calcein-am and Neuroblastoma

Prion diseases are characterized by the conversion of the normal cellular prion protein PrP(C) into a pathogenic isoform, PrP(Sc). The mechanisms involved in neuronal cell death in prion diseases are largely unknown, but accumulating evidence has demonstrated oxidative impairment along with metal imbalances in scrapie-infected brains. In this study, we report changes in cellular iron metabolism in scrapie-infected mouse neuroblastoma N2a cells (ScN2a). We detected twofold lower total cellular iron and calcein-chelatable cytosolic labile iron pool (LIP) in ScN2a cells as compared to the N2a cells. We also measured in ScN2a cells significantly lower activities of iron regulatory proteins 1 and 2 (IRP1 and IRP2, respectively), regulators of cellular iron by sensing cytosolic free iron levels and controlling posttranscriptionally the expression of the major iron transport protein transferrin receptor 1 (TfR1) and the iron sequestration protein ferritin. IRP1 and IRP2 protein levels were decreased by 40% and 50%, respectively, in ScN2a cells. TfR1 protein levels were fourfold reduced and ferritin levels were threefold reduced in ScN2a cells. TfR1 and ferritin mRNA levels were significantly reduced in ScN2a cells. ScN2a cells responded normally to iron and iron chelator treatment with respect to the activities of IRP1 and IRP2, and biosynthesis of TfR1 and ferritin. However, the activities of IRP1 and IRP2, and protein levels of TfR1 and ferritin, were still significantly lower in iron-depleted ScN2a cells as compared to the N2a cells, suggesting lower need for iron in ScN2a cells. Our results demonstrate that scrapie infection leads to changes in cellular iron metabolism, affecting both total cellular and cytosolic free iron, and the activities and expression of major regulators of cellular iron homeostasis.

Topics: Animals; Blotting, Southern; Blotting, Western; Cell Line, Tumor; Deferoxamine; Electrophoretic Mobility Shift Assay; Ferric Compounds; Ferritins; Fluoresceins; Gene Expression Regulation; Infections; Iron; Iron Regulatory Protein 1; Iron Regulatory Protein 2; Mice; Neuroblastoma; Quaternary Ammonium Compounds; Receptors, Transferrin; RNA, Messenger; Scrapie; Time Factors

Coenzyme Q10 (CoQ10) is an essential biological cofactor which increases brain mitochondrial concentration and exerts neuroprotective effects. In the present study, we exposed SHSY5Y neuroblastoma cells to neurotoxic beta amyloid peptides (Abeta) and oxygen glucose deprivation (OGD) to investigate the neuroprotective effect of 10 microM CoQ10 by measuring (i) cell viability by the MTT assay, (ii) opening of the mitochondrial permeability transition pore via the fluorescence intensity of calcein-AM, and (iii) superoxide anion concentration by hydroethidine. Cell viability (mean +/- S.E.M.) was 55.5 +/- 0.8% in the group exposed to Abeta + OGD, a value lower than that in the Abeta or OGD group alone (P < 0.01). CoQ10 had no neuroprotective effect on cell death induced by either Abeta or OGD, but increased cell survival in the Abeta + OGD group to 57.3 +/- 1.7%, which was higher than in the group treated with vehicle (P < 0.05). The neuroprotective effect of CoQ10 was blocked by administration of 20 microM atractyloside. Pore opening and superoxide anion concentration were increased in the Abeta + OGD group relative to sham control (P < 0.01), and were attenuated to the sham level (P > 0.05) when CoQ10 was administered. Our results demonstrate that CoQ10 protects neuronal cells against Abeta neurotoxicity together with OGD by inhibiting the opening of the pore and reducing the concentration of superoxide anion.

Topics: Amyloid beta-Peptides; Atractyloside; Cell Hypoxia; Cell Line, Tumor; Cell Survival; Coenzymes; Fluoresceins; Glucose; Humans; Lactate Dehydrogenases; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Neuroblastoma; Neurons; Superoxides; Ubiquinone

The mitochondrial permeability transition event is implicated in the activation phase of apoptosis and necrosis, and is therefore postulated to play a role in many disease states. Mitochondrial permeability transition is therefore of increasing pharmaceutical interest. Drug discovery requires the rapid screening of compound libraries to identify functionally active ligands. We report the development of two fluorescence-based approaches for screening compound libraries for effects on mitochondrial function. These assays use the fluorometric imaging plate reader in 96-well format, and two commercially available dyes: JC-1 and calcein-AM. We show here that a JC-1 assay proved highly amenable to HTS implementation. By combining this with a calcein-based assay, these approaches gave complementary information: JC-1 facilitates the discovery of modulators of mitochondrial polarization from a library of approximately 100,000 compounds screened at 8 microM, and the calcein assay identifies permeability transition pore-specific inhibitors.

Topics: Benzimidazoles; Carbocyanines; Fluoresceins; Fluorescence; Fluorescent Dyes; Humans; Microscopy, Confocal; Mitochondria; Neuroblastoma; Permeability; Tumor Cells, Cultured

Neutrophils and mononuclear cells (MNC) can mediate antibody-dependent cellular cytotoxicity (ADCC) against cancer cells. To study cytotoxicity and growth inhibition of neuroblastoma cells by neutrophils and MNC with chimeric anti-disialoganglioside (GD2) monoclonal antibody (mAb) ch14.18, we developed digital image microscopy scanning (DIMSCAN) assays that measure fluorescence of target cells in 96-well plates after 6-18 h (cytotoxicity assay) or 7 days (growth assay). Neuroblastoma cell lines (GD2-positive: SMS-KCN, SMS-LHN, LA-N-1; GD2-negative: SK-N-SH) were preloaded with calcein acetoxymethyl ester for the cytotoxicity assay or labeled in situ after 7 days of culture with fluorescein diacetate in the growth assay. Fluorescence, as quantified by DIMSCAN, was correlated with neuroblastoma cell number in both assays (100-2000 cells/well). In the cytotoxicity test, both neutrophils and MNC effectively mediated ADCC of GD2-positive but not GD2-negative neuroblastoma cell lines. Cytotoxicity of both neutrophils and MNC increased with effector to target cell (E:T) ratio (5-50:1) and mAb ch.14.18 dose (0.1-10 microg/ml). ADCC of neutrophils, but not MNC, increased with addition of GM-CSF. Neutrophils, especially with rhGM-CSF, significantly suppressed growth of GD2-positive cell lines at a high E:T ratio (50:1) and mAb dose (10 microg/ml). Without antibody, neutrophils inhibited growth of one cell line (LA-N-1) but stimulated growth of two others (SMS-KCN, SMS-LHN). If neuroblastoma cells did not express GD2 (SK-N-SH), neutrophils stimulated growth whether or not antibody was present. Neutrophil culture supernatants increased growth of SK-N-SH, LA-N-1, and SMS-KCN cells, and MNC culture supernatants increased growth of SK-N-SH. In conclusion, neutrophils can mediate cytotoxicity and growth inhibition with a chimeric anti-GD2 antibody but also can promote tumor cell growth if antibody is not present or if GD2 is not expressed.

Topics: Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Antigens, Neoplasm; Cell Count; Cell Division; Culture Media, Conditioned; Dose-Response Relationship, Immunologic; Eosine Yellowish-(YS); Fluoresceins; Fluorescent Dyes; Gangliosides; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Monocytes; Neuroblastoma; Neutrophils; Recombinant Fusion Proteins; Tumor Cells, Cultured

Effects of the heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) were investigated on cytotoxicity in clonal NG108-15 neuroblastoma-glioma hybrid cells. Three min after addition of 100 microM TPEN, cells began to retract their neurites and lose their characteristic multipolar shape; by 3-4 hr of exposure, most cells detached from the substrate, either singly or as variable-sized aggregates. Viability was assessed by monitoring uptake of calcein AM and propidium iodide, fluorescent dyes that served as markers for live and dead cells, respectively. Incubation of cultures in 100 microM TPEN led to a gradual decrease in the population exhibiting calcein fluorescence (viable cells) and a corresponding increase in the population displaying propidium iodide fluorescence (nonviable cells). Loss of cell viability reached 12% at 8 hr, 61% at 24 hr and 83% by 48 hr. Ultrastructural examination of TPEN-treated cells revealed condensed chromatin and fragmented nuclei, characteristic of apoptosis, as well as plasma membrane defects and organelle swelling, generally associated with necrosis. Addition of an equimolar concentration of Zn2+ or Cu2+ but not Fe2+ or Mn2+ prevented morphological abnormalities and cell death.

Topics: Animals; Apoptosis; Biomarkers; Cell Aggregation; Cell Nucleus; Cell Survival; Chelating Agents; Chromatin; Copper; Ethylenediamines; Fluoresceins; Fluorescent Dyes; Glioma; Iron; Manganese; Metals, Heavy; Mice; Microscopy, Electron, Scanning; Necrosis; Neuroblastoma; Propidium; Rats; Time Factors; Tumor Cells, Cultured; Zinc