Prevented DNA Fragmentation Research Articles

Granzyme B–Mediated Death of Pancreatic β-Cells Requires the Proapoptotic BH3-Only Molecule Bid

Perforin-deficient NOD mice are protected from diabetes, suggesting that cytotoxic granule contents of CD8(+) T-cells have a significant role in killing beta-cells. Despite this, cytotoxic granule effects on human or mouse pancreatic islets have not been reported. We tested the susceptibility of human and mouse islet cells to purified recombinant perforin and granzyme B and measured apoptotic death using a number of assays. Perforin and granzyme B impaired insulin secretion from islet cells, and this was accompanied by cytochrome c release, caspase activation, and DNA fragmentation. Granzyme B-mediated apoptotic changes only occurred in the presence of perforin. When compared with hemopoietic cells, traditionally used as targets to measure cytotoxic T-cell function in vitro, islet cells were relatively resistant to perforin and granzyme B. Inhibition of caspases prevented DNA fragmentation but not cytochrome c release, indicating that mitochondrial disruption due to granzyme B is independent of caspase activation. Consistent with this, islet cells from mice deficient in the BH3-only protein Bid were resistant to cytochrome c release and were protected from apoptosis after exposure to perforin/granzyme B. Our data suggest that Bid cleavage by granzyme B precedes mitochondrial disruption and apoptosis in pancreatic islets.

Ebp1 isoforms distinctively regulate cell survival and differentiation

Ebp1, an ErbB3 receptor-binding protein, inhibits the proliferation and induces the differentiation of human cancer cells. Ebp1 binds nuclear Akt and prevents DNA fragmentation by inhibiting caspase-activated DNase. Here, we show that Ebp1 possesses two different isoforms, p48 and p42, which differentially mediate PC12 cell survival and differentiation. The longer-form p48 localizes in both the cytoplasm and the nucleus and suppresses apoptosis, whereas the shorter-form p42 predominantly resides in the cytoplasm and promotes cell differentiation. EGF strongly stimulates p42 to bind ErbB3, and the association depends on PKC-mediated phosphorylation of Ebp1. By contrast, p48 does not bind to ErbB3 regardless of EGF treatment. Overexpression of p48 provokes cell proliferation, which is inhibited by p42. Moreover, nerve growth factor elicits extensive sprouting in p42 stably transfected PC12 cells, whereas p48 cells reveal modest neurite outgrowth. Although mitogen-activated protein kinase cascade remains similar in both cells, Akt is more active in p48 cells than in p42 cells. Thus, Ebp1 might regulate cell survival and differentiation through two distinctive p48 and p42 isoforms.

Chromatin condensation of ovarian nurse and follicle cells is regulated independently from DNA fragmentation during Drosophila late oogenesis

Programmed cell death constitutes a common fundamental incident occurring during oogenesis in a variety of different organisms. In Drosophila melanogaster, it plays a significant role in the maturation process of the egg chamber. In the present study, we have used an in vitro development system for studying the effects of inducers and inhibitors of programmed cell death during the late stages of oogenesis. Treatment of the developing egg chambers with two widely used inducers of cell death, etoposide and staurosporine, blocks further development and induces chromatin condensation but not DNA fragmentation in nurse and follicle cells, as revealed by propidium iodide staining and terminal transferase-mediated dUTP nick-end labeling assay. Moreover, incubation of the developing egg chambers with the caspase-3 inhibitor Z-DEVD-FMK significantly delays development, prevents DNA fragmentation, but does not affect chromatin condensation. The above results demonstrate, for the first time, that chromatin condensation in Drosophila ovarian nurse and follicle cells is a caspase-3-like independent process and is regulated independently from DNA fragmentation.

Nuclear Akt associates with PKC-phosphorylated Ebp1, preventing DNA fragmentation by inhibition of caspase-activated DNase

Akt promotes cell survival through phosphorylation. The physiological functions of cytoplasmic Akt have been well defined, but little is known about the nuclear counterpart. Employing a cell-free apoptotic assay and NGF-treated PC12 nuclear extracts, we purified Ebp1 as a factor, which contributes to inhibition of DNA fragmentation by CAD. Depletion of Ebp1 from nuclear extracts or knockdown of Ebp1 in PC12 cells abolishes the protective effects of nerve growth factor, whereas overexpression of Ebp1 prevents apoptosis. Ebp1 (S360A), which cannot be phosphorylated by PKC, barely binds Akt or inhibits DNA fragmentation, whereas Ebp1 S360D, which mimics phosphorylation, strongly binds Akt and suppresses apoptosis. Further, phosphorylated nuclear but not cytoplasmic Akt interacts with Ebp1 and enhances its antiapoptotic action independent of Akt kinase activity. Moreover, knocking down of Akt diminishes the antiapoptotic effect of Ebp1 in the nucleus. Thus, nuclear Akt might contribute to suppressing apoptosis through interaction with Ebp1.

Treatment with the Non-ionic Surfactant Poloxamer P188 Reduces DNA Fragmentation in Cells from Bovine Chondral Explants Exposed to Injurious Unconfined Compression

Excessive mechanical loading to a joint has been linked with the development of post-traumatic osteoarthritis (OA). Among the suspected links between impact trauma to a joint and associated degeneration of articular cartilage is an acute reduction in chondrocyte viability. Recently, the non-ionic surfactant poloxamer 188 (P188) has been shown to reduce by approximately 50% the percentage of non-viable chondrocytes 24 h post-injury in chondral explants exposed to 25 MPa of unconfined compression. There is a question whether these acutely 'saved' chondrocytes will continue to degrade over time, as P188 is only thought to act by acute repair of damaged cell membranes. In order to investigate the degradation of traumatized chondrocytes in the longer term, the current study utilized TUNEL staining to document the percentage of cells suffering DNA fragmentation with and without an immediate 24 h period of exposure of the explants to P188 surfactant. In the current study, as in the previous study by this laboratory, chondral explants were excised from bovine metacarpophalangeal joints and subjected to 25 MPa of unconfined compression. TUNEL staining was performed at 1 h, 4 days, and 7 days post-impact. The current study found that P188 was effective in reducing the percentage of cells with DNA fragmentation in impacted explants by approximately 45% at 4 and 7 days post-impact. These data suggest that early P188 intervention was effective in preventing DNA fragmentation of injured chondrocytes. The current hypothesis is that this process was mitigated by the acute repair of damaged plasma membranes by the non-ionic surfactant P188, and that most repaired cells did not continue to degrade as measured by the fragmentation of their DNA.

Activated RhoA Induces Cardiomyocyte Apoptosis via a Mitochondrial Death Pathway

The small G-protein RhoA is a transducer of signals from both GPCRs and the extracellular matrix, influencing cell morphology as well as gene expression. A role for RhoA has been firmly established in cultured cardiomyocyte hypertrophy, however the possibility that RhoA can influence myocyte survival or apoptosis has not been examined. Previous work from our lab employing cardiac specific RhoA transgenic mice demonstrated that RhoA overexpression leads to premature death, apparently secondary to the development of heart failure. We have now determined that adenoviral overexpression of activated RhoA in neonatal rat cardiomyocytes induces early changes indicative of hypertrophy which transitions to apoptosis, with the accompanying hallmarks of caspase activation and nucleosomal DNA fragmentation. To delineate the mechanism of RhoA-mediated apoptosis we tested the effects of the Rho kinase inhibitors Y-27632 and HA-1077. Treatment with Y-27632 or HA-1077 prevented nucleosome fragmentation and caspase-3 cleavage. Activated RhoA increases caspase-9 cleavage but not that of caspase-8, and a caspase-9 selective inhibitor prevented DNA fragmentation whereas inhibition of caspase-8 did not, consistent with involvement of a mitochondrial death pathway. Further, RhoA induces a 3-4 fold upregulation in protein levels of the proapoptotic Bcl family protein Bax, which is sensitive to Rho kinase inhibition. Bax involvement in this apoptotic effect was tested by treatment with a Bax inhibitory peptide, which markedly attenuated RhoA-induced DNA fragmentation and caspase-9 and -3 activation. The mechanism of Bax upregulation by RhoA is currently under investigation.

Ca2+ Dysregulation Induces Mitochondrial Depolarization and Apoptosis: ROLE OF Na+/Ca2+ EXCHANGER AND AKT

We previously reported that constitutively activated Galpha(q) (Q209L) expression in cardiomyocytes induces apoptosis through opening of the mitochondrial permeability transition pore. We assessed the hypothesis that disturbances in Ca(2+) handling linked Galpha(q) activity to apoptosis because resting Ca(2+) levels were significantly increased prior to development of apoptosis. Treating cells with EGTA lowered Ca(2+) and blocked both loss of mitochondrial membrane potential (an indicator of permeability transition pore opening) and apoptosis (assessed by DNA fragmentation). When cytosolic Ca(2+) and mitochondrial membrane potential were simultaneously measured by confocal microscopy, sarcoplasmic reticulum (SR)-driven slow Ca(2+) oscillations (time-to-peak approximately 4 s) were observed in Q209L-expressing cells. These oscillations were seen to transition into sustained increases in cytosolic Ca(2+), directly paralleled by loss of mitochondrial membrane potential. Ca(2+) transients generated by caffeine-induced release of SR Ca(2+) were greatly prolonged in Q209L-expressing cells, suggesting a decreased ability to extrude Ca(2+). Indeed, the Na(+)/Ca(2+) exchanger (NCX), which removes Ca(2+) from the cell, was markedly down-regulated at the mRNA and protein levels. Adenoviral NCX expression normalized cytosolic Ca(2+) levels and prevented DNA fragmentation in cells expressing Q209L. Interestingly, constitutively activated Akt, which rescues cells from Q209L-induced apoptosis, prevented the decrease in NCX expression, normalized cytosolic Ca(2+) levels and spontaneous Ca(2+) oscillations, shortened caffeine-induced Ca(2+) transients, and prevented loss of the mitochondrial membrane potential. Our findings demonstrate that NCX down-regulation and consequent increases in cytosolic and SR Ca(2+) can lead to Ca(2+) overloading-induced loss of mitochondrial membrane potential and suggest that recovery of Ca(2+) dysregulation is a target of Akt-mediated protection.

Cadmium inhibits both intrinsic and extrinsic apoptotic pathways in renal mesangial cells

Cadmium is a potent nephrotoxin that has been shown to induce apoptosis in some cells but also to prevent it under certain circumstances. In several clinical situations and experimental models of injury to the renal glomerulus, pathological proliferation of mesangial cells is followed by resolution involving mesangial cell apoptosis. We investigated the effects of Cd(2+) on rat mesangial cells induced to undergo apoptosis through either the extrinsic receptor-mediated pathway or the intrinsic mitochondrial-dependent pathway. Camptothecin initiated the intrinsic pathway with activation of caspase-9 and caspase-dependent cleavage of procaspase-3. Tumor necrosis factor-alpha (TNF-alpha) initiated caspase-8 activity and cleavage of pro-caspase-3 at the convergence point of the two pathways. However, pro-caspase-8 levels were low, and caspase-9 was also activated in response to TNF-alpha, characteristic of what have been termed type II cells. With both TNF-alpha and camptothecin, concurrent exposure to 10 microM CdCl(2) suppressed DNA laddering, nuclear condensation, and pro-caspase-3 cleavage. It also decreased activity of both caspase-8 and caspase-9, prevented caspase-8-dependent cleavage of the proapoptotic factor Bid, and suppressed release of cytochrome c from mitochondria. At this 10-microM concentration, Cd(2+) was unique among a number of metal ions in preventing DNA fragmentation. We conclude that Cd(2+) is anti-apoptotic in rat mesangial cells, acting by a mechanism that may involve general caspase inhibition. This may have consequences for the resolution of nephritis in situations of mesangial cell hyperproliferation.

Nitric oxide involvement in pancreatic β cell apoptosis by glibenclamide

Glibenclamide as a second-generation compound of sulfonylurea has widely been used in the treatment of type 2 diabetes patients. It has been shown that it induces apoptosis in beta cells, which is partially mediated by Ca(2+) influx. Here, we investigated the role of nitric oxide (NO) and nitric oxide synthase (NOS) isoforms on glibenclamide-induced apoptosis in rat insulinoma cells. Our results showed that glibenclamide induces NO generation (measured as nitrite) that is accompanied with decrease of cell viability in a defined concentration of glibenclamide. The effects of glibenclamide on cell viability were partially inhibited after treatment with N(G)-nitro-L-arginine methyl ester (L-NAME), inhibitor more selective for constitutive nitric oxide synthase, and in the presence of D600--a blocker of voltage-gated L-type Ca(2+) channels inhibited Ca(2+) influx into beta cells, whereas aminoguanidine (AG), a preferential inhibitor of inducible NOS, was significantly less effective. Analysis of DNA fragmentation by electrophoresis and staining with Hoechest 33342 and propidium iodide showed that L-NAME, but not AG, prevented DNA fragmentation and decreased the number of cells with condensed and fragmented nuclei. It revealed that the effects of glibenclamide on apoptosis were partially inhibited by treatment with L-NAME. In conclusion, we have shown that NO production in glibenclamide treated cells may be involved in the induction of apoptotic cell death in pure beta cell line and it may be due to Ca(2+) dependent activation of constitutive NOS isoforms.

Cyclin-dependent Kinase Inhibitors Uncouple Cell Cycle Progression from Mitochondrial Apoptotic Functions in DNA-damaged Cancer Cells

DNA damage results in transcriptional induction of p53 target genes, including the cyclin-dependent kinase (CDK) inhibitor p21(Cip1) (CDKN1A) and the proapoptotic Bcl-2 family member p53 up-regulated modulator of apoptosis (PUMA). Depending on the cellular context, p21(Cip1) and PUMA mediate cell cycle arrest and apoptosis, respectively. By imposing cell cycle arrest at the expense of apoptosis, p21(Cip1) can sharply reduce the effectiveness of DNA-damaging anticancer agents in colorectal cancer cells. We investigated the link between cell cycle progression and the onset of apoptosis in DNA-damaged cells by analyzing the activation of the apoptotic cascade in p21(Cip1)-deficient HCT116 colorectal cancer cells. DNA damage induced a similar level of p53 activation and PUMA induction in p21(Cip1)-deficient cells compared with wild-type isogenic counterparts. p21(Cip1) did not act as a direct blocker of PUMA. However, only p21(Cip1)-deficient cells showed extensive cytochrome c release, mitochondrial membrane depolarization, and caspase activation. An increase in caspase activation occurred as these cells reached M-phase and incurred polyploidy. When ectopically expressed in p21(Cip1)-deficient HCT116 cells, p21(Cip1), its family member p27(Kip1), and the structurally unrelated CDK inhibitor p16(Ink4a) were similarly effective at causing cell cycle arrest and inhibiting DNA damage-induced apoptotic events such as cytochrome c release, mitochondrial membrane depolarization, and activation of the caspase cascade. These observations suggest that by blocking dysregulated cell cycle progression, CDK inhibitors can influence the sensitivity of the mitochondria to proapoptotic signals in DNA damage-induced cancer cells.

In vitro properties of 5-(benzylsulfonyl)-4-bromo-2-methyl-3(2H)-pyridazinone: A novel permeability transition pore inhibitor

Despite the increasing implication of the permeability transition pore (PTP) in the pathophysiology of neurodegenerative diseases, few selective PTP inhibitors have been reported so far. Here, we evaluate the pharmacological properties of a novel PTP inhibitor, BBMP (5-(benzylsulfonyl)-4-bromo-2-methyl-3(2H)-pyridazinone). This drug was discovered from the screening of a compound library against the PTP using a functional assay with isolated mitochondria. Similarly to cyclosporin A, the drug prevented Ca2+-induced permeability transition and mitochondrial depolarization. BBMP appeared more potent that minocycline in both swelling and membrane potential assays displaying pIC50 values of 5.5±0.1 and 5.6±0.0, respectively. Unlike minocycline, BBMP dose-dependently prevented DNA fragmentation induced by KCl 25/5 mM shift and serum deprivation in cerebellar granule neurons with a pIC50 of 5.7±0.6. The inhibition of PTP-mediated cytochrome c release observed in isolated mitochondria at 10 and 100 μM may explain its neuroprotective properties in vitro. These data suggest that the mitochondrial PTP is potentially involved in neuronal cell death and that PTP inhibitors, like BBMP, may possess a therapeutic potential in neurodegenerative disorders.

Attenuation of apoptosis in enterocytes by blockade of potassium channels

Apoptosis plays an important role in maintaining the balance between proliferation and cell loss in the intestinal epithelium. Apoptosis rates may increase in intestinal pathologies such as inflammatory bowel disease and necrotizing enterocolitis, suggesting pharmacological prevention of apoptosis as a therapy for these conditions. Here, we explore the feasibility of this approach using the rat epithelial cell line IEC-6 as a model. On the basis of the known role of K+ efflux in apoptosis in various cell types, we hypothesized that K+ efflux is essential for apoptosis in enterocytes and that pharmacological blockade of this efflux would inhibit apoptosis. By probing intracellular [K+] with the K+-sensitive fluorescent dye and measuring the efflux of 86Rb+, we found that apoptosis-inducing treatment with the proteasome inhibitor MG-132 leads to a twofold increase in K+ efflux from IEC-6 cells. Blockade of K+ efflux with tetraethylammonium, 4-aminopyridine, stromatoxin, chromanol 293B, and the recently described K+ channel inhibitor 48F10 prevents DNA fragmentation, caspase activation, release of cytochrome c from mitochondria, and loss of mitochondrial membrane potential. Thus K+ efflux occurs early in the apoptotic program and is required for the execution of later events. Apoptotic K+ efflux critically depends on activation of p38 MAPK. These results demonstrate for the first time the requirement of K+ channel-mediated K+ efflux for progression of apoptosis in enterocytes and suggest the use of K+ channel blockers to prevent apoptotic cell loss occurring in intestinal pathologies.

Nucleophosmin/B23, a Nuclear PI(3,4,5)P 3 Receptor, Mediates the Antiapoptotic Actions of NGF by Inhibiting CAD

Phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P(3)] is an essential second messenger implicated in various cellular processes. Cytoplasmic PI(3,4,5)P(3) has been well characterized, but little is known about the physiological role of nuclear PI(3,4,5)P(3). Here, we describe a nuclear PI(3,4,5)P(3) receptor, nucleophosmin (NPM)/B23, that mediates the antiapoptotic effects of NGF by inhibiting DNA fragmentation activity of caspase-activated DNase (CAD). Employing PI(3,4,5)P(3) column and NGF-treated PC12 nuclear extracts, we identified B23 as a nuclear PI(3,4,5)P(3) binding protein. Purification from nuclear extract demonstrates that B23 contributes to DNA fragmentation inhibitory activity. Depletion of B23 from nuclear extracts or knockdown B23 in PC12 cells abolishes NGF-provoked protective effect, whereas overexpression of B23 in PC12 cells prevents apoptosis. Further, hydrolyzing PI(3,4,5)P(3) with PTEN or SHIP abrogates its antiapoptotic activity. Moreover, B23 mutants that can not associate with PI(3,4,5)P(3) fail to prevent DNA fragmentation. Thus, the nuclear B23-PI(3,4,5)P(3) complex regulates the antiapoptotic activity of NGF in the nucleus.

Identification of Three Mutations in the Cu,Zn‐Superoxide Dismutase (Cu,Zn‐SOD) Gene with Familial Amyotrophic Lateral Sclerosis: Transduction of Human Cu,Zn‐SOD into PC12 Cells by HIV‐1 TAT Protein Basic Domain

The most frequent genetic causes of amyotrophic lateral sclerosis (ALS) determined so far are mutations occurring in the gene coding for copper/zinc superoxide dismutase (Cu,Zn-SOD). The mechanism may involve the formation of hydroxyl radicals or malfunctioning of the SOD protein. Wild-type SOD1 was constructed into a transcription-translation expression vector to examine the SOD1 production in vitro. Wild-type SOD1 was highly expressed in Escherichia coli. Active SOD1 was expressed in a metal-dependent manner. To investigate the possible roles of genetic causes of ALS, a human Cu,Zn-SOD gene was fused with a gene fragment encoding the nine amino acid transactivator of transcription (Tat) protein transduction domain (RKKRRQRRR) of human immunodeficiency virus type 1 in a bacterial expression vector to produce a genetic in-frame Tat-SOD1 fusion protein. The expressed and purified Tat-SOD1 fusion proteins in E. coli can enter PC12 neural cells to observe the cellular consequences. Denatured Tat-SOD1 was successfully transduced into PC12 cells and retained its activity via protein refolding. Three point mutations, E21K, D90V, and D101G, were cloned by site-directed mutagenesis and showed lower SOD1 activity. In undifferentiated PC12 cells, wild-type Tat-SOD1 could prevent DNA fragmentation due to superoxide anion attacks generated by 35 mM paraquat, whereas mutant Tat-D101G enhanced cell death. Our results demonstrate that exogenous human Cu,Zn-SOD fused with Tat protein can be directly transduced into cells, and the delivered enzymatically active Tat-SOD exhibits a cellular protective function against oxidative stress.

Antimycin A–induced killing of HL‐60 cells: Apoptosis initiated from within mitochondria does not necessarily proceed via caspase 9

Antimycin A (AMA) inhibits mitochondrial electron transport, collapses the mitochondrial membrane potential, and causes the production of reactive oxygen species. Previous work by me and my colleagues has demonstrated that AMA causes an array of typical apoptotic phenomena in HL-60 cells. The hypothesis that AMA causes HL-60 apoptosis by the intrinsic apoptotic pathway has now been tested. Z-LEHD-FMK and Z-IETD-FMK were used as specific inhibitors of the initiator caspases 9 and 8, respectively. Caspase 3 activation, DNA fragmentation, and cellular disintegration were measured by flow cytometry. Cytochrome c release, chromatin condensation, and nuclear fragmentation were measured by microscopy. AMA caused mitochondrial cytochrome c release and neither Z-LEHD-FMK nor Z-IETD-FMK inhibited that. In the absence of caspase inhibition there was a very close correlation between cytochrome c release and caspase 3 activation. Z-LEHD-FMK blocked caspase 3 activation but enhanced DNA fragmentation and failed to stop nuclear or cellular disintegration. Z-IETD-FMK also blocked caspase 3 activation but, in contrast to Z-LEHD-FMK, delayed DNA fragmentation and disintegration of the nucleus and the cell. The hypothesis to explain AMA-induced HL-60 apoptosis was clearly inadequate because: (a) caspase 9 inhibition did not prevent DNA fragmentation or cell death, (b) apoptosis proceeded in the absence of caspase-3 activation, (c) the main pathway leading to activation of the executioner caspases was by caspase-8 activation, but caspase 8 inhibition only delayed apoptosis, and (d) activation of caspases 8 and 9 may be necessary for caspase-3 activation. Thus, in this cell model, apoptosis triggered from within the mitochondria does not necessarily proceed by caspase 9, and caspase 3 is not critical to apoptosis. The results provide further evidence that, when parts of the apoptotic network are blocked, a cell is able to complete the program of cell death by alternate pathways.

Carvedilol prevents doxorubicin-induced free radical release and apoptosis in cardiomyocytes in vitro

The clinical use of doxorubicin, a highly active anticancer drug, is limited by its severe cardiotoxic side effects. Increased oxidative stress and apoptosis have been implicated in the cardiotoxicity of doxorubicin. Carvedilol is an adrenergic blocking agent with potent anti-oxidant activity. In this study we investigated whether carvedilol has protective effects against doxorubicin-induced free radical production and apoptosis in cultured cardiac muscle cells, and we compared the effects of carvedilol to atenolol, a β-blocker with no anti-oxidant activity. Reactive oxygen species (ROS) generation in cultured cardiac muscle cells (H9c2 cells) was evaluated by flow cytometry using dichlorofluorescein (DCF) and hydroethidine (HE). Apoptosis was assessed by measuring annexin V–FITC/propidium iodide double staining, DNA laddering, levels of expression of the pro-apoptotic protein Bax-α and the anti-apoptotic protein Bcl-2, and caspase-3 activity. Pre-treatment with carvedilol significantly attenuated the doxorubicin-induced increases in DCF ( P < 0.001 compared to cells not pre-treated with carvedilol) and HE ( P < 0.01) fluorescence. Doxorubicin increased the fraction of annexin V–FITC-positive fluorescent cells, while pre-treatment with carvedilol reduced the number of positive fluorescent cells ( P < 0.01). Doxorubicin-induced DNA fragmentation to a clear ladder pattern, while carvedilol prevented DNA fragmentation. Doxorubicin-induced a fall in mRNA expression of the anti-apoptotic Bcl-2 and an increase in the expression of the pro-apoptotic Bax-α. Carvedilol pre-treatment blunted both the decrease of Bcl-2 ( P < 0.01) and the increase of Bax-α mRNA expression ( P < 0.01). Caspase-3 activity significantly increased after the addition of doxorubicin. Concurrently, carvedilol partially inhibited the doxorubicin-induced activation of caspase-3 ( P < 0.01). Atenolol did not produce any effect in preventing doxorubicin-induced ROS generation and cardiac apoptosis. Our results suggest that carvedilol is potentially protective against doxorubicin cardiotoxicity by decreasing free radical release and apoptosis in cardiomyocytes.

Differential Efficacy of Caspase Inhibitors on Apoptosis Markers during Sepsis in Rats and Implication for Fractional Inhibition Requirements for Therapeutics

A rodent model of sepsis was used to establish the relationship between caspase inhibition and inhibition of apoptotic cell death in vivo. In this model, thymocyte cell death was blocked by Bcl-2 transgene, indicating that apoptosis was predominantly dependent on the mitochondrial pathway that culminates in caspase-3 activation. Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers. Inhibition of DNA fragmentation required substantially higher levels of caspase-3 attenuation than that required for blockade of other apoptotic events such as spectrin proteolysis and phosphatidylserine externalization. These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase. These findings suggest that putative caspase-independent apoptosis may be overestimated in some systems since blockade of spectrin proteolysis and other cell death markers does not accurately reflect the high degrees of caspase-3 inhibition needed to prevent DNA fragmentation. Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.

Ceramide-1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages

It was reported previously that ceramide-1-phosphate (Cer-1-P) is mitogenic for fibroblasts (Gómez-Muñoz, A., P. A. Duffy, A. Martin, L. O'Brien, H-S. Byun, R. Bittman, and D. N. Brindley. 1995. Mol. Pharmacol. 47: 883-889; Gómez-Muñoz, A., L. M. Frago, L. Alvarez, and I. Varela-Nieto. 1997. Biochem. J. 325: 435-440). We now show that Cer-1-P prevents cell death in bone-marrow-derived macrophages (BMDMs) after withdrawal of macrophage colony-stimulating factor (M-CSF). Removal of M-CSF is known to induce apoptosis in these cells. Cer-1-P blocked activation of the caspase-9/caspase-3 pathway and prevented DNA fragmentation, indicating that the enhancement of cell survival was due to inhibition of apoptosis. M-CSF deprivation resulted in activation of acid sphingomyelinase (A-SMase), increased ceramide levels, and a decrease in intracellular Cer-1-P. Exogenously added Cer-1-P inhibited A-SMase in intact BMDMs at concentrations that also prevented apoptosis. Cer-1-P also inhibited A-SMase in cell homogenates, suggesting a possible direct physical interaction of Cer-1-P with the enzyme. In conclusion, these data demonstrate that Cer-1-P blocks apoptosis in BMDMs through inhibition of A-SMase, thereby reducing ceramide generation. This adds a new dimension to the understanding of the metabolic interrelationship of ceramides and Cer-1-P, and shows how altering the balance of intracellular levels of these mediators can affect cell survival.

328MEIOTIC COMPETENCE AND DNA FRAGMENTATION OF PORCINE OOCYTES FROM OVARIES STORED IN VARIOUS TEMPERATURES

The aim of this study was to investigate the effects of storage of porcine ovaries at different temperatures before oocyte collection on the nuclear maturation and DNA fragmentation of cumulus-oocyte complexes (COCs). Ovaries were collected at a local abattoir and randomly kept in physiological saline at 4°C, 15°C, 25°C and 35°C. Ovaries were stored for 6 hours prior to follicle aspiration. After storage at each temperature (about 80 oocytes each group), COCs were fixed immediately after aspiration and stained by the terminal deoxynucleotidyl transferase (TdT) nick-end labeling (TUNEL) method to examine the DNA fragmentation under fluorescein microscope. To investigate meiotic competence of the oocytes, some COCs of each treatment group (about 100 oocytes each group) were matured in vitro for 45 hours in a modified North Carolina State University (NCSU)-37 solution supplemented with 10% (v/v) porcine follicular fluid, 0.6mM cysteine, 10IUmL−1 eCG and 10IUmL−1 hCG. After maturation culture, the cumulus cells were removed from COCs and fixed in acetic acid-ethanol (1:3, v/v) for 48–72h. The fixed oocytes were stained with acetic-orcein (1% orcein in 45% acetic acid) and examined under a phase-contrast microscope. Data were subjected to arc-sin transform before analyzing by ANOVA. The proportions of oocytes with DNA fragmentation increased with increasing storage temperature of ovaries (25.2% in 4°C, 31.8% in 15°C, 37.4% in 25°C and 54.7% in 35°C, respectively). There was no significant difference between the proportions of germinal vesicle breakdown (GVBD) of 25°C and 35°C storage groups (74.7 and 83.6%, respectively), but the proportions of 25°C and 35°C storage groups were significantly higher (P&amp;lt;0.05) than those of 4°C and 15°C storage groups (58.1 and 59.6%, respectively). The proportions of oocytes reaching metaphase II (MII) was significantly higher (P&amp;lt;0.05) in the 25°C storage group than in other groups (48.0% in 25°C v. 0% in 4°C, 0% in 15°C and 40.1% in 35°C). Moreover, none of oocytes in 4°C and 15°C storage groups reached MII. These results indicate that 25°C is the most suitable temperature for long-term storage of ovaries to maintain meiotic competence and prevent DNA fragmentation of porcine oocytes.

HSP25 overexpression attenuates oxidative stress–induced apoptosis: roles of ERK1/2 signaling and manganese superoxide dismutase

HSP25 has been shown to induce resistance to radiation and oxidative stress; however, its exact mechanisms remain unclear. In the present study, a high concentration of H2O2 was found to induce DNA fragmentation in L929 mouse fibroblast cells, and HSP25 overexpression attenuated this phenomenon. To elucidate the mechanisms of H2O2-mediated cell death, ERK1/2, p38 MAPK, and JNK1/2 phosphorylation in the cells after treatment with H2O2 were examined. ERK1/2 and JNK1/2 were activated by H2O2; ERK1/2 activation was inhibited in HSP25-overexpressed cells, while JNK1/2 was indifferent. Inhibition of ERK1/2 activation by treatment of the cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced cell death; similarly treated HSP25-overexpressed cells were not at all affected. Moreover, inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 transfection did not affect H2O2-mediated cell death in control cells. Dominant-negative Ras or Raf transfection inhibited H2O2-mediated ERK1/2 activation and cell death in control cells. On the contrary, HSP25-overexpressed cells did not show any differences. Upstream pathways of H2O2-mediated ERK1/2 activation and cell death involved both tyrosine kinase (PDGFβ receptor and Src) and PKCδ, while in HSP25-overexpressed cells these kinases did not respond to H2O2 treatment. Since HSP25 overexpression reduced reactive oxygen species (ROS), increased manganese superoxide dismutase (MnSOD) gene expression, and increased enzyme activity, involvement of MnSOD in HSP25-mediated attenuation of H2O2-mediated ERK1/2 activation and cell death was examined. Blockage of MnSOD with antisense oligonucleotides prevented DNA fragmentation and returned the ERK1/2 activation to the control level. Indeed, when MnSOD was overexpressed in L929 cells, similar to in HSP25-overexpressed cells, DNA fragmentation and ERK1/2 activation were reduced. From the above results, we suggest for the first time that reduced oxidative damage by HSP25 was due to MnSOD-mediated downregulation of ERK1/2.