Mitosis-dependent protein expression in neuroblastoma cell line N1E-115

Systematic work on differential protein expression in mitosis is limited, and we therefore used neuroblastoma cells (N1E-115) incubated with either colcemid or nocodazole to arrest mitosis. Proteins were identified by MALDI-TOF/TOF and nano-LC-ESI-MS/MS with subsequent quantification of spot volumes with specific software. Immunoblotting was used for verification of selected proteins. Levels of 10 individual proteins were increased and levels of 6 proteins were decreased concordantly by both treatments. These proteins were constituents of heat shock and chaperone, cytoskeleton, proteasomal, heterochromatin, and DNA replication signaling as well as housekeeping and metabolic systems. Identification of mitosis-dependent proteins is of importance for the interpretation of previous work and for designing future experiments.     

Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line

Summary. No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.     

Reserpine sensitivity of catecholamine metabolism in murine neuroblastoma clone N1E-115.

—Neuroblastoma cells of clone NIE-115, originally obtained from the murine tumor C1300, resemble normal noradrenergic neurons in that they have high levels of tyrosine 3-monooxygenase (EC 1.14.16.2; l -tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating)) and dopamine β-monooxygenase (EC 1.14.17.1; 3,4-dihydroxyphenylethylamine, ascorbate: oxygen oxidoreductase (β-hydroxylating)) activities, dense core versicles (100–300 nm in dia), long neurites and excitable membranes. These studies show that reserpine, a blocker of vesicular uptake in noradrenergic neurons, inhibits the accumulation and storage of catecholamines, as well as the conversion of dopamine to NE in neuroblastoma cells. Differentiated monolayer cultures took up [³H]dopamine [10⁻⁴] at a rate of 37 pmol/min per mg protein. Reserpine [5 × 10⁻⁵m ] did not affect the initial rate of uptake, but reduced the extent of uptake at saturation by 60%. Chromatographic examination of cell extracts showed that dopamine was converted to NE in control cultures, but not in reserpine treated cultures. Cells labelled with [³H]dopamine for 60 min and then exposed to release buffer without dopamine for an additional 60 min, retained approximately 40% of the label, 10% as dopamine and 30% as NE. Thirty-five per cent of the radioactivity retained was found, after homogenization and high speed centrifugation, to be associated with a particulate, subcellular fraction. Reserpine, present during release incubations, also reduced the ability of cells to store catecholamines. These results show that N1E-115 cells synthesize and store NE by reactions similar to those in normal noradrenergic neurons.     

Lack of effect of the opioid antagonist, naltrexone, on the in situ growth of C-1300, N1E-115 and NS206 murine neuroblastoma tumor cell lines

Attempts have been made to confirm previously reported results which demonstrated that the opioid antagonist, naltrexone, altered the in situ growth of murine neuroblastoma tumors. Adult male A/J mice were injected with tumor cells from three different cell lines of murine neuroblastoma; the spontaneously arising C-1300 line, the adrenergic clonal line N1E-115, and the cholinergic clonal line NS20Y. Naltrexone was administered daily in doses of 0.1, 0.4, or 10.0 mg/kg subcutaneously, to replicate the reported experimental design. In contrast to previous studies, we were unable to demonstrate any effect of naltrexone on in situ growth or other characteristics of tumors produced by the C-1300, N1E-115 or NS20Y murine neuroblastoma cell lines. Ligand binding studies have demonstrated the presence of high levels of opiate binding sites on membranes prepared from the NS20Y clonal cell line and low levels on the membranes of the C1300 tumor line.     

Development of resting membrane potentials in differentiating murine neuroblastoma cells (N1E-115) evaluated by flow cytometry

With the aid of a voltage-sensitive oxonol dye, flow cytometry was used to measure relative changes in resting membrane potential (V_m) and forward angle light scatter (FALS) profiles of a differentiating/differentiated murine neuroblastoma cell line (N1E-115). Electrophysiological differentiation was characterized by V_m establishment. The (V_m)-time profile was found to be seed cell concentration-dependent for cell densities of less than 2 × 10⁴ cells/cm². At higher initial cell densities, under differentiating culture conditions, V_m development commenced on day 2 and reached a steady-state on day 12. The relative distribution of differentiated cells between low and high FALS has been proposed as a potential culture electrophysiological differentiation state index. These experiments offer a general methodology to characterize cultured excitable cells of nervous system origin, with respect to electrophysiological differentiation. This information is valuable in studies employing neuroblastoma cells as in vitro screening models for safety/hazard evaluation and/or risk assessment of therapeutical and industrial chemicals under development. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differentiation-dependent expression of hypothetical proteins in the neuroblastoma cell line N1E-115

Several protein cascades, including signaling, cytoskeletal, chaperones, metabolic, and antioxidant proteins, have been shown to be involved in the process of neuronal differentiation (ND) of neuroblastoma cell lines. No systematic approach to detect hitherto unknown and unnamed proteins or structures that have been predicted upon nucleic acid sequences in ND has been published so far. We therefore decided to screen hypothetical protein (HP) expression by protein profiling. Two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF) identification was used for expression analysis of undifferentiated and dimethylsulfoxide-induced neuronally differentiated N1E-115 cells. We unambiguously identified six HPs: Q8C520, Q99LF4, Q9CXS1, Q9DAF8, Q91WT0, and Q8C5G2. A prefoldin domain in Q91WT0, a t-SNARE domain in Q9CXS1, and a bromodomain were observed in Q8C5G2. For the three remaining proteins, no putative function using Pfam, BLOCKS, PROSITE, PRINTS, InterPro, Superfamily, CoPS, and ExPASy could be assigned. While two proteins were present in both cell lines, Q9CXS1 was switched off (i.e., undetectably low) in differentiated cells only, and Q9DAF8, Q91WT0, and Q8C5G2 were switched on in differentiated cells exclusively. Herein, using a proteomic approach suitable for screening and identification of HP, we present HP structures that have been only predicted so far based upon nucleic acid sequences. The four differentially regulated HPs may play a putative role in the process of ND.     

Acute changes in myo-inositol uptake and 22Na+ flux in murine neuroblastoma cells (N1E-115) following insulin

myo-Inositol uptake was investigated in a murine neuroblastoma clone (N1E-115) to determine the effect of altered Na+,K+-ATPase activity. The Na+ ionophore monensin, and veratridine, an alkaloid affecting voltage-dependent Na+ entry, increased acute 22Na+ uptake and 22Na+ efflux from pre-loaded cells, concomitant with enhanced myo-inositol uptake. This effect was also seen following insulin. Insulin-stimulated myo-inositol uptake was inhibited by amiloride, ouabain and pyrithiamine. Amiloride inhibition suggests that activation of Na+/H+ exchange preceding Na+,K+-ATPase activation is involved in insulin stimulation of myo-inositol uptake. Pyrithiamine inhibition is an indication of prior activation of the Na+,K+-ATPase alpha + catalytic subunit by insulin. The results provide evidence that insulin contributes to the maintenance of Na+,K+-ATPase in neuronal tissue.     

Activation of cyclic nucleotide formation in murine neuroblastoma N1E-115 cells by modified human thrombins

The activity of alpha-thrombin and chemically modified derivatives of this enzyme in stimulating cGMP formation in murine neuroblastoma clone N1E-115 cells is reported. The rank order potency of the compounds falls into three classes: 1) alpha-thrombin is the most potent and effective; 2) the catalytically active enzymes gamma-thrombin, trypsin, and nitro-alpha-thrombin are approximately 50-fold less potent than alpha-thrombin; and 3) active site blocked derivatives of alpha-thrombin are 100 to 1000-fold less potent than alpha-thrombin. Native alpha-thrombin consistently produces the most effective response, usually 1.5 to 3-fold greater than any of the other compounds tested. Preincubation of cells with quinacrine, an inhibitor of phospholipase A2, or with the lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid prior to thrombin challenge resulted in a concentration-dependent attenuation of the response. Indomethacin was without effect in modifying the response. These results suggest that thrombin stimulation of neuroblastoma cells involves the release of arachidonic acid and its metabolism by lipoxygenase. These results clearly demonstrate the activity of alpha-thrombin in stimulating a receptor-mediated response in cultured nerve cells. The results are discussed in relation to the interaction of endogenous alpha-thrombin with nerve cells following invasive trauma and to the possible presence of endogenous proteinases with a neurotransmitter-like function.     

Catabolism of neurotensin by neural (neuroblastoma clone N1E115) and extraneural (HT29) cell lines

The mechanisms by which neurotensin (NT) was inactivated by differentiated neuroblastoma and HT29 cells were characterized. In both cell lines, the sites of primary cleavages of NT were Pro⁷-Arg⁸, Arg⁸-Arg⁹ and Pro¹⁰-Tyr¹¹ bonds. The cleavage at the Pro⁷-Arg⁸ bond was totally inhibited by N-benzyloxycarbonyl-Prolyl-Prolinal and therefore resulted from the action of proline endopeptidase. This peptidase also contributed in a major way to the cleavage at the Pro¹⁰-Tyr¹¹ bond. However the latter breakdown was partly due to an NT-degrading neutral metallopeptidase. Finally, we demonstrated the involvement of a recently purified rat brain soluble metalloendopeptidase at the Arg⁸-Arg⁹ site by the use of its specific inhibitor N-[1(R,S)-carboxy-2-Phenylethyl]-alanylalanylphenylalanine-p-aminobenzoate. The secondary processing of NT degradation products revealed differences between HT29 and N1E115 cells. Angiotensin converting enzyme was shown to degrade NT_1–10 and NT_1–7 in N1E115 cells but was not detected in HT29 cells. A post-proline dipeptidyl aminopeptidase activity converted NT_9–13 into NT_11–13 in HT29 cells but not in N1E115 cells. Finally bestatin-sensitive aminopeptidases rapidly broke down NT_11–13 to Tyr in both cell lines. Models for the inactivation of NT in HT29 and N1E115 cells are proposed and compared to that previously described for purified rat brain synaptic membranes.     

Intracellular distribution of functional M(1) -muscarinic acetylcholine receptors in N1E-115 neuroblastoma cells

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.     

Properties of tyrosine hydroxylation in living mouse neuroblastoma clone N1E-115

Tyrosine hydroxylation was studied in intact cells of mouse neuroblastoma clone N1E-115 which have high levels of tyrosine 3-monooxygenase (EC 1.14.16.2) and which have been fully characterized for tyrosine transport. Measurement of [3H]OH formed from L-[3,5(-3)H]tyrosine in the medium was the method of assay and [3H]OH formed was stoichiometric with the formation of L-[3H]3,4-dihydroxyphenylalanine. Tyrosine hydroxylation was dependent on time of incubation, cell number, and the concentration of [3H]tyrosine in the medium. From velocity vs. [3H]tyrosine concentration experiments, two apparent Km values were obtained: Km1 = 10 +/- 2 microM; Km2 = 140 +/- 10 microM. Substrate inhibition occurred with tyrosine concentrations between 20 and 50 microM. The reaction was twice as fast at pH 5.5 as at pH 7.4. alpha,alpha'-Dipyridyl (1 mM) caused major inhibition (75%) when [3H]tyrosine concentration was 10 microM. L-3-Iodotyrosine was a competitive inhibitor with Ki = 0.3 microM. Dopamine was a non-competitive inhibitor with Ki = 500 microM. 1-Norepinephrine had no effect. These results show that the hydroxylation of tyrosine by living N1E-115 cells has many of the properties of the reaction catalyzed by purified tyrosine 3-monooxygenase from normal tissue.     

Identification and characterisation of arsenite (+3 Oxidation State) methyltransferase (AS3MT) in mouse neuroblastoma cell line N1E-115

Handling and detoxification of metals by enzymes is a major issue that is not in the focus of current biomedical research concepts. The finding of the presence of arsenic (+3 Oxidation State) methyltransferase (AS3MT) in neuroblastoma cells NE-115 as a high abundance protein made us investigate primary structure of AS3MT reflecting an example of metal-handling in eucaryotes. Proteins extracted from NE-115 cells were run on 2-DE followed by two different mass spectrometrical methods. High sequence coverage was obtained by multiple protease digestion and a sequence conflict was solved at arginine 335. These findings are important when future studies on this enzyme are designed at the protein level and in particular, when antibodies against this protein will be generated.     

Up-regulation of Jun/AP-1 during differentiation of N1E-115 neuroblastoma cells.

Neuroblastoma cell lines isolated from neuroblastoma tumors can be induced to differentiate into neuronal cell types by treatment with chemical agents, such as dimethyl sulfoxide and retinoic acid. The molecular mechanisms underlying this differentiation process, however, are completely obscure. In this paper, we show that neuronal differentiation of mouse N1E-115 neuroblastoma cells by dimethyl sulfoxide is accompanied by a prolonged rise in c-jun, junB, and junD expression and AP-1 activity. Multiple sequence elements in the Jun promoters are involved in this process. Furthermore, we show that c-jun and junD, but not junB, are expressed at high levels in the neuronal cell types obtained after dimethyl sulfoxide treatment. These results suggest an important role for c-jun and junD in neuronal differentiation of N1E-115 cells.     

Production of an EDRF-like activity in the cytosol of N1E-115 neuroblastoma cells

Accumulation of cyclic GMP in cultured rat lung fibroblasts was used to test the hypothesis that N1E-115 neuroblastoma cells produce an endothelium-derived relaxing factor (EDRF)-like activity. By using this assay, the production of an EDRF-like activity in homogenates and cytosolic fractions of N1E-115 neuroblastoma cells was observed. Detection of the activity required the presence of superoxide dismutase and was inhibited by hemoglobin. Production of the EDRF-like factor was dependent on L-arginine and NADPH. The apparent Km for L-arginine was 1.25 microM and the apparent Km for NADPH was 1.67 microM. The production of the EDRF-like activity was inhibited by the L-arginine analogs, NG-monomethyl-L-arginine and NG-nitro-L-arginine, with apparent Ki values of 1.0 and 0.3 microM, respectively.     

Activation of phospholipase C increases intramembrane electric fields in N1E-115 neuroblastoma cells

We imaged the intramembrane potential (a combination of transmembrane, surface, and dipole potential) on N1E-115 neuroblastoma cells with a voltage-sensitive dye. After activation of the B(2) bradykinin receptor, the electric field sensed by the dye increased by an amount equivalent to a depolarization of 83 mV. The increase in intramembrane potential was blocked by the phospholipase C (PLC) inhibitors U-73122 and neomycin, and was invariably accompanied by a transient rise of [Ca(2+)](i). A depolarized inner surface potential, as the membrane loses negative charges via phosphatidylinositol 4,5-bisphosphate (PIP(2)) hydrolysis, and an increase in the dipole potential, as PIP(2) is hydrolyzed to 1,2-diacylglycerol (DAG), can each account for a small portion of the change in intramembrane potential. The primary contribution to the measured change in intramembrane potential may arise from an increased dipole potential, as DAG molecules are generated from hydrolysis of other phospholipids. We found bradykinin produced an inhibition of a M-type voltage-dependent K(+) current (I(K(M))). This inhibition was also blocked by the PLC inhibitors and had similar kinetics as the bradykinin-induced modulation of intramembrane potential. Our results suggest that the change in the local intramembrane potential induced by bradykinin may play a role in mediating the I(K(M)) inhibition.     

Effects of heat shock on neuroblastoma (N1E 115) cell proliferation and differentiation.

Heat shock (44 degrees C) applied for only 15 min induced the development of neurites in neuroblastoma cells 3-6 days later. During the first day after heat shock a transient increase in the rate of cytokinesis together with a synchronizing effect was observed, which led to waves of cytokinesis 14.5 h apart. Individual cell cycles were determined and showed a lengthening in the minimal cell cycle duration and a decrease in the cell cycle variance after shock. Two to 3 days after heat shock the proliferation rate decreased and then recovered. During the 6 days after heat shock, total protein synthesis was lower compared to the untreated cultures. The synthesis of heat shock proteins (100, 90, 84, 70, 68 kDa and some of lower MW) reached a maximum 6 h after heat shock. Parallel changes in the phosphorylation state of proteins were observed in an in vitro assay. Four proteins (100, 89, 67, and 15 kDa) increased and two proteins (97, 73 kDa) decreased their phosphorylation state significantly. Six days after heat shock two proteins (89, 55 kDa) increased their phosphorylation state; the 55-kDa phosphoprotein was identified as tubulin. The effect of heat shock on the intracellular calcium level was determined by measuring Fura 2 fluorescence. Six hours after shock, the Ca2+ level increased to a maximum (about three times the control value) and then dropped during the following days below the control values. We conclude from these results that a decrease in the calcium level may be causally involved in the differentiation process. The calcium effect is probably mediated by changes in the activity of different kinases. This assumption is compatible with the results of experiments with cyclic nucleotides when 10(-5) M cAMP and cGMP were added to in vitro assays of protein phosphorylation. They had different stimulating effects in heat-shocked, differentiating, and growing (control) cells.