Expression of A and B Types of Monoamine Oxidase in Differentiated Neuroblastoma Hybrid Cells

The total activities of monoamine oxidase (MAO) and the ratio of type B/type A activities were determined in mouse neuroblastoma N1E-115 cells, and in NX31T and NG108-15 hybrid cells derived from mouse neuroblastoma X rat sympathetic ganglion hybrid or mouse neuroblastoma X rat glioma hybrid cells. N1E-115 and NX31T cells possessed type A activities exclusively, although NG108-15 cells showed both type A (65-90%) and type B (10-35%) MAO activities. The activity of type A MAO in NX31T and N1E-115 cells was relatively constant during culturing periods in the presence or absence of dibutyryl cyclic AMP (Bt2cAMP), whereas total MAO activity and the ratio of type B MAO/type A MAO in NG108-15 cells increased as a function of culture periods. Prostaglandin E1 (PGE1) and theophylline, the best known combination to increase intracellular cyclic AMP content of NG108-15 cells, caused similar increases of MAO and of the type B/type A ratio in NG108-15 cells. The results suggest that MAO activity and expression of MAO B activity are regulated in NG108-15 cells in a cyclic AMP-dependent manner.     

Ect2, an Ortholog of <Emphasis Type="Italic">Drosophila’s</Emphasis><Emphasis Type="Italic">Pebble</Emphasis>, Negatively Regulates Neurite Outgrowth in Neuroblastoma × Glioma Hybrid NG108-15 Cells

To identify genes required for brain development, we previously performed in vivo RNA interference (RNAi) screening in Drosophila embryos. We identified pebble as a gene that disrupts development of the Drosophila nervous system. Although pebble has been shown to be involved in neuronal development of Drosophila in several screens, the involvement of Ect2, a mammalian ortholog of pebble, in mammalian neuronal development has not been addressed. To examine the role of Ect2 in neuronal differentiation, we performed Ect2 RNAi in the mouse neuroblastoma × rat glioma NG108-15 cell line. Depletion of Ect2 resulted in an increased proportion of binucleate cells and morphological differentiation of NG108-15 cells characterized by the outgrowth of neurites. These morphological changes were correlated with an increased level of acetylcholine esterase mRNA. In addition, expression of Ect2 was decreased in differentiated NG108-15 cells induced by dibutyryl cyclic AMP. These findings indicate that Ect2 negatively regulates the differentiation of NG108-15 cells and suggest that Ect2 may play a role in neuronal differentiation and brain development in vivo.     

Overexpression of Adhesion Molecule L1 in NG108-15 Neuroblastoma × Glioma Hybrid Cells Enhances Dibutyryl Cyclic AMP-Induced Neurite Outgrowth and Functional Synapse Formation with Myotubes

Abstract: The role of adhesion molecule L1 in synapse formation was examined by transient transfection of L1 cDNA in neuroblastoma × glioma hybrid NG108-15 cells. L1 overexpression was found in ∼50% of the transfected NG108-15 cell population. Neurite outgrowth induced by 0.25 m M dibutyryl cyclic AMP (cAMP) was much greater in L1-transfected NG108-15 cells than that in nontransfected and mock-transfected cells. The proportion of cells with neurites and the number of neurites per cells were increased in L1-transfected cells after 2 days of dibutyryl cAMP treatment. The proportion of cells with branched neurites and the average length of neurites were higher at day 4. A significantly higher rate of synapse formation with myotubes was apparent in the late phase of coculture (days 4–7) in L1-transfected cells than in control cells. The miniature end-plate potential frequency in myotubes was the same for the three types of NG108-15 cells. These results show that overexpression of L1 in NG108-15 cells facilitates synaptic connections by enhancing branching and elongation of neurites induced with dibutyryl cAMP, rather than by increasing probability of acetylcholine release.     

Polyoma virus and cyclic AMP-mediated control of dihydrofolate reductase mRNA abundance in methotrexate-resistant mouse fibroblasts.

As a model cell culture system for studying polyoma-mediated control of host gene expression, we isolated methotrexate-resistant 3T6 cells in which one of the virus-induced enzymes, dihydrofolate reductase, is a major cellular protein. In highly methotrexate-resistant cell lines dihydrofolate reductase synthesis accounts for over 10% that of soluble portein, corresponding to an increase of approximately 100-fold over the level in parental cells. This increase in dihydrofolate reductase synthesis is due to a corresponding increase in the abundance of dihydrofolate reductase mRNA and gene sequences. We have used these cells to show that infection with polyoma virus results in a 4- to 5-fold increase in the relative rate of dihydrofolate reductase synthesis and a corresponding increase in dihydrofolate reductase mRNA abundance. The increase in dihydrofolate reductase synthesis begins 15 to 20 h after infection and continues to increase until cell lysis. These observations represent the first direct evidence that viral infection of eukaryotic cells results in the increased synthesis of a specific cellular enzyme and an increase in the abundance of a specific cellular mRNA. In order to gain additional insight into the control of dihydrofolate reductase synthesis we examined other parameters affecting dihydrofolate reductase synthesis. We found that the addition of fresh serum to stationary phase cells results in a 2-fold stimulation of dihydrofolate reductase synthesis, beginning 10 to 12 h after serum addition. Serum stimulation of dihydrofolate reductase synthesis is completely inhibited by the presence of dibutyryl cyclic AMP as well as by theophylline or prostaglandin E1, compounds which cause an increase in intracellular cyclic AMP levels. In fact, the presence of dibutyryl cyclic AMP and theophylline results in a 2- to 3-fold decrease in the rate of dihydrofolate reductase synthesis and the abundance of dihydrofolate reductase mRNA. However, in contrast to the effect on serum stimulation, dibutyryl cyclic AMP and theophylline do not inhibit polyoma virus induction of dihydrofolate reductase synthesis or dihydrofolate reductase mRNA levels. These observations suggest that dihydrofolate reductase gene expression is controlled by at least two regulatory pathways: one involving serum that is blocked by high levels of cyclic AMP and another involving polyoma induction that is not inhibited by cyclic AMP.     

Increased resistance to nitric oxide cytotoxicity associated with differentiation of neuroblastoma-glioma hybrid (NG108-15) cells

Nitric oxide (NO), synthesized by the enzyme nitric oxide synthase (NOS), acts as an intercellular messenger associated with various physiological and pathological events. In this study, we investigated whether there exits a difference in the vulnerability to NO-induced cytotoxicity between undifferentiated and differentiated NG108-15 cells, and if so, the mechanisms responsible for the difference. Following a 7- to 8-day exposure to dibutyryl cAMP (dbcAMP), NG108-15 cells exhibited a neuron-like morphology associated with the expression of the neuronal protein, synaptophysin, and with increased NADPH-d activity. Neuron-like differentiated NG108-15 cells acquired resistance to exogenously applied NO. This increased resistance to NO toxicity in differentiated cells was almost completely cancelled out by inhibiting the activity of superoxide dismutase (SOD), but not by inhibiting the activity of NOS. The present study suggested that the activity of SOD increased in parallel with the activity of NOS associated with differentiation and was crucial for the acquired resistance to NO toxicity in differentiated cells.     

Increased Resistance to Nitric Oxide Cytotoxicity Associated with Differentiation of Neuroblastoma-Glioma Hybrid (NG108-15) Cells

Nitric oxide (NO), synthesized by the enzyme nitric oxide synthase (NOS), acts as an intercellular messenger associated with various physiological and pathological events. In this study, we investigated whether there exits a difference in the vulnerability to NO-induced cytotoxicity between undifferentiated and differentiated NG108-15 cells, and if so, the mechanisms responsible for the difference. Following a 7- to 8-day exposure to dibutyryl cAMP (dbcAMP), NG108-15 cells exhibited a neuron-like morphology associated with the expression of the neuronal protein, synaptophysin, and with increased NADPH-d activity. Neuron-like differentiated NG108-15 cells acquired resistance to exogenously applied NO. This increased resistance to NO toxicity in differentiated cells was almost completely cancelled out by inhibiting the activity of superoxide dismutase (SOD), but not by inhibiting the activity of NOS. The present study suggested that the activity of SOD increased in parallel with the activity of NOS associated with differentiation and was crucial for the acquired resistance to NO toxicity in differentiated cells.     

Modification of the indolamine content in neuroblastoma × glioma hybrid NG108-15 cells upon induced differentiation

1. The neuroblastoma x glioma hybrid NG108-15 cell line has been widely studied as a neuronal model for its serotonergic, cholinergic, and peptidergic properties. 2. The catecholamine and serotonin content and that of their major metabolites have been determined by high-performance liquid chromatography with electrochemical detection (HPLC-EC) in NG108-15 cells under differentiated and undifferentiated conditions. 3. Cellular contents of L-DOPA, norepinephrine, (NE), L-epinephrine (EPI), and dopamine (DA) in differentiated cells, induced by 1 mM dibutyryl cyclic AMP (dBcAMP), are 149, 40, 129, and 124%, respectively, higher than those in undifferentiated cells. 4. 3,4-Dihydroxyphenethylacetic acid (DOPAC), the major metabolite of DA, is detectable only in differentiated cells. Similarly, DOPAC is present only in culture medium from differentiated cells, and not that of undifferentiated cells. 5. Serotonin (5-HT) is detectable only in undifferentiated cells; and the level of 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of 5-HT, is also 12.7% higher is undifferentiated cells. 6. Comparative analyses of differentiated and undifferentiated cells in monolayer cultures and undifferentiated cells cultured in the presence of 1 mM dBcAMP under suspension conditions suggest that change in the indolamine content is due to cellular changes upon morphological differentiation. 7. The clonal NG108-15 cell line is also catecholaminergic, in addition to cholinergic and serotonergic; and a shift of neurotransmitter pattern from serotonin to dopamine production occurs during morphological differentiation.     

Identification and characterization of the beta-adrenergic receptor on neuroblastoma × glioma hybrid NG108-15 cells

1. Using [3H]DHA and unlabeled L-alprenolol, a substantial amount of over 64% specific binding of beta-adrenergic receptor has been identified on the neuroblastoma x glioma hybrid NG108-15 cell, which has been proven to display numerous functional characteristics of intact neurons. 2. Beta-adrenergic receptor binding on intact NG108-15 cells does not change significantly upon morphological differentiation, induced by 1 mM dibutyryl cyclic AMP (dBcAMP). 3. The [3H]DHA binding on intact NG108-15 cells is rapid, saturable, and reversible, having a t1/2 of 1.0 min for association and 3.5 min for dissociation. 4. The affinity constant (Kd) and maximum binding capacity (Bmax) for binding of [3H]DHA to beta-adrenergic receptors on NG108-15 cells have been estimated by Scatchard plot analysis to be 2.5 and 0.23 nM, respectively. Further analysis indicates a single class of receptors for [3HDHA binding on NG108-15 cells. 5. Studies on kinetic properties have revealed on-rate (K + 1) and off-rate (K - 1) constants of 0.7 X 10(-9) M min-1 and 0.19 min-1, respectively. Further, the IC50 value and inhibition constant (Ki) for unlabeled L-alprenolol to inhibit [3HDHA binding on NG108-15 cells have been estimated to be 10(-5) and 8.9 X 10(-6) M, respectively. 6. The rank-order potency of catecholamine agonists, (-)ISO greater than (+)ISO greater than EPI greater than NE, reveals the presence of type 2 receptor for the beta-adrenergic binding on both differentiated and undifferentiated NG108-15 cells. 7. The present study indicates that the clonal neuroblastoma x glioma hybrid NG108-15 cell line possesses substantial amounts of beta-adrenergic receptors with characteristics similar to those on neuronal cells.     

Cyclic AMP induces activity increase of kinase FA (a transmembrane signal of insulin) during NG108-15 hybrid cell differentiation

The ATP.Mg-dependent protein phosphatase activating factor (protein kinase FA) has been identified to exist in neuroblastoma x glioma hybrid 108-15 cells (NG108-15 cells). More importantly, when NG cells were induced to differentiate with N6, O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (dibutyryl cAMP), the cellular activity of kinase FA was found to increase dramatically. Time course study further revealed that induction of differentiation in NG cells by dibutyryl cAMP treatment increased the FA activity to over 3 times the levels found in undifferentiated cells and in a linear day-dependent manner, indicating that the FA activity level is correlated with the state of differentiation of NG108-15 cells. This is the first report providing initial evidence that protein kinase FA (a transmembrane signal of insulin) is involved in the induction of neuronal cell differentiation.     

Decreased Cyclic AMP Degradation in NG 108-15 Neuroblastoma × Glioma Hybrid Cells and S49 Lymphoma Cells Chronically Treated with Drugs that Inhibit Adenylate Cyclase

The increase in hormone-stimulated cyclic AMP accumulation observed in a variety of intact cells after chronic pretreatment with drugs that inhibit adenylate cyclase activity has been attributed to an increase in adenylate cyclase activity following withdrawal of the inhibitory drug. In NG 108-15 mouse neuroblastoma X rat glioma hybrid cells (NG cells) chronically treated with the muscarinic cholinergic agonist carbachol, we have found a significant decrease in the apparent degradation rate constant for cyclic AMP, in addition to an increase in the prostaglandin E1 (PGE1)-stimulated cyclic AMP synthesis rate in intact cells. In carbachol-pretreated NG cells that were stimulated with a maximally effective dose of PGE1, and that accumulated steady-state cyclic AMP concentrations fourfold or more higher than in control cells, the apparent rate constant for degradation was about 53% lower than the value for control cells. In carbachol-pretreated cells stimulated with a submaximal dose of PGE1 to yield a steady-state cyclic AMP concentration comparable to control cells, the apparent rate constant was 31% lower than the value for control cells. In S49 mouse lymphoma cells (S49 cells) chronically treated with an analog of the inhibitory agonist somatostatin, the first-order rate constant for cyclic AMP degradation in intact cells following isoproterenol stimulation was 29% lower than the value for control cells. Despite these changes in the kinetics of cyclic AMP degradation in intact NG cells and S49 cells, there was either no change or a minimal change (less than 10%) in phosphodiesterase activities assayed in extracts of cells chronically exposed to inhibitory drugs.(ABSTRACT TRUNCATED AT 250 WORDS)