Morphological differentiation of neuroblastoma cells induced by dimethylsulfoxide

Morphological features of neuroblastoma cells grown in culture in the presence of dimethylsulfoxode (DMSO) were studied. Morphological differentiation, expressed as the appearance of long axon-like processes (neurites), an increase in size of the cells, and inhibition of cell division, was observed in neuroblastoma cells of line C 1300, subline N-18-TG₂A₁, incubated in medium containing 1% DMSO. In the early stages of culture in normal growth medium the cells possess primary features of morphological differentiation. Quantitative criteria for the development of these features depending on duration of culture in modified medium were worked out. An increase in the total length of the neurites of cells differentiating under the influence of DMSO is a linear function of time. The rate of growth of the neurites is 20.0±3.0 µ/h. The area of cross-section of the soma of the differentiated cells is 6–7 times greater than the corresponding parameter in the control. An increase in the DMSO concentration in the culture medium (1.5 and 2.0%) does not induce rapid growth of the neurites or an increase in size of the cell soma, but it does block mitosis. Characteristics of morphological differentiation of neuroblastoma cells are compared with probable functional changes in these cells.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 519–527, July–August, 1984.     

Changes in surface charge of mouse neuroblastoma cells during growth and morphological differentiation of the cell population

The surface charge of neuroblastoma cells (clone C1300-N18TG₂) was studied by microelectrophoresis. The surface charge of these cells was shown to be determined mainly by anionic groups of the membrane, located in a layer about 10 mm thick, with a density of 0.2 e/nm³, covering its outer surface. These groups interact with Ca ions with a binding constant of K_Ca=10–50 liters/mole and titrate corresponding to pK=3.8. The electrophoretic mobility of the neuroblastoma cells is reduced by trypsin, neuraminidase, and N-bromosuccinimide, which irreversibly neutralizes carboxyl groups, and is increased on treatment of the cells with tosyl chloride — a specific reagent for amino groups. The value of the surface charge also depends on the conditions of culture of the cell population. The process of morphological differentiation of the cells (termination of division, dendrite formation), induced by removal of serum from the medium, leads to an increase of about 30% in their electrophoretic mobility. If cells are cultured in medium containing 10 and 50% of blood serum, enabling them to multiply, variations are observed in the mean electrophoretic mobility, which are opposite in phase to the 24-hourly increase in the number of cells. It is suggested that these effects are determined by partial "self-synchronization" of the cell population. It is concluded that the surface charge of neuroblastoma cells, measured by the microelectrophoresis method, is determined mainly by carboxyl groups of peripheral proteins and gangliosides of the membrane, and that the content of these compounds in the membrane depends on the phase of cell development.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR. Translated from Neirofiziologiya, Vol. 17, No. 2, pp. 168–174, March–April, 1985.     

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.     

Structural changes induced by diethyl ether in cultured neuroblastoma cells

It has been found that the more differentiated growing cells of a neuroblastoma culture retract their neurites under the action of diethyl ether (for anesthesia) in a dose of 1.0 ml per 15 ml of culture medium for 2 h; as a result, their cytoplasm is gradually drawn into the cell bodies. Under these conditions the coefficient K, which reflects the ratio of the number of neuroblastoma cell bodies to the number of their processes and is 9.94±2.12 in the initial state, gradually increases to 19.66±1.93 (P<0.002). There is also a substantial change in the shape of the cells and a decrease in their volume. This reaction is characteristic both of relatively undifferentiated neuroblastoma cells and for more differentiated cells in various phases of individual cellular development and morphological differentiation. The data obtained create the prerequisites for a search for drugs capable of preventing the destruction of neurons under anesthesia.A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. Kiev Institute for the Advanced Training of Physicians, Ukrainian Ministry of Health. Translated from Neirofiziologiya, Vol. 24, No. 3, pp. 279–285, May–June, 1992.     

Sodium and calcium channels in the somatic membrane of artificially differentiated neuroblastoma cells

Using an intracellular dialysis technique a study was made on calcium and sodium inward currents at the neuroblastoma somatic cell membrane in suspension and during the course of artificial morphological differentiation produced by raising the pH of the culture medium to 8.0–8.2. The density of sodium currents in the somata of cells cultured in the suspension averaged 7.3±0.8 µA/µF, while this value varied from 37±5.2 to 54.7±3.6 µA/µF at various stages of culture. These values equalled 1.4±0.2 and 1.1±0.2 to 2.8±0.4 µA/µF in the case of calcium currents. Reciprocal changes were produced in the density of sodium and calcium channels by altering the culture medium.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 207–214, March–April, 1986.     

Murine neuroblastoma cells absorb anti-cytoplasmic tetrodotoxin-sensitive protein antibodies

The properties were investigated of polyclonal antibodies obtained by immunizing with a fraction of cytoplasmic glycoproteins forming sodium channels in liposomes. It was shown that these antibodies can be absorbed by intact murine neuroblastoma cells. Graphs plotting intensity of absorption against numbers of absorbant cells follow a characteristic course dependent on life of the cells in culture and serum concentration in the culture medium.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 98–105, January–February, 1988.     

Role of glycoproteins in neuronal differentiation : Inhibition of neurite outgrowth and the major cell surface glycoprotein of murine neuroblastoma cells by a purified tunicamycin homologue

Mouse neuroblastoma cells in culture can be induced to differentiate morphologically by serum deprivation or by dibutyryl cyclic AMP (db-cAMP), e.g. they appear flattened, adhere more firmly to the culture substratum and extend long neuritic processes, and thus represent a widely used model system for neuronal cells. This differentiation is accompanied by modulation of cell surface components, such as the induction of a high molecular weight (HMW) glycoprotein (200 kD). We have studied the role of glycoproteins in the process of neuronal differentiation, using a purified homologue of the antibiotic tunicamycin (Al-tunicamycin) and neuroblastoma N115 cells grown in culture. Al-tunicamycin markedly inhibited (up to 60–75%) the incorporation of radioactively labelled sugars into cellular proteins of differentiating neuroblastoma cells. Concomitantly, the cells altered their morphology, they became rounded and less adhesive and retracted their neurites. Changes in the appearance, glycosylation and electrophoretic mobility of several cellular and secreted glycoproteins were observed, when cells were incubated in the presence of Al-tunicamycin. The most striking effect of Al-tunicamycin on the composition of cellular glycoproteins was the marked reduction in appearance of the 200 kD glycoprotein. The findings suggest that glycoproteins and in particular the neuron-specific 200 kD glycoprotein, are related to morphological differentiation processes, mainly to cellular adhesion and neurite outgrowth.     

Storage and growth of neuroblastoma cells immobilized in calcium-alginate beads

Summary Mouse neuroblastoma cells (N18) were immobilized in calcium-alginate gel beads. Under standard culture conditions (37° C; 5% CO₂), cell growth was observed inside the beads. The number of cells increased threefold during 7 days of culture with cell division and differentiation visualized by electron microscopy. Cell properties maintained after short-term storage (2–3 days at 4° C) included: (i) properties of voltage-dependent ionic channels tested by patch-clamp electrophysiological techniques; (ii) expression of cell-adhesion membrane proteins tested by immunohistochemistry (iii) morphological differentiation obtained by depletion of foetal calf serum in culture medium. The advantages of such an immobilization technique as applied to neurone cells are discussed. Offprint requests to: M. Simonneau     

α2-Interferon- and oligoadenylate-induced morphological differentiation and modulation of the ion channels in neuroblastoma cells

The experiments were carried out on the IMR-32 human neuroblastoma and NIE-115 murine neuroblastoma cultured cells. Peculiarities of the ion channel expression and their correlation with the main morphological parameters characterizing neuronal differentiation were studied under conditions of incubation of the cells with 2-interferon and 2,5-oligoadenylate, 2–5A. Twenty-four hours after addition of 1000 U act./ml interferon to the culture medium, a 56% increase in the mean projective surface of the IMR-32 cells was observed, and after a nine-day-long exposition this increase was 132%, as compared with the control. Mean total length of the cellular protrusions nearly doubled after nine-day-long incubation. Morphological and electrophysiological properties of the N1E-115 murine neuroblastoma cells showed practically no changes after incubation with human 2-interferon. Cultivation of the IMR-32 human neuroblastoma cells in the presence of 2–5A evoked insignificant changes in their morphological parameters. By contrast, the mean total length of neurites of the N1E-115 neuroblastoma protrusion-supplied cells increased more than a factor of five after eight-day-long incubation with 2–5A in a 1.0 µM concentration, and at a 0.01 µM 2–5A concentration this increase was about fourfold. At the same time, the projective surface exhibited no significant changes either in the neurite-supplied or the neurite-free cellular subpopulations. Twenty-four hours after the incubation with human 2-interferon had been begun, the density of sodium current in the IMR-32 human neuroblastoma cells increased by 250% compared with the control. A similar effect was observed after the addition of 2–5A to the medium: the density of sodium current approximately doubled. Cultivation of the neurite-supplied N1E-115 murine neuroblastoma cells was followed by a gradual increase in the density of fast sodium current both in control and 2–5A-influenced samples, but in the latter case this increase was significantly faster. In the neurite-free cells, the density of sodium current was 27% higher after their 24-h-long incubation with 2–5A, as compared with the control values (11.05±0.9 and 8.7±0.9 pA/pF, respectively). Longer incubation resulted in a sharp decrease in the density of potassium current. The results of our study are in agreement with the data about species-related individuality of 2-interferon and different intensity of its effects on the cells passing different stages of cellular differentiation.Neirofiziologiya/Neurophysiology, Vol. 27, No. 3, pp. 199–207, May–June, 1995.     

Decrease in the density of DNA topological turns during in vitro aging of Syrian hamster cells

Primary cultures of Syrian hamster embryonal fibroblasts were grown until the culture flask bottom was completely covered, then passed into a fresh medium with the dilution 1:2 and grown again until complete sealing of the bottom glass surface was achieved. The period required to attain cellular confluency and the density of topological turns (‘titratable superhelical turns’) in nuclear chromosomal superhelical DNA loops were measured in the course of cultivation. Embryonal cells of Syrian hamsters were found to undergo up to twelve culture doublings (passages) in a medium containing 10% bovine serum. The period required to reach confluency increased from 1–2 days for early passage cells, to 4–8 days for late passage cells, which were often able to reach confluency only after change of growth medium. The density of topological turns in nuclear DNA loops was found to decrease, from a range of −0.076 to −0.083 turns per 10 base pairs (bp) in cells of 1–3 passages, to −0.062 turns per 10 bp in cells of 11–12 passages. This decrease in density of DNA-topological turns may play a part in switching the activity of the genes responsible for cellular proliferation and differentiation in the course of aging of primary cell culture.     

The reaction of the neuroblastoma cells in the culture on the influence of tretionine and neurotoxine

Effect of the tretionine (retinoid) and aluminum chloride (neurotoxin) on the growth and differentiation of neuroblastoma cells in culture after their introduction into the medium separately and in combination was studied. The introduction of these substances creates a new information field in the medium, which becomes apparent by the reactions of neuroblastoma found on the populational and cellular levels of its organization. The presence of tretionine stimulates proliferation and induces differentiation of the cells into astrocytes. Aluminum chloride inhibits cell proliferation and enhances the process of their destruction in the monolayer. The variety of the reactions of neuroblastoma cells to the presence of these substances in the medium indicates the existence and functioning of a mechanism that selects from the information introduced only the portion which may contribute to adaptation of neuroblastoma cells to the changed culture conditions.     

Neuron-Like Differentiation of PC12 Cells Treated With Media Conditioned by Either Sciatic Nerves,Optic Nerves,or Schwann Cells

In previous works we reported the finding of neurotrophic activity in a serum-free Dulbeccos modified Eagles medium conditioned by rat sciatic nerves, previously maintained in culture for 11 days. This medium produces rapid neuron-like differentiation of cultured PC12 cells, as revealed by an increase in the size of the cell body and by the extension of short and/or long neurites by most of the cells. Neuregulin present in the conditioned medium was demonstrated to play a key role in the observed differentiation.In the present work, taking into consideration those latter results, the neurotrophic activity of conditioned media prepared with sciatic and optic nerves cultured during days 1–4 and 9–12 were studied.Evaluation of the trophic activities of those media revealed an opposite timing in the activities of sciatic and optic nerves conditioned media. The activity of the sciatic nerve was not observed in the 1–4-day period, increasing then up to the 9–12-day period. On the contrary, the optic nerve conditioned medium was active in the 1–4-day period, decreasing down to the 9–12-day period.These results led us to explore the contribution of the different cellular constituents of those nerves to their neurotrophic properties. As a first step in that direction we also investigated the neurotrophic activity of media conditioned during 12 days by cultured Schwann cells isolated from rat sciatic nerves. The Schwann cell conditioned media did produce a rapid differentiation of the PC12 cells similar to that caused by the sciatic nerve conditioned medium, though of a lower magnitude.Variations in the trophic activities of the conditioned media used in the present work is discussed taking into consideration the production of trophic and inhibitory factors by the peripheral and central glial cells. The role played by the optic nerve glia and myelin is being investigated at present.     

Morphological differentiation of a murine neuroblastoma clone in monolayer culture induced by dexamethasone

Dexamethasone induces morphological differentiation in murine neuroblastoma cells in culture. The percentage of differentiated cells depends on the concentration of dexamethasone in the medium and duration of treatment. After drug removal (with or without replating), many cells maintain their differentiated phenotype. Dexamethasone-treated cells have larger soma and contain more protein. Dexamethasone also produces a concentration-dependent inhibition of population growth. Growth inhibition is complete by 2 days following treatment with dexamethasone 50 micrograms/ml. Complete growth inhibition is achieved prior to the complete expression of morphological differentiation. Androstenedione, testosterone, and 17-beta-estradiol--all steroids without glucocorticoid activity--have no differentiating effect, although they inhibit growth or cause cell death at higher concentrations.     

Effect of verapamil on morphological differentiation and low-threshold calcium current of murine neuroblastoma cells

Changes in electrophysiological and morphological characteristics of N1E-115 murine neuroblastoma cells induced by their incubation in a medium containing verapamil, a blocker of high-threshold calcium channels, and/or increased concentrations of KCl were investigated. On the 3th day of cultivation with 1.0 µM verapamil, the cells showed morphological differentiation and increased density of low-threshold calcium current. Incubation of cells in a high-potassium solution (25 mM) produced inhibition of morphological differentiation and reduction of the density of this current. Morphological and electrophysiological parameters of cells cultivated in a high-potassium medium with verapamil added were close to those of control cells. Verapamil taken at a lower concentration (0.01 µM) did not affect the characteristics of calcium current and morphological differentiation of the cells. Higher concentrations of verapamil (100 µM) substantially suppressed viability of cells (a decrease in cells' projective area, retraction of cell processes, and sharp reduction of calcium current density were observed). The results are consistent with suppositions about functional significance of intracellular calcium level for cell differentiation.Neirofiziologiya/Neurophysiology, Vol. 27, No. 4, pp. 261–267, July–August, 1995.     

Metabolism of glycosaminoglycans in cultured smooth muscle cells from pig aorta

Arterial wall smooth muscle cells, originating from the inner layer (media) of pig aortas, were grown in culture. The synthesis and secretion of proteoglycans by these cells were investigated. These cells were incubated in the presence of [³⁵S] sulfate or [¹⁴C] glucosamine and these precursors incorporation into glycosaminoglycans was followed.Proteoglycans synthesized by media cells exhibit different glycosaminoglycan distribution patterns according to their localization. The glycosaminoglycan components are largely confined to the medium (80 per cent) and exhibit a distribution pattern that ressembles closely that found in pig aorta tissue. In comparison with the extracellular and intracellular pools, the pericellular pool (trypsin released material) contains proportionally more heparan sulfate.Isotopic chase experiments demonstrated that glycosaminoglycans leave the intracellular and pericellular compartments with initial half-lives of 7 – 8 h and 13 – 14 h, respectively.About half of the labelled glycosaminoglycans was released into the medium, in an apparently undegraded form, while the rest was degraded.The production of proteoglycans is not affected by modifying the exogenous concentration of hyaluronic acid or chondroitin sulfate present in the culture medium. The synthesis of proteoglycans, but not their secretion is inhibited with cytochalasin-B, a microfilament modifying agent. The secretion of proteoglycans and also — in part — their synthesis is inhibited by antimicrotubular agents: colchicine and vinblastine, with observed intracellular accumulation of proteoglycans.These data suggest that, in arterial cells, the intracellular movement of proteoglycans during the secretory process is mediated by microtubular elements.In conclusion, our results provide evidence for the responsiveness of cultured mediacytes to antimicrotubular and antimicrofilamentar drugs, the utilization of which allows modification in the metabolism and secretion of arterial proteoglycans.     

Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release

The death of the hybridoma VO 208 in a continuous culture at pH 7 and 6.8 was investigated by measuring both the appearance of visible dead cells which do not exclude the trypan blue dye and the release of lactate dehydrogenase (LDH) in the culture medium. The intracellular LDH was found to be completely released either when live cells lysed or when they were transformed into visible dead cells. No significant lysis of blue dead cells could be observed at the two different pH. Using a LDH balance over the culture system, cell lysis was found negligible at pH 7, but accounted for 20% of the total cell death at pH 6.8. A methodology is proposed to evaluate the rate constants of hybridoma lysis and total death. For the investigated cell line in continuous culture, the calculated total cell death rate constant was found to increase from 0.002 h^–1 to 0.01 h^–1 when decreasing the pH from 7 to 6.8.Abbreviations D dilution rate (h^–1) - k_b specific trypan-blue dead cells appearance rate (h^–1) - k_L specific lysis rate of viable cells (h^–1) - k_d specific death rate (h-1) - LDH₀ lactate dehydrogenase activity in the feed culture medium (IU.l^–1) - LDH lactate dehydrogenase activity in the outlet culture medium (IU.l^–1) - LDH_i intracellular lactate dehydrogenase activity of viable cells (IU.10^–9 cells) - r_LDH total rate of LDH release (IU.h^–1.L^–1) - r_b transformation rate of viable cells into blue dead cells (10⁹ cells.h^–1.L^–1) - x_v viable cell concentration (10⁹ cells.l^–1) - x_b trypan-blue dead cell concentration (10⁹ cells.l^–1)     

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.     

Surface glycosaminoglycans as a differentiation cofactor in neuroblastoma cell cultures.

The possible role of surface glycosaminoglycans (GAGs) in neuronal maturation occuring in neuroblastoma cultures has been investigated. GAGs of neuroblastoma cells, grown in suspension and monolayer, were labelled with 3H-glucosamine and 35S-sulfate. Neuron maturation, following cell adhesion to culture dishes, is accompanied by an increased ability of the cells to retain heparan sulfate (HS) on their surface, which is otherwise lost into the culture medium. The role of surface HS as a cofactor of cellular differentiation is discussed.     

Microenvironment Promotes Tumor Cell Reprogramming in Human Breast Cancer Cell Lines

The microenvironment drives mammary gland development and function, and may influence significantly both malignant behavior and cell growth of mammary cancer cells. By restoring context, and forcing cells to properly interpret native signals from the microenvironment, the cancer cell aberrant behavior can be quelled, and organization re-established. In order to restore functional and morphological differentiation, human mammary MCF-7 and MDA-MB-231 cancer cells were allowed to grow in a culture medium filled with a 10% of the albumen (EW, Egg White) from unfertilized chicken egg. That unique microenvironment behaves akin a 3D culture and induces MCF-7 cells to produce acini and branching duct-like structures, distinctive of mammary gland differentiation. EW-treated MDA-MB-231 cells developed buds of acini and duct-like structures. Both MCF-7 and MDA-MB-231 cells produced β-casein, a key milk component. Furthermore, E-cadherin expression was reactivated in MDA-MB-231 cells, as a consequence of the increased cdh1 expression; meanwhile β-catenin – a key cytoskeleton component – was displaced behind the inner cell membrane. Such modification hinders the epithelial-mesenchymal transition in MDA-MB-231 cells. This differentiating pathway is supported by the contemporary down-regulation of canonical pluripotency markers (Klf4, Nanog). Given that egg-conditioned medium behaves as a 3D-medium, it is likely that cancer phenotype reversion could be ascribed to the changed interactions between cells and their microenvironment.