Cultured Rat Glioma Research Articles

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.

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.

New method for the evaluation of the response of cultured cell lines to continuous low-dose-rate irradiation from encapsulated iridium-192 seeds with hyperthermia.

A new method was designed to investigate and evaluate the biological effectiveness of hyperthermia combined with continuous low-dose-rate irradiation (CLDRI) from encapsulated iridium-192 seed sources on glioma cells in vitro using the MTT assay. The system consists of 10 iridium seeds contained in a catheter bent into a circle, which is placed on a culture plate containing the cells. The effects of CLDRI and CLDRI combined with hyperthermia on a cultured rat glioma cell line (C-6) were studied. The number of surviving cells decreased as the total radiation dose increased. There was no significant difference in survival rates at dose rates of 0.1 Gy/hr and of 0.2 Gy/hr (p = 0.2811). An additive effect was observed in the cells treated with hyperthermia at 41 degrees C and 42 degrees C, combined with CLDRI, and synergistic effect between the two treatment modalities was observed at 43 degrees C. This new device is less expensive, easily reproducible, and can also be performed easily enough to examine a large number of samples in a short time period for sensitivity testing.

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.

Effects of three distinct natriuretic peptides on receptor binding and guanylate cyclase activities in rat glioma cells

Receptor binding and cyclic GMP generation by three distinct natriueretic peptides (ANP, BNP, CNP) were studied in a cultured rat glioma cell line (C6). Binding studies revealed the presence of high-affinity binding sites for three natriuretic peptides with almost comparable affinities. In contrast, CNP and BNP were almost equipotent in stimulating intracellular cyclic GMP generation over the low concentration range, but CNP caused further elevation in the high concentration range, whereas ANP was minimally effective. Our data suggest that the glioma cells possess receptors more responsive to CNP than ANP and BNP despite no apparent correlation between receptor binding affinities and cyclic GMP responses.

Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis by cytochalasin B in cultured glioma cells: Potential regulation of biosynthesis by Ca 2+-dependent mechanisms

The major route of phosphatidylcholine (PtdCho) biosynthesis in mammalian cells is the sequence: choline (Cho)----phosphocholine (PCho)----cytidinediphosphate choline (CDP-Cho)----PtdCho. Recently, we have found that intermediates of this pathway are not freely diffusible in cultured rat glioma (C6) cells but are channeled towards PtdCho biosynthesis (George et al. (1989). Biochim. Biophys. Acta. 1004, 283-291). Channeling of intermediates in other mammalian systems is thought to be mediated through adsorption of enzymes to membranes and cytoskeletal elements to form multienzyme complexes. In this study, agents which perturb the structure and function of cytoskeletal elements were tested for effects on phospholipid metabolism in glioma cells. The filament-disrupting agent cytochalasin B (CB), but not other cytochalasins or the microtubule depolymerizer colchicine inhibited PtdCho and phosphatidylethanolamine (PtdEtn) biosynthesis as judged by dose-dependent reduction of labeling from [3H]Cho and [14C]ethanolamine (Etn). 32Pi pulse-labeling indicated that CB selectively decreased PtdCho and PtdEtn biosynthesis without affecting synthesis of other phospholipids. Synthesis of water-soluble intermediates of PtdCho metabolism was unaffected but the conversion of phosphoethanolamine to CDP-ethanolamine was reduced by CB. Effects of CB on phospholipid biosynthesis were not due to inhibition of glucose uptake as shown by experiments with 2-deoxyglucose, glucose-starved cells and other cytochalasins. Experiments with Ca(2+)-EGTA buffers and digitonin-permeabilized cells, and the Ca(2+)-channel blocker verapamil suggest that effects of CB on PtdCho and PtdEtn biosynthesis are due to alteration of intracellular Ca2+. Taken together, these results suggest that CB acts at sites distinct from glucose transport and cellular microfilaments to specifically inhibit PtdCho and PtdEtn biosynthesis by mechanisms dependent on intracellular Ca2+.

Protein kinase A- and protein kinase C-regulated interaction of plectin with lamin B and vimentin.

Solid-phase binding assays with protein species purified from cultured rat glioma C6 cells and Ehrlich ascites revealed that plectin bound specifically to lamin B but not to lamins A and C. Lamin B interaction was significantly decreased upon in vitro phosphorylation of either lamin B or plectin with protein kinase A or C. In contrast, phosphorylation of plectin with kinase A increased its binding to vimentin, suggesting a different regulation of plectin interactions by this kinase. 32P-radiolabeling of rat glioma C6 cells revealed plectin as a major in vivo target of protein kinase A and protein kinase C. Plectin, present in lysates of dibutyryladenosine 3',5'-cyclic monophosphate-treated cells, showed a 2.5 times higher binding affinity to vimentin than plectin from phorbol ester-treated cells. Furthermore, the relative amounts of plectin in 1% Triton X-100/high salt-insoluble cell fractions decreased to one-fourth of control values upon treating cells with phorbol esters, whereas vimentin was unaffected. This finding suggested a protein kinase C-dependent weakening of plectin interaction with intermediate filaments in vivo. Taken together, these results point to a role of plectin in interlinking cytoskeletal and nuclear elements and suggest that specific protein kinases are involved in regulating these interactions.

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.

Reduction in β‐Adrenoceptor Density in Cultured Rat Glioma C6 Cells After Incubation with Antidepressants Is Dependent upon the Culturing Conditions Used

The hydrophilic beta-adrenoceptor ligand (-)-[3H]CGP-12177 binds to intact C6 cells with a high affinity (KD approximately 0.1 nM) and with a high degree of specificity. The binding was inhibited by DL-propranolol (Ki approximately 1 nM). Treatment of cells cultured in Dulbecco's modified Eagle medium (DMEM) without fetal calf serum for 4 days with desipramine reduced the (-)-[3H]CGP-12177 specific binding in a concentration-dependent manner, a reduction from 127 to 102 fmol/mg of protein being found at a ligand concentration of 1 nM after treatment with 10 microM desipramine. Lesser effects were seen after treatment for 1 day. A similar result was found with maprotiline, and reductions in specific binding were seen after 4 days of treatment with amitriptyline, iprindole, and citalopram. The reduction in binding-site density (measured per milligram of protein to compensate for variability in cell density per well), however, was paralleled in all cases by a reduction in the rate of cell proliferation. When C6 glioma cells were cultured in Ham's medium without fetal calf serum during the antidepressant treatment period, a higher specific binding was observed than for the DMEM-cultured cells, and 10 microM desipramine was without effect on either the (-)-[3H]CGP-12177 specific binding or cell proliferation. It is concluded that the effects of the antidepressants tested upon the density of (-)-[3H]CGP-12177 specific binding sites in intact C6 cells may be secondary to the toxicity of the compounds under the conditions used.

Transcriptional regulation of ferritin heavy chain messenger RNA expression by thyroid hormone

The effect of 3,5,3′-triiodo-L-thyronine (T3) on the steady state levels of ferritin heavy chain (ferritin H) mRNA in cultured rat glioma C6 cells and various rat tissues was examined. Addition of T3 to cultured C6 cells showed the time and dose-dependent increase in the steady-state level of ferritin H mRNA. Invitro nuclear run-on assay revealed that the stimulatory effect was due to the increase in the transcription rate of ferritin H gene. T3 had no effect on the half live of ferritin H mRNA. In hyperthyroid rats, the level of ferritin H mRNA in the kidney was elevated. On the contrary, that was decreased in hypothyroid rats. The results suggest the involvement of T3 in the regulation of ferritin H gene expression.

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.

Effect of the Tricyclic Antidepressant Desipramine on β‐Adrenergic Receptors in Cultured Rat Glioma C6 Cells

Rat glioma C6 cells, cultured in the presence of the tricyclic antidepressant desipramine, lost a significant number of beta-adrenergic receptors in a time- and dose-dependent manner. A similar loss was observed whether binding was determined on intact cells with the hydrophilic beta-adrenergic antagonist (+/-)-[3H]4-(3-tert-butylamino-2-hydroxypropoxyl)benzimidazole-2-o n HCl ([3H]CGP-12177) or on cell lysates with the more hydrophobic antagonists [125I]iodocyanopindolol or [3H]dihydroalprenolol. When stimulated with the agonist isoproterenol, desipramine-treated cells accumulated less cyclic AMP than control cells. The affinity of the beta-adrenergic receptors for either antagonist or agonist was unchanged after desipramine treatment. Desipramine interacted only weakly with the receptors and competed for [125I]iodocyanopindolol binding with a Ki of 30 microM. The presence in the culture medium of alprenolol or propranolol, potent beta-adrenergic antagonists, however, did not prevent the reduction in receptors by desipramine. Desipramine also caused a loss of beta-adrenergic receptors from cells maintained in serum-free medium and the cells themselves did not contain or secrete endogenous catecholamines. Although desipramine is a potent inhibitor of catecholamine uptake, it appears unlikely that the observed loss of beta-adrenergic receptors in rat glioma C6 cells exposed to the drug is due to an increase in extracellular catecholamine levels or to a direct interaction with the receptors.

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.

Exfoliation of the β-adrenergic receptor and the regulatory components of adenylate cyclase by cultured rat glioma C6 cells

Cultured rat glioma C6 cells exfoliate membrane vesicles which have been termed ‘exosomes’ into the culture medium. The exosomes contained both stimulatory and inhibitory GTP-binding components of adenylate cyclase (the stimulatory, G s, and the inhibitory, G i, regulatory components) and β-adrenergic receptors but were devoid of adenylate cyclase activity. It was therefore apparent that the catalytic component of adenylate cyclase was either not exfoliated or was inactivated during the exfoliation process. The presence of G s or G i in the exosomes was detected by ADP ribosylation using [α- 32P]NAD in the presence of cholera or pertussis toxins, respectively. The exosomal concentration of each of the two components was estimated to be about one fifth of that of the cell membrane when expressed on a per mg protein basis. Exosomal G s was almost as active as the membrane-derived G s in its ability to reconstitute NaF- and guanine nucleotide-stimulated adenylate cyclase activity in membranes of S49 cyc − cells, which lack a functional G s. The ability of exosomal G s to reconstitute isoproterenol-stimulated activity, however, was much lower than that of membrane G s. The density of β-adrenergic receptors in the exosomes was much less than that found in the membranes. Although the exosomal receptors bound the antagonist iodocyanopindolol with the same affinity as receptors from the cell membrane, the affinity for the agonist isoproterenol was 13- to 18-fold lower in the exosomes. In addition, this affinity was not modulated by GTP in the exosomes. Thus, exfoliated β-adrenergic receptors seem to be impaired in their ability to couple to and activate G s. This was directly tested by coupling the receptors to a foreign adenylate cyclase using membrane fusion. The fusates were then assayed for agonist-stimulated activity. While significant stimulation of the acceptor adenylate cyclase was obtained using C6 membrane receptors, the exosomal receptors were completely inactive. Thus during exfoliation, there appear to be changes in the components of the β-adrenergic-sensitive adenylate cyclase that results in a nonfunctional system in the exosomes.

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.