Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0027819 (neuroblastoma)
27,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Calpains and calpastatin in SH-SY5Y neuroblastoma cells during retinoic acid-induced differentiation and neurite outgrowth: comparison with the human brain calpain system. 916 Feb 41

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.
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PMID:Restriction of microM-calcium-requiring calpain activation to the plasma membrane in human neuroblastoma cells: evidence for regionalized influence of a calpain activator protein. 921 May 24

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.
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PMID:Beta-amyloid and ionophore A23187 evoke tau hyperphosphorylation by distinct intracellular pathways: differential involvement of the calpain/protein kinase C system. 933 63

We report the pharmacological characterization and cytoprotective effect of DY-9760e, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-( 4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, a novel antagonist of calmodulin. DY-9760e inhibited calmodulin-dependent enzymes, including calmodulin-dependent protein kinase II and IV, calcineurin, [corrected] calmodulin-dependent phosphodiesterase and myosin light chain kinase with Ki values of 1.4, 12, 2.0, 3.8 and 133 microM, respectively. These antagonistic effects of DY-9760e were more potent than those of W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, another calmodulin antagonist. This compound showed little or no effect on calmodulin-independent enzymes, such as protein kinase A and C and calpain I and II. Analysis of the hydrophobic interaction of DY-9760e with calmodulin by using 2-p-toluidinylnaphthalene-6-sulfonate and 9-anthroylcholine revealed that, like W-7, DY-9760e bound to the hydrophobic regions of calmodulin. The [14C]DY-9760e binding assay indicated that DY-9760e bound to calmodulin at one class of binding site. Finally, DY-9760e substantially protected N1E-115 neuroblastoma cells from cytotoxicity induced by the Ca2+ ionophore, A23187. These results indicate that DY-9760e, a novel calmodulin antagonist, possesses a cytoprotective action and suggest that calmodulin plays a critical role in mediating some of the biochemical events leading to cell death following Ca2+ overload.
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PMID:DY-9760e, a novel calmodulin antagonist with cytoprotective action. 938 59

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro studies have demonstrated that the transamidating activity of tTG requires calcium and is inhibited by GTP. To investigate the endogenous regulation of tTG, a quantitative in situ transglutaminase (TG) activity assay was developed. Treatment of human neuroblastoma SH-SY5Y cells with retinoic acid (RA) resulted in a significant increase in tTG levels and in vitro TG activity. In contrast, basal in situ TG activity did not increase concurrently with RA-induced increased tTG levels. However, stimulation of cells with the calcium-mobilizing drug maitotoxin (MTX) resulted in increases in in situ TG activity that correlated (r2 = 0.76) with increased tTG levels. To examine the effects of GTP on in situ TG activity, tiazofurin, a drug that selectively decreases GTP levels, was used. Depletion of GTP resulted in a significant increase in in situ TG activity; however, treatment of SH-SY5Y cells with a combination of MTX and tiazofurin resulted in significantly less in situ TG activity compared with treatment with MTX alone. This raised the possibility of calcium-dependent proteolysis due to the effects of tiazofurin, because in vitro GTP protects tTG against proteolysis by trypsin. Studies with a selective membrane permeable calpain inhibitor indicated that tTG is likely to be an endogenous substrate of calpain, and that depletion of GTP increases tTG degradation after elevation of intracellular calcium levels. TG activity was also increased in response to activation of muscarinic cholinergic receptors, which increases intracellular calcium through inositol 1,4,5-trisphosphate generation. The results of these experiments demonstrate that selective changes in calcium and GTP regulate the activity and levels of tTG in situ.
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PMID:Modulation of the in situ activity of tissue transglutaminase by calcium and GTP. 944 73

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.
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PMID:Calcium ionophore-induced degradation of neurofilament and cell death in MSN neuroblastoma cells. 948 52

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.
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PMID:In vivo degradation of N-myc in neuroblastoma cells is mediated by the 26S proteasome. 952 55

In this study, the effects of oxidative stress on calpain-mediated proteolysis and calpain I autolysis in situ were examined. Calpain activity was stimulated in SH-SY5Y human neuroblastoma cells with the calcium ionophore, ionomycin. Calpain-mediated proteolysis of the membrane-permeable fluorescent substrate N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcouma rin, as well as the endogenous protein substrates microtubule-associated protein 2, tau and spectrin, was measured. Oxidative stress, induced by addition of either doxorubicin or 2-mercaptopyridine N-oxide, resulted in a significant decrease in the extent of ionophore-stimulated calpain activity of both the fluorescent compound and the endogenous substrates compared with control, normoxic conditions. Addition of glutathione ethyl ester, as well as other antioxidants, resulted in the retention/recovery of calpain activity, indicating that oxidation-induced calpain inactivation was preventable/reversible. The rate of autolytic conversion of the large subunit of calpain I from 80 to 78 to 76 kDa was decreased during oxidative stress; however, the extent of calpain autolysis was not altered. These data indicate that oxidative stress may reversibly inactivate calpain I in vivo.
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PMID:Oxidative stress inhibits calpain activity in situ. 958 80

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.
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PMID:Calcium/calmodulin-dependent protein kinase IV is cleaved by caspase-3 and calpain in SH-SY5Y human neuroblastoma cells undergoing apoptosis. 968 36

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.
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PMID:Caspase-mediated fragmentation of calpain inhibitor protein calpastatin during apoptosis. 970 9


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