calpain and Neuroblastoma

Neovascular inflammatory vitreoretinopathy (NIV) is a rare eye disease that ultimately leads to complete blindness and is caused by mutations in the gene encoding calpain-5 (CAPN5), with six pathogenic mutations identified. In transfected SH-SY5Y cells, five of the mutations resulted in decreased membrane association, diminished S-acylation, and reduced calcium-induced autoproteolysis of CAPN5. CAPN5 proteolysis of the autoimmune regulator AIRE was impacted by several NIV mutations. R243, L244, K250 and the adjacent V249 are on β-strands in the protease core 2 domain. Conformational changes induced by Ca

Topics: Calpain; Humans; Mutation; Neuroblastoma; Vitreoretinopathy, Proliferative

Undecylenic acid (UDA), a naturally occurring 11-carbon unsaturated fatty acid, has been used for several years as an economical antifungal agent and a nutritional supplement. Recently, the potential usefulness of UDA as a neuroprotective drug has been suggested based on the ability of this agent to inhibit μ-calpain activity. In order to verify neuroprotective potential of UDA, we tested protective efficacy of this compound against cell damage evoked by pro-apoptotic factors (staurosporine and doxorubicin) and oxidative stress (hydrogen peroxide) in human neuroblastoma SH-SY5Y cells. We showed that UDA partially protected SH-SY5Y cells against the staurosporine- and doxorubicin-evoked cell death; however, this effect was not connected with its influence on caspase-3 activity. UDA decreased the St-induced changes in mitochondrial and cytosolic AIF level, whereas in Dox-model it affected only the cytosolic AIF content. Moreover, UDA (1-40 μM) decreased the hydrogen peroxide-induced cell damage which was connected with attenuation of hydrogen peroxide-mediated necrotic (PI staining, ADP/ATP ratio) and apoptotic (mitochondrial membrane potential, caspase-3 activation, AIF translocation) changes. Finally, we demonstrated that an inhibitor of PI3-K/Akt (LY294002) but not MAPK/ERK1/2 (U0126) pathway blocked the protection mediated by UDA in all tested models of SH-SY5Y cell injury. These in vitro data point to UDA as potentially effective neuroprotectant the utility of which should be further validated in animal studies.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Calpain; Cell Line, Tumor; Chromones; Doxorubicin; Humans; Morpholines; Neuroblastoma; Neurons; Neuroprotective Agents; Oxidative Stress; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Staurosporine; Undecylenic Acids

Endoplasmic reticulum (ER) stress is involved in neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Therefore, interventions that attenuate ER stress may contribute to induction in apoptotic cell death. This study aimed to evaluate the potential involvement of O-demethyldemethoxycurcumin, an analog of curcuminoids, on thapsigargin-induced apoptosis in cultured neuroblastoma (SK-N-SH) cells through the ER stress signaling pathway. The results showed that O-demethyldemethoxycurcumin reduced thapsigargin induced cell death in SK-N-SH cells and the release of lactate dehydrogenase (LDH) by decreasing the apoptotic cell death induced by thapsigargin. Consistent with these findings, O-demethyldemethoxycurcumin inhibited the thapsigargin-induced activation of cleavagecaspase-12. Moreover, O-demethyldemethoxycurcumin attenuated the intracellular Ca(2+) level and the expression of the calpain protein. O-demethyldemethoxycurcumin also downregulated the expression of ER stress signaling proteins, including the phosphorylation of PKR-like endoplasmic reticulum kinase (p-PERK), the phosphorylation of inositol-requiring enzyme 1 (p-IRE1), activating transcription factor 6 (ATF6), binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP). Our findings suggest that O-demethyldemethoxycurcumin could protect against thapsigargin-induced ER stress in SK-N-SH cells.

Topics: Activating Transcription Factor 6; Annexin A5; Apoptosis; Calcium; Calpain; Cell Line, Tumor; Cell Survival; Curcumin; Diarylheptanoids; Dose-Response Relationship, Drug; eIF-2 Kinase; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Extracellular Fluid; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; L-Lactate Dehydrogenase; Neuroblastoma; Thapsigargin; Time Factors; Transcription Factor CHOP

Insulin-like growth factor-1 (IGF-1) is a neuroprotective growth factor that promotes neuronal survival by inhibition of apoptosis. To examine whether IGF-1 exerts cytoprotective effects against extracellular inflammatory stimulation, ventral spinal cord 4.1 (VSC4.1) motoneuron cells were treated with interferon-gamma (IFN-γ). Our data demonstrated apoptotic changes, increased calpain:calpastatin and Bax:Bcl-2 ratios, and expression of apoptosis-related proteases (caspase-3 and -12) in motoneurons rendered by IFN-γ in a dose-dependent manner. Post-treatment with IGF-1 attenuated these changes. In addition, IGF-1 treatment of motoneurons exposed to IFN-γ decreased expression of inflammatory markers (cyclooxygenase-2 and nuclear factor-kappa B:inhibitor of kappa B ratio). Furthermore, IGF-1 attenuated the loss of expression of IGF-1 receptors (IGF-1Rα and IGF-1Rβ) and estrogen receptors (ERα and ERβ) induced by IFN-γ. To determine whether the protective effects of IGF-1 are associated with ERs, ERs antagonist ICI and selective siRNA targeted against ERα and ERβ were used in VSC4.1 motoneurons. Distinctive morphological changes were observed following siRNA knockdown of ERα and ERβ. In particular, apoptotic cell death assessed by TUNEL assay was enhanced in both ERα and ERβ-silenced VSC4.1 motoneurons following IFN-γ and IGF-1 exposure. These results suggest that IGF-1 protects motoneurons from inflammatory insult by a mechanism involving pivotal interactions with ERα and ERβ.

Topics: Animals; Apoptosis; Calpain; Cell Line; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Estrogen Receptor alpha; Estrogen Receptor beta; Insulin-Like Growth Factor I; Interferon-gamma; Mice; Motor Neurons; Neuroblastoma; Neuroprotective Agents; Rats; Receptor Cross-Talk; RNA, Small Interfering; Spinal Cord

Anesthetic treatment has been associated with widespread apoptotic neurodegeneration in the neonatal rodent brain. It has recently been suggested that propofol, a short-acting intravenous anesthetic agent, may have a potential as a neuroprotective agent. An apoptotic pathway mediated through endoplasmic reticulum (ER) stress has been attracting attention. ER stress is associated with accumulation of unfolded or misfolded proteins in ER, and ER stress-induced apoptosis is implicated in a wide range of diseases, including ischemia/reperfusion injury, neurodegeneration, and diabetes. We investigated whether thapsigargin-induced ER stress is prevented by propofol in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were pretreated with various concentrations of propofol (1-10 μM) for 3h before co-treatment with 0.5 μM thapsigargin and propofol for 20 h. Levels of ssDNA, specific evidence of apoptosis, and biomarkers of ER stress (mRNA expression of Chop and sXbp-1) were determined. We also assayed calpain and caspase-4 activities and intracellular Ca(2+) ([Ca(2+)]i) levels. Thapsigargin-induced increases in ssDNA levels, expressions of ER stress biomarkers, activities of caspase-4 and calpain, and level of [Ca(2+)]i were suppressed by co-incubation with propofol. Our data indicate the possibility that propofol inhibits the Ca(2+) release from ER at clinically employed dose levels. These results demonstrate that propofol suppresses the ER stress-induced apoptosis in this cell system, and may have the neuroprotective potency. It may also be a promising agent for preventing damage from cerebral ischemia or edema.

Topics: Apoptosis; Calcium; Calpain; Caspases, Initiator; Cell Line, Tumor; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Gene Expression Regulation; Humans; Intracellular Space; Neuroblastoma; Neuroprotective Agents; Phosphorylation; Propofol

Kallikrein-6 and calpain-1 are amongst a small group of proteases that degrade α-synuclein. We have explored the possibility that reduction in the level or activity of these enzymes contributes to the accumulation of α-synuclein in Lewy body diseases. We measured calpain-1 activity by fluorogenic activity assay, kallikrein-6 level by sandwich ELISA, and levels of α-synuclein and α-synuclein phosphorylated at serine 129 (α-synuclein-P129), in post-mortem brain tissue in pure dementia with Lewy bodies (DLB, n=12), Alzheimer's disease (AD, n=20) and age-matched controls (n=19). Calpain-1 activity was significantly reduced in DLB within the cingulate and parahippocampal cortex, regions with highest α-synuclein and α-synuclein-P129 load, and correlated inversely with the levels of α-synuclein and α-synuclein-P129. Calpain-1 was unaltered in the thalamus and frontal cortex, regions with less α-synuclein pathology. Kallikrein-6 level was reduced in the cingulate cortex in the DLB cohort, and correlated inversely with α-synuclein and α-synuclein-P129. Kallikrein-6 was also reduced in DLB in the thalamus but not in relation to α-synuclein or α-synuclein-P129 load and was unaltered in the frontal and parahippocampal cortex. In SH-SY5Y cells overexpressing wild-type α-synuclein there was partial co-localisation of kallikrein-6 and calpain-1 with α-synuclein, and siRNA-mediated knock-down of kallikrein-6 and calpain-1 increased the amount of α-synuclein in cell lysates. Our results indicate that reductions in kallikrein-6 and calpain-1 may contribute to the accumulation of α-synuclein in DLB.

Topics: alpha-Synuclein; Alzheimer Disease; Brain; Calpain; Case-Control Studies; Cell Line, Tumor; Child; Child, Preschool; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation; Humans; Kallikreins; Lewy Body Disease; Male; Neuroblastoma; Phosphorylation; RNA, Small Interfering; Serine

Glucocorticoids (GCs) have a significant role in the adaptive response of the brain to stress. Increasing evidence has demonstrated that an increase of GC levels may induce neuronal cell death via apoptotic pathways. There is a correlation between over-production of reactive oxygen species (ROS) and an elevation in cytosolic calcium that causes a subsequent increase in the calcium-dependent death-process activation in GC-induced toxicity. Consequently, melatonin, via its antioxidant activity, exhibits a neuroprotective effect against apoptosis induced by intracellular calcium overload. Therefore, in the present study, we explored the protective effect of melatonin in GC-induced toxicity in dopaminergic SH-SY5Y cells. Cellular treatment with the synthetic GCs, dexamethasone (DEX), resulted in a marked decrease in cell viability and in the level of the calpain-inhibitor protein, calpastatin. DEX-induced toxicity also caused an increase in ROS production and the activation of the calcium-dependent cysteine protease, calpain, along with an increase in caspase-3 activation. Pretreatment of the cells with melatonin substantially prevented the decrease in cell viability, over-production of ROS and the activation of calpain and caspase-3, and reversed the depletion in calpastatin levels. These results suggest that melatonin may exert its protective effects against the calpain- and caspase-dependent death process in DEX-induced neurotoxicity.

Topics: Blotting, Western; Calpain; Caspases; Cell Line, Tumor; Cell Survival; Dexamethasone; Humans; Melatonin; Neuroblastoma; Oxidative Stress; Reactive Oxygen Species

Calpain (intracellular Ca(2+)-dependent protease) and calpastatin (calpain specific endogenous inhibitor) are widely distributed in biological systems, and have been implicated in many cellular physiological and pathological processes. Calpastatin level is of central importance to the control of calpain activity. We demonstrated for the first time that calpastatin is overexpressed in mycoplasma-contaminated cultured cells (SH-SY5Y cells that are infected by a strain of Mycoplasma hyorhinis (NDMh)). We have found that the calpastatin-upregulating activity resides in the mycoplasmal membrane lipoproteins, and is associated with NF-κB activation. Calpain-promoted proteolysis is attenuated in the NDMh lipoprotein-treated cells. Here we show that the NDMh lipoproteins promoted an increase in calpastatin in SH-SY5Y cells via the TLR2/TAK1/NF-κB pathway. The synthetic mycoplasmal lipopeptide MALP-2 and the bacterial lipopeptide PAM3CSK4 (TLR2 agonists) also promoted calpastatin upregulation. LPS (TLR4 agonist) activated NF-κB without calpastatin increase in the cell. In contrast, lipoteichoic acid (TLR2 agonist) upregulated calpastatin not via NF-κB activation, but via the MEK1/ELK1 pathway. Zymosan and peptidoglycan, TLR2 agonists that lack lipids, did not induce calpastatin upregulation. Cell treatment with a calpastatin-upregulating agonist (lipoteichoic acid) led to the attenuation of Ca(2+)-promoted calpain activity, whereas agonists that do not upregulate calpastatin (LPS, Zymosan) were ineffective. Overall, the results indicate that in these non-immune cells, calpastatin is upregulated by TLR2-agonists containing lipids, with more than one downstream pathway involved. Such agonists may be useful for studying mechanisms and factors involved in calpastatin regulation. In addition, suitable TLR2 agonists may be of interest in devising treatments for pathological processes involving excessive calpain activation.

Topics: Calcium-Binding Proteins; Calpain; ets-Domain Protein Elk-1; Humans; Immunoblotting; Lipopeptides; Lipopolysaccharides; Lipoproteins; MAP Kinase Kinase 1; Mycoplasma hyorhinis; Neuroblastoma; NF-kappa B; Proteolysis; Teichoic Acids; Toll-Like Receptor 2; Tumor Cells, Cultured; Up-Regulation

Calpains are involved in calcium-induced neuronal cell toxicity, which is associated with the pathophysiology of Alzheimer's disease (AD). The activity of calpains is regulated by the inhibitor calpastatin, and increased activity of calpains and decreased calpastastin are often found in AD. Histone deacetylase (HDAC) inhibitors are implicated in AD treatment through the improvement of learning and memory but the underlying mechanism is yet to be understood. Here, using SH-SY5Y neuroblastoma cells and a calcium ionophore ionomycin, we examined whether and how HDAC inhibitor trichostatin A (TSA) inhibits calcium-induced neuronal cell death. TSA increased both the mRNA and protein levels of calpastatin, with no alterations in those of calpain 1 and calpain 2. Furthermore, TSA-stimulated increase of calpastatin was accompanied by a significant attenuation of ionomycin-induced autolysis of calpain 1, but not of calpain 2, and calpain-dependent 150 kDa αII spectrin cleavage. Under these conditions, however, caspase activity was unaltered. Moreover, ectopic expression of small interfering RNA of calpastatin reversed the inhibitory effect of TSA on ionomycin-induced calpain 1 autolysis and αII spectrin cleavage. Chromatin immunoprecipitation assay revealed the increased levels of acetylation at lysine 5 of histone H4 (H4K5-Ac), H3K9-Ac and H3K14-Ac within the calpastatin promoter region in TSA-treated cells relative to control cells. Finally, TSA significantly decreased ionomycin-induced cell toxicity. This study demonstrates that TSA attenuates calcium-induced neuronal cell death by the inhibition of calpain activity which is mediated in part by increased calpastatin expression via histone hyperacetylation within the calpastatin promoter region. Our study provides a novel mechanism for the neuroprotective effect of HDAC inhibitors on AD.

Topics: Acetylation; Apoptosis; Blotting, Western; Calcium; Calcium-Binding Proteins; Calpain; Cell Proliferation; Chromatin Immunoprecipitation; Epigenomics; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Ionomycin; Luciferases; Neuroblastoma; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured

Peroxiredoxin-5 (PRDX5) is an antioxidant enzyme which differs from the other peroxiredoxins with regards to its enzymatic mechanism, its high affinity for organic peroxides and peroxynitrite and its wide subcellular distribution. In particular, the mitochondrial isoform of PRDX5 confers a remarkable cytoprotection toward oxidative stress to mammalian cells. Mitochondrial dysfunction and disruption of Ca²⁺ homeostasis are implicated in neurodegeneration. Growing evidence supports that endoplasmic reticulum (ER) could operate in tandem with mitochondria to regulate intracellular Ca²⁺ fluxes in neurodegenerative processes. Here, we overexpressed mitochondrial PRDX5 in SH-SY5Y cells to dissect the role of this enzyme in 1-methyl-4-phenylpyridinium (MPP)⁺-induced cell death. Our data show that mitochondria-dependent apoptosis triggered by MPP⁺, assessed by the measurement of caspase-9 activation and mitochondrial DNA damage, is prevented by mitochondrial PRDX5 overexpression. Moreover, PRDX5 overexpression blocks the increase in intracellular Ca²⁺, Ca²⁺-dependent activation of calpains and Bax cleavage. Finally, using Ca²⁺ channel inhibitors (Nimodipine, Dantrolene and 2-APB), we show that Ca²⁺ release arises essentially from ER stores through 1,4,5-inositol-trisphosphate receptors (IP3 R). Altogether, our results suggest that the MPP⁺ mitochondrial pathway of apoptosis is regulated by mitochondrial PRDX5 in a process that could involve redox modulation of Ca²⁺ transporters via a crosstalk between mitochondria and ER.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Adenosine Triphosphate; Animals; Apoptosis; Boron Compounds; Calcium; Calpain; Caspase 3; Caspase 9; Cell Line, Tumor; DNA, Mitochondrial; Dopamine Agents; Endoplasmic Reticulum; Enzyme Inhibitors; Gene Expression Regulation; Humans; Hydro-Lyases; Mice; Mitochondria; Neuroblastoma; Peroxiredoxins; Reactive Oxygen Species; RNA, Small Interfering; Subcellular Fractions; Transfection; Tyrosine

Methamphetamine (METH) is an abused psychostimulant drug that can cause neurotoxicity to dopaminergic cells. It has been demonstrated that METH can induce caspase- and calpain-dependent death cascades. The purpose of the present study was to investigate the functional role of calpastatin, a specific endogenous calpain inhibitor protein, on caspase and calpain activation in METH-induced degeneration in neuroblastoma SH-SY5Y cell cultures. In this study, we found that METH significantly decreased cell viability, tyrosine hydroxylase phosphorylation and calpastatin levels. Supplementation of cells with exogenous calpastatin was able to reverse the toxic effect of METH on reduction in cell viability and tyrosine hydroxylase phosphorylation. METH also significantly increased calpain levels, the formation of calpain-specific breakdown products and cleaved caspase-3 levels; once again, these effects were diminished by pretreating the cells with calpastatin. These data suggest the contribution of calpastatin as a potential regulatory factor for calpain- and caspase-dependent death processes.

Topics: Calcium-Binding Proteins; Calpain; Caspase 3; Caspases; Cell Line, Tumor; Cell Survival; Central Nervous System Stimulants; Enzyme Activation; Humans; Methamphetamine; Neuroblastoma; Neuroprotective Agents; Phosphorylation; Tyrosine 3-Monooxygenase

Time-lapse imaging analysis was previously used to show that spontaneous proteolysis of PrP(C), which is fluorescence-labeled at both NH(2)- and COOH-termini, occurred in mouse neuroblastoma neuro2a (N2a) cells susceptible to PrP(Sc). We demonstrated that, unlike other protease inhibitors, a calpain inhibitor, calpastatin, drastically inhibited endoproteolysis of PrP(C), as observed with time-lapse imaging in living cells, suggesting calpain-like activity. Calpastatin also inhibited cleavage of endogenous PrP(C), and unprocessed molecules and the double-labeled PrP(C) accumulated around the perinuclear region. The molecular weight of PrP(C) fragments generated by spontaneous proteolysis was identical to those produced when PrP(C) synthesized in vitro was exposed to exogenous calpain. These results suggest that a calpain-like activity mediates normal processing of PrP(C) in N2a cells.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Enzyme Activation; Mice; Neuroblastoma; Peptide Hydrolases; Protein Processing, Post-Translational; PrPC Proteins

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

Valproate (VPA) is a widely used anticonvulsant and mood-stabilizing drug. Recent studies have shown that VPA could reduce amyloid-β generation, and improve memory deficits in transgenic mouse models of Alzheimer's disease (AD). However, whether VPA affects tau phosphorylation and the underlying mechanism has not been established. Here, we showed that systemic treatment of APP and presenilin 1 double transgenic mice with VPA (50mg/kg, once a day for 12 weeks), significantly reduced the levels of tau phosphorylation at the sites of Thr205, Ser396 and Thr231. Meanwhile, VPA treatment markedly reduced the activities of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3β (GSK3β), two protein kinases involved in abnormal hyperphosphorylation of tau. In an okadaic acid-induced tau hyperphosphorylation SH-SY5Y cell model, the anti-tau-phosphorylation effect of VPA was further confirmed, accompanied by a marked decrease in the activities of CDK5 and GSK3β. Our present data suggest that the inhibitory effects of VPA on tau hyperphosphorylation might be mediated through both CDK5 and GSK3β signaling pathways.

Topics: Amyloid beta-Protein Precursor; Animals; Brain; Calpain; Cell Line, Tumor; Cyclin-Dependent Kinase 5; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Humans; Mice; Mice, Transgenic; Neuroblastoma; Phosphorylation; Presenilin-1; Signal Transduction; tau Proteins; Threonine; Valproic Acid

Paclitaxel (Taxol) is one of the most effective treatment options for patients suffering from a variety of cancers. A major side effect seen in a high percentage of patients treated with paclitaxel is irreversible peripheral neuropathy. We previously reported that prolonged treatment with paclitaxel activates a calcium-dependent enzyme, calpain, which degrades neuronal calcium sensor 1 (NCS-1) and subsequent loss of intracellular calcium signaling. Because it appears that activation of calpain is an early step in this destructive cascade, we proposed that inhibition of calpain will protect against the unwanted side effects of paclitaxel treatment. First, NCS-1 levels and intracellular calcium signaling were found to be protected by the presence of lactacystin, a protesome inhibitor. To reinforce the role of calpain in this process, we showed that increased concentrations of calpastatin, a naturally occurring calpain inhibitor, were protective. Next, we tested two mutated versions of NCS-1 developed with point mutations at the P2 position of the calpain cleavage site of NCS-1 to decrease the likelihood of NCS-1 degradation. One mutant was cleaved more favorably by calpain compared with NCS-1 WT, whereas the other mutant was less favorably cleaved. Expression of either mutated version of NCS-1 in neuroblastoma cells protected intracellular calcium signals from paclitaxel-induced changes. These results support our hypothesis that it is possible to protect cells from paclitaxel-induced degradation of NCS-1 by inhibiting calpain activity.

Topics: Acetylcysteine; Calcium Signaling; Calpain; Cell Line, Tumor; Humans; Mutation; Neuroblastoma; Neuronal Calcium-Sensor Proteins; Neurons; Neuropeptides; Paclitaxel; Point Mutation; Protease Inhibitors; Protein Isoforms; Signal Transduction; Thermodynamics

Methamphetamine (METH) is a potent psychostimulant drug that may cause neuronal cell degeneration. The underlying mechanisms of METH-induced neuronal toxicity remains poorly understood. In this study, we investigated an important role of calpain-dependent cascades in methamphetamine-induced toxicity in human dopaminergic neuroblastoma SH-SY5Y cultured cell lines. In addition, the protective effect of melatonin against METH-induced calpain-dependent death pathway was also investigated. The results of this study show that METH significantly decreased cell viability and tyrosine hydroxylase phosphorylation in SH-SY5Y cultured cells. Melatonin reversed the toxic effect of METH by inducing cell viability. In addition, melatonin was able to restore the reduction in mitochondrial function and phosphorylation of tyrosine hydroxylase in SH-SY5Y treated cells. An induction of calpain expression and activity but a reduction of calpain inhibitor (calpastatin) protein levels were observed in SH-SY5Y cells treated with METH but these effects were diminished by melatonin. These results implicated calpain-dependent death pathways in the processes of METH-induced toxicity and also indicated that melatonin has the capacity to reverse this toxic effect in SH-SY5Y cultured cells.

Topics: Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Cell Survival; Humans; Melatonin; Methamphetamine; Neuroblastoma

Mycoplasmas often contaminate cultured cells, leading to alterations in cellular gene expression, protein synthesis, signal transduction and metabolic pathways. Mycoplasmal contamination is often unnoticed, so that mycoplasma-induced alterations in cell functions may not be appreciated, unless specifically studied. Here, we show for the first time that contamination of SH-SY5Y cells by Mycoplasma hyorhinis leads to increased levels of calpastatin (the endogenous inhibitor of the Ca(2+)-dependent protease calpain), resulting in inhibition of Ca(2+)-induced calpain activation and inhibition of calpain-promoted proteolysis in the mycoplasmal-infected cells. Calpain activity is recovered upon calpastatin removal from extracts of contaminated cells. The calpain-calpastatin system has been implicated in a variety of physiological and pathological processes (signal transduction, motility, cell cycle, cell differentiation, membrane damage and apoptosis). Because the ratio of calpastatin to calpain is an important factor in the control of calpain activity within the cell, the elevated calpastatin may protect the mycoplasma-infected cells against certain types of damage (e.g. caused by high Ca(2+)). Thus, our results are important for studies on the modulation of host cells by mycoplasmas, and relevant to the pathobiology of processes involving mycoplasmal infections. The mycoplasma-infected cells provide a system for identifying factors that participate in the regulation of cellular calpastatin.

Topics: Calcium; Calcium-Binding Proteins; Calpain; Cell Differentiation; Equipment Contamination; Host-Pathogen Interactions; Mycoplasma hyorhinis; Neuroblastoma; Neurons; Proteins; Tumor Cells, Cultured; Up-Regulation

Galantamine, which is currently used in the treatment of patients with Alzheimer's disease (AD), has been shown to have a neuroprotective effect against beta-amyloid (Abeta) peptide-induced toxicity, which is involved in the pathogenesis of AD. In this study, we investigated the mechanism underlying the protective effect of galantamine on Abeta-induced toxicity in human neuroblastoma cells (SH-SY5Y). Using MTT and LDH leakage assays, we observed that galantamine pretreatment significantly prevented Abeta1-40-induced cell death. Abeta1-40-induced overexpression and increased cleavage of both calpain and calcineurin were observed by Western blotting and double immunofluorescent staining. Increased calcineurin phosphatase activity and decreased level of pSer112 BAD were also observed in Abeta1-40-damaged cells. However, all these alterations were found to be reversed by galantamine pretreatment. We also found that the neuroprotection of galantamine can be blocked by an alpha7 nAChR antagonist. Overall, our results suggest that galantamine may prevent the neuronal damage induced by Abeta1-40 through a mechanism related to the regulation of calpain-calcineurin activation and BAD phosphorylation, which may involve the participation of alpha7 nAChR.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Calcineurin; Calcineurin Inhibitors; Calpain; Cell Line, Tumor; Cholinesterase Inhibitors; Galantamine; Humans; Nerve Degeneration; Neuroblastoma; Peptide Fragments; Signal Transduction

Human malignant neuroblastoma is characterized by poor differentiation and uncontrolled proliferation of immature neuroblasts. Retinoids such as all-trans-retinoic acid (ATRA), 13-cis-retinoic acid (13-CRA), and N-(4-hydroxyphenyl) retinamide (4-HPR) at low doses are capable of inducing differentiation, while flavonoids such as (-)-epigallocatechin-3-gallate (EGCG) and genistein (GST) at relatively high dose can induce apoptosis. We used combination of retinoid and flavonoid for controlling growth of malignant neuroblastoma SH-SY5Y cells. Cells were treated with a retinoid (1 microM ATRA, 1 microM 13-CRA, or 0.5 microM 4-HPR) for 7 days and then with a flavonoid (25 microM EGCG or 25 microM GST) for 24 h. Treatment of cells with a low dose of a retinoid for 7 days induced neuronal differentiation with downregulation of telomerase activity and N-Myc but overexpression of neurofilament protein (NFP) and subsequent treatment with a relatively high dose of a flavonoid for 24 h increased apoptosis in the differentiated cells. Besides, retinoids reduced the levels of inflammatory and angiogenic factors. Apoptosis was associated with increases in intracellular free [Ca2+], Bax expression, cytochrome c release from mitochondria and activities of calpain and caspases. Decreases in expression of calpastatin (endogenous calpain inhibitor) and baculovirus inhibitor-of-apoptosis repeat containing (BIRC) proteins (endogenous caspase inhibitors) favored apoptosis. Treatment of SH-SY5Y cells with EGCG activated caspase-8, indicating induction of the receptor-mediated pathway of apoptosis. Based on our observation, we conclude that combination of a retinoid and a flavonoid worked synergistically for controlling the malignant growth of human neuroblastoma cells.

Topics: Apoptosis; Blotting, Western; Calcium; Calpain; Caspase 3; Caspase 8; Cell Differentiation; Cell Line, Tumor; Down-Regulation; Drug Synergism; Enzyme Activation; Flavonoids; Humans; Mitochondria; Neuroblastoma; Proto-Oncogene Proteins c-myc; Retinoids; Telomerase

Caspase-8 is a proapoptotic protease that suppresses neuroblastoma metastasis by inducing programmed cell death. Paradoxically, caspase-8 can also promote cell migration among nonapoptotic cells; here, we show that caspase-8 can promote metastasis when apoptosis is compromised. Migration is enhanced by caspase-8 recruitment to the cellular migration machinery following integrin ligation. Caspase-8 catalytic activity is not required for caspase-8-enhanced cell migration; rather, caspase-8 interacts with a multiprotein complex that can include focal adhesion kinase and calpain 2 (CPN2), enhancing cleavage of focal adhesion substrates and cell migration. Caspase-8 association with CPN2/calpastatin disrupts calpastatin-mediated inhibition of CPN2. In vivo, knockdown of either caspase-8 or CPN2 disrupts metastasis among apoptosis-resistant tumors. This unexpected molecular collaboration provides an explanation for the continued or elevated expression of caspase-8 observed in many tumors.

Topics: Alstrom Syndrome; Animals; Calcium-Binding Proteins; Calpain; Caspase 8; Cell Line, Tumor; Cell Movement; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Humans; Lung Neoplasms; Mice; Mice, Transgenic; Neoplasm Metastasis; Neuroblastoma; Talin

In resting human neuronal cells, nitric oxide synthase (nNOS) is present in its native 160 kDa form in a quiescent state predominantly co-localized on the plasma membrane, via its PDZ (Psd-95/Discs-large/Zona Occludens) domain, with NMDA receptor (NMDA-R) and in tight association with heat shock protein 90 (HSP90). Following exposure of the cells to Ca(2+)-ionophore or to NMDA, nNOS undergoes proteolytic removal of the PDZ domain being converted into a fully active 130 kDa form. The newly generated nNO synthase form dissociates from NMDA-R and extensively diffuses into the cytosol in direct correlation with NO production. Intracellular redistribution and activation of nNOS are completely prevented in cells preloaded with calpain inhibitor-1, indicating that these processes are triggered by a concomitant activation of calpain. The role of calpain has been confirmed by immunoprecipitation experiments revealing that also mu-calpain is specifically recruited into the NMDA-R-nNOS-HSP90 complex following calcium loading. Thus, the formation of clusters containing HSP90, mu-calpain, nNOS and NMDA-R represents the limiting step for the operation of the mechanism that links an efficient synthesis of NO to a local increase in Ca(2+) influx.

Topics: Calcium; Calcium Signaling; Calpain; Cell Line, Tumor; Cell Membrane; Cytosol; Diffusion; Enzyme Activation; Enzyme Inhibitors; HSP90 Heat-Shock Proteins; Humans; Ionophores; Neuroblastoma; Neurons; Nitric Oxide; Nitric Oxide Synthase Type I; Peptide Hydrolases; Protein Structure, Tertiary; Receptors, N-Methyl-D-Aspartate

Chronic alcohol consumption causes pathological changes in the brain and neuronal loss. Ethanol toxicity may partially result from the perturbation of microtubule-associated proteins, like tau. Tau dysfunction is well known for its involvement in certain neurodegenerative diseases, such as Alzheimer's disease. In the present study, the effect of ethanol on tau was examined using differentiated human neuroblastoma cells that inducibly express the 4R0N isoform of tau via a tetracycline-off expression system. During tau induction, ethanol exposure (1.25-5mg/ml) dose-dependently increased tau protein levels and reduced cell viability. The increase in cell death likely resulted from tau accumulation since increased levels of tau were sufficient to reduce cell viability and ethanol was toxic to cells expressing tau but not to non-induced controls. Tau accumulation did not result from greater tetracycline-off induction since ethanol increased neither tau mRNA expression nor the expression of the tetracycline-controlled transactivator. Additionally, ethanol increased endogenous tau protein levels in neuroblastoma cells lacking the tetracycline-off induction system for tau. Ethanol delayed tau clearance suggesting ethanol impedes its degradation. Though ethanol inhibited neither cathepsin B, cathepsin D, nor chymotrypsin-like activity, it did significantly reduce calpain I expression and activity. Calpain I knockdown by shRNA increased tau levels indicating that calpain participates in tau degradation in this model. Moreover, the activation of calpain, by the calcium ionophore A23187, partially reversed the accumulation of tau resulting from ethanol exposure. Impaired calpain-mediated degradation may thus contribute to the increased accumulation of tau caused by ethanol.

Topics: Calpain; Cell Line, Tumor; Central Nervous System Depressants; Ethanol; Gene Expression; Humans; Neuroblastoma; Neurons; Polymerase Chain Reaction; RNA, Messenger; tau Proteins

Glutathione (GSH) depletion is widely used to sensitize cells to anticancer treatment inducing the progression of programmed cell death and overcoming chemoresistance. It has been reported that neuroblastoma cells with MYCN amplification are unable to start TRAIL-dependent death and MYCN, in concert with cytotoxic drugs, efficiently induces the mitochondrial pathway of apoptosis through oxidative mechanisms. In this study, we show that GSH loss induced by L-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH biosynthesis, leads to overproduction of reactive oxygen species (ROS) and triggers apoptosis of MYCN-amplified neuroblastoma cells. BSO susceptibility of SK-N-BE-2C, a representative example of MYCN-amplified cells, has been attributed to stimulation of total SOD activity in the absence of changes in the level and the activity of catalase. Therefore, the unbalanced intracellular redox milieu has been demonstrated to be critical for the progression of neuroblastoma cell death that was efficiently prevented by antioxidants and rottlerin. These results describe a novel pathway of apoptosis dependent on ROS formation and PKC-delta activation and independent of p53, bcl-2, and bax levels; the selective redox modulation of PKC-delta might be suggested as a potential strategy for sensitizing MYCN-amplified cells to therapeutic approaches.

Topics: Acetophenones; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Ascorbic Acid; Benzopyrans; Biphenyl Compounds; Buthionine Sulfoximine; Calpain; Caspases; Catalase; Cell Line, Tumor; DNA Damage; Glutathione; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Onium Compounds; Protein Kinase C; Reactive Oxygen Species; Superoxide Dismutase; Superoxide Dismutase-1

In order to elucidate underlying mechanism of cell death pathways in neuronal cells in humans, we studied responsible pathways involved in the endoplasmic reticulum (ER) stress-induced cell death in neuroblastoma cells, SK-N-SH and its neuroblast-type subclone SH-SY5Y cells. A time-dependent induction of ER chaperons, glucose regulated protein (GRP)78 and GRP94, was observed after treatment with tunicamycin (TM), and cell death was also induced concomitantly in both cells. Although the pro-caspase-12-like protein was defined in both cells, a decrease in the protein was observed in only SH-SY5Y cells after exposure to TM. In contrast, pro-caspase-4 was detected in only SK-N-SH cells, and the cleaved-form was induced by the treatment with TM. A caspase-4 inhibitor, Z-LEVD-FMK attenuated TM-induced cell death in SK-N-SH cells. Calpain- and caspase-3-mediated proteolysis of alpha II-spectrin was also increased after the treatment with TM in both cells. A calpain inhibitor, calpeptin, repressed TM-induced cell death in only SK-N-SH cells. GADD153/C/EBP homologous protein (CHOP) was significantly induced after exposure to TM in only SH-SY5Y cells and RNA interference to GADD153/CHOP repressed TM-induced cell death. These results demonstrate that induction of GADD153/CHOP plays a pivotal role in mechanism of ER stress-induced cell death in SH-SY5Y cells, on the other hand, cleavage of pro-caspase-4 by activation of calpain play a crucial role in SK-N-SH cells. It is also suggested that the relevance of caspase-4 to ER stress is cell-specific even between human-origin cell lines.

Topics: Anti-Bacterial Agents; Calpain; Caspase 12; Caspases, Initiator; Cell Death; Cell Line, Tumor; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Heat-Shock Proteins; Humans; Membrane Glycoproteins; Molecular Chaperones; Neuroblastoma; Neurons; Oxidative Stress; Transcription Factor CHOP; Tunicamycin

p120-catenin contributes to the cadherin-mediated adhesion and aggregation of cells. mu-Calpain was activated and p120-catenin was degraded after 36 h of ischemia in differentiated SH-SY5Y cells. Calpain inhibitors Cbz-Val-Phe-H (MDL28170, 20 microM) and N-acetyl-leucyl-leucyl-norleucinal (ALLN, 20 microM) increased the levels of dephosphorylated p120-catenin, aggregation, and cell survival as detected by reduced LDH release in ischemic cells. However, a proteasome inhibitor lactacystin had no such effects. This is the first report of the calpain-mediated degradation of p120-catenin and an association between the level of dephosphorylated p120-catenin and cell aggregation in ischemic neuronal cells.

Topics: Acetylcysteine; Calpain; Catenins; Cell Adhesion Molecules; Cell Aggregation; Cell Death; Cell Line, Tumor; Delta Catenin; Dipeptides; Humans; Ischemia; Leupeptins; Neuroblastoma; Phosphoproteins

Malignant (N-type) neuroblastoma continues to defy current chemotherapeutic regimens. We tested the garlic compounds diallyl sulfide (DAS) and diallyl disulfide (DADS) for induction of apoptosis in human malignant neuroblastoma SH-SY5Y cells. Viability of human primary neurons was unaffected after 24 h treatment with 50 and 100 microM DAS and 50 microM DADS but slightly affected with 100 microM DADS. Treatment with 50 and 100 microM DAS or DADS significantly decreased viability in SH-SY5Y cells. Wright staining showed morphological features of apoptosis in SH-SY5Y cells treated with 50 and 100 microM DAS or DADS for 24 h. ApopTag assay demonstrated DNA fragmentation in apoptotic cells. Apoptosis was associated with an increase in [Ca(2+)](i), increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, increase in cytosolic Smac/Diablo, and down regulation of inhibitor-of-apoptosis proteins and nuclear factor kappa B (NFkappaB). Activation of caspase-9 and caspase-3 indicated involvement of intrinsic pathway of apoptosis. Calpain and caspase-3 activities produced 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Also, caspase-3 activity cleaved inhibitor of caspase-activated DNase (ICAD). Results strongly suggested that the garlic compounds DAS and DADS suppressed anti-apoptotic factors and activated calpain and intrinsic caspase cascade for apoptosis in SH-SY5Y cells.

Topics: Allyl Compounds; Anticarcinogenic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Calcium; Calpain; Caspases; Cell Line, Tumor; Cell Survival; Cells, Cultured; Cytochromes c; Disulfides; Enzyme Activation; Enzyme Inhibitors; Garlic; Humans; I-kappa B Proteins; Intracellular Signaling Peptides and Proteins; Mitochondrial Proteins; Neuroblastoma; Neurons; NF-kappa B; NF-KappaB Inhibitor alpha; Plant Oils; Proto-Oncogene Proteins c-bcl-2; Spectrin; Sulfides

The formation of intraneuronal inclusions is a common feature of neurodegenerative polyglutamine disorders, including Spinocerebellar ataxia type 3. The mechanism that triggers inclusion formation in these typically late onset diseases has remained elusive. However, there is increasing evidence that proteolytic fragments containing the expanded polyglutamine segment are critically required to initiate the aggregation process. We analyzed ataxin-3 proteolysis in neuroblastoma cells and in vitro and show that calcium-dependent calpain proteases generate aggregation-competent ataxin-3 fragments. Co-expression of the highly specific cellular calpain inhibitor calpastatin abrogated fragmentation and the formation of inclusions in cells expressing pathological ataxin-3. These findings suggest a critical role of calpains in the pathogenesis of Spinocerebellar ataxia type 3.

Topics: Animals; Antibodies; Ataxin-3; Calpain; Cell Line, Tumor; Enzyme Inhibitors; Humans; Ionomycin; Ionophores; Kidney; Machado-Joseph Disease; Mice; Nerve Tissue Proteins; Neuroblastoma; Nuclear Proteins; Peptides; Rabbits; Rats; Repressor Proteins; Transcription Factors

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

Microtubule-stabilizing and -destabilizing proteins play a crucial role in regulating the dynamic instability of microtubules during neuronal development and synaptic transmission. The microtubule-destabilizing protein SCG10 is a neuron-specific protein implicated in neurite outgrowth. The SCG10 protein is significantly reduced in mature neurons, suggesting that its expression is developmentally regulated. In contrast, the microtubule-stabilizing protein tau is expressed in mature neurons and its function is essential for the maintenance of neuronal polarity and neuronal survival. Thus, the establishment and maintenance of neuronal polarity may down-regulate the protein level/function of SCG10. In this report, we show that treatment of PC12 cells and neuroblastoma cells with the microtubule-stabilizing drug Taxol induced a rapid degradation of the SCG10 protein. Consistently, overexpression of tau protein in neuroblastoma cells also induced a reduction in SCG10 protein levels. Calpain inhibitor MDL-28170, but not caspase inhibitors, blocked a significant decrease in SCG10 protein levels. Collectively, these results indicate that tau overexpression and Taxol treatment induced a calpain-dependent degradation of the microtubule-destabilizing protein SCG10. The results provide evidence for the existence of an intracellular mechanism involved in the regulation of SCG10 upon microtubule stabilization.

Topics: Animals; Blotting, Western; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Activation; Gene Expression Regulation; Humans; Leupeptins; Membrane Proteins; Neuroblastoma; Paclitaxel; Rats; Stathmin; tau Proteins; Transfection; Tubulin Modulators

Neuroblastoma is the most common extracranial solid tumor in children causing death at pre-school age, as no cure has yet been developed. We investigated the proteolytic mechanisms for apoptosis in human malignant (N-type) neuroblastoma SH-SY5Y cells following exposure to flavonoids such as apigenin (APG), (-)-epigallocatechin (EGC), (-)-epigallocatechin-3-gallate (EGCG) and genistein (GST). We found decrease in viability of SH-SY5Y cells with an increase in dose of APG, EGC, EGCG and GST. Predominantly apoptosis occurred following exposure of SH-SY5Y cells to 50 microM APG, 50 microM EGC, 50 microM EGCG and 100 microM GST for 24 hr. Apoptosis was associated with increases in intracellular free [Ca(2+)] and Bax:Bcl-2 ratio, mitochondrial release of cytochrome c and activation of caspase-9, calpain and caspase-3. Induction of apoptosis with APG and GST showed activation of caspase-12 as well. Activation of caspase-3 could cleave the inhibitor-of-caspase-activated DNase (ICAD) to release and translocate caspase-3-activated DNase (CAD) to the nucleus. Activation of caspase-8 cleaved Bid to truncated Bid (tBid) in cells treated with EGC and EGCG. EGC and EGCG induced apoptosis with caspase-8 activation and mitochondria-mediated pathway, whereas APG and GST caused apoptosis via an increase in intracellular free [Ca(2+)] with calpain activation and mitochondria-mediated pathway. Activation of different proteases for cell death was confirmed using caspase-8 inhibitor II, calpeptin (calpain inhibitor), caspase-9 inhibitor I and caspase-3 inhibitor IV. Thus, plant-derived flavonoids cause cell death with activation of proteolytic activities of calpain and caspases in SH-SY5Y cells, and therefore serve as potential therapeutic agents for controlling the growth of neuroblastoma.

Topics: Apoptosis; Calpain; Caspases; Cell Line, Tumor; Colorimetry; Flavonoids; Humans; Neuroblastoma

Impaired tau catabolism may contribute to tau accumulation and aggregation in Alzheimer's disease and neurofibrillary tangle formation. This study examined the effects of proteasome and calpain inhibition on tau levels and turnover in primary rat hippocampal neurons and differentiated SH-SY5Y human neuroblastoma cells. Administration of proteasome (MG-115, lactacystin) or calpain (MDL28170) inhibitors for up to 24 hours did not alter tau levels in differentiated SH-SY5Y cells or rat hippocampal neurons. Addition of 1 microM and 10 microM MG-115 did not change total tau levels, but did result in increased reactivity of phosphorylation-dependent tau antibodies (PHF-1, CP-13) and decreased Tau-1 immunoreactivity. Administration of cycloheximide to inhibit de novo protein synthesis also did not alter tau levels in the presence or absence of lactacystin. These results demonstrate that although the proteasome and calpain protease systems are capable of degrading tau in cell-free assays, their inhibition does not alter cellular tau levels in primary neurons or differentiated neuroblastoma cells.

Topics: Alzheimer Disease; Aminopeptidases; Animals; Antibodies, Monoclonal; Calpain; Cell Aggregation; Hippocampus; In Vitro Techniques; Neural Inhibition; Neuroblastoma; Neurofibrillary Tangles; Neurons; Proteasome Endopeptidase Complex; Rats; tau Proteins; Tumor Cells, Cultured

Glutamate toxicity has been implicated in cell death in neurodegenerative diseases and injuries. Glutamate-induced Ca2+ influx may mediate activation of calpain, a Ca2+-dependent cysteine protease, which in turn may degrade key cytoskeletal proteins. We investigated glutamate-mediated apoptosis of VSC4.1 motoneurons and functional neuroprotection by calpain inhibition. Exposure of VSC4.1 cells to 10 microM glutamate for 24 hr caused significant increases in intracellular free [Ca2+], as determined by fura-2 assay. Pretreatment of cells with 10 or 25 microM calpeptin (a cell-permeable calpain-specific inhibitor) for 1 hr prevented glutamate-induced Ca2+ influx. Western blot analyses showed an increase in Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and calpain and caspase-3 activities during apoptosis. Cell morphology, as evaluated by Wright staining, indicated predominantly apoptotic features following glutamate exposure. ApopTag assay further substantiated apoptotic features morphologically as well as biochemically. Our data showed that calpeptin mainly prevented calpain-mediated proteolysis and apoptosis and maintained whole-cell membrane potential, indicating functional neuroprotection. The results imply that calpeptin may serve as a therapeutic agent for preventing motoneuron degeneration, which occurs in amyotrophic lateral sclerosis and spinal cord injury. In this investigation, we also examined glutamate receptor subtypes involved in the initiation of apoptosis in VSC4.1 cells following exposure to glutamate. Our results indicated that the N-methyl-D-aspartate (NMDA) receptors contributed more than alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors to glutamate-mediated Ca2+ influx and cell death mechanism. Inhibition of the activities of both NMDA and AMPA receptors protected VSC4.1 cells from glutamate toxicity and preserved whole-cell membrane potential.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Calcium; Calpain; Caspase 3; Caspases; Cell Fusion; Cysteine Proteinase Inhibitors; Cytochromes c; Dipeptides; Glutamic Acid; Humans; Ionomycin; Ionophores; Mice; Mitochondria; Motor Neurons; Neuroblastoma; Neuroprotective Agents; Patch-Clamp Techniques; Proto-Oncogene Proteins c-bcl-2; Rats; Spectrin; Spinal Cord

Calcium-dependent cysteine proteases, calpains, have physiological roles in cell motility and differentiation but also play a pathological role following insult or disease. The ubiquitous calpains are widely considered to be cytosolic enzymes, although there has been speculation of a mitochondrial calpain. Within a highly enriched fraction of mitochondria obtained from rat cortex and SH-SY5Y human neuroblastoma cells, immunoblotting demonstrated enrichment of the 80kDa mu-calpain large subunit and 28kDa small subunit. In rat cortex, antibodies against domains II and III of the large mu-calpain subunit also detected a 40kDa fragment, similar to the autolytic fragment generated following incubation of human erythrocyte mu-calpain with Ca(2+). Mitochondrial proteins including apoptosis inducing factor and mitochondrial Bax are calpain substrates, but the mechanism by which calpains gain access to these proteins is uncertain. Mitochondrial localization of mu-calpain places the enzyme in proximity to its mitochondrial substrates and to Ca(2+) released from mitochondrial stores.

Topics: Animals; Calpain; Cell Line, Tumor; Humans; Male; Mitochondria; Neuroblastoma; Rats; Rats, Sprague-Dawley; Tissue Distribution

On the basis of clinical studies showing that arsenic trioxide (As(2)O(3)), via an apoptotic mechanism, and with minimal toxicity induces complete remission in patients with refractory acute promyelocytic leukemia and that multidrug-resistant and p53-mutated neuroblastoma cells are sensitive to As(2)O(3) both in vitro and in vivo, we searched for molecular mechanisms involved in the As(2)O(3)-induced neuroblastoma cell death.. We have studied the effect of As(2)O(3) on the expression and cellular localization of proteins involved in drug-induced death in two neuroblastoma cell lines with intact p53 and two with mutated p53, the latter two displaying multidrug resistance.. As(2)O(3) provoked Bax expression in all tested neuroblastoma cell lines, including SK-N-BE(2) cells with mutated p53 and LA-N-1 cells, which have a deleted p53. In all cell lines exposed to As(2)O(3), p21 Bax was proteolytically cleaved in a calpain-dependent way into the more proapoptotic p18 Bax, which was detected exclusively in a mitochondria-enriched subcellular fraction. As(2)O(3) also caused an increase of cytoplasmic cytochrome c, translocation of antiapoptosis-inducing factor to the nuclei, and a slight activation of caspase 3. However, inhibition of caspase 3 did not prevent cell death, whereas inhibition of Bax cleavage was associated with a decreased As(2)O(3)-induced cell death.. We show that multidrug-resistant neuroblastoma cells die after exposure to As(2)O(3), independent of functional p53, suggesting activation of a cytotoxic pathway different from that induced by conventional chemotherapeutic agents. We further propose that proteolytic activation of Bax is an important event in As(2)O(3)-induced cell death.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; bcl-2-Associated X Protein; Blotting, Western; Calpain; Carboplatin; Caspase 3; Caspases; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance, Neoplasm; Etoposide; Humans; Mutation; Neuroblastoma; Oxides; Proto-Oncogene Proteins c-bcl-2; Time Factors; Tumor Suppressor Protein p53; Vincristine

The 94 kDa glucose-regulated protein (GRP94), the endoplasmic reticulum (ER) resident molecular chaperone, has a role in cell death due to endoplasmic reticulum stress (ER stress). Here, we report that expression of GRP94 was increased in human neuroblastoma cells (SH-SY5Y (SY5Y) cells) exposed to hypoxia/reoxygenation (H/R). H/R mediated death of SY5Y cells was associated with the activation of major cysteine proteases, caspase-3 and calpain, along with an elevated intracellular calcium concentration. Pretreatment with adenovirus-mediated antisense GRP94 (AdGRP94AS) led to reduced viability of SY5Y cells after being subjected to H/R compared with wild-type cells or cells with adenovirus-mediated overexpression of GRP94 (AdGRP94S). These results indicate that suppression of GRP94 is associated with accelerated apoptosis and that expression of GRP94 (as a stress protein) suppresses oxidative stress-mediated neuronal death and stabilizes calcium homeostasis in the ER. We also used gerbils with transient forebrain ischemia to study the role of GRP94 in vivo. Neurons with adenovirus-mediated overexpression of GRP94 were resistant to ischemic damage. These results confirmed that GRP94 could suppress ischemic injury to neurons, suggesting that gene transfer of GRP94 into the brain may have therapeutic potential in the treatment of cerebrovascular disease.

Topics: Animals; Apoptosis; Calcium; Calpain; Caspase 3; Caspases; Cell Survival; Cerebrovascular Disorders; Cysteine Endopeptidases; DNA, Recombinant; Endoplasmic Reticulum; Gene Transfer Techniques; Gerbillinae; HSP70 Heat-Shock Proteins; Humans; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Membrane Proteins; Models, Animal; Neuroblastoma; Neurons; Reperfusion Injury; Tumor Cells, Cultured

Cultured mammalian PC12 or B104 cells do not instantaneously restore a plasmalemmal barrier (seal) after neurite transection, as measured using fluorescent dye probes of various sizes and saline solutions with different [Ca(2+)](o). Rather, transected cells gradually (from 15 to 60 min postseverance) exclude probes (dye molecules) of progressively smaller size. Furthermore, an inhibitor (calpeptin) of a Ca(2+)-activated cysteine protease (calpain) and antibodies or toxins to a Ca(2+)-regulated protein (synaptotagmin) and other membrane fusion proteins (syntaxin and synaptobrevin) inhibit plasmalemmal sealing. These data obtained using molecular probes on mammalian cell lines are consistent with previous data on invertebrate giant axons indicating that Ca(2+) plays many roles in the formation, accumulation, and fusion/interaction of vesicles gradually forming a seal at a site of plasmalemmal damage.

Topics: Animals; Axotomy; Calcium; Calcium-Binding Proteins; Calpain; Cell Membrane; Coloring Agents; Cysteine Proteinase Inhibitors; Dipeptides; Extracellular Fluid; Membrane Fusion; Membrane Glycoproteins; Membrane Proteins; Nerve Regeneration; Nerve Tissue Proteins; Neurites; Neuroblastoma; PC12 Cells; Qa-SNARE Proteins; R-SNARE Proteins; Rats; Synaptotagmins; Tumor Cells, Cultured

Activation of calpain results in the breakdown of alpha II spectrin (alpha-fodrin), a neuronal cytoskeleton protein, which has previously been detected in various in vitro and in vivo neuronal injury models. In this study, a 150 kDa spectrin breakdown product (SBDP150) was found to be released into the cell-conditioned media from SH-SY5Y cells treated with the calcium channel opener maitotoxin (MTX). SBDP150 release can be readily quantified on immunoblot using an SBDP150-specific polyclonal antibody. Increase of SBDP150 also correlated with cell death in a time-dependent manner. MDL28170, a selective calpain inhibitor, was the only protease inhibitor tested that significantly reduced MTX-induced SBDP150 release. The cell-conditioned media of cerebellar granule neurons challenged with excitotoxins (NMDA and kainate) also exhibited a significant increase of SBDP150 that was attenuated by pretreatment with an NMDA receptor antagonist, R(-)-3-(2-carbopiperazine-4-yl)-propyl-1-phosphonic acid (CPP), and MDL28170. In addition, hypoxic/hypoglycemic challenge of cerebrocortical cultures also resulted in SBDP150 liberation into the media. These results support the theory that an antibody-based detection of SBDP150 in the cell-conditioned media can be utilized to quantify injury to neural cells. Furthermore, SBDP150 may potentially be used as a surrogate biomarker for acute neuronal injury in clinical settings.

Topics: Analysis of Variance; Animals; Blotting, Western; Calpain; Cell Death; Cells, Cultured; Cerebellum; Cerebral Cortex; Dipeptides; Dose-Response Relationship, Drug; Glucose; Humans; Hypoxia; Marine Toxins; Nerve Tissue Proteins; Neuroblastoma; Neurons; Neurotoxins; Oxocins; Rats; Rats, Sprague-Dawley; Spectrin; Time Factors

In neuroblastoma LAN-5 cells during calpain activation, in addition to the two expressed 70 kDa and 30 kDa calpastatin forms, other inhibitory species are produced, having molecular masses of 50 kDa and 15 kDa. At longer times of incubation, both native and new calpastatin species disappear. The formation of these new calpastatins as well as the decrease in intracellular total calpastatin activity are mediated by calpain itself, as indicated by the effect of the synthetic calpain inhibitor I, which prevents both degradative processes. Analysis of the calcium concentrations required for the two processes indicates that the first conservative proteolytic event is mediated by micro-calpain, whereas the second one is preferentially carried out by m-calpain. The appearance of the 15 kDa form, containing only the calpastatin repetitive inhibitory domain and identified also in red cells of hypertensive rats as the major inhibitor form, can be considered a marker of intracellular calpain activation, and it can be used for the monitoring of the involvement of calpain in pathological situations.

Topics: Animals; Calcium; Calcium-Binding Proteins; Calpain; Humans; Neuroblastoma; Rats; Tumor Cells, Cultured

2,5-Hexanedione (2,5-HD), the neurotoxic metabolite of n-hexane, can structurally modify neurofilaments (NF) by pyrrole adduct formation and subsequent covalent cross-linking. 2,5-HD also induces accumulations of NF within the pre-terminal axon. We examined whether exposure of NF to 2,5-HD affected NF degradation. Two different models were used: (1) NF-enriched cytoskeletons isolated from human sciatic nerve were incubated with 2,5-HD in vitro and (2) differentiated human neuroblastoma cells (SK-N-SH) were exposed to 2, 5-HD in culture prior to isolation of cytoskeletal proteins. The cytoskeletal preparations were subsequently incubated with calpain II. The amount of NF-H and NF-L remaining after proteolysis was determined by SDS-PAGE and quantitative immunoblotting. NF-M proteolysis could not be quantified. Incubation of sciatic nerve cytoskeletal preparations with 2,5-HD resulted in cross-linking of all three NF proteins into high molecular weight (HMW) material with a range of molecular weights. Proteolysis of the NF-H and NF-L polypeptides was not affected by 2,5-HD-exposure. Degradation of the HMW material containing NF-H or NF-L was retarded when comparing with degradation of the NF-H and NF-L polypeptides, respectively, from control samples, but not as compared to the corresponding NF polypeptides from 2,5-HD-treated samples. Exposure of SK-N-SH cells to 2,5-HD also resulted in considerable cross-linking of NF. No differences were found between the proteolytic rates of NF-L and NF-H from exposed cells as compared with those subunits from control cells. Moreover, degradation of cross-linked NF-H was not different from monomeric NF-H. In conclusion, whether 2,5-HD affects calpain-mediated degradation of cross-linked NF proteins will depend on which model better reflects NF cross-linking as occurring in 2, 5-HD-induced axonopathy. However, with both models it was demonstrated that exposure of NF proteins to 2,5-HD without subsequent cross-linking is not adequate to inhibit NF proteolysis in vitro by added calpain.

Topics: Adult; Calpain; Cross-Linking Reagents; Cytoskeletal Proteins; Cytoskeleton; Female; Hexanones; Humans; Kinetics; Male; Neuroblastoma; Neurofibrils; Neurofilament Proteins; Neurotoxins; Sciatic Nerve; Tumor Cells, Cultured

Tissue transglutaminase (tTG) is up-regulated in Alzheimer's disease brain and localizes to neurofibrillary tangles with the tau protein. Tau is an in vitro tTG substrate, being cross-linked and/or polyaminated. Further, the Gln and Lys residues in tau that are modified by tTG in vitro are located primarily within or adjacent to the microtubule-binding domains. Considering these and other previous findings, this study was carried out to determine if tau is modified in situ by tTG in human neuroblastoma SH-SY5Y cells, and whether tTG-catalyzed tau polyamination modulates the function and/or metabolism of tau in vitro. For these studies, SH-SY5Y cells stably overexpressing tTG were used. tTG coimmunoprecipitated with tau, and elevating intracellular calcium levels with maitotoxin resulted in a 52 +/- 4% increase in the amount of tTG that coimmunoprecipitated with tau. The increase in association of tTG with tau after treatment with maitotoxin corresponded to a coimmunolocalization of tTG, tTG activity, and tau in the cells. Further, tau was modified by tTG in situ in response to maitotoxin treatment. In vitro polyaminated tau was significantly less susceptible to micro-calpain proteolysis; however, tTG-mediated polyamination of tau did not significantly alter the microtubule-binding capacity of tau. Thus, tau interacts with and is modified by tTG in situ, and modification of tau by tTG alters its metabolism. These data indicate that tau is likely to be modified physiologically and pathophysiologically by tTG, and tTG may play a role in Alzheimer's disease.

Topics: Alzheimer Disease; Animals; Calcium; Calpain; Guinea Pigs; Humans; Immunosorbent Techniques; Marine Toxins; Microtubules; Neuroblastoma; Neurofibrillary Tangles; Oxocins; Polyamines; Recombinant Proteins; tau Proteins; Transglutaminases; Tumor Cells, Cultured

Topics: Apoptosis; Calcium; Calpain; Cell Survival; Cholesterol; Humans; Hydroxycholesterols; Ketocholesterols; L-Lactate Dehydrogenase; Lipid Peroxides; Necrosis; Neuroblastoma; Reactive Oxygen Species; Tumor Cells, Cultured

Extensive necrotic death of MSN neuroblastoma cells could be induced after incubation with the calcium ionophore, A23187. The reaction was concentration-dependent and time course-dependent. Levels of the 66 kd/alpha-internexin neurofilament protein (NF-66) and the cognate heat shock protein 70 (Hsc 70) decreased during the Ca2+-activated cell death. Addition of the calcium chelator, ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) restored the normal level of NF-66 and partially that of the Hsc 70. Use of either calpain I or calpain II inhibitor could alleviate the reduction of 66 kd protein during the ionophore treatment whereas only calpain I inhibitor treatment was effective in restoring the normal level of the Hsc 70. Neither of these calpain inhibitors could block the ionophore triggered cell death. EGTA was toxic to cells in a wide range of concentration suggesting a calcium-independent activation of cell death mechanism.

Topics: Calcimycin; Calpain; Carrier Proteins; Cell Death; HSC70 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Intermediate Filament Proteins; Leupeptins; Nerve Tissue Proteins; Neuroblastoma; Neurofibrils; Oligopeptides; Tumor Cells, Cultured

N-myc is a short-lived transcription factor, frequently amplified in human neuroblastomas. The ubiquitin-proteasome system is involved in the degradation of many short-lived cellular proteins and previous studies have shown that ubiquitin-dependent proteolysis is implicated in the turn-over of N-myc in vitro. However, calpain has also been implicated in N-myc degradation in vitro. Here we report that, in vivo, N-myc is a sensitive substrate for the 26S proteasome in N-myc amplified neuroblastoma cells. We observed that inhibition of the 26S proteasome with two inhibitors, ALLnL and lactacystin, led to an elevation of the N-myc protein steady-state and increased N-myc protein polyubiquitination, as revealed by ubiquitin Western blotting. Pulse-chase experiments have shown that the increased N-myc levels resulted from stabilization of the protein. In contrast treatment with several calpain and cathepsin inhibitors failed to block N-myc degradation in vivo. Furthermore, fluorescence microscopy of ALLnL-treated cells localized N-myc exclusively to the nuclear compartment, suggesting the absence of a requirement for transport to the cytoplasm prior to degradation.

Topics: Acetylcysteine; Calpain; Cysteine Proteinase Inhibitors; Humans; Lysosomes; Neuroblastoma; Peptide Hydrolases; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-myc; Substrate Specificity; Tumor Cells, Cultured; Ubiquitins

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the transamidation of specific polypeptide-bound glutamine residues, a reaction that is inhibited by GTP. There is also preliminary evidence that, in situ, calpain and GTP may regulate tTG indirectly by modulating its turnover by the calcium-activated protease calpain. In the present study, the in vitro and in situ proteolysis of tTG by calpain, and modulation of this process by GTP, was examined. tTG is an excellent substrate for calpain and is rapidly degraded. Previously it has been demonstrated that GTP binding protects tTG from degradation by trypsin. In a similar manner, guanosine-5'-O-(3-thiotriphosphate) protects tTG against proteolysis by calpain. Treatment of SH-SY5Y cells with 1 nM maitotoxin, which increases intracellular calcium levels, resulted in a significant increase in in situ TG activity, with only a slight decrease in tTG protein levels. In contrast, when GTP levels were depleted by pretreating the cells with tiazofurin, maitotoxin treatment resulted in an approximately 50% decrease in tTG protein levels, and a significant decrease in TG activity, compared with maitotoxin treatment alone. Addition of calpain inhibitors inhibited the degradation of tTG in response to the combined treatment of maitotoxin and tiazofurin and resulted in a significant increase in in situ TG activity. These studies indicate that tTG is an endogenous substrate of calpain and that GTP selectively inhibits the degradation of tTG by calpain.

Topics: Antineoplastic Agents; Calcium; Calpain; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Proteinase Inhibitors; Diazomethane; Enzyme Inhibitors; Enzyme Precursors; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Marine Toxins; Neuroblastoma; Oligopeptides; Oxocins; Ribavirin; Substrate Specificity; tau Proteins; Transglutaminases; Tumor Cells, Cultured

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

Two cysteine protease families (caspase and calpain) participate in apoptosis. Here we report that the endogenous calpain inhibitor calpastatin is fragmented by caspase(s) to various extents during early apoptosis in two cell types. In anti-fas or staurosporine-treated Jurkat T-cells, the high-molecular-weight form (HMW) of calpastatin (apparent Mr 110 K) was extensively degraded to immunoreactive fragments of Mr 75 K and 30 K In apoptotic SH-SY5Y human neuroblastoma cells, HMW calpastatin was degraded to a major immunoreactive fragment of 75 K. In both cell types, fragmentation of HMW calpastatin was blocked by a caspase-specific inhibitor carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene. In vitro translated HMW calpastatin was sensitive to proteolysis by recombinant caspase-1, -3, and -7. By contrast, in vitro translated LMW calpastatin (which lacks domains L and I) was cleaved into multiple fragments only by caspase-1 and was relatively resistant to caspase-3, -7, and other caspases tested. Consistently with that, purified erythroid LMW calpastatin was also highly susceptible to caspase-1 digestion. Recombinant human calpastatin spanning domain I through III (CAST(DI-III)) was found cleaved by caspase-1 at at least three sites, located in either the A or the C helix of domains I and III (ALDD137*L, LSSD203*F and ALAD404*S), while only a single site (ALDD137*L) was cleaved by caspase-3. These findings suggest that both HMW and LMW calpastatins are more vulnerable to caspase-1 than to caspase-3. Surprisingly, both erythroid LMW calpastatin and recombinant CAST(DI-III) fragmented by caspase-1 suffered only a less than twofold reduction of inhibitory activity toward calpain. We propose that the proteolysis of calpastatin in early apoptosis might have yet unidentified effects on the cross-talk between the two protease systems.

Topics: Apoptosis; Binding Sites; Calcium-Binding Proteins; Calpain; Caspase 1; Caspase 3; Caspases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Humans; Hydrolysis; Jurkat Cells; Molecular Weight; Neuroblastoma; Recombinant Proteins; Tumor Cells, Cultured

DAN gene was first isolated by differential screening between rat 3Y1 and v-src-transformed 3Y1 cells and showed a tumor-suppressive activity toward v-src-transformed 3Y1 cells. When DAN-transfected neuroblastoma cells were treated with a tumor promoter phorbol ester, TPA, neurite-like processes appeared within 2 h whereas no apparent change was observed in the parent and vector-transfected cells up to 8 h. This suggests some difference in TPA-receptor, protein kinase C (PKC), between DAN-transfectants and the control cells. DAN-transfected SH-SY5Y cells showed complete loss in PKCalpha and a large decrease in PKCgamma. Similar down-regulation in PKCalpha and PKCgamma was also observed in DAN-transfected Ha-ras-transformed NIH 3T3 cells. The decreased level of PKCalpha was partially recovered after treatment with a calpain inhibitor, ZLLH. A 150-kDa proteolytic product of a calpain-specific substrate, non-erythroid alpha-spectrin, was detectable in DAN-transfected SH-SY5Y cells but not in the parent or vector-transfected control cells. This suggests that DAN-transfected cells contain activated calpain which may cause down-regulation of PKC and hence induce the altered TPA response.

Topics: 3T3 Cells; Animals; Blotting, Western; Calpain; Carrier Proteins; Cell Cycle Proteins; Cytokines; Dexamethasone; Down-Regulation; Fibroblasts; Humans; Isoenzymes; Mice; Microfilament Proteins; Molecular Weight; Nerve Tissue Proteins; Neurites; Neuroblastoma; Neurons; Protein Kinase C; Protein Kinase C-alpha; Proteins; Rats; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured

Calpains have importance in human neurodegenerative disease pathogenesis, but these mechanisms are difficult to study in postmortem tissues. To establish a cellular model of the human calpain and calpastatin system, we characterized calpain I, calpain II, and calpastatin in SH-SY5Y human neuroblastoma cells in relation to their counterparts in human brain and investigated their expression and activity after inducing cellular differentiation with retinoic acid (RA), a physiological effector of normal brain development. Calpain I in both SH-SY5Y cells and human brain existed in the cytosolic and particulate fractions as three isoforms (80, 78, and 76 kDa) and exhibited atypical isoelectric focusing behavior. Calpain II in SH-SY5Y cells, as in human brain, migrated as a single predominantly cytosolic 76-kDa protein with an isoelectric point ranging from 5.9 to 6.3. Calpastatin from both sources was also 90% cytosolic. In the cells it was composed of four discrete bands, ranging in molecular weight from 110 to 127 kDa. Levels of activated (76 and 78 kDa) and precursor (80 kDa) calpain I isoforms rose 54% (P < 0.0001) in the particulate fraction and 26% (P < 0.0001) in the soluble fraction after 3 days of RA exposure. Because levels and activity of calpastatin remain unchanged during the first 7 days of RA exposure, the increased abundance of calpain I implies a net activation of the calpain system during differentiation. Calpain I activation may contribute to the remodeling of cell shape and neurite extension/retraction associated with neuronal differentiation.

Topics: Brain; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cysteine Proteinase Inhibitors; Humans; Isomerism; Molecular Weight; Neurites; Neuroblastoma; Tretinoin; Tumor Cells, Cultured

Regulation of the microM-calcium-requiring form of calpain (mu calpain) was studied in SH-SY-5Y human neuroblastoma cells. Immunoblot analysis demonstrated that the vast majority of mu calpain is localized within cytosolic pools. Calpain activation was monitored as a function of autolysis within intact cells following calcium influx from the culture medium by calcium ionophores A23187 or ionomycin, or following release of calcium from intracellular stores by thapsigargin. Within intact neuronal cells, following an influx of calcium into the cytosolic from either extracellular or intracellular sources, mu calpain is preferentially activated at the plasma membrane as evidenced by autolytic generation of faster-migrating isoforms. By contrast, similar autolytic profiles for mu calpain in membrane or cytosolic fractions following addition of calcium were observed under cell-free conditions and within cells following death due to extended ionophore-mediated calcium influx. These differential activation profiles for cytosolic mu calpain within living cells and following cellular fractionation/cell death indicate the presence of a regulatory system within neuronal cells. As in previous studies in other systems, we demonstrate the presence of a calpain activator protein. Cycloheximide treatment depleted the autolytic capacity of membrane-associated mu calpain within 4-6 hr without a corresponding decline in total mu calpain protein levels, indicating that the activator protein undergoes rapid turnover in comparison to calpain; pulse-chase radiolabeling confirmed the half-life of mu calpain to exceed 24 hr. Our data suggest that this labile protein represents a major rate-limiting step for in situ calpain activation within neuronal cells, and that, given the tremendous latent mu calpain activity within the cytosol, the interplay of the activator protein and the endogenous inhibitor calpastatin are crucial for maintaining neuronal homeostasis.

Topics: Calcimycin; Calcium; Calpain; Cell Membrane; Cycloheximide; Cytosol; Enzyme Activation; Enzyme Inhibitors; Enzyme Precursors; Half-Life; Humans; Ionomycin; Ionophores; Isoenzymes; Membrane Proteins; Neoplasm Proteins; Nerve Tissue Proteins; Neuroblastoma; Neurons; Protein Synthesis Inhibitors; Thapsigargin; Tumor Cells, Cultured

SH-SY-5Y human neuroblastoma cells were treated with 22 microM of a synthetic peptide corresponding to amino acid residues 25-35 of beta-amyloid (betaA) or 3 microM calcium ionophore A23187 in culture medium containing 1.8 mM extracellular calcium. Both agents increased tau immunoreactivity towards antibodies (PHF-1, ALZ-50) that recognize epitopes common with paired helical filaments (PHFs) and towards an antibody (5E2) that recognized a phosphate-independent tau epitope. However, only ionophore increased immunoreactivity with an additional phosphate-dependent antibody (AT-8) that recognized an epitope of tau when phosphorylated, and induced a corresponding decrease in immunoreactivity towards an additional antibody (Tau-1) that recognizes the same site when that site is not phosphorylated. Moreover, the ionophore-mediated increase in PHF-1 was blocked by EGTA, by the calpain inhibitor calpeptin and by the PKC inhibitor H7, while that evoked by betaA treatment was not inhibited by any of these treatments. Since ionophore-mediated calpain activation induces proteolytic PKC activation, we further examined the influence of PKC inhibition on betaA and ionophore-mediated PHF-1 induction. Antisense oligonucleotide-mediated downregulation of PKCepsilon in a stable transfectant SH-SY-5Y subclone diminished the ionophore-mediated, but not the betaA-mediated, increase in PHF-1 immunoreactivity. These data indicate specific differences in the intracellular cascade of events invoked by betaA and ionophore A23187. Moreover, although betaA invoked calcium influx in these cells, our findings further suggest that the induction of tau hyperphosphorylation by betaA may not be due to calcium influx.

Topics: Amyloid beta-Peptides; Antibodies, Monoclonal; Antibody Specificity; Biomarkers; Calcimycin; Calcium; Calpain; Epitopes; Humans; Ionophores; Neuroblastoma; Phosphoproteins; Phosphorylation; Protein Kinase C; tau Proteins; Tumor Cells, Cultured

Calcium influx into SH-SY5Y human neuroblastoma cells after ionophore treatment or transient permeabilization in calcium-containing medium increased ALZ-50 immunoreactivity markedly. This increase was prevented by inhibitors active against calpain or against protein kinase C (PKC), suggesting that both of these enzymes were required to mediate the effect of calcium influx on ALZ-50 immunoreactivity. Treatment with PKC activator TPA increased ALZ-50 immunoreactivity in the absence of calcium influx or after intracellular delivery of the specific calpain inhibitor calpastatin, indicating that the influence of PKC was downstream from that of calpain. Calcium influx also resulted in mu-calpain autolysis (one index of calpain activation) and the transient appearance of PKM (i.e., free PKC catalytic subunits, generated by calpain-mediated cleavage of the regulatory and catalytic PKC domains). Inhibition of calpain within intact cells resulted in a dramatic increase in steady-state levels of total tau (migrating at 46-52 kDa) but resulted in a relatively minor increase in 68-kDa ALZ-50-immunoreactive tau isoforms. Although calcium influx into intact cells resulted in accumulation of ALZ-50 immunoreactivity, total tau levels were, by contrast, rapidly depleted. Incubation of isolated fractions with calpain in the presence of calcium indicated that ALZ-50-immunoreactive tau isoforms were more resistant to calpain-mediated proteolysis than were non-ALZ-50 reactive tau isoforms. These data therefore indicate that calpain may regulate tau levels directly via proteolysis and indirectly through PKC activation. A consequence of the latter action is altered tau phosphorylation, perhaps involving one or more kinase cascades, and the preferential accumulation of ALZ-50-immunoreactive tau isoforms due to their relative resistance to degradation. These findings provide a basis for the possibility that disregulation of calcium homeostasis may contribute to the pathological levels of conversion of tau to A68 by hyperactivation of the calpain/PKC system.

Topics: Antigens; Calcimycin; Calcium; Calpain; Endopeptidases; Humans; Hydrolysis; Immunoblotting; Isomerism; Nerve Tissue Proteins; Neuroblastoma; Phosphorylation; Protein Kinase C; tau Proteins; Tumor Cells, Cultured

The cytoskeletal protein non-erythroid alpha-spectrin is well documented as an endogenous calpain substrate, especially under pathophysiological conditions. In cell necrosis (e.g. maitotoxin-treated neuroblastoma SH-SY5Y cells), alpha-spectrin breakdown products (SBDPs) of 150 kDa and 145 kDa were produced by cellular calpains. In contrast, in neuronal cells undergoing apoptosis (cerebellar granule neurons subjected to low potassium and SH-SY5Y cells treated with staurosporine), an additional SBDP of 120 kDa was also observed. The formation of the 120 kDa SBDP was insensitive to calpain inhibitors but was completely blocked by an interleukin 1 beta-converting-enzyme (ICE)-like protease inhibitor, Z-Asp-CH2OC(O)-2,6-dichlorobenzene. Autolytic activation of both calpain and the ICE homologue CPP32 was also observed in apoptotic cells. alpha-Spectrin can also be cleaved in vitro by purified calpains to produce the SBDP doublet of 150/145 kDa and by ICE and ICE homologues [ICH-1, ICH-2 and CPP32(beta)] to produce a 150 kDa SBDP. In addition, CPP32 and ICE also produced a 120 kDa SBDP. Furthermore inhibition of either ICE-like protease(s) or calpain protects both granule neurons and SH-SY5Y cells against apoptosis. Our results suggest that both protease families participate in the expression of neuronal apoptosis.

Topics: Amino Acid Sequence; Animals; Apoptosis; Calpain; Caspase 1; Cell Line; Cells, Cultured; Cerebellum; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Humans; Kinetics; Marine Toxins; Molecular Sequence Data; Neuroblastoma; Neurons; Oxocins; PC12 Cells; Rats; Spectrin; Staurosporine; Substrate Specificity; Tumor Cells, Cultured

Treatment of neuroblastoma cultures with N-methyl-D-aspartate (NMDA) or human immunodeficiency virus type 1 (HIV-1) coat protein, gp120, induces significant cytotoxic effects which are reduced by leupeptin, E-64, N-Ac-Leu-Leu-norleucinal (ALLnL) as well as by N-Ac-Leu-Leu-normethioninal (ALLnM) and this suggests that activation of the Ca(2+)-dependent protease, calpain, is involved. The cell death induced by NMDA and gp120 appears to be of the necrotic type; in fact, analysis of DNA fragmentation by flow cytometry or agarose gel electrophoresis failed to demonstrate signs of apoptosis, such as the presence of apoptotic bodies or internucleosomal cleavage. Similar negative results were also obtained by studying the nuclear morphology of the cells with Hoechst 33258 staining. Altogether the data indicate that neuroblastoma cell death induced by NMDA and gp120 is of the necrotic type and this implicates calpain protease.

Topics: Calcium; Calpain; Cell Death; Cell Nucleus; Cysteine Proteinase Inhibitors; Enzyme Activation; HIV Envelope Protein gp120; Humans; N-Methylaspartate; Necrosis; Neuroblastoma; Neurons; Neurotoxins; Receptors, N-Methyl-D-Aspartate; Tumor Cells, Cultured

Both ice-like protease and calpain have been shown to be involved in apoptosis in non-neuronal cells. Cultured rat cerebellar granule neurons undergo apoptosis when exposed to low potassium-containing medium. Calpain inhibitors 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) and N-acetyl-Leu-Leu-Met-CHO (calpain inhibitor II) as well as interleukin-beta 1 converting enzyme (ICE)-like protease inhibitor Z-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-D-DCB) protect against such apoptotic death. They also reduce DNA laddering and the number of apoptotic nuclei. Staurosporine treatment also evokes apoptosis in human neuroblastoma SH-SY5Y. While Z-D-DCB is again anti-apoptotic, calpain inhibitors only provide modest effects in this model. Our results suggest that ICE-like protease plays a critical role in neuronal apoptosis whereas the contributions of calpain are more cell-type dependent.

Topics: Animals; Apoptosis; Brain Neoplasms; Calpain; Caspase 1; Coloring Agents; Cysteine Endopeptidases; DNA Fragmentation; Humans; L-Lactate Dehydrogenase; Neuroblastoma; Neurons; Protease Inhibitors; Rats; Tumor Cells, Cultured

The protein kinase C inhibitor bisindolylmaleimide GF109203X has a dual effect on the behavior of the neuroblastoma cell line Neuro-2A; when the inhibitor is added in conditions that induce differentiation (absence of serum), neurite outgrowth is potentiated in a dose-dependent manner. However, if the inhibitor is added in growth-promoting conditions (presence of serum), programmed cell death (apoptosis) is induced, as assessed by internucleosomal DNA cleavage and specific immunoassays. This effect is also seen with other specific protein kinase C inhibitors. Bcl2 gene overexpression protects Neuro-2A cells from apoptosis, as has been found in other systems. We also show that calpain I, a neutral Ca(2+)-activated proteinase, participates in this apoptotic pathway. Our results point to a key role of protein kinase C in the regulation of growth and differentiation in Neuro-2A cells.

Topics: Animals; Apoptosis; Blood Proteins; Calpain; Cell Differentiation; Cell Division; Enzyme Inhibitors; Gene Expression; Indoles; Maleimides; Mice; Neuroblastoma; Protein Kinase C; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

Changes in expression of calpains and calpastatin during differentiation in GOTO cells were examined using antibodies specific to calpains and calpastatin. Neuronal differentiation induced by dibutyryl cyclic AMP elicited a remarkable decrease in m-calpain. No marked changes in the levels of calpains were found in bromodeoxyuridine-induced Schwannian differentiation. Calpastatin was detected as a single band of 110k in undifferentiated and in neuronally differentiated cells by Western blot analysis. However, the appearance of a 120k species was detected in Schwannian differentiation associated with morphological change. The data show that marked changes in m-calpain and calpastatin occur in a differentiation-specific manner.

Topics: Bromodeoxyuridine; Bucladesine; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cytosol; Humans; Neuroblastoma; Neurons; Schwann Cells; Tumor Cells, Cultured

Proteolytic cleavage of protein kinase C (PKC) under cell-free conditions generates a co-factor independent, free catalytic subunit (PKM). However, the difficulty in visualizing PKM in intact cells has generated controversy regarding its physiological relevance. In the present study, treatment of SH-SY-5Y cells with 2-O-tetradecanoylphorbol 13-acetate resulted in complete down-regulation of PKC within 24 h without detection of PKM. By contrast, low levels of PKM were transiently detected following ionophore-mediated calcium influx under conditions which induced no detectable PKC loss. PKM was not detected during rapid cell-free degradation of partially purified SH-SY-5Y PKC alpha by purified human brain mM calpain. However, when the kinetics of PKC degradation were slowed by lowering levels of calpain, PKM was transiently detected. PKM was also only transiently observed following calpain-mediated degradation of purified rat brain PKC alpha. Densitometric analyses indicated that, once formed, PKM was degraded approximately 10 times faster than PKC. These data provide an explanation as to why PKM is difficult to observe in situ, and indicate that PKM should not be considered as an 'unregulated' kinase, since its persistence is apparently strictly regulated by proteolysis.

Topics: Calpain; Cell-Free System; Humans; In Vitro Techniques; Isoenzymes; Neuroblastoma; Protein Kinase C; Protein Kinase C-alpha; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured

Mouse NB2a/dl neuroblastoma cells elaborate axonal neurites in response to various chemical treatments including dibutyryl cyclic AMP and serum deprivation. Hirudin, a specific inhibitor of thrombin, initiated neurite outgrowth in NB2a/dl cells cultured in the presence of serum; however, these neurites typically retracted within 24 h. The cysteine protease inhibitors leupeptin and N-acetyl-leucyl-leucyl-norleucinal (CI; preferential inhibitor of micromolar calpain but also inhibits millimolar calpain) at 10(-6) M considerably enhanced neurite outgrowth induced by serum deprivation, but could not induce neuritogenesis in the presence of serum. A third cysteine protease inhibitor, N-acetyl-leucyl-leucyl-methional (CII; preferential inhibitor of millimolar calpain but also inhibits micromolar calpain), had no detectable effects by itself. Cells treated simultaneously with hirudin and either leupeptin, CI, or CII elaborated stable neurites in the presence of serum. Cell-free enzyme assays demonstrated that hirudin inhibited thrombin but not calpain, CI and CII inhibited calpain but not thrombin, and leupeptin inhibited both proteases. These results imply that distinct proteolytic events, possibly involving more than one protease, regulate the initiation and subsequent elongation and stabilization of axonal neurites. Since the addition of exogenous thrombin or calpain to serum-free medium did not modify neurite outgrowth, the proteolytic events affected by these inhibitors may be intracellular or involve proteases distinct from thrombin or calpain.

Topics: Animals; Axons; Blood; Calpain; Endopeptidases; Hirudins; Leupeptins; Neuroblastoma; Osmolar Concentration; Protease Inhibitors; Thrombin; Tumor Cells, Cultured

Calpain is known to play a variety of cellular functions in various cells by Ca2(+)-dependent limited proteolysis. Protein kinase C (PK-C) is a key enzyme in signal transduction. It is known that treatment of a cell with 12-0-tetradecanoylphorbol 13-acetate (TPA) causes down-regulation of PK-C, and that calpain can cleave PK-C into catalytic and regulatory fragments in vitro. In vivo involvement of calpain in down-regulation of PK-C was studied with neuroblastoma cells using various drugs, a synthetic peptide fragment of calpastatin and inhibitors against calpain. TPA-dependent down-regulation of PK-C was partially inhibited by pre-treatment with calpastatin peptide and inhibitors, suggesting in vivo involvement of calpain in down-regulation of PK-C during signal transduction.

Topics: Amino Acid Sequence; Calpain; Enzyme Activation; Humans; Molecular Sequence Data; Neoplasm Proteins; Neuroblastoma; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured