g(m1)-ganglioside and Neuroblastoma

Our studies with cultured cells have provided new insight into the particular role of GM1 in regulating excitatory opioid responses. GM1 is significantly elevated in chronic opioid-treated cells via Gs/adenylyl cyclase activation. Such GM1 elevation promotes coupling of opioid receptor with Gs, resulting in attenuation of inhibitory opioid effects and induction of a sustained excitatory response. Application of exogenous GM1, but not other gangliosides, induces excitatory opioid responses not only in neurons and neuroblastoma cells that bear intrinsic opioid receptors but also in nonneuronal cells that are transfected with delta-opioid receptor. The latter system provides evidence that allosteric binding of GM1 changes receptor conformation from a Gi-coupled to a Gs-coupled mode. This is supported by preliminary experiments with a mutated delta-opioid receptor.

Topics: Adenylyl Cyclases; Animals; G(M1) Ganglioside; GTP-Binding Proteins; Humans; Narcotics; Neuroblastoma; Neurons; Receptors, Opioid; Receptors, Opioid, delta; Signal Transduction; Transfection; Tumor Cells, Cultured

The pronounced increases in gangliosides belonging to the gangliotetraose family during the neurite outgrowth phase of neuronal differentiation have suggested a functional requirement for these substances related to process extension, arborization, and possibly synaptogenesis. Support for this hypothesis has come from a variety of experimental paradigms utilizing neuroblastoma cell lines, primary neuronal cultures, and observations on the developing nervous system. We have recently observed that differentiation of both primary neurons and neuroblastoma cells by Ca(2+)-elevating stimulants is characterized by upregulation of GM1 in the nuclear membrane. Immunostaining revealed these Ca(2+)-induced neurites to have axonal characteristics. Recent work has indicated that nuclear GM1 facilitates efflux of nuclear Ca2+, thereby contributing to the reduced level of nuclear Ca2+ that characterizes the differentiated neuron. Thus, while GM1 is generally recognized as a pluripotent molecule with several modulatory roles in the plasma membrane of developing and mature neurons, regulation of Ca2+ flux across the nuclear membrane is proposed as another critical function of this ganglioside in neuronal development, with special relevance to axonogenesis.

Topics: Animals; Cell Differentiation; G(M1) Ganglioside; Gangliosides; Humans; Membrane Lipids; Neurites; Neuroblastoma; Neurons; Nuclear Envelope; Tumor Cells, Cultured

Alzheimer's disease is a progressive degenerative condition that mainly affects cognition and memory. Recently, distinct clinical and neuropathological phenotypes have been identified in AD. Studies revealed that structural variation in Aβ fibrillar aggregates correlates with distinct disease phenotypes. Moreover, environmental surroundings, including other biomolecules such as proteins and lipids, have been shown to interact and modulate Aβ aggregation. Model membranes containing ganglioside (GM1) clusters are specifically known to promote Aβ fibrillogenesis. This study unravels the modulatory effect of non-micellar GM1, a glycosphingolipid frequently released from the damaged neuronal membranes, on Aβ

Topics: Alzheimer Disease; Amyloid; Amyloid beta-Peptides; G(M1) Ganglioside; Humans; Neuroblastoma; Peptide Fragments

G-protein-coupled receptors (GPCRs) are commonly pharmacologically modulated due to their ability to translate extracellular events to intracellular changes. Previously, studies have mostly focused on protein-protein interactions, but the focus has now expanded also to protein-lipid connections. GM1, a brain-expressed ganglioside known for neuroprotective effects, and GPR37, an orphan GPCR often reported as a potential drug target for diseases in the central nervous system, have been shown to form a complex. In this study, we looked into the functional effects. Endogenous GM1 was downregulated when stably overexpressing GPR37 in N2a cells (N2a

Topics: Animals; Cytoprotection; Down-Regulation; G(M1) Ganglioside; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Mice; Neuroblastoma; Neuroprotective Agents; Receptors, G-Protein-Coupled; Signal Transduction

The crucial role of ganglioside GM1 in the regulation of neural homeostasis has been assessed by several studies. Recently we shed new light on the molecular basis underlying GM1 effects demonstrating that GM1 oligosaccharide directly binds TrkA receptor and triggers MAPK pathway activation leading to neuronal differentiation and protection. Following its exogenous administration, proteomic analysis revealed an increased expression of proteins involved in several biochemical mechanisms, including mitochondrial bioenergetics. Based on these data, we investigated the possible effect of GM1 oligosaccharide administration on mitochondrial function. We show that wild-type Neuro2a cells exposed to GM1 oligosaccharide displayed an increased mitochondrial density and an enhanced mitochondrial activity together with reduced reactive oxygen species levels. Interestingly, using a Neuro2a model of mitochondrial dysfunction, we found an increased mitochondrial oxygen consumption rate as well as increased complex I and II activities upon GM1 oligosaccharide administration. Taken together, our data identify GM1 oligosaccharide as a mitochondrial regulator that by acting at the plasma membrane level triggers biochemical signaling pathway inducing mitochondriogenesis and increasing mitochondrial activity. Although further studies are necessary, the capability to enhance the function of impaired mitochondria points to the therapeutic potential of the GM1 oligosaccharide for the treatment of pathologies where these organelles are compromised, including Parkinson's disease.

Topics: G(M1) Ganglioside; Humans; Mitochondria; Neuroblastoma; Oligosaccharides; Proteomics

Recently, we highlighted that the ganglioside GM1 promotes neuroblastoma cells differentiation by activating the TrkA receptor through the formation of a TrkA-GM1 oligosaccharide complex at the cell surface. To study the TrkA-GM1 interaction, we synthesized two radioactive GM1 derivatives presenting a photoactivable nitrophenylazide group at the end of lipid moiety, 1 or at position 6 of external galactose, 2; and a radioactive oligosaccharide portion of GM1 carrying the nitrophenylazide group at position 1 of glucose, 3. The three compounds were singly administered to cultured neuroblastoma Neuro2a cells under established conditions that allow cell surface interactions. After UV activation of photoactivable compounds, the proteins were analyzed by PAGE separation. The formation of cross-linked TrkA-GM1 derivatives complexes was identified by both radioimaging and immunoblotting. Results indicated that the administration of compounds 2 and 3, carrying the photoactivable group on the oligosaccharide, led to the formation of a radioactive TrkA complex, while the administration of compound 1 did not. This underlines that the TrkA-GM1 interaction directly involves the GM1 oligosaccharide, but not the ceramide. To better understand how GM1 relates to the TrkA, we isolated plasma membrane lipid rafts. As expected, GM1 was found in the rigid detergent-resistant fractions, while TrkA was found as a detergent soluble fraction component. These results suggest that TrkA and GM1 belong to separate membrane domains: probably TrkA interacts by 'flopping' down its extracellular portion onto the membrane, approaching its interplay site to the oligosaccharide portion of GM1.

Topics: Animals; Cell Differentiation; Cell Line; G(M1) Ganglioside; Membrane Microdomains; Mice; Neuroblastoma; Receptor, trkA; Signal Transduction

In the search of a potent candidate for neurotherapy, we designed and synthesized various analogues of ganglioside Hp-s1. The modification includes the change in hydrophobicity by varying the carbon chain length, altering the number of hydrogen bonds, and replacing the anomeric atom. The chemical synthesis was carried out by using various methods and discussed in details. The neuritogenic activities of these analogues are confirmed in a human neuroblastoma cell line SH-SY5Y. A higher activity of ganglioside Hp-s1 analogue on IL-17A transcript upregulation than ganglioside Hp-s1 was found.

Topics: Biological Assay; Cell Line; G(M1) Ganglioside; Gangliosides; Humans; Neurites; Neuroblastoma; Neurogenesis; Tumor Cells, Cultured

Mounting evidence indicates soluble Aβ

Topics: Amyloid; Amyloid beta-Peptides; Cell Line, Tumor; Circular Dichroism; Drug Evaluation, Preclinical; Fluorescence Resonance Energy Transfer; G(M1) Ganglioside; Humans; Lawsonia Plant; Membrane Microdomains; Microscopy, Electron; Molecular Structure; Neuroblastoma; Peptide Fragments; Plant Leaves

Topics: Animals; Cell Differentiation; Cell Line; G(M1) Ganglioside; Mice; Molecular Docking Simulation; Neurites; Neuroblastoma; Oligosaccharides; Receptor, trkA; Signal Transduction

Ganglioside Hp-s1 is isolated from the ovary of sea urchin Diadema setosum. It exhibited better neuritogenic activity than GM1 in pheochromocytoma 12 cells. To explore the roles of glucosyl moiety of Hp-s1 in contributing to the neurogenic activity, we developed feasible procedures for synthesis of Hp-s1 analogues (2a-2f). The glucosyl moiety of Hp-s1 was replaced with α-glucose, α-galactose, β-galactose, α-mannose, and β-mannose, and their biological activities on SH-SY5Y cells and natural killer T (NKT) cells were evaluated. We found that the orientation of C-2 hydroxyl group at glucosyl moiety of Hp-s1 plays an important role to induce neurite outgrowth of SH-SY5Y cells. Surprisingly, compound 2d could activate NKT cells to produce interleukin 2, although it did not show great activity on neurite outgrowth of SH-SY5Y cells. In general, the Hp-s1 might be considered as a lead compound for the development of novel drugs aimed at modulating the activity of neuronal cells.

Topics: Animals; Carbohydrate Conformation; Cell Differentiation; Cell Line, Tumor; Cytokines; G(M1) Ganglioside; Glycosylation; Humans; Killer Cells, Natural; Magnetic Resonance Spectroscopy; Mass Spectrometry; Mice; Models, Chemical; Molecular Structure; Neurites; Neuroblastoma; Neurogenesis

Studies indicate that bupivacaine-induced neurotoxicity results from apoptosis. Gangliosides have been shown to promote neuronal repair and recovery of neurological function after spinal cord injury. Previously, we confirmed that in vivo administration of the ganglioside GM-1 attenuated bupivacaine-induced neurotoxicity in various animal models; however, the underlying mechanism remains unclear.. Cells of the neuroblastoma line N2a (Neuro2a cells) were divided into three experimental groups: control, bupivacaine-treated, and bupivacaine-treated with GM-1 pretreatment. Cell viability and apoptosis were assessed through CCK-8 assays, Hoechst staining, and flow cytometry analysis of Annexin-V/propidium iodide double labeling. Real-time polymerase chain reaction and western blotting assessed the expression of caspase-3, caspase-8, and caspase-9.. Bupivacaine-induced apoptosis worsened with increasing dose and exposure time. Bupivacaine induced increased expression of caspase-3 and caspase-9, but not caspase-8, indicating that the mitochondrial pathway but not the death receptor apoptosis pathway was activated. GM-1 pretreatment inhibited bupivacaine-induced apoptosis and the expression of caspase-3 and caspase-9 in a dose-dependent manner.. Bupivacaine induced neurotoxicity by activating apoptosis via the mitochondrial pathway, and this was inhibited by GM-1 pretreatment.

Topics: Animals; Apoptosis; Bisbenzimidazole; Blotting, Western; Bupivacaine; Cell Line, Tumor; Cell Survival; Flow Cytometry; G(M1) Ganglioside; Mice; Neuroblastoma; Neuroprotection; Neurotoxins; Real-Time Polymerase Chain Reaction; RNA, Messenger

Hemagglutinating virus of Japan-envelope (HVJ-E) is a drug delivery vector based on inactivated Sendai virus. Recently, antitumor activities were found for HVJ-E itself and clinical trials of HVJ-E for some malignant tumors are now ongoing. We investigated the in vitro and in vivo antitumor effects of HVJ-E against neuroblastoma, which is one of the most common malignant solid tumors in childhood. The sensitivity of human neuroblastoma cell lines to HVJ-E correlated with the expression level of gangliosides, Sialylparagloboside (SPG) and GD1a, receptors for HVJ. Among the cell lines, SK-N-SH was the most sensitive to HVJ-E in vitro and total SPG and GD1a expression was the highest. Complete eradication of subcutaneous tumors derived from SK-N-SH cells was achieved by intratumoral injection of HVJ-E in SCID mice and no recurrence was observed for more than 300 days after HVJ-E inoculation. In contrast, NB1 cells expressed the lowest amount of GD1a and SPG and were resistant to HVJ-E in vitro. The expression of GD1a increased by 13-cis retinoic acid (13cRA), which is a therapeutic drug for high risk neuroblastoma, thus leading to an improved sensitivity to HVJ-E in vitro. Only growth inhibition of the subcutaneous tumors derived from NB1 cells was achieved by HVJ-E in the SCID mice, but the combination of 13cRA and HVJ-E could achieve partial eradication of the xenograft and also lead to an improved prognosis. In conclusion, HVJ-E is a promising therapeutic modality for neuroblastoma and 13cRA can be used as an adjuvant to HVJ-E.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Chemotherapy, Adjuvant; Female; G(M1) Ganglioside; Gangliosides; Genetic Vectors; Humans; Isotretinoin; Mice; Mice, SCID; Neuroblastoma; Oncolytic Virotherapy; Sendai virus; Viral Envelope Proteins

Orchestrated upregulation of cell surface presentation of ganglioside GM1 and homodimeric galectin-1 is the molecular basis for growth regulation of human neuroblastoma (SK-N-MC) cells. Further study led to the discovery of competitive inhibition by galectin-3, prompting us to test tandem-repeat-type galectin-4 (two different lectin domains connected by a 42-amino-acid linker). This lectin bound to cells at comparably high affinity without involvement of the ganglioside, as disclosed by assays in the presence of cholera toxin B-subunit or galectin-1 and blocking glucosylceramide synthesis. Notably, when tested separately, binding of both lectin domains showed partial sensitivity to the bacterial agglutinin. Despite its ability for cross-linking surface association of galectin-4 did not affect proliferation, in contrast to homodimeric galectins. The truncation of linker length from 42 to 16 amino acids altered binding properties to let partial sensitivity to the bacterial lectin emerge. Cross-competition between parental and engineered proteins did not exceed 40%. No effect on cell growth was detected. This study reveals complete functional divergence between galectins differing in the spatial mode of lectin-site presentation and dependence of reactivity to distinct counter-receptor(s) on linker length. Due to the documented presence of galectin-4 in the nervous system and its affinity for sulfatide these in vitro results indicate the potential for a distinct functionality profile of this lectin in vivo, giving further research direction.

Topics: Cell Line, Tumor; Cell Proliferation; Cholera Toxin; G(M1) Ganglioside; Galectin 1; Galectin 4; Galectins; Humans; Ligands; Neuroblastoma; Structure-Activity Relationship

Galectins are potent effectors with conspicuous cell-type-specific activity profile. Its occurrence poses the question on the nature of the underlying biochemical determinants, in human SK-N-MC neuroblastoma cells involved in negative growth regulation. Since increase of surface presentation of ganglioside GM1 and homodimeric galectin-1 precedes growth inhibition, a direct interaction is suggested. We thus examined cell binding depending on glucosylceramide synthesis. It was drastically reduced by N-butyldeoxynojirimycin and threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, adding decisive evidence for the assumed galectin/ganglioside binding. Glycoproteins do not compensate ganglioside depletion which was verified by measuring lipid-bound sialic acid. Binding affinity is significantly lowered by disrupting microdomain integrity, also effective for the competitive inhibitor galectin-3. This was caused by cell treatment with either 2-hydroxypropyl-beta-cyclodextrin or filipin III. In this cell system, target specificity and topology of ligand presentation act together to enable high-affinity binding.

Topics: 1-Deoxynojirimycin; 2-Hydroxypropyl-beta-cyclodextrin; Anti-Bacterial Agents; beta-Cyclodextrins; Cell Adhesion; Cell Membrane; Enzyme Inhibitors; Excipients; Filipin; G(M1) Ganglioside; Galectin 1; Galectin 3; Glycoside Hydrolase Inhibitors; Humans; Membrane Microdomains; Morpholines; Neuroblastoma; Tumor Cells, Cultured

Membrane rafts are discrete microdomains found in cell membranes that contain cholesterol and glycosphingolipids such as gangliosides. As cholesterol is a major component of membrane rafts, its sequestration by the polyene filipin can be used to disrupt them. In previous work we observed that membrane raft disruption by filipin treatment of murine neuroblastoma N2a cells led to changes in expression of cell processes. In this study, we determined the type of process formation induced by filipin treatment as well as whether their expression was accompanied by changes in ganglioside content or subcellular distribution. The results indicate that the processes formed were axonal in nature and their expression was accompanied by changes in both ganglioside content as well as the subcellular localization of GM1.

Topics: Animals; Axons; Cell Nucleus; Filipin; G(M1) Ganglioside; Membrane Microdomains; Mice; Neurites; Neuroblastoma; Tumor Cells, Cultured

GM1 ganglioside was reported to mediate the amyloid beta-protein (Abeta) secretion and accumulation in the pathogenesis of Alzheimer's disease (AD). The objective of this project was to comprehend the underlying molecular changes related to amyloid beta-protein precursor (APP) processing pathway induced by GM1. Using suppression subtractive hybridisation (SSH), we detected one prominent sequence with increased expression in human neuroblastoma cells that stably transfected with human APP695 cDNA treated with GM1. This transcript has high identity to human Ubiquilin 1 gene. Differential expression was initially confirmed by dot blot hybridization. This result was further authenticated with quantitative real-time polymerase chain reaction (RT-PCR) analysis. Furthermore, using Western blots, we discovered that GM1 stimulated the expression of Ubiquilin 1 in human neuroblastoma cells and rat cortical neurons while other gangliosides Asialo-GM1 and GD1b did not. Ubiquilin 1 is one of the candidate genes of AD, which have been shown to modulate the gamma-secretase components in the proteolytic processing of APP, and is therefore a putative candidate for further investigation of GM1 mechanisms in the etiology and pathology of AD.

Topics: Adaptor Proteins, Signal Transducing; Amyloid beta-Protein Precursor; Analysis of Variance; Animals; Animals, Newborn; Autophagy-Related Proteins; Blotting, Western; Carrier Proteins; Cell Cycle Proteins; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; G(M1) Ganglioside; Gene Expression; Gene Expression Regulation; Humans; Neuroblastoma; Neurons; Nucleic Acid Hybridization; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection

The growth-regulatory interplay between ganglioside GM1 on human SK-N-MC neuroblastoma cells and an endogenous lectin provides a telling example for glycan (polysaccharide) functionality. Galectin-1 is the essential link between the sugar signal and the intracellular response. The emerging intrafamily complexity of galectins raises the question on defining extent of their structural and functional overlap/divergence. We address this problem for proto-type galectins in this system: ganglioside GM1 as ligand, neuroblastoma cells as target. Using the way human galectin-1 interacts with this complex natural ligand as template, we first defined equivalent positioning for distinct substitutions in the other tested proto-type galectins, e.g., Lys63 vs. Leu60/Gln72 in galectins-2 and -5. As predicted from our in silico work, the tested proto-type galectins have affinity for the pentasaccharide of ganglioside GM1. In contrast to solid-phase assays, cell surface presentation of the ganglioside did not support binding of galectin-5, revealing the first level of regulation. Next, a monomeric proto-type galectin (CG-14) can impair galectin-1-dependent negative growth control by competitively blocking access to the shared ligand without acting as effector. Thus, the quaternary structure of proto-type galectins is an efficient means to give rise to functional divergence. The identification of this second level of regulation is relevant for diagnostic monitoring. It might be exploited therapeutically by producing galectin variants tailored to interfere with galectin activities associated with the malignant phenotype. Moreover, the given strategy for comparative computational analysis of extended binding sites has implications for the rational design of galectin-type-specific ligands.

Topics: Amino Acid Sequence; Animals; Chickens; G(M1) Ganglioside; Galectin 1; Galectin 2; Galectin 3; Galectin 4; Galectins; Humans; Ligands; Models, Molecular; Molecular Sequence Data; Neuroblastoma; Protein Structure, Quaternary; Rats; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Tumor Cells, Cultured

In flow cytometry using two detecting methods, we have found that amyloid-beta-protein(1-40) [Abeta(1-40)] has high affinity to IMR-32 neuroblastoma cell membrane when it is aggregated to form beta-sheet conformation, whereas random coil small Abeta-species has low affinity. The difference in the binding ability to the cell membranes well accounts for the cytotoxicity of Abeta(1-40); namely, aggregated beta-sheet Abeta(1-40) gives cytotoxicity higher than random coil Abeta(1-40). Specific binding between Abeta(1-40) and ganglioside GM1 of the raft-like domain in lipid membrane is suggested from a surface plasmon resonance (SPR) experiment.

Topics: Amyloid beta-Peptides; Cell Membrane; Circular Dichroism; Flow Cytometry; G(M1) Ganglioside; Humans; Membrane Lipids; Membrane Microdomains; Neuroblastoma; Peptide Fragments; Protein Conformation; Surface Plasmon Resonance; Toxicity Tests; Tumor Cells, Cultured

Plaques containing amyloid beta-peptides (Abeta) are a major feature in Alzheimer's disease (AD), and GM1 ganglioside is an important component of cellular plasma membranes and especially enriched in lipid raft. GM1-bound Abeta (GM1/Abeta), found in brains exhibiting early pathological changes of AD including diffuse plaques, has been suggested to be involved in the initiation of amyloid fibril formation in vivo by acting as a seed. However, the role of GM1 in amyloid beta-protein precursor (APP) processing is not yet defined. In this study, we report that exogenous GM1 ganglioside promotes Abeta biogenesis and decreases sAPPalpha secretion in SH-SY5Y and COS7 cells stably transfected with human APP695 cDNA without affecting full-length APP and the sAPPbeta levels. We also observe that GM1 increases extracellular levels of Abeta in primary cultures of mixed rat cortical neurons transiently transfected with human APP695 cDNA. These findings suggest a regulatory role for GM1 in APP processing pathways.

Topics: Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Cell Line, Tumor; Cells, Cultured; Cerebral Cortex; Chlorocebus aethiops; COS Cells; DNA, Complementary; Endopeptidases; Extracellular Fluid; G(M1) Ganglioside; Humans; Neuroblastoma; Neurons; Peptide Fragments; Rats; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Transfection; Up-Regulation

Hypoxia-inducible factor 1 alpha (HIF-1alpha), a component of HIF-1, is expressed in human tumors and renders cells able to survive and grow under hypoxic (low-oxygen) conditions. YC-1, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole, an agent developed for circulatory disorders that inhibits platelet aggregation and vascular contraction, inhibits HIF-1 activity in vitro. We tested whether YC-1 inhibits HIF-1 and tumor growth in vivo.. Hep3B hepatoma, NCI-H87 stomach carcinoma, Caki-1 renal carcinoma, SiHa cervical carcinoma, and SK-N-MC neuroblastoma cells were grown as xenografts in immunodeficient mice (69 mice total). After the tumors were 100-150 mm(3), mice received daily intraperitoneal injections of vehicle or YC-1 (30 microg/g) for 2 weeks. HIF-1 alpha protein levels and vascularity in tumors were assessed by immunohistochemistry, and the expression of HIF-1-inducible genes (vascular endothelial growth factor, aldolase, and enolase) was assessed by reverse transcription-polymerase chain reaction. All statistical tests were two-sided.. Compared with tumors from vehicle-treated mice, tumors from YC-1-treated mice were statistically significantly smaller (P<.01 for all comparisons), expressed lower levels of HIF-1 alpha (P<.01 for all comparisons), were less vascularized (P<.01 for all comparisons), and expressed lower levels of HIF-1-inducible genes, regardless of tumor type.. The inhibition of HIF-1 alpha activity in tumors from YC-1-treated mice is associated with blocked angiogenesis and an inhibition of tumor growth. YC-1 has the potential to become the first antiangiogenic anticancer agent to target HIF-1 alpha.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Carcinoma; Carcinoma, Hepatocellular; Cell Hypoxia; Culture Media, Conditioned; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Female; G(M1) Ganglioside; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoblotting; Indazoles; Intercellular Signaling Peptides and Proteins; Kidney Neoplasms; Killer Cells, Natural; Liver Neoplasms; Lymphokines; Male; Mice; Mice, SCID; Neoplasms; Neovascularization, Pathologic; Neuroblastoma; Platelet Endothelial Cell Adhesion Molecule-1; Precipitin Tests; Rats; Reverse Transcriptase Polymerase Chain Reaction; Stomach Neoplasms; Transcription Factors; Transplantation, Heterologous; Tumor Cells, Cultured; Uterine Cervical Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The extracellular functions of galectin-7 (p53-induced gene 1) are largely unknown. On the surface of neuroblastoma cells (SK-N-MC), the increased GM1 density, a result of upregulated ganglioside sialidase activity, is a key factor for the switch from proliferation to differentiation. We show by solid-phase and cell assays that the sugar chain of this ganglioside is a ligand for galectin-7. In serum-supplemented proliferation assays, galectin-7 reduced neuroblastoma cell growth without the appearance of features characteristic for classical apoptosis. The presence of galectin-3 blocked this effect, which mechanistically resembles that of galectin-1. By virtue of carbohydrate binding, galectin-7 thus exerts neuroblastoma growth control similar to galectin-1 despite their structural differences. In addition to p53-linked proapoptotic activity intracellularly, galectin-7, acting as a lectin on the cell surface, appears to be capable of reducing cancer cell proliferation in susceptible systems.

Topics: Carbohydrate Metabolism; Cell Division; Dimerization; G(M1) Ganglioside; Galectin 3; Galectins; Gene Expression Regulation, Neoplastic; Humans; Ligands; Mass Spectrometry; Neuroblastoma; Tumor Cells, Cultured

The role of endogenous GM1 ganglioside in neurite outgrowth has been studied in N18 and NG108-15 neuroblastoma cells with the GM1-specific ligand cholera toxin B subunit (Ctx B), which stimulates Ca(2+) influx together with neuritogenesis. Our primary goal has been to identify the nature of the calcium channel that is modulated by GM1. An L-type voltage-operated Ca(2+) channel (VOCC) was previously proposed as the mediator of this phenomenon. This investigation, employing fura-2 fluorescent measurements and specific channel blockers and other agents, revealed that GM1 modulates a hitherto unidentified Ca(2+) channel not of the L type. It was opened by Ctx B; was permeable to Ca(2+) and Ba(2+) but not Mn(2+); and was blocked by Ni(2+), Cd(2+), and La(3+). Although most dihydropyridines inhibited Ctx B-induced Ca(2+) influx as well as neurite outgrowth at higher concentrations, they and other VOCC blockers at normally employed concentrations failed to do so, suggesting uninvolvement of VOCC. In addition, Ca(2+) influx induced by Ctx B was not mediated by cGMP-dependent or G-protein-coupled nonselective cation channels, as demonstrated by the cGMP antagonist Rp-cGMPS or the G-protein/receptor uncoupling agent suramin, respectively. Finally, Ca(2+) influx was unlikely to be due to inhibition or reversal of Na(+)-Ca(2+) exchanger via Ctx B induction of Na(+) uptake, insofar as no effect was seen on blocking Na(+) channels, inhibiting Na(+)-K(+)-ATPase, or eliminating extracellular Na(+). The results suggest that this novel channel is gated by interaction with GM1, which, when associated with the channel and bound by appropriate ligand, promotes Ca(2+) influx. This in turn induces signaling for the onset of neuritogenesis.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Differentiation; Cholera Toxin; Fura-2; G(M1) Ganglioside; Lidocaine; Neuroblastoma; Suramin; Tumor Cells, Cultured

The inhibitory action of gangliosides GT1B, GD1A, GM3 and GM1 on cell proliferation and epidermal growth factor receptor (EGFR) phosphorylation was determined in the N-myc amplified human neuroblastoma cell line NBL-W. The IC50 of each ganglioside was estimated from concentration-response regressions generated by incubating NBL-W cells with incremental concentrations (5-1000 microm) of GT1B, GD1A, GM3 or GM1 for 4 days. Cell proliferation was quantitatively determined by a colourimetric assay using tetrazolium dye and spectrophotometric analysis, and EGFR phosphorylation by densitometry of Western blots. All gangliosides assayed, with the exception of GM1, inhibited NBL-W cell proliferation in a concentration-dependent manner. The IC50s for gangliosides GT1B [molecular weight (MW) 2129], GM3 (MW 1236), and GD1A (MW 1838) were (mean +/- SEM) 117 +/- 26, 255 +/- 29, and 425 +/- 44 m, respectively. In contrast, the IC50 for GM1 (MW 1547) could not be determined. Incubation of NBL-W cells with epidermal growth factor (EGF) concentrations ranging from 0.1 to 1000 ng/ml progressively increased cell proliferation rate, but it plateaued at concentrations above 10 ng/ml. EGFR tyrosine phosphorylation, however, was incrementally stimulated by EGF concentrations from 1 to 100 ng/ml. The suppression of EGF-induced EGFR phosphorylation differed for each ganglioside, and their respective inhibitory potencies were as follows: EGFR phosphorylation [area under curve (+ EGF)/area under curve (- EGF)]: control (no ganglioside added) = 8.2; GM1 = 8.3; GD1A = 6.7; GM3 = 4.87, and GT1B = 4.09. The lower the ratio, the greater the inhibitory activity of the ganglioside. Gangliosides GD1A and GT1B, which have terminal N-acetyl neuraminic acid moieties, as well as one and two N-acetyl neuraminic acid residues linked to the internal galactose, respectively, both inhibited cell proliferation and EGFR phosphorylation. However, GD1A was a more potent suppressor of cell proliferation and GT1B most effective against EGFR phosphorylation. GM3, which only has a terminal N-acetyl neuraminic acid, inhibited cell proliferation and EGFR phosphorylation almost equivalently. These data suggest that gangliosides differ in their potency as inhibitors of NBL-W neuroblastoma cell proliferation and EGFR tyrosine phosphorylation, and that perturbations in the differential expression of membrane glycosphingolipids may play a role in modulating neuroblastoma growth.

Topics: Animals; Carbohydrate Sequence; Cell Division; Dogs; Epidermal Growth Factor; ErbB Receptors; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Humans; Molecular Sequence Data; Neuroblastoma; Phosphorylation; Structure-Activity Relationship; Tumor Cells, Cultured; Tyrosine

High-affinity anti-GM1 antibodies are frequently described in several nervous system diseases, mainly in multifocal motor neuropathy (MMN) and some acute neuropathies. These antibodies are currently detected using enzyme-linked immunosorbent assay (ELISA) and immuno-thin-layer-chromatography (immuno-TLC) methods. We report in this article a new method based on the incorporation of exogenous GM1 in a selected cell line to detect anti-GM1 antibodies using flow cytometry (FC). This method is evaluated on 80 sera from normal blood donors (NBD) and patients suffering various nervous system diseases. It appears to be as sensitive as our method ELISA for the diagnosis of some motor neuron syndromes.

Topics: Autoantibodies; Flow Cytometry; G(M1) Ganglioside; Glioblastoma; Humans; Motor Neuron Disease; Neuroblastoma; Sensitivity and Specificity; Tumor Cells, Cultured

Gangliosides located in the outer leaflet of the plasma membrane are important modulators of cellular functions. Our previous work has shown that in cultured human SK-N-MC neuroblastoma cells a sialidase residing in the same membrane selectively desialylates gangliosides with terminal sialic acid residues, causing a shift from higher species to GM1 and a conversion of GM3 to lactosylceramide. Inhibition of this sialidase by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en) resulted in increased cell proliferation and a loss of differentiation markers. In this study, we examined the occurrence and function of this ganglioside sialidase in other neuronal cells. Subcellular fractionation showed the sialidase to be located in the plasma membrane of all cell lines studied. The presence of the inhibitor NeuAc2en led to a profound decrease in the amount of the differentiation marker 200 kDa/70 kDa neurofilaments and an increase in cell proliferation in the cholinergic SK-N-MC and mixed cholinergic/adrenergic SK-N-FI and SK-N-DZ neuroblastoma lines, but had little or no effect in the human adrenergic SK-N-SH and SK-N-AS and the adrenergic/cholinergic PC12 cells from rat. The influence of the inhibitor on cell behaviour was paralleled by a diminished number of cholera toxin B-binding GM1 sites. The findings demonstrate that the plasma membrane ganglioside sialidase is an important element of proliferation and differentiation control in some, but not all, neuroblastoma cells and suggest that there might be a relationship between plasma membrane sialidase activity and cholinergic differentiation.

Topics: Adrenergic Fibers; Animals; Antigens, Differentiation; Azides; Cell Differentiation; Cell Division; Cell Fractionation; Cell Membrane; Cholinergic Fibers; G(M1) Ganglioside; Gangliosides; Humans; Neuraminidase; Neuroblastoma; Rats; Sialic Acids; Subcellular Fractions; Tumor Cells, Cultured

Increased titers of IgM anti-GM1 antibodies are present in some patients with Lower Motor Neuron Disease (LMND) or Motor Neuropathy (MN), but their pathogenic role and the mechanism of action are unclear. Previous studies have shown that the B subunit of Cholera Toxin (CT), which binds and crosslinks ganglioside GM1, modulate intracellular calcium in murine neuroblastoma cells via the activation of L-type voltage-dependent calcium channels (VGCC). Therefore, using a fluorimetric approach, we have examined the hypothesis that the pentameric IgM anti-GM1 antibodies, could similarly alter calcium concentration in N18 neuroblastoma cells. Sera with human IgM anti-GM1 antibodies were obtained from 5 patients with LMND and 2 patients with MN. Human IgG anti-GM1, IgM anti-Myelin Associated Glycoprotein (MAG), IgM anti-sulfatide antibodies and lectin peanut agglutinin (PNA), that recognizes specifically the Gal(betal-3)GalNAc epitope, were used as control sera. Direct application of either human IgM anti-GM1 antibodies or the B subunit of CT to N18 neuroblastoma cells induced a sustained influx of manganese ions, as indicated by a quench of the intracellular fura-2 fluorescence. Furthermore, the dihydropyridine L-type channel antagonists completely inhibited the manganese influx, suggesting that it is due to activation of an L-type VGCC. The magnitude of the influx was correlated with antibody titers. None of human IgG anti-GM1, IgM anti-MAG, IgM anti-sulfatide antibodies or PNA induce an ion influx, pointing to the selective participation of the pentameric IgM isotype of anti-GM1 in the modulation of L-type calcium channels opening. Given that L-type calcium channels are present on motor neurons, the modulation of L-type calcium channels by IgM GM1 antisera may have important implications in diseases such as LMND and MN.

Topics: Adult; Autoantibodies; Calcium; Calcium Channels; Cholera Toxin; G(M1) Ganglioside; Homeostasis; Humans; Immunoglobulin G; Immunoglobulin M; Middle Aged; Motor Neuron Disease; Neuroblastoma; Neurons; Tumor Cells, Cultured

Lysosphingolipids, which lack the fatty acid moiety of sphingolipids, are known to be accumulated in some variants of sphingolipid storage diseases. Here, we report that lysosphingolipids with naturally occurring stereochemical configurations induce apoptosis in mouse neuroblastoma Neuro2a cells. The intracellular dehydrogenase activity and [3H]thymidine incorporation of Neuro2a cells were strongly suppressed by the addition of lysosphingolipids in a dose-dependent manner, whereas the parental sphingolipids had no effect. Intranucleosomal DNA fragmentation, chromatin condensation, and phosphatidylserine externalization, which are typical features of apoptosis, were observed when the cells were cultured with 40-80 microM of lysosphingolipids for 24-48 h in the presence of 5% fetal calf serum. Activation of caspase-3-like enzyme occurred after addition of lysosphingolipids followed by incubation at 37 degrees C for 24 h. The addition of an inhibitor of caspases, ZVAD-fmk, to the Neuro2a cell culture completely inhibited the elevation of caspase-3 activity but not the DNA fragmentation. These results may indicate that a caspase-3 independent signaling pathway is involved in the lysosphingolipid-induced apoptosis and suggest that accumulation of lysosphingolipids, but not parental sphingolipids, triggers the apoptotic cascade in neuronal cells of patients with sphingolipidoses.

Topics: Amidohydrolases; Animals; Apoptosis; Caspase 3; Caspases; Cattle; Chromatin; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; G(M1) Ganglioside; G(M2) Ganglioside; Glycosphingolipids; Humans; Mice; Neuroblastoma; Oxidoreductases; Phosphatidylserines; Pseudomonas; Psychosine; Signal Transduction; Sphingolipids; Sphingosine; Tumor Cells, Cultured

To investigate mechanisms of neurite outgrowth, murine Neuro-2a neuroblastoma cells were exposed to ganglioside GM1 in the presence or absence of specific protein kinase inhibitors. Isoquinolinesulfonamide (H-89), an inhibitor of cyclic AMP dependent protein kinase A (PKA), and bisindolylmaleimide I (BIM), which inhibits protein kinase C, each stimulated neurite outgrowth in a dose-dependent manner in the absence of exogenous GM1. Minimally effective (threshold) concentrations of H-89 or BIM potentiated outgrowth when they were used in combination with GM1. To search for a shared component in the mechanisms of GM1, H-89 and BIM, phosphorylation of ERK1/2 was examined. Inhibition of the activation of extracellular signal regulated kinases (ERK1/2) by U0126, prevented neuritogenesis of Neuro-2a by all the three agents. Pretreatment of serum-depleted Neuro-2a cultures with GM1 or BIM enhanced ERK1/2 phosphorylation when the serum level was restored to 10%. In contrast, H-89 did not alter the serum-mediated response. In cells exposed to GM1 or BIM without additional serum, a transitory decrease in ERK phosphorylation occurred. These data suggest that GM1 influences two neuritogenic pathways, one modulated by PKC and the other regulated by PKA. Therefore, GM1 may have the potential to stimulate alternate pathways resulting in outgrowth.

Topics: Animals; Enzyme Inhibitors; G(M1) Ganglioside; Indoles; Isoquinolines; Maleimides; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neurites; Neuroblastoma; Phosphorylation; Sulfonamides; Tumor Cells, Cultured

Recent work has demonstrated that induced neurite outgrowth in neuroblastoma cells and spontaneous differentiation of primary neurons in culture are accompanied by upregulation of GM1 ganglioside in the nuclear envelope. Previous reports have depicted morphological variations in the nature of stimulated neurites resulting from different neuritogenic agents, and a recent study by this laboratory demonstrated that such stimulants could be divided into two categories: those which induce axon-like neurites (group I) as opposed to those that stimulate dendrite-like outgrowths (group II). The former includes KCl, ionomycin, neuraminidase, and cholera toxin B subunit (all agents which elevate intracellular Ca2+), while the latter group is comprised of retinoic acid, dibutyryl cAMP, exogenous GM1, and low serum treatment. The present study was undertaken to determine whether differences in neuritic phenotype could be correlated with upregulation of nuclear GM1. The neuroblastoma x glioma NG108-15 cell line was employed because of its ability to respond robustly to a variety of neuritogenic stimuli. It was found that although both groups of stimulants are capable of inducing stable neurites (terminal differentiation) in this cell line, nuclear GM1 is elevated only in the presence of group I stimulants. Thus, a correlation is indicated between axonogenesis and upregulation of GM1 in the nuclear envelope. Additionally, these two events appear to coincide with elevation of intracellular Ca2+. Conversion of cells to the differentiated phenotype, with or without nuclear GM1 elevation, was found to depend in some cases on concentration of stimulant and duration of treatment.

Topics: Animals; Axons; Cell Division; Cell Nucleus; Dendrites; G(M1) Ganglioside; Mice; Neurites; Neuroblastoma; Tumor Cells, Cultured; Up-Regulation

Interaction of antibodies to ganglioside GM1 with Neuro2a cells was studied to investigate the role of GM1 in cell signaling. Binding of anti-GM1 to Neuro2a cells induced the formation of 3H-inositol phosphates (3H-IPs) and elevated the intracellular Ca2+ concentration [Ca2+]i. The rise in [Ca2+]i was due to the influx of Ca2+ from the extracellular medium and release from intracellular Ca2+ pools. The Ca2+ influx pathway did not allow the permeation of Na+ or K+. The influx was inhibited by amiloride, a specific blocker of T-type Ca2+ channels, whereas nifedipine and diltiazem, blockers of L-type Ca2+ channels, did not have any effect. Thus, anti-GM1 appears to activate a T-type Ca2+ channel in Neuro2a cells. The intracellular Ca2+ release was inhibited by pretreatment of cells with neomycin sulfate, phorbol dibutyrate, and pertussis toxin (PTx), which also inhibited the 3H-IP formation in Neuro2a cells. Addition of caffeine neither elevated the [Ca2+]i nor affected the anti-GM1-induced [Ca2+]i rise. The data reveal that the binding of anti-GM1 to Neuro2a cells activates phospholipase C via a PTx-sensitive G protein, which leads to formation of IPs and release of Ca2+ from inositol trisphosphate-sensitive pool of endoplasmic reticulum. Anti-GM1 also arrested the differentiation of Neuro2a cells in culture and significantly stimulated their proliferation. This stimulatory effect of anti-GM1 on cell proliferation was blocked by amiloride but not by PTx, suggesting that the influx of Ca2+ was essentially required for cell proliferation. Our data suggest a role for GM1 in the regulation of transmembrane signaling events and cell growth.

Topics: Amiloride; Antibodies; Calcium; Calcium Channels; Cell Division; Chelating Agents; Diuretics; Egtazic Acid; G(M1) Ganglioside; GTP-Binding Proteins; Inositol Phosphates; Neomycin; Neuroblastoma; Neurons; Pertussis Toxin; Potassium; Protein Synthesis Inhibitors; Signal Transduction; Sodium; Tritium; Tumor Cells, Cultured; Virulence Factors, Bordetella

Neuroblastoma cells are widely utilized models for the study of the neuritic outgrowth phase of neuronal differentiation, but relatively few such studies have attempted to identify the nature of the process outgrowths. This identification will be necessary in developing strategies for utilizing these models to distinguish the underlying mechanisms involved in axonogenesis vs dendritogenesis. In an effort to identify procedures for inducing specific types of neurite outgrowth, and for distinguishing axon- from dendrite-like processes, we have subjected two neuroblastoma cell lines to a variety of stimuli previously shown to induce neurite outgrowth in these cells. These include neuraminidase, ionomycin, KCl+dibutyryl cAMP, cholera toxin B subunit, retinoic acid, dibutyryl cAMP (alone), GM1 ganglioside, and low serum. The first four of these (group 1) gave rise to neurites with axon-like characteristics, including immunostaining that was positive for phosphorylated high molecular weight neurofilament protein (NF-H) and synaptic vesicle protein-2 (SV2), but negative for microtubule-associated protein-2 (MAP2). The next three treatments (group 2) resulted in dendrite-like processes, as evidenced in immunostaining that was positive for MAP2 and negative for NF-H and SV2. Neurites produced by low serum had mixed properties. These cytoskeletal differences were supported by immunoblot analysis with antisera to the above cytoskeletal proteins. Striking morphological differences were also noted, group 2-induced neurites being significantly shorter with more branch points than those generated by group 1 stimulants. Time of exposure to stimulatory agent was crucial in determining expression of the neuritic phenotype. Correlation with previous studies suggests that axon-like neurites result from stimulants which elevate intracellular Ca2+, a dependence not previously reported to our knowledge. Dendrite-like process outgrowth, on the other hand, does not appear to depend on altered intracellular Ca2+.

Topics: Animals; Axons; Bucladesine; Cholera Toxin; Dendrites; G(M1) Ganglioside; Ionomycin; Membrane Glycoproteins; Microtubule-Associated Proteins; Nerve Tissue Proteins; Neuraminidase; Neuroblastoma; Neurofilament Proteins; Neurotransmitter Agents; Potassium Chloride; Rats; Tretinoin; Tumor Cells, Cultured

Cell density-dependent inhibition of growth and neural differentiation in the human neuroblastoma cell line SK-N-MC are associated with a ganglioside sialidase-mediated increase of GM1 and lactosylceramide at the cell surface. Because these glycolipids expose galactose residues, we have initiated the study of the potential role of galectins in such cellular events. Using specific antibodies, galectin-1 but not galectin-3 was found to be present at the cell surface. Assessment of carbohydrate-dependent binding revealed a saturable amount of ligand sites approaching 2.6 x 10(6) galectin-1 molecules bound/cell. Presence during cell culture of the sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid or of the GM1-binding cholera toxin B subunit effected a decrease of the presentation of galectin-1 ligands by 30-50%. The assumption that GM1 is a major ligand for galectin-1 was reinforced by the correlation between the number of carbohydrate-dependent 125I-iodinated GM1-neoganglioprotein binding sites and the amount of immunoreactive surface galectin-1, the marked sensitivity of probe binding to the presence of anti-galectin-1 antibody, and the inhibition of cell adhesion to surface-immobilized GM1 by the antibody. The results open the possibility that the carbohydrate-dependent interaction between ganglioside GM1 and galectin-1 may relay sialidase-dependent alterations in this cell system.

Topics: Cell Division; Cell Membrane; G(M1) Ganglioside; Galectin 1; Hemagglutinins; Humans; Membrane Proteins; Neuraminidase; Neuroblastoma; Tumor Cells, Cultured

A human IgG1.k monoclonal antibody (MAb) designated GMA1 was developed by fusing pooled lymph node lymphocytes from cancer patients with the human lymphoblastoid cell line, SHFP-1. The GMA1 MAb reacted with several melanoma and neuroblastoma cell lines. Normal tissue derived from human brain and tumor-cell lines derived from colon, ovary, and breast were not reactive. FACS analysis performed using live cells demonstrated that the antibody recognizes a cell-surface antigen. Enzyme immunoassay (EIA) and thin layer chromatography (TLC) immunostaining with purified gangliosides indicated that the antibody has specificity for the major tumor associated gangliosides GD3, GM3, and GD2. GMA1 heavy and light chain genes were isolated by RT-PCR and a recombinant derivative of this human antibody was expressed in Chinese hamster ovary (CHO) cells. High-level antibody synthesis and secretion was achieved using a vector designed to maximize expression. FACS analysis and TLC immunostaining indicated recombinant GMA1 reacted with human tumor cell lines and gangliosides GD3, GM3, GD2 in a manner similar to the antibody produced by the hybridoma cell line, demonstrating that the specificity of the antibody was not altered during molecular cloning.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; Antigens, Surface; Base Sequence; G(M1) Ganglioside; Gangliosides; Humans; Hybridomas; Immunoglobulin G; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Melanoma; Molecular Sequence Data; Neuroblastoma; Recombinant Proteins; Tumor Cells, Cultured

Addition of DL-threo-1-phenyl-2-decanolylamine-3-morpholino-1-propanol HCl (PDMP; 7-24 microM) or Fumonisin B1 (FB1; 30-50 microM) to SH-SY5Ytrk-A human neuroblastoma cells results within 4 days in a 40% decrease of the ganglioside content and in a reduction of nerve-growth-factor (NGF)-induced outgrowth of neuritic processes. NGF-induced enhancement of GAP-43 expression was not affected. However, unlike controls, immunostained GAP-43 appeared concentrated in defined areas of cell perikarya and mostly absent from cell processes. Presence of 20-microM exogenous GM1 for 4 days in NGF and PDMP containing cell cultures led to an increase of cell-associated GM1(15-fold), GM2 (10-fold), GM3 (15 fold), GD1a (4-fold), GD2, GD1b, and GT1b (all 3-fold), and partially reversed the PDMP (and FB1) effects on neurite growth and GAP-43 distribution. In a newly developed neuronal tissue culture system, PDMP and FB1 led to a comparable dose-dependent inhibition of neurite outgrowth from embryonic chicken spinal cord explants, which had been embedded into a fibrin matrix. In this system, addition of GM1 led to a further inhibition of neurite growth, probably due to an interaction with growth-promoting components present in the surrounding fibrin matrix.

Topics: Animals; Carboxylic Acids; Cell Differentiation; Chick Embryo; Enzyme Inhibitors; Fumonisins; G(M1) Ganglioside; Gangliosides; GAP-43 Protein; Humans; Morpholines; Mycotoxins; Nerve Growth Factors; Neurites; Neuroblastoma; Neurons; Spinal Cord; Tumor Cells, Cultured

Topics: Becaplermin; G(M1) Ganglioside; Humans; Neurites; Neuroblastoma; Oligonucleotides, Antisense; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Recombinant Proteins; Thionucleotides; Thymidine; Tumor Cells, Cultured

Addition of gangliosides to neuroblastoma cells maintained in vitro has been shown to enhance neuritogenesis. Although the mechanism by which they exert this effect is unknown, it has been postulated that they may act by adhering to cell surface proteins. In this article, we describe the isolation and identification of an S20Y murine neuroblastoma cell protein recognized by a monoclonal antibody that was prepared against putative GM1-binding proteins and shown to inhibit GM1-enhanced neuritogenesis. The protein identified was nonmuscle myosin heavy chain B, which appears to function in neurite formation but may not adhere to gangliosides.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibody Specificity; Blotting, Western; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; G(M1) Ganglioside; Isoenzymes; Mice; Myosin Heavy Chains; Neurites; Neuroblastoma; Tumor Cells, Cultured

Exogenously added gangliosides are known to promote neurite outgrowth in a variety of cell types, including some neuroblastoma cell lines. To study neuritogenesis in SH-SY5Y human neuroblastoma we serum starved the cells for 24 hr and exposed them to gangliosides (GM1, GM3, or GT1b), platelet-derived growth factor (PDGF), insulin, nerve growth factor (NGF), insulin-like growth factor I (IGF-I), or combinations of these for 3 days. We measured four parameters of neurite outgrowth using image analysis. PDGF induced neurite outgrowth in SH-SY5Y and GM1 inhibited this. Both phenomena were dose-dependent with neurites/cell and neurite length being below controls with 100 microM GM1, and percent of neurite-bearing cells being below controls with 25, 50, and 100 microM GM1. Similar but more inhibitory results were obtained with GM3 and GT1b. Insulin and IGF-I induced a neuritogenic response that was less potent than that of PDGF and was also inhibited by gangliosides. NGF had no effect on neurite outgrowth but gangliosides were still inhibitory even in cells not treated with growth factors. From this we conclude that gangliosides inhibit spontaneous and trophic factor-induced neurite outgrowth in SH-SY5Y cells. For GM1 and GT1b, but not GM3, this probably involves inhibition of trophic factor receptor function.

Topics: Culture Media, Serum-Free; Dose-Response Relationship, Drug; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Growth Inhibitors; Growth Substances; Humans; Insulin; Insulin-Like Growth Factor I; Kinetics; Nerve Growth Factors; Neurites; Neuroblastoma; Platelet-Derived Growth Factor; Tumor Cells, Cultured

GM1 ganglioside was previously shown to function as a specific regulator of excitatory opioid activity in dorsal root ganglion neurons and F11 hybrid cells, as seen in its facilitation of opioid-induced activation of adenylyl cyclase and its ability to dramatically reduce the threshold opioid concentration required to prolong the action potential duration. The elevated levels of GM1 resulting from chronic opioid exposure of F11 cells were postulated to cause the ensuing opioid excitatory supersensitivity. We now show that GM1 promotes opioid (DADLE)-induced activation of adenylyl cyclase in NG108-15 cells which possess the delta-type of receptor. In keeping with previous studies of other systems, this can be envisioned as conformational interaction of GM1 with the receptor that results in uncoupling of the receptor from Gi and facilitated coupling to Gs. This would also account for the observation that DADLE-induced attenuation of forskolin-stimulated adenylyl cyclase was reversed by GM1, provided the cells were not pretreated with pertussis toxin. When the cells were so pretreated, GM1 evoked an unexpected attenuation of forskolin-stimulated adenylyl cyclase attributed to GM1-promoted influx of calcium which was postulated to inhibit a calcium-sensitive form of adenylyl cyclase. This is concordant with several studies showing GM1 to be a potent modulator of calcium flux. Pertussis toxin in these experiments exerted dual effects, one being to promote interaction of the delta-opioid receptor with Gs through inactivation of Gi, and the other to enhance the GM1-promoted influx of calcium by inactivation of Go; the latter is postulated to function as constitutive inhibitor of the relevant calcium channel. NG108-15 cells thus provide an interesting example of competitive interaction between two GM1-regulated systems involving enhancement of both opioid receptor excitatory activity and calcium influx.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Aminopterin; Binding, Competitive; Calcium Channels; Cholera Toxin; Cyclic AMP; Enkephalin, Leucine-2-Alanine; Fluorescent Dyes; G(M1) Ganglioside; Glioma; Hybrid Cells; Hypoxanthine; Neuroblastoma; Pertussis Toxin; Receptors, Opioid; Thymidine; Tumor Cells, Cultured; Virulence Factors, Bordetella

The effect of ganglioside GM1 on components of the neuronal cytoskeleton was studied in Neuro-2a neuroblastoma cells using immunofluorescent, immunogold-labeled, and Western-blot analysis. Exposure of cells to GM1 for 24 h resulted in an increased microtubular network and level of tubulin, a redistribution of MAP2 immunoreactivity from perikarya to distal neuritic processes, and an increased MAP2 gold label in the subplasmalemmal cytoplasm, neuritic spines, and growth cones. A similar change in the distribution of actin-positive fluorescent immunoreactivity was observed. In contrast to the redistribution of MAP2, immunolocalization of MAP5 and tau did not change following 24 h GM1 exposure. Our results suggest that gangliosides enhance neuritogenesis by selectively altering the distribution of MAP2 from perikaryon to neuritic spines. Furthermore, the enhanced presence of MAP2 in regions known to be rich in microfilaments following GM1 treatment suggests that an interaction of MAP2 with microfilaments may be necessary for early neurite formation.

Topics: Actins; Animals; Base Sequence; Blotting, Western; Fluorescent Antibody Technique; G(M1) Ganglioside; Mice; Microscopy, Immunoelectron; Microtubule-Associated Proteins; Microtubules; Molecular Sequence Data; Neuroblastoma; Neurons; Tumor Cells, Cultured

Various glycolipid-binding toxins are internalized from the cell surface to the Golgi apparatus. Prominent among these is cholera toxin (CT), which consists of a pentameric B subunit that binds to ganglioside GM1 and an A subunit that mediates toxicity. We now demonstrate that rhodamine (Rh)-CT can be further internalized from the Golgi apparatus to the endoplasmic reticulum (ER) in cultured hippocampal neurons and in neuroblastoma N18TG-2 cells and that the A subunit is essential for retrograde transport to the ER. In addition, the rate of internalization of Rh-CT to the Golgi apparatus and ER decreases dramatically as hippocampal neurons mature. The Golgi apparatus was labeled in almost all 1-day-old neurons after < 1 h of incubation with Rh-CT but was labeled in < 10% of 14-day-old neurons after 1 h. During the first 14 days in culture, there was a 15-fold increase in the number of 125I-CT-binding sites per cell, indicating that the decrease in the rate of internalization of Rh-CT is not due to reduced levels of cell surface GM1 in older neurons. These results imply that the rate of retrograde transport of CT from the plasma membrane to the Golgi apparatus and ER is regulated during neuronal development and differentiation.

Topics: Animals; Axonal Transport; Binding Sites; Cell Membrane; Cells, Cultured; Cholera Toxin; Endoplasmic Reticulum; G(M1) Ganglioside; Golgi Apparatus; Hippocampus; Kinetics; Neuroblastoma; Neurons; Rats; Rhodamines; Tumor Cells, Cultured

We have examined ganglioside compositions and the presence of sulfated glucuronyl glycolipids of immortalized motor neuron-like cell lines, neuroblastoma-spinal cord (NSC) hybrid cell lines established by fusing mouse neuroblastoma N18TG2 with motor neuron-enriched embryonic spinal cord cells. Among NSC cell lines, only NSC-34 aggregates acetylcholine receptors on co-cultured myotube and expresses a receptor for S-laminin, a neuromuscular junction specific basal lamina protein. GM2, which is only a minor ganglioside component of CNS, was the major component in NSC-34 occupying almost 75% of total gangliosides, whereas GD1a and GM3 were major species in the parental N18TG2, which had only 8.5% GM2. These results indicated that NSC lines have unique ganglioside pattern that is distinctive from other nervous tissues, and this pattern, especially that of NSC-34 cells, might reflect the characteristics of mouse spinal motor neuron gangliosides. Sulfated glucuronyl paragloboside was demonstrated to be present in N18TG2, however, it could not be detected in either of NSC cell lines. Even though the pathogenesis of amyotrophic lateral sclerosis remains unknown, autoimmunological participation has been suggested. Because high-titered antibody against GM2 has been observed in a patient with amyotrophic lateral sclerosis-like disease, GM2 which is possibly expressed on the surface of motor neurons might serve as a potential target antigen in this disorder.

Topics: Animals; Cell Line; Cholera Toxin; Chromatography, Thin Layer; G(M1) Ganglioside; G(M2) Ganglioside; Immunohistochemistry; Mice; Motor Neurons; Neuroblastoma; Phenotype; Protein Binding; Spinal Cord

Neuro-2a neuroblastoma cells can be stimulated to extend neurites with a number of agents, one of which, neuraminidase, induces terminal differentiation by a mechanism involving enhanced Ca2+ influx. Permeabilization of such differentiated cells with saponin and treatment with cholera toxin B subunit linked to horseradish peroxidase revealed intense staining of the nuclear membrane, indicating the presence of GM1 ganglioside. Unstimulated cells had barely detectable levels of nuclear GM1. Nuclei isolated by sucrose density gradient centrifugation similarly showed intense staining with fluorescently labeled cholera toxin B subunit, in contrast to nuclei from undifferentiated controls. Treatment with chloroform-methanol removed most of the fluorogenic material. Chemical analysis of such nuclei from neuraminidase-treated cells confirmed significant elevation of GM1 above control levels, along with virtual absence of markers for plasma membrane and Golgi apparatus. Cerebellar granule cells from neonatal rats revealed a similar phenomenon following spontaneous neurite outgrowth in culture.

Topics: Animals; Calcium; Cell Differentiation; Cell Line; Cell Nucleus; Cerebellum; Cholera Toxin; G(M1) Ganglioside; Horseradish Peroxidase; Membrane Lipids; Mice; Neuraminidase; Neurites; Neuroblastoma; Neurons; Nuclear Envelope; Rats; Rats, Sprague-Dawley; Saponins; Tumor Cells, Cultured

Gangliosides GM1 [3H-labeled at the sphingosine (Sph) moiety] and GM2 [3H-labeled at the Sph or N-acetylgalactosamine (GalNAc) moiety] were administered to cultured Neuro2a cells for varying pulse (1-4 h) and chase (up to 4 h) periods, and their metabolic processing was followed. The main and earliest formed 3H-metabolites of [Sph-3H]GM1 were GM2, asialo-GM1 asialo-GM2, and lactose-ceramide, and those of [Sph-3H]GM2 were asialo-GM2 and lactose-ceramide. The asialo-GM1 and asialo-GM2 formed were isolated and chemically characterized. [3H]Asialo-GM2 was produced in identical amounts after treatment with equimolar [Sph-3H]GM2 and [GalNAc-3H]GM2. At low temperature or in the presence of chloroquine, the formation of all 3H-metabolites, including asialo-GM2 and asialo-GM1, was undetectable, indicating that ganglioside metabolic processing was an endocytosis- and lysosome-dependent process. These results demonstrate that in Neuro2a cells exogenous GM1 (and GM2) is mainly degraded through the pathway GM1-->GM2-->asialo-GM2-->-->Sph, with a minor fraction of GM1 undergoing degradation with the sequence GM1-->asialo-GM-1-->asialo-GM2-->-->Sph. These findings are consistent with the hypothesis that Neuro2a cells contain a sialidase (likely of lysosomal nature) affecting ganglioside GM1 and GM2. The sialidase-mediated degradative pathway of GM1 and GM2 in Neuro2a cells might be related to the tumoral nature of these cells.

Topics: Animals; G(M1) Ganglioside; G(M2) Ganglioside; Mice; Neuraminidase; Neuroblastoma; Tumor Cells, Cultured

Prolongation of the action potential duration of dorsal root ganglion (DRG) neurons by low (nM) concentrations of opioids occurs through activation of excitatory opioid receptors that are positively coupled via Gs regulatory protein to adenylate cyclase. Previous results suggested GM1 ganglioside to have an essential role in regulating this excitatory response, but not the inhibitory (APD-shortening) response to higher (microM) opioid concentrations. Furthermore, it was proposed that synthesis of GM1 is upregulated by prolonged activation of excitatory opioid receptor functions. To explore this possibility we have utilized cultures of hybrid F11 cells to carry out closely correlated electrophysiological and biochemical analyses of the effects of chronic opioid treatment on a homogeneous population of clonal cells which express many functions characteristic of DRG neurons. We show that chronic opioid exposure of F11 cells does, in fact, result in elevated levels of GM1 as well as cyclic adenosine monophosphate (AMP), concomitant with the onset of opioid excitatory supersensitivity as manifested by naloxone-evoked decreases in voltage-dependent membrane K+ currents. Such elevation of GM1 would be expected to enhance the efficacy of excitatory opioid receptor activation of the Gs/adenylate cyclase/cyclic AMP system, thereby providing a positive feedback mechanism that may account for the remarkable supersensitivity of chronic opioid-treated neurons to the excitatory effects of opioid agonists as well as antagonists. These in vitro findings may provide novel insights into the mechanisms underlying naloxone-precipitated withdrawal syndromes and opioid-induced hyperalgesia after chronic opiate addiction in vivo.

Topics: Animals; Cells, Cultured; Cyclic AMP; Electrophysiology; Enkephalin, Leucine-2-Alanine; G(M1) Ganglioside; Ganglia, Spinal; Gangliosides; Hybrid Cells; Naloxone; Neuroblastoma; Neurons; Potassium; Potassium Channels; Time Factors; Tumor Cells, Cultured

Murine Neuro-2A neuroblastoma cells were exposed to ethanol in culture under two experimental paradigms: (1) short-term (24 hr or less) and low concentrations (0.05 to 0.5%; 8.5 to 86 mM) and (2) long-term (48 hr at 0.5%; 86 mM). Long-term ethanol exposure did not affect Neuro-2A viability, determined by DNA synthesis or the ability to exclude Trypan Blue. Similarly, long-term ethanol treatment did not inhibit differentiation, exhibited by the extension of neurites, promoted by either dibutyryl-cyclic-AMP or by incubation with exogenous ganglioside GM1. The incorporation of exogenous ganglioside GM1 into plasma membranes was not influenced by varying concentrations of ethanol (up to 1.2%; 204 mM). In contrast, ethanol did influence Neuro-2A cell attachment to collagen in a dualistic manner. During short-term ethanol exposure, cell attachment was enhanced. However, when cells were initially exposed to ethanol for 48 hr a marked inhibition of subsequent attachment was observed. Long-term ethanol exposure also inhibited attachment to other substrata, including laminin, fibronectin and vitronectin. Incubation of Neuro-2A cells with either exogenous ganglioside GM1 or a mixture of brain gangliosides partially reversed the inhibition of attachment to collagen. This reversal did not appear to be due to any one particular ganglioside structure, however. Mixed brain gangliosides were fractionated into three fractions, according to the number of sialic acid residues. Each of the three fractions were equally effective in partially restoring Neuro-2A cell attachment to collagen after long-term ethanol treatment. The results suggest that the mechanism by which these effects occur is at the level of plasma membrane fluidity, because both ethanol and glycosphingolipid content are known to influence membrane lateral mobility, although other mechanisms, such as changes in headgroup hydration, are possible.

Topics: Animals; Brain Neoplasms; Cattle; Cell Adhesion; Cell Membrane; Cell Survival; Central Nervous System Depressants; Ethanol; G(M1) Ganglioside; Gangliosides; Mice; Neurites; Neuroblastoma; Tumor Cells, Cultured

Digital imaging fluorescence microscopy was used to investigate the effect of the B subunit of cholera toxin on calcium homeostasis in neuroblastoma N18 cells. The B subunit, which binds specifically to ganglioside GM1 in the outer leaflet of the cell membrane, was found to induce a sustained increase of intracellular calcium concentration ([Ca2+]i). The increase in [Ca2+]i was not observed in the absence of extracellular calcium, or in the presence of the calcium chelator EGTA, and was blocked by nickel. The B subunit was also found to induce an influx of manganese ions, as indicated by a quench of the intracellular fura-2 fluorescence. These data suggest that the B subunit induces an increase in calcium influx in N18 cells. Potassium-induced depolarization also stimulated manganese influx; however, after the onset of depolarization-induced influx, the B subunit had no further effect. This occlusion suggests involvement of voltage-dependent calcium channels. Treatment with BayK8644, a dihydropyridine agonist selective for L-type calcium channels, induced manganese influx that was not altered by the B subunit and apparently blocked the effect of the B subunit itself. Furthermore, the dihydropyridine L-type channel antagonists niguldipine or nicardipine completely inhibited B subunit-induced manganese influx. Thus, the B subunit-induced manganese influx is likely due to activation of an L-type voltage-dependent calcium channel. Spontaneous influx of manganese ions was also inhibited by nicardipine or niguldipine and by exogenous gangliosides. Ganglioside GM1 was more potent than GM3, but globoside had no significant effect. The modulation of L-type calcium channels by endogenous ganglioside GM1 has important implications for its role in neural development, differentiation, and regeneration and also for its potential function in the electrical excitability of neurons.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Line; Cholera Toxin; Dihydropyridines; Egtazic Acid; Fura-2; G(M1) Ganglioside; G(M3) Ganglioside; Manganese; Mice; Microscopy, Fluorescence; Neuroblastoma; Nicardipine; Nickel; Potassium; Tumor Cells, Cultured

Human neuroblastoma SH-SY5Y cell is a cloned cell line which has many attractive features for the study of neuronal proliferation and neurite outgrowth, because it has receptors for insulin, IGF-I and PDGF. Gangliosides are sialic acid containing glycosphingolipids which form an integral part of the plasma membrane of many mammalian cells. They inhibit cell growth mediated by tyrosine kinase receptors and ligand-stimulated tyrosine kinase activity, and autophosphorylation of EGF(epidermal growth factor) and PDGF receptors. The experiment was designed to study the effects of GM1 ganglioside on growth of human neuroblastoma SH-SY5Y cells stimulated with trophic factor in vitro. The cells were plated in Eagle's minimum essential medium without serum. The number and morphologic change of SH-SY5Y cells were evaluated in the serum free medium added GM1 ganglioside with insulin or PDGF. SH-SY5Y cells were maintained for six days in serum-free medium, and then cultured for over two weeks in serum-free medium containing either insulin or PDGF. The effect of insulin on cell proliferation developed earlier and was more potent than that of PDGF. These proliferative effects were inhibited by GM1 ganglioside, and the cells showed prominent neurites outgrowth. These findings suggest that GM1 ganglioside inhibits the cell proliferation mediated by tyrosine kinase receptors and directly induces neuritogenesis as one of the neurotrophic factors.

Topics: Cell Differentiation; Cell Division; G(M1) Ganglioside; Humans; Insulin; Neuroblastoma; Neurons; Platelet-Derived Growth Factor; Receptor Protein-Tyrosine Kinases; Tumor Cells, Cultured

Neuro-2a murine neuroblastomal cells exposed to exogenous ganglioside undergo increased neuritogenesis in vitro. To determine if the distribution of exogenous ganglioside (GM1) in neuronal membranes is related to neuritogenesis, the surface topography of exogenous ganglioside in these cells was examined by localization with cholera toxin B-FITC. Following exposure to exogenous ganglioside, levels of fluorescent label appeared similar on perikaryal and neuritic surfaces. Scanning electron microscopic studies using protein G-gold to label antibody against exogenous ganglioside confirmed these observations at higher magnification. Within the general labelling pattern, occasionally labelled material was observed which seemed to form short linear arrays. This suggested that elements of the cytoskeleton might be influencing the surface distribution of exogenous ganglioside. To examine this possibility, Neuro-2a cells were exposed to agents known to alter the stability of specific cytoskeletal components, after which the general distribution of exogenous ganglioside was determined. Treatment with Colcemid, which disrupted microtubules, resulted in restriction of most exogenous ganglioside-positive label to the perikaryal surfaces. In contrast, exposure to taxol which enhanced microtubule stability diminished perikaryal fluorescence and increased neuritic labelling. The disruption of cytochalasin D-sensitive microfilaments did not influence the topographic distribution of exogenous ganglioside. Under the experimental conditions employed, mean neuritic lengths for Colcemid- and taxol-treated cells were nearly equal, indicating that altered neuritic length resulting from treatment with cytoskeletal agents was not a major factor in the redistribution of exogenous ganglioside. These studies suggest that microtubules play a role in determining the distribution of recently incorporated ganglioside in neuronal plasma membranes.

Topics: Animals; Cell Membrane; Cholera Toxin; Cytochalasin D; Cytoskeleton; Demecolcine; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique; Fluorescent Dyes; G(M1) Ganglioside; Gangliosides; Mice; Microscopy, Electron, Scanning; Microtubules; Neurites; Neuroblastoma; Paclitaxel; Tumor Cells, Cultured

A serum containing a monoclonal IgM lambda with anti-GM1 and anti-GD1b activity was obtained from a patient with upper motor neuron syndrome. By indirect immunocytochemical techniques with double staining, the patient's IgM strongly stained membranes of neurons in primary cultures of fetal central and peripheral nervous system. It was cytotoxic for neurons in two human neuroblastoma established cell lines in a complement-dependent chromium release assay. These results are in keeping with the hypothesis of a direct pathogenetic role of such monoclonal anti-GM1 and GD1b IgM antibodies.

Topics: Antibodies, Monoclonal; Cells, Cultured; Female; G(M1) Ganglioside; Gangliosides; Humans; Immunoglobulin M; Neuroblastoma; Neurons; Pregnancy; Tumor Cells, Cultured

S20Y murine neuroblastoma cells appear to express a protein component(s) able to adhere specifically to the oligosaccharide portion of GM1 (oligo-GM1). To identify proteins with which the oligo-GM1 becomes closely associated, a radiolabeled (125I), photoactivatable derivative of oligo-GM1 was prepared. This was accomplished by reductive amination of the glucosyl moiety of oligo-GM1 to 1-deoxy-1-aminoglucitol, followed by reaction of the amine with sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD). Crosslinking studies using the photoactivatable probe indicated that it came in close proximity to a protein with an apparent molecular mass of approximately 71 kDa. In competition experiments, as little as a 10-fold molar excess of oligo-GM1 resulted in a selective reduction in labeling of this protein; preincubation with a 200-fold molar excess of siayllactose was necessary to observe the same change in the labeling pattern, lending additional support to the hypothesis that the approximately 71-kDa protein specifically associates with oligo-GM1. Cell surface location of the oligo-GM1 binding protein was confirmed using subcellular fractionation and morphological analyses.

Topics: Animals; Cell Fractionation; Cells, Cultured; Fibroblasts; G(M1) Ganglioside; Iodine Radioisotopes; Mice; Mice, Inbred BALB C; Neuroblastoma; Oligosaccharides; Protein Binding; Tumor Cells, Cultured

The present study uses the B subunit of cholera toxin, a protein that binds specifically to ganglioside GM1, to examine the role of endogenous GM1 in the process of growth and differentiation of mouse neuroblastoma N18 cells. Binding of the B subunit to neuroblastoma N18 cells inhibited DNA synthesis with concomitant induction of differentiation. The B subunit induced pronounced morphological changes: an increase in neurite outgrowth with branched neurites and spinelike processes. The distinct morphological alterations and neuritogenesis in response to the B subunit were also revealed by immunofluorescence with fluorescein-labeled B subunit. The mechanism of the B subunit-induced differentiation is different than that of spontaneous differentiation. Thrombin, a serine protease present in normal serum, inhibits neurite outgrowth induced by the removal of serum from the medium. In contrast, thrombin did not cause retraction of the neurites induced by the B subunit. Thus, thrombin or a thrombin-like protease is not involved in the process of neurite outgrowth mediated through endogenous GM1. The biological effects of the B subunit are due to the binding of the B subunit to ganglioside GM1 and not due to changes in cAMP levels resulting from contaminating A subunit. We used highly purified cloned B subunit that cannot contain any A subunit because it was isolated from a Vibrio cholerae mutant that only expresses the B subunit. Neither the cloned nor commercial preparations of the B subunit induced increases of cAMP in these cells. There was a good correlation between the amount of B subunit bound to the cells and the biological effect. Finally, treatment with neuraminidase, which caused a fourfold increase in the level of membrane GM1 as determined by iodinated cholera toxin binding, enhanced the biological effect of the B subunit. However, neuraminidase treatment alone did not have significant effects, either on DNA synthesis or on morphology of the cells, indicating that elevations in the level of GM1 per se are not sufficient by themselves to cause significant changes in cell growth or differentiation. It seems most likely that the aggregation of endogenous GM1 on the cell surface by the B subunit is responsible for these effects on mouse neuroblastoma N18 cells.

Topics: Animals; Cell Differentiation; Cell Division; Cholera Toxin; Cyclic AMP; DNA; Drug Synergism; G(M1) Ganglioside; Intracellular Membranes; Neuraminidase; Neuroblastoma; Thrombin; Tumor Cells, Cultured

Gangliosides of 11 different neuroblastoma cell lines, grown to confluence, were extracted and quantified with respect to: (a) total lipid-bound sialic acid, (b) total gangliotetraose family, and (c) GM1 content. The cultured cells were induced to grow neurites in 3 ways: (a) serum reduction, (b) exogenous ganglioside, and (c) retinoic acid. Neurite outgrowth was quantified in terms of % of cells bearing neurites and average number of neurites per cell. No correlation was observed between neurite outgrowth and total ganglioside concentration, but a reasonably good correlation was observed with respect to neuritogenesis and gangliotetraose content. When exogenous ganglioside was the stimulant the best correlation was with GM1, whereas retinoic acid-stimulated outgrowth was approximately proportional to GD1a content. The 'neurite minus' N1A-103 line, which had the lowest level of GM1, GD1a, and total gangliotetraose gangliosides, showed little if any response to any of the stimuli.

Topics: Animals; Brain; Cattle; Cell Differentiation; Cholera Toxin; Chromatography, Thin Layer; G(M1) Ganglioside; Horseradish Peroxidase; Humans; Mice; Neurites; Neuroblastoma; Rats; Staining and Labeling; Tretinoin; Tumor Cells, Cultured

Several lines of evidence suggest that gangliosides may play a role in the regulation of growth in many cell types. Here we describe the effects on growth of two different cell lines by the addition of two different chemicals which have been reported to elevate the cellular ganglioside content through different mechanisms. Growth of neuroblastoma (Neuro 2a) cells in medium containing fetal bovine serum was inhibited in a dose-dependent fashion by both exogenous GM1 ganglioside and NeuAc2en, an inhibitor of sialidase activity. In contrast, growth of glioma cells (U-1242 MG) was not affected by exogenous GM1 or NeuAc2en in the presence of as little as 1% calf serum. However, NeuAc2en inhibited growth of U-1242 MG cells stimulated by platelet-derived growth factor in serum-free medium. These results demonstrate that the growth inhibitory effects of ganglioside on U-1242 MG but not Neuro 2a cells can be counteracted by serum, suggesting that the mechanisms through which gangliosides affect cell growth may be different for different growth factors and cell types.

Topics: Animals; Azides; Brain Neoplasms; Cell Division; Culture Media; Culture Media, Serum-Free; Depression, Chemical; G(M1) Ganglioside; Glioma; Humans; Mice; Neuraminidase; Neuroblastoma; Platelet-Derived Growth Factor; Sialic Acids; Tumor Cells, Cultured

Subclones of F11 neuronal hybrid cells (neuroblastoma x dorsal root ganglion neurons) have segregated differing and/or overlapping neuritogenic mechanisms on three substrata--plasma fibronectin (pFN) with its multiple receptor activities, cholera toxin B subunit (CTB) for binding to ganglioside GM1, and platelet factor-4 (PF4) for binding to heparan sulfate proteoglycans. In this study, specific cell surface receptor activities for the three substrata were tested for their modulation during neuritogenesis by several experimental paradigms, using F11 subclones representative of three differentiation classes (neuritogenic on pFN only, on CTB only, or on all three substrata). When cycloheximide was included in the medium to inhibit protein synthesis during the active period, neurite formation increased significantly for all subclones on all three substrata, virtually eliminating substratum selectivity for differentiation mediated by cell surface integrin, ganglioside GM1, or heparan sulfate proteoglycans. Therefore, one or more labile proteins (referred to as disintegrins) must modulate functions of matrix receptors (e.g., integrins) mediating neurite formation. To verify whether cycloheximide-induced neuritogenesis was also regulated by integrin interaction with cell surface GM1, two approaches were used. When (Arg-Gly-Asp-Ser)-containing peptide A was added to the medium, it completely inhibited cycloheximide-induced neuritogenesis on all three substrata of all subclones, indicating stringent requirement for cell surface integrin function in these mechanisms. In contrast, when CTB or a monoclonal anti-GM1 antibody was also added to the medium, cycloheximide-induced neuritogenesis was amplified further on pFN and sensitivity to peptide A inhibition was abolished. Therefore, in some contexts ganglioside GM1 must complex with integrin receptors at the cell surface to modulate their function. These results also indicate that (a) cycloheximide treatment leads to loss of substratum selectivity in neuritogenesis, (b) this negative regulation of neurite outgrowth is affected by integrin receptor association with labile regulatory proteins (disintegrins) as well as with GM1, and (c) complexing of GM1 by multivalent GM1-binding proteins shifts neuritogenesis from an RGDS-dependent integrin mechanism to an RGDS-independent receptor mechanism.

Topics: Amino Acid Sequence; Animals; Axons; Cell Differentiation; Cell Line; Cholera Toxin; Chondroitin Sulfate Proteoglycans; Cycloheximide; Fibronectins; G(M1) Ganglioside; Ganglia, Spinal; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Hybrid Cells; Integrins; Mice; Molecular Sequence Data; Neuroblastoma; Neurons; Oligopeptides; Platelet Factor 4; Rats; Receptors, Fibronectin; Receptors, Immunologic

Treatment of three neuroblastoma cell types in culture with neuraminidase resulted in enhanced neurite outgrowth. These included the mouse Neuro-2A and rat B104 and B50 lines. The morphological changes depended on the presence of exogenous Ca2+ and were accompanied by modest but statistically significant increases in 45Ca2+ influx. Neuraminidase-stimulated neuritogenesis was blocked by the B subunit of cholera toxin (cholera B) and anti-GM1 antibody, a finding suggesting the effect was due to an increased amount of GM1 on the cell surface. Cholera B also blocked the increase in 45Ca2+ influx. The mouse N1A-103 line, previously characterized as "neurite minus," did not respond to neuraminidase with either neurite outgrowth or enhanced Ca2+ influx. These results point to an influence of GM1 on neuritogenesis in cells with differentiation potential and suggest a mechanism involving modulation of Ca2+ flux.

Topics: Animals; Antibodies; Axons; Calcium; Calcium Radioisotopes; Cholera Toxin; G(M1) Ganglioside; Lanthanum; Mice; Neuraminidase; Neuroblastoma; Peptide Fragments; Rats; Tumor Cells, Cultured

Cultures of mouse Neuro-2a neuroblastoma cells treated with 3-6 mM extracellular Ca2+ exhibited enhanced neurite extension characterized by increased neurite numbers and lengths. The ganglioside GM1 potentiated the effect of extracellular Ca2+ by increasing further the number and length of the neurites formed in response to exogenous Ca2+. Maximal neuritic numbers were achieved with 4 mM Ca2+ while the longest neurites were observed in medium containing 4-6 mM Ca2+. Stimulation of the Ca2+ influx with the ionophore A23187 or the amino acid taurine also enhanced neurite formation and GM1 potentiated these actions. Transmission electron microscopy revealed numerous microtubules and neurofilaments in neurites and microfilaments with the spine-like processes along fine neuritic branches and in the filopodia of growth cones. Neuritic varicosities and growth cones contained a variety of vesicles. All of these structures were increased in the presence of GM1 and were increased further by extracellular Ca2+ or A23187. The ability of GM1 to enhance neuritogenesis was diminished by EGTA or Ruthenium red. Similarly, the effect of GM1 was diminished or abolished by Ca2+ channel blockers such as CdCl2 or LaCl3. X-ray microprobe analysis revealed that GM1 alone enhanced intracellular levels of total ionic and membrane bound Ca2+, perhaps accounting for the increased neuritogenesis observed under conditions in which Ca2+ was manipulated. The present study suggest that the neuritogenic action of GM1 is Ca2+ dependent.

Topics: Animals; Axons; Cadmium; Cadmium Chloride; Calcimycin; Calcium; Cell Differentiation; Cell Line; Egtazic Acid; G(M1) Ganglioside; Intermediate Filaments; Lanthanum; Mice; Microtubules; Neuroblastoma; Neurons; Ruthenium Red; Taurine

The enzymatic basis for ganglioside regulation during differentiation of NG108-15 mouse neuroblastoma x rat glioma hybrid cells was studied. This cell line contains four gangliosides that lie along the same biosynthetic pathway: GM3, GM2, GM1, and GD1a. Chemically induced neuronal differentiation of NG108-15 cells led to an 80% drop in the steady-state level of their major ganglioside, GM3, a sixfold increase in the level of a minor ganglioside, GM2 (which became the predominant ganglioside of differentiated cells); and relatively little change in the levels of GM1 and GD1a, which lie further along the same biosynthetic pathway. The enzymatic basis for this selective change in ganglioside expression was investigated by measuring the activity of two glycosyltransferases involved in ganglioside biosynthesis. UDP-N-acetylgalactosamine: GM3 N-acetylgalactosaminyltransferase (GM2-synthetase) activity increased fivefold during butyrate-induced differentiation, whereas UDP-galactose: GM2 galactosyltransferase (GM1-synthetase) activity decreased to 10% of its control level. Coordinate regulation of these two glycosyltransferases appears to be primarily responsible for the selective increase of GM2 expression during NG108-15 differentiation.

Topics: Alprostadil; Animals; Bucladesine; Cell Differentiation; G(M1) Ganglioside; G(M2) Ganglioside; G(M3) Ganglioside; Galactosyltransferases; Ganglioside Galactosyltransferase; Gangliosides; Glioma; Hybrid Cells; Kinetics; Mass Spectrometry; Mice; N-Acetylgalactosaminyltransferases; Neuroblastoma; Polypeptide N-acetylgalactosaminyltransferase; Rats; Theophylline; Tumor Cells, Cultured

Since exogenous gangliosides are known to promote neuritogenesis, the incorporation of exogenous GM1 into neuroblastoma membranes was examined. Neuro-2A cells, synchronized in the G1/G0 phase, were suspended in HEPES buffered saline containing 10(-4) M [3H]GM1, and membrane incorporation was measured as radioactivity remaining with the cell pellet following incubation with serum-containing medium and trypsin. Calcium ion (0.01 to 10 mM) reduced incorporation of exogenous GM1, due to its interaction with GM1 micelles in solution. When cells were treated with proteases prior to incubation with GM1, the inhibitory effect of Ca2+ was lost and total incorporation into membranes was lowered by approximately one order of magnitude. Pretreatment of cells with 0.05% trypsin resulted in an inhibition of GM1 incorporation within 5 minutes. When trypsinized cells were resuspended in complete growth medium, the cells recovered the ability to incorporate GM1 with time, and this paralleled labeling of cellular protein with [3H]leucine. The role of membrane protein in the incorporation of exogenous GM1 could not be explained by the lytic release of cytosolic transfer proteins nor the artifactual coating of the cell surface by serum proteins. These results suggest that the incorporation of exogenous gangliosides into cellular membrane lipid bilayers cannot be fully explained by considerations of lipophilicity alone, and leads us to propose that initial recognition by membrane protein(s) is necessary.

Topics: Animals; Calcium; Cell Membrane; G(M1) Ganglioside; Hyaluronoglucosaminidase; Hydrogen-Ion Concentration; Immunologic Techniques; In Vitro Techniques; Membrane Lipids; Membrane Proteins; Mice; Neuroblastoma; Peptide Hydrolases; Solubility; Tumor Cells, Cultured

Human neuroblastoma cells (Platt and La-N1) adhere and extend neurites on a ganglioside GM1-binding substratum provided by cholera toxin B (CTB). These adhesive responses, similar to those on plasma fibronectin (pFN), require the mediation of one or more cell-surface proteins [G. Mugnai and L. A. Culp (1987) Exp. Cell Res. 169, 328]. The involvement of two pFN receptor molecules in ganglioside GM1-mediated responses on CTB have now been tested. In order to test the role of cellular FN binding to its glycoprotein receptor integrin, a soluble peptide containing the Arg-Gly-Asp-Ser (RGDS) sequence was added to the medium. It did not inhibit attachment on CTB but completely inhibited formation of neurites; in contrast, the RGDS peptide minimally inhibited attachment or neurite formation on pFN. Once formed, neurites on CTB became resistant to the peptide. In order to test the role of cell-surface heparan sulfate proteoglycan (HS-PG), two approaches were used. First, the HS-binding protein platelet factor-4 (PF4) was used to dilute CTB or pFN on the substratum or, alternatively, added to the medium. Diluting the substratum ligand with PF4 had no effects on attachment on either CTB or pFN. However, neurite formation on CTB was readily inhibited and on pFN partially inhibited; the effects of PF4 were far greater than a similar dilution with nonbinding albumin. When PF4 was added to the medium of cells, attachment on either substratum was unaffected as was neurite outgrowth on pFN, revealing differences in PF4's inhibition as the substratum-bound or medium-borne component. In contrast, PF4 in the medium at low concentrations (1 microgram/ml) was highly inhibitory for neurite formation on CTB. The second approach utilized the addition of bovine cartilage dermatan sulfate proteoglycan (DS-PG), shown to bind to pFN as well as to substratum-bound CTB by ELISA, or cartilage chondroitin sulfate/keratan sulfate proteoglycan (CS/KS-PG) to the substratum or to the medium. At low concentrations, DS-PG but not CS/KS-PG actually stimulated neurite formation on CTB while at higher concentrations DS-PG completely inhibited attachment and neurite formation. While DS-PG partially inhibited attachment on pFN, it had no effect on neurite formation of the attached cells. Neuroblastoma cells adhered to some extent to substrata coated only with DS-PG, indicating "receptors" for PGs that permit stable interaction.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Axons; Cell Adhesion; Cholera Toxin; Chondroitin Sulfate Proteoglycans; Dermatan Sulfate; Enzyme-Linked Immunosorbent Assay; Fibronectins; G(M1) Ganglioside; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Neuroblastoma; Neurons; Oligopeptides; Platelet Aggregation Inhibitors; Proteoglycans; Receptors, Fibronectin; Receptors, Immunologic; Tumor Cells, Cultured

A variety of naturally occurring ganglioside structures were previously shown to be effective agents for inducing neurite outgrowth of primary neurons and neuroblastoma lines. We report here the results of similar experiments with a synthetic epimer of GM3 (epi-GM3) possessing a neuraminidase-resistant beta-ketosidic linkage. This substance was found to enhance neuritogenesis toward two transformed cell lines (neuro-2A, PC-12) and one primary neuronal tissue (dorsal root ganglia). The results indicate that the stereochemistry of the ketoside linkage is not critical and that metabolism of exogenous ganglioside by the treated cells is not involved directly in the neuritogenic phenomenon.

Topics: Animals; Cell Count; Cell Line; Chick Embryo; Dendrites; G(M1) Ganglioside; G(M3) Ganglioside; Ganglia, Spinal; Gangliosides; Mitogens; Molecular Conformation; Neuroblastoma; Stereoisomerism; Tumor Cells, Cultured

The oligosaccharide portion of ganglioside GM1 was found to enhance neuritogenesis by S20Y murine neuroblastoma cells grown in vitro. The average length of the neurites produced by cells grown in the presence of the oligosaccharide portion of GM1 was comparable to that of cells grown in the presence of intact GM1. The processes of these cells were significantly longer (p less than 0.005, pooled t test) than those of cells grown in the presence of comparable concentrations of sialic acid, lactose, sialyllactose, GD1a, or the oligosaccharide moiety of GD1a. These results suggest that it is the oligosaccharide portion of GM1 that is responsible for the ability of GM1 to enhance process outgrowth by S20Y neuroblastoma cells.

Topics: Animals; Axons; Cholera Toxin; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; G(M1) Ganglioside; Gangliosides; Mice; Neuroblastoma; Oligosaccharides; Resorcinols; Tumor Cells, Cultured

The potential involvement of gangliosides in the adherence and neurite extension of human neuroblastoma cells (Platt and La-N1) was investigated on tissue culture substrata coated with the ganglioside GM1-binding protein, cholera toxin B (CTB) subunit, for comparison with similar processes on plasma fibronectin (pFN)-coated substrata. Cells attached with reduced efficiency on CTB substrata as compared with pFN substrata and required a much longer time to form neurite processes for a small percentage of cells on CTB. The specificity of these processes for GM1 binding was tested in a variety of ways. Supplementation of the cells with exogenous GM1, but not GD1a, identified a larger population of cells adherent on CTB (comparable to pFN-adherent cells) and dramatically increased the proportion of cells capable of forming neurites without reducing the time requirement. In ultrastructural studies using the scanning electron microscope (SEM) and immunofluorescence (IF) analyses to discriminate microtubule distributions, neurites of GM1-supplemented cells on CTB were virtually identical with pFN-adherent neurites, whereas unsupplemented cells on CTB generated processes with fine-structural differences. Treatment of cells during the GM1 supplementation period with cycloheximide completely abolished the ability of cells to generate neurites on CTB and decreased the adhesive capacity of cells as well; a similar treatment of cells had no adverse effect on adherence or neurite extension on pFN. The importance of one or more proteins in GM1-dependent processes was further confirmed by demonstrating the trypsin sensitivity of a cell surface component(s) required to achieve maximal attachment on CTB; in contrast, adherence and neurite extension on pFN were much more resistant to this treatment process. Therefore, these experiments demonstrate that certain cell surface gangliosides are capable of mediating adherence and neurite outgrowth of human neuroblastoma cells on a suitable ganglioside-binding substratum; this ganglioside dependence is cooperative with one or more cell surface proteins which can now be analysed. These results are discussed in light of the identification in ref. [16] (Exp cell res 169 (1987) 311) of a second 'cell-binding' domain on the pFN molecule competent for adherence and neurite extension of these neuroblastoma cells, as well as the potential role of pFN binding to a complex ganglioside on the surface of these neural tumor cells in these proce

Topics: Axons; Cell Adhesion; Cell Line; Cholera Toxin; Cycloheximide; Egtazic Acid; G(M1) Ganglioside; Gangliosides; Humans; Microscopy, Electron, Scanning; Neuroblastoma; Trypsin

An abnormal circulating ganglioside was found in patients with neuroblastoma. This ganglioside appeared as a single band by resorcinol-HCl staining of thin-layer chromatograms of purified total gangliosides isolated from as little as 1 ml of patient plasma. It is a major ganglioside of neuroblastoma tumour tissue and was present (250-1500 pmol lipid-bound sialic acid/ml) in the plasma of five patients with widespread neuroblastoma. In contrast, the ganglioside was not detected (less than 50 pmol/ml) in plasma samples of six patients in complete remission, nor in plasma samples of seventeen healthy children and adults. Measurement of this circulating tumour-associated ganglioside should be clinically useful in neuroblastoma, offering a new approach to the detection of tumour and the evaluation of therapy.

Topics: Adolescent; Adult; Child; Child, Preschool; Chromatography, Thin Layer; Female; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Humans; Male; Middle Aged; Neuroblastoma

Individual ganglioside species (possessing the gangliotetrose oligosaccharide) were purified from bovine brain gray matter and applied in varying concentrations to the culture medium of mouse neuroblastoma cells (N2A) in vitro. After 48 hr of incubation, the cells were stained, and the neuritogenic response quantitated with a video analysis system, employing a program to measure three parameters of neuroblastoma differentiation: neurites per cell (sprouting), neurite length (extension), and degree of neurite branching (arborization). All the individual gangliosides tested promoted neurite extension in a dose-dependent fashion. Asialogangliosides ("neutral" glycosphingolipids) were without effect, which suggests that sialic acid (N-acetylneuraminic acid) is necessary to elicit this cellular response. With increasing concentrations of GM1 (5 to 500 micrograms/ml), the average cellular neurite length increased significantly, whereas the number of neurites per cell decreased. With the trisialoganglioside GT1b, neurite length did not increase to the extent seen with GM1, but an increase in the number of neurites per cell (sprouting) and branch points per neurite (arborization) was observed. These results suggest that the in vitro neuronal response to exogenous gangliosides may combine specific responses to individual species making up the total.

Topics: Animals; Cell Line; G(M1) Ganglioside; Gangliosides; Glycosphingolipids; Mice; Neuroblastoma; Neurons

The effects of exogenous GM1 and GD1a on S20Y murine neuroblastoma cells were assessed by monitoring morphology, tumorigenicity, mitotic index, and plating efficiency. S20Y cells were seeded at a density equivalent to 5 X 10(4) cells per 35-mm tissue culture dish; 38-42 hr after seeding (preconfluent stage) the cells were treated for 12 hr with 100 micrograms of ganglioside per ml of medium in which the serum content was reduced from 10% to 0.5%. Analysis of the cell lipids indicated that added ganglioside became tightly associated with the membrane during the 12-hr exposure. GM1 treatment resulted in increased projections on the cell surface and fine structures projecting from the cell processes. GD1a treatment resulted in a reduction in the cellular mitotic index. Plating efficiency was reduced by both GM1 and GD1a. Neither ganglioside affected tumorigenicity of the S20Y cells. Twelve hours after removal of the added ganglioside and exposure of the cells to normal medium, the ganglioside composition of the membranes from treated cells approached that of the controls, and the ganglioside-induced effects had been reversed. These results suggest that addition of specific gangliosides induces different cellular responses and that these changes are dependent upon the continued presence of the ganglioside.

Topics: Animals; Brain Chemistry; Cell Division; Clone Cells; Female; G(M1) Ganglioside; Gangliosides; Growth; Humans; Mice; Microscopy, Electron, Scanning; Middle Aged; Neuroblastoma; Neurons

Ganglioside GM1 promoted neuritogenesis of neuroblastoma cells, neuro-2a clone, in monolayer culture. GM1 bound to neuro-2a cells in three distinct forms, one removable by treatment with serum-containing solutions, one serum-resistant and labile to trypsin treatment, and one resistant to serum and trypsin treatments. The proportions among the three forms of cell-associated GM1 varied in relation to duration of exposure to ganglioside, ganglioside concentration in the medium, and number of cells in culture. The form removable by serum was predominant at the initial stages of association and at the highest ganglioside concentrations (over 10(-6)M); the trypsin-labile and -stable forms tended to increase with increasing cell number and decreasing ganglioside concentration. The neuritogenic effect of GM1 was higher when neuro-2a cells were incubated for 24 h in the presence of GM1 and fetal calf serum. Under this condition the percentage of neurite-bearing cells increased from 11% of control to 62% at the optimal ganglioside concentration of 10-4M. The effect was still present, although to a lower extent (from 11% to 28% of neurite-bearing cells), when cells were first exposed for only 2 h to GM1, then washed and incubated for 24 h in the presence of fetal calf serum. The trypsin-labile and -stable forms of cell-associated GM1 had a fundamental role in the effect, whereas the form removable by serum was not involved. The preparation of GM1 used was extremely pure (99%) and, in particular, had a peptide contamination, if any, less than 1:20,000-1:50,000.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Axons; Cell Line; Culture Media; G(M1) Ganglioside; Gangliosides; Kinetics; Mice; Neuroblastoma

Cholera toxin (CT) covalently linked to horseradish peroxidase (HRP) is a specific cytochemical marker for its receptor, the monosialoganglioside GM1. The binding and endocytosis of exogenous [3H]GM1 by cultured murine neuroblastoma cells (line 2A [CCl-131] ), which contain predominantly GM3, was examined by quantitative electron microscope autoradiography. The relationship between exogenous receptor, [3H]GM1, and CT HRP was studied in double labeling experiments consisting of autoradiographic demonstration of [3H]GM1 and cytochemical visualization of HRP. Exogenous [3H]GM1 was not degraded after its endocytosis by cells for 2 h at 37 degrees C. Quantitative studies showed similar grain density distributions in cells treated with [3H]GM1 alone and in cells treated with [3H]GM1 followed by CT-HRP. Qualitative studies conducted in double labeling experiments showed autoradiographic grains over the peroxidase-stained plasma membrane, lysosomes, and vesicles at the trans aspect of the Golgi apparatus. The findings indicate that exogenous glycolipid is associated with the plasmid membrane of deficient cells and undergoes endocytosis. The quantitative ultra-structural autoradiographic studies are consistent with the hypothesis that the spontaneous endocytosis of exogenous [3H]GM1 controls the subsequent uptake of CT-HRP.

Topics: Animals; Autoradiography; Cell Line; Cholera Toxin; Endocytosis; G(M1) Ganglioside; Gangliosides; Histocytochemistry; Horseradish Peroxidase; Mice; Microscopy, Electron; Neoplasms, Experimental; Neuroblastoma

The adsorptive endocytosis of conjugates of the marker enzyme horseradish peroxidase (HRP) with lectins and cholera toxin was studied in cultured neurons, neuroblastoma cells, and, in vivo, in the rat, by electron microscopic cytochemical (HRP), quantitative autoradiographic, morphometric, and biochemical techniques. The uptake of ricin-HRP by cultured neuroblastoma cells is 100 to 200 times greater than free HRP; furthermore, ricin does not stimulate the fluid phase endocytosis of HRP. Conjugates of wheat germ agglutinin and cholera toxin with HRP are 10 to 40 times more sensitive than free HRP in tracing retrograde connections in the rat central and peripheral nervous system. Conjugates of various ligands with HRP undergo endocytosis into the cisternae of the Golgi apparatus (or Golgi-Endoplasmic-Reticulum-Lysosome-GERL) and in residual bodies of cultured neurons and in vivo, while free HRP is found only in residual bodies. We conclude that: 1) various ligands with affinities to plasma membrane moieties are more sensitive, and probably more reliable, markers of "membrane" flow than free HRP, and 2) the pathway involved in the adsorptive endocytosis of ligands is different from the pathway of fluid phase uptake of HRP. These findings are consistent with the view that, in addition to its established role in the centrifugal traffic of various moieties, the neuronal Golgi apparatus is also involved in a centripetal vesicular membrane traffic. We spectulate that the neuronal Golgi apparatus, and probably the Golgi apparatus of other cells, is at the "crossroads" of a vesicular membrane traffic, and may thus exert significant controls on biologic membrane equilibria.

Topics: Adsorption; Animals; Biological Transport; Cell Membrane; Cholera Toxin; Endocytosis; Endoplasmic Reticulum; G(M1) Ganglioside; Golgi Apparatus; Horseradish Peroxidase; Lysosomes; Muridae; Neoplasms, Experimental; Neuroblastoma; Neurons

Choleragen (cholera toxin) activates adenylate cyclase in HeLa cells, which contain less than 15,000 toxin receptors per cell, in a time- and concentration-dependent manner. Activation is blocked by the addition of the oligosaccharide chain of the ganglioside GM1, the receptor for the toxin. When the cells are preincubated with choleragen at 4 degrees C and then incubated with oligosaccharide at 37 degrees C, adenylate cyclase is activated less than 10%. When the preincubation phase is above 18 degrees C, adenylate cyclase becomes activated and the amount of activation depends on the time of preincubation. This inhibitory effect of the oligosaccharide is also observed with human lymphocytes and rat glial C6 cells but not with Friend erythroleukemic and mouse neuroblastoma N18 cells. The latter two cell lines have large numbers ot toxin receptors, whereas the former two cell lines have few receptors. When the number of toxin receptors in HeLa and C6 cells is increased by treating the cells with GM1, activation of adenylate cyclase by choleragen is no longer blocked by the oligosaccharide. The oligosaccharide has a corresponding effect on the displacement of bound 125I-choleragen. When bound to cells at 4 degrees C, most of the radiotoxin is displaced from HeLa, C6, and lymphocytes but not from Friend, N18, or HeLa cells pretreated with GM1. In untreated HeLa cells, dissociation of toxin-receptor complexes by the oligosaccharide depends on the time and temperature of complex formation; above 18 degrees C, the toxin rapidly becomes stably bound to the cells. The inhibitory effect of GM1 oligosaccharide us reversible, as, once it is removed, the small amount of toxin that remains bound can activate adenylate cyclase. These results are consistent with a model in which choleragen, which is multivalent, must bind to several GM1 molecules on the cell surface in order to subsequently activate adenylate cyclase. Lateral mobility of toxin-receptor complexes may be required only to achieve multivalent binding in cells with few receptors.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cholera Toxin; Enzyme Activation; G(M1) Ganglioside; HeLa Cells; Humans; Leukemia, Erythroblastic, Acute; Lymphocytes; Mice; Neoplasms, Nerve Tissue; Neuroblastoma; Rats; Receptors, Cell Surface; Receptors, Immunologic

The lag period for activation of adenylate cyclase by choleragen was shorter in mouse neuroblastoma N18 cells than in rat glial C6 cells. N18 cells have 500-fold more toxin receptors than C6 cells. Treatment of C6 cells with ganglioside GM1 increased the number of toxin receptors and decreased the lag phase. Choleragen concentration also effected the lag phase, which increased as the toxin concentration and the amount of toxin bound decreased. The concentration, however, required for half-maximal activation of adenylate cyclase depended on the exposure time; at 1.5, 24, and 48 hr, the values were 200, 1.1, and 0.35 PM, respectively. Under the latter conditions, each cell was exposed to 84 molecules to toxin. The length of the lag period was temperature-dependent. When exposed to choleragen at 37, 24, and 20 degrees C, C6 cells began to accumulate cyclic AMP after 50, 90, and 180 min, respectively. In GM1-treated cells, the corresponding times were 35, 60, and 120 min. Cells treated with toxin at 15 degrees C for up to 22 hr did not accumulate cAMP, whereas above this temperature they did. Antiserum to choleragen, when added prior to choleragen, completely blocked the activation of adenylate cyclase. When added after the toxin, the antitoxin lost its inhibitory capability in a time and temperature-dependent manner. Cells, however, could be preincubated with toxin at 15 degrees C, and the antitoxin was completely effective when added before the cells were warmed up. Finally, cells exposed to choleragen for less than 10 min at 37 degrees C accumulated cyclic AMP when shifted to 15 degrees C. Under optimum conditions at 37 degrees C, the minimum lag period for adenylate cyclase activation in these cells was 10 min. These findings suggest that the lag period for choleragen action represents a temperature-dependent transmembrane event, during which the toxin (or its active component) gains access to adenylate cyclase.

Topics: Adenylyl Cyclases; Animals; Cell Line; Cholera Toxin; Dose-Response Relationship, Drug; Enzyme Activation; G(M1) Ganglioside; Glycolipids; Kinetics; Neoplasms, Nerve Tissue; Neuroblastoma; Rats; Receptors, Cell Surface; Receptors, Immunologic

Cholera toxin (CT), covalently attached to horseradish peroxidase (HRP), is a specific cytochemical marker for GM1 ganglioside (GM1) and retains the ability of the native toxin to raise levels of cyclic AMP in avian erythrocytes. Using a cytochemical stain for HRP, we found that 9% of control cultured murine neuroblastoma cells bound cholera toxin-horseradish peroxidase conjugates (CT-HRP) on their surfaces after incubations for 1 h at 4 degrees C. Exogenous GM1, the natural receptor of CT, becomes associated in the culture medium with the plasma membranes of these cells so that 96% of cells are stained. Cells preincubated with GM1 at 4 degrees C were exposed to CT-HRP for 1 h at 4 degrees C. After washing, cells were incubated at 37 degrees C for 30 min-24 h. Endocytosis of CT-HRP occurred within 30 min and CT-HRP remained, throughout the 24-h period, in tubules, vesicles, and cisternae often found near the Golgi apparatus; this aggregate of peroxidase-positive elements probably corresponds to Golgi apparatus-endoplasmic reticulum-lysosomes (GERL) of neurons. In metaphase cells, CT-HRP was observed in aggregates of vesicles and tubules clustered near the centriole. Conjugates of HRP with subunit B, the GM1 binding component of CT, were internalized by cells pretreated with GM1 as was CT-HRP. The 9% of neuroblastoma cells binding CT-HRP in the absence of exogenous GM1 internalized the ligand in a manner indistinguishable from that of the treated cells. These findings indicate that, in neuroblastoma cells, a system of vesicles, tubules, and cisternae, analogous to GERL of neurons, is the primary recipient of adsorptive endocytosis of CT bound to endogenous or exogenously introduced GM1.

Topics: Animals; Cholera Toxin; Endocytosis; Endoplasmic Reticulum; G(M1) Ganglioside; Gangliosides; Golgi Apparatus; Horseradish Peroxidase; Lysosomes; Metaphase; Mice; Neoplasms, Experimental; Neuroblastoma

Topics: Animals; Astrocytes; Cells, Cultured; Cricetinae; Fucosyl Galactose alpha-N-Acetylgalactosaminyltransferase; G(M1) Ganglioside; G(M2) Ganglioside; G(M3) Ganglioside; Galactose; Gangliosides; Glucosyltransferases; Humans; Mice; Neuroblastoma; Neuroglia; Sialic Acids

The reaction sequence for the biosynthesis of gangliosides by mouse neuroblastoma cells has been investigated by studying the pattern of incorporation of labeled precursors into sialoglycosphingolipids. Cultured NB41A cells incorporated N-[3H]acetylmannosamine into the sialic acid moiety of GM3 in less than 10 min. Labeled GM2 was not detected in cells incubated for less than 30 min, while measurable radioactivity did not appear in GM1 until after 60 to 90 min. Analogous experiments were carried out using [14C]galactose. No significant amount of labeled hexose was incorporated into asialo-GM2 during 60 min of culture. These studies are in accord with results of previous studies on glycosyltransferases of NB41A cells (Kemp, S. F., and Stoolmiller, A. C. (1976), J. Neurochem. 26, 723-732), and further support the concept that the pathway of synthesis of gangliosides proceeds via GM3 leads to GM2 leads to GM1.

Topics: Animals; Cell Line; Culture Media; G(M1) Ganglioside; G(M2) Ganglioside; G(M3) Ganglioside; Galactose; Glucose; Glycosphingolipids; Hexosamines; Kinetics; Mice; Neuroblastoma; Sialic Acids