Translocation of human glial and glioma cells in culture

Two normal glial lines and four selected malignant glioma lines--considered as representative of the varying morphologies previously found among these lines--were studied quantitatively as to their translocatory ability on glass under standard culture conditions. The two glial lines showed very similar characteristics with almost identical results for total and net translocation, as well as in their directional persistence. The glioma lines gave values for these criteria which were either greater or lower than those of the glial cells. These values could to a certain degree be related to phenotypic traits, such as degree of cell polarization and amounts of cytoplasmic microfilament bundles. The findings once again display the heterogeneity of the human malignant glioma lines and raise doubts as to the general applicability of findings in studies of single lines of tumor cells, or of experimentally transformed cells, to all malignant cells.     

Effects of N-monobutyryl-cyclic AMP on glutamate decarboxylase activity in fetal rat brain cells and glial tumor cells in culture

Cultures of fetal rat brain cells were treated chronically with 1 mM N⁶-monobutyryl-3′,5′-cyclic adenosine monophosphate and assayed at various times for protein and for activity of glutamate decarboxylase. Treated cultures increased their protein content very slowly, but showed a very early 2.5-fold stimulation of enzyme specific activity. This degree of stimulation persisted during temporal development of the enzyme activity. The stimulated levels of specific activity were nearly as great as those in adult rat brain homogenates. Similar treatment of cells from two rat gliomas, which also contain the enzyme activity, did not mimic the effect found in brain cultures. The effects on glutamate decarboxylase were very different from those on another enzyme of neurotransmitter synthesis, choline acetyltransferase, suggesting that different cell types may have been responsible for the two dissimilar effects of the treatment with the cyclic nucleotide.     

Biochemically differentiated clonal human glial cells in tissue culture

A clonal line of astrocytes (designated CHB) derived from a brain tumor of human origin has been established in tissue culture. This cell line is capable of synthesizing an acid protein unique to the nervous system (S100-protein). The S100-protein synthesized by CHB cells is immunologically indistinguishable (by micro-complement fixation) from S100-protein present in human brain.     

Occurrence of phenolsulfotransferase in primary glial culture cells of rat

Phenolsulfotransferase (PST) activity towards phenol and monoamines was determined in rat brain and in primary cultures of rat astrocytes. The pH requirement.K _m values and the proportion of PST activity with respect to phenol and dopamine as substrates were similar between PST from the glial cells and the rat cortex. The enzyme activity increased with age in the brain of older animals, and also with increasing incubation time in the primary culture of astroglia. The specific PST activity of the astroglia appeared to be higher than that of the brain enzyme. In glial cultures treated with 0.25 mM dibutyryl cyclic AMP in the same culture conditions, PST activity is suppressed to about 25% of its untreated counterpart, even though dibutyryl cyclic AMP at concentrations of 1 mM only slightly inhibited PST activity in vitro.     

Relation of C-6 glial cells in culture to myelin.

C-6 glial cells were shown to contain enriched in their plasma membranes an enzyme, 2':3'-cyclic nucleotide 3'-phosphohydrolase, characteristic of myelin, and, in addition, proteolipid protein and two basic proteins that are identical in their electrophoretic mobilities with the respective proteins found in myelin. The data indicate that C-6 cells exhibit features of myelin-producing glia as well as astrocytes.     

Selenoprotein W in overexpressed and underexpressed rat glial cells in culture

Selenium deficiency results in undetectable levels of selenoprotein W (SeW) in muscle but has very little effect upon its content in the brain and thus rat glial cells were studied. Previous work showed that glutathione (GSH) is bound to SeW and this study was undertaken to elucidate its possible antioxidant functions. Full length cDNA of SeW was cloned to inducible LacSwitch expression vector and stably transfected in C6 rat glial cells. After induction, SeW and its mRNA were expressed 22- and 11-fold higher respectively than control. The cDNA coding region of SeW was cloned to the vector in the antisense direction and stably transfected in C6 cells for underexpression of the protein. After induction, SeW expression was reduced to 20% of the control cells. Glutathione peroxidase activity and GSH levels were not significantly different between induced and control cells. There was a greater survival rate of overexpressed than control cells when incubated with 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH), suggesting SeW possibly has an antioxidant function.     

Release of nerve growth factor by human glial cells in culture

Non-transformed human glial cells obtained from brain biopsies (lines U-787 CG, U-1169 CG and U-1508 CG) release to their culture medium a factor which, in bioassay, induces neurite outgrowth in spinal and sympathetic embryonic chick ganglia. The neurite-stimulating activity, which was enhanced after pressure dialysis of glial-conditioned medium, is inhibited by specific antiserum prepared to mouse βnerve growth factor (NGF). The glial factor was partially purified by gel filtration on Sephadex G-200 of concentrated, serum-containing, conditioned medium. The activity eluted close to a molecular weight of 30000, as did mouse NGF run under identical conditions. Ion-exchange chromatography on DEAE-Sepharose CL-6B and flat-bed electrofocusing of conditioned medium showed the activity to be associated with a heat-labile entity having an isoelectric point of about 4.1. All purified preparations were blocked by anti(mouse)-βNGF. The results demonstrate the existence of a human glial NGF which in several respects resembles the mouse submandibular gland NGF.     

Phagocytotic and iron-storing capacities of stromal cells in the rat endometrium

Summary Cytoplasmic pigment inclusions of rat endometrial stromal cells were studied by histology, histochemistry, fluorescence microscopy, electron microscopy and X-ray microprobe analysis. It is shown that a number of endometrial perivascular stromal cells contain numerous free cytoplasmic ferritin particles as well as hemosiderin vacuoles. The larger pigment inclusions reveal also positive PAS- and Schmorl reactions indicating that they contain polysaccharide and lipofuscin material, respectively. These pigmentstoring stromal cells also display acid phosphatase activity; they avidly phagocytose instillated latex particles. No pigment-storing cells occur within the surface or glandular epithelium, either in the basal endometrium or in the myometrium.It is demonstrated that the endometrial iron-storing cells function as iron depots; they take part in the phagocytosis and endocytosis of extracellular tissue components and therefore can be named phagocytes. Our data show that fibroblastoid endometrial stromal cells may differentiate into endometrial resident phagocytes, which ensure interstitial proteolysis and hence facilitate the drainage of extracellular fluid into the venous blood capillaries.Supported by a grant from the Nationaal Fonds voor Wetenschappelijk Onderzoek — Fonds voor Geneeskundig Wetenschappelijk Onderzoek, Belgium.     

Biological effects of bovine glia maturation factor on glial cells in culture

Optimal bioassay conditions for bovine glia maturation factor (GMF) were determined among glial cells from normal glioblasts to glioma cells. Rat glioblasts 4–8 days after subculture show the highest response to GMF with regard to morphological transformation and mitogenic activity. Bovine GMF enhances DNA synthesis of rat glioblasts at 12 hr after stimulation; maximum incorporation of [methyl-³H]thymidine was detected at 18 hr. GMF increases twofold the saturation density of rat glioblasts but does not alter that of C6 astrocytoma cells. The apparent inhibition of mitogenic activity of high doses of GMF is seen in both normal and malignant glial cells.     

Early and late passage C-6 glial cell growth: Similarities with primary glial cells in culture

Earlier studies in our laboratory have shown that C-6 glial cells in culture exhibit astrocytic properties with increasing cell passage. In this study, we tested the responsiveness of early and late passage C-6 glial cells to various cultures conditions: culture substrata (collagen, poly-L-lysine, plastic), or supplements for the culture medium, DMEM, [fetal calf, or heat inactivated (HI) serum, or media conditioned from mouse neuroblastoma cells (NBCM) or primary chick embryo cultured neurons (NCM)]. Glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP), astrocytic and oligodendrocytic glial markers, were used. Cell numer and protein content increased exponentially with days in culture regardless of the type of the substratum or cell passage. Differences in cell morphology among the three types of substratum were also reflected on GS activity, which rose by three-fold on culture day 3 for cells grown on collagen; thereafter, GS profiles were similar for all substrata. This early rise in GS is interpreted to reflect differential cell adhesion processes on the substrata; specifically, cell adhesion on the collagen stimulated differentiation into astrocytic phenotype.Analogous to immature glia cells in primary cultures, early passage C-6 glial cells responded to neuronal factors supplied either from NCM or NBCM by expressing reduced GS activity, the astrocytic marker and enhanced CNP activity, the oligodendrocytic marker. Thus, early passage cells can be induced to express either astrocytic or oligodendrocytic phenotype. In accordance with our previous reports on primary glial cells, late passage C-6 cells exhibit their usual astrocytic behavior, responding to serum factors with GS activity. Moreover, whereas NCM or NBCM alone markedly lowered GS activity, a combination with serum restored activity. The present findings confirm our previous observations and further establish the C-6 glial cells as a reliable model to study immature glia.Special issue dedicated to Dr. Paola S. Timiras.     

Behaviour of neuroblasts in the presence of glial cells, fibroblasts and meningeal cells in culture

Dissociated nerve cells from 7-day-old chick embryo cerebral hemispheres were cultivated on plastic surfaces, astroblast layers, fibroblast layers and meningeal cell layers. The cell suspensions obtained by mechanical dissociation and plated on these layers contained primarily neuroblasts. The neuroblasts cultured on astroblast layers behaved differently from those cultured on fibroblast or on meningeal cell layers. They adhered within 2 h to the preformed astroblast monolayers and remained scattered over it. In contrast, in the two other cases, the neuroblasts formed floating aggregates which adhered to the layers only after 24 h. Neuroblasts behaved on monolayers made of fibroblasts or meningeal cells as on plastic surfaces.The neuronal cells grown on astroblast layers were much more differentiated than those plated on plastic or on the two other layers studied. After 2–3 weeks of culture the neurons were large and the fibres were longer, thicker and more ramified. However, the fibroblast and the meningeal cells enhanced slightly the growth of the neuroblasts relative to plastic surfaces. These results support the possibility of specific interactions between astroblasts and neuroblasts.     

Alcohol inhibits morphological and biochemical differentiation of C6 glial cells in culture

Cultures of glial cell lines (C6) were exposed to 10-micromilligram Dexamethasone which is known to cause morphological differentiation and induction of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) in these cells. Ethanol in a concentration of 1.5% abolished these responses, and at 1% diminished them.     

Acid sphingomyelinase activity triggers microparticle release from glial cells

We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL-1β, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X₇, microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATP-induced shedding and IL-1β release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1β release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1β release, thus, opening new strategies for the treatment of neuroinflammatory diseases.     

Uptake and metabolism of choline in primary isolated neurons and glial cells in culture

The influx and metabolism of choline have been studied in primary cultures of isolated neurons and glial cells from chick embryo dissociated cerebral hemispheres. The results showed a correlation between both influx and metabolism of choline and the exogenous concentrations of choline. When neurons and glial cells were preincubated (10 min) and incubated in Krebs-Ringer phosphate solution with concentrations of choline lower (0.5 μM) or higher (150 μM) than the one present in the growth medium, the metabolism of choline, as a function of time, approached saturation following unusual kinetics. This suggests a non steady state of the endocellular concentrations of free choline. Moreover, when both neurons and glial cells were preincubated (10 min) with 50 μM choline and then incubated (2 min) with various concentrations of choline, only one uptake mechanism was measured, while the preincubation in the absence of choline followed by the incubation of the cells with various concentrations of choline showed the presence of two apparent K_m's with different affinities.The results also indicate the capacity of glial cells to incorporate choline suggesting a storage function for the cells.