calpain and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

NS5A is a multifunctional non-structural protein of classical swine fever virus (CSFV) that plays an important role in viral replication, but how it exerts its functions is unknown. Here, we report the cleavage of NS5A of the vaccine C-strain, resulting in two truncated forms (b and c). Further experiments using calpain- and caspase-family-specific inhibitors, followed by a caspase-6-specific shRNAs and inhibitor, showed that the cleavage of C-strain NS5A to produce truncated form c is mediated by caspase-6, mapping to

Topics: Amino Acid Chloromethyl Ketones; Amino Acid Sequence; Animals; Calpain; Caspase 6; Caspase Inhibitors; Cell Line; Classical Swine Fever Virus; Leydig Cells; Male; Plasmids; Protease Inhibitors; Proteolysis; RNA-Dependent RNA Polymerase; RNA, Small Interfering; Swine; Testis; Viral Nonstructural Proteins; Virus Replication

This research aimed to investigate whether glutamate induced spiral ganglion neurons (SGNs) apoptosis through apoptosis inducing factor (AIF) pathway. And verify whether PD150606, a calpain inhibitor could prevent apoptosis by inhibiting cleaving and releasing AIF in mitochondrion.. SGNs of postnatal days 0-3 were harvested and cultured in dishes. 20 mM Glu, the caspase inhibitor Z-VAD-FMK and calpain inhibitor PD150606 were added into cultured dishes separately. We used optical microscope and immunofluoresence staining to observe cell morphology and AIF distribution, RT-PCR and Westernblot to analyse AIF and calpain expression in SGNs. TUNEL assay was used to test cell apoptosis.. Cell morphology and nuclear translocation of AIF were altered in SGNs by 20 mM Glu treated in vitro. The axon of SGN shortened, more apoptosis SGN were observed and the expression of AIF and calpain were up-regulated in Glu-treated group than the normal one (P<0.05). The same experiments were conducted in 20 mM+PD150606 treated group and 20 mM+Z-VAD-FMK group. Obviously AIF were located from cytoplasm to the nuclear and the expressions of AIF and calpain were down-regulated by PD150606 (P<0.05). Positive cells in TUNEL staining decreased after PD150606 treating. However, Z-VAD-FMK had no influence on AIF, calpain expression or cell apoptosis.. The AIF-related apoptosis pathway is involved in the process of Glu-induced SGN injury. Furthermore, the inhibition of calpain can prevent AIF from releasing the nuclear or inducing SGN apoptosis.

Topics: Acrylates; Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Apoptosis; Apoptosis Inducing Factor; Calpain; Caspase Inhibitors; Caspases; Cell Nucleus; Gene Expression Regulation; Glutamic Acid; Glycoproteins; Mitochondria; Neurons; Primary Cell Culture; Protein Transport; Rats; Rats, Sprague-Dawley; Signal Transduction; Spiral Ganglion

Rosin, the traditional Chinese medicine, is reported to be able to inhibit skin cancer cell lines. In this report, we investigate the inhibitory effect against HCC cells of QC2, the derivative of rosin's main components dehydroabietic acid.. MTT assay was used to determine the cytotoxicity of QC2. Morphological changes were observed by time-lapse microscopy and transmission electron microscopy and the cytoskeleton changes were observed by laser-scanning confocal microscopy. Cytomembrane integrity and organelles damage were confirmed by detection of the reactive oxygen (ROS), lactate dehydrogenase (LDH), and mitochondrial membrane potential (Δψm). The underlying mechanism was manifested by Western blotting. The oncotic cell death was further confirmed by detection of oncosis related protein calpain.. Swelling cell type and destroyed cytoskeleton were observed in QC2-treated HCC cells. Organelle damage was visualized by transmission electron microscopy. The detection of ROS accumulation, increased LDH release, and decreased ATP and Δψm confirmed the cell death. The oncotic related protein calpain was found to increase time-dependently in QC2-treated HCC cells, while its inhibitor PD150606 attenuated the cytotoxicity.. Dehydroabietic acid derivative QC2 activated oncosis related protein calpain to induce the damage of cytomembrane and organelles which finally lead to oncosis in HCC cells.

Topics: Abietanes; Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Calpain; Carcinoma, Hepatocellular; Caspase 3; Caspase Inhibitors; Cell Death; Cell Line, Tumor; Cell Membrane; Cytoskeleton; Hepatocytes; Humans; Imidazoles; Indoles; Liver Neoplasms; Membrane Potential, Mitochondrial; Reactive Oxygen Species

Brain-derived neurotrophic factor (BDNF) protects hippocampal neurons from glutamate excitotoxicity as determined by analysis of chromatin condensation, through activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3-K) signaling pathways. However, it is still unknown whether BDNF also prevents the degeneration of axons and dendrites, and the functional demise of synapses, which would be required to preserve neuronal activity. Herein, we have studied the time-dependent changes in several neurobiological markers, and the regulation of proteolytic mechanisms in cultured rat hippocampal neurons, through quantitative western blot and immunocytochemistry. Calpain activation peaked immediately after the neurodegenerative input, followed by a transient increase in ubiquitin-conjugated proteins and increased abundance of cleaved-caspase-3. Proteasome and calpain inhibition did not reproduce the protective effect of BDNF and caspase inhibition in preventing chromatin condensation. However, proteasome and calpain inhibition did protect the neuronal markers for dendrites (MAP-2), axons (Neurofilament-H) and the vesicular glutamate transporters (VGLUT1-2), whereas caspase inhibition was unable to mimic the protective effect of BDNF on neurites and synaptic markers. BDNF partially prevented the downregulation of synaptic activity measured by the KCl-evoked glutamate release using a Förster (Fluorescence) resonance energy transfer (FRET) glutamate nanosensor. These results translate a time-dependent activation of proteases and spatial segregation of these mechanisms, where calpain activation is followed by proteasome deregulation, from neuronal processes to the soma, and finally by caspase activation in the cell body. Moreover, PI3-K and PLCγ small molecule inhibitors significantly blocked the protective action of BDNF, suggesting an activity-dependent mechanism of neuroprotection. Ultimately, we hypothesize that neuronal repair after a degenerative insult is initiated at the synaptic level.

Topics: Amino Acid Chloromethyl Ketones; Animals; Axons; Brain-Derived Neurotrophic Factor; Calpain; Cells, Cultured; Down-Regulation; Drug Interactions; Embryo, Mammalian; Enzyme Inhibitors; Glutamic Acid; Hippocampus; Nerve Tissue Proteins; Neurons; Neuroprotective Agents; Rats; Signal Transduction; Time Factors; TRPC Cation Channels

Research on the mechanisms of bilirubin-induced neurological dysfunction focuses mainly on neuronal death, astrocyte-mediated events and microglia activation. Although myelin damage by unconjugated bilirubin (UCB) has been documented in neonatal kernicterus cases, the events leading to myelination impairment were never explored. This condition may occur by reduced oligodendrocyte precursor cells (OPC) number, or failure of OPC to differentiate in myelinating oligodendrocytes. We have shown that UCB elicits an inflammatory response, glutamate release and reactive oxygen species (ROS) generation in neurons and glial cells, biomolecules with toxic properties on OPC. Hence, we propose to examine whether UCB determines OPC demise and, if so, which signaling pathways are involved. Our results show that OPC display increased apoptosis and necrosis-like cell death upon UCB exposure, mediated by early signals of endoplasmic reticulum (ER) stress [e.g. upregulation of glucose-regulated protein (GRP)78, inositol-requiring enzyme (IRE)-1α and activation transcription factor (ATF)-6, as well as activation of caspase-2 and c-Jun N-terminal kinase (JNK)], followed by mitochondrial dysfunction (e.g. loss of mitochondria membrane potential and caspase-9 activation). The later calpain activation points to intracellular Ca(2+) overload and intervention of both ER and mitochondria. Downstream production of ROS may derive from mitochondria damage and secondary injuries, possibly determining the second cycle of GRP78, IRE-1α, caspase-2 and JNK activation. Moreover, inhibition of caspases, calpains and oxidative stress, by using specific inhibitors, prevented UCB-induced OPC death. UCB did not induce the release of cytokines or glutamate by OPC. These results indicate that UCB by reducing OPC survival, through a cascade of programmed intracellular events triggered by ER stress and mitochondria dysfunction, can compromise myelinogenesis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Apoptosis; Bilirubin; Calpain; Caspases; Cells, Cultured; Cerebral Cortex; Cytokines; Endoplasmic Reticulum Stress; Enzyme Activation; Glutamic Acid; JNK Mitogen-Activated Protein Kinases; Membrane Potential, Mitochondrial; Mitochondria; Neural Stem Cells; Oligodendroglia; Primary Cell Culture; Rats; Rats, Wistar; Reactive Oxygen Species; Signal Transduction

Cisplatin injury to renal tubular epithelial cells (RTEC) is accompanied by autophagy and caspase activation. However, autophagy gradually decreases during the course of cisplatin injury. The role of autophagy and the mechanism of its decrease during cisplatin injury are not well understood. This study demonstrated that autophagy proteins beclin-1, Atg5, and Atg12 were cleaved and degraded during the course of cisplatin injury in RTEC and the kidney. zVAD-fmk, a widely used pancaspase inhibitor, blocked cleavage of autophagy proteins suggesting that zVAD-fmk would promote the autophagy pathway. Unexpectedly, zVAD-fmk blocked clearance of the autophagosomal cargo, indicating lysosomal dysfunction. zVAD-fmk markedly inhibited cisplatin-induced lysosomal cathepsin B and calpain activities and therefore impaired autophagic flux. In a mouse model of cisplatin nephrotoxicity, zVAD-fmk impaired autophagic flux by blocking autophagosomal clearance as revealed by accumulation of key autophagic substrates p62 and LC3-II. Furthermore, zVAD-fmk worsened cisplatin-induced renal dysfunction. Chloroquine, a lysomotropic agent that is known to impair autophagic flux, also exacerbated cisplatin-induced decline in renal function. These findings demonstrate that impaired autophagic flux induced by zVAD-fmk or a lysomotropic agent worsened renal function in cisplatin acute kidney injury (AKI) and support a protective role of autophagy in AKI. These studies also highlight that the widely used antiapoptotic agent zVAD-fmk may be contraindicated as a therapeutic agent for preserving renal function in AKI.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 12; Autophagy-Related Protein 5; Beclin-1; Calpain; Caspase Inhibitors; Cathepsin B; Cells, Cultured; Cisplatin; Kidney; Lysosomes; Microtubule-Associated Proteins; Proteins

Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was >95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels. Bid cleavage was caspase-dependent (55-60%) and calcium-dependent (40-45%). Intracellular calcium as an intrinsic mechanism and extracellular calcium as an extrinsic mechanism were responsible for about 30 and 70% of calcium dependence for Bid cleavage, respectively. The results reveal electric field-mediated cell death induction and progression, activating pro-apoptotic-like mechanisms and affecting plasma membrane and intracellular functions, primarily through extrinsic-like pathways with smaller contributions from intrinsic-like pathways. Nanosecond second pulsed electric fields trigger heterogeneous cell death mechanisms in E4 SCC populations to delete them, with caspase-associated cell death as a predominant, but not an unaccompanied event.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Calpain; Carcinoma, Squamous Cell; Caspases; Cell Line, Tumor; Cell Membrane; Cytochromes c; Egtazic Acid; Electric Stimulation; Electricity; Enzyme Activation; Humans; Membrane Potential, Mitochondrial; Protease Inhibitors; Reactive Oxygen Species; Time Factors

In the present study dopaminergic neuroblastoma B65 cells were exposed to Camptothecin (CPT) (0.5-10 μM), either alone or in the presence of roscovitine (ROSC). The results show that CPT induces apoptosis through the activation of ataxia telangiectasia mutated (ATM)-induced cell-cycle alteration in neuroblastoma B65 cells. The apoptotic process is mediated through the activation of cystein proteases, namely calpain/caspases. However, whereas a pan-caspase inhibitor, zVADfmk, inhibited CPT-mediated apoptosis, a calpain inhibitor, calpeptin, did not prevent cell death. Interestingly, CPT also induces CDK5 activation and ROSC (25 μM) blocked CDK5, ATM activation and apoptosis (as measured by caspase-3 activation). By contrast, selective inhibition of ATM, by KU55933, and non-selective inhibition, by caffeine, did not prevent CPT-mediated apoptosis. Thus, we conclude that CDK5 is activated in response to DNA damage and that CDK5 inhibition prevents ATM and p53ser15 activation. However, pharmacological inhibition of ATM using KU55933 and caffeine suggests that ATM inhibition by ROSC is not the only mechanism that might explain the anti-apoptotic effects of this drug in this apoptosis model. Our findings have a potential clinical implication, suggesting that combinatory drugs in the treatment of cancer activation should be administered with caution.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Ataxia Telangiectasia Mutated Proteins; Calpain; Camptothecin; Cell Cycle Proteins; Cell Line, Tumor; Cyclin-Dependent Kinase 5; Dipeptides; DNA Damage; DNA-Binding Proteins; Humans; Morpholines; Neuroblastoma; Protein Serine-Threonine Kinases; Purines; Pyrones; Roscovitine; Tumor Suppressor Proteins

Glycogen synthase kinase-3beta (GSK-3beta) is a crucial component in the cascade of events that culminate in a range of neurodegenerative diseases. It is controlled by several pathways, including calpain-mediated cleavage. Calpain mediates in cell death induced by 3-nitropropionic acid (3-NP), but GSK-3beta regulation has not been demonstrated. Here we studied changes in total GSK-3beta protein levels and GSK-3beta phosphorylation at Ser-9 in this model. The 3-NP treatment induced GSK-3beta truncation. This regulation was dependent on calpain activation, since addition of calpeptin to the medium prevented this cleavage. While calpain inhibition prevented 3-NP-induced neuronal loss, inhibition of GSK-3beta by SB-415286 did not. Furthermore, inhibition of cdk5, a known target of calpain involved in 3-NP-induced cell death, also failed to rescue neurons in our model. Our results point to a new target of calpain and indicate possible cross-talk between calpain and GSK-3beta in the 3-NP toxicity pathway. On the basis of our findings, we propose that calpain may modulate 3-NP-induced neuronal loss.

Topics: Amino Acid Chloromethyl Ketones; Aminophenols; Animals; Calpain; Caspases; Cell Survival; Cells, Cultured; Convulsants; Disease Models, Animal; Embryo, Mammalian; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hippocampus; Male; Maleimides; Mice; Neurodegenerative Diseases; Neurons; Neuroprotective Agents; Nitro Compounds; Propionates; Purines; Rats; Roscovitine; Signal Transduction; Time Factors

Autophagy is a process to engulf aberrant organelles or protein aggregates into double-membrane vesicles for lysosomal breakdown. Autophagy is a protective process against some intracellular bacteria and viruses; however, it is also used for replication by some viruses, such as poliovirus. We recently found that coxsackievirus B4 (CVB4) also induces the autophagy pathway and activates the calpain system for replication in neurons. Notably, the inhibition of autophagy with 3-methyladenine (3MA) reduced calpain activation and virus replication. Calpain inhibitors also reduced autophagosome formation and virus replication. This finding indicates that calpain and the autophagy pathway are closely connected with each other during the infection. Interestingly, we also found that 3MA and calpain inhibitors enhanced the caspase-3 specific cleavage of spectrin during CVB4 infection, suggesting that autophagy inhibition by these drugs triggered apoptosis. Thus, autophagy and apoptosis may balance each other in CVB4-infected neurons. Here, we show that inhibition of caspase with zVAD increased autophagosome formation, further proposing the cross-talk between autophagy and apoptosis in CVB4-infected neurons.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Autophagy; Calpain; Caspases; Coxsackievirus Infections; Cysteine Proteinase Inhibitors; Models, Biological; Neurons; Rats; Sirolimus; Virus Replication

Caspases and apoptosis are thought to play a role in infection-associated preterm-delivery. We have shown that in vitro treatment with pancaspase inhibitor Z-VAD-FMK protects trophoblasts from microbial antigen-induced apoptosis. Objective. To examine whether in vivo administration of Z-VAD-FMK would prevent infection-induced preterm-delivery. Methods. We injected 14.5 day-pregnant-mice with heat-killed group B streptococcus (HK-GBS). Apoptosis within placentas and membranes was assessed by TUNEL staining. Calpain expression and caspase-3 activation were assessed by immunohistochemistry. Preterm-delivery was defined as expulsion of a fetus within 48 hours after injection. Results. Intrauterine (i.u.) or intraperitoneal (i.p.) HK-GBS injection led to preterm-delivery and induced apoptosis in placentas and membranes at 14 hours. The expression of calpain, a caspase-independent inducer of apoptosis, was increased in placenta. Treatment with the specific caspase inhibitor Z-VAD-FMK (i.p.) prior to HK-GBS (i.p.) delayed but did not prevent preterm-delivery. Conclusion. Caspase-dependent apoptosis appears to play a role in the timing but not the occurrence of GBS-induced preterm delivery in the mouse.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calpain; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Disease Models, Animal; Extraembryonic Membranes; Female; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Ovarian Follicle; Placenta; Pregnancy; Premature Birth; Streptococcal Infections; Streptococcus agalactiae

Gossypol, a cottonseed extract derivative, acts as a BH3-mimetic, binding to the BH3 pocket of antiapoptotic proteins and displacing pro-death partners to induce apoptosis. However, knowledge on the molecular underpinnings of its downstream effects is limited. Since chronic lymphocytic leukemia (CLL) cells express high levels of antiapoptotic proteins that act as a survival mechanism for these replicationally quiescent lymphocytes, we investigated whether gossypol induces apoptosis in these cells and what mechanism underlies gossypol-mediated cytotoxicity. Gossypol induced cell death in a concentration- and time-dependent manner; 24-hour incubation with 30 microM gossypol resulted in 50% cell death (median; range, 10%-80%; n = 47) that was not abrogated by pan-specific caspase inhibitor. Starting at 4 hours, the mitochondrial outer membrane was significantly permeabilized (median, 77%; range, 54%-93%; n = 15). Mitochondrial outer membrane permeabiliztaion (MOMP) was concurrent with increased production of reactive oxygen species (ROS); however, antioxidants did not abrogate gossypol-induced cell death. Mitochondrial membrane permeabilization was also associated with loss of intracellular adenosine triphosphate (ATP), activation of BAX, and release of cytochrome c and apoptosis-inducing factor (AIF), which was translocated to the nucleus. Blocking AIF translocation resulted in a decreased apoptosis, suggesting that AIF contributes to gossypol-mediated cytotoxicity in CLL lymphocytes.

Topics: Acetylcysteine; Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Antioxidants; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Biological Transport, Active; Calpain; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Dipeptides; Gossypol; Humans; In Vitro Techniques; Leukemia, Lymphocytic, Chronic, B-Cell; Mitochondrial Membranes; Molecular Mimicry; Reactive Oxygen Species; Superoxides

In most retinal degenerations in humans and in animal models, photoreceptor cells die by apoptosis. Although the biochemical features are similar in all apoptotic cells, different molecular events lead the cell to death. In the present study we used a rat model of inherited retinal degeneration, the RCS rats, to investigate the involvement of the proteases, caspases and/or calpains, in photoreceptor apoptosis. In the first experiments, rats were untreated or injected intravitreally at post natal day 27 (P27) with the large broad spectrum caspase inhibitor, ZVAD, the calpain inhibitor, MuhPhe, or with the vehicle, DMSO. Retinal status was evaluated at P35 and P42 by electroretinography, morphometry and apoptotic nuclei detection. DMSO and MuhPhe had no effect on RCS retinas as evidenced by equivalent loss of function and equivalent number of apoptotic cells than in untreated group. ZVAD transiently reduced apoptotic cells and preserved photoreceptor function at P35 but not at P42. These results suggest that caspases but not calpains are involved in retinal degeneration in the RCS. In the second experiments, RCS rats were injected twice at P27 and P35 with ZVAD or DMSO. Although ZVAD-treated retinas were preserved at P35 compared to the DMSO controls, the second injection of ZVAD did not extend the preserving effect to P42. Moreover, a single injection of ZVAD at P35 had no preserving effect at P42. All these data taken together suggest that caspases do not play a pivotal role after P35. In a fourth set of experiments, we used specific caspase inhibitors to elucidate which caspase was activated. The caspase-1/4 inhibitor (YVAD) or the caspase-3/7 inhibitor (DEVD) were injected intravitreally at P27 and retinal status was evaluated at P35 and P42. Electroretinograms and apoptotic nuclei detection demonstrated that YVAD and DEVD preserved photoreceptors at P35 but not at P42. These results suggest that both caspase-1/4 and caspase-3/7 play a major role in the apoptotic pathway between P27 and P35 in retinal degeneration of RCS rats. In this study, we show that 1/ the photoreceptor apoptotic process in the RCS rat involves caspases but not calpains, and 2/ the retinal degeneration seems to be composed of different phases involving different molecular players. Indeed, we have demonstrated that caspases are playing a major role at P35, but not at P42.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calpain; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Disease Models, Animal; Drug Administration Schedule; Electroretinography; Optic Nerve; Photoreceptor Cells, Vertebrate; Rats; Rats, Mutant Strains; Retinal Degeneration; Time Factors

Abuse of 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) and methamphetamine (Meth or Speed) is a growing international problem with an estimated 250 million users of psychoactive drugs worldwide. It is important to demonstrate and understand the mechanism of neurotoxicity so potential prevention and treatment therapies can be designed. In this study rat primary cerebrocortical neuron cultures were challenged with MDMA and Meth (1 or 2 mM) for 24 and 48 h and compared to the excitotoxin N-methyl-D-aspartate (NMDA). The neurotoxicity of these drugs, as assessed by microscopy, lactate dehydrogenase release and immunoblot, was shown to be both dose- and time-dependent. Immunoblot analysis using biomarkers of cell death showed significant proteolysis of both alphaII-spectrin and tau proteins. Breakdown products of alphaII-spectrin (SBDPs) of 150, 145, and 120 kDa and tau breakdown products (TBDPs) of 45, 32, 26, and 14 kDa were observed. The use of the protease inhibitors calpain inhibitor SJA6017 and caspase inhibitors z-VAD-fmk and Z-D-DCB, attenuated drug-induced alphaII-spectrin and tau proteolysis. The calpain inhibitor reduced the calpain-induced breakdown products SBDP145 and TBDP14, but there was an offset increase in the caspase-mediated breakdown products SBDP120 and TBDP45. The caspase inhibitors, on the other hand, decreased SBDP120 and TBDP45. These data suggest that both MDMA and Meth trigger concerted proteolytic attacks of the structural proteins by both calpain and caspase family of proteases. The ability of the protease inhibitors to reduce the damage caused by these drugs suggests that the treatment arsenal could include similar drugs as possible tools to combat the drug-induced neurotoxicity in vivo.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Aspartic Acid; Calpain; Caspase Inhibitors; Caspases; Cell Death; Cells, Cultured; Cerebral Cortex; Cysteine Proteinase Inhibitors; Dipeptides; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Methamphetamine; Microfilament Proteins; Molecular Weight; N-Methyl-3,4-methylenedioxyamphetamine; N-Methylaspartate; Neurons; Peptide Fragments; Psychotropic Drugs; Rats; Rats, Sprague-Dawley; tau Proteins; Time Factors; Vesicular Transport Proteins

Renal cell carcinoma is the most common neoplasm occurring in the kidney and is largely resistant to current chemotherapy. Understanding the mechanisms involved in renal carcinoma cell death may lead to novel and more effective therapies. In Cak(i)-1 renal cancer cells, using phosphatidylserine externalization as a marker of apoptosis, the anti-cancer drugs 5-fluorouracil (5-FU), and its pro-drugs, doxifluridine (Dox) and floxuridine (Flox) proceeds via a caspase-dependent mechanism. In contrast, phosphatidylserine externalization produced by staurosporine in the renal cancer cell lines Cak(i)-1 and A-498 proceeds via a caspase-independent mechanism. That is, the pan caspase inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (ZVAD) did not ameliorate annexin V binding, cell shrinkage or changes in nuclear morphology. Subsequent experiments were conducted to determine mediators of phosphatidylserine externalization, using annexin V binding, when caspases were inhibited. Prior treatment of A-498 cells with cathepsin B (CA74 methyl ester), cathespsin D (pepstatin A) or calpain inhibitors (calpeptin, E64d) in the presence or absence of ZVAD did not ameliorate annexin V binding. The endonuclease inhibitor aurintricarboxylic acid (ATA), phospholipase A(2) inhibitor bromoenol lactone (BEL), protein synthesis inhibitor cycloheximide (CH) and chloride channel blockers niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) all had no effect on staurosporine-induced annexin V binding in A-498 cells either in the presence or absence of ZVAD. We also modulated sphingomyelin and the de novo pathways of ceramide synthesis and found no amelioration of staurosporine-induced annexin V binding in A-498 cells either in the presence or absence of ZVAD. These results indicate that 5-FU, Dox and Flox induce externalization of phosphatidylserine during apoptosis in Cak(i)-1 renal cancer cells primarily through a caspase-dependent mechanism and that externalization of phosphatidylserine during apoptosis produced by staurosporine in the renal cancer cell line A-498 is independent of many of the common signaling pathways known to be involved in this process.

Topics: Amino Acid Chloromethyl Ketones; Aniline Compounds; Antimetabolites, Antineoplastic; Apoptosis; Benzoic Acid; Benzylidene Compounds; Calpain; Caspases; Cathepsin B; Cathepsin D; Cell Line, Tumor; Cell Size; Ceramides; Cisplatin; Exocytosis; Fumonisins; Humans; Kidney Neoplasms; Naphthalenes; Niflumic Acid; Nitrobenzoates; Phosphatidylserines; Pyrones; Sphingomyelin Phosphodiesterase; Staurosporine; Triterpenes

To elucidate mechanism of cell death in response to hypoxia, we attempted to compare hypoxia-induced cell death of HepG2 cells with cisplatin-induced cell death, which has been well characterized as a typical apoptosis. Cell death induced by hypoxia turned out to be different from cisplatin-mediated apoptosis in cell viability and cleavage patterns of caspases. Hypoxia-induced cell death was not associated with the activation of p53 while cisplatin-induced apoptosis is p53 dependent. In order to explain these differences, we tested involvement of micro-calpain and m-calpain in hypoxia-induced cell death. Calpains, especially micro-calpain, were initially cleaved by hypoxia, but not by cisplatin. Interestingly, the treatment of a calpain inhibitor restored PARP cleavage that was absent during hypoxia, indicating the recovery of activated caspase-3. The inhibition of calpains prevented proteolysis induced by hypoxia. In addition, hypoxia resulted in a necrosis-like morphology while cisplatin induced an apoptotic morphology. The calpain inhibitor prevented necrotic morphology induced by hypoxia and converted partially to apoptotic morphology with nuclear segmentation. Our result suggests that calpains are involved in hypoxia-induced cell death that is likely to be necrotic in nature and the inhibition of calpain switches hypoxia-induced cell death to apoptotic cell death without affecting cell viability.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Calpain; Caspase Inhibitors; Caspases; Cell Death; Cell Line, Tumor; Cell Shape; Cisplatin; Cysteine Proteinase Inhibitors; Humans; Hypoxia; Poly(ADP-ribose) Polymerases; Tumor Suppressor Protein p53

Apoptosis of endothelial cells is considered an initial step in the development of atherosclerosis. Recent studies have indicated that depletion of the endoplasmic reticulum (ER) Ca(2+) content plays an important role in apoptosis. Caspase-12 is a key signal in ER stress-induced apoptosis. However, it is not known whether the depletion of ER Ca(2+) is linked to caspase-12 signalling in endothelial cells. Here we have investigated the interaction of Ca(2+) signalling and caspase-12 cleavage in apoptosis of endothelial cells.. Cytosolic Ca(2+) concentration ([Ca(2+)](i)) of primary porcine aortic endothelial cells was measured using fura-2/AM. Apoptosis was assessed by DNA fragmentation, and cleavage of caspase-12 using Western blotting techniques.. Thapsigargin (5 microM), an inhibitor of the ER Ca(2+)-ATPase, depleted ER Ca (2+) content, increased [Ca(2+)](i), cleaved caspase-12, and induced apoptosis. Bradykinin (10 nM) also increased [Ca(2+)](i) but did not cleave caspase-12 or induce apoptosis. However, when intracellular Ca(2+) was chelated with BAPTA/AM (100 microM), bradykinin caused ER Ca(2+) depletion and apoptosis without accompanying caspase-12 cleavage. A non-selective caspase inhibitor, z-VAD.fmk (100 microM), inhibited apoptosis and cleavage of caspase-12 stimulated by thapsigargin, while a calpain inhibitor, MDL 28170 (120 microM), inhibited caspase-12 cleavage but not apoptosis.. Thus, increases in intracellular Ca(2+) concentration are not sufficient for the induction of apoptosis in endothelial cells, and ER Ca(2+) depletion appears to induce apoptosis independently of caspase-12.

Topics: Amino Acid Chloromethyl Ketones; Animals; Aorta; Apoptosis; Blotting, Western; Bradykinin; Calcium; Calcium-Transporting ATPases; Calpain; Caspase 12; Caspase Inhibitors; Caspases; Cells, Cultured; Chelating Agents; Cytosol; Dipeptides; DNA Fragmentation; Egtazic Acid; Endoplasmic Reticulum; Endothelial Cells; Endothelium, Vascular; Enzyme Inhibitors; Signal Transduction; Swine; Thapsigargin

Glutamate toxicity in traumatic brain injury, ischemia, and Huntington's disease causes cortical neuron death and dysfunction. We tested the efficacy of calpain and caspase-3 inhibitors alone and in combination to prevent neuronal death and preserve electrophysiological functions in rat primary cortical neurons following glutamate exposure. Cortical neurons exposed to 0.5 microM glutamate for 24 h committed mostly apoptotic death as determined by Wright staining and ApopTag assay. Levels of expression, formation of active forms, and activities of calpain and caspase-3 were increased following glutamate exposure. Also, in situ double labeling identified conformationally active caspase-3-p20 fragment and chromatin condensation in apoptotic neurons. Pretreatment of cortical neurons with 0.2 microM N-benzyloxylcarbonyl-Leu-Nle-aldehyde (calpain-specific inhibitor) and 100 microM N-benzyloxylcarbonyl-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-fluoromethyl ketone (caspase-3-specific inhibitor) provided strong neuroprotection. Standard patch-clamp techniques were used to measure the whole-cell currents associated with Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors. The lack of a change in capacitance indicated that neurons treated with inhibitor(s) plus glutamate did not undergo apoptotic shrinkage and maintained the same size as the control neurons. Whole-cell currents associated with Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors were similar in amplitude and activation/inactivation kinetics for cells untreated and treated with inhibitor(s) and glutamate. Spontaneous synaptic activity as observed by miniature end-plate currents was also similar. Prevention of glutamate-induced apoptosis by calpain and caspase-3 inhibitors preserved normal activities of crucial ion channels such as Na+ channels, N-methyl-D-aspartate receptors, and kainate receptors in neurons. Our studies strongly imply that calpain and caspase-3 inhibitors may also provide functional neuroprotection in the animal models of traumatic brain injury and neurodegenerative diseases.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Apoptosis; Calpain; Caspase 3; Caspase Inhibitors; Caspases; Cell Survival; Cells, Cultured; Cerebral Cortex; Chromatin; Cysteine Proteinase Inhibitors; DNA Fragmentation; Drug Interactions; Female; Glutamic Acid; Ion Channel Gating; Membrane Potentials; Neurons; Oligopeptides; Pregnancy; Rats; Rats, Sprague-Dawley

In this study, a significant increase by 50% in intracellular free calcium concentration ([Ca(2+)](i)) was observed in differentiated human neuroblastoma (SH-SY5Y) cells after exposure to 0.25microM of the fungal metabolite gliotoxin for 72h. Further, the involvement of caspases and calpains was demonstrated to underlie the gliotoxin-induced cytotoxic and neurite degenerative effects. The caspase inhibitor Z-VAD-fmk almost completely reduced the neurite degeneration from 40% degeneration of neurites to 5% as compared to control. Inhibition of calpains with calpeptin significantly attenuated gliotoxin-induced cytotoxicity, determined as reduction in total cellular protein content, from 43% to 14% as compared to control cells. Western blot analyses of alphaII-spectrin breakdown fragments confirmed activity of the proteases, and that alphaII-spectrin was cleaved by caspases in gliotoxin-exposed cells. These results show that calpains and caspases have a role in the toxicity of gliotoxin in differentiated SH-SY5Y cells and that the process may be Ca(2+)-mediated.

Topics: Amino Acid Chloromethyl Ketones; Calcium; Calpain; Caspases; Cell Differentiation; Cell Line, Tumor; Enzyme Inhibitors; Gliotoxin; Humans; Immunosuppressive Agents; Neurites; Neuroblastoma; Spectrin

Alveolar hypoxia, a common feature of many respiratory disorders, has been previously reported to induce functional changes, particularly a decrease of transepithelial Na and fluid transport. In polarized epithelia, cytoskeleton plays a regulatory role in transcellular and paracellular transport of ions and fluid. We hypothesized that exposure to hypoxia could damage cytoskeleton organization, which in turn, may adversely affect ion and fluid transport. Primary rat alveolar epithelial cells (AEC) were exposed to either mild (3% O(2)) or severe (0.5% O(2)) hypoxia for 18 h or to normoxia (21% O(2)). First, mild and severe hypoxia induced a disorganization of actin, a major protein of the cytoskeleton, reflected by disruption of F-actin filaments. Second, alpha-spectrin, an apical cytoskeleton protein, which binds to actin cytoskeleton and Na transport proteins, was cleaved by hypoxia. Pretreatment of AEC by a caspase inhibitor (z-VAD-fmk; 90 microM) blunted hypoxia-induced spectrin cleavage as well as hypoxia-induced decrease in surface membrane alpha-ENaC and concomitantly induced a partial recovery of hypoxia-induced decrease of amiloride-sensitive Na transport at 3% O(2). Finally, tight junctions (TJs) proteins, which are linked to actin and are a determinant of paracellular permeability, were altered by mild and severe hypoxia: hypoxia induced a mislocalization of occludin from the TJ to cytoplasm and a decrease in zonula occludens-1 protein level. These modifications were associated with modest changes in paracellular permeability at 0.5% O(2,) as assessed by small 4-kD dextran flux and transepithelial resistance measurements. Together, these findings indicate that hypoxia disrupted cytoskeleton and TJ organization in AEC and may participate, at least in part, to hypoxia-induced decrease in Na transport.

Topics: Actins; Amino Acid Chloromethyl Ketones; Animals; Calpain; Cell Membrane; Cell Polarity; Cells, Cultured; Cytoskeleton; Epithelial Cells; Epithelial Sodium Channels; Hypoxia; Male; Membrane Proteins; Occludin; Patch-Clamp Techniques; Permeability; Phosphoproteins; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Spectrin; Tight Junctions; Zonula Occludens-1 Protein

Activation of protein kinase C delta (PKCdelta) is believed to be pro-apoptotic. PKCdelta is reported to be reduced in colon cancers. Using a colon cancer cell line, COLO 205, we have examined the roles of PKCdelta in apoptosis and of caspase-3 in the activation and inhibition of PKCdelta. PKCdelta activation with bistratene A and its inhibition with rottlerin induced apoptosis. Effects of PKC activators and inhibitors were additive, suggesting that PKCdelta down-regulation was responsible for the effects on apoptosis. Different apoptotic pathways induced PKCdelta cleavage, but the fragment produced was inactive in kinase assays. Caspase-3 inhibition did not block DNA fragmentation or PKCdelta proteolysis despite blocking intracellular caspase-3 activity. Calpain inhibition with calpeptin did not prevent TPA-induced PKCdelta cleavage. We conclude that in colonocytes, inhibition of PKCdelta is sufficient to lead to caspase-3-independent apoptosis. Caspase-3 does not cleave PKCdelta to an active form, nor does caspase-3 inhibition block apoptosis.

Topics: Acetamides; Acetophenones; Alkaloids; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Benzophenanthridines; Benzopyrans; Calpain; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Dipeptides; DNA Fragmentation; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; Histones; Humans; Indomethacin; Kinetics; Phenanthridines; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Pyrans; Spiro Compounds; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

In this study, we have evaluated the cytotoxic effect of combining two HDAC inhibitors, SAHA and TSA, with TRAIL in human multiple myeloma cell lines. Low doses of SAHA or TSA enhanced the cytotoxic and apoptotic effects of TRAIL and upregulated the surface expression of TRAIL death receptors (DR4 and/or DR5). SAHA and TSA induced G1 phase cell cycle growth arrest by upregulating p21(WAF1) and p27(Kip1) expression and by inhibiting E2F transcriptional activity. The enhanced TRAIL effect after pretreatment with HDAC inhibitors was consistent with the upregulation of the proapoptotic Bcl-2 family members (Bim, Bak, Bax, Noxa, and PUMA), the downregulation of the anti-apoptotic members of the Bcl-2 family (Bcl-2 and Bcl-X(L)), and IAPs. SAHA and TSA dissipated the mitochondrial membrane potential and enhanced the release of Omi/HtrA2 and AIF from the mitochondria to the cytosol. The cytotoxic effect of both SAHA and TSA was caspase- and calpain-independent. Inhibition of NF(kappa)B activation by the proteasome inhibitor, MG132, enhanced the apoptotic effect of TSA. Our study demonstrated the enhancing effects of HDAC inhibitors on apoptosis when combined with TRAIL and, for the first time, emphasized the role of AIF in mediating the cytotoxic effects of HDAC inhibitors.

Topics: Amino Acid Chloromethyl Ketones; Annexin A5; Apoptosis; Apoptosis Regulatory Proteins; Calpain; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; E2F Transcription Factors; Enzyme Inhibitors; Flow Cytometry; G1 Phase; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Immunoblotting; Inhibitory Concentration 50; Leupeptins; Luciferases; Membrane Glycoproteins; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Multiple Myeloma; NF-kappa B; Phosphorylation; Propidium; Protein Binding; Ribonucleases; Subcellular Fractions; Time Factors; TNF-Related Apoptosis-Inducing Ligand; Transcription Factors; Tumor Necrosis Factor-alpha; Tumor Suppressor Proteins; Up-Regulation

Caspases, members of the cysteine protease family, execute UVB-induced apoptosis in several cell lines and keratinocytes. Several researchers investigating UVB-induced apoptosis have demonstrated a dose-dependent protective effect of the synthetic peptide caspase inhibitor zVAD-fmk. However, zVAD-fmk displays a dose-dependent protective effect against UVB-induced apoptosis, even at doses higher than those required to block all known proapoptotic caspases. In addition, it is known that zVAD-fmk also inhibits other cysteine proteases including cathepsins and calpains, and these proteases have recently been demonstrated to play a role in the execution of programmed cell death induced by other stimuli, e.g. TNF-alpha. The purpose of the present study was therefore to investigate whether inhibitors of cysteine cathepsins and calpains could prevent UVB-induced apoptosis in HeLa cells and keratinocytes. This was done by investigating the effect of the irreversible cysteine protease inhibitor zFA-fmk, the cathepsin B inhibitor CA-074-Me and the calpain inhibitor ALLN on the viability of UVB-irradiated human keratinocytes and HeLa cells. At concentrations of 10 microM and above zVAD-fmk conferred partial dose-dependent protection against UVB-induced apoptosis in HeLa cells and keratinocytes. Moreover, caspase-3 activity was completely blocked at zVAD-fmk concentrations of 1 microM in HeLa cells. This indicates that caspase-independent mechanisms could be involved in UVB-induced apoptosis. However, the protease inhibitors zFA-fmk, CA-074-Me and ALLN all failed to prevent UVB-induced apoptosis in HeLa cells and keratinocytes. In conclusion, the protective effect of zVAD-fmk at high concentrations indicates that other proteases than caspases are active in the execution of UVB-induced apoptosis but further studies are needed to identify these proteases.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Calpain; Caspase Inhibitors; Cathepsins; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; HeLa Cells; Humans; Keratinocytes; Ketones; Leupeptins; Phospholipases A; Ultraviolet Rays

The endoplasmic reticulum (ER) resident-94 kDa glucose-regulated protein (GRP94), plays a pivotal role in cell death due to ER stress. In our study expression of GRP94 was increased in human neuroblastoma SH-SY5Y cells due to exposure to calcium ionophore A23187. A23187-mediated cell death was associated with activation of the major cysteine proteases, caspase-3 and calpain. Pretreatment with adenovirus-mediated antisense GRP94 (AdGRP94AS) reduced viability of SH-SY5Y cells subjected to A23187 treatment compared with wild type cells or cells with adenovirus-mediated overexpression of GRP94 (AdGRP94S). These results indicated that suppression of GRP94 is associated with accelerated cell death. Moreover, expression of GRP94 suppressed A23187-induced cell death and stabilized calcium homeostasis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Calcimycin; Calcium; Calpain; Caspase 3; Caspases; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Dantrolene; DNA, Antisense; Endoplasmic Reticulum; Gene Expression; Histocytochemistry; Homeostasis; HSP70 Heat-Shock Proteins; Humans; In Situ Nick-End Labeling; Lac Operon; Membrane Proteins; Neurons; Thapsigargin; Transfection

A critical role for reactive oxygen species (ROS) in photoreceptor apoptosis has been established. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. This study delineates the molecular events that occur after treatment of the photoreceptor cell line 661W with the nitric oxide donor sodium nitroprusside (SNP). Cytosolic calcium levels increased during photoreceptor apoptosis, leading to activation of the calcium-dependent proteases calpains. Furthermore, caspase activation also occurred following SNP insult. However, although treatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone inhibited caspase activity per se in SNP-treated 661W cells, it did not prevent apoptosis. On the other hand, CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) acted as a scavenger of ROS and reduced 661W photoreceptor apoptosis induced by SNP by preventing the activation of a pathway in which calpains have a key role. In summary, we report for the first time that both caspases and calpains are involved in 661W photoreceptor apoptosis and that calpain activation can be prevented by the ROS scavenger CR-6.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Benzopyrans; Calcium; Calpain; Caspase 12; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cell Line; Cysteine Proteinase Inhibitors; Free Radical Scavengers; Mice; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Oxidative Stress; Photoreceptor Cells, Vertebrate; Reactive Oxygen Species

The involvement of PKC isoform in the methamphetamine (MA)-induced death of neuron-like PC12 cell was studied. The death and the enhanced terminal dUTP nick end labeling (TUNEL) staining were inhibited by a caspase inhibitor, z-Val-Ala-Asp- (OMe)-CH(2)F (z-VAD-fmk). However, the cell death shows neither morphological nor biochemical features of apoptosis or necrosis. The cell death was suppressed by a protein kinase C (PKC) activator, 12,13-phorbol myristate acetate, but was enhanced by PKC specific inhibitor calphostin C or bisindolylmaleimide, not by PKC inhibitor relatively specific for PKC-alpha (safingol) or PKC-delta (rottlerin). Western blotting demonstrated the expression of PKC-alpha, gamma, delta, epsilon and zeta, of which PKC-epsilon translocated from the soluble to the particulate fraction after MA-treatment. Antisense to PKC-epsilon enhanced MA-induced death. A glutamate receptor antagonist MK801 abrogated the cell death, which is reversed by PKC inhibition. These data suggest that PKC-epsilon promotes PC12 cell survival through glutamate receptor suppression.

Topics: Adrenergic Agents; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blotting, Western; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Calpain; Caspase Inhibitors; Cell Survival; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme Inhibitors; In Situ Nick-End Labeling; Isoenzymes; Methamphetamine; Naphthalenes; Oligonucleotides, Antisense; PC12 Cells; Protein Kinase C; Protein Kinase C-epsilon; Rats; Receptors, Glutamate; Tetradecanoylphorbol Acetate; Transfection

Oxidative stress plays an important role in the development of ischemia/reperfusion (I/R)-induced apoptosis of hepatocytes. We aimed to examine the involvement of caspases and calpains in H2O2-induced hepatic cell apoptosis. TUNEL-positive apoptotic cells appeared in parallel with poly(ADP-ribose) polymerase (PARP) cleavage and procaspase-3 proteolysis by H2O2 treatment in a dose-dependent manner (250-1,000 micro M). Bcl-xL and intact Bax expression levels decreased when H2O2 was >250 micro M. The cleaved form of Bax appeared prior to caspase-3 activation, increasing in a dose-dependent manner. A pan-caspase inhibitor, Z-VAD-fmk, completely blocked H2O2-induced procaspase-3 proteolysis and PARP cleavage without changing Bax cleavage, but partially attenuated H2O2-induced apoptosis. Calpeptin, a calpain inhibitor, did not inhibit caspase-3 activation, Bax cleavage or apoptosis. Our results indicate that Bax cleavage is upstream signal of caspase-dependent apoptosis in hepatocytes exposed to H2O2, but not independent upon calpain. Molecular targeting of Bax cleavage may allow the development of strategies to prevent hepatic I/R injury.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Calpain; Caspase 3; Caspase Inhibitors; Caspases; Cell Line; Cysteine Proteinase Inhibitors; Dipeptides; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Precursors; Hepatocytes; Humans; Hydrogen Peroxide; Oxidants; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

Mature mouse oligodendrocytes (OLs) are susceptible to death in demyelinating diseases such as multiple sclerosis and in brain injury following neurotrauma, ischemia, or stroke. To understand mechanisms leading to death of mature OLs and develop strategies for protection, we utilized cultures of mature mouse OLs to investigate the role of caspases and calpains in OL cell death mediated by different mechanisms. The agents used were (i) staurosporine, which induces apoptotic death via inhibition of protein kinases; (ii) kainate, which activates non-NMDA glutamate receptors; (iii) thapsigargin, which releases intracellular calcium stores; and (iv) SNAP, which releases active NO species and causes necrotic cell death. Inhibitors blocking primary effector caspases (including caspase 3), the FAS (death receptor)-mediated initiator caspases (including caspase 8), and stress-induced caspases (including caspase 9), were tested for their protective effects. Inhibition of caspases 3, 8, and 9 each robustly protected OLs following insult with staurosporine, thapsigargin, or kainate when added at optimal times. The time of addition of the inhibitors for maximal protection varied with the agent, from 1 h of preincubation before addition of staurosporine to 6 h after addition of kainate. Much less protection was seen for the NO generator SNAP under any condition. The role of calcium in OL death in each model was investigated by chelating extracellular Ca++ with EGTA, and by inhibiting the Ca++-activated calpain proteases. Calcium chelation did not protect against staurosporine, but decreased OL death initiated by kainate, thapsigargin, or NO. The calpain inhibitors PD150606 and calpain inhibitor I protected from cell death initiated by staurosporine, kainate, and thapsigargin, but not from cell death initiated by the NO donor SNAP.

Topics: Amino Acid Chloromethyl Ketones; Animals; Calcium; Calpain; Caspase Inhibitors; Cell Death; Cells, Cultured; Cysteine Proteinase Inhibitors; Kainic Acid; Mice; Mice, Inbred BALB C; Oligodendroglia; Penicillamine; Staurosporine; Thapsigargin

Members of both calpain and caspase protease families can degrade several components of focal adhesions, leading to disassembly of these complexes. In this report, we investigated the disappearance of tensin from cell adhesion sites of chicken embryonic fibroblast cells (CEFs) exposed to etoposide and demonstrated that loss of tensin from cell adhesions during etoposide-induced apoptosis may be due to degradation of tensin by caspase-3. Tensin cleavage by caspase-3 at the sequence DYPD(1226)G separates the amino-terminal region containing the actin binding domain and the carboxyl-terminal region containing the SH2 domain. The resultant carboxyl-terminal fragment of tensin is unable to bind phosphoinositide 3-kinase (PI3-kinase) transducing cell survival signaling. We also demonstrated that overexpression of the amino-terminal tensin fragment induced disruption of actin cytoskeleton in chicken embryonic fibroblasts. Therefore, caspase-mediated cleavage of tensin contributes to the disruption of actin organization and interrupts ECM-mediated survival signals through phosphatidylinositol 3-kinase.

Topics: Actins; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calpain; Caspase 3; Caspases; Cell Adhesion; Cell Culture Techniques; Cell Survival; Chick Embryo; Cytoskeleton; DNA; DNA, Complementary; Dose-Response Relationship, Drug; Etoposide; Fibroblasts; Green Fluorescent Proteins; Immunoblotting; Luminescent Proteins; Microfilament Proteins; Microscopy, Fluorescence; Mutagenesis, Site-Directed; Nucleic Acid Synthesis Inhibitors; Phosphatidylinositol 3-Kinases; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; src Homology Domains; Tensins; Time Factors; Transfection

Galectin-9 (Gal-9) induced the apoptosis of not only T cell lines but also of other types of cell lines in a dose- and time-dependent manner. The apoptosis was suppressed by lactose, but not by sucrose, indicating that beta-galactoside binding is essential for Gal-9-induced apoptosis. Moreover, Gal-9 required at least 60 min of Gal-9 binding and possibly de novo protein synthesis to mediate the apoptosis. We also assessed the apoptosis of peripheral blood T cells by Gal-9. Apoptosis was induced in both activated CD4(+) and CD8(+) T cells, but the former were more susceptible than the latter. A pan-caspase inhibitor (Z-VAD-FMK) inhibited Gal-9-induced apoptosis. Furthermore, a caspase-1 inhibitor (Z-YVAD-FMK), but not others such as Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-AEVD-FMK (caspase-10 inhibitor), inhibited Gal-9-induced apoptosis. We also found that a calpain inhibitor (Z-LLY-FMK) suppresses Gal-9-induced apoptosis, that Gal-9 induces calcium (Ca(2+)) influx, and that either the intracellular Ca(2+) chelator BAPTA-AM or an inositol trisphosphate inhibitor 2-aminoethoxydiphenyl borate inhibits Gal-9-induced apoptosis. These results suggest that Gal-9 induces apoptosis via the Ca(2+)-calpain-caspase-1 pathway, and that Gal-9 plays a role in immunomodulation of T cell-mediated immune responses.

Topics: Adjuvants, Immunologic; Amino Acid Chloromethyl Ketones; Apoptosis; B-Lymphocytes; Calcium; Calcium Signaling; Calpain; Caspase 1; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Immunologic; Galectins; HL-60 Cells; Humans; Jurkat Cells; Monocytes; Myeloid Cells; Oligopeptides; Signal Transduction; T-Lymphocytes; Tumor Cells, Cultured

3-Nitropropionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase, has been used to model features of neurodegenerative disorders including Huntington disease, as well as acute neuronal insults such as cerebral ischemia. 3NP induces rapid necrosis and delayed apoptosis in primary cultures of rat hippocampal neurons. Low levels of extracellular glutamate shift the cell death mechanism to necrosis, whereas antagonism of NMDA receptors results in predominately apoptotic death. In the present study, the involvement of cysteine proteases in the morphologic and biochemical alterations accompanying 3NP-induced neuron death was investigated. Immunoblots of spectrin breakdown products indicated Ca(2+)-dependent cysteine protease (calpain) activation within the 8 hours of 3NP administration, whereas caspase-3 activation was not evident until 16 to 48 hours after treatment. The NMDA receptor antagonist MK-801 (dizocilpine) decreased 3NP-induced calpain activity, but did not alter caspase-3 activity. Similar to MK-801, calpain inhibitors (Z-Val-Phe.H and Z-Leu-Phe-CONHEt) shifted the cell death morphology towards apoptosis and delayed, but did not prevent, the 3NP-induced cell death. Together, the results indicate that following 3NP administration, increased calpain activity precedes caspase-3 activation, contributes to the necrotic morphology, and facilitates and accelerates the cell death.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calpain; Caspases; Cell Death; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Dizocilpine Maleate; Drug Synergism; Embryo, Mammalian; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Immunoblotting; Immunohistochemistry; Necrosis; Neurons; Neurotoxins; Nitro Compounds; Oligopeptides; Propionates; Rats; Spectrin; Time Factors

Apoptosis is the mode of photoreceptor cell death in many retinal dystrophies. Exposure of Balb/c mice to excessive levels of light induces photoreceptor apoptosis and represents an animal model for the study of retinal degenerations. Caspases have emerged as central regulators of apoptosis, executing this tightly controlled death pathway in many cells. Previously we have reported that light-induced photoreceptor apoptosis occurs independently of one the key executioners of apoptosis, caspase-3. This present study extends these results reporting on the lack of activation of other caspases in this model including caspases-8, -9, -7, and -1. Furthermore, photoreceptor apoptosis cannot be inhibited with the broad range caspase inhibitor zVAD-fmk indicating that light-induced retinal degeneration is caspase-independent. We demonstrate that cytochrome c does not translocate from mitochondria to the cytosol during photoreceptor apoptosis. We also show that during retinal development apoptotic protease activating factor (Apaf-1) protein levels are markedly decreased and this is associated with the inability to activate the mitochondrial caspase cascade in the mature retina. In addition, there is also a significant reduction in expression of caspases-3 and -9 during retinal maturation and these levels do not increase following light exposure. Finally, we show that the calcium-dependent proteases calpains are active during light-induced retinal degeneration and establish that the calcium channel blocker D-cis-diltiazem completely inhibits photoreceptor apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Calcium; Calcium Channel Blockers; Calpain; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Cytochrome c Group; Diltiazem; Light; Mice; Photoreceptor Cells; Protein Biosynthesis; Proteins; Retina

Tumor necrosis factor-alpha (TNF) is well known for its cytotoxic effect on malignant cells. Its role in cell cycle control is relatively less known. In this study, we found that TNF induced G(1) arrest of TF-1 and MV4-11 cells while simultaneously causing apoptosis. Treatment of the cells with TNF for 48 h caused cell cycle arrest, accompanied by dephosphorylation of pRb and reduction in D-type cyclin expression. The down-regulation of the D-type cyclins resulted in approximately 50-80% decrease of the cyclin-dependent kinase activities. Cells treated with calpain-dependent inhibitor ALLN and apoptosis inhibitor zVAD-FMK suppressed degradation of IkappaBalpha and activation of caspase 3, respectively. However, treatment of cells with these two inhibitors was not able to prevent TNF-induced down-regulation of the D-type cyclins. In contrast, proteasome inhibitor MG-132 and lactacystin blocked both TNF-induced degradation of IkappaBalpha and down-regulation of D-type cyclins. These data suggest that down-regulation of D-type cyclins by TNF may be proteasome-proteolysis dependent. Additional support for this conclusion was obtained from experiments showing an increase of proteasome activity in TNF-treated cells and in vitro degradation of cyclin D3 by 26 S proteasome.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Calpain; Caspase 3; Caspases; Cell Cycle; Cell Line; Cyclin D; Cyclin D3; Cyclins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; G1 Phase; Humans; I-kappa B Proteins; Leupeptins; Multienzyme Complexes; NF-KappaB Inhibitor alpha; Peptide Hydrolases; Plasmids; Precipitin Tests; Proteasome Endopeptidase Complex; Protein Binding; Protein Biosynthesis; Ribonucleases; RNA; RNA, Messenger; Spectrometry, Fluorescence; Time Factors; Transcription, Genetic; Tumor Necrosis Factor-alpha; Ubiquitin

Apoptosis participates in the development of the nervous system and in neurodegeneration. The aim of this study was to investigate the mechanisms of detachment of neuronal cells from the extracellular matrix (ECM) during apoptosis. Detachment of Ntera2 neuronal cells was accompanied by decreased surface expression of the beta1 integrin and redistribution of proteins from focal adhesions (FA). FA proteins were cleaved in a discrete sequence: p130cas, then paxillin, then talin. Caspase inhibition prevented detachment and cleavage of paxillin and p130cas, whilst calpain inhibition blocked talin cleavage. Neuronal cells therefore detach as a result of redistribution and caspase-dependent cleavage of focal adhesion proteins. Cleavage occurs sequentially such that critical ECM-integrin survival signalling cascades are severed before disruption of focal adhesion-cytoskeletal links.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Calpain; Caspase Inhibitors; Caspases; Cell Line; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Dipeptides; Extracellular Matrix; Focal Adhesions; Neurons; Paxillin; Phosphoproteins; Proteins; Retinoblastoma-Like Protein p130; Talin

Immunofluorescence microscopy revealed the rearrangement and gradual dissociation of paxillin from focal adhesion sites during apoptosis. In vitro, cleavage of paxillin by caspase-3 generated a 42-kDa fragment, among other products, while cleavage by calpain generated a different set of fragments. In Rat-1 cells, cleavage of paxillin by caspase-3 was suppressed by zVAD-fmk or zDEVD-cmk, making caspase-3 a likely executioner during etoposide-induced apoptosis. In contrast, the cleavage of paxillin and p130cas in apoptotic L929 cells was blocked by calpain-specific inhibitors, which also reduced the death rate by 23 to 44%. Therefore, The disassembly and degradation of p130cas and paxillin during apoptosis may controlled by both caspases and calpains, depending upon their cellular contexts. Our findings also suggest that focal adhesion proteins paxillin and p130cas take part in integrin-mediated signaling for cell survival, and that their cleavage by caspase and/or calpain may not only disrupt focal adhesion complexes, but may also impede cell survival signaling.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calpain; Caspase 3; Caspase Inhibitors; Caspases; Cell Line; Crk-Associated Substrate Protein; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Etoposide; Fluorescent Antibody Technique; Focal Adhesions; Oligopeptides; Paxillin; Phosphoproteins; Proteins; Rats; Retinoblastoma-Like Protein p130; Tumor Cells, Cultured

Ethanol is known to induce apoptosis in hepatocytes. However, intracellular signaling events of ethanol-induced death are still only partially understood. We studied such processes in ethanol-induced apoptosis in HepG2 cells as a model system for human liver cells. We determined the incidence of apoptosis by DNA fragmentation and tested the effects of various known inhibitors. Ethanol induces apoptosis in HepG2 cells in a dose- and time-dependent manner as well as in rat primary hepatocytes. This effect was not mediated through the death receptor CD95 and the tumor necrosis factor receptors. It was efficiently inhibited by the caspase inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zVAD-fmk), the Ca(2+) chelator EGTA, and the serine protease inhibitor N-p-tosyl-l-lysine chloromethyl ketone (TLCK). Upon ethanol treatment, the intracellular calcium ion concentration was increased and cytochrome c was released from the mitochondria, and caspases were activated. EGTA and TLCK could inhibit cytochrome c release from the mitochondria. Furthermore, overexpression of Bcl-x(L) saved cells from ethanol-induced apoptosis. These data suggest that ethanol-induced apoptosis in liver cells is initiated by the intracellular Ca(2+) elevation in the cytoplasm and activation of TLCK-sensitive serine proteases. Our data provide new insight into ethanol-induced apoptosis in liver cells and may lead to therapeutic strategies to prevent liver damage.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-X Protein; Blotting, Western; Calcium; Calmodulin; Calpain; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Caspases; Cell Separation; Cytochrome c Group; Densitometry; Dose-Response Relationship, Drug; Egtazic Acid; Endopeptidases; Enzyme Activation; Ethanol; Fas Ligand Protein; fas Receptor; Flow Cytometry; Humans; Ions; Liver Neoplasms; Membrane Glycoproteins; Membrane Potentials; Mitochondria; Models, Biological; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Tosyllysine Chloromethyl Ketone; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

We have previously reported the activation of procalpain mu (precursor for low-calcium-requiring calpain) in apoptotic cells using a cleavage-site-directed antibody specific to active calpain [Kikuchi, H. and Imajoh-Ohmi, S. (1995) Cell Death Differ. 2, 195-199]. In this study, calpastatin, the endogenous inhibitor protein for calpain, was cleaved to a 90-kDa polypeptide during apoptosis in human Jurkat T cells. The limited proteolysis of calpastatin preceded the autolytic activation of procalpain. Inhibitors for caspases rescued the cells from apoptosis and simultaneously inhibited the cleavage of calpastatin. The full-length recombinant calpastatin was also cleaved by caspase-3 or caspase-7 at Asp-233 into the same size fragment. Cys-241 was also targeted by these caspases in vitro but not in apoptotic cells. Caspase-digested calpastatin lost its amino-terminal inhibitory unit, and inhibited three moles of calpain per mole. Our findings suggest that caspases trigger the decontrol of calpain activity suppression by degrading calpastatin.

Topics: Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Apoptosis; Aspartic Acid; Calcium-Binding Proteins; Calpain; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; fas Receptor; Humans; Jurkat Cells; Leupeptins; Oligopeptides; Poly(ADP-ribose) Polymerases; Recombinant Proteins; Tumor Necrosis Factor-alpha

The differentiated cells seem to share the ability to induce their own death by the activation of an internally encoded suicide program. When activated, this suicide program initiates a characteristic form of cell death called apoptosis. A central challenge in apoptosis research is understanding the mechanisms by which apoptotic cascades are initiated and affected. We tested a potential role for calpain in the programmed cell death under ischemic conditions and found that calpain is (1) activated at a time preceding morphological changes, DNA fragmentation and death, (2) that calpain is translocated to the nucleus before DNA laddering, (3) pretreatment with caspase inhibitors and/or calpain inhibitors block not only the proteolytic actions of the enzyme, but also the cell death process itself in the CA1 subfield after transient global ischemia in a synergistic manner. In conclusion, the present results contribute additional evidence that proteases may play a functional role in apoptotic cell death and extend them to include the possibility that endogenous proteases are capable of inducing the striking DNA fragmentation and chromatin condensation, which are the principle criteria currently used to define apoptotic death. Moreover, the synergistic effect of caspase and calpain inhibitors in protecting neurons form ischemic damage suggests that there is a cross-talk between caspase and calpain during apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Benzenesulfonates; Brain Ischemia; Calpain; Caspase Inhibitors; Caspases; Coloring Agents; Cysteine Proteinase Inhibitors; Disease Models, Animal; DNA Fragmentation; Drug Combinations; Drug Synergism; Glycoproteins; Hippocampus; Immunohistochemistry; In Situ Nick-End Labeling; Male; Neurons; Neuroprotective Agents; Oxazines; Rats; Rats, Wistar; Reperfusion Injury

Inhibitors of proteases are currently emerging as a potential anti-cancer modality. Nonselective protease inhibitors are cytotoxic to leukemia and cancer cell lines and we found that this cytotoxicity is correlated with their potency as inhibitors of the proteasome but not as inhibitors of calpain and cathepsin. Highly selective inhibitors of the proteasome were more cytotoxic and fast-acting than less selective inhibitors (PS341>>ALLN>>ALLM). Induction of wt p53 correlated with inhibition of the proteasome and antiproliferative effect in MCF7, a breast cancer cell line, which was resistant to apoptosis caused by proteasome inhibitors. In contrast, inhibitors of the proteasome induced apoptosis in four leukemia cell lines lacking wt p53. The order of sensitivity of leukemia cells was: Jurkat>HL60> or =U937>>K562. The highly selective proteasome inhibitor PS-341 induced cell death with an IC50 as low as 5 nM in apoptosis-prone leukemia cells. Cell death was preceded by p21WAF1/CIP1 accumulation, an alternative marker of proteasome inhibition, and by cleavage of PARP and Rb proteins and nuclear fragmentation. Inhibition of caspases abrogated PARP cleavage and nuclear fragmentation and delayed, but did not completely prevent cell death caused by PS-341. Reintroduction of wt p53 into p53-null PC3 prostate carcinoma cells did not increase their sensitivity to proteasome inhibitors. Likewise, comparison of parental and p21-deficient cells demonstrated that p21WAF1/CIP1 was dispensable for proteasome inhibitor-induced cytotoxicity. We conclude that accumulation of wt p53 and induction of apoptosis are independent markers of proteasome inhibition.

Topics: Acetylcysteine; Acrylates; Amino Acid Chloromethyl Ketones; Apoptosis; Boronic Acids; Bortezomib; Calpain; Cathepsins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Drug Synergism; Genes, p53; Humans; Jurkat Cells; Leupeptins; Multienzyme Complexes; Neoplasm Proteins; Neoplasms; Oligopeptides; Protease Inhibitors; Proteasome Endopeptidase Complex; Pyrazines; Tumor Cells, Cultured; Tumor Suppressor Protein p53; U937 Cells

Apoptosis induced by cadmium has been shown in many tissues in vivo and in cultured cells in vitro. However, its molecular mechanism is not fully understood. When the human histiocytic lymphoma cell line U937 was treated with cadmium for 12 h, evidence of apoptotic features, including change in nuclear morphology, DNA fragmentation, formation of DNA ladder in agarose gel electrophoresis, and phosphatidylserine externalization, were obtained. Moreover, loss of the mitochondrial membrane potential (Deltapsi(m)) was observed in the cadmium-treated cells and was inhibited by a broad caspase inhibitor (Z-VAD-FMK). Caspase inhibitors suppressed the DNA fragmentation in the order of Z-VAD-FMK > caspase-8 inhibitor > caspase-3 inhibitor. Expression of Bcl-x(L) and Bid decreased significantly in the cadmium-treated cells, although no apparent change in Bcl-2 and Bax expression was found. Tetrakis-(2-pyridylmethyl) ethylendiamine, a cell-permeable heavy metal chelator, partially reversed the increase of fluorescence of Fura-2 in the cadmium-treated cells. In addition, verapamil (70 microm), a voltage-dependent Ca(2+) channel blocker, inhibited the DNA fragmentation induced by cadmium less than 100 microm and decreased the fluorescence of Fura-2. Cadmium up-regulated the expression of type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R) but not type 2 or type 3 IP(3)R. Calpain inhibitors I and II partially prevented DNA fragmentation. No effects of Z-VAD-FMK on the expression of type 1 IP(3)R or of calpain inhibitors on the loss of Deltapsi(m) were observed. These results suggest that cadmium possibly induced apoptosis in U937 cells through two independent pathways, the Ca(2+)-calpain-dependent pathway and the caspase-mitochondria-dependent pathway.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Cadmium; Calcium Channel Blockers; Calcium Channels; Calpain; Carrier Proteins; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Nucleus; Chelating Agents; DNA Fragmentation; Dose-Response Relationship, Drug; Ethylenediamines; Humans; Inositol 1,4,5-Trisphosphate Receptors; Lymphoma; Membrane Potentials; Mitochondria; Models, Biological; Phosphatidylserines; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; Time Factors; U937 Cells; Up-Regulation; Verapamil

There are at least three types of inositol 1,4,5-trisphosphate receptor (IP(3)R) [IP(3)-gated Ca(2+)channels], which are expressed in different cell types and mammalian tissues. In this study, we have identified three IP(3)R subtypes in human Jurkat T-lymphoma cells. All three subtypes have a molecular mass of about 260 kDa, and display Ca(2+)channel properties in an IP(3)-dependent manner. We have also demonstrated that TNFalpha promotes the activity of different proteases (e.g. caspase-8, caspase-3 and calpain), alters the TCR-mediated Ca(2+)response and subsequently induces apoptosis in Jurkat cells. During the first 6 h of incubation with TNFalpha, several IP(3)R subtype-related changes occur (e.g. proteolysis of IP(3)R subtypes, inhibition of IP(3)binding and impairment of IP(3)-mediated Ca(2+)flux) concomitantly with an elevation of protease (caspase-8, caspase-3 and calpain) activity. Furthermore, the caspase inhibitor, Z-VAD-fmk, significantly reduces TNFalpha-mediated perturbation of IP(3)R1 and IP(3)R2 (but not IP(3)R3) function; whereas the calpain inhibitor I, ALLN, is capable of blocking the inhibitory effect of TNFalpha on IP(3)R3 function. These findings suggest that IP(3)R1 and IP(3)R2 serve as cellular substrates for caspases, and IP(3)R3 is a substrate for calpain. We propose that the selective down-regulation of IP(3)R subtype-mediated Ca(2+)function by caspase-dependent and calpain-sensitive mechanisms may be responsible for the early onset of the apoptotic signal by TNFalpha in human T-cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Binding Sites; Calcium; Calcium Channels; Calpain; Caspase Inhibitors; Caspases; Down-Regulation; Glycoproteins; Humans; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Jurkat Cells; Lymphoma, T-Cell; Microscopy, Confocal; Receptors, Cytoplasmic and Nuclear; Tumor Necrosis Factor-alpha

Cerebellar granule cells (CGC) have provided a reliable model for studying the toxicity of methylmercury (MeHg), a well-known neurotoxicant contaminating the environment. In the present study we report that doses of MeHg ranging from 0.1 microM to 1.5 microM activated apoptosis, as shown by cell shrinkage, nuclear condensation, and formation of high-molecular-weight DNA fragments. Nevertheless, caspase-3-like activity was not significantly induced, and the broad caspase inhibitor Z-VAD-FMK was not capable of protecting the cells. This argues for a minor role of caspases in the intracellular pathways leading to MeHg-induced cell death in CGC. Instead, proteolytic fragments obtained by specific calpain cleavage of procaspase-3 and alpha-fodrin were increased consistently in samples exposed to MeHg, pointing to a substantial activation of calpain. Notably, two antioxidants, 17beta-estradiol (10 microM) and the Delta(8,9)-dehydro derivative of 17alpha-estradiol J811 (10 microM), protected from MeHg damage, preventing morphological alterations, chromatin fragmentation, and activation of calpain. These findings underscore the key role of oxidative stress in MeHg toxicity, placing it upstream of calpain activation. The shielding effect of the 17beta-estradiol and the radical scavenger J811 is potentially relevant for the development of therapeutic strategies for MeHg intoxication.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Antioxidants; Apoptosis; Calpain; Caspase 3; Caspases; Cell Size; Cells, Cultured; Cerebellum; Drug Interactions; Environmental Exposure; Estradiol; Female; Free Radical Scavengers; Methylmercury Compounds; Neurons; Neuroprotective Agents; Neurotoxins; Pregnancy; Rats; Rats, Sprague-Dawley; Signal Transduction

The porin (PorB) of Neisseria gonorrhoeae is an intriguing bacterial factor owing to its ability to translocate from the outer bacterial membrane into host cell membranes where it modulates the infection process. Here we report on the induction of programmed cell death after prolonged infection of epithelial cells with pathogenic Neisseria species. The underlying mechanism we propose includes translocation of the porin, a transient increase in cytosolic Ca2+ and subsequent activation of the Ca2+ dependent protease calpain as well as proteases of the caspase family. Blocking the porin channel by ATP eliminates the Ca2+ signal and also abolishes its pro-apoptotic function. The neisserial porins share structural and functional homologies with the mitochondrial voltage-dependent anion channels (VDAC). The neisserial porin may be an analogue or precursor of the ancient permeability transition pore, the putative central regulator of apoptosis.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Apoptosis; Bacterial Outer Membrane Proteins; Calcium; Calpain; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; HeLa Cells; Humans; Ion Transport; Mutation; Neisseria gonorrhoeae; Porins

We have previously demonstrated that calpain is responsible for the cleavage of Bax, a proapoptotic protein, during drug-induced apoptosis of HL-60 cells (Wood, D. E., Thomas, A., Devi, L. A., Berman, Y., Beavis, R. C., Reed, J. C., and Newcomb, E. W. (1998) Oncogene 17, 1069-1078). Here we show the sequential activation of caspases and calpain during drug-induced apoptosis of HL-60 cells. Time course experiments using the topoisomerase I inhibitor 9-amino-20(S)-camptothecin revealed that cleavage of caspase-3 substrates poly(ADP-ribose) polymerase (PARP) and the retinoblastoma protein as well as DNA fragmentation occurred several hours before calpain activation and Bax cleavage. Pretreatment with the calpain inhibitor calpeptin blocked calpain activation and Bax cleavage but did not inhibit PARP cleavage, DNA fragmentation, or 9-amino-20(S)-camptothecin-induced morphological changes and cell death. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) inhibited PARP cleavage, DNA fragmentation, calpain activation, and Bax cleavage and increased cell survival by 40%. Interestingly, Z-VAD-fmk-treated cells died in a caspase- and calpain-independent manner that appeared morphologically distinct from apoptosis. Our results suggest that excessive or uncontrolled calpain activity may play a role downstream of and distinct from caspases in the degradation phase of apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Calpain; Caspase 3; Caspases; Coumarins; Cysteine Proteinase Inhibitors; Dipeptides; DNA Fragmentation; Enzyme Activation; HL-60 Cells; Humans; Oligopeptides

The retinoblastoma protein plays a critical role in regulating the G1/S transition. Less is known about the function and regulation of the homologous pocket protein p107. Here we present evidence for the posttranslational regulation of p107 by the Ca2+-activated protease calpain. Three negative growth regulators, the HMG-CoA reductase inhibitor lovastatin, the antimetabolite 5-fluorouracil, and the cyclic nucleotide dibutyryl cAMP were found to induce cell type-specific loss of p107 protein which was reversible by the calpain inhibitor leucyl-leucyl-norleucinal but not by the serine protease inhibitor phenylmethylsulfonylfluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26S proteasome. Purified calpain induced Ca2+-dependent p107 degradation in cell lysates. Transient expression of the specific calpain inhibitor calpastatin blocked the loss of p107 protein in lovastatin-treated cells, and the half-life of p107 was markedly lengthened in lovastatian-treated cells stably transfected with a calpastatin expression vector versus cells transfected with vector alone. The data presented here demonstrate down-regulation of p107 protein in response to various antiproliferative signals, and implicate calpain in p107 posttranslational regulation.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Bucladesine; Calpain; Cyclin B; Cyclin B1; Cysteine Proteinase Inhibitors; Dipeptides; Fluorouracil; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Ketones; Leupeptins; Lovastatin; Nuclear Proteins; Protein Processing, Post-Translational; Retinoblastoma Protein; Retinoblastoma-Like Protein p107; Tumor Cells, Cultured

The caspase-mediated cleavage of a limited number of cellular proteins is a common feature of apoptotic cell death. This cleavage usually inhibits the function of the target protein or generates peptides that actively contribute to the death process. In the present study, we demonstrate that the cyclin-dependent kinase inhibitor p27Kip1 is cleaved by caspases in human leukemic cells exposed to apoptotic stimuli. We have shown recently that p27Kip1 overexpression delayed leukemic cell death in response to cytotoxic drugs. In transient transfection experiments, the p23 and the p15 N-terminal peptides generated by p27Kip1 proteolysis demonstrate an anti-apoptotic effect similar to that induced by the wild-type protein, whereas cleavage-resistant mutants have lost their protective effect. Moreover, stable transfection of a cleavage-resistant mutant of p27Kip1 sensitizes leukemic cells to drug-induced cell death. Altogether, these results indicate that proteolysis of p27Kip1 triggered by caspases mediates the anti-apoptotic activity of the protein.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Base Sequence; Calpain; Caspase 3; Caspase 6; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cysteine Proteinase Inhibitors; Etoposide; Humans; Leukemia; Leupeptins; Microtubule-Associated Proteins; Molecular Sequence Data; Mutation; Nucleic Acid Synthesis Inhibitors; Oligopeptides; Protein Serine-Threonine Kinases; Thimerosal; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

We have previously demonstrated cleavage of alpha-spectrin by caspase-3 and calpain during apoptosis in SH-SY5Y neuroblastoma cells (Nath, R., Raser, K. J., Stafford, D., Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R. V., Yuen, P., Gilbertsen, R. B., and Wang, K. K. (1996) Biochem. J. 319, 683-690). We demonstrate here that calcium/calmodulin-dependent protein kinase IV (CaMK IV) is cleaved during apoptosis by caspase-3 and calpain. We challenged SH-SY5Y cells with the pro-apoptotic agent thapsigargin. Western blot analysis revealed major CaMK IV breakdown products of 40, 38, and 33 kDa. Digestion of control SH-SY5Y lysate with purified caspase-3 produced a 38-kDa CaMK IV fragment; digestion with purified calpain produced a major fragment of 40 kDa. Pretreatment with carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene or Z-Val-Ala-Asp-fluoromethylketone was able to block the caspase-3-mediated production of the 38-kDa fragment both in situ and in vitro. Calpain inhibitor II similarly blocked formation of the calpain-mediated 40-kDa fragment both in situ and in vitro. Digestion of recombinant CaMK IV by other caspase family members revealed that only caspase-3 produces a fragmentation pattern consistent to that seen in situ. The major caspase-3 and calpain cleavage sites are respectively identified as PAPD176*A and CG201*A, both within the CaMK IV catalytic domain. Furthermore, calmodulin-stimulated protein kinase activity decreases within 6 h in thapsigargin-treated SH-SY5Y. The loss of activity precedes cell death.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Aspartic Acid; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Calcium-Calmodulin-Dependent Protein Kinases; Calpain; Caspase 3; Caspases; Chlorobenzoates; Cysteine Endopeptidases; Enzyme Inhibitors; Humans; Mice; Neuroblastoma; Peptide Fragments; Recombinant Proteins; Staurosporine; Thapsigargin; Tumor Cells, Cultured

Chromatin degradation into oligonucleosomal and approximately 30-50 Kb fragments is a hallmark of apoptosis. Crude nuclear extract from apoptotic rat thymocytes is able to recapitulate both types of DNA fragmentation in an assay using HeLa cell nuclei as an exogenous substrate. Using size exclusion chromatography we have identified a novel activity (approximately 260 Kd) that produces only approximately 30-50 Kb DNA fragments, and a 25 Kd activity that generates both approximately 30-50 Kb and oligonucleosomal fragments. Both activities produced DNA fragments with 3'-OH termini, are dependent on Ca2+ and Mg2+ and are inhibited by N-ethyl-maleimide, sodium tetrathionate, aurintricarboxylic acid and sodium chloride, similar to other nucleases implicated in apoptosis. These activities were inhibited by the serine protease inhibitors N-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone, but not by the serine protease inhibitor diisopropyl fluorophosphate, or by calpain inhibitors I or II, or the capsase inhibitors Ac-Asp-Glu-Val-Asp-aldehyde, Ac-Tyr-Val-Ala-Asp-aldehyde, or Z-Val-Ala-Asp-fluoromethyl ketone. Both activities were insensitive to protease inhibitors when extracts were incubated with naked linear DNA, indicating the presence of both nuclease and protease activities in the preparation. Together, these observations suggest the involvement of non-caspase proteases in apoptosis which perhaps function by altering chromatin substructure and exposing it to nucleolytic attack.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calcium; Calpain; Caspase 1; Caspase 3; Caspase Inhibitors; Caspases; Cell Nucleolus; Chromatin; Cysteine Proteinase Inhibitors; DNA Fragmentation; Electrophoresis, Gel, Pulsed-Field; Endonucleases; HeLa Cells; Humans; Isoflurophate; Magnesium; Male; Oligopeptides; Peptide Fragments; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Serine Endopeptidases; Serine Proteinase Inhibitors; Thymus Gland; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone

The molecular events involved in apoptosis induced by ionizing radiation remain unresolved. In this paper we show that the cleavage of fodrin to a 150 kDa fragment is an early proteolytic event in radiation-induced apoptosis in the Burkitts' Lymphoma cell line BL30A and requires 100 microM zVAD-fmk for inhibition. Caspases-1, -3, -6 and -7 were shown to cleave fodrin to the 150 kDa fragment in vitro and all were inhibited by 10 microM zVAD-fmk. We also show that the in vitro cleavage of fodrin by calpain is inhibited by 100 microM zVAD-fmk as was the calpain-mediated hydrolysis of casein. We demonstrate that calpain is activated within 15 min after radiation exposure, concomitant with the cleavage of fodrin to the 150 kDa fragment whereas caspase-3 is activated at 2 h correlating with the cleavage of fodrin to the 120 kDa fragment. These results support a role for calpain in the early phases of the radiation-induced apoptosis pathway, upstream of the caspases.

Topics: Amino Acid Chloromethyl Ketones; Amino Acid Sequence; Apoptosis; Burkitt Lymphoma; Calpain; Carrier Proteins; Caspase 1; Caspase 3; Caspase 6; Caspase 7; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Enzyme Activation; fas Receptor; Humans; Membrane Proteins; Microfilament Proteins; Molecular Sequence Data; Peptide Fragments; Tumor Cells, Cultured

Proteases play an important role in the programme of cell death by apoptosis but little is known of the substrates cleaved, particularly in constitutive models of this type of cell death. Neutrophils spontaneously undergo apoptosis in culture without requiring external stimuli. During this process we found biochemical and immunochemical evidence for the cleavage of membrane-associated actin, a component of the cytoskeleton that links polymerized actin to the plasma membrane. Cleavage occurred at a single site at the N-terminus, between residues Val43-Met44, a site devoid of a consensus motif for cleavage by cysteine proteases of the interleukin-1beta-converting enzyme (ICE)-family. Whereas actin cleavage and nuclear/cell surface markers of apoptosis were co-ordinately diminished by zVAD-fmk, an inhibitor of the ICE-like family of proteases, only acetyl-leucyl-leucylnormethional, an inhibitor of calpains, was capable of completely inhibiting actin cleavage. Our results suggest that actin is not a direct substrate for the ICE-like family of proteases. By disabling the cytoskeleton, actin cleavage may be an important component in the capacity of apoptosis to reduce the injurious potential of neutrophils.

Topics: Actins; Amino Acid Chloromethyl Ketones; Amino Acid Sequence; Apoptosis; Calpain; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Flow Cytometry; Humans; Leupeptins; Molecular Sequence Data; Neutrophils; Protease Inhibitors