Cyclic AMP-induced differentiated neuroblastoma cells: changes in total nucleic acid and protein contents

The total DNA contents of neuroblastoma cells “differentiated” by dibutyryl cyclic AMP, prostaglandin E₁ and 4-(-3-butoxy-4-methoxybenzyl)-2-imidazolidinone treatment was about 50 percent that of control cells, indicating that cells were accumulated in the G₁-phase of the cell cycle. Sodium butyrate-treated cells were also accumulated in the G₁-phase; however, the expression of “differentiated” phenotype did not occur indicating that inhibition of cell division is not sufficient for morphological differentiation. A marked increase in RNA and protein contents of cyclic AMP-induced “differentiated” cells is consistent with an increase in the size of soma and nucleus.     

A further study on the regulation of cyclic nucleotide phosphodiesterase activity in neuroblastoma cells: Effect of growth

Summary Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) phosphodiesterase activity in mouse neuroblastoma cells in culture markedly increased during exponential growth and reached a maximal level at confluency; whereas guanosine 3′, 5′-cyclic monophosphate (cyclic GMP) phosphodiesterase activity only slightly but significantly increased under a similar experimental condition. The increase in cyclic AMP phosphodiesterase activity was blocked by both cycloheximide and dactinomycin, whereas the increase in cyclic GMP phosphodiesterase was blocked by only cycloheximide. When the confluent cells were replated at low density, the cyclic nucleotide phosphodiesterase activity decreased; however, when they were plated at high cell density which equaled confluency, the enzyme activity did not decrease. Unlike cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity did not change significantly in prostaglandin E₁-treated cells, but decreased in cells treated with the inhibitor of phosphodiesterase. Like cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity also did not change in cells treated with serum-free medium, X-irradiation, sodium butyrate and 6-thioguanine. This work was supported by USPHS NS-09230, and DRG-1273 from Damon Runyon-Walter Winchell Cancer Fund.     

DIFFERENTIATION OF NEUROBLASTOMA CELLS IN CULTURE

1. An elevation of the intracellular level of cyclic AMP in neuroblastoma cells by prostaglandin E₁ by an inhibitor of cyclic AMP phosphodiesterase, or by analogues of cyclic AMP irreversibly induces many differentiated functions which are characteristic of mature neurones. These include formation of long neurites, increase in size of soma and nucleus associated with a rise in total RNA and protein contents, increase in activities of specific neural enzymes, loss of malignancy, increase in sensitivity of adenylate cyclase to catecholamines and blockade of cells in G₁-stage of the cell cycle. 2. Other agents, including serum-free medium, X-irradiation, 6-thioguanine, cytosine arabinoside, methotrexate, 5-bromodeoxyuridine, nerve growth factor, glial extract and hypertonic medium can induce some of the differentiated functions which are induced by high intracellular cyclic AMP. 3. Morphological differentiation and differentiated biochemical functions can each be expressed in the absence of the other. 4. Many of the responses of normal embryonic nerve cells to cyclic AMP are similar to those of neuroblastoma cells. 5. A working hypothesis for the malignancy of nerve cells has been proposed. This states that an abnormal regulation of cyclic AMP phosphodiesterase activity which allows the expression of high amounts of this enzyme in neuroblastoma cells, may be one of the early lesions during a malignant transformation of nerve cells. 6. A new experimental therapeutic model for the treatment of neuroblastoma is proposed. This involves the administration of sodium butyrate followed by the injection of l -dihydroxyphenylalanine (l-dopa) and prostaglandin E₁ in the presence of cyclic AMP phosphodiesterase inhibitor. 7. Recent studies have elucidated the control mechanisms of some differentiated functions in neuroblastoma cells. Cyclic AMP may become an important biological tool to probe the regulation and expression of many other differentiated functions in these cells. In addition to neuroblastoma cells, other neuronal culture systems are now available for investigating the problems of differentiation and maturation in nerve cells.     

The effect of cyclic AMP and prostaglandins on the fibrinolytic activity of mouse neuroblastoma cells.

The C1300 mouse neuroblastoma cell line was found to produce plasminogen activator which is secreted into the growth medium. The intra- and extracellular activities of this enzyme were markedly increased (up to 14 fold) by treatment with cyclic AMP agents. Prostaglandins E₁ and E₂ and butyric acid were the most efficient inducers followed by propionic acid and dibutyryl cyclic AMP. Theophylline was found to be ineffective. The highest enzyme activities were found in cells exposed simultaneously to prostaglandin E₁ and dibutyryl cyclic AMP.     

Identification and characterization of functional D1 dopamine receptors in a human neuroblastoma cell line

Dopamine stimulated human neuroblastoma SK-N-MC cells to accumulated cyclic AMP. The D1 agonist SKF (R)-38393 also stimulated cyclic AMP production whereas the response to dopamine was inhibited by the D1 antagonist SCH (R)-23390. Membranes from SK-N-MC cells bound the D1 ligand [125I]SCH 23982 with a Kd of 2.1 nM and a Bmax of 102 fmol/mg protein. Binding was displaced by dopamine, SKF 38393, and SCH 23390. Up to 40% of the receptors were in an agonist high affinity, guanine nucleotide-sensitive state, compared to only 6% in rat striatum. A D1 photoaffinity probe labeled a 72 kDa protein in both SK-N-MC and rat striatal membranes. Thus, SK-N-MC human neuroblastoma cells contain D1 dopamine receptors which are similar to those found in mammalian striatum, but which are more tightly coupled to adenylate cyclase. SK-N-MC cells may be a useful model to investigate the properties and regulation of D1 dopamine receptors.     

Difference in the Cyclic Adenosine 3′,5′-Monophosphate Levels in Normal and Transformed Cells

CYCLIC 3′5′-adenosine monophosphate (cyclic AMP) regulates many physiological phenomena^1,2. Cellular morphology changes when the dibutyryl derivative of cyclic AMP is added in vitro to the nutrient media of cultured mammalian cells^3–6. Dibutyryl cyclic AMP has also been shown to restore controlled growth to transformed cells³, change the cell's surface architecture^3,7 and induce axon formation⁸ with an accompanied increase in acetylcholinesterase activity⁹ in neuroblastoma cells growing in culture. These effects suggest that the cyclic AMP moiety may have some basic regulatory action on cell growth and cell specialization.     

Regulation of glycogen metabolism in astrocytoma and neuroblastoma cells in culture.

The regulation of glycogen metabolism in C-6 astrocytoma and C-1300 neuroblastoma cells in culture has been investigated. Two modes of control of glycogen metabolism appear to be operative. The regulation of intracellular glycogen concentrations and the predominant forms of glycogen phosphorylase and glycogen synthase vary with (a) the available energy supply, and (b) altered intracellular concentration of cyclic adenosine 3':5'-monophosphate (cyclic AMP). Both cell lines respond to glucose in the medium; when glucose levels are high, glycogen is synthesized, glycogen phosphorylase a decreases, and glycogen synthase a increases. When glucose in the medium decreases to a critical level, the phosphorylase a increases and glycogen concentrations in the cells decrease in aprallel with the medium glucose. The critical glucose concentration is 2.5 mM for the astrocytoma cells and 4 mM for the neuroblastoma cells. Insulin promotes the conversion of phosphorylase to the b form and synthase to the a form in both cell lines. All of these changes occur without alteration in the intracellular cyclic AMP concentrations. When cyclic AMP concentrations are increased in either cell line, phosphorylase a is increased, synthase a is decreased, and glycogen concentrations decrease. Isobutyl methylxanthine is effective in promoting glycogenolysis in both cell lines. Norepinephrine is effective with the astrocytoma cells, and prostaglandin E1 is effective with the neuroblastoma cells.     

Differentiation of neuroblastoma cells by chick gizzard extract

Cholinergic neuroblastoma NS20Y cells were differentiated by the chicken gizzard extract. They were first inoculated into a glass culture bottle and the aggregated cells which grew in the suspension culture were collected. The aggregated cells (round and immature neuroblastoma cells) were seeded on a polyornithinecoated plastic dish, and the effect of various agents on the differentiation of the neuroblastoma was investigated. When gizzard extract from chicken was added to the culture, many flat cells with neurites emerged around the cell aggregates within 24 h. The flat cells could evoke action potentials with high frequency (in 70% cells). Cyclic GMP levels in the treated cultures were much lower than that in the control culture, and remained continuously lower during 2 days culture. The factor responsible for the differentiation of neuroblastoma cells was rich in the chick gizzard among extracts or conditioned media from various tissues tested. A similar effect was observed by the addition of dibutyryl cyclic AMP or prostaglandin E₁ plus theophylline over a slower time course. The factor in gizzard extract was trypsin-sensitive and heat-labile. The molecular size was estimated to be about 12 s.     

Changes of ornithine decarboxylase activity and polyamine content upon differentiation of mouse NB-15 neuroblastoma cells

The possible functions of ornithine decarboxylase (ODC) and polyamines in the differetiation of mouse NB-15 neuroblastoma cells were investigated by examining the changes of these parameters in the differentiaton and nondifferentiating NB-15 cells over a 5-day culture period. Differentiation of NB-15 cells was induced by the addition of dibutyryl cyclic AMP and 3-isobutyl-1-methylxanthin (IBMX) to the growth medium and was monitored by neurite outgrowth, increase of acetylcholinesterase (AChE), and R_I cAMP-binding protein. Plating of NB-15 cells in fresh serum-containing growth medium was accompanied by rapid growth and a marked increase of ODC activity, this early increase of ODC activity was attenuated, both in duration and in magnitude, in the differentiating cells. The spermidine content of the differentiating neuroblastoma cell was significantly lower than that of the nondifferentiating cells. In the fully differentiated neuroblastoma cells, the ODC activity and spermidine content were lower than that of the undifferentiated cells by approximately 15-fold and five-fold, respectively. Based on these results it is proposed that changes of polyamine metabolism may be of significance in the differentiation of mouse neuroblastoma cells.     

A further study on the regulation of cyclic nucleotide phosphodiesterase activity in neuroblastoma cells: effect of growth.

Adenosine 3',5'-cyclic monophosphate (cyclic AMP) phsophodiesterase activity in mouse neuroblastoma cells in culture markedly increased during exponential growth and reached a maximal level at confluency; whereas guanosine 3'5'-cyclic monophosphate (cyclic GMP) phosphodiesterase activity only slightly but significantly increased under a similar experimental condition. The increase in cyclic AMP phosphodiesterase activity was blocked by both cycloheximide and dactinomycin, whereas the increase in cyclic GMP phosphodiesterase was blocked by only cycloheximide. When the confluent cells were replated at low density, the cyclic nucleotide phosphodiesterase activity decreased; however, when they were plated at high cell density which equaled confluency, the enzyme activity did not decrease. Unlike cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity did not change significantly in prostaglandin E1-treated cells, but decreased in cells treated with the inhibitor of phosphodiesterase. Like cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity also did not change in cells treated with serum-free medium, X-irradiation, sodium butyrate and 6-thioguanine.     

Sodium and calcium currents in neuroblastoma x glioma hybrid cells before and after morphological differentiation by dibutyryl cyclic AMP

Sodium and calcium inward currents (INa and ICa) were measured in neuroblastoma X glioma hybrid cells of clones 108CC5 and 108CC15 by a single suction pipette method for internal perfusion and voltage clamp. Morphologically undifferentiated, exponentially growing cells were compared with cells differentiated by cultivation with 1 mmol/l dibutyryl cyclic AMP. Outward currents were eliminated by perfusing the cells with a K+-free solution. Voltage dependence and ion selectivity as well as steady state inactivation characteristics of INa and ICa resembled those of differentiated mouse neuroblastoma cells, clone N1E-115 (Moolenaar and Spector 1978, 1979). These parameters were identical in undifferentiated and differentiated cells of both clones. After differentiation the average density of the peak sodium and calcium currents was increased two and four-fold, respectively, in both cell lines. Our data indicate that exponentially growing, morphologically undifferentiated 108CC5 and 108CC15 neuroblastoma X glioma hybrid cells possess functional Na+ and Ca2+ channels undistinguishable from those of non-proliferating cells of these clones differentiated morphologically by treatment with dibutyryl cyclic AMP. That Na+ and Ca2+ spikes were not detected by other authors in these cells prior to morphological differentiation by dibutyryl cyclic AMP may be attributed to the fact that at the low resting membrane potential measured the Na+ and Ca2+ channels are inactivated.     

Morphological Differentiation induced by Prostaglandin in Mouse Neuroblastoma Cells in Culture

PROSTAGLANDIN is known to affect concentrations of cyclic AMP in some cells¹. Dibutyryl cyclic AMP induces irreversible differentiation of mouse neuroblastoma cells in vitro², which raises the question of whether prostaglandin would mimic this effect. I report here that prostaglandins (PG)E₁ and PGE₂ induce irreversible morphological differentiation of mouse neuroblastoma cells in culture as shown by axon formation, whereas PGF₂α does not.     

Neurotransmitter metabolism in murine neuroblastoma cells.

Murine neuroblastoma cells in culture are able to synthesize the putative neurotransmitters--acetylcholine, dopamine, norepinephrine, tyramine, octopamine, histamine, serotonin and γ-aminobutyric acid (GABA). They possess not only synthetic, but also degradative enzymes involved in metabolism of these transmitters, and many of these enzymes increase in activity as the cells “differentiate”. Catecholamines, and perhaps other transmitters, appears to be stored within membrane-limited vesicles which accumulate within the process endings of these cells. Uptake of some transmitters, GABA, glycine, dopamine and norepinephrine, shows characteristics of the high affinity transport systems observed in other neuronal populations; uptake of choline and other amino acids is similar to that in non-neuronal populations. Cells show receptor sensitivities to acetyl-choline, dopamine, norepinephrine, prostaglandins E₁ and morphine, as demonstrated by electrophysiologic, toxin binding and cyclic nucleotide studies.     

Nerve growth factor (NGF) induced differentiation of human neuroblastoma cells

1. Nerve growth factor (NGF) induced differentiation of human neuroblastoma cell line. 2. The differentiated cells had a relatively high activity of adenylate cyclase and cyclic AMP phosphodiesterase, and a high intracellular level of cyclic AMP. 3. These cells synthesized a higher amount of met5-o-enkephalin than undifferentiated cells. 4. Undifferentiated cells bound less met5-enkephalin than differentiated cells. The maximum number of [3H]met5-enkephalin receptor sites per mg of membrane protein increased more in differentiated cells. 5. Previous observations taken together with our results suggests that increased intracellular levels of cyclic AMP after treatment with NGF induced differentiation of human neuroblastoma cells. Reversal of undifferentiated tumor cells into the differentiated changes the capacity of synthesis of met5-enkephalin and its interaction with receptors.     

Inhibition of neuroblastoma adenylate cyclase by cannabinoid and nantradol compounds

This study was undertaken to ascertain the effects of cannabinoid drugs on prostanoid-stimulated adenylate cyclase in neuroblastoma cells. This report demonstrates that Δ⁹-tetrahydrocannabinol (THC) and levonantradol could decrease initial rate cyclic AMP accumulation in response to prostacyclin in intact cells. Basal accumulation was also diminished. Prostanoid-stimulated adenylate cyclase in a membrane preparation from these cells was inhibited by cannabinoid and nantradol compounds. However, this inhibition was not competitive with prostaglandin E₁ or prostacyclin. Further, inhibition was also observed when the enzyme was stimulated by peptide hormones at the secretin receptor. In contrast, enzyme activated by NaF was not inhibited by cannabinoid compounds. Cyclic AMP phosphodiesterase activity in subcellular fractions was unaltered by these agents. These data demonstrate that cannabinoid and nantradol compounds decrease cyclic AMP accumulation in neuronally derived cells, and that this results from an inhibition of basal and hormone-stimulated adenylate cyclase activity.     

Homologous Desensitization of the D1 Dopamine Receptor

Preincubation of D384 cells, derived from the human astrocytoma cell line G-CCM, with dopamine resulted in a time-dependent attenuation of cyclic AMP responsiveness to subsequent dopamine stimulation. This effect was agonist specific because the prostaglandin E1 (PGE1) stimulation of cyclic AMP of similarly treated cells remained unchanged. The attenuation by dopamine was concentration dependent with a maximum observed at 100 microM. A comparison of dopamine concentration-response curves of control and dopamine-preincubated cells revealed no change in the Ka apparent value, but a marked attenuation of the maximal response. Preincubation of cells with dopamine in the presence of D1 but not D2 selective antagonists partially prevented the observed attenuation. Attenuations in dopamine responsiveness were also obtained when D384 cells were preincubated with D1 but not D2 receptor agonists. The level of attenuation attained related to agonist efficiency in stimulating cyclic AMP: SKF38393 less than 3,4-dihydroxynomifensine less than fenoldopam less than 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene = dopamine. However, increasing the efficiency of 3,4-dihydroxynomifensine stimulation of cyclic AMP, using the synergistic effect of adding a low concentration of forskolin, produced no further change in the attenuation of the subsequent response to dopamine. Thus, the D1 dopamine receptors expressed by D384 cells undergo homologous desensitization. Uncoupling of the D1 dopamine receptor appears to be independent of cyclic AMP formation, analogous to a mechanism proposed for the beta-adrenergic receptor.     

Modification of response of mouse neuroblastoma cells in culture by serum type

Summary The effect of various types of serum on morphological and biochemical changes in mouse neuroblastoma cells (clone NBP₂) in culture was studied. The extent of spontaneous morphological differentiation varied markedly depending upon the type of serum and was maximal in agammaglobulin calf serum (CS). The extent of morphological differentiation after treatment of cells with cyclic AMP-stimulating agents was also dependent upon serum type and was least pronounced in fetal calf serum. The doubling time and extent of clumping varied with the type, of serum. The activity of tyrosine hydroxylase (TH) in NB cells was dependent upon serum type and it was highest in newborn CS and agammaglobulin CS. Although elevation of intracellular levels of cyclic AMP in NBP₂ clone invariably stimulates neurite formation and TH activity, these functions were increased in certain sera without a significant increase in the cellular cyclic AMP levels. The present study shows that neurite formation, growth rate and TH activity are regulated by more than one mode, one of which is mediated by cyclic AMP. The above changes are independently regulated in the sense that the expression of one can be increased in the absence of others. Preliminary reports of this work were presented at the Symposium on Cell Differentiation and Neoplasia, March 1976; American Society for Neurochemistry, March 1978; and the FASEB meetings, April 1978. This work was supported in part by NIH Grant ROESNS 01576, NIH Training Grant 4007072 and Research Scientist Career Development Award MH 42479.     

Cyclic AMP in the cell cycle of Physarum polycephalum

Cyclic AMP, [³H]thymidine incorporation, and DNA content were measured in the cell cycle of Physarum polycephalum. A sensitive radioimmunoassay was employed to assay cyclic AMP so that plasmodia could be assayed individually. In contrast to previously published results (Lovely, J.R. and Threlfall, R.J. (1976) Biochem. Biophys. Res. Commun. 71, 789–795), no pre-mitotic peak of cyclic AMP was detected. In seven experiments levels of cyclic AMP showed only small changes in individual experiments and ranged from 1–6 pmol/mg protein in different experiments. When plasmodia in the immediate premitotic period were collected on the basis of nuclear mitotic morphology, no evidence of a peak of cyclic AMP was found. Light was found to increase plasmodial cyclic AMP in a rapid, transient fashion. However, the brief exposure of cell cycle samples to light during collection did not induce any apparent cell cycle specific peaks of cyclic AMP. Although the occurrence of extremely rapid transient peaks of cyclic AMP in the cell cycle cannot be ruled out, it appears likely that the P. polycephalum cell cycle can proceed normally without major changes in cyclic AMP.