Cultured Mouse Neuroblastoma Cells Research Articles

Regulation of muscarinic receptor-mediated cyclic GMP synthesis by cultured mouse neuroblastoma cells.

Mouse neuroblastoma clone N1E-115 has muscarinic acetylcholine receptors that mediate cyclic GMP synthesis. This receptor-mediated response is not significantly higher than background until the cells have been maintained in the stationary phase for at least 1 week. The basis of the influence of time in culture on the cyclic GMP response was investigated. The relative amount of cyclic GMP synthesized by intact cells was measured by radioactively labeling the GTP pool with [3H]guanine, incubating cells with agonists, and then chromatographically isolating [3H]cyclic GMP. Carbamylcholine-, ionophore X-537A-, and sodium azide-induced cyclic GMP formation increased with time in culture to a maximum of 13-, 9-, and 2.5-fold above basal, respectively. There was no change in the number or the apparent affinity of the muscarinic receptors as measured by [3H]quinuclidinyl benzylate ([3H]QNB) binding. In addition, there was no change in the apparent affinity of the receptors for agonist as measured by the ability of carbamylcholine to displace the specific binding of [3H]QNB. Guanylate cyclase activity per milligram protein and per cell increased six- and sevenfold, respectively, from day 0 to day 22. However, this increase in guanylate cyclase appeared to precede the marked increase in sensitivity of the cells to agonists. These data suggest that, in addition to guanylate cyclase and muscarinic receptors, there is another factor which is responsible for the development of this muscarinic receptor-mediated response.

Endocytosis and compartmentation of peroxidases and cationized ferritin in neuroblastoma cells

To investigate further the selectivity of retrograde axonal transport of horseradish peroxidase (HRP) isoenzymes previously observed in the rat central visual pathways, cultured mouse neuroblastoma cells (clone N18) were examined for selectivity of endocytosis of peroxidases and cationized ferritin in vitro. Differentiating N18 cells were incubated with proteins in various timing and pulse-chase experiments, and examined for the ultrastructural localization of endocytosed protein using cytochemical techniques. Semi-quantitative morphometry was performed on some of the samples. Major findings were as follows. (1) The protein was internalized into vesicles (coated and uncoated), short tubules and occasional small cup-shaped bodies within the first few minutes, and transferred via tubules and uncoated vesicles to secondary lysosome-like organelles (vacuoles, multivesicular bodies and dense bodies) from 5 to 15 min, with dense bodies representing the final site of protein accumulation. All the proteins tested were endocytosed through the same pathways in the soma and neurite of the cell. (2) There was no indication of a net orthograde or retrograde neuritic transport proteins. (3) There were induced increases in both vesicle and tubule formation as a result of protein endocytosis, with HRP isoenzyme C showing the greatest effect. (4) The apparent rate of internalization of HRP isoenzyme C into vesicles during the initial 5 min was significantly greater than for other peroxidases amd cationized ferritim. (5) Relatively more tubules than vesicles were involved in the uptake of protein as endocytosis was prolonged from 5 min to 8 h, with HRP isoenzyme C showing the largest effect. (6) There were indications of preferential compartmentation of endocytosed protein into certain organelles.

The cytotoxicity of methylmercuric hydroxide and colchicine in cultured mouse neuroblastoma cells

After a 24-hr growth period in unsealed flasks, uncloned C1300 mouse neuroblastoma cells were exposed to either methylmercuric hydroxide (CH 3HgOH) (3 × 10 −9 –3 × 10 −6 m) or colchicine (3 × 10 −10–1 × 10 −7 m) in nutrient mixture F-12 (Ham) for 24–72 hr in sealed flasks. After 48 hr, CH 3HgOH (1 × 10 −6 m) produced a 50% decrease in the number of morphologically differentiated cells and a significant increase in cell sloughing without inhibiting cell division or decreasing cell viability. However, after 48 hr, colchicine inhibited cell division and morphological differentiation at concentrations lower than those that caused increased cell sloughing or decreased cell viability. When CH 3HgOH (1 × 10 −6 m) was replaced with control medium after 24 hr, most cytotoxic effects were reversed. The toxic effects were not as readily reversed after exposure to CH 3HgOH for 48 hr. Sloughed cells did not survive 48 hr exposure to CH 3HgOH when replaced in the control medium although they did survive after a 24-hr exposure. Recovery from the cytotoxic effects of CH 3HgOH was reduced in the presence of 3 × 10 −6 m cycloheximide. It is concluded that CH 3HgOH exerts its toxicity at several sites within the neuroblastoma cell. Although the organomercurials have been shown to bind to neurotubles, the cytotoxicity of CH 3HgOH in neuroblastoma cells can not be ascribed solely to this mechanism. Recovery from the effects of short-term exposure may involve the synthesis of new protein.

Neurovirulence of influenza virus in mice II. Mechanism of virulence as studied in a neuroblastoma cell line

The difference between neurovirulent WSN and nonneurovirulent Hong Kong (HK) virus strains in intracerebral infection of mice was attributed to NA, M, and NS genes ( Sugiura and Ueda, 1980, Virology 101, 440–449). The mechanism by which NA, M, and NS proteins were involved in the virulence was studied by infection of mouse neuroblastoma cells in culture. Replication of prototype and recombinant viruses in neuroblastoma cells reflected in many respects the infection of the mouse brain in situ. Viruses containing WSN neuraminidase multiplied productively in the neuroblastoma cells. Progeny viruses released from neuroblastoma cells infected with viruses containing HK neuraminidase had uncleaved HA polypeptide, and were, as a result, noninfectious. Treatment with trypsin in vitro conferred the infectivity to the noninfectious progeny. The role of WSN neuraminidase was considered to facilitate the cleavage of HA polypeptide. Indeed, the brains of mice infected intracerebrally with HK virus on the previous day contained noninfectious viruses which were activated by trypsin. When either M or NS gene was derived from HK virus, the growth rate of recombinants was decreased in multicycle infection. These recombinants infected neuroblastoma cells with similar efficiency as virulent viruses. M and NS proteins from HK virus probably affected the replicative process in neuroblastoma cells at the stage later than the initiation of infection, resulting in diminished growth potential of the virus.

Differentiation of mouse neuroblastoma cells under increased oxygen tension.

After exposure to increased oxygen tension for 3 days, cultured mouse neuroblastoma cells were found to undergo morphological differentiation and to significantly increase their ecto-acetylcholinesterase activity. Differentiation could not be blocked by KCN or lipid-soluble antioxidants, indicating that neither an increased respiration, nor an increased oxidation of membrane lipids was responsible for the effect.

Effects of methylmercuric chloride on high affinity uptake of certain putative neurotransmitters in neuroblastoma and glioma cell cultures

The effect of methylmercuric chloride (CH 3HgCl) on high affinity uptake of certain putative neurotransmitters on mouse neuroblastoma (NBE −) and rat glioma (C-6) cells in culture 3 days after treatment was studied. The energydependent accumulation of radioactive choline was decreased in NB cells afte treatment with CH 3HgCl (1 μm), whereas the accumulation of radioactive glycine or glutamate was increased. The uptake of radioactive glycine or glutamate in glioma cells was decreased after treatment with CH 3HgCl (0.3 μM). High affinity uptake of choline was not demonstrable in glioma cells. Low concentrations of CH 3HgCl affect the accumulation of glycine and glutamate in glioma and NB cells in an opposite manner.

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

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.

Effect of sodium butyrate in combination with X0irradiation, chemotherapeutic and cyclic AMP stimulating agents on neuroblastoma cells in culture

Sodium butyrate, X-irradiation, chemotherapeutic agents and cyclic AMP-stimulating agents cuased reduction in the cell number (due to cell death and reduction in cell division) when added individually to mouse neuroblastoma cells in culture. However, the combination of sodium butyrate with X-irradiation, chemotherapeutic and cyclic AMP-stimulating agents produced a greater reduction in the cell number than that produced by the individual agents.

Differential sensitivities of glioma cells and neuroblastoma cells to methylmercury toxicity in cultures

The effects of mercury compounds in rat glioma (C-6) and mouse neuroblastoma (NBP 2) cells in culture were studied. Glioma cells were more sensitive to methylmercuric chloride (CH 3HgCl) than neuroblastoma (NB) cells for the criterion of growth inhibition (due to cell death and inhibition of cell division). This may be due in part to the fact that glioma cells accumulated more CH 3 203HgCl than NB cells. The biological half-life of CH 3 203HgCl in glioma cells was less (3.2–5.2 hr) than that in NB cells (7 – 7.3 hr). Although a much higher concentration of mercuric chloride (HgCl 2) was required to produce effects similar to those produced by CH 3HgCl, it produced no differential effects on glioma and NB cells.

Hallucinogens antagonize histamine H 1 receptors of cultured mouse neuroblastoma cells

Hallucinogens were competitive antagonists of histamine at the H 1-receptor of cultured mouse neuroblastoma cells. Their rank order of potency at this receptor was similar to that for their potency at eliciting subjective effects in vivo. Comparison with studies of other receptors, however, suggests that no single model of drug—receptor interaction adequately accounts for the known subjective effects of these compounds.

Induction of differentiation in cultured mouse neuroblastoma cells by N-methyl-N′-nitro-N-nitrosoguanidine

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a potent mutagen and carcinogen, induces differentiation uniquely in N-18 mouse neuroblastoma cells, when compared with that of dibutyryl cyclic adenosine monophosphate. After treatment with 10 μM MNNG for only 2 h, RNA and protein synthesis are stimulated together with neurite formation, while DNA synthesis and growth of the cells are inhibited indefinitely. Induction of neurite formation by MNNG is irreversible, being inhibited by actinomycin D or cycloheximide but recovering after withdrawal of these inhibitors.

Sodium ascorbate potentiates the growth inhibitory effect of certain agents on neuroblastoma cells in culture.

Mouse neuroblastoma (NB) cells in culture were more sensitive to sodium L-ascorbate than were rat glioma cells by the criterion of growth inhibition (due to cell death and reduction in cell division). Sodium L-ascorbate at nonlethal concentrations potentiated the effect of 5-fluorouracil (FUra), x-irradiation, bleomycin, RO20-1724, prostaglandin E1, and sodium butyrate on NB cells but did not produce such an effect on glioma cells. Sodium L-ascorbate did not enhance the effect of vincristine, 6-thioguanine, or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) except at higher drug doses and it reduced the cytotoxic effect of methotrexate and 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) on NB cells. Sodium D-ascorbate produced effects similar to those produced by sodium L-ascorbate on NB cells. L-Ascorbic acid-2-sulfate (barium salt) affected neither the growth rate nor the effect of 5-FUra on NB cells. Glutathione, a reducing agent, was more toxic to NB cells in comparison to D- OR L-ascorbate; however, at a similar concentration it failed to potentiate the effect of 5-FUra on NB cells.

Cyclic-AMP-induced differentiation in neuroblastoma is independent of cell division rate.

CLONED, cultured mouse neuroblastoma cells, when grown in conditions which restrict cell division, extend neurites1–6 and increase the activity of neurotransmitter-related enzymes7–9. Treatments that can induce this differentiation include lowered serum concentrations3, prostaglandins4, phosphodiesterase inhibitors5 and analogues of cyclic-3′,5′-AMP (cyclic-AMP)6,10–11. These treatments have been proposed to act by elevating intracellular cyclic-AMP levels, but because they also inhibit cell division it has been difficult to determine whether their effects are due directly to cyclic-AMP or indirectly to inhibition of cell division12. As neurite growth and enzyme elevation occur during the interphase portion of the cell cycle (and are retarded during cell division), cyclic-AMP may simply allow differentiation by stopping the cell cycle in interphase3. The butyryl analogues of cyclic-AMP, in particular, markedly restrict cell division (ref. 10; see also Fig. 1a). Because butyrate, which can dissociate from the dibutyryl analogue of cyclic-AMP, can itself markedly inhibit cell division we reasoned that other analogues may exist which are not inhibitors of cell division but which effectively mimic the action of cyclic-AMP. We report here that 8-bromo-cyclic-3′,5′-AMP (8Br-cyclic-AMP) effects biochemical and morphological changes in the neuroblastoma clone NBD-2 (ref. 9) without concomitant inhibition of cell division.

Histamine H1 receptor-mediated guanosine 3',5'-monophosphate formation by cultured mouse neuroblastoma cells.

Incubation of cultured mouse neuroblastoma cells with histamine caused a rapid and marked increase in the formation of guanosine 3',5'-monophosphate (cyclic GMP) by these cells. Receptor agonists for H1, but not H2, caused this effect which was reduced by H1 but not by H2 or muscarinic acetylcholine receptor antagonists. These results indicate that activation of H1 receptors in these cultured nerve cells stimulated cyclic GMP formation.

Haemin promotes rapid neurite outgrowth in cultured mouse neuroblastoma cells.

MOUSE neuroblastoma cells grown in suspension culture are round and anaplastic, but when grown in culture attached to a surface and in the presence of various inducing agents such as dibutyryl cyclic AMP1, bromodeoxyuridine2, prostaglandin E1 (ref. 3), cytosine arabinoside4, papaverine5, dimethyl sulphoxide (DMSO)6,7, hexamethylene bisacetamide6,7, or serum-free medium8,9, the cells will extend neurites and assume a morphology resembling that of the mature neurone. The mechanism of neurite outgowth is unknown. Neurite outgrowth develops slowly in the presence of the defined agonists, usually requiring one to several days to attain the optimum outgrowth. Thus, the possible importance of cell-cycle associated events and cell division can complicate the interpretation of experimental results. And, with serum deprivation, nonspecific alterations may result from the removal of numerous factors in the complex mixture comprising serum. Therefore, the availability of a rapidly acting agonist effective in the presence of the normal complement of serum would be a useful tool. We report here that haemin can rapidly and reversibly induce neurite outgrowth in cultured mouse neuroblastoma cells and suggest that haemin may have a wider role in cells than previously thought.

Antioxidants in neoplastic cells: II. Isolation and partial characterization of a phenolic antioxidant from differentiated mouse neuroblastoma cells.

The generation of an antioxidant has been shown to be associated with the dramatic increase in resistance to lipid peroxidation which occurs during the differentiation of mouse neuroblastoma cells in culture. The antioxidant has been isolated from the neuroblastoma neutral lipid fraction and partially characterized by means of low-resolution and high-resolution mass spectrometry and other lines of evidence. All presently available information suggests that this antioxidant is a highly aromatic, monosubstituted phenol having the molecular formula C19H14O2.

Stimulation of tyrosine hydroxylase activity in cultured mouse neuroblastoma cells by monocarboxylic acids.

Stimulation of tyrosine hydroxylase activity in cultured mouse neuroblastoma cells by monocarboxylic acids.

Antioxidants in neoplastic cells: I. Changes in the antioxidative capacity of mouse neuroblastoma cells measured by a single-phase assay.

Cultured mouse neuroblastoma cells exhibit a striking increase in antioxidative capacity during the transition from logarithmically dividing cells to nondividing, neurite-bearing cells. Two physically separable phenomena are involved: (a) the membrane pellet of neurite-bearing cells is highly resistant to lipid peroxidation, and (b) the postmicrosomal supernatant of these cells inhibits peroxidation in rat liver mitochondria and other biological membranes. A precise, single-phase assay has been developed for assessing antioxidant levels in lipid extracts. By means of this assay, the increase in membrane resistance to lipid peroxidation has been correlated with a threefold increase in the antioxidant activity of the neuroblastoma neutral lipid fraction. This finding implies that generations of a neutral lipid antioxidant (or antioxidants) is involved in the profound increase in antioxidative capacity which occurs in differentiating neuroblastoma cells.

Tumor promoters inhibit morphological differentiation in cultured mouse neuroblastoma cells.

When added to mouse neuroblastoma cultures, the potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) inhibits spontaneous neurite formation as well as that induced in response to serum deprivation, prostaglandin E1, 5-bromo-2'-deoxyuridine, and papaverine. Other tumor-promoting macrocyclic plant diterpenes also inhibit neurite formation, whereas nonpromoting diterpenes do not. Inhibition by TPA was reversible and was unrelated to toxicity.

Ionic currents in cultured mouse neuroblastoma cells under voltage-clamp conditions.

1. Ionic currents in differentiated cells of mouse neuroblastoma clone N1E-115 have been studied under voltage-clamp conditions. 2. Depolarizing voltage steps from a holding potential of -85 mV to levels more positive than -40 mV produced fast transient inward currents followed by delayed outward currents. 3. The fast inward current is carried by Na+: it is blocked by tetrodotoxin and is absent in Na+-free solutions. Its kinetic behaviour resembles that of the Na+ current in squid giant axon. A mean value of 85 mmho/cm2 was found for the maximum Na+ conductance (GNa).4. The delayed outward current is carried primarily by K+: it is blocked by externally applied tetraethylammonium (TEA, 15 mM) and has a reversal potential (mean -71 mV) close to the theoretical K+ equilibrium potential. Its instantaneous I--V curve is linear. By analogy with the formulation of Hodgkin & Huxley (1952c), the outward current can be described by IK = -GKn2(V--EK) where GK = 12 mmho/mc2. 5. During prolonged depolarizations the delayed outward current declines. This decline, which occurs in two phases, represents a partial inactivation of the K+ conductance. 6. A weak inward current with slow activation and inactivation kinetics appears in Na+-free solution containing 10 mM-Ca2+. It is activated at a membrane potential of -55 mV and reaches its maximum at -20 mV with a time to peak of about 10 msec. This current is tetrodotoxin-resistant, reversibly blocked by Co2+ (5mM) and is suggested to be carried by Ca2+. 7. An increase in the external divalent cation concentration results in a parallel shift of the steady-state I--V curve along the voltage axis in positive direction. The activation of delayed outward currents is suggested not to depend on Ca2+ influx. 8. It is concluded that separate voltage-dependent Na+, K+ and Ca2+ channels exist in the differentiated neuroblastoma membrane with kinetic and pharmacological properties similar to those observed in non-mammalian preparations.