3-nitrotyrosine and Neuroblastoma

Mitochondria are the major site of adenosine triphosphate (ATP) production in mammalian cells. Moreover, mitochondria produce most of the reactive oxygen species (ROS) in nucleated cells. Redox and bioenergetic abnormalities have been seen in mitochondria during the onset and progression of neurodegenerative diseases. In that context, excitotoxicity induced by glutamate (GLU) plays an important role in mediating neurotoxicity. Several drugs have been used in the treatment of diseases involving excitotoxicity. Nonetheless, some patients (20-30%) present drug resistance. Thus, it is necessary to find chemicals able to attenuate mitochondrial dysfunction in the case of excitotoxicity. In this work, we treated the human neuroblastoma SH-SY5Y cell line with the diterpene carnosic acid (CA) at 1 μM for 12 h prior to the exposure to GLU for further 24 h. We found that CA prevented the GLU-induced mitochondrion-related redox impairment and bioenergetic decline in SH-SY5Y cells. CA also downregulated the pro-apoptotic stimulus elicited by GLU in this experimental model. CA exerted mitochondrial protection by a mechanism associated with the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), since silencing of this protein with small interfering RNA (siRNA) suppressed the CA-induced protective effects. Future directions include investigating whether CA would be able to modulate mitochondrial function and/or dynamics in in vivo experimental models of excitotoxicity.

Topics: Abietanes; Apoptosis; Cell Line, Tumor; Cell Survival; Glutamic Acid; Humans; Membrane Potential, Mitochondrial; Mitochondria; Neuroblastoma; Neurons; Neuroprotective Agents; NF-E2-Related Factor 2; Nitric Oxide; Oxidation-Reduction; Reactive Oxygen Species; Tyrosine

The aim of this study was to elucidate whether glutathione is involved in lithium's ability to decrease carbonylation and nitration produced by complex I inhibition, which is consistently found in BD. Neuroblastoma cells were treated with rotenone, a complex I inhibitor. Our results suggest that glutathione is essential for lithium's ability to ameliorate rotenone-induced protein carbonylation, but not nitration.

Topics: Cell Line, Tumor; Cell Survival; Electron Transport Complex I; Glutathione; Humans; Immunohistochemistry; Lithium Compounds; Neuroblastoma; Protein Carbonylation; Rotenone; Tyrosine; Uncoupling Agents

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

Alzheimer's disease neuropathology is characterized by neuronal death, amyloid beta-peptide deposits and neurofibrillary tangles composed of paired helical filaments of tau protein. Although crucial for our understanding of the pathogenesis of Alzheimer's disease, the molecular mechanisms linking amyloid beta-peptide and paired helical filaments remain unknown. Here, we show that amyloid beta-peptide-induced nitro-oxidative damage promotes the nitrotyrosination of the glycolytic enzyme triosephosphate isomerase in human neuroblastoma cells. Consequently, nitro-triosephosphate isomerase was found to be present in brain slides from double transgenic mice overexpressing human amyloid precursor protein and presenilin 1, and in Alzheimer's disease patients. Higher levels of nitro-triosephosphate isomerase (P < 0.05) were detected, by Western blot, in immunoprecipitates from hippocampus (9 individuals) and frontal cortex (13 individuals) of Alzheimer's disease patients, compared with healthy subjects (4 and 9 individuals, respectively). Triosephosphate isomerase nitrotyrosination decreases the glycolytic flow. Moreover, during its isomerase activity, it triggers the production of the highly neurotoxic methylglyoxal (n = 4; P < 0.05). The bioinformatics simulation of the nitration of tyrosines 164 and 208, close to the catalytic centre, fits with a reduced isomerase activity. Human embryonic kidney (HEK) cells overexpressing double mutant triosephosphate isomerase (Tyr164 and 208 by Phe164 and 208) showed high methylglyoxal production. This finding correlates with the widespread glycation immunostaining in Alzheimer's disease cortex and hippocampus from double transgenic mice overexpressing amyloid precursor protein and presenilin 1. Furthermore, nitro-triosephosphate isomerase formed large beta-sheet aggregates in vitro and in vivo, as demonstrated by turbidometric analysis and electron microscopy. Transmission electron microscopy (TEM) and atomic force microscopy studies have demonstrated that nitro-triosephosphate isomerase binds tau monomers and induces tau aggregation to form paired helical filaments, the characteristic intracellular hallmark of Alzheimer's disease brains. Our results link oxidative stress, the main etiopathogenic mechanism in sporadic Alzheimer's disease, via the production of peroxynitrite and nitrotyrosination of triosephosphate isomerase, to amyloid beta-peptide-induced toxicity and tau pathology.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Blotting, Western; Case-Control Studies; Cell Line; Cell Line, Tumor; Frontal Lobe; Humans; Immunohistochemistry; Mice; Mice, Transgenic; Microscopy, Atomic Force; Microscopy, Confocal; Microscopy, Electron; Models, Molecular; Neuroblastoma; Neurofibrillary Tangles; Oxidative Stress; Peroxynitrous Acid; Phosphorylation; tau Proteins; Triose-Phosphate Isomerase; Tyrosine

Diabetic complications can often affect the central nervous system since the chronic exposure to hyperglycemia can result in the production of high concentration of reactive oxygen species with subsequent damage of several cell structures such as the cytoskeleton. In order to antagonize the oxidative status many substances have been tested as antioxidants. In the present work attention has been focused on the possible nitrosative effect of hyperglycemia on microtubular network of neuroblastoma and glioma mortalized cell lines, testing the possible neuroprotective effect of testosterone.. Neuroblastoma (C1300) and glioma (C6) cell lines were cultured in the presence of 300 mM (C1300) or 310 mM (C6) D-glucose, with or without 50 nM testosterone. After 72 hrs, morphology, growth rate, cell viability and catalase activity were evaluated. In addition, with the aim to detect any changes in the amount of tubulin isoforms, Western blot analysis was performed.. In D-glucose-exposed cells, it was found a down-regulation of tubulin isoforms and an increase in 3-nitro-L-tyrosine and subsequent modifications in cell morphology, growth rate, viability and catalase activity. All these changes were more severe in neuroblastoma than in glioma cell line. When testosterone was added to the medium, all the parameters were very similar to controls. This neuroprotective action was well-detectable in C1300 cells, whereas testosterone was not able to recover significantly in C6 cells.. Our results displayed: i) a selective action of high glucose on microtubules; ii) a different sensitivity to oxidative stress in neuronal and glial cells; iii) a different neuroprotective action of testosterone on neuronal and glial cells.

Topics: Androgens; Animals; Catalase; Cell Division; Cell Line, Transformed; Cell Survival; Down-Regulation; Drug Interactions; Glioma; Glucose; Mice; Microtubules; Neuroblastoma; Neuroglia; Neurons; Neuroprotective Agents; Oxidative Stress; Rats; Reactive Nitrogen Species; Testosterone; Tubulin; Tyrosine

Parkinson's disease is a progressive neurodegenerative disorder characterized by selective degeneration of nigrostriatal dopaminergic neurons. Long-term systemic mitochondrial complex I inhibition by rotenone induces selective degeneration of dopaminergic neurons in rats. We have reported dopamine redistribution from vesicles to the cytosol to play a crucial role in selective dopaminergic cell apoptosis. In the present study, we investigated how rotenone causes dopamine redistribution to the cytosol using an in vitro model of human dopaminergic SH-SY5Y cells. Rotenone stimulated nitration of the tyrosine residues of intracellular proteins. The inhibition of nitric-oxide synthase or reactive oxygen species decreased the amount of nitrotyrosine and attenuated rotenone-induced apoptosis. When we examined the intracellular localization of dopamine immunocytochemically using anti-dopamine/vesicular monoamine transporter 2 (VMAT2) antibodies and quantitatively using high-performance liquid chromatography, inhibiting nitration was found to suppress rotenone-induced dopamine redistribution from vesicles to the cytosol. We demonstrated rotenone to nitrate tyrosine residues of VMAT2 using an immunocytochemical method with anti-nitrotyrosine antibodies and biochemically with immunoprecipitation experiments. Rotenone inhibited the VMAT2 activity responsible for the uptake of dopamine into vesicles, and this inhibition was reversed by inhibiting nitration. Moreover, rotenone induced the accumulation of aggregate-like formations in the stained image of VMAT2, which was reversed by inhibiting nitration. Our findings demonstrate that nitration of the tyrosine residues of VMAT2 by rotenone leads to both functional inhibition and accumulation of aggregate-like formations of VMAT2 and consequently to the redistribution of dopamine to the cytosol and apoptosis of dopaminergic SH-SY5Y cells.

Topics: Apoptosis; Cell Line, Tumor; Cytosol; DNA Fragmentation; Dopamine; Dose-Response Relationship, Drug; Electron Transport Complex I; Fluorescent Antibody Technique, Direct; Humans; Mitochondria; Neuroblastoma; NG-Nitroarginine Methyl Ester; Nitrogen; Piperazines; Rotenone; Time Factors; Tyrosine; Uncoupling Agents; Vesicular Monoamine Transport Proteins

Oxidative stress is one of the hypotheses involved in the etiology of Alzheimer's disease (AD). Considerable attention has been focused on increasing the intracellular glutathione (GSH) levels in many neurodegenerative diseases, including AD. Pycnogenol (PYC) has antioxidant properties and stabilizes intracellular antioxidant defense systems including glutathione levels. The present study investigated the protective effects of PYC on acrolein-induced oxidative cell toxicity in cultured SH-SY5Y neuroblastoma cells. Decreased cell survival in SH-SY5Y cultures treated with acrolein correlated with oxidative stress, increased NADPH oxidase activity, free radical production, protein oxidation/nitration (protein carbonyl, 3-nitrotyrosine), and lipid peroxidation (4-hydroxy-2-nonenal). Pretreatment with PYC significantly attenuated acrolein-induced cytotoxicity, protein damage, lipid peroxidation, and cell death. A dose-response study suggested that PYC showed protective effects against acrolein toxicity by modulating oxidative stress and increasing GSH. These findings provide support that PYC may provide a promising approach for the treatment of oxidative stress-related neurodegenerative diseases such as AD.

Topics: Acrolein; Aldehydes; Analysis of Variance; Blotting, Western; Cell Line, Tumor; Cell Survival; Cytotoxins; Flavonoids; Free Radicals; Glutathione; Humans; Lipid Peroxidation; Luminescence; NADPH Oxidases; Neuroblastoma; Neurons; Neuroprotective Agents; Oxidative Stress; Plant Extracts; Protein Carbonylation; Tyrosine

The microtubular network of neurons is involved in several functions such as formation and tropism of cellular processes, cell division and intracellular transport. A lot of evidences testify that the microtubular network of neurons can be impaired by oxidative stress. A condition of oxidative stress is often possible when D-glucose overloads its metabolic pathway, resulting in an increase in reactive oxygen species and subsequent neurological disorders. The aim of this work was to check in undifferentiated mouse neuroblastoma cells (C1300) the possible oxidative effects of D-glucose on microtubules. Using a concentration of 110mM D-glucose, cell morphology, growth rate, viability and catalase activity were seriously altered. Noteworthy, an increase in 3-nitro-L-tyrosine and a downregulation of tubulins was found in D-glucose-exposed cells, whereas another cytoskeletal proteins, namely actin, did not show any changes. In conclusion, microtubular network can be impaired by D-glucose through specific nitrosative effects, suggesting a possible mechanism at the basis of hyperglycemia-induced neuronal damage.

Topics: Actins; Animals; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytoskeleton; Down-Regulation; Energy Metabolism; Glucose; Hyperglycemia; Mice; Microtubules; Nerve Degeneration; Neuroblastoma; Neurons; Oxidative Stress; Tubulin; Tyrosine

3-nitro-L-tyrosine is formed by nitric oxide following different pathways such as NADPH oxidase, xanthine oxidase or glutamate NMDA receptor activation and is involved in the pathology of different neurological disorders. Unlike estradiol, a neuroprotective role of androgens against oxidative cell injury has not been fully investigated. This work targets the possible effects of testosterone on neuroblastoma cells exposed to 3-nitro-L-tyrosine. C1300 mouse undifferentiated neuroblastoma cells exposed to 3-nitro-L-tyrosine were cultured in the presence of testosterone. Morphological examination, proliferation and nuclear viability assays were performed. The expression of tyrosinated alpha-tubulin and incorporation of 3-nitro-L-tyrosine into protein were also estimated. Cells exposed to 3-nitro-L-tyrosine showed globular shape, reduced cytoplasmic processes and growth inhibition in comparison with controls. When testosterone was added to the medium, these changes were not evident. In addition, testosterone induced an upregulation of tyrosinated alpha-tubulin, a marker of neuronal plasticity, and a decrease in 3-nitro-L-tyrosine incorporation into tubulin. Our results suggest that testosterone exposure can diminish 3-nitro-L-tyrosine toxic effects on the morphology and growth rate of neuroblastoma cells. The upregulation of tyrosinated alpha-tubulin in testosterone-exposed cells would be consistent with concurrent plasticity events. Failure in alpha-tubulin nitration detected in cells exposed to both 3-nitro-L-tyrosine and testosterone, may support the idea that testosterone interferes with 3-nitro-L-tyrosine protein incorporation. Moreover, testosterone-induced neuroprotection likely entails a linkage with the androgen receptor as is suggested by the flutamide-induced inhibition of the hormone activity. Finally, the neuroprotective effects of testosterone in neuroblastoma cells could deal with the cellular antioxidant defence system, as shown by testosterone-induced increase in catalase activity.

Topics: Androgen Antagonists; Animals; Brain; Catalase; Cell Line, Tumor; Cell Proliferation; Cell Shape; Flutamide; Free Radicals; Mice; Neuroblastoma; Neuronal Plasticity; Neurons; Neuroprotective Agents; Oxidative Stress; Receptors, Androgen; Testosterone; Tubulin; Tyrosine; Up-Regulation

Using undifferentiated mouse neuroblastoma cells (C1300), we have previously observed that testosterone (T) exerts a neuroprotective action against oxidative stress. Nitrogen intermediates induce the production of 3-nitro-L-tyrosine (3NT), an amino acid analogue involved in many neurodegenerative disorders. The aim of our work is to investigate T capability on C1300 cell differentiation. It is also evaluated whether differentiation could mitigate the nitrosative effects of 3NT.. The effects of both T and 3NT were studied on an undifferentiated cell line of neural origin (C1300). For this purpose, cell cultures underwent morphometric investigation, blot analyses and catalase activity assay. All data obtained were expressed as mean+/-SD and tested by one-way ANOVA or Student's t test.. The results were compared with those gathered by means of N6,2'-O-dibutyryl-adenosine-3',5'-cyclic-mono-phosphate (db-cAMP), a well-known differentiating agent. T-exposed cells showed an irregular shape and exhibited long branching cytoplasmic extensions, which were longer than in db-cAMP cells. Moreover, T-exposure induced an increase in the expression of tyrosinated and acetylated alpha-tubulin while 3NT-incorporation into tubulin was markedly reduced. The action of antioxidant defence systems, namely catalase activity, was enhanced in cells exposed to T.. This work highlighted the effects of db-cAMP on differentiation and neuroprotection, but even indicated that T exposure induced differentiation in C1300 cells and this process matches a significant neuroprotective effect. This action seemed to be more effective than in db-cAMP-treated cells. T is suggested, like other substances having antioxidant properties, to be of potential interest in the experimental therapy of neuropathological conditions.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cell Shape; Mice; Neuroblastoma; Neurons; Neuroprotective Agents; Oxidative Stress; Stem Cells; Testosterone; Tyrosine

Many models of Parkinson's disease (PD) have succeeded in replicating dopaminergic neuron loss or alpha-synuclein aggregation but not the formation of classical Lewy bodies, the pathological hallmark of PD. Our cybrid model of sporadic PD was created by introducing the mitochondrial genes from PD patients into neuroblastoma cells that lack mitochondrial DNA. Previous studies using cybrids have shown that information encoded by mitochondrial DNA in patients contributes to many pathogenic features of sporadic PD. In this paper, we report the generation of fibrillar and vesicular inclusions in a long-term cybrid cell culture model that replicates the essential antigenic and structural features of Lewy bodies in PD brain without the need for exogenous protein expression or inhibition of mitochondrial or proteasomal function. The inclusions generated by PD cybrid cells stained with eosin, thioflavin S, and antibodies to alpha-synuclein, ubiquitin, parkin, synphilin-1, neurofilament, beta-tubulin, the proteasome, nitrotyrosine, and cytochrome c. Future studies of these cybrids will enable us to better understand how Lewy bodies form and what role they play in the pathogenesis of PD.

Topics: Aged; alpha-Synuclein; Blotting, Western; Carrier Proteins; Case-Control Studies; Cell Line; Cysteine Endopeptidases; Cytochromes c; DNA, Mitochondrial; Electron Transport Complex I; Female; Humans; Immunohistochemistry; Lewy Bodies; Male; Microscopy, Confocal; Microscopy, Electron; Middle Aged; Multienzyme Complexes; Nerve Tissue Proteins; Neuroblastoma; Neurofilament Proteins; Neurons; Parkinson Disease; Precipitin Tests; Proteasome Endopeptidase Complex; Staining and Labeling; Synucleins; Transgenes; Tubulin; Tyrosine; Ubiquitin; Ubiquitin-Protein Ligases

The amino acid analogue 3-nitrotyrosine (3-NT) is formed in neural cells as a result of the intense stimulation of NMDA glutamate receptors. 3-NT is involved in the pathology of diverse neurodegenerative disorders. The aim of our work is to investigate the sensitivity of cultured neural and glial cells to 3-NT. We report the morphological changes detected on mouse neuroblastoma (C1300) and rat glioma (C6) cell lines cultured in a medium supplemented with different 3-NT concentrations. Western blot displayed a selective incorporation of 3-NT into a single protein that co-migrated with tubulin. Both cell lines showed morphological changes, nuclear suffering, decreased viability and growth inhibition (starting from 90 and 360 microM for C1300 and C6, respectively). Such effects were dose-dependent, though glioma cells showed severe alterations at higher 3-NT concentrations. Our results point out a higher 3-NT sensitivity in the neural cells studied in comparison with those of glial origin. The dramatic toxicity of 3-NT in neural cells suggests further investigations focused on the biochemical mechanisms at the roots of neurodegenerative diseases.

Topics: Animals; Cell Division; Cell Line; Cell Nucleus; Cell Size; Cell Survival; Dose-Response Relationship, Drug; Glioma; Mice; Neuroblastoma; Neurodegenerative Diseases; Neuroglia; Neurons; Rats; Tubulin; Tyrosine

Patients with Down's syndrome (DS) show elevated levels of copper, zinc-containing superoxide dismutase (SOD1) and appear to have increased lipid peroxidation and oxidative damage to DNA as well as elevated glutathione peroxidase activity. Increasing SOD1 levels by gene transfection in NT-2 and SK-N-MC cell lines also led to a rise in glutathione peroxidase activity, but this was nevertheless accompanied by decreased proliferation rates, increased lipid peroxidation and protein carbonyls, and a trend to a rise in 8-hydroxyguanine and protein-bound 3-nitrotyrosine. Transfection of these cell lines with DNA encoding two mutant SOD1 enzymes (G37R and G85R) associated with familial amyotrophic lateral sclerosis (FALS), produced similar, but more severe changes, i.e. even lower growth rates, higher lipid peroxidation, 3-nitrotyrosine and protein carbonyl levels, decreased GSH levels, raised GSSG levels and higher glutathione peroxidase activities. Since G85R has little SOD activity, these changes cannot be related to increased O(2)(-) scavenging. In no case was SOD2 (mitochondrial Mn-SOD) level altered. Our cellular systems reproduce many of the biochemical changes observed in patients with DS or ALS, and in transgenic mice overexpressing mutant SOD1. They also show the potentially deleterious effects of SOD1 overexpression on cellular proliferation, which may be relevant to abnormal development in DS.

Topics: Aldehydes; Amyotrophic Lateral Sclerosis; Antioxidants; Cell Division; Cell Line; Cell Survival; Down Syndrome; Gene Expression; Glutathione; Glutathione Disulfide; Guanine; Humans; Ketones; Lipid Peroxidation; Mutation; Neuroblastoma; Oxidative Stress; Superoxide Dismutase; Superoxide Dismutase-1; Teratocarcinoma; Transfection; Tyrosine

Mutations in alpha-synuclein (A30P and A53T) are involved in some cases of familial Parkinson's disease (FPD), but it is not known how they result in nigral cell death. We examined the effect of alpha-synuclein overexpression on the response of cells to various insults. Wild-type alpha-synuclein and alpha-synuclein mutations associated with FPD were overexpressed in NT-2/D1 and SK-N-MC cells. Overexpression of wild-type alpha-synuclein delayed cell death induced by serum withdrawal or H(2)O(2), but did not delay cell death induced by 1-methyl-4-phenylpyridinium ion (MPP(+)). By contrast, wild-type alpha-synuclein transfectants were sensitive to viability loss induced by staurosporine, lactacystin or 4-hydroxy-2-trans-nonenal (HNE). Decreases in glutathione (GSH) levels were attenuated by wild-type alpha-synuclein after serum deprivation, but were aggravated following lactacystin or staurosporine treatment. Mutant alpha-synucleins increased levels of 8-hydroxyguanine, protein carbonyls, lipid peroxidation and 3-nitrotyrosine, and markedly accelerated cell death in response to all the insults examined. The decrease in GSH levels was enhanced in mutant alpha-synuclein transfectants. The loss of viability induced by toxic insults was by apoptosic mechanism. The presence of abnormal alpha-synucleins in substantia nigra in PD may increase neuronal vulnerability to a range of toxic agents.

Topics: 1-Methyl-4-phenylpyridinium; Aldehydes; alpha-Synuclein; Cell Division; Cell Line; Cell Survival; Clone Cells; Culture Media, Serum-Free; Enzyme Inhibitors; Gene Expression; Glutathione; Guanine; Humans; Hydrogen Peroxide; Ketones; Lipid Peroxidation; Mitochondria; Mutation; Nerve Tissue Proteins; Neuroblastoma; Oxidants; Oxidative Stress; Parkinsonian Disorders; Synucleins; Teratocarcinoma; Transfection; Tyrosine

Mutations in Cu/Zn-superoxide dismutase (SOD1) are associated with some cases of familial amyotrophic lateral sclerosis (ALS). We overexpressed Bcl-2, wild-type SOD1 or mutant SOD1s (G37R and G85R) in NT-2 and SK-N-MC cells. Overexpression of Bcl-2 rendered cells more resistant to apoptosis induced by serum withdrawal, H2O2 or 4-hydroxy-2-trans-nonenal (HNE). Overexpression of Bcl-2 had little effect on levels of protein carbonyls, lipid peroxidation, 8-hydroxyguanine (8-OHG) or 3-nitrotyrosine. Serum withdrawal or H2O2 raised levels of protein carbonyls, lipid peroxidation, 8-OHG and 3-nitrotyrosine, changes that were attenuated in cells overexpressing Bcl-2. Overexpression of either SOD1 mutant tended to increase levels of lipid peroxidation, protein carbonyls, and 3-nitrotyrosine and accelerated viability loss induced by serum withdrawal, H2O2 or HNE, accompanied by greater rises in oxidative damage parameters. The effects of mutant SOD1s were attenuated by Bcl-2. By contrast, expression of wild-type SOD1 rendered cells more resistant to loss of viability induced by serum deprivation, HNE or H2O2. The levels of lipid peroxidation in wild-type SOD1 transfectants were elevated. Overexpression of mutant SOD1s makes cells more predisposed to undergo apoptosis in response to several insults. Our cellular systems appear to mimic events in patients with ALS or transgenic mice overexpressing mutant SOD1.

Topics: Aldehydes; Amino Acid Substitution; Cell Death; Cell Survival; Cross-Linking Reagents; Culture Media, Serum-Free; Genes, bcl-2; Guanine; Humans; Hydrogen Peroxide; Kinetics; Lipid Peroxidation; Motor Neuron Disease; Mutagenesis, Site-Directed; Neuroblastoma; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Superoxide Dismutase; Superoxide Dismutase-1; Teratocarcinoma; Tumor Cells, Cultured; Tyrosine

We found that the exposure of human neuroblastoma SH-SY5Y cells to the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) induced tyrosine nitration of a 130-kDa protein, and prevented tyrosine phosphorylation of the 130-kDa protein. The focal adhesion protein p130cas was identified as a component of the 130-kDa protein using specific antibody. These results suggest that p130cas is a new target protein for nitration induced by SIN-1.

Topics: Crk-Associated Substrate Protein; Humans; Molsidomine; Neuroblastoma; Nitrates; Nitric Oxide Donors; Oxidants; Phosphoproteins; Phosphorylation; Proteins; Retinoblastoma-Like Protein p130; Tumor Cells, Cultured; Tyrosine