Phosphatase activity in cultured glioma and neuroblastoma cells

Acid phosphatase activity in human glioma cells (138 MG) and mouse neuroblastoma cells (C 1300) was associated with structures accumulating neutral red and acridine orange. Only neuroblastoma cells gave a significant positive histochemical reaction for alkaline phosphatase. Glioma and neuroblastoma cell homogenates exhibited maximal phosphatase activity at pH 5 as measured by spectrophotometer. The specific activity; μmoles phosphate released per hour/mg protein was 1.1 in glioma and 0.9 in neuroblastoma. At pH 8, glioma cells lacked activity whereas neuroblastoma cells showed another maximum. The acid phosphatase activity of both cell types was strongly inhibited by CuCl₂ (0.3 mM) and NaF (10 mM) and moderately by -tartaric acid (10 mM). cGMP (1 mM) stimulated the phosphatase activity of both cell lines. db-cAMP, in serum-free medium, induced characteristic morphological changes of the cells studied. This process was unaffected by CuCl₂, c-GMP and -tartaric acid. db-cAMP (1 mM) inhibited proliferation in both glioma and neuroblastoma cells during a 48 h incubation in serum-containing medium. This growth inhibition was associated with an increase in acid phosphatase activity of the glioma but not of the neuroblastoma cells.     

Enhancement of BDNF production by co-cultivation of human neuroblastoma and fibroblast cells

It has been proved that co-cultivation of human neuroblastoma cells and human fibrolast cells can enhance nerve cell growth and the production of BDNF in perfusion cultivation. In batch co-cultivation, maximum cell density was increased up to 1.76×10⁶ viable cells/mL from 9×10⁵ viable cells/mL of only neuroblastoma cell culture. The growth of neuroblastoma cells was greatly improved by culturing both nerve and fibroblast cells in a perfusion process, maintaining 1.5×10⁶ viable cells/mL, which was much higher than that from fed-batch cultivation. The nerve cell growth was greatly enhanced in both fed-batch and perfusion cultivations while the growth of fibroblast cells was not. It strongly implies that the factors secreted from, human fibroblast cells and/or the environments of co-culture system can enhance both cell growth and BDNF secretion. Specific BDNF production rate was not enhanced in co-cultures; however, the production period was increased as the cell growth was lengthened in the co-culture case. Competitive growth between nerve cells and fibroblast cells was not observed in all cases, showing no changes of fibroblast cell growth and only enhancement of the neuroblastoma cell growth and overall BDNF production. It was also found that the perfusion cultivation was the most appropriate process for cultivating two cell lines simultaneously in a bioreactor.     

Lipopolysaccharide treatment modifies pH- and cation-dependent ecto-ATPase activity of endothelial cells.

The aim of this study was to investigate the distribution pattern of Ca2+- and Mg2+-dependent ecto-ATPases on the surface of rat brain capillary endothelial cells (ECs) in control and lipopolysaccharide (LPS)-treated animals. Ecto-ATPases in the membrane of vascular endothelial cells are suggested to play a crucial role in thromboregulation. Loss of this enzyme activity after oxidative stress and upregulation of the enzyme chain hydrolyzing extracellular ATP after transient forebrain ischemia have also been reported. We used histochemistry to localize the activities of this enzyme on ECs and found pH- and cation-dependent changes in the localization of enzyme activity both in control and in LPS-treated animals. These findings suggest the presence of more than one ecto-ATPase enzyme on the surface of rat capillary ECs. The different behavior of ECs after LPS treatment is the target of further investigations. The increased ecto-nucleotidase activity might play a role in nucleotide-mediated cellular responses after bacterial infections.     

Turnover of succinyl-CoA:3-oxoacid CoA-transferase in glioma and neuroblastoma cells. Specific influence of acetoacetate in neuroblastoma cells.

The specific activity of succinyl-CoA:3-oxo-acid CoA-transferase (3-oxoacid CoA-transferase, EC 2.8.3.5) increases significantly during growth in culture in both mouse neuroblastoma N2a and rat glioma C6 cells. To investigate the mechanism(s) responsible for this, antibody specific for rat brain 3-oxoacid CoA-transferase was raised in rabbits. Immunotitrations of 3-oxoacid CoA-transferase from neuroblastoma and glioma cells on days 3 and 7 of growth after subculture showed that the ratio of 3-oxoacid CoA-transferase activity to immunoprecipitable enzyme protein remained constant, indicating that differences in specific activity of the enzyme at these times in both cell types reflect differences in concentration of enzyme protein. In glioma cells, the relative rate of 3-oxoacid CoA-transferase synthesis was about 0.04-0.05% throughout 9 days in culture. In contrast, the relative rate of synthesis of 3-oxo-acid CoA-transferase in neuroblastoma cells was about 0.07-0.08% on days 3, 5 and 7 after subculture, but fell to 0.052% on day 9. The degradation rates of total cellular protein (t1/2 = 28 h) and 3-oxoacid CoA-transferase (t1/2 = 46-50 h) were similar in both cell lines. The rise in specific activity of the enzyme in both cell lines from days 3 to 7 without a significant increase in the relative rate of synthesis reflects a slow approach to steady-state conditions for the enzyme secondary to its slow degradation. Differences in 3-oxoacid CoA-transferase specific activity between the two cell lines are apparently due to a difference of about 60% in relative rates of enzyme synthesis. The presence of 0.5 mM-acetoacetate in the medium significantly increased the specific activity of 3-oxoacid CoA-transferase in neuroblastoma cells during the early exponential growth phase. This treatment increased the relative rate of synthesis of 3-oxoacid CoA-transferase by 23% (P less than 0.025) in these cells on day 3, suggesting that substrate-mediated induction of enzyme synthesis is a mechanism of regulation of 3-oxoacid CoA-transferase.     

The effect of hydrogen peroxide upon beta-adrenoceptor density and function in C6 rat glioma cells

Hydrogen peroxide has been suggested to play an important role in the pathogenesis of Alzheimer's disease. In the present study, the effects of hydrogen peroxide upon the functional integrity of beta-adrenoceptors have been investigated in C6 glioma cells. Treatment of cells for 24 h with hydrogen peroxide in serum-free medium produced a concentration-dependent cell toxicity, seen both using cell counting and LDH release into medium as end point. There were no large nor consistent changes in either the density of cell surface beta 1, or beta 2-adrenoceptors, measured using the hydrophilic ligand [3H](-)-CGP 12177, nor in either basal, forskolin and isoprenaline-stimulated cAMP responses, following hydrogen peroxide treatment. It is concluded that the decreased adenylyl cyclase activity and responsiveness to Gs stimulation found in post-mortem brain samples from Alzheimer's disease autopsy cases is unlikely to be mediated by hydrogen peroxide.     

Differences in surface membrane ecto-ATPase and ecto-AMPase in normal and malignant cells. I. Decrease in ecto-ATPase in myeloid leukemic cells and the independent regulation of ecto-ATPase and ecto-AMPase.

The hydrolysis of ATP and AMP by enzymes located on the external side of the plasma membrane (ecto-ATPase and ecto-AMPase) was studied in mouse myeloid leukemic cells, normal early myeloid cells, and normal mature granulocytes and macrophages. Nine clones of myeloid leukemic cells were used belonging to three groups that differ in their ability to be induced to differentiate by the differentiation-inducing protein MGI. These three groups consisted of MGI+D+ that can be induced to undergo complete differentiation, MGI+D- that can be induced to partially differentiate and MGI-D- with no induction of differentiation. The ecto-ATPase activity of normal early myeloid cells was similar to that of normal mature granulocytes and macrophages and higher than that of any of the leukemic cells. Among the leukemic cells, the MGI-D- cells had the highest level of ecto-ATPase activity. The behaviour of ecto-AMPase differed from that of ecto-ATPase. Some MGI-D- clones had a higher ecto-AMPase activity than normal cells and MGI+D- and MGI+D+ cells showed no detectable activity. Neither the ecto-ATP-ase nor ecto-AMPase activities changed after induction of differentiation in normal early myeloid or MGI+D+ leukemic cells. The results indicate that the myeloid leukemic cells had a decreased ability to hydrolyse external ATP, that there can be an independent regulation of ecto-ATPase and ecto-AMPase and that neither of these enzyme activities changed during differentiation.     

Differences in metabolite levels upon differentiation of intact neuroblastoma X glioma cells observed by proton NMR spectroscopy

Proton NMR spectroscopy was used to study the effect of differentiation with prostaglandin E1 and theophylline on intact hybrid neuroblastoma X glioma cells. The standard proton NMR method showed more resolvable signals than the spin echo NMR spectra. The differentiated cells were found to contain significantly higher levels of glutamine than the undifferentiated precursors. Observations on cell extracts confirmed these results.     

Metabolism of biogenic amines in neuroblastoma and glioma cells in culture

The kinetic parameters of monoamine oxidase (MAO; E.C 1.4.3.4) and catechol-O-methyltransferase (COMT; EC 2.1.1.6) were evaluated in extracts of adrenergic and non-adrenergic mouse neuroblastoma cells and in rat glioma cells. Using the naturally-occurring substrates tyramine, tryptamine, serotonin and norepinephrine, the affinity of MAO for a given substrate was independent of the presence of the catecholaminergic pathway or cell type used, with apparent Km values ranging from 8-14 microM for tryptamine to 510-580 microM for norepinephrine. The MAO activity in glioma cells was substantially greater than in either neuroblastoma clone, but Vmax values varied little with substrate among cell lines. Both the neuronal and glial COMT had a similar Km for 1-norepinephrine (200 microM); the corresponding Vmax values were also similar among the different cell lines, but represented only 2-10% of the maximal MAO activity. Neuroblastoma and glioma cells, when grown from early logarithmic to stationary phase, showed no significant changes in specific activity of either MAO or COMT. Growth of cells for 3 days with 1 mM-N6,O2'-dibutyryl adenosine-3',5'-cyclic monophosphate resulted in no marked change in either MAO or COMT activity. These results suggest that in neurons neither MAO nor COMT plays a major role in the type of transmitter inactivation that is analogous to that of acetylcholinesterase in cholinergic synapses. The occurrence of considerable MAO and acetylcholinesterase activities in glioma cells may indicate a role for these cells in neurotransmitter inactivation.     

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.     

Desensitization of opioid-stimulated GTPase in neuroblastoma x glioma hybrid cells

NG108-15 cells were pretreated with the opioid peptide [D-Ala2, D-Leu5]enkephalin and the opioid-dependent low Km GTPase was assayed in membranes. Pretreatment resulted in a small decrease in basal GTPase activity and led to a concentration-dependent reduction in opioid-mediated stimulation of the enzyme. These effects were observed whether the agonist was present or absent throughout all the experimental procedures, but, in the second condition, the desensitization was smaller. The addition of naloxone had no effect on basal GTPase activity, in either control or pretreated cell membranes. Both Na+ and Mg++ were required for the opioid-induced stimulation of the GTPase. Mg++ enhanced basal enzymatic activity in controls, whereas in membranes from pretreated cells, it produced an inhibition. Thus, desensitization of the opioid-dependent low Km GTPase occurs upon chronic opioid treatment and a Mg++ regulatory site might be altered in the course of this process.     

Internalization of ecto-ATPase activity in human neutrophils upon stimulation with phorbol ester or formyl peptide

 We have demonstrated the alteration of the localization of ecto-ATPase activity in human neutrophils after stimulation with phorbol myristate acetate or N-formylmethionyl-leucyl-phenylalanine using a cerium-based cytochemical method. In unstimulated cells, the enzyme activity was observed on the plasma membrane. Both the diazonium salt of sulfanilic acid and diethylpyrocarbonate inhibited the production of the reaction precipitates. Within 2–3 min of stimulation, cells developed cytoplasmic projections (ruffles). The ecto-ATPase activity on the plasma membrane of these ruffles was, however, weaker than that at the non-ruffle-forming side. The ruffle-forming side (RFS) was also the site where elongated tubular structures positive for the enzyme reaction tended to concentrate and associated with the plasma membrane. The enzyme activity was also detected in intracellular compartments, which appeared predominantly in the RFS concomitantly with the disappearance of the enzyme activity from the plasma membrane. Using a series of thick sections and computer-assisted three-dimensional reconstruction, the enzyme reaction-positive internalized membranes were visualized as a complicated mass formed by enzyme reaction-positive vesicles which gathered together and were, at least in part, interconnected. The present results indicate that the detected enzyme reaction is a product of the ecto-ATPase activity, and that RFS possibly serves the membrane flow with respect to endocytosis. Accepted: 25 February 1997     

Phosphoinositides and synaptic function in NG108-15 neuroblastoma x glioma hybrid cells.

1. The second-messengers system of bradykinin (BK) receptors was examined in NG108-15 neuroblastoma x glioma hybrid cells. 2. An application of BK induced an immediate outward (K+) current and acetylcholine (ACh) release, which are generated through inositol 1,4,5-trisphosphate (InsP3)-dependent Ca2+ ions. 3. Application of phorbol dibutyrate (a protein kinase C activator) produced a voltage-dependent inward current and inhibited another K+ (M)-current. 4. A similar current response has been produced by ACh in NG108-15 cells transfected with rodent muscarinic ACh receptor I and III subtype genes. 5. These results suggest a dual and time-dependent role for these two intracellular messengers in the control of neuronal signalling by BK and ACh.     

Metabolic consequences of drug-induced inhibition of the pentose phosphate pathway in neuroblastoma and glioma cells.

6-Aminonicotinamide leads to a considerable accumulation of 6-phosphogluconate, which is 3 times higher in C-6 glial cells than it is in C-1300 neuroblastoma cells. Dephosphorylation of the accumulated 6-phosphogluconate causes a rise of intracellular gluconate, which can be released from the cells. The higher dephosphorylating capacity of neuroblastoma cells leads to an intracellular gluconate content which is 4 times that found in C-6 glial cells. Although 6-phosphogluconate is a potent competitive inhibitor of glucose phosphate isomerase, no reduction of glycolytic flux and ATP content in stationary phase neuroblastoma cells was found in contrast to observations in C-6 glial cells. Morphological changes are only found in C-6 glial cells during the experimental period.     

Density-dependent inhibition of 2-deoxy-d-glucose uptake into glioma and neuroblastoma cells in culture

The transport of 2-deoxy-d-glucose (2-DG) into cultured human glioma (138 MG) and mouse neuroblastoma (C1300) cells has been studied in relation to the growth curves of the cells. An initial increase in the uptake of 2-DG into exponentially growing 138 MG cells, could be attributed to the number of days the cells were kept in culture rather than to their increased density. As the 138 MG cells reached confluency after 3 days the 2-DG uptake became density-dependent inhibited. In still denser cultures the cell growth was inhibited. This was accompanied by morphological ‘normalization’ of the cells and increased uptake of 2-DG. Uptake of 2-DG into C1300 cells was density-dependent inhibited throughout the cell growth cycle. As the cell density increased from 15 × 10³ to 130× 10³ cells/cm² the rate of uptake/cell decreased to one-fourth. At the latter cell density the cells entered stationary phase, without any major changes in morphology. The results suggest that spontaneously occurring tumour cells, such as glioma and neuroblastoma cells, can regulate the sugar transport in relation to cell density. This could be due to newly-acquired differentiated properties of the cells or to true contact-inhibitory phenomena.     

Ultrastructural changes in differentiating neuroblastoma × glioma hybrid cells

The ultrastructure of differentiating neuroblastoma × glioma hybrid cells NG108-15 was observed. Cells cultured in growth medium showed undifferentiated features, while cells treated with dBcAMP became round and large, and extended thick long neurites. After 1 week in culture, cells showed features similar to those of normal neurons. The dense cored vesicles with diameters ranging from 60 to 170 nm were observed in differentiated NG108-15 cells, but clear vesicles were usually rare. However, in the case of co-culture with striated myotubes, clusters of clear vesicles appeared in the neurites and terminals. The timecourse of the differentiation process was correlated with results obtained by the electrophysiology and freeze-fracture.     

The effect of explosive blast loading on human neuroblastoma cells

Diagnosis of mild to moderate traumatic brain injury is challenging because brain tissue damage progresses slowly and is not readily detectable by conventional imaging techniques. We have developed a novel in vitro model to study primary blast loading on dissociated neurons using nitroamine explosives such as those used on the battlefield. Human neuroblastoma cells were exposed to single and triple 50-psi explosive blasts and single 100-psi blasts. Changes in membrane permeability and oxidative stress showed a significant increase for the single and triple 100-psi blast conditions compared with single 50-psi blast and controls.     

The effect of medium pH on rate of growth, neurite formation and acetylcholinesterase activity in mouse neuroblastoma cells in culture.

Cell division, neurite formation and acetylcholinesterase activity were examined in a clone (NBA2) of mouse neuroblastoma cells maintained for up to 120 hours in medium with pH values between 6.6 and 8.0. Growth rate decreased as pH was reduced from 7.8 to 6.6. Generation time at pH 7.4 was 25 hours, while the rate of cell division was negligible at pH 6.6. The total number of cells at stationary phase was less at the lower pH values. Neurite formation was enhanced markedly as the pH was reduced from 7.4 to 6.6. Acetylcholinesterase activity was 5- to 8-fold greater in cells exposed to medium at pH 6.6 than in cells maintained in medium at pH 7.4. The reduction in the rate of cell division and increases in neurite formation and acetylcholinesterase activity at pH 6.6 were reversible upon exposure of the cells to pH 7.4 medium. Cell viability was greater than 90% at all medium pH values over a period of 120 hours. Uncloned T-59 mouse neuroblastoma cells were affected similarly by changes in pH. These results show that manipulation of the environmental pH can reversibly alter growth, neurite formation, and acetylcholinesterase activity of mouse neuroblastoma cells in culture.