Cultured Rat Glioma Cells Research Articles

13C-labelling studies indicate compartmentalized synthesis of triacylglycerols in C6 rat glioma cells

NMR-visible mobile lipid (ML) signals have been detected in 1H-NMR spectra of tissues in vivo, ex vivo and in vitro, and have been shown to change in apparent intensity in association with pathology (necrosis in brain tumours) and normal processes (cell differentiation, cell growth arrest and apoptosis). Although it is widely accepted that ML signals originate mainly from fatty-acyl chains in triacylglycerols (TAG) contained in cytosolic lipid droplets (LD), the dynamics of TAG in LD is not yet fully understood. In order to better understand the synthesis of cellular TAG and its relationship to ML dynamics we carried out a set of labelling experiments with C6 rat glioma cells in culture. TAG and phospholipid metabolism was monitored by incubating C6 cells with [1- 13C]-glucose at two time points during cell growth curve –24 h incubation starting at log-phase; 48 h incubation starting at saturation density– and by acquiring the 2D-HMQC NMR spectra of the respective total lipid extracts. The resulting TAG, diacylglycerol (DAG) and phospholipid labelling patterns can only be explained if TAG synthesis takes place in two different subcellular compartments. One compartment would be the endoplasmic reticulum, which is known to be involved in TAG metabolism, while the other compartment could be the plasma membrane and/or the LD. This possible role of LD is further supported by the recent description of diacylglycerolacyltranferase-activity associated with LD. Accordingly, we postulate the existence of a carbon-shuttling mechanism between plasma membrane phospholipids and endoplasmic reticulum by way of LD content. The results we have obtained with C6 cells may also apply to other cellular systems and should be taken into account when interpreting ML dynamics detected by NMR in vivo.

A comparative study of ectonucleotidase and P2 receptor mRNA profiles in C6 cell line cultures and C6 ex vivo glioma model

Glioblastoma multiforme is the most common type of primary brain tumour and has the worst clinical outcome. Nucleotides represent an important class of extracellular molecules involved in cell proliferation, differentiation and apoptosis. Alterations in purinergic signalling have been implicated in pathological processes, such as cancer, and glioma cell lines are widely employed as a model to study the biology of brain tumours. Increasing evidence, however, suggests that glioma cell lines may not present all the phenotypic and genetic characteristics of the primary tumours. We have compared the biological characteristics of C6 rat glioma cells in culture and the same cells after their implantation in the rat brain and growth in culture (denominated as the C6 ex vivo culture model). Parameters evaluated included cell morphology, differentiation, angiogenic markers, purinergic receptors and ecto-nucleotidase mRNA profile/enzymatic activity. Analysis of the C6 glioma cell line and C6 ex vivo glioma cultures revealed distinct cell morphologies, although cell differentiation and angiogenic marker expressions were similar. Both glioma models co-expressed multiple P2X and P2Y receptor subtypes with some differences. In addition, the C6 glioma cell line and C6 ex vivo glioma cultures exhibited similar extracellular ATP metabolism and cell proliferation behaviour when exposed to cytotoxic ATP concentrations. Thus, the disruption of purinergic signalling is a feature shown not only by glioma cell lineages, but also by primary glioma cultures. Our results therefore suggest the participation of the purinergic system in glioma malignancy.

Differentiation-inducing activity of neomycin in cultured rat glioma cells

Induction of cellular differentiation is an attractive therapeutic strategy against glioma cell proliferation and tumorigenicity. Preliminary in vitro studies have indicated that neomycin inhibits the proliferation of cultured glioma cells and induces changes in cellular morphology, making it potentially useful as a therapeutic agent for gliomas. The purpose of this work was to expand on the preliminary research by investigating the differentiation effect of neomycin in rat C6 glioma cells, using glial fibrillary acidic protein (GFAP) staining as a reliable marker of differentiation for normal astrocytes and for tumors of astrocytic lineage. Cell cultures were grown in the absence or presence of 10 mM neomycin sulfate for 48 hours. Neomycin treatment produced changes in cell morphology and GFAP expression indicative of cellular differentiation. These results suggest that neomycin is an attractive differentiation agent for the treatment of gliomas.

Cell Culture Studies of a Carborane Cholesteryl Ester with Conventional and PEG Liposomes

A new cholesterol-carborane conjugate (BCH) has been synthesized as a potential targeting agent for boron neutron capture therapy (BNCT) of cancers. The compound is extremely water insoluble and was formulated in two liposomal formulations to determine if the compound could be adequately taken up by 9L rat glioma cells in cell culture. Several factors potentially affecting the cellular uptake were evaluated, such as concentration of BCH in the incubation medium, incubation time, cell confluence, and the addition of polyethylene glycol (PEG) phospholipids to the liposomal formulation. The studies indicated that the cellular uptakes of BCH in the conventional and PEG liposomal formulations were 49.1 and 45.9 μg boron/g cells, respectively. Therefore, this compound, formulated in both liposomal formulations, delivered sufficient levels of boron to cancer cells in vitro, indicating that BCH is a promising approach for use in BNCT. The uptake appeared to depend upon BCH concentration in the media as well as the confluence of the cells. The greater boron uptake by nonconfluent cells indicated that active growth of cells was a factor in the uptake of this compound.

Brain contains inhibiting factors specific to the large T-antigen gene

SV40 large T-antigen (LTa) gene-induced immortalized rat dopamine-producing nerve cells (1RB 3AN 27), which produce LTa protein and divide in vitro, do not divide and do not produce LTa protein when transplanted into striatum of adult rats. This suggests the presence of LTa gene-inhibiting factors in brain. Here we report that rat brain soluble fraction (SF) contains factors which specifically inhibit LTa gene activity in vitro. The brain SF inhibited LTa protein levels and the growth of 1RB 3AN 27 cells and 2RSG cells (LTa gene-induced immortalized rat parotid acinar cells) in vitro, but it stimulated the growth of spontaneously immortalized human parotid acinar cells (2HPC8) and had no effect on the proliferation of murine neuroblastoma cells (NBP 2) and rat glioma cells (C-6) in culture. In contrast, the liver SF inhibited the growth of all cell lines tested at varying degrees and thus lacked specificity with respect to LTa gene activity. The presence of specific LTa gene-inhibiting factors in the brain and general tumor growth-inhibiting factors in the liver may provide some of the mechanisms of protection against in vivo carcinogenesis.

Proliferative potential and apoptosis in rat glioma cell lines after hyperthermia.

The proliferative potential of cultured rat glioma cells (C6 and 9L) was evaluated after hyperthermia using immunohistochemical staining with bromodeoxyuridine (BrdU) and Ki-67 monoclonal antibodies. Apoptosis was assessed by in situ end-labeling of deoxyribonucleic acid breaks. Both BrdU and Ki-67 labeling indexes decreased with increasing hyperthermia time. The decrease of the Ki-67 labeling index was not as great as that of the BrdU labeling index. The number of apoptotic cells increased with time after hyperthermia. These results indicate that the antitumor effect of hyperthermia may reflect the induction of apoptosis in the cells within the cell cycle, and the resultant reduction of the proliferative potential of surviving cells, especially in the S phase.

Inhibitory Effect of (−)-Epicatechin 4-Benzylthioether on the Growth of Glioma Cells in Culture

The effect of (-)-epicatechin 4-benzylthioether (EC-BE) on the growth of cultured rat glioma cells was investigated. EC-BE was found to produce a concentration-dependent inhibitory effect on the growth of glioma cells. Inhibition was also observed in samples treated with (-)-epicatechin acetoxymethyl ester (EC-AM). The original compound (-)-epicatechin, which gave rise to EC-AM and EC-BE, failed to produce a similar result. Because both EC-BE and EC-AM are membrane permeable, while (-)-epicatechin is not, the results indicated that the (-)-epicatechin and its derivatives need to be intracellular in order to produce an effect. Moreover, it is demonstrated that EC-BE inhibited the incorporation of [ 3 H]thymidine into DNA while the uptake of extracellular [ 3 H]thymidine by glioma cells was not affected. The activities of redox enzymes such as glutathione peroxidase and glutathione reductase were attenuated in cells treated with EC-BE. These data suggest that EC-BE can inhibit the growth of glioma cells by affecting a redox pathway related to the formation of DNA.

Effects of intracerebroventricular administration of atrial natriuretic peptide on subcortical EEG activity in conscious rabbits.

Atrial natriuretic peptide (ANP) influences the activity of rat hypothalamic neurons, modifies the membrane excitability of the rat forebrain neurons, and induces changes in membrane potentials in cultured rat glioma cells. In order to explore whether these effects are reflected in the electrical activity of larger subcortical brain areas, we investigated the electroenceophalographic activity (EEG) recorded from 20 male albino (New Zealand White) rabbits. Recordings of EEG were made on restrained, conscious animals 1 week after the implantation of an indwelling intracerebroventricular (i.c.v.) cannula (lateral right ventricle) and two stainless steel electrodes, implanted in the paraventricular (PVN) and supraoptic (SON) nuclei. Animals were classified into two main groups: those with water available ad libitum (group A) and those which were dehydrated for 24 h before EEG recordings (group B). Each group was divided into two subgroups (1 and 2) of five animals each. EEG was recorded at 0 min (control) and 30, 60, and 90 min following the i.c.v. injection of either 25 microliters artificial cerebrospinal fluid (aCSF; subgroup 1) or 1 microgram alpha-human ANP in 25 microliters a CSF (subgroup 2). Each EEG record duration was 6 s. For each EEG record the power spectrum of the digitized waveform was estimated in the frequencies 0.5-48 Hz using the fast Fourier transform, and the energy of each waveform was subsequently calculated. The results were analyzed by repeated-measures ANOVA and by the t-test. The analysis revealed that (1) water deprivation does not affect mean EEG energy and value (2) ANP attenuates (P < 0.05; in comparison with zero time) the mean energy value of EEG recorded from SON at 30 min and 60 min in the frequencies 8-48 Hz, whilst it tends to decrease (P < 0.1) the mean energy of EEG recorded from PVN at 30 min in the frequencies 8-15 Hz. Mean EEG energy changes caused by ANP would reflect its various (mainly inhibitory) effects on the electrical activity recorded from PVN and SON neurons in in vitro and in vivo studies.

Lactate Transport in Cultured Glial Cells

Uptake of L-lactate was investigated with a radioactive tracer method in cultured rat glioma cells and in astroglia-rich primary cultures derived from rat brain. In the glioma cells, a saturable component of uptake was identified with half-maximal uptake occurring at 1.0 +/- 0.4 mM lactate. In addition, a non-saturable component dominated the uptake at high concentrations of lactate. In astroglia-rich primary cultures, no saturable component of uptake could be detected in the concentration range studied (0.1-15 mM). In conclusion, lactate uptake at physiological concentrations is predominantly mediated in the glioma cells by a carrier-dependent process, whereas in astroglial cells, simple diffusion appears to be the dominant way of lactate transport.

Stable expression in rat glioma cells of sense and antisense nucleic acids to a human multifunctional DNA repair enzyme, APEX nuclease

We have cloned mouse and human cDNAs for a multifunctional DNA repair enzyme (APEX nuclease) having apurinic/apyrimidinic (AP) endonuclease, 3′–5′ exonuclease, DNA 3′ repair diesterase and DNA 3′-phosphatase activities. To investigate the biological role of APEX nuclease, sense or antisense APEX RNA was stably expressed at a high level in cultured rat glioma cells by introducing plasmids (pABWN-HAPX1F for expression of sense RNA or pABWN-HAPX2R for expression of antisense RNA) constructed from the human APEX cDNA and an expression vector pABWN. Multiple copies of the construct were integrated into the glioma cells tranfected with pABWN-HAPX1F or pABWN-HAPX2R. These transfectants showed markedly high expression of RNA hybridizable to human APEX cDNA, indicating the expression of the sense or antisense RNA. Activity blotting analyses of salt extracts of these transfectants showed that the sense RNA-expressed cells had higher AP endonuclease activity and that the antisense RNA-expressed cells had extremely lower AP endonuclease activity than the control cells. The APEX nuclease-depressed glioma cells became more sensitive to methyl methanesulfonate and hydrogen peroxide than the control cells or the APEX nuclease-overexpressed cells. The results indicate that APEX nuclease plays an important role in repair of DNA damage caused by these genotoxic agents. The present stable expression systems for the sense and antisense APEX RNAs should be useful for analyzing the biological functions such as an antimutagenic function of the enzyme.

Influence of glucose on metabolism and growth of rat glioma cells (C6) in multicellular spheroid culture.

The metabolism and growth of rat glioma C6 cells in multicellular spheroid culture depended strongly on the glucose supply. A low glucose level (0.1 g/l) in the culture medium reduced lactate production, increased oxygen consumption and diminished hydrogen ion production under normoxia as well as hypoxia. A high glucose level (10 g/l glucose) increased lactate production, had no significant influence on oxygen consumption and increased the hydrogen ion production under hypoxia. Hydrogen ion release from cells under normoxic and hypoxic conditions could be significantly diminished by amiloride (l mM), indicating the involvement of the Na+/H+ exchanger. The growth of the C6 spheroids was enhanced under low glucose conditions, possibly due to the more physiological extracellular pH in the deeper regions of the spheroids. The growth was inhibited under high glucose conditions, which seemed to be toxic due to a massive hydrogen production giving acidosis. The glucose supply strongly influenced the local hydrogen ion production inside the C6 spheroids and this might in turn lead to changes in the response to different therapeutic modalities.

Transcriptional Regulation of Ferritin Messenger Ribonucleic Acid Levels by Insulin in Cultured Rat Glioma Cells

Recent data have shown that ferritin, a ubiquitous protein, has a role as a regulator of cellular differentiation. In the present study we have investigated the expression of ferritin mRNAs in cultured C6 cells, a rat glioma cell line, in response to insulin, which has an important role in cellular growth and differentiation. Insulin stimulated steady state levels of both ferritin heavy chain and ferritin light chain mRNAs. An increase in the level of ferritin heavy or light chain mRNA was detected after 2 h of incubation with insulin, and a plateau was reached after 48 h for heavy chain mRNA and after 72 h for light chain mRNA. The responses were dose-dependent and were maximal at 100 nM for both mRNAs. Treatment of cells with actinomycin-D showed that insulin had no effect on the posttranscriptional stability of these mRNAs. Actinomycin-D inhibited insulin-induced accumulation of both mRNAs, suggesting transcriptional stimulation of ferritin genes by insulin. A nuclear run-on assay showed that the insulin-induced increase in ferritin heavy chain mRNA was due to an increase in the rate of gene transcription. We also demonstrated that insulin-like growth factor-I (IGF-I) increased ferritin heavy and light chain mRNA levels in a dose-dependent fashion, and that the maximum effect was obtained at a concentration of 10 nM on both mRNA levels. IGF-I was not only 10-fold more potent, but the absolute level of maximum stimulation was also about 2-fold greater than that for insulin. The combination of insulin (100 nM) and IGF-I (10 nM) showed no additive effect. The results suggested that the ferritin heavy and light chain genes are transcriptionally regulated by insulin and influenced by IGF-I.

Isolation and characterization of a metalloproteinase secreted by rat glioma cells in serum-free culture

The isolation of a metalloproteinase secreted by a rat glioma cell line (BT5C) in serum-free media is described. After affinity purification, the activity was present as a double band with Mr 86000 and 76000 both of which required CaCl 2 for activity. The enzyme was able to degrade gelatin but not casein. It was unable to degrade native types I, III, IV and V collagens but their denatured counterparts were degraded. Using a radiolabel release assay the enzyme was inhibited by EDTA, 1:10 phenanthroline and TIMP confirming that it belongs to the family of metalloproteinases. Its activity was not affected by either serine or cysteine protease inhibitors. The proteinase was activated by APMA but was unaffected by trypsin treatment.

Organic cations substituted for sodium are toxic to cultured rat glioma cells

As a prelude to studying the sodium dependence of choline-transport systems, a number of organic compounds plus LiCl, CsCl and RbCl as sodium substitutes were tested for toxicity to maintain isoosmolality or ionic strength on cultured rat astrocytoma cells. In short term experiments (1 hour), tetramethylammonium chloride, triethanolammonium chloride, guanidinium chloride, tris hydrochloride, mannitol, sucrose, LiCl, RbCl and CsCl were well tolerated. In long term exposure (4 days), no compound was completely nontoxic as a sodium substitute, but sucrose, mannitol, LiCl, and RbCl allowed maximum cell survival.

Increased 5′-nucleotidase activity induced by dibutyryl cyclic AMP treatment of cultured glial cells

In an attempt to clarify the relationship between ecto-5′-nucleotidase (5′-N) activity and cell differentiation of glial cells, dibutyryl cyclic AMP (dBcAMP), which induces cell differentiation, was administrated to cultured rat glioma cells in logarithmic and confluent phases of cultivation. To evaluate the cellular differentiation, cell morphology and the number of glial fibrillary acidic protein (GFAP) positive cells were examined. Treatment with 1 mM dBcAMP decreased cell proliferation and induced cell differentiation in both the logarithmic and the confluent phases. The number of GFAP-positive cells increased with cellular aging and this tendency was enhanced by dBcAMP administration. Ecto-5′-N activity was higher in dBcAMP treated cells than in non-treated cells in both the logarithmic and the confluent phases. These findings suggest that ecto-5′-N activity of C6 glioma cells is increased by dBcAMP actions and is accompanied by cell differentiation.

A rapid and sensitive bioassay involving cultured rat glioma cells to screen for substances capable of elevating intracellular cyclic AMP concentration.

Cultured rat glioma (ASK) cells are morphologically converted from a spindle to an astrocyte form when treated with dibutyryl cAMP. This morphologic transformation is discernible by light microscopy and can be visually quantitated. As described herein, dose-dependent astrocyte generation was demonstrated by treatment of confluent monolayers with forskolin [1], a compound known to activate adenylate cyclase, and the potency of four forskolin derivatives was found to correlate with previously established biologic potential. Neither a crude ginseng extract nor purified ginsenosides were active in the process, but supplementation of the otherwise inactive ginseng extract with 1 demonstrated 50% of the cells were morphologically converted to the astrocyte form at a concentration of approximately 0.0008%. Retinoic acid was also active in this test system; the morphologic transformation was reversed on treatment with colchicine, and intracellular cAMP concentration was elevated approximately 10-fold. Evaluation of 15 retinoids established a general correlation between the activity in this system and other systems reported in the literature. Thus, the astrocyte formation assay appears to provide several advantages that make it attractive as a screen for the detection or evaluation of substances capable of elevating intracellular cAMP concentration. In addition to technical ease, the procedure is rapid, sensitive, and relatively inexpensive.

Atrial natriuretic polypeptide hormones induce membrane potential responses in cultured rat glioma cells

Atrial natriuretic hormones (ANHs) applied to polyploid rat glioma cells induced hyperpolarizations of about 30 s duration, followed by depolarizations lasting 1–2 min. Repeated applications of the peptide resulted in desensitization. The reversal potential of −87 mV at an extracellular K + concentration of 5 mM and the decrease of membrane resistance during the hyperpolarization indicate that K + channels were activated by ANH. In these cells the fluorescence signal of 2-[(2-bis[car☐ymethyl]amino-5-methylphenoxy)-methyl]-6-methoxy-8-bis[car☐ymethyl]aminoquinoline (quin2) was not affected by ANH suggesting that ANH did not change the cytosolic Ca 2+-activity.

Glial culture on artificial capillaries: electron microscopic comparisons of C6 rat glioma cells and rat astroglia.

The functional association of astroglial footplates with blood vessels is important because astrocytes may provide a channel between the blood and neurons deeper in the brain parenchyma for the passage of ions and metabolites. This hypothesized function is very difficult to study in vivo or in monolayer cultures. We have produced a three-dimensional cell culture model of perivascular astroglia by means of an artificial capillary system. Conventional primary cultures of astroglia were first prepared from neonatal rat cerebral hemispheres in 75-cm2 tissue culture flasks. After 25 days, the cells were seeded in Amicon Vitafiber hollow fiber culture vessels. Direct seeding of brain cell suspensions was not successful. A culture unit consists of a bundle of hollow, semi-permeable polysulfone fibers encased in a plastic shell. The fibers were coated with fibronectin and bovine serum albumin, and astroglia were seeded on their outer surfaces. Warmed medium was pumped through the lumina of the fibers. After 13 days the cells were fixed with paraformaldehyde and examined. Scanning electron microscopy revealed the tubes to be uniformly covered with astroglia with short processes that contacted nearby cells. Transmission electron microscopy showed glial filaments and gap junctions. Astrocyte cultures were compared morphologically to C6 rat glioma cells in hollow fiber culture. The astrocytes formed a monolayer, whereas C6 cells formed a stratified culture. Furthermore, C6 cells did not form gap junctions. Astrocytes have been hypothesized to take up K+ discharged to the extracellular space by depolarizing neurons and move it to areas of low concentration, i.e., to act as a K+ spatial buffer. Our culture system should permit direct testing of this hypothesis.

Enrichment of high mannose-type glycans in nervous tissue glycoproteins in neuronal ceroid-lipofuscinosis

In view of the hypothesis that a biochemical abnormality in the childhood forms of neuronal ceroid-lipofuscinosis may lie in the utilization of dolichols in glycoprotein synthesis, we analyzed the oligosaccharide structures of brain glycoproteins in infantile neuronal ceroid-lipofuscinosis (INCL). Lectin affinity chromatography of purified glycopeptides and of oligosaccharides prepared by hydrazinolysis showed an increase in high mannose-type glycopeptides and a decrease in tri- and tetra-antennary glycopeptides in INCL brain when compared with control brain. These changes were more pronounced in the storage cytosomes than in whole brain. Methylation analysis of the isolated glycopeptide fractions did not reveal differences in substitution patterns of the individual sugar residues between INCL and control brain. In INCL the core disaccharide of the O-glycosidically-linked oligosaccharides was sialylated to a higher degree than in control brain indicating that the structural changes were not confined only to N-glycosidically-linked carbohydrate chains. The observed structural changes in the carbohydrate chains of brain glycopeptides in INCL could be explained by a defect in the biosynthesis of glycoproteins. Alternatively, the changes may reflect the increased glial cell population in the degenerating brain. In fact, the elution profiles in lectin chromatography of oligosaccharides prepared from cultured rat glioma cells resembled those from INCL brain.

Effect of Calmodulin Antagonist on the Growth of Rat Glioma Cells in Culture

Calcium ions have been implicated in the regulation or modulation of various cell functions. Calmodulin, a calcium modulator protein in the cell, may play an important role in cell proliferation. If the calcium ion level in the cell can be altered by the addition of calmodulin antagonist, the aberrant growth of glioma may be regulated. The authors, therefore, intended to examine the effect of a calmodulin antagonist W-7, on a cultured glioma cell strain (GA-1), which is chemically induced from a rat glioblast. The results are as follows: 1) Inhibition dose of W-7 to 50% of normal cell growth, was 50 μM. W-5, a dechlorinated structural analog of W-7, which had a lower affinity to calmodulin showed no definite inhibition on GA-1 growth. 2) At lower concentrations of W-7 (12.5 μM) in the cultured medium, deoxyribonucleic acid (DNA) synthesis in GA-1 was not markedly inhibited, but ribonucleic acid (RNA) and protein syntheses were strongly suppressed. 3) At higher concentrations of W-7 (25 to 75 μM), DNA, RNA, and protein syntheses were all suppressed. 4) Calmodulin content of GA-1 cell (58.0±3.0 pg/mg soluble protein) was lowered to half of the control level by the addition of W-7 within 2 hours and was maintained afterwards at the lower level. These results indicate that administration of calmodulin antagonist W-7 may be effective for nonsurgical treatment of glioma.