2010).

Small Molecule Inhibitor of Neutrophil Elastase and Severe Congenital Neutropenia.

Abstract 552Severe congenital neutropenia (SCN) is a rare heritable hematopoietic disorder characterized by maturation arrest at the promyelocytes, recurring severe infections, and evolution to leukemia. Heterozygous mutations in the neutrophil elastase (NE or ELANE) gene (sporadic or autosomal-dominant SCN) or homozygous mutations in the HAX1 gene (autosomal-recessive SCN) are associated with similar clinical phenotype and a block of myeloid differentiation or “maturation arrest” in the marrow. We and others reported that human myeloid progenitor cells expressing mutant elastase exhibit impaired cell survival (Aprikyan et al, 2003, Massullo et al, 2005, Kollner et al, 2006, Grenda et al, 2007). The hetero- and homozygous deletion of NE as well as the knock-in of mutant NE identified in SCN/AML patient failed to produce severe neutropenia phenotype in mice. Thus, the pathomechanism of severe neutropenia remains largely unclear due to the lack of cellular or animal model of SCN with characteristic block of myeloid differentiation and accelerated apoptosis.We established a cellular model of SCN with inducible tet-regulated expression of del.145-152 NE mutant (identified in SCN/AML patients) in human promyelocytic tet-off HL60 cells. The ratio of normal/mutant NE products in these cells is approximately 1:1, which is similar to that expected in SCN patients with heterozygous NE mutation. Expression of mutant NE in the promyelocytic cells results in a characteristic block of myeloid differentiation with ∼70% decline in differentiated neutrophils which is similar to that observed in SCN, whereas induced expression of control wild type NE has no effect on myeloid differentiation. Reduced production of myeloid cells and accelerated apoptosis are also observed upon DMSO or retinoic acid induced granulocytic differentiation of the cells in response to mutant, but not wild type NE expression. Thus, this cellular model of SCN appears to closely recapitulate the human phenotype. Expression of mutant NE resulted in approximately 40% increase in total NE-specific proteolytic activity, suggesting that mutant elastase exhibits at least some proteolytic activity. To date there are more than 50 heterozygous mutations in the NE gene have been identified in pre-leukemic SCN patients. Molecular modeling of the NE tertiary structure revealed that these mutations predominantly affect the N-glycosylation sites or the binding pocket of neutrophil elastase. Importantly, the active site of the mutant protease appears to be intact, which suggests that NE-specific small molecule inhibitors may be useful in preventing accelerated apoptosis and the characteristic block of myeloid differentiation of myeloid progenitor cells. Screening this cellular model of SCN, we identified a proprietary cell-penetrant elastase-specific small molecule inhibitor (compound A, Merck, USA), which inhibits the proteolytic activity of NE by more than 80%. When treated with compound A, control cells with induced expression of wtNE exhibit normal myeloid differentiation and production of myeloid cells, similar to that in untreated cells. These data suggest that human NE is dispensable and that accelerated apoptosis and impaired myeloid differentiation in SCN is due to a gain-of-function effect of pro-apoptotic mutant elastase. Importantly, treatment of human promyelocytic cells expressing del.145-152 mutant NE with this small molecule inhibitor restores impaired production of myeloid cells and improves myeloid differentiation to near normal levels, thus neutralizing the pro-apoptotic effect of mutant NE. These data suggest that small molecule inhibitors of NE may represent a promising therapy in severe congenital neutropenia. We have examined the effect of the inhibitor on bone marrow cells from an SCN patient positive for NE mutation. At the time of bone marrow aspiration the patient was on G-CSF and the patient's freshly isolated bone marrow CD33+ progenitor cells exhibited ∼21% apoptosis, which was gradually increased reaching 43% at 3 days of culture. However, daily treatment of SCN cells with compound A preserved the cell survival rate at the initial value resulting in approximately 2-fold reduction in apoptotic cell death at 3 days of culture. These data demonstrate that the small molecule inhibitor of NE and its analogs should be considered for clinical trials in patients with SCN that is attributable to mutant NE. Disclosures:Dale:Merck: Research support; Amgen: Consultancy, Research Funding, Speaker.

Application of Zinc-bis-(DL-Hydrogensaspartate) Does Not Reduce Apoptotic Cell Death in Myocardial Infarction in the Rat Heart

Early studies in different stress models have shown potential beneficial effects of exogenous zinc application with reduction in the rate of apoptotic cell death. This has not been shown in models of myocardial infarction. Rats were exposed to either brief episodes of acute ischemia followed by reperfusion (phase 1) or chronic coronary occlusion (phase 2). Animals were either treated with zinc or vehicle. Groups 1 and 3 received zinc-bis-(DL-hydrogenaspartate) 10 mg/kg body weight as a single 5-mL bolus administered intraperitoneally 24 hours prior to coronary occlusion, groups 2 and 4 received saline. The infarct sizes were determined by triphenyltetrazolium chloride staining and expressed at relative areas to areas of ischemia. Histological slices of the rat's myocardium at the border zones of the infarcts were stained with the TUNEL method to assess for apoptosis. Animals in groups 5, 7, and 9 received zinc, given once before and then repeated every 4 days after coronary occlusion, whereas groups 6, 8, and 10 received saline. Animals were observed for observation periods of 13 (groups 9 and 10), 16 (groups 7 and 8), or 19 weeks (groups 5 and 6), respectively. Two-dimensional echocardiography was performed to measure ejection fraction (EF) at baseline and at the end of the observation periods. TUNEL staining was used to detect and quantify apoptosis rate in the border zones of infarcts after the hearts were excised. Infarct sizes were 49% + 22% in group 1 (zinc + 30 minutes ischemia + 30 minutes reperfusion); 48% + 10% in group 2 (vehicle + 30 minutes ischemia + 30 minutes reperfusion); 42% + 11% in group 3 (zinc + 60 minutes ischemia + 30 minutes reperfusion); and 41% + 23% in group 4 (vehicle + 60 minutes ischemia + 60 minutes reperfusion). In group 1, 11% + 6% of cells were apoptotic compared to 12% + 4% in group 2, 16% + 9% in group 3, and 17% + 7% in group 4 (P > .05). In phase 2, echocardiography revealed a significant reduction in EF in all groups after coronary occlusion. There were no significant differences in EF between the 5 groups at baseline and at follow-up. TUNEL staining did not reveal any significant apoptosis after 13 to 19 weeks. Application of zinc failed to result in reduction of infarct size after temporary coronary occlusion followed by reperfusion and did not demonstrate any reduction in apoptotic cell death. In chronic coronary occlusion, zinc also did not improve EF compared to controls in the presented model in rats. The mechanisms involved in antiapoptotic effects seem to be more complex and might not be inducible by simple zinc injections.

Propofol limits rat myocardial ischemia and reperfusion injury with an associated reduction in apoptotic cell death in vivo

Propofol, a rapidly acting, short duration, intravenous hypnotic anesthetic induction agent, is often used in clinical situations where myocardial ischemia/ reperfusion (I/R) injury is a threat. The aim of the present study was to evaluate the protective effect of propofol on myocardial I/R injury in rat due to apoptosis. Myocardial I/R injury were induced by occluding the left anterior descending (LAD) coronary artery for 25 min followed by either 2 h or 6 h reperfusion. Apoptosis was evaluated by Western blot analysis (Bcl-2, Bax expression), DNA strand breaks, TUNEL analysis and measuring myocardial caspase-3 activity. Propofol significantly reduced infarct size and improved I/R-induced myocardial contractile dysfunction by improving left ventricular diastolic pressure and positive and negative maximal values of the first derivative (+ d p/d t) of left ventricular pressure. Propofol increased Bcl-2/Bax expression ratio and decreased caspase-3 activity in I/R rat hearts, which resulted in reduction of myocardial apoptosis as evidenced by TUNEL analysis and DNA laddering experiments. In an in vitro study, propofol increased H9c2 cell viability against oxidative stress induced by glucose oxidase (GOX) in a dose-dependent manner. These data suggest propofol limits I/R injury with an associated reduction in apoptotic cell death in vivo.

Effect of propofol on calcium homeostasis in hypoxia-reoxygenated neonatal rat cardiomyocytes

Intracellular Ca 2+ overload induced by hypoxia–reoxygenation alters Ca 2+ homeostasis, which plays an important role in myocardial cell injury. Even though propofol is known as a radical scavenger with Ca 2+ channel blocking properties, little is known about cardioprotective effect associated with Ca 2+ homeostasis in cardiomyocytes. In the present study, we showed that propofol protects cardiomyocytes against hypoxia–reoxygenation injury. In propofol-treated cardiomyocytes, we observed a decrease in the expression of pro-apoptotic protein Bax, cytochrome c, caspase-3 activation and intracellular Ca 2+ content. We also found that propofol treatment enhanced expression of anti-apoptotic protein Bcl-2 and activation of ERK concerned with survival. Propofol attenuated alterations of genes involving Ca 2+-regulatory mechanism and significantly modulated abnormal changes of SERCA2a genes in hypoxia-reoxygenated neonatal cardiomyocytes. These results suggest that propofol modulates the expression of genes involved in Ca 2+ homeostasis, thereby producing cardioprotective effects through a reduction in apoptotic cell death.

Mesenchymal Stem Cells Derived from Human Adipose Tissues Favor Tumor Cell Growth in vivo

Mesenchymal stem cells (MSCs) have generated a great deal of interest in clinical situations, due principally to their potential use in regenerative medicine and tissue engineering applications. However, the therapeutic application of MSCs remains limited, unless the favorable effects of MSCs for tumor growth in vivo and the long-term safety of the clinical applications of MSCs can be understood more thoroughly. In this study, MSCs derived from human adipose tissues (hASCs) together with tumor cells were transplanted subcutaneously or intracranially into BALB/c nude mice to observe tumor outgrowth. The results indicated that hASCs with H460 or U87MG cells promoted tumor growth in nude mice. Our histopathological analyses indicated that the co-injection of tumor cells with hASCs exerted no influence on the formation of intratumoral vessels. Co-culture of tumor cells with hASCs or the addition of conditioned medium (CM) from hASCs effected an increase in the proliferation of H460 or U87MG cells. Co-injection of hASCs with tumor cells effected an increase in tumor cell viability in vivo, and also induced a reduction in apoptotic cell death. CM from hASCs inhibited hydrogen peroxide-induced cell death in H460 or U87MG cells. These findings indicated that MSCs could favor tumor growth in vivo. Thus, it is necessary to conduct a study concerning the long-term safety of this technique before MSCs can be used as therapeutic tools in regenerative medicine and tissue engineering.

Guanosine reduces apoptosis and inflammation associated with restoration of function in rats with acute spinal cord injury

Spinal cord injury results in progressive waves of secondary injuries, cascades of noxious pathological mechanisms that substantially exacerbate the primary injury and the resultant permanent functional deficits. Secondary injuries are associated with inflammation, excessive cytokine release, and cell apoptosis. The purine nucleoside guanosine has significant trophic effects and is neuroprotective, antiapoptotic in vitro, and stimulates nerve regeneration. Therefore, we determined whether systemic administration of guanosine could protect rats from some of the secondary effects of spinal cord injury, thereby reducing neurological deficits. Systemic administration of guanosine (8 mg/kg per day, i.p.) for 14 consecutive days, starting 4 h after moderate spinal cord injury in rats, significantly improved not only motor and sensory functions, but also recovery of bladder function. These improvements were associated with reduction in the inflammatory response to injury, reduction of apoptotic cell death, increased sparing of axons, and preservation of myelin. Our data indicate that the therapeutic action of guanosine probably results from reducing inflammation resulting in the protection of axons, oligodendrocytes, and neurons and from inhibiting apoptotic cell death. These data raise the intriguing possibility that guanosine may also be able to reduce secondary pathological events and thus improve functional outcome after traumatic spinal cord injury in humans.

Therapeutic potential of erythropoietin in cardiovascular disease: Erythropoiesis and beyond

Erythropoietin (EPO) is a glycoprotein hormone implicated in the regulation of red blood cell production. Anemia is common in chronic heart failure (CHF) patients and associated with an inappropriately low EPO-production, suggesting a role for its recombinant human form (rhEPO) in treatment. Although safety concerns have been raised regarding treatment with rhEPO in patients with chronic kidney disease, treatment with rhEPO in patients with CHF has so far been safe and well tolerated. The effect of rhEPO on outcome in anemic CHF patients is under investigation in a phase III clinical trial. In addition to its erythropoietic effects, EPO has been detected in the cardiovascular system, fueling intense research into possible non-hematopoietic effects. EPO has been shown to exert protective effects on the heart during acute myocardial ischemia and improve cardiac function in experimental CHF. Acute protection is mediated through reduction of apoptotic cell death. Improvement of cardiac function in CHF is related to myocardial neovascularization. EPO exhibits a vast array of beneficial effects in cardiovascular disease. In addition to the correction of anemia in CHF, rhEPO might benefit patients with cardiovascular disease.

Characterization of Soluble Fas Receptor as a Neuroprotective Agent following Acute Spinal Cord Injury

Spinal cord injury (SCI) is a devastating form of neurotrauma, affecting any population, and is a major cause of morbidity and mortality in children and young adults. The pathophysiology of SCI consists of a primary mechanical insult that triggers a secondary cascade of cellular damage. A key event in this pathology is delayed apoptotic cell death at and adjacent to the injury site, leading to progressive neurodegeneration. Several molecular pathways have been attributed to apoptosis after SCI. The Fas receptor pathway plays an integral role in the initiation of apoptosis through receptor-ligand binding of target cells and has been observed after CNS trauma and in several neurodegenerative diseases. Previous work in our lab has revealed that subarachnoid infusion of a soluble form of the Fas receptor (sFasR) is neuroprotective, as shown by long-term behavioral evaluation and neuronal tracing. We hypothesize that inhibition of the Fas receptor pathway is neuroprotective in the acutely injured spinal cord and results in reduction of neuronal and oligodendroglial cell death and enhanced axonal integrity across the lesion site. In this study, we used a 35g and 50g clip compression injury model at C7-T1 followed by intrathecal administration of sFasR using osmotic minipumps and catheterization at the site of injury. Using Western blotting and immunohistochemistry at 5 and 7 days following injury, our results reveal enhanced axonal preservation, enhanced survival of oligodendrocytes, and reduction in apoptotic cell death in sFasR-treated animals compared with controls. These results indicate that disruption of the Fas pathway in acute injury can lead to enhanced axonal and tissue preservation, which mirrors well with the improved long-term recovery observed previously in our lab. Moreover, this work shows the potential of soluble Fas receptor administration to be a therapeutic option for individuals suffering from acute SCI.

Metallothionein expression and roles in the central nervous system

Metallothioneins (MTs) are low-molecular-weight (6-7kD) nonenzymatic proteins (60-68 amino acid residues, 25-30% being cysteine) ubiquitously expressed in the animal kingdom. In the central nervous system (CNS), three MT isoforms are known, namely MT-I, MT-II, andMT-III. MT-I andMT-II (MT-I+II) are regulated and expressed coordinately and are currently the best characterized MT isoforms. This review will focus on the expression and roles of MT-I+II in the CNS. MT-I+II are implicated in diverse physiological and pathophysiological functions, such as metal ion metabolism, regulation of the CNS inflammatory response, protection against reactive oxygen species and oxidative stress, reduction of apoptotic cell death, and stimulation of neuroregeneration and brain tissue repair in vivo. Accordingly, brain tissue damage and neuro degeneration during pathological conditions and the accompanying mortality and clinical symptoms are altogether significantly increased in MT-I+II deficient mice, while the opposite is observed after medical MT-II treatment and in MT-I overexpressing mice. Consequently, MT-I+II are likely essential factors in CNS disorders, which suggests a potential therapeutic use of these proteins. Biomedical Reviews 2002; 13: 1-15.

Increased expression and activation of c-Jun contributes to human amylin-induced apoptosis in pancreatic islet beta-cells.

The role of c-Jun in apoptosis evoked by human amylin was investigated using human and rat insulinoma beta-cell lines. Two transient increases in the levels of c-jun mRNA were detected at 30 minutes and eight hours after treatment with human amylin. The level of c-Jun protein was also up-regulated in a time-dependent manner, reaching maximal levels after eight hours of exposure. However, no c-Jun induction was detected in cells treated with vehicle only or with rat amylin, indicating that the amyloidogenic feature of the human peptide may be important for c-Jun induction. We found that c-Jun was activated by phosphorylation specifically at Ser63 at four hours, but not at Ser73, after treatment with human amylin, preceding increased c-Jun protein. Furthermore, expression of an antisense c-jun (AS-c-jun), which suppressed protein levels of both c-Jun and phosphorylated-c-Jun, caused a marked reduction in apoptotic cell death, whereas the corresponding sense c-jun (S-c-jun) had no effect on changes of either c-Jun production or apoptosis. This indicated that increased expression and activation of c-Jun is required for human amylin-induced apoptosis. Immunocytochemical studies showed a significant increase in nuclear staining for c-Jun, phosphorylated-c-Jun (Ser63) and phosphorylated-JNK, suggesting that c-Jun may be activated through activation of JNK. In addition, electrophoretic mobility-shift assays showed that the increase in expression and phosphorylation of c-Jun was associated with increased AP-1 DNA binding activity. Supershift assays demonstrated that c-Jun, c-Fos and ATF-2 are part of the AP-1 complex, indicating that activated c-Jun is dimerized with c-Fos or ATF-2 for control of its target gene expression. Finally, we showed that human amylin triggers AP-1-mediated transcriptional activation. Our results suggest strongly that human amylin induces apoptosis through stimulation of expression and activation of c-Jun, and that co-expression and dimerization of c-Jun and c-fos or ATF-2 may be important for activation of the downstream apoptotic process.

DNA damage response pathway in radioadaptive response

Radioadaptive response is a biological defense mechanism in which low-dose ionizing irradiation elicits cellular resistance to the genotoxic effects of subsequent irradiation. However, its molecular mechanism remains largely unknown. We previously demonstrated that the dose recognition and adaptive response could be mediated by a feedback signaling pathway involving protein kinase C (PKC), p38 mitogen activated protein kinase (p38MAPK) and phospholipase C (PLC). Further, to elucidate the downstream effector pathway, we studied the X-ray-induced adaptive response in cultured mouse and human cells with different genetic background relevant to the DNA damage response pathway, such as deficiencies in TP53, DNA-PKcs, ATM and FANCA genes. The results showed that p53 protein played a key role in the adaptive response while DNA-PKcs, ATM and FANCA were not responsible. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), mimicked the priming irradiation in that the inhibitor alone rendered the cells resistant against the induction of chromosome aberrations and apoptosis by the subsequent X-ray irradiation. The adaptive response, whether it was afforded by low-dose X-rays or wortmannin, occurred in parallel with the reduction of apoptotic cell death by challenging doses. The inhibitor of p38MAPK which blocks the adaptive response did not suppress apoptosis. These observations indicate that the adaptive response and apoptotic cell death constitute a complementary defense system via life-or-death decisions. The p53 has a pivotal role in channeling the radiation-induced DNA double-strand breaks (DSBs) into an adaptive legitimate repair pathway, where the signals are integrated into p53 by a circuitous PKC-p38MAPK-PLC damage sensing pathway, and hence turning off the signals to an alternative pathway to illegitimate repair and apoptosis. A possible molecular mechanism of adaptive response to low-dose ionizing irradiation has been discussed in relation to the repair of DSBs and implicated to the current controversial observations on the expression of adaptive response.

Non-specific caspase inhibition reduces infarct size and improves post-ischaemic recovery in isolated ischaemic/reperfused rat hearts.

Myocardial ischaemia and reperfusion lead to myocardial cell death due, at least in part, to apoptotic mechanisms. Although cysteinyl aspartate-specific proteinase (caspase) activation is a major event and the most-cited culprit in the development of apoptosis, its potential contribution to ischaemic myocardial cell death is largely unknown. To study the role of caspase activation, isolated rat hearts (n=6 per group) were subjected to 30 min coronary artery occlusion followed by 120 min reperfusion. A non-selective [0.1 or 0.5 microM acetyl-Tyr-Val-Ala-Asp chloromethylketone (YVAD-cmk)] or selective caspase inhibitors [0.07 or 0.2 microM acetyl-Asp-Glu-Val-Asp-cmk (Ac-DEVD-cmk, caspase-3 inhibitor); 0.07 or 0.2 microM benzoxycarbonyl-Leu-Glu-OMe-His-Asp(OMe)-fluoromethylketone (z-LEHD-fmk, caspase-9 inhibitor)] were added to the perfusate at the start of reperfusion. Non-selective caspase inhibition with 0.1 or 0.5 microM YVAD-cmk limited infarct size: (21 +/- 4%, P<0.05; 17 +/- 3%, P<0.05, respectively) compared with the ischaemic/reperfused control (32 +/- 5%). In hearts treated with 0.1 or 0.5 microM caspase II non-selective inhibitor, the fraction of terminal-deoxynucleotidyl-transferase deoxyuridine nick end labelling (TUNEL)-positive myocyte nuclei in the infarcted zone was reduced from the ischaemic/reperfused non-treated control of 11.2 +/- 2.1% to 6.2 +/- 1.6% (P<0.05) and 1.2 +/- 0.2% (P<0.05), respectively. The recovery of post-ischaemic cardiac function (coronary flow, aortic flow and left-ventricular developed pressure) improved significantly with the application of the non-selective caspase inhibitor as well. In hearts perfused with specific caspase inhibitors (caspase-3 and caspase-9) there was no significant reduction in the infarct size, no improvement in post-ischaemic cardiac function and no reduction of apoptotic cell death. We conclude that non-specific inhibition of caspases may be therapeutically beneficial in myocardial ischaemia/reperfusion-induced damage, while selective caspase inhibitors may fail to prevent such reperfusion-induced injury in our model system.

Death of Dopaminergic Neurons in Vitro and in Nigral Grafts: Reevaluating the Role of Caspase Activation

Caspases are cysteine proteases involved in apoptotic cell death, and pharmacological caspase inhibition has been demonstrated to prevent neuronal cell death in certain experimental paradigms. In this study, the role of caspase-1 and -3 in the death of dopaminergic neurons derived from the E14 rat ventral mesencephalon (VM) has been examined in two model systems using peptide caspase inhibitors. First, cell death was induced in vitro by withdrawing serum after 2 days. Different doses of caspase-1 (IL-1β converting enzyme) and caspase-3 inhibitors (Ac-DEVD-cmk) were added to the medium at the time of serum withdrawal, and the ability of the inhibitors to promote dopaminergic neuronal survival and prevent activation of caspase-3 was assessed at 7 days. Immunostaining using tyrosine hydroxylase (TH) and cleaved caspase-3 antibodies demonstrated that caspase-1 and -3 inhibitors reduce caspase-3 activation as well as overall cell death. This did not, however, improve the survival of TH-positive neurons, although it did appear to promote their maturation. The second paradigm investigated the effects of these inhibitors in the 6-hydroxydopamine rat model of PD, and similarly, addition of caspase-1 or -3 inhibitor during tissue preparation or immediately prior to grafting of VM tissue did not promote dopaminergic neuronal survival. These results demonstrate that the reduction of apoptotic cell death by pharmacological inhibition of caspase-1 and -3 does not increase dopaminergic neuronal survival in these paradigms and suggest either that caspase-3 activation is not the major determinant of dopaminergic neuronal death in vitro and in grafts or that the ability of caspase inhibitors to rescue cells depends upon the degree of apoptotic stress. This implies that strategies to improve dopaminergic cell survival in clinical programmes of transplantation for PD will need to target other pathways of cell death.

Oxidative stress developed during open heart surgery induces apoptosis: reduction of apoptotic cell death by ebselen, a glutathione peroxidase mimic.

Apoptosis, a genetically controlled programmed cell death, has been found to play a role in ischemic reperfusion injury in several animal species including rats and rabbits. To examine whether this also is true for other animals, a surgically relevant model was established using an isolated in situ swine heart. Hearts were subjected to 15 min of normothermic regional ischemia by left anterior descending artery (LAD) occlusion followed by 30 min of normothermic cardioplegic arrest and 3 h of reperfusion. Oxygen free radicals have been shown to be the inducers of apoptosis and because reperfusion of ischemic myocardium is associated with the generation of free radicals, an additional group of hearts was preperfused with three different doses (5, 10, and 25 nM) ebselen, a glutathione peroxidase mimic, for 15 min before 15 min of LAD occlusion. Hearts were then subjected to 30 min of normothermic cardioplegic arrest followed by 3 h of reperfusion at normothermia. Control experiments were performed by perfusing the hearts for 4 h at normothermia. Two other groups of hearts were subjected to either 30 or 60 min of LAD occlusion followed by 30 min of cardioplegic arrest without subjecting them to reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis and DNA laddering. The in situ end-labeling (ISEL) technique was used to detect apoptotic cardiomyocyte nuclei while DNA laddering was evaluated by subjecting the DNA obtained from the cardiomyocytes to 1.8% agarose gel electrophoresis followed by photographing under UV illumination. The apoptotic cells appeared only after 90 min of reperfusion, as demonstrated by the intense fluorescence of the immunostained genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation showing increased ladders of DNA bands in the same reperfused hearts. The presence of apoptotic cells and DNA fragmentation in the myocardium was abolished by preperfusing the hearts in the presence of 10 nM ebselen, which also moderated the oxidative stress developed in the heart. Apoptotic cells and DNA ladders were completely absent in the hearts subjected to either 30 or 60 min of LAD occlusion. The results demonstrate that reperfusion of the ischemic heart induces apoptosis, which can be reduced with ebselen by reducing the oxidative stress associated with ischemia/reperfusion.

Correlation of p53 protein expression with apoptotic incidence in colorectal neoplasia.

The wild-type p53 gene suppresses cell proliferation and induces apoptosis when it is transfected into human colon cancer cell lines. Therefore, mutation of the p53 gene, which correlates closely with p53 protein overexpression, would be predicted to activate cell proliferation and limit apoptosis. We tested this hypothesis by correlating p53 protein expression with cell proliferation and apoptosis in 70 neoplasms (29 adenomas and 41 carcinomas) using p53 and Ki-67 immunohistochemical staining and DNA nick end labelling. The p53 immunoreactivity was independent of the Ki-67 positivity. The apoptotic incidence was less frequent (P < 0.005) in tumours with diffuse p53 protein overexpression than in those with the sporadic overexpression, defined as p53 staining of isolated or scattered expression. In addition, apoptotic incidence only correlated directly (P < 0.05) with Ki-67 positivity in tumours with sporadic p53-protein expression. These results indicate that p53 protein that is expressed sporadically in colorectal neoplasms is probably wild-type protein and induces apoptosis in response to active cell proliferation. In contrast, diffusely overexpressed p53 protein in colorectal neoplasms is probably mutant and correlates with a reduction in apoptotic cell death independently of cell proliferation.