Morphogenesis of measles virus on C6 rat glioma cells

Rat glioma cells (C₆) persistently infected with measles virus show a locally dissociated distribution of budding processes at the cell surface.     

Secretion of S-100 from rat C6 glioma cells

Conditioned media from rat C6 glioma cells contain significant levels of S-100 immunoreactivity as determined by radioimmunoassay. The levels of extracellular S-100 detected could not be accounted for by the release of intracellular S-100 into the media from lysed cells. The extracellular form of S-100 exhibits fractionation properties and immunological characteristics that are different from those of the intracellular form of S-100 in C6 cells. While the intracellular S-100 levels increase as a function of days in culture, the extracellular S-100 levels are high until the cells reach confluency and are lower in postconfluent cultures. Altogether, our data suggest that C6 glioma cells secrete S-100, and that the quantitative levels of the intracellular and secreted forms of S-100 are differentially regulated.     

Regulation of cAMP levels by protein kinase C in C6 rat glioma cells

Cultures of rat C6 rat glioma cells exhibit a diminished response to isoproterenol and forskolin after being treated with phorbol 12,13-dibutyrate (PDbU). An IC50 for PDbU of 38 +/- 5 nM and 62 +/- 8 nM was observed in the isoproterenol and forskolin response, respectively. Similarly, C6 cultures exhibited a diminished response to isoproterenol and forskolin after an overnight incubation with phospholipase C. We previously demonstrated that this treatment will increase diacylglycerol levels in these cells (Bressler: J Neurochem 48:181-186, 1987). An IC50 for phospholipase C of 6.0 +/- 0.1 x 10(-1) and 7.0 +/- 0.1 x 10(-1) units/ml was observed for the isoproterenol and forskolin response, respectively. A kinetic analysis suggests that the site of PDbU-mediated inhibition to beta-adrenergic and forskolin stimulation was different. Degradation of cAMP was a contributory factor since elevated cAMP levels decreased faster in PDbU treated cells than in nontreated cells. In addition, PDbU treated cells exhibited a significantly higher level of phosphodiesterase activity. We conclude that activation of protein kinase C and subsequent stimulation of phosphodiesterase activity contributes to the inhibition of the beta-adrenergic and forskolin mediated increase in cAMP levels in intact C6 rat glioma cells. The consequences of lower cAMP levels in sustaining differentiated function in the C6 rat glioma cell line will be discussed.     

Intracellular pH regulation in primary rat astrocytes and C6 glioma cells

We used the pH-sensitive fluorescent dye BCECF to study intracellular pH (pH_i) regulation in primary cultures of rat astrocytes and C6 glioma cells. Both cell types contain three pH-regulating transporters: (1) alkalinizing Na⁺/H⁺ exchange; (2) alkalinizing Na⁺ + HCO₃ ?/Cl_? exchange; and (3) acidifying Cl^?/HCO₃ ^? exchange. Na⁺/H⁺ exchange was most evident in the absence of CO₂; recovery from acidification was Na⁺ dependent and amiloride sensitive. Exposure to CO₂ caused a cell alkalinization that was inhibited by DIDS, dependent on external Na⁺, and inhibited 75% in the absence of Cl^? (thus mediated by Na⁺ + HCO₃ ^?/Cl^? exchange). When pH_i was increased above the normal steady-state pH_i, a DIDS-inhibitable and Na⁺ -independent acidifying recovery was evident, indicating the presence of Cl^? /HCO₃ ^? exchange. Astrocytes, but not C6 cells, contain a fourth pH-regulating transporter, Na⁺ ?HCO₃ ^? cotransport; in the presence of CO₂, depolarization caused an alkalinization of 0.12 ⁺_? 0.01 (n = 8) and increased the rate of CO₂-induced alkalinization from 0.23 ± 0.02 to 0.42 ± 0.03 pH unit/min. Since C6 cells lack the Na⁺ -HCO₃ ⁺ cotransporter, they are an inferior model of pH_i regulation in glia. Our results differ from previous observations in glia in that: (1) Na⁺ /H⁺ exchange was entirely inhibited by amiloride; (2) Na⁺ + HCO₃ ^?/Cl^? exchange was present and largely responsible for CO₂?induced alkalinization; (3) Cl^? /HCO₃ ^? exchange was only active at pH_i values above steady state; and (4) depolarization-induced alkalinization of astrocytes was seen only in the presence of CO₂.     

Tunneling nanotubes between rat primary astrocytes and C6 glioma cells alter proliferation potential of glioma cells

The tunneling nanotube(TNT) is a newly discovered, long and thin tubular structure between cells. In this study, we established a co-culture system for rat primary astrocytes and C6 glioma cells and found that TNTs formed between them. Most of the TNTs initiated from astrocytes towards C6 glioma cells. The formation of TNTs depended on p53. In addition, hydrogen peroxide increased the number of TNTs in the co-culture system. Established TNTs reduced the proliferation of C6 glioma cells. Our data suggest that TNTs between astrocytes and glioma cells facilitate substance transfer and therefore alter the properties, including the proliferation potential, of glioma cells.     

FLIP蛋白抑制顺铂诱导大鼠C6胶质瘤细胞凋亡

目的探讨自杀相关因子（Fas）相关死亡结构域样白介素1（IL-1）β转化酶抑制蛋白（FLIP）对于顺铂（CDDP）诱导大鼠脑胶质瘤细胞（C6细胞株）凋亡的抑制作用,为进一步研究胶质瘤的耐药性奠定分子生物学基础。方法利用由Ad—Max腺病毒包装系统成功构建的携载大鼠FLIP基因的腺病毒表达载体Ad—FLIP感染大鼠C6胶质瘤细胞,24h后经逆转录酶一多聚酶链反应（RT—PCR）及Westernblot检测感染组及对照组细胞中FLIP基因的mRNA及蛋白表达水平;分别给予Ad—FLIP感染组及对照组细胞不同浓度的CDDP（0,1,2,4,8mg／ml）,药物处理48h后,经流式细胞仪（FCM）分析细胞凋亡状况;四唑蓝显色法（MTF）测定并比较两组细胞活力。结果Ad—FLIP感染组细胞与对照组细胞相比,FLIPmRNA和蛋白表达水平明显增高;流式细胞仪检测结果显示Ad—FLIP感染组细胞凋亡率明显低于对照组。MTT法结果提示经CDDP处理后,Ad—FLIP感染组与对照组细胞活力均有下降,但FLIP蛋白具有明显的抑制作用。结论FLIP蛋白在大鼠c6胶质瘤细胞中具有抵抗化疗药物的作用。     

Lithium chloride inhibits thrombin-induced intracellular calcium mobilization in C6 rat glioma cells

In this study, the authors have demonstrated the effect of lithium, a typical mood stabilizer, on thrombin-evoked Ca2+ mobilization in C6 cells to elucidate the action mechanisms of the drug. Thrombin-induced Ca2 mobilization was reduced 24 hr after 1 or 10 mM lithium chloride (LiCl) pretreatment. The Ca2+ rise was reduced in a time-dependent manner, and the significant inhibition was observed 9 hr pretreatment with 10 mM LiCl. On the other hand, pretreatment of the cells with 10 mM LiCl for 24 hr did not alter the amount of Galphaq/11 significantly. Pretreatment with 10 mM LiCl for 24 hr failed to reduce the 5-HT-induced Ca2+ mobilization or to affect the desensitization of the 5-HT signal. Finally, thrombin-elicited Ca2+ rise was markedly inhibited in the presence of 0.05 U/ml plasmin, however, the Ca2+ rise was not further attenuated in the presence of plasmin in C6 cells pretreated with LiCl for 24 hr. These results indicate that pretreatment with LiCl attenuated thrombin-evoked intracellular Ca2+ mobilization in plasmin sensitive manner in C6 rat glioma cells. Thus, it is important to investigate the effect of lithium on thrombin-induced cellular responses to clarify the action mechanism of lithium in relation to some abnormality in thrombin-evoked Ca2+ rise observed in bipolar disorders.     

Comparison of endothelin binding and calcium mobilization in C6-BU-1 rat glioma and N18TG2 mouse neuroblastoma cells.

We have previously reported that endothelin (RT) receptor activation increases intracellular calcium concentrations ([Ca2+]i) in NG108-15 cells, a hybrid of rat glioma C6-BU-1 and mouse neuroblastoma N18TG2 cells. This study was designed to further explore the origin of the ET receptor and [Ca2+]i mobilization in the parent cell lines hybridized to form the NG108-15 cells. [125I]ET-1 bound to a single class of high affinity sites in C6-BU-1 cells with a KD value of 108pM and Bmax of 12,400 sites/cell. ET-1, ET-2, ET-3 and big ET inhibited [125I]ET-1 binding to C6-BU-1 cells with KD values of 0.074, 0.167, 261 and 187 nM, respectively. All ETs produced a rapid increase in [Ca2+]i in C6-Bu-1 cells. EC50 values for ET-1, ET-2, ET-3 and big ET were 0.71, 1.14, 120 and 243 nM respectively. There was a significant correlation between the KD values obtained from competition binding experiments and the EC50 values from [Ca2+]i response curves in C6-BU-1 cells (r = 0.996, p less than 0.004). Ten nM ET-1 produced about 85% of the maximal [Ca2+]i increase in C6-BU-1 cells which was reduced by 96% in the absence of extracellular calcium. Furthermore, diltiazem (10 microM) and nifedipine (1 microM) failed to block ET-induced [Ca2+]i mobilization. None of the ETs elevated [Ca2+]i or displayed any specific [125I]ET-1 binding in N18TG2 cells. These data suggest that ET binds to a specific ET receptor in C6-BU-1 cells, and elevates [Ca2+]i through dihydropyridine-insensitive, receptor-mediated calcium influx. Further, the ability of ETs to elevate [Ca2+]i in NG108-15 hybrid cells is due to the ET receptor inherent to the C6-BU-1 glioma parent line.     

Induction of GDNF mRNA expression by melatonin in rat C6 glioma cells

In order to determine the physiological effect of melatonin on glial cell line-derived neurotrophic factor (GDNF), which is reportedly up-regulated by high doses of this hormone, concentration-dependent studies were carried out in cultured cells. RT-PCR studies indicated that, in addition to GDNF, rat C6 glioma cells express both of the G protein-coupled melatonin receptor subtypes, MT1 and MT2. When C6 cells were treated with physiological (0.05-1 nM) or higher (10 and 100 nM) concentrations of melatonin for 24 h, a significant induction of relative GDNF mRNA levels (n = 4) was detected by semi-quantitative RT-PCR. These findings suggest that induction of GDNF is involved in physiological neuroprotection by melatonin. Given the potency of GDNF in maintaining nigrostriatal dopaminergic integrity, understanding the mechanisms of its induction by melatonin could provide novel therapies for Parkinson's disease.     

Individual C6 glioma cells migrate in adult rat brain after neural homografting

Cultured C6 glioma cells were prelabeled with the plant lectin Phaseolus vulgaris leuco-agglutinin (PHAL) and grafted as a cell suspension (10(6) cells in 5.0 microliters) into freshly made cortical implantation pockets in adult host rats. Animals were killed 1-21 days post-implantation (DPI). The brains were removed, dehydrated, embedded in paraffin and sectioned at 8 microns. Paraffin sections were processed for light level immunofluorescent double labeling for PHAL, a marker for graft derived cells, and glial fibrillary acidic protein (GFAP), a specific marker for C6 glioma cells and astrocytes. Cells positive for both PHAL and GFAP were graft-derived C6 cells. By 7 DPI a large mass developed which extended above the surface of the brain and invaded (displacement of host tissue by a cell mass) the host parenchyma. This mass increased in size over the next 14 days. The invading tumor mass contained double labeled cells at all time periods examined. In addition to the invasion process, grafted C6 cells spread through the host parenchyma by migration (movement of single cells). Individual graft-derived C6 (GFAP/PHAL positive) cells migrated into host cortex surrounding the implantation pocket, corpus callosum ventral to the implantation pocket, ipsilateral internal capsule and bilaterally in the habenula.     

Mechanotransducing ion channels in C6 glioma cells

Mechanotransducing (MS) ion channels and images of the patch membrane were studied in cell-attached patches in C6 glioma cells. MS channel density was ∼0.08 to 0.5 channels/μm², channel conductance was ∼40 pS (at -40 mV), and the reversal potential was +15 mV. Replacement of NaCl with KCl, CsCl, or Na gluconate in the pipette solution was without substantial effect on the current-voltage relationship. Replacement of NaCl with NMDG (N-Methyl-D-Glucamine) Cl or reducing NaCl decreased the amplitude of inward currents at negative membrane potentials and caused the reversal potential to shift in the negative direction. Rapid application of suction to the back of the pipette usually elicited a fast (<0.1 s) appearance of channel activity. The peak (phasic) in channel activity was followed by a decrease to a constant (tonic) level of activity. The reduction in channel activity—called adaptation—was reduced at depolarizing membrane potentials and disappeared if too much pressure was applied. Positive pressure caused the patch membrane to curve toward the pipette tip, move in the direction of the tip, and evoke MS channel activity. Removal of the positive pressure caused the patch to move back to the original position. Conversely, negative pressure caused the patch membrane to curve away from the pipette tip, move away from the tip, and elicit MS channel activity. Gigohm seal resistances were always maintained during translational movement of the patch membrane. Tonic MS channel activity was not associated with translational movements of the patch membrane. Phasic and tonic channel activity were independent of the sign of curvature of the patch membrane. C6 glioma cells have rapidly adapting voltage-dependent MS ion channels, which are non-selective for monovalent cations, and belong to the stretch-activating class of mechanosensory ion channels. Adaptation in MS channels may allow the cell to limit the influx of cations in response to mechanical input. The selective loss of adaptation suggests that the MS channel's gate receives input from two sources. A minimal viscoelastic mechanical model of adaptation and two alternative models for translational movement of the patch are presented. © 1996 Wiley-Liss, Inc.     

大鼠脑干胶质瘤模型的建立

目的 建立大鼠脑干胶质瘤模型,为脑干胶质瘤的研究提供动物实验平台.方法 将1×10~6个大鼠C6胶质瘤细胞通过立体定向头架注射到大鼠脑桥,种植后2周对大鼠行磁共振扫描观察,然后行灌流固定取脑,HE染色.结果 磁共振检查均发现肿瘤生长,肿瘤为长T1和T2信号,经大鼠舌静脉注射Gd-DTPA信号明显增强.HE染色显示肿瘤为胶质母细胞瘤,呈浸润性生长,瘤内新生血管丰富,假栅栏样坏死明显.结论 大鼠C6胶质瘤细胞株可以建立脑干胶质瘤模型,成瘤率高,重复性好.     

Endothelin stimulates 86Rb efflux in rat glioma C6-Bu-1 cells.

Endothelin-1 (ET) elevates intracellular calcium ([Ca2+]i) and increased [Ca2+]i has been associated with K+ efflux. Therefore, we investigated ET stimulation of K+ efflux in rat glioma C6-BU-1 cells. K+ efflux was measured by monitoring the release of 86Rb+ from cells pre-loaded with 86RbCl. ET stimulated 86Rb+ efflux with an EC50 of 5.9 nM. ET-stimulated 86Rb+ efflux was insensitive to Ca2+ channel blockade, however it was reduced by 68% in Ca(2+)-free buffer, suggesting a sizable dependence on an extracellular source of Ca2+ influx through non voltage-operated Ca2+ channels. ET-stimulated 45Ca2+ efflux slightly preceded 86Rb+ efflux, again suggesting the presence of Ca2+ dependent K+ channels. ET-stimulated 86Rb+ efflux was insensitive to glyburide suggesting that efflux is not through ATP-sensitive K+ channels. ET-stimulated 86Rb+ efflux was insensitive to pertussis toxin (PTX) pre-treatment. Pre-incubation with the protein kinase C (PKC) inhibitor, staurosporine, inhibited 86Rb+ efflux by 66%, suggesting the involvement of PKC activation in ET-mediated 86Rb+ efflux. In summary, in C6-BU-1 cells, ET stimulates Ca2+ dependent K+ efflux which is mediated in part by protein kinase C activation, but not a PTX sensitive G-protein, nor through an ATP-sensitive K+ channel. These data extend the intracellular mechanisms initiated by ET to include Ca2+ dependent K+ efflux in glial cells and further support a neuromodulatory role for ET.     

Growth inhibition and induction of differentiation by panaxydol in rat C6 glioma cells

OBJECTIVES: Panaxydol is a naturally occurring non-peptidyl small molecule isolated from the lipophilic fractions of Panax notoginseng, a well-known Chinese traditional medicine. In this study, we aimed to investigate the effects of panaxydol on growth inhibition and its mechanisms in C6 rat glioma cells. METHODS: The effects of panaxydol on cell proliferation, morphologic changes, glial fibrillary acidic protein (GFAP) expression and cell cycle regulation in rat C6 cells were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, hematoxylin and eosin (HE) staining, immunocytochemistry, flow cytometric analysis and Western blot respectively. RESULTS: Panaxydol markedly inhibited the proliferation of C6 cells in a dose-dependent manner with IC50 of 39.5 +/- 2.3 microM. In addition, the cell morphologic changes and increased expression of GFAP in C6 cells in the presence of panaxydol implied a cellular differentiation. Flow cytometric analysis revealed that panaxydol-treated cells accumulated in G0/G1 phase with a marked decrease in the number of C6 cells at S phase. Western blot analysis demonstrated that panaxydol resulted in an increase in the protein expression of p27 in C6 cells as early as 3 hours after treatment consistent with the differentiation response, but protein expression of p53, p21, p16 and pRb remained unchanged. CONCLUSION: These findings suggest that panaxydol inhibits the proliferation of C6 cells via G0/G1 cell cycle arrest in association with induction of p27 expression and differentiation.     

骨髓间充质干细胞干预大鼠C6胶质瘤的试验研究

目的 探讨骨髓间充质干细胞(BMSCs)干预对大鼠脑胶质瘤C6细胞分化的影响及其相关机制.方法 采用Transwell小室共培养骨髓间充质干细胞及C6细胞,并以细胞计数法对C6细胞增值水平进行检测,以流式细胞术对C6细胞的细胞周期进行检测;以免疫荧光对波形蛋白(vimentin)的表达情况进行检测,以免疫组化法对胶质纤...     

苯乙酸对胶质瘤C6细胞增殖和凋亡的影响

目的观察诱导分化剂苯乙酸（PA）对胶质瘤C6细胞增殖和凋亡的影响。方法体外培养胶质瘤C6细胞，用0、2．5、5．0和7．5mmol／L不同浓度的PA，分别在PA诱导24、48、72、96h后。甲基噻唑基四唑（MTT）比色法检测细胞增殖抑制率，进一步用免疫细胞化学检测胶质纤维酸性蛋白（GFAP）的表达变化，末端脱氧核糖核酸转移酶介导的脱氧尿嘧啶核苷酸缺口末端标记（TUNEL）检测细胞凋亡。结果PA显著抑制c6细胞增殖，随药物浓度的增加和作用时间的延长，细胞增殖抑制率增加。PA作用后GFAP表达增强。随PA浓度和作用时间的增加，细胞凋亡率增加。结论PA对胶质瘤C6细胞有显著的增殖抑制和诱导凋亡作用，呈时间剂量依赖性。     

Arachidonic acid metabolism in cultured astrocytes from rat embryo and in C6 glioma cells

Arachidonic acid metabolism in primary cultures of astroglial cells prepared from cerebra of rat embryos was examined. Arachidonic acid was mainly metabolized through the lipoxygenase pathway and the major metabolites formed were 12-hydroxyeicosatetraenoic acid (12-HETE), 11-HETE and 15-HETE. By contrast, in C6 cells, which are considered to be of astroglial origin, arachidonic acid was mainly metabolized through the cyclooxygenase pathway and the major metabolites formed were prostaglandin (PG)E2, PGF2 alpha and thromboxane B2.     

Regulation of c-fos gene expression by lipopolysaccharide and cycloheximide in C6 rat glioma cells

The effect of lipopolysaccharide (LPS) on the expression of immediate early genes, such as c-fos and c-jun, was examined in C6 rat glioma cells. LPS (1 microg/ml) alone did not affect c-fos mRNA level. LPS, however, transiently increased c-jun mRNA level. Cycloheximide (CHX, 20 microM), a protein synthesis inhibitor, alone caused increases of c-fos and c-jun mRNA levels. LPS showed a potentiating effect in the regulation of c-fos mRNA level, whereas LPS showed an additive action for the regulation of CHX-induced c-jun mRNA expression. To determine if CREB and mitogen-activated protein kinases (MAPKs) are involved in the regulation of c-fos mRNA expression by LPS and CHX, Western blot was carried out using the phosphorylated form of antibodies against ERK, JNK, p38, and CREB. LPS transiently increased the phosphorylation of p38-MAPK and CREB. In addition, LPS alone elevated phosphorylation of ERK (p44/p42) MAPK in a time-dependent manner. Furthermore, LPS plus CHX enhanced phosphorylation of ERK, p38, and CREB in a synergistic manner. Our results suggest that the phosphorylation of ERK, p38, and CREB may be involved in the regulation of synergistic c-fos mRNA expression induced by LPS plus CHX in C6 rat glioma cells.     

Dehydroepiandrosterone inhibits the death of immunostimulated rat C6 glioma cells deprived of glucose

Pretreatment of interferon-gamma and lipopolysaccharides made C6 glioma cells highly vulnerable to glucose deprivation. Neither 12 h of glucose deprivation nor 2-day treatment with interferon-gamma (100 U/ml) and lipopolysaccharides (1 microg/ml) altered the viability of C6 glioma cells. However, significant death of immunostimulated C6 glioma cells was observed after 5 h of glucose deprivation. The augmented death was prevented by dehydroepiandrosterone (DHEA) treatment during immunostimulation, but not by DHEA treatment during glucose deprivation. DHEA reduced the rise in nitrotyrosine immunoreactivity, a marker of peroxynitrite, and superoxide production in glucose-deprived immunostimulated C6 glioma cells. DHEA, however, did not protect glucose-deprived C6 glioma cells from the exogenously produced peroxynitrite by 3-morpholinosydnonimine. Further, DHEA did not alter the production of total reactive oxygen species and nitric oxide in immunostimulated C6 glioma cells. Superoxide dismutase (SOD) and the synthetic SOD mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin inhibited the death of glucose-deprived immunostimulated C6 glioma cells. In addition, a superoxide anion generator paraquat reversed the protective effect of DHEA on the augmented death. The data indicate that DHEA prevents the glucose deprivation-evoked augmented death by inhibiting the production of superoxide anion in immunostimulated C6 glioma cells.