Activity In Neuroblastoma Cells Research Articles

Dose dependence of acetylcholinesterase activity in neuroblastoma cells exposed to modulated radio-frequency electromagnetic radiation.

Radio-frequency electromagnetic radiation (RFR) at 915 and 147 MHz, when sinusoidally amplitude modulated (AM) at 16 Hz, has been shown to enhance release of calcium ions from neuroblastoma cells in culture. The dose-response relation is unusual, consisting of two power-density "windows" in which enhanced efflux occurs, separated by power-density regions in which no effect is observed. To explore the physiological importance of these findings, we have examined the impact of RFR exposure on a membrane-bound enzyme, acetylcholinesterase (AChE), which is intimately involved with the acetylcholine (ACh) neurotransmitter system. Neuroblastoma cells (NG108), exposed for 30 min to 147-MHz radiation, AM at 16 Hz, demonstrated enhanced AChE activity, as assayed by a procedure using 14C-labeled ACh. Enhanced activity was observed within a time window between 7.0 and 7.5 h after the cells were plated and only when the exposure occurred at power densities identified in a previous report as being effective for altering the release of calcium ions. Thus RFR affects both calcium-ion release and AChE activity in nervous system-derived cells in culture in a common dose-dependent manner.

Effect of increased glucose levels on Na+/K+-pump activity in cultured neuroblastoma cells.

Neuroblastoma cells were used to analyze the effect of elevated glucose levels on myo-inositol metabolism and Na+/K+-pump activity. The activity of the Na+/K+ pump in neuroblastoma cells is almost totally sensitive to ouabain inhibition. Culturing neuroblastoma cells in 30 mM glucose caused a significant decrease in Na+/K+-pump activity, myo-inositol metabolism, and myo-inositol content, compared to cells grown in the presence of 30 mM fructose. Glucose supplementation also caused a large intracellular accumulation of sorbitol. The aldose reductase inhibitor sorbinil prevented the abnormalities in myo-inositol metabolism and partially restored Na+/K+-pump activity in neuroblastoma cells cultured in the presence of elevated glucose levels. These results suggest that the accumulation of sorbitol by neuroblastoma cells exposed to elevated concentrations of extracellular glucose causes a decrease in myo-inositol metabolism and these abnormalities are associated with a reduction in Na+/K+-pump activity.

Biochemical pharmacology of N-acetyl- N-(methylcarbamoyloxy)- N'-methylurea (caracemide; NSC-253272)

Preclinical pharmacologic studies of caracemide [ N-acetyl- N-(methylcarbamoyloxy)- N'- methylurea; CAR] have demonstrated a marked instability of this compound in the presence of either phosphate buffer (pH 7.4) or human plasma. Using [1- 14C- acetyl]CAR and [ 3H- methyl- carbamoyloxy] CAR, three CAR degradation products were identified: product A, N-(methyl-carbamoyloxy) acetamide; product B: N-(methylcarbamoyloxy)-N'-methylurea; and product C: N-hydroxy- N'-methylurea. CAR degradation in human plasma was demonstrated by high-performance liquid chromatography (HPLC) to occur in a time- and temperature-dependent manner. A 30-min incubation (37°) of CAR (10 −4M) with human plasma resulted in degradation of more than 55% of parent compound; at 1 hr, more than 75% of original CAR was degraded. Incubation of [1- 1+C- acetyl] CAR with rat brain homogenate resulted in the formation of 14CO 2. This reaction was partially inhibited by coincubation with physostigmine (10 −3M). CAR inhibited acetylcholinesterase activity in neuroblastoma cells with an ic 50 of 14 μM. In mechanism of action studies, CAR was found to inhibit ribonucleotide reductase activity but only at nine times the ic 50 of hydroxyurea. In contrast to hydroxyurea, CAR was found to be non-cell-cycle phase-specific and non-cross-resistant with two CHO cell lines resistant to hydroxyurea. These data demonstrate the instability of CAR; moreover, they suggest that its mechanism of cytotoxicity is distinctly different from that of hydroxyurea and that the neurotoxicity associated with CAR administration may be caused in part by inhibition of acetyl-cholinesterase activity.

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.

Cyclic nucleotides in the regulation of expression of differentiated functions in neuroblastoma cells.

Adenosine 3',5'-cyclic monophosphate (cAMP) may be one of the important factors in regulating the expression of many differentiated functions in neuroblastoma cells, but some of these functions can be induced by agents that do not increase the intracellular level of cAMP. An elevation of the intracellular level of guanosine 3',5'-cyclic monophosphate (cGMP) neither induced differentiation nor antagonized the effects of cAMP. Neuroblastoma cells increased the level of cAMP-binding proteins during differentiation, whereas glial cells and L-cells did not. This might have accounted in part for an increase in the intracellular level of cAMP even in the presence of high phosphodiesterase activity in neuroblastoma cells, since the protein-bound with the same proteins, but cAMP had about 10 times higher affinity than did cGMP. cAMP promoted the organization of microtubules and microfilaments necessary for the expression of differentiated phenotypes. The extension of neurites required the synthesis of new protein, but it did not need the synthesis of new RNA. cAMP induced differentiation in neuroblastoma cells by increasing the expression of some genetic information while suppressing the expression of others; e.g., the activities of neural enzymes increased, whereas the synthesis of histone and the phosphorylation of H1-histone markedly decreased in differentiated cells. A hypothesis was offered: An increase in cAMP phosphodiesterase activity as a result of mutation in the regulatory gene for phosphodiesterase in a single, or group of, dividing nerve cell(s) is the primary lesion that leads to malignancy. Based on the concept that selective cytocytoxic drugs should be used with agents that cause differentiation, a new therapeutic approach was suggested for the treatment of neuroblastoma. This involved administration of sodium butyrate followed by L-DOPA or prostaglandin E1 in the presence of cAMP phosphodiesterase inhibitor followed by the less immunosuppressive vincristine and 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide.

The inhibition of the induction of ornithine decarboxylase by cations

L1210 cells reinitiate growth after dilution with fresh medium; during this time there occurs a transient increase in ornithine decarboxylase (EC4.1.1.17) (ODC) activity. The addition of 10 to 20 mM Na +, K + or Mg ++ completely inhibits this induction of ODC activity with no effect on cell growth. These cations also inhibit the increase of ODC activity in neuroblastoma cells and in H-35 cells which is induced by prostaglandin E 1 plus 3-isobutyl-1-methyl-xanthine and by 15% fetal calf serum respectively. This inhibitory effect of low levels of cations on the induction of ODC activity in different cell lines suggests that the intracellular function of ODC, and of the products of the reaction it catalyzes (putrescine, spermidine and spermine), may be intimately involved with changes in cation pools.

Different mechanisms for the induction of acetylcholinesterase in neuroblastoma cells

Neuroblastoma cells, incliding clones selected for resistance to dibutyryl-cAMP or for their ability to survive and multiply at 40°C, were used to study differences in the induction of acetylcholinesterase activity by dibutyryl-cAMP and 5-bromodeoxyuridine. In nonselected neuroblastoma cells both of these compounds induced this enzyme activity. Actinomycin D inhibited induction by 5-bromodeoxyuridine but did not inhibit inducion by dibutyryl-cAMP. Enzyme activity in dibutyryl-cAMP-resistant cells was induced by 5-bromodeoxyuridine and not by dibutyryl-cAMP. In the temperature-resistant cells, induction by 5-bromodeoxyuridine was lower at 40 than at 37°C and induction by dibutyryl-cAMP was higher at 40 than at 37°C. This difference in induction at the two temperatures was associated with a higher inhibition of cell multiplication at 40°C by both compounds. The results indicate that 5-bromodeoxyuridine and dibutyryl-cAMP induce acetylcholinesterase activity in neuroblastoma cells by different mechanisms.

Manganese inhibits adenylate cyclase activity and stimulates phosphodiesterase activity in neuroblastoma cells: Its possible implication in manganese-poisoning

The effect of manganese on adenylate cyclase and cAMP phosphodiesterase activities in homogenates of mouse malignant and adenosine 3′,5′-cyclic monophosphate (cAMP)-induced “differentiated” neuroblastoma cells was studied. High concentrations of manganese inhibited adenylate cyclase activities, but they stimulated cAMP phosphodiesterase activity. These enzymes of cAMP-induced “differentiated” cells were more sensitive to manganese than were those of malignant cells. These data indicate that excess of manganese may markedly reduce the intracellular level of cAMP by inhibiting adenylate cyclase activity and stimulating phosphodiesterase activity. We speculate that some of the neurological syndromes in patients with manganese-poisoning may be related to the decreased level of cAMP which may result from manganese-induced inhibition of adenylate cyclase and manganese-induced stimulation of phosphodiesterase activities.

Cytochemical demonstration of catecholamines and acetylcholinesterase activity in neuroblastoma cells in culture.

Cytochemical demonstration of catecholamines and acetylcholinesterase activity in neuroblastoma cells in culture.