phalloidine and Neuroblastoma

Activated microglial cells are an important pathological component in brains of patients with neurodegenerative diseases. The purpose of this study was to investigate the effect of He-Ne (632.8 nm, 64.6 mW/cm2) low-level laser therapy (LLLT), a non-damaging physical therapy, on activated microglia, and the subsequent signaling events of LLLT-induced neuroprotective effects and phagocytic responses.. To model microglial activation, we treated the microglial BV2 cells with lipopolysaccharide (LPS). For the LLLT-induced neuroprotective study, neuronal cells with activated microglial cells in a Transwell™ cell-culture system were used. For the phagocytosis study, fluorescence-labeled microspheres were added into the treated microglial cells to confirm the role of LLLT.. Our results showed that LLLT (20 J/cm2) could attenuate toll-like receptor (TLR)-mediated proinflammatory responses in microglia, characterized by down-regulation of proinflammatory cytokine expression and nitric oxide (NO) production. LLLT-triggered TLR signaling inhibition was achieved by activating tyrosine kinases Src and Syk, which led to MyD88 tyrosine phosphorylation, thus impairing MyD88-dependent proinflammatory signaling cascade. In addition, we found that Src activation could enhance Rac1 activity and F-actin accumulation that typify microglial phagocytic activity. We also found that Src/PI3K/Akt inhibitors prevented LLLT-stimulated Akt (Ser473 and Thr308) phosphorylation and blocked Rac1 activity and actin-based microglial phagocytosis, indicating the activation of Src/PI3K/Akt/Rac1 signaling pathway.. The present study underlines the importance of Src in suppressing inflammation and enhancing microglial phagocytic function in activated microglia during LLLT stimulation. We have identified a new and important neuroprotective signaling pathway that consists of regulation of microglial phagocytosis and inflammation under LLLT treatment. Our research may provide a feasible therapeutic approach to control the progression of neurodegenerative diseases.

Topics: Actins; Analysis of Variance; Animals; Animals, Newborn; Brain; Cells, Cultured; Chromones; Cyclic N-Oxides; Cytokines; Cytotoxicity Tests, Immunologic; Enzyme Inhibitors; Free Radical Scavengers; Gene Expression Regulation; Humans; Imidazoles; Lipopolysaccharides; Low-Level Light Therapy; Mice; Mice, Inbred C57BL; Microglia; Microscopy, Confocal; Morpholines; Myeloid Differentiation Factor 88; Neuroblastoma; Nitric Oxide; Phagocytosis; Phalloidine; Proto-Oncogene Proteins pp60(c-src); rac1 GTP-Binding Protein; Signal Transduction; Statistics as Topic; Time Factors; Transfection; Tyrosine

Loss of chromosome 11q defines a subset of high-stage aggressive neuroblastomas. Deletions are typically large and mapping efforts have thus far not lead to a well defined consensus region, which hampers the identification of positional candidate tumour suppressor genes. In a previous study, functional evidence for a neuroblastoma suppressor gene on chromosome 11 was obtained through microcell mediated chromosome transfer, indicated by differentiation of neuroblastoma cells with loss of distal 11q upon introduction of chromosome 11. Interestingly, some of these microcell hybrid clones were shown to harbour deletions in the transferred chromosome 11. We decided to further exploit this model system as a means to identify candidate tumour suppressor or differentiation genes located on chromosome 11.. In a first step, we performed high-resolution array CGH DNA copy-number analysis in order to evaluate the chromosome 11 status in the hybrids. Several deletions in both parental and transferred chromosomes in the investigated microcell hybrids were observed. Subsequent correlation of these deletion events with the observed morphological changes lead to the delineation of three putative regions on chromosome 11: 11q25, 11p13-->11p15.1 and 11p15.3, that may harbour the responsible differentiation gene.. Using an available model system, we were able to put forward some candidate regions that may be involved in neuroblastoma. Additional studies will be required to clarify the putative role of the genes located in these chromosomal segments in the observed differentiation phenotype specifically or in neuroblastoma pathogenesis in general.

Topics: Alleles; Cell Differentiation; Cell Line; Chromosome Deletion; Chromosome Mapping; Chromosomes, Artificial, Bacterial; Chromosomes, Human, Pair 11; Gene Deletion; Genes, Tumor Suppressor; Heterozygote; Humans; In Situ Hybridization, Fluorescence; Microsatellite Repeats; Neuroblastoma; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Phalloidine; Phenotype; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Cells of various lines assume similar shapes when grown attached to substrates like coverslips. In contrast, cells cultured in a collagen and/or laminin matrix often assume a relatively normal morphology in comparison with their in situ counterparts. During investigations of neuroblastoma SH-SY5Y cells, an attempt was made to identify culture conditions which would cause the cells to assume a more regular shape. SH-SY5Y cells cultured on bare coverslips, on coverslips coated with rat-tail collagen, and in approximately 1 mm thick gels containing extracellular matrix components were compared. Striking differences were apparent when comparing the gel-cultured cells with cells cultured on coverslips. Cells grown in the gel formed ganglia-like clusters which generated bundles of neurites which targeted other 'ganglia'. The same cells grown on coverslips, whether or not they were collagen-coated, appeared unaware of the presence of other cells, and did not cluster, nor did they generate neurites.

Topics: Axons; Cell Culture Techniques; Cell Differentiation; Cell Division; Cell Size; Collagen; Extracellular Matrix Proteins; Fluorescent Dyes; Gels; Glass; Humans; Microscopy, Confocal; Neuroblastoma; Neurons; Phalloidine; Rhodamines; Tumor Cells, Cultured

Two actin isoforms, gamma and beta, are contained within neuroblastoma cells. However, the relative amount and distribution of both isoforms within the cells are differentially regulated during neurite extension. The proportion of gamma-actin isoform became about four times greater than that of beta actin during neuroblastoma cell differentiation. Additionally, whereas beta actin appears to be concentrated in the cell cortex, gamma actin is also present throughout the cell body. Upon differentiation, neuroblastoma cells reorganize their actin cytoskeleton and gamma actin is induced to polymerize whereas beta actin polymers are partially disassembled. Moreover, both actin isoforms are differentially distributed within differentiated cells. Thus, gamma actin polymers are located both in the soma and proximal regions of extended neurites, whereas beta actin is enriched in the terminal tip of the neurites. Our results strongly suggest that both actin isoforms are involved in a different way in neuroblastoma cell differentiation.

Topics: Actins; Animals; Cell Differentiation; Cytochalasin D; Cytoskeleton; Mice; Nerve Tissue Proteins; Neurites; Neuroblastoma; Neurons; Nocodazole; Paclitaxel; Phalloidine; Tumor Cells, Cultured

Phenotypic variability of the human neuroblastoma cell line SK-N-SH was studied with the use of three subclones that interconvert at a slower rate than the parent cell line, i.e., a neuroblast-type subclone (SH-SY5Y), a nonneuronal, strongly substrate adherent subclone (SH-EP), and an intermediate type subclone (SH-IN). Rhodamine-phalloidin staining of actin fibers revealed differences in the cytoskeleton morphology of the three subclones, while the clathrin subunit proteins (heavy and light chains), components of coated vesicles, were invariant. Dramatic differences were observed for the expression of neurotransmitter systems, i.e., the mu and delta opioid receptor, the muscarinic cholinergic receptor and its effect on phosphatidylinositol turnover, and the uptake1 transporter for catecholamines. While these systems were strongly expressed in the parent line and the neuroblast-like clones SH-SY5Y and SH-IN, they were absent or barely detectable in the nonneuronal EP clone. Furthermore, the protooncogenes N- and c-myc were only expressed in the neuroblast containing lines, consistent with their growth characteristics of fully transformed cells. The strong c-myc expression in the absence of c- or N-myc amplification in SK-N-SH, adds a new form of high protooncogene activity in neuroblastoma cell lines. The remarkable differences of neurotransmitter systems and myc expression among the various phenotypes of human neuroblastoma cells should be considered in the therapy of neuroblastoma.

Topics: Actins; Cell Line; Clathrin; Humans; Neuroblastoma; Phalloidine; Proto-Oncogenes; Receptors, Dopamine; Receptors, Muscarinic; Receptors, Opioid; Rhodamines