Human Neuroblastoma NB-1 Cells Research Articles

Identification of a biologically active fragment of ALK and LTK-Ligand 2 (augmentor-α)

Elucidating the physiological roles and modes of action of the recently discovered ligands (designated ALKAL1,2 or AUG-α,β) of the receptor tyrosine kinases Anaplastic Lymphoma Kinase (ALK) and Leukocyte Tyrosine Kinase (LTK) has been limited by difficulties in producing sufficient amounts of the two ligands and their poor stability. Here we describe procedures for expression and purification of AUG-α and a deletion mutant lacking the N-terminal variable region. Detailed biochemical characterization of AUG-α by mass spectrometry shows that the four conserved cysteines located in the augmentor domain (AD) form two intramolecular disulfide bridges while a fifth, primate-specific cysteine located in the N-terminal variable region mediates dimerization through formation of a disulfide bridge between two AUG-α molecules. In contrast to AUG-α, the capacity of AUG-α AD to undergo dimerization is strongly compromised. However, full-length AUG-α and the AUG-α AD deletion mutant stimulate similar tyrosine phosphorylation of cells expressing either ALK or LTK. Both AUG-α and AUG-α AD also stimulate a similar profile of MAP kinase response in L6 cells and colony formation in soft agar by autocrine stimulation of NIH 3T3 cells expressing ALK. Moreover, both AUG-α and AUG-α AD stimulate neuronal differentiation of human neuroblastoma NB1 and PC12 cells in a similar dose-dependent manner. Taken together, these experiments show that deletion of the N-terminal variable region minimally affects the activity of AUG-α toward LTK or ALK stimulation in cultured cells. Reduced dimerization might be compensated by high local concentration of AUG-α AD bound to ALK at the cell membrane and by potential ligand-induced receptor-receptor interactions.

PKA, novel PKC isoforms, and ERK is mediating PACAP auto-regulation via PAC1R in human neuroblastoma NB-1 cells

The neuropeptide PACAP is expressed throughout the central and peripheral nervous system where it modulates diverse physiological functions including neuropeptide gene expression. We here report that in human neuroblastoma NB-1 cells PACAP transiently induces its own expression. Maximal PACAP mRNA expression was found after stimulation with PACAP for 3h. PACAP auto-regulation was found to be mediated by activation of PACAP specific PAC1Rs as PACAP had >100-fold higher efficacy than VIP, and the PAC1R selective agonist Maxadilan potently induced PACAP gene expression. Experiments with pharmacological kinase inhibitors revealed that both PKA and novel but not conventional PKC isozymes were involved in the PACAP auto-regulation. Inhibition of MAPK/ERK kinase (MEK) also impeded the induction, and we found that PKA, novel PKC and ERK acted in parallel and were thus not part of the same pathways. The expression of the transcription factor EGR1 previously ascribed as target of PACAP signalling was found to be transiently induced by PACAP and pharmacological inhibition of either PKC or MEK1/2 abolished PACAP mediated EGR1 induction. In contrast, inhibition of PKA mediated increased PACAP mediated EGR1 induction. Experiments using siRNA against EGR1 to lower the expression did however not affect the PACAP auto-regulation indicating that this immediate early gene product is not part of PACAP auto-regulation in NB-1 cells. We here reveal that in NB-1 neuroblastoma cells, PACAP induces its own expression by activation of PAC1R, and that the signalling is different from the PAC1R signalling mediating induction of VIP in the same cells. PACAP auto-regulation depends on parallel activation of PKA, novel PKC isoforms, and ERK, while EGR1 does not seem to be part of the PACAP auto-regulation.

Activation of the BRCA1/Chk1/p53/p21Cip1/Waf1 pathway by nitric oxide and cell cycle arrest in human neuroblastoma NB69 cells

Nitric oxide (NO) works as a bi-modal effector of cell proliferation, inducing either the increase or decrease of cell growth when cells are exposed, respectively, to low or high NO concentrations. To get further insight into the action of NO, we tested the effect of short- and long-lived NO donors on the control of the cell cycle in human neuroblastoma NB69 cells. We demonstrated that long-time exposure of cells to NO not only decreased the expression and/or the phosphorylation of elements involved in the control of the G1/S transition, such as the transcriptional repressor pRb and cyclin D1, but also down-regulated systems controlling the S and G2/M phases, such as the phosphorylation of Cdk1(cdc2) and the expression of cyclins A and B1. Increasing concentrations of NO also induced a biphasic effect on the expression of cyclins D1, A and B1, while this effect was less pronounced for cyclin E expression, but the levels of mRNAs of those cyclins changed in a distinct and complex manner. NO also changed the phosphorylation pattern of cyclin E and decreased the levels of phospho-cyclins D1 and B1. Moreover, NO decreased the expression of the Cdk inhibitors p16Ink4a and p19Ink4d, without affecting p27Kip1. In contrast, NO induced a biphasic effect on p21Cip1/Waf1 expression. The BRCA1/Chk1/p53 pathway mediated the upregulation of p21Cip1/Waf1. We also demonstrated that the NO-mediated up-regulation of p21Cip1/Waf1 was inversely correlated with the activation status of the p38MAPK pathway.

Nitric oxide changes distinct aspects of the glycophenotype of human neuroblastoma NB69 cells

It is an open question whether the presence of nitric oxide (NO) affects the cell glycophenotype. A panel of six plant lectins was used in this study to monitor distinct aspects of cell surface glycosylation under nitrosative stress. We determined that treating human neuroblastoma NB69 cells with the long-lived NO donor 2,2′-(hydroxynitrosohydrazono)bis-ethanimine (DETA/NO) and monitoring the non-apoptotic adherent cell population significantly increases the presentation of N-glycans as detected by concanavalin A. Examining fine-structural features, bisected N-glycans and branch-end tailoring including α2,6-sialylation were found to be enhanced. Confocal fluorescence microscopy and cell permeabilization experiments pointed to a major effect of NO on the extent of cell surface N-glycan presentation. We also show that NO increases the level of protein O-GlcNAcylation, a multifunctional post-translational modification. Our results thus establish the first evidence for NO as modulator of distinct aspects of cell glycosylation.

Differential p38 mitogen-activated protein kinase-controlled hypophosphorylation of the retinoblastoma protein induced by nitric oxide in neuroblastoma cells

In this report we show that exogenous NO added to human neuroblastoma NB69 cells inhibits cell proliferation and downregulates the epidermal growth factor receptor (EGFR) and its downstream signaling pathways. These comprise the 3-phosphoinositide-dependent kinase 1/Akt/glycogen synthase kinase-3β pathway, the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinases 1 and 2 pathway, and the phospholipase Cγ pathway. In contrast, NO enhances the EGFR-controlled p38MAPK pathway. We also show that NO enhances the activation of the cAMP-responsive element binding protein, a transcription factor controlled by p38MAPK, as demonstrated using 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1 H-imidazole (SB202190), a p38MAPK inhibitor. These processes are accompanied by the NO-mediated hypophosphorylation of the retinoblastoma protein (pRb), preferentially at Ser795 compared to Ser780 and Ser807/811, and the downregulation of p27 KIP1, p21 CIP1/WAF1, and p16 INK4a, although NO downregulated p16 INK4a only when the p38MAPK activity was suppressed. The p38MAPK pathway controls the phosphorylation status of pRb as SB202190 enhances the hypophosphorylation of pRb. We reverted the inhibitory action of NO on EGFR and pRb phosphorylation in living cells using cell-permeable reducing agents, which suggested that reversible S-nitrosation controls these proteins. Our results support the notion that NO negatively modulates the p38MAPK-controlled phosphorylation of pRb, inducing the subsequent arrest of the cell cycle at the G1/S transition.

Novel Bioassay for the Assessment of Neurotoxicity of Chemicals Based on the Neurite Extension in Human Neuroblastoma NB-1 Cells

A new testing method for the assessment of hazardous environmental chemicals using cultured human neuronal cells has been developed. This method is based on the image analysis of the neurite extension in human neuroblastoma NB-1 cells: the length of the extended neurites was determined using image analysis software. Using this system, 255 chemicals including methylmercury and endocrine disrupting chemicals were tested. Methylmercury and several chemicals inhibited neurite extension, while cadmium chloride, phthalates, and many other chemicals promoted it. These results suggest that the extended neurite length is a useful biological marker for the effects of neurotoxic environmental chemicals, especially on the developing nervous system.

Assessment of Potential Neurotoxic Actions of Organoarsenic Compounds Using Human Neuroblastoma NB-1 Cells and Rat Cerebellar Neurons in Primary Culture

The potential neurotoxic effects of diphenylarsinic acid (DPAA) was investigated using two in vitro assay systems, since DPAA could be formed from phenylarsenic compounds as chemical warfare agents in the environment and pose potential health risks to the population using ground water contaminated with DPAA as a source for drinking water. DPAA showed inhibitory effects on the neurite extension in human neuroblastoma NB-1 cells similar to that of methylmercury, although it required a several hundred times higher dose than that of methylmercury. In addition, DPAA affected the expression and localization of 440-kDa ankyrinB, a neuron-specific marker protein, in rat cerebellar neurons in primary culture. These results suggest that DPAA may have weak neurotoxic actions similar to those of methylmercury, even though it requires a 1000 times higher dose than methylmercury.

Gas1 Is Induced during and Participates in Excitotoxic Neuronal Death

We have performed differential screening to identify genes participating in NMDA-induced neuronal death. The gas1 (growth arrest-specific gene 1) gene, whose product is known to inhibit cell cycle progression, was induced in cultured corticohippocampal neurons committed to die after a brief exposure to NMDA. Overexpression of Gas1 in cultured hippocampal neurons and in human neuroblastoma NB69 cells produced a marked reduction in the number of viable cells. Furthermore, gas1 antisense oligodeoxynucleotide or antisense mRNA protected hippocampal neurons or NB69 cells from neuronal death. Importantly, Gas1-induced neuronal death was attenuated by coexpression of the human Bcl-2 protein or the baculoviral caspase inhibitor OpIAP2. While Gas1 does not directly interact with Bcl-2, OpIAP2 coimmunoprecipitates with Gas1. In addition, induction of gas1 also occurred in rat brain in two models of excitotoxicity: delayed neuronal death after intraperitoneal kainate injection and neuronal death in hippocampal slices after ischemia. These results indicate that Gas1 is induced by activation of glutamate receptors and is part of the gene expression program directing neuronal death after mild excitotoxic insults.

Modulation of neuritogenesis by ganglioside-specific sialidase (Neu 3) in human neuroblastoma NB-1 cells.

Plasma membrane-associated sialidase (Neu 3), which specifically hydrolyzes gangliosides, is relatively abundantly present in the nervous system. To understand the role of Neu 3 in neuronal differentiation, we studied the relationship between neurite outgrowth and Neu 3 expression in human neuroblastoma NB-1 cells. The expression of Neu 3 in NB-1 cells increased when neurite outgrowth in these cells was induced by dibutyryl cAMP. While treatment with dibutyryl cAMP alone enhanced the outgrowth of dendrite-like processes, transfection of the Neu 3 gave rise to a more prominent outgrowth of neurites with axon-like characteristics, even in the absence of dibutyryl cAMP. Neu 3 induction by dibutyryl cAMP is probably attributable, in part, to transactivation of the Neu 3 gene through cAMP responsive elements in the 5'-upstream region, as revealed by the promotor activity assay using Neu 3 promotor expression plasmid. These results indicate that Neu 3 regulates neurite formation in NB-1 cells, and suggest that this effect may be enhanced by dibutyryl cAMP via a cAMP-dependent pathway.

Methylmercury-Mediated Down-Regulation of mtHSP70 and Phospholipase A2 mRNA Expression in Human Neuroblastoma NB-1 Cells Identified by cDNA Macroarray Analysis.

bCREST, Methylmercury (MeHg) is a well-known environmental pollutant that causes severe neurological damage, especially in the developing nervous system. When human neuroblastoma NB-1 cells were treated with MeHg at sublethal concentrations, down-regulation of two apoptosis-associated genes, mtHSP70 (GRP75/ PBP74/ mtHSP75 /HSPA9B/mortalin) and phospholipase A2 (PLA2), was identified using the cDNA macroarray technique. These observations were further confirmed by RT-PCR and Northern blotting experiments. Northern blotting data also demonstrated that only groups III and VI B of PLA2 were downregulated, while group IIE remained unchanged. These results suggest that the mtHSP70 and PLA2 may be involved in the MeHg-mediated degeneration of neuronal cells.

Effects of Cadmium Chloride on Neurite Outgrowth and Gene Expression in Human Neuroblastoma NB-1 Cells.

Cadmium is a well-studied environmental toxicant. The neurotoxicity of cadmium is a concern, especially in the developing human brain, because it is thought that cadmium exerts long-term and irreversible effects. In the present study cadmium toxicity using human neuroblastoma NB-1 cells was investigated in relation to gene expression and subsequent neurite extension using cDNA macroarray and image analysis techniques. The neurite outgrowth in NB-1 cells was stimulated significantly by the presence of dibutyryl-cyclicAMP (db-cAMP) and by cadmium chloride at sublethal concentrations. Db-cAMP-stimulated neurite outgrowth was associated with a three-fold increased expression of axonal membrane protein growth-associated protein-43 (GAP-43), but cadmium chloride did not affect GAP-43 expression. Db-cAMP also increased dopamine β-hydroxylase gene expression eight-fold and down-regulated muscle/brain cAMP-dependent protein kinase inhibitor gene expression. However, cadmium chloride had no effect on gene expression except for that of metallothionein II (mtII) gene among 1764 genes analyzed. The results demonstrate that the increased neurite outgrowth with cadmium chloride is not associated with the same gene expression profile of that with db-cAMP.

Effect of Dominant-Negative Expression of L1 Cytoplasmic Domain on the Localization of Brain Ankyrins in Human Neuroblastoma NB-1 Cells.

Two isoforms of brain ankyrin, 440 kD and 220 kD ankyrinB, and neuronal cell adhesion molecule L1, which can bind to brain ankyrin, are both expressed in human neuroblastoma NB-1 cells. To examine the potential importance of the interaction between brain ankyrins and L1 in neuronal cell functions, an expression vector encoding a cytoplasmic domain of L1 has been constructed and transfected in NB-1 cells. The results obtained show that the expressed L1 cytoplasmic domain is ineffective in perturbing the interaction between brain ankyrins and L1, possibly because it exists in a monomeric form.

Nitric oxide induces differentiation in the NB69 human catecholamine-rich cell line

The nitric oxide (NO) donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), induced differentiation of human neuroblastoma NB69 cells to a dopamine phenotype, as shown by phase-contrast microscopy and tyrosine hydroxylase (TH) immunocytochemistry. NB69 cells were treated with 50 to 750 μM SNAP in serum-free-defined medium for 24 h. SNAP treatment did not increase the number of necrotic or apoptotic cells. However, a decrease in the number of viable cells was observed at 750 μM SNAP. In addition, a decrease in 3H-thymidine uptake was detected at the highest dose of SNAP. An increase in the antiapoptotic Bcl-2 and Bcl-xL protein levels and a decrease in the proapoptotic Bax and Bcl-xS protein levels were also detected by Western blot analysis after SNAP treatment. At low doses (50–125 μM), SNAP induced an increase in catecholamine levels, 3H-dopamine uptake, TH activity and monoamine metabolism, while a decrease in all these parameters was observed at high doses (250–750 μM). The TH protein content, analyzed by Western blot, remained unchanged in SNAP-treated cells throughout the range of doses studied, when compared with the control group. SNAP produced a dose-dependent decrease in the glutathione (GSH) content of the culture medium, without altering intracellular GSH. In addition, cGMP levels and nitrite concentration, measured in the supernatant of SNAP-treated cells, increased in a dose-dependent manner, as compared to control levels. The guanylate cyclase inhibitor lH-[1,2,4]oxadiazolo[4,3a]quinoxaline-l-one (ODQ) did not revert the SNAP-induced effect on 3H-dopamine uptake to control values. These results suggest that NO, released from SNAP, induces differentiation of NB69 cells and regulates TH protein at the post-transcriptional level through a cGMP-independent mechanism.

Expression of stanniocalcin in zona glomerulosa and medulla of normal human adrenal glands, and some adrenal tumors and cell lines.

Stanniocalcin (STC) is a calcium (Ca)-regulating hormone that was originally discovered in the fish Stannius body, which is a unique endocrine organ. Hypercalcemia increases STC secretion, which inhibits Ca uptake by the gills and normalizes serum Ca level. In this study we investigated the STC expression in human normal and abnormal adrenal cells. Immunohistochemistry using monoclonal antibody against STC revealed specific staining in zona glomerulosa and medulla of normal human adrenal glands. STC was also detected in human adrenal tumors, such as pheochromocytoma, differentiated neuroblastoma, and aldosterone-producing adenoma, and cultured adrenal tumor cells (rat pheochromocytoma PC-12 cells and human neuroblastoma NB-1 cells). However, undifferentiated human adrenal neuroblastoma was negative for STC staining. Reverse transcription polymerase chain reaction demonstrated STC mRNA expression in cultured PC-12 cells and NB-1 cells. Following several studies indicating that zona glomerulosa cells of adrenal glands express neuroendocrine properties, STC expression in normal and abnormal adrenal cells provides additional evidence to support the neuroendocrine differentiation of these cells. In conclusion, STC may be useful as a new cell marker of adrenal glands under physiological and pathological conditions.

Neurotrophin regulation of sodium and calcium channels in human neuroblastoma cells

Neurotrophins, acting through tyrosine kinase family genes, are essential for neuronal differentiation. The expression of tyrosine kinase family genes is prognostic in neuroblastoma, and neurotrophins reduce proliferation and induce differentiation, indicating that neuroblastomas are regulated by neurotrophins. We tested the effects of nerve growth factor and brain-derived neurotrophic factor on Na + and Ca 2+ currents, using the whole-cell patch-clamp technique, in human neuroblastoma NB69 cells. Control cells exhibited a slow tetrodotoxin-resistant ( ic 50=98 nM) Na + current and a high-voltage-activated Ca 2+ current. Exposure to nerve growth factor (50 ng/ml) and/or brain-derived neurotrophic factor (5 ng/ml) produced the expression of a fast tetrodotoxin-sensitive ( ic 50=10 nM) Na + current after day 3, and suppressed the slow tetrodotoxin-resistant variety. The same type of high-voltage-activated Ca 2+ current was expressed in control and treated cells. The treatment increased the surface density of both Na + and Ca 2+ currents with time after plating, from 17 pA/pF at days 3–5 and 1–5 to 34 and 30 pA/pF after days 6–10, respectively. Therefore, both nerve growth factor and brain-derived neurotrophic factor, acting through different receptors of the tyrosine kinase family and also possibly the tumor necrosis factor receptor-II, were able to regulate differentiation and the expression of Na + and Ca 2+ channels, partially reproducing the modifications induced by diffusible astroglial factors. We show that neurotrophins induced differentiation to a neuronal phenotype and modified the expression of Na + and Ca 2+ currents, partially reproducing the effects of diffusible astroglial factors.

Biosynthesis of d-aspartate in mammalian cells

In this communication, we demonstrate that d-aspartate ( d-Asp) is synthesized in pheochromocytoma cells (PC12). To our knowledge this is the first report of biosynthesis of d-Asp in mammalian cells. Synthesis of d-Asp was demonstrated by its time-dependent accumulation in the cell culture, and by the fact that this accumulation was proportional to the number of inoculated cells. d-Asp in PC12 cells was identified by (i) co-elution with authentic d-Asp on two different HPLC columns, an octadesyl silica column and a Pirkle-type chiral column, (ii) reversed elution order of d-Asp and l-Asp on another Pirkle-type chiral column with an opposite configuration, and (iii) sensitivity to d-Asp oxidase. In the cells the amount of d-Asp was approx. 12–14% of total Asp and no other investigated d-amino acid was detected. The amount of d-Asp did not increase during the culture of mouse 3T3 fibroblasts and human neuroblastoma NB-1 cells. Immunocytochemical staining with anti- d-Asp antiserum demonstrated that d-Asp synthesized is present in the cytoplasm of the cells.

Differential expression of voltage‐gated Ca2 conductances in human neuroblastoma NB69 cells cultured in defined serum‐free and astrocyte‐conditioned media

Differential expression of voltage‐gated Ca2 conductances in human neuroblastoma NB69 cells cultured in defined serum‐free and astrocyte‐conditioned media

Differential expression of voltage-gated Ca2+ conductances in human neuroblastoma NB69 cells cultured in defined serum-free and astrocyte-conditioned media

Voltage-gated Ca2+ conductances were investigated with the whole-cell patch-clamp technique-either using Ca2+ or Ba2+ as charge carriers-in NB69 human neuroblastoma cells plated in "defined" serum-free (DM) and in "astroglial-conditioned" media (CM). Cells expressed the microtubule associated protein 1A when plated in both media, indicating neuronlike differentiation. Cells of similar sizes and shapes were selected for recordings. Different sets of voltage-gated Ca2+ current types were usually expressed in DM- and CM-plated cells. DM-plated cells exhibited a high-voltage-activated current (HVAC) in isolation, whereas 43% of the CM-plated cells also displayed the low-voltage-activated current (LVAC). The membrane surface density of the HVAC was about twofold higher in CM than in DM-plated cells and increased with plating time from 10 and 16pA/pF (days 1-4) to 24 and 37 pA/pF (days 5-10) in DM- and CM-plated cells, respectively. However, the amplitude of the LVAC did not change significantly with culture age. In conclusion, NB69 cells expressed HVAC in isolation when plated in DM, whereas both HVAC and LVAC were present in many CM-plated cells, suggesting that the CM contained diffusible factors secreted by astroglial cells which: (1) could induce the appearance of the LVAC and (2) increased HVAC current expression.

Selective down-regulation of 440 kDa ankyrinBassociated with neurite retraction

Brain-specific isoforms of ankyrin, 440 kDa and 220 kDa ankyrinB, which are generated from a single gene by alternative splicing of pre-mRNA, are both expressed in human neuroblastoma NB-1 cells and the expression of the larger isoform is increased upon induction of neurite outgrowth. Exposure to methylmercury, a potent neurotoxic substance, at a sublethal dose induced dramatic retraction of neurites in NB-1 cells. Concomitantly, synthesis of 440 kDa ankyrinB polypeptide and mRNA were selectively attenuated in methylmercury-treated cells, while the 220 kDa isoform was not affected. These results indicate that the expression of 440 kDa ankyrinB is intimately associated not only with the neurite outgrowth but also with neurite retraction in neuronal cells, and is regulated at mRNA level.

Possible involvement of the 440 kDa isoform of ankyrin B in neuritogenesis in human neuroblastoma NB-1 cells

Two isoforms of brain ankyrin, 440 kDa and 220 kDa ankyrin B, which are products of alternatively spliced pre-mRNA encoded by a single gene, are both expressed in human neuroblastoma NB-1 cells. Expression of the polypeptide and mRNA of the larger isoform increased upon induction of neurite outgrowth in NB-1 cells by dibutyryl cAMP, while those of the smaller isoform were unaffected. The expressed 440 kDa ankyrin B was concentrated at the tip of growing neurites and was partly co-localized with GAP-43. These results suggest that 440 kDa ankyrin B is one of the neuronal growth-associated proteins and provides an interesting example of gene regulation by alternative splicing in neuronal cells.