Potentiation by BL191 of differentiation of neuroblastoma cells induced by dibutyryl cAMP and prostaglandin E1

BL191, a newly developed phosphodiesterase inhibitor, markedly potentiated a differentiation of neuroblastoma cell clones (Neuro2a, NS-20Y, and N1E115) induced by dibutyryl cyclic adensoine 3′:5′-monophosphate(dibutyryl cAMP) and prostaglandin E₁ (PGE₁). BL191 (1 mM) inhibited DNA synthesis more strongly when used together with PGE₁ (0.5 μg/ml) and dibutyryl cAMP (0.5 mM) than papaverine (1.6 μg/ml) alone did. The inhibition rates of DNA synthesis were 72.5% for N1E-115, 75.3% for Neuro2a, and 82.5% for NS-20Y. After the treatment with BL191. PGE₁, and dibutyryl cAMP for 48 h all of three cell lines became enlarged and flattened, and extended long processes. The specific activities of choline acetyl transferase (EC 2.3.1.9) of NS-20Y and dopamine β-hydroxylase (EC 1.14.17.1) of N1E-115 increased about 3-fold as compared to the controls. The tumorigenicities of Neuro2a and N1E-115 cells were decreased, but not of NS-20Y. These data suggest the heterogenous responsiveness in neuroblastoma cells to drug treatment.     

Adriamycin promotes neurite outgrowth in the "neurite-minus" N1A-103 mouse neuroblastoma cell line.

Adriamycin, an anticancer agent acting on topoisomerase II, promotes the arrest of cell division and neurite extension in a "neurite-minus" murine neuroblastoma cell line, N1A-103. This morphological differentiation is accompanied by a blockade in the S phase of the cell cycle, modification of the amount of peripherin, and appearance of the beta 7-tubulin isoform. Yet, adriamycin-induced N1A-103 cells fail to express other neuronal markers, such as long-lasting Ca2+ channels, synaptophysin, and the shift in the proportion of the beta'1 tubulin isoform to the beta'2 isoform, whose appearance parallels the terminal differentiation of the wild type neuroblastoma cell line N1E-115. Hence, a comparison of the behavior of these two cell lines leads to the proposal that there are two programs of neuroblastoma differentiation: one where expression is triggered by the arrest of cell division and which is observed in adriamycin-induced N1A-103 variant cells, and the other, presumably occurring further downstream, which would involve further changes in morphogenesis and acquisition of new electrophysiological properties.     

Post-transcriptional regulation of cAMP-dependent protein kinase activity by cAMP in GH3 pituitary tumor cells. Evidence for increased degradation of catalytic subunit in the presence of cAMP

The effects of cyclic AMP treatment on total cAMP-dependent protein kinase activity in GH3 pituitary tumor cells have been studied. Incubation of cells for 24 h with 1 microM forskolin resulted in a 50% decrease in total cAMP-dependent protein kinase activity which was reversible upon removal of forskolin from culture media. A similar response was observed in GH3 cells treated with 5 ng/ml cholera toxin and 0.5 mM dibutyryl cAMP but not 0.5 mM dibutyryl cGMP. Northern blot analysis demonstrated that the steady-state level of the mRNA for each of the six kinase subunit isoforms studied was not detectably altered after treatment with 1 microM forskolin for 24 h. The concentration of catalytic subunit was also assessed by binding studies using a radiolabeled heat-stable protein kinase inhibitor. Treatment of GH3 cells with 1 microM forskolin for 24 h reduced protein kinase inhibitor binding activity by 50%, consistent with the observed forskolin-induced decrease in total kinase activity. Analysis of endogenous heat-stable protein kinase inhibitor activity in GH3 cell extracts showed no significant difference between forskolin-treated cells and cells maintained under control conditions. To assess possible effects on catalytic subunit degradation, pulse-chase experiments were performed and radiolabeled catalytic subunit was isolated by affinity chromatography. The results demonstrated that treatment of cells with chlorophenylthio-cAMP detectably increased the apparent degradation of radiolabeled catalytic subunit. The increased degradation of the catalytic subunit was sufficient to account for the observed decreases in kinase activity. These results suggest that relatively long term cAMP treatment can alter total cAMP-dependent protein kinase activity through effects to alter the degradation of the catalytic subunit of the enzyme.     

Vasoactive intestinal polypeptide stimulates cyclic AMP production in mouse N1E-115 neuroblastoma cells: Modulation by a protein kinase C activator and ionomycin

In this study, we investigated the vasoactive intestinal polypeptide (VIP)-stimulated cAMP production and its interaction with protein kinase C activation and elevation of intracellular Ca²⁺ in N1E-115 neuroblastoma cells. VIP treatment caused a 55-fold increase in cAMP accumulation. Addition of 4β-phorbol 12-myristate 13-acetate reduced VIP-but not forskolin-stimulated cAMP response. In comparison, ionomycin potentiated both VIP- and forskolin-induced cAMP accumulation. Our results indicate that VIP stimulates cAMP accumulation in N1E-115 cells, and that although activation of protein kinase C inhibits the VIP-stimulated cAMP response, elevation of intracellular Ca²⁺ potentiates this signaling pathway.     

Neurite Extension and Increased Expression of R1 Cyclic AMP-Binding Protein in Ouabain-Resistant Neuroblastoma Mutants

Morphological and biochemical parameters of neuroblastoma differentiation were assessed in 12 clonal derivatives of the N-18 mouse neuroblastoma cell line selected for their ouabain-resistant (ouar) property. When cultured in a normal growth medium, nine of the 12 ouar cell lines exhibited a more complex pattern of neurite outgrowth than the parental N-18 cells. The morphological pattern most frequently observed with the ouar cells was the extension of several branched processes per cell. This pattern of spontaneous neurite outgrowth in the ouar cell lines can be correlated with an increase in expression of the 47,000-dalton RI cyclic AMP (cAMP)-binding protein. The growth rate, intracellular level of cAMP, and acetylcholinesterase activity of the ouar cell lines were not significantly different from those of the parental N-18 neuroblastoma cells. Treatment of the parental and ouar neuroblastoma cell lines with 1 mM N6, O2-dibutyryl cAMP promoted an elaborate pattern of neurite outgrowth and marked increases in acetylcholinesterase and RI cAMP-binding activities. The distinctive pattern of differentiation phenotype exhibited by the ouar cells and the dibutyryl cAMP-induced differentiated neuroblastoma cell suggests that these two protocols yielded different degrees of differentiation. Furthermore, our results suggest a linkage of the biochemical events underlying ouabain resistance and expression of differentiation phenotypes in the mouse neuroblastoma cells.     

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.     

Melatonin activates PKC-alpha but not PKC-epsilon in N1E-115 cells.

Previous reports have revealed that calmodulin antagonism by melatonin is followed by microtubule enlargements and neurite outgrowths in neuroblastoma N1E-115 cells. In addition, activation of protein kinase C (PKC) by this neurohormone is also followed by increased vimentin phosphorylation, and reorganization of vimentin intermediate filaments (IFs) in N1E-115 cells. In this work, we further characterize the activation of PKC by melatonin in neuroblastoma N1E-115 cells. We studied the Ca(2+)-dependent effects of melatonin on PKC activity and distribution of PKC-alpha in isolated N1E-115 cell IFs. Also, the effects of melatonin on PKC-alpha translocation in comparison to PKC-epsilon, were studied in intact N1E-115 cells. The results showed that both melatonin and the PKC agonist phorbol-12-myristate-13-acetate increased PKC activity in isolated IFs. The effects of the hormone were Ca(2+)-dependent, while those caused by the phorbol ester were produced with or without Ca(2+). Also, in isolated in situ IFs, the hormone changed the distribution of PKC-alpha. In intact N1E-115 cells, melatonin elicited PKC-alpha translocation and no changes were detected in PKC-epsilon. Phorbol-12-myristate-13-acetate modified the subcellular distribution of both PKC isoforms. The results showed that melatonin selectively activates the Ca(2+)-dependent alpha isoform of PKC and suggest that PKC-alpha activation by melatonin underlies IF rearrangements and participates in neurite formation in N1E-115 cells.     

Ethanol differentially regulates G proteins in neural cells

Long-term incubation of clonal neural cell lines with ethanol differentially reduces the stimulation of cAMP accumulation by hormones and cholera toxin. In the NG108-15 neuroblastoma chi glioma hybrid cell line, this heterologous desensitization was associated with a 42% reduction in the expression of Gs alpha and no significant change in Gi alpha. By contrast, ethanol treatment of the parental neuroblastoma cell line N18TG2 caused little loss of response to hormones or cholera toxin and no significant change in Gs alpha or Gi alpha. Ethanol induced heterologous desensitization in N1E-115 neuroblastoma cells; however, this cell line showed a dose-dependent increase in Gi alpha and a later decrease in Gs alpha. Thus, ethanol causes heterologous desensitization of hormone-stimulated cAMP accumulation by different mechanisms in related neural cell lines.     

Differential Expression of MARCKS and Other Calmodulin-Binding Protein Kinase C Substrates in Cultured Neuroblastoma and Glioma Cells

Abstract: Expression of the protein kinase C substrate MARCKS and other heat-stable myristoylated proteins have been studied in four cultured neural cell lines. Amounts of MARCKS protein, measured by [³H]myristate labeling and western blotting, were severalfold higher in rat C6 glioma and human HTB-11 (SK-N-SH) neuroblastoma cells than in HTB-10 (SK-N-MC) or mouse N1E-115 neuroblastoma cells. Higher levels of MARCKS mRNA were also detected in the former cell lines by S1 nuclease protection assay. At least two additional ³H-myristoylated proteins of 50 and 40–45 kDa were observed in cell extracts heated to >80°C or treated with perchloric acid. The 50-kDa protein, which bound to calmodulin in the presence of Ca²⁺, was more prominent in cells (N1E-115 and HTB-10) with less MARCKS, whereas neuromodulin (GAP-43) was detected in N1E-115 and HTB-11 cells only. Heating resulted in a fourfold increase in the detection of MARCKS by western blotting; this was not paralleled by a similar increase in [³H]myristate-labeled MARCKS and may be due to a conformational change affecting the C-terminal epitope or enhanced retention of the protein on nitrocellulose. Addition of β-12- O -tetradecanoylphorbol 13-acetate resulted in three- to fourfold increased phosphorylation of MARCKS in HTB-11 cells, with little increase noted in HTB-10 cells. These results indicate that MARCKS, neuromodulin, and other calmodulin-binding protein kinase C substrates exhibit distinct levels of expression in cultured neurotumor cell lines. Of these proteins, only MARCKS appears to be correlated with phorbol ester stimulation of phosphatidylcholine turnover in these cells.     

Regulation of neurite outgrowth in N1E-115 cells through PDZ-mediated recruitment of diacylglycerol kinase zeta

Syntrophins are scaffold proteins that regulate the subcellular localization of diacylglycerol kinase zeta (DGK-zeta), an enzyme that phosphorylates the lipid second-messenger diacylglycerol to yield phosphatidic acid. DGK-zeta and syntrophins are abundantly expressed in neurons of the developing and adult brain, but their function is unclear. Here, we show that they are present in cell bodies, neurites, and growth cones of cultured cortical neurons and differentiated N1E-115 neuroblastoma cells. Overexpression of DGK-zeta in N1E-115 cells induced neurite formation in the presence of serum, which normally prevents neurite outgrowth. This effect was independent of DGK-zeta kinase activity but dependent on a functional C-terminal PDZ-binding motif, which specifically interacts with syntrophin PDZ domains. DGK-zeta mutants with a blocked C terminus acted as dominant-negative inhibitors of outgrowth from serum-deprived N1E-115 cells and cortical neurons. Several lines of evidence suggest DGK-zeta promotes neurite outgrowth through association with the GTPase Rac1. DGK-zeta colocalized with Rac1 in neuronal processes and DGK-zeta-induced outgrowth was inhibited by dominant-negative Rac1. Moreover, DGK-zeta directly interacts with Rac1 through a binding site located within its C1 domains. Together with syntrophin, these proteins form a tertiary complex in N1E-115 cells. A DGK-zeta mutant that mimics phosphorylation of the MARCKS domain was unable to bind an activated Rac1 mutant (Rac1(V12)) and phorbol myristate acetate-induced protein kinase C activation inhibited the interaction of DGK-zeta with Rac1(V12), suggesting protein kinase C-mediated phosphorylation of the MARCKS domain negatively regulates DGK-zeta binding to active Rac1. Collectively, these findings suggest DGK-zeta, syntrophin, and Rac1 form a regulated signaling complex that controls polarized outgrowth in 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.     

Cyclic AMP responses are suppressed in mammalian cells expressing the yeast low Km cAMP-phosphodiesterase gene.

A genomic DNA fragment from Saccharomyces cerevisiae which contains the SRA5 (=PDE2) gene, coding for a low Km cAMP-phosphodiesterase, was transfected into Chinese hamster ovary cells. Clones carring the cAMP-phosphodiesterase gene were capable of growth in the presence of cholera toxin, which slows the growth of untransfected cells by elevating their cAMP levels. The cholera toxin-resistant transfected cell lines expressed high levels of cAMP-phosphodiesterase mRNA and cAMP-phosphodiesterase activity. Basal intracellular cAMP levels were not significantly affected by the presence of the yeast cAMP-phosphodiesterase gene, but elevation of cAMP levels in response to cholera toxin or prostaglandin E1 was suppressed. Induction of the cAMP-responsive tyrosine aminotransferase promoter by cholera toxin was also blocked in cell lines carrying the yeast cAMP-phosphodiesterase gene. Cholera toxin-resistant transfected cell lines were sensitive to the growth inhibitory effects of N6,02'-dibutyryladenosine 3',5'-monophosphate, which can be used to bypass the effects of the yeast cAMP-phosphodiesterase.     

Neurotensin, bradykinin and somatostatin inhibit cAMP production in neuroblastoma N1E115 cells via both pertussis toxin sensitive and insensitive mechanisms

Neurotensin, bradykinin and somatostatin inhibited in a time- and concentration-dependent manner prostaglandin E1- or forskolin-stimulated cAMP production in neuroblastoma N1E115 cells. Cell treatment with 1 microgram/ml pertussis toxin for 6 hours reversed the inhibition elicited by peptides after short incubation periods (less than or equal to 1 min) but, in contrast, had no effect after longer incubation periods (greater than or equal to 3 min). Fluoroaluminate also inhibited prostaglandin E1-stimulated cAMP production in N1E115 cells, and this effect was not reversed by pertussis toxin. The 6 hour treatment with pertussis toxin was shown to be sufficient to ADP ribosylate virtually all of the 41 kD protein substrate corresponding to the alpha subunit of Gi. Protein kinase C activation with phorbol ester did not inhibit basal or stimulated cAMP production. Our data point to the existence of both pertussis toxin sensitive and insensitive mechanisms of neuropeptide-mediated inhibition of cAMP formation in N1E115 cells. The toxin insensitive response is not mediated by protein kinase C. The possibility is discussed that it results from the activation of a pertussis toxin insensitive G protein.     

Effects of an inhibitor of glucosylceramide synthase on glycosphingolipid synthesis and neurite outgrowth in murine neuroblastoma cell lines.

1-Phenyl-2-decanolyamino-3-morpholino-1-propanol (PDMP), an effective inhibitor of UDP-glucose:ceramide glucosyltransferase, caused inhibition of cell growth in murine neuroblastoma cell lines. Metabolic labeling of glycosphingolipids with [14C]galactose in NS-20Y, Neuro2a, and N1E-115 cells showed reduced incorporation of radioactivity into gangliosides and neutral glycosphingolipids when threo-PDMP was present in the medium. Treatment of NS-20Y cells with threo-PDMP resulted in a time-dependent decrease in mass levels of gangliosides and neutral glycosphingolipids. After 24 h in the presence of 50 microM threo-PDMP, neutral glycosphingolipid mass was reduced to 32%, where glucosylceramide was the most affected (90% decrease). The ganglioside mass was reduced to 57% of the original content. Neurite outgrowth from neuroblastoma cells in serum-free medium was significantly inhibited by threo-PDMP in a dose-dependent manner. Threo-PDMP also caused retraction of neurites which had been induced to extend in serum-free medium. Pretreatment of cells with GM1 partially restored the ability of NS-20Y cells for neurite outgrowth in the medium containing threo-PDMP. These results suggest a possible role for glycosphingolipids in neurite outgrowth of murine neuroblastoma cells.     

Tetrahydrobiopterin Biosynthesis Enhanced by Lipopolysaccharide Stimulation in Murine Neuroblastoma Cell Line N1E-115

Abstract: We investigated for the first time the effect of lipopolysaccharide and the signal transduction pathway on the biosynthesis of tetrahydrobiopterin [(6 R - l - erythro -1',2'-dihydroxypropyl)-2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine], the cofactor for the enzymatic hydroxylation of the aromatic amino acids, in the murine neuroblastoma cell line N1E-115, which synthesizes tetrahydrobiopterin constitutively. Activation of N1E-115 cells with 1 µg/ml lipopolysaccharide resulted in statistically significant increases in both intracellular tetrahydrobiopterin contents and the activity ( V _max) of GTP cyclohydrolase I, a rate-limiting enzyme in tetrahydrobiopterin de novo biosynthesis. Following simultaneous addition of the inhibitors of protein tyrosine kinases and GTP-binding proteins into serum-free culture media with lipopolysaccharide, we analyzed the transduction pathway of lipopolysaccharide signal toward the tetrahydrobiopterin biosynthetic system in N1E-115 cells. Our data indicate the following conclusions: (a) Protein tyrosine kinase systems are involved in mediating lipopolysaccharide signal to tetrahydrobiopterin production, and (b) there may be a cross-talk between GTP-binding protein and the protein tyrosine kinase system in mediating lipopolysaccharide signal. These observations suggest that a neuronal cell such as N1E-115, which barely expresses CD14 on its cell surface, responds to lipopolysaccharide like macrophages and monocytes in the absence of soluble CD14.     

Endogenous expression of nNOS protein in several neuronal cell lines

Several neuronal cell lines were screened for endogenous expression of neuronal nitric oxide synthase (nNOS) protein using Western blot analysis. Detectable levels of the nNOS protein were evident in the SK-N-SH, SH-SY5Y, and N1E-115 neuroblastoma cell lines, as well as the NG108-15 neuroblastoma x glioma hybrid. Only trace amounts were visible in Neuro2A human neuroblastoma cells. The presence of endogenously expressed nNOS in these cells may allow for the study of the interaction between nNOS and the endogenous receptor systems expressed in the same cells.     

Clonal variants of PC12 pheochromocytoma cells with defects in cAMP-dependent protein kinases induce ornithine decarboxylase in response to nerve growth factor but not to adenosine agonists.

We have isolated and partially characterized three mutants of the pheochromocytoma line PC12 by using dibutyryl cyclic AMP (cAMP) as a selective agent. Each of these variants, A126-1B2, A208-4, and A208-7, was resistant to both dibutyryl cAMP and cholera toxin when cell growth was measured. In comparison to wild-type PC12 cells, each of these mutants was deficient in the ability to induce ornithine decarboxylase (ODC) in response to agents that act via a cAMP-dependent pathway. In contrast, each of these mutants induced ODC in response to nerve growth factor. To understand the nature of the mutations, the cAMP-dependent protein kinases of the wild type and of each of these mutants were studied by measuring both histone kinase activity and 8-N3-[32P]cAMP labeling. Wild-type PC12 cells contained both cAMP-dependent protein kinase type I (cAMP-PKI) and cAMP-dependent protein kinase type II (cAMP-PKII). Regulatory subunits were detected in both soluble and particulate fractions. The mutant A126-1B2 contained near wild-type PC12 levels of cAMP-PKI but greatly reduced levels of cAMP-PKII. Furthermore, when compared with wild-type PC12 cells, this cell line had an altered distribution in ion-exchange chromatography of regulatory subunits of cAMP-PKI and cAMP-PKII. The mutant A208-4 demonstrated wild-type-level binding of 8-N3-[32P]cAMP to both type I and type II regulatory subunits, but only half the wild-type level of type II catalytic activity. The mutant A208-7 had type I and type II catalytic activities equivalent to those in wild-type cells. However, the regulatory subunit of cAMP-PKI occurring in A208-7 demonstrated decreased levels of binding 8-N3-[32P]cAMP in comparison with the wild type. Furthermore, all mutants were defective in their abilities to bind 8-N3-[32P]cAMP to the type II regulatory protein in the particulate fraction. Thus, cAMP-PK was altered in each of these mutants. We conclude that both cAMP-PKI and cAMP-PKII are apparently required to induce ODC in response to increases in cAMP. Finally, since all three mutants induced ODC in response to nerve growth factor, the nerve growth factor-dependent induction of OCD was not mediated by an increase in cAMP that led to an activation of cAMP-PK. These mutants will be useful in the elucidation of the many functions controlled by cAMP and nerve growth factor.     

Evidence that the Modulation of Membrane-Associated Protein Kinase C Activity by an Endogenous Inhibitor Plays a Role in N1E-115 Murine Neuroblastoma Cell Differentiation

Abstract: Murine neuroblastoma cells, N1E-115, were induced to differentiate into neuron-like cells by serum deprivation for 18 h. As previous studies have shown that the suppression of protein kinase C (PKC) activity by selective inhibitors or neutralizing antibodies induces neuroblastoma cells to differentiate, we tested the hypothesis that serum deprivation may cause a rapid loss in membrane PKC activity that occurs well before the morphological changes that are characteristic of cell differentiation. A significant reduction in particulate (membrane) PKC activity was indeed observed within 3 h of serum withdrawal when enzyme activity was measured in intact native membranes by the recently described in vitro "direct" assay. This rapid reduction in enzyme activity was confirmed by the decreased phosphorylation of the MARCKS protein, an endogenous PKC-selective substrate, in intact cells. The decrease in membrane PKC activity occurred without any loss in the amount of membrane-associated enzyme, suggesting that some factor(s) resident in neuroblastoma membranes was suppressing PKC activity. Indeed, results indicate the presence of an endogenous inhibitor of PKC tightly associated with neuroblastoma membranes. This inhibitory activity increased in the membranes of cells subjected to serum deprivation, raising the possibility that it was likely responsible for the decline in membrane PKC activity in differentiating N1E-115 cells. Preliminary characterization indicated that the inhibitory activity is a protein and is localized mainly in the membrane fraction. Thus, these results demonstrate directly that endogenous inhibitor can regulate membrane-associated PKC activity in cells and thereby modulate PKC-related neuronal functions.