oligomycins and Neuroblastoma

The conserved Blm10/PA200 proteins are proteasome activators. Previously, we identified PA200-enriched regions in the genome of SH-SY5Y neuroblastoma cells by chromatin immunoprecipitation (ChIP) and ChIP-seq analysis. We also found that selective mitochondrial inhibitors induced PA200 redistribution in the genome. Collectively, our data indicated that PA200 regulates cellular homeostasis at the transcriptional level. In the present study, our aim is to investigate the impact of stable PA200 depletion (shPA200) on the overall transcriptome of SH-SY5Y cells. RNA-seq data analysis reveals that the genetic ablation of PA200 leads to overall changes in the transcriptional landscape of SH-SY5Y neuroblastoma cells. PA200 activates and represses genes regulating metabolic processes, such as the glycolysis and mitochondrial function. Using metabolic assays in live cells, we showed that stable knockdown of PA200 does not change basal respiration. Spare respiratory capacity and proton leak however are slightly, yet significantly, reduced in PA200-deficient cells by 99.834% and 84.147%, respectively, compared to control. Glycolysis and glycolytic capacity show a 42.186% and 26.104% increase in shPA200 cells, respectively, compared to control. These data suggest a shift from oxidative phosphorylation to glycolysis especially when cells are exposed to oligomycin-induced stress. Furthermore, we observed a preserved long and compact tubular mitochondrial morphology after inhibition of ATP synthase by oligomycin, which might be associated with the glycolytic change of shPA200 cells. The present study also demonstrates that the proteolytic cleavage of Opa1 is affected, and that the level of OMA1 is significantly reduced in shPA200 cells upon oligomycin-induced mitochondrial insult. Together, these findings suggest a role for PA200 in the regulation of metabolic changes in response to selective inhibition of ATP synthase in an in vitro cellular model.

Topics: Cell Line, Tumor; Chromatin Immunoprecipitation; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Glycolysis; GTP Phosphohydrolases; Humans; Mitochondria; Neuroblastoma; Nuclear Proteins; Oligomycins; Oxidative Phosphorylation; RNA, Small Interfering; Sequence Analysis, RNA

The conserved Blm10/PA200 activators bind to the proteasome core and facilitate peptide and protein turnover. Blm10/PA200 proteins enhance proteasome peptidase activity and accelerate the degradation of unstructured proteasome substrates. Our knowledge about the exact role of PA200 in diseased cells, however, is still limited. Here, we show that stable knockdown of PA200 leads to a significantly elevated number of cells in S phase after treatment with the ATP synthase inhibitor, oligomycin. However, following exposure to the complex I inhibitor rotenone, more PA200-depleted cells were in sub-G1 and G2/M phases indicative of apoptosis. Chromatin immunoprecipitation (ChIP) and ChIP-seq data analysis of collected reads indicate PA200-enriched regions in the genome of SH-SY5Y. We found that PA200 protein peaks were in the vicinity of transcription start sites. Gene ontology annotation revealed that genes whose promoters were enriched upon anti-PA200 ChIP contribute to the regulation of crucial intracellular processes, including proliferation, protein modifications and metabolism. Selective mitochondrial inhibitors induced PA200 redistribution in the genome, leading to protein withdrawal from some gene promoters and binding to others. Collectively, the results support a model in which PA200 potentially regulates cellular homeostasis at the transcriptional level, in addition to its described role as an alternative activator of the proteasome.

Topics: Apoptosis; Cell Cycle; Cell Death; Cell Line, Tumor; Cell Survival; Chromatin; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Neuroblastoma; Nuclear Proteins; Oligomycins; Reproducibility of Results; Rotenone

We herein report the synthesis, antioxidant power and neuroprotective properties of nine homo-bis-nitrones HBNs 1-9 as alpha-phenyl-N-tert-butylnitrone (PBN) analogues for stroke therapy. In vitro neuroprotection studies of HBNs 1-9 against Oligomycin A/Rotenone and in an oxygen-glucose-deprivation model of ischemia in human neuroblastoma cell cultures, indicate that (1Z,1'Z)-1,1'-(1,3-phenylene)bis(N-benzylmethanimine oxide) (HBN6) is a potent neuroprotective agent that prevents the decrease in neuronal metabolic activity (EC

Topics: Animals; Apoptosis; Brain Ischemia; Cell Line, Tumor; Cyclic N-Oxides; Disease Models, Animal; Drug Evaluation, Preclinical; Free Radical Scavengers; Glucose; Infarction, Middle Cerebral Artery; Lipid Peroxidation; Lipoxygenase Inhibitors; Male; Mice; Mice, Inbred C57BL; Molecular Structure; Neuroblastoma; Neurons; Neuroprotection; Neuroprotective Agents; Nitrogen Oxides; Oligomycins; Oxygen; Rotenone

Parkinson's disease (PD) is characterized in part by the presence of alpha-synuclein (alpha-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the alpha-synuclein gene (SNCA) are associated with familial PD. Since Ca2+ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca2+ homeostasis in cells stably transfected with either wild-type alpha-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca2+ influx evoked by exposure of cells to 50 mM K+ was enhanced in cells expressing all three forms of alpha-syn, an effect which was due specifically to increased Ca2+ entry via L-type Ca2+ channels. Mobilization of Ca2+ by muscarine was not strikingly modified by any of the alpha-syn forms, but they all reduced capacitative Ca2+ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca2+](i) in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca2+ content was unaffected by any form of alpha-synuclein. However, only WT alpha-syn transfected cells displayed significantly impaired viability. Our findings suggest that alpha-syn regulates Ca2+ entry pathways and, consequently, that abnormal alpha-syn levels may promote neuronal damage through dysregulation of Ca2+ homeostasis.

Topics: alpha-Synuclein; Amino Acids; Analysis of Variance; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium Signaling; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Fura-2; Gene Expression Regulation; Humans; Indoles; Mutation; Neuroblastoma; Nifedipine; Oligomycins; omega-Conotoxin GVIA; Potassium Chloride; Serine; Transfection

Apoptosis has been shown to be induced by some pathological stimuli. MPP+ is a neurotoxin and an inducer of parkinsonism. When SH-SY5Y cells, human neuroblastoma cell line, were treated with MPP+, cell death estimated by lactate dehydrogenase (LDH) leakage assay occurred. The cell death was associated with the DNA fragmentation into nucleosomal fragments at 180 bp, suggesting that MPP(+)-induced cell death of SH-SY5Y cells occurs through apoptosis. Although SH-SY5Y cells natively express Bcl-2 protein, which inhibits apoptosis, the level of Bcl-2 protein in SH-SY5Y cells increased with increases in the treatment periods of MPP+. MPP+ inhibits the mitochondrial respiratory chain. The other inhibitors of the mitochondrial respiratory chain, antimycin A and oligomycin, also caused cell death associated with DNA fragmentation, but did not increase the Bcl-2 protein level, suggesting that an MPP(+)-induced apoptosis may be due to the inhibition of the mitochondrial respiratory chain but the MPP(+)-induced increase in the Bcl-2 protein level is not due to it. A protein kinase inhibitor, staurosporine, inhibited the MPP(+)-induced increase in the Bcl-2 protein level, but not the MPP(+)-induced cell death. These results also suggest that the mechanism by which MPP+ increases the Bcl-2 protein level is different from that of MPP(+)-induced cell death.

Topics: 1-Methyl-4-phenylpyridinium; Anti-Bacterial Agents; Antimycin A; Blotting, Western; Cell Death; DNA Damage; Enzyme Inhibitors; Genes, bcl-2; Humans; L-Lactate Dehydrogenase; Neuroblastoma; Oligomycins; Staurosporine; Tumor Cells, Cultured

The intracellular nonmitochondrial calcium pools of saponin-permeabilized NG108-15 cells were characterized using inositol 1,4,5-trisphosphate (IP3) and GTP. IP3 or GTP alone induced release of 47 and 68%, respectively, of the calcium that was releasable by A23187. GTP induced release of a further 24% of the calcium after IP3 treatment, whereas IP3 induced release of a further 11% of the calcium after GTP treatment. Guanosine 5'-O-(3-thio)triphosphate had little effect on IP3-induced calcium release but completely inhibited GTP-induced calcium release. In contrast, heparin inhibited the action of IP3 but not that of GTP. The results imply the existence of at least three nonmitochondrial pools: (a) 31% is releasable by IP3 and GTP, (b) 11% is releasable by IP3 alone, and (c) 24% is releasable by GTP alone. GTP enhanced calcium uptake in the presence of oxalate with an EC50 of 0.6 microM and stimulated calcium release in the absence of oxalate with an EC50 of 0.32 microM. The similar EC50 values for these dual effects of GTP on calcium movement suggest that GTP exerts its dual action by the same mechanism.

Topics: Adenosine Triphosphate; Animals; Biological Transport, Active; Calcimycin; Calcium; Calcium-Transporting ATPases; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Cell Membrane Permeability; Glioma; Guanosine Triphosphate; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Kinetics; Mice; Neuroblastoma; Oligomycins; Rats

The mechanism of T3 entry into cells was studied in undifferentiated NB41A3 neuroblasts and after differentiation with 0.5 mM sodium butyrate. In undifferentiated neuroblasts, cell uptake of labeled L-T3 at 2 h was reduced to 43% of the control value by excess L-T3, whereas only 5.9% of D-T3 uptake was saturable. After incubation of intact cells with labeled hormone, the nuclei contained 3.8% of the total cellular L-T3 and 4.3% of D-T3. Thus, L-T3 nuclear uptake was 3 times higher than D-T3. Kinetic analysis of the initial rate of uptake of L-T3 by the cells gave a Km of 1.25 nM and a maximum velocity of 2.38 fmol/min.10(6) cells. The initial rate of D-T3 uptake was not saturable. Inhibitors of ATP production (antimycin and oligomycin) as well as monodansylcadaverine virtually abolished saturable cell uptake and decreased nuclear uptake more than total cell uptake, suggesting that the saturable component of uptake into the cells is the major source of nuclear L-T3. After differentiation by butyrate, both cell uptake and nuclear uptake of L-T3 increased. The apparent affinity (Ka) of the nuclear T3 receptors was determined in intact cells and compared with the Ka measured with isolated nuclei. In undifferentiated cells, the apparent Ka was 3-fold higher than the true Ka, presumably due to a step-up in the free T3 concentration in the cytosol compared to that in the incubation medium. After butyrate exposure, the apparent nuclear Ka was decreased to less than 1.5 times the control value, but Ka in isolated nuclei was unchanged. The number of nuclear receptors, however, was increased by butyrate in both intact cell and isolated nuclei experiments. These results indicate that butyrate exerts separate effects on the number of nuclear receptors and saturable T3 transport in mouse neuroblasts.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Biological Transport; Butyrates; Butyric Acid; Cadaverine; Cell Differentiation; Cell Nucleus; Cytosol; Glucose; Kinetics; Mice; Neuroblastoma; Oligomycins; Stereoisomerism; Triiodothyronine; Tumor Cells, Cultured

Tetanus toxin is known to bind neuronal tissue selectively. To study the interactions of this potent neurotoxin in an intact cell system, the binding of 125I-tetanus toxin was characterized in a neuroblastoma retina hybrid cell line, N18-RE-105. The binding of 125I-tetanus toxin to membranes prepared from N18-RE-105 cells showed many similarities to the interactions of 125I-toxin with rat synaptic membranes. The binding was decreased with increasing temperature, ionic strength, and pH. 125I-Toxin bound to membranes with high affinity: KD = 0.62 +/- 0.05 nM; Bmax = 196 +/- 45 pmol/mg protein. Quantitative thin-layer chromatography and acid-degradation analysis revealed that N18-RE-105 cells contained polysialogangliosides GD1a and GT1b in high concentrations. An assay was developed to quantitate surface-bound and internalized 125I-tetanus toxin by exploiting the observation that surface-bound 125I-toxin is susceptible to pronase digestion. When cells were incubated with 125I-tetanus toxin at 0 degree C, all of the bound 125I-toxin could be degraded with pronase. In contrast, when the incubations were performed at 37 degrees C, within 10 min about 50% of the total cell-associated 125I-toxin was pronase-resistant. Temperature pulse experiments demonstrated that 125I-tetanus toxin that was bound to cells at 0 degree C rapidly disappeared from the surface when the cells were warmed to 37 degrees C, as revealed by the appearance of pronase-resistant radioactivity. This internalization was sensitive to metabolic inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Binding, Competitive; Cell Line; Chromatography, Thin Layer; Drug Resistance; Gangliosides; Hybrid Cells; Membrane Proteins; Mice; Neuroblastoma; Oligomycins; Pronase; Rats; Receptors, Cholinergic; Rotenone; Temperature; Tetanus Toxin