tetramethylrhodamine and Neuroblastoma

Nanoparticles (NPs) offer promise for a multitude of biological applications including cellular probes at the bio-interface for targeted delivery of anticancer substances, Raman and fluorescent-based imaging and directed cell growth. Nanodiamonds (NDs), in particular, have several advantages compared to other carbon-based nanomaterials - including a rich surface chemistry useful for chemical conjugation, high biocompatibility with little reactive oxygen species (ROS) generation, physical and chemical stability that affords sterilization, high surface area to volume ratio, transparency and a high index of refraction. The visualization of ND internalization into cells is possible via photoluminescence, which is produced by direct dye conjugation or high energy irradiation that creates nitrogen vacancy centers. Here, we explore the kinetics and mechanisms involved in the intracellular uptake and localization of novel, highly-stable, fluorophore-conjugated NDs. Examination in a neuronal cell line (N2A) shows ND localization to early endosomes and lysosomes with eventual release into the cytoplasm. The addition of endocytosis and exocytosis inhibitors allows for diminished uptake and increased accumulation, respectively, which further corroborates cellular behavior in response to NDs. Ultimately, the ability of the NDs to travel throughout cellular compartments of varying pH without degradation of the surface-conjugated fluorophore or alteration of cell viability over extended periods of time is promising for their use in biomedical applications as stable, biocompatible, fluorescent probes.

Topics: Brefeldin A; Cell Line, Tumor; Endocytosis; Exocytosis; Fluorescent Dyes; Humans; Hydrazones; Microscopy, Confocal; Nanodiamonds; Neuroblastoma; Photoelectron Spectroscopy; Rhodamines

The cytotoxicity of hPrP[173-195] prion peptide against a neuroblastoma cell model was found independent of its tendency to aggregate over time. Cytosolic and nuclear inclusions of peptide were highlighted by confocal microscopy, suggesting a role as a transcription factor in activating signal transduction pathways involved in cell toxicity.

Topics: Animals; Cell Line, Tumor; Cell Survival; Fluorescein; Microscopy, Confocal; Neuroblastoma; Peptide Fragments; Prion Diseases; Protein Multimerization; Protein Structure, Secondary; PrPC Proteins; Rats; Rhodamines; Signal Transduction; Tetracycline

3-Aminopropanal (3-AP), a degradation product of polyamines such as spermine, spermidine and putrescine, is a lysosomotropic small aldehyde that causes apoptosis or necrosis of most cells in culture, apparently by inducing moderate or extensive lysosomal rupture, respectively, and secondary mitochondrial changes. Here, using the human neuroblastoma SH-SY5Y cell line, we found simultaneous occurrence of apoptotic and necrotic cell death when cultures were exposed to 3-AP in concentrations that usually are either nontoxic, or only cause apoptosis. At 30 mM, but not at 10 mM, the lysosomotropic base and proton acceptor NH3 completely blocked the toxic effect of 3-AP, proving that 3-AP is lysosomotropic and suggesting that the lysosomal membrane proton pump of neuroblastoma cells is highly effective, creating a lower than normal lysosomal pH and, thus, extensive intralysosomal accumulation of lysosomotropic drugs. A wave of internal oxidative stress, secondary to changes in mitochondrial membrane potential, followed and gave rise to further lysosomal rupture. The preincubation of cells for 24 h with a chain-breaking free radical-scavenger, alpha-tocopherol, before exposure to 3-AP, significantly delayed both the wave of oxidative stress and the secondary lysosomal rupture, while it did not interfere with the early 3-AP-mediated phase of lysosomal break. Obviously, the reported oxidative stress and apoptosis/necrosis are consequences of lysosomal rupture with ensuing release of lysosomal enzymes resulting in direct/indirect effects on mitochondrial permeability, membrane potential, and electron transport. The induced oxidative stress seems to act as an amplifying loop causing further lysosomal break that can be partially prevented by alpha-tocopherol. Perhaps secondary brain damage during a critical post injury period can be prevented by the use of drugs that temporarily raise lysosomal pH, inactivate intralysosomal 3-AP, or stabilize lysosomal membranes against oxidative stress.

Topics: Aldehydes; alpha-Tocopherol; Ammonium Chloride; Analysis of Variance; Annexin A5; Apoptosis; Cell Line, Tumor; Cell Survival; Chromatography, High Pressure Liquid; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Electrochemistry; Flow Cytometry; Fluoresceins; Glutathione; Humans; Leucine; Lysosomes; Mitochondria; Necrosis; Neuroblastoma; Pepstatins; Propylamines; Protease Inhibitors; Reactive Oxygen Species; Rhodamines; Time Factors

A genetic defect in complex I of the mitochondrial electron transport chain (ETC) is implicated in the etiology of Parkinson's disease (PD), and has been studied in hybrid mitochondrial transgene cells based on the SH-SY5Y neuroblastoma. We sought to characterize further the mechanisms and time course of cell death in cultures of human SH-SY5Y neuroblastoma cells exposed to the ETC complex I inhibitor methylpyridinium ion (MPP+). We verify previous reports that apoptosis occurs after MPP+ exposure in SH-SY5Y cells. Nuclear pyknosis, the end stage of apoptosis, is evident after 18-hr exposure to 5 mM MPP+ and reversible until 10 hr, providing a temporal window within which to look for molecular and physiological correlates of MPP+-induced apoptosis. We then looked for mitochondrial correlates of MPP+ induced apoptosis in SH-SY5Y cells. Using flow cytometry, we found that MPP+ -induced increased reactive oxygen species (ROS) and lactate production consistent with inhibition of the ETC. Rho(o) cells, lacking a functional ETC, showed no ROS production, compensatory lactate production or apoptosis after exposure to MPP+. Finally, we show a collapse in ROS production and mitochondrial potential that is temporally correlated with irreversibility of MPP+ -induced apoptosis.

Topics: 1-Methyl-4-phenylpyridinium; Apoptosis; Cell Cycle; Cell Membrane; Cell Nucleus; DNA Fragmentation; Dose-Response Relationship, Drug; Electron Transport; Flow Cytometry; Fluoresceins; Humans; Lactic Acid; Membrane Potentials; Microscopy, Electron, Scanning; Mitochondria; NAD(P)H Dehydrogenase (Quinone); Neuroblastoma; Neurons; Reactive Oxygen Species; Rhodamines; Time Factors; Tumor Cells, Cultured

Multi-step signal transducing events, such as those mediated by G proteins, have been difficult to study in intact cells. We prepared fluorescently labelled G protein subunits, tetramethylrhodamine-alpha o (TMR-alpha o) and TMR-beta gamma, in order to study their subcellular distribution and lateral mobility. Heterotrimeric G proteins labelled in the alpha (TMR-alpha o/beta gamma) or beta (TMR-beta gamma/alpha o) subunit were reconstituted into lipid vesicles and fused to NG-108-15 cells using polyethylene glycol (PEG). Vesicles fused completely to the cells as determined by dequenching of a fluorescent lipid probe, octadecyl rhodamine B. The orientation of G protein beta gamma subunits after fusion followed the expected random distribution; the quenching of surface fluorescence with anti-fluorescein antibodies showed that about 50% of the label was accessible extracellularly. G proteins incorporated by the fusion method were able to couple to endogenous alpha 2 adrenergic receptors based on the restoration of high affinity agonist binding to pertussis toxin-treated cells. The subcellular localization of TMR-alpha o and TMR-beta gamma determined by differential centrifugation and confocal microscopy indicated that TMR-alpha o was present in the plasma membrane and in intracellular membranes, whereas TMR-beta gamma was mainly localized in the plasma membrane. The lateral mobility of TMR-alpha o and TMR-beta gamma measured using fluorescence recovery after photobleaching (FRAP) demonstrated low mobile fractions of 0.34 +/- 0.03 and 0.16 +/- 0.03, respectively. The translational diffusion coefficients of the mobile components were similar, 4.0 x 10(-9) and 2.0 x 10(-9) cm2/s, for alpha and beta gamma respectively. Neither activation of Gi-linked receptors nor cytoskeletal disruption with nocodozole or cytochalasin D changed the mobile fraction or diffusion coefficient of the alpha or beta gamma subunits. The FRAP data combined with the localization of fluorescent subunits by confocal microscopy suggest that the beta gamma subunits are highly constrained to localized regions of the plasma membrane while the alpha subunit may diffuse in intracellular regions to transmit signals from receptors to effector proteins.

Topics: Cell Membrane; Fluorescent Dyes; Glioma; GTP-Binding Proteins; Humans; Hybrid Cells; Membrane Fluidity; Microscopy, Confocal; Neuroblastoma; Rhodamines; Tumor Cells, Cultured