Identification of caveolae and caveolin in C6 glioma cells

Caveolae (CAV) constitute a novel subcellular transport vesicle that has received special attention based on its proven and postulated participation in transcytosis, potocytosis, and in cell signaling events. One of the principal components of CAV are caveolin protein isoforms. Here, we have undertaken the immunochemical identification of CAV and the known caveolin isoforms (1alpha, 1beta, 2 and 3) in cultured rat C6 glioma cells. Immunoblot analysis revealed that particulate fractions from rat C6 glioma cells express caveolin-1 and caveolin-2. The relative detergent-insolubility of these caveolin isoforms was also determined by Western blot analysis. Indirect immunofluorescence analysis with caveolin-1 and -2 antibodies revealed staining patterns typical of CAV's known subcellular distribution and localization. For both caveolin isoforms immunocytochemical staining was characterized by intensely fluorescent puncta throughout the cytoplasm and diffuse micropatches at the level of the plasmalemma. Perinuclear staining was also detected, consistent and suggestive of caveolin's localization in the trans Golgi region. The caveolin-1 and -2 immunoreactivity seen in Western blots and immunocytochemically is related to structurally relevant CAV as supported by the isolation of caveolin-enriched membrane complexes using two different methods. Light-density, Triton X-100-insoluble caveolin-1- and caveolin-2-enriched fractions were obtained after fractionation of rat C6 glioma cells and their separation over 5-40% discontinuous sucrose-density gradients. Similar fractions were obtained using a detergent-free, sodium carbonate-based fractionation method. These results further support the localization of CAV and caveolins in glial cells. In addition, they demonstrate that cultured C6 glioma cells can be useful as a model system to study the role of CAV and caveolins in subcellular transport and signal transduction events in glial cells and the brain.     

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.     

自噬在ALA-PDT诱导C6胶质瘤细胞死亡中的作用机制

目的 探讨自噬在光敏剂5-氨基乙酰丙酸(aminolaevulinic acid,ALA)介导的光动力治疗(photodynamic therapy,PDT)C6胶质瘤细胞死亡中作用机制.方法 ALA-PDT处理后不同时间,用自噬特异染料MDC和免疫荧光法观察自噬水平变化,Western blotting检测自噬相关蛋白cathepsin B的表达水平.结果 MDC染色和免疫荧光观察可见ALA-PDT后C6胶质瘤细胞内有大量含点状荧光颗粒的自噬泡和自噬体标志物LC3绿色荧光蛋白,Western-blot示自噬相关蛋白cathepsin B表达水平明显升高,其白噬水平随AIA-PDT后时间而逐渐增强.结论 ALA-PDT可诱导C6胶质瘤细胞发生自噬,其诱导胶质瘤细胞发生自噬的机制可能与溶酶体酶cathepsin B大量释放有关.     

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...     

Regulation of kynurenic acid synthesis in C6 glioma cells

Studies with brain slices have provided evidence that synthesis of kynurenic acid (KYNA) from kynurenine (KYN), which occurs in astrocytes, is modulated by changes in the ionic composition of the medium and the presence of depolarizing agents or the excitatory amino acid glutamate (Glu). The present study analyzed the effects of changes in incubation medium on KYNA synthesis in cultured C6 glioma cells. The synthesis was not affected by omission of Na(+) and raising K(+) concentration to 50 mM, conditions that in brain slices stimulate or inhibit KYNA formation, respectively. KYNA synthesis in C6 cells was inhibited by the absence of Ca(2+), which contrasts with its Ca(2+) independence in brain slices. Also, lack of Mg(2+) and addition of a chloride channel blocker, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonate (SITS), did not affect the synthesis. KYNA synthesis in C6 cells was dose dependently inhibited by Glu. The inhibitory effect of Glu was not affected by GDPbetaS, an antagonist of metabotropic Glu receptors, the receptor class prevailing in C6 cells, suggesting that Glu acted intracellularly. NH(4)Cl and veratridine decreased KYNA production, mirroring the effects noted in brain slices. KYNA synthesis was strongly reduced in the presence of leucine (Leu), and the uptake of [(14)C]Leu was inhibited by the KYNA precursor KYN, which points to Leu as a potential endogenous modulator of KYNA formation in CNS cells.     

骨髓间充质干细胞源性神经细胞对C6胶质瘤的趋向性

目的探索骨髓间充质干细胞(BMSCs)源性神经细胞对C6胶质瘤的趋向性。方法阿尔法最低必需培养基(α-MEM)、神经细胞生长添加剂B27、碱性成纤维细胞生长因子(bFGF)、表皮细胞生长因子(EGF)和全反式维甲酸(ATRA)诱导BMSCs向神经细胞分化。体外通过Transwell系统共培养BMSCs源性神经细胞与C6胶质瘤细胞培养基,HE染色分析穿过聚醋酸纸膜微孔的细胞数量;体内在C6胶质瘤模型中移植5-溴脱氧尿嘧啶核苷(BrdU)标记的BMSCs源性神经细胞,免疫组化分析移植细胞的迁移能力。结果条件培养基诱导BMSCs分化的细胞表达神经元核抗原(NeuN)为47%±0.08%,高分子量神经丝蛋白(NF-H)为45%±0.07%及部分巢蛋白(Nestin)为10%±0.04%,体外Transwell结果显示BMSCs源性神经细胞透过聚碳酸酯膜微孔的数量明显高于对照组(P<0.05),体内移植结果显示BMSCs源性神经细胞与BMSCs对胶质瘤的迁移无明显差异(P>0.05)并呈现时间特异性(P<0.01)。结论 BMSCs源性神经细胞对C6胶质瘤有明显的趋向性。     

大鼠骨髓基质干细胞治疗C6胶质瘤实验研究

目的观察骨髓基质干细胞(BMSCs)在体内环境下对C6胶质瘤的治疗效果。方法流式细胞术检测培养的BMSCs表面标记;SD大鼠生存周期绘成Kaplan-Meier图,行Log-rank Test,HE染色和BrdU免疫组化染色观察BMSCs对胶质瘤的趋向性和治疗情况。结果流式实验显示所培养细胞为BMSCs;HE染色结果示呈瘤率为100%,实验组较对照组生存时间明显延长;BrdU免疫组化染色示BMSCs存在于肿瘤组织及其周边。结论侧脑室移植BMSCs后,BMSCs可向肿瘤组织迁移,并可改善荷瘤大鼠的生存质量,明显延长其晚期生存周期。     

大鼠骨髓间充质干细胞分化的神经细胞在体外 对C6胶质瘤细胞的趋向性

目的研究大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)诱导分化后的神经细胞在体外对C6胶质瘤细胞的趋向性。方法首先采用梯度离心分离BMSCs,在条件培养基中添加合适诱导因子诱导BMSCs分化为神经细胞,分化后的细胞进行免疫荧光细胞化学分析后与C6胶质瘤细胞采用Transwell培养板共培养,最后对迁移的细胞做统计学分析。结果 BMSC成功诱导分化为神经细胞Nestin(24.3±5.2)%、NSE(33.6±3.8)%和NeuN(41.9±4.7)%,共培养实验显示实验组迁移细胞明显多于对照组迁移细胞(p<0.05)。结论大鼠骨髓间充质干细胞分化后的神经细胞在体外对C6胶质瘤细胞具有明显趋向性。     

Improving lipoplex-mediated gene transfer into C6 glioma cells and primary neurons

The development of methodologies for gene transfer into the central nervous system is crucial for gene therapy of neurological disorders. In this study, different cationic liposome formulations were used to transfer DNA into C6 glioma cells and primary hippocampal and cortical neurons by varying the nature of the helper lipid (DOPE, Chol) or a mixture of DOPE and cholesterol (Chol) associated to DOTAP. In addition, the effect of the lipid/DNA (+/-) charge ratio, the association of the ligand transferrin to the lipoplexes, and the stage of differentiation of the primary cells on the levels of transfection activity, transfection efficiency, and duration of gene expression were evaluated. Mechanistic studies were also performed to investigate the route of delivery of the complexes into neurons. Our results indicate that DOTAP:Chol (1:1 mol ratio) was the best formulation to transfer a reporter gene into C6 glioma cells, primary hippocampal neurons, and primary cortical neurons. The use of transferrin-associated lipoplexes resulted in a significant enhancement of transfection activity, as compared to plain lipoplexes, which can be partially attributed to the promotion of their internalization mediated by transferrin. While for hippocampal neurons the levels of luciferase gene expression are very low, for primary cortical neurons the levels of transgene expression are high and relatively stable, although only 4% of the cells has been transfected. The stage of cell differentiation revealed to be critical to the levels of gene expression. Consistent with previous findings on the mechanisms of cell internalization, the experiments with inhibitors of the endocytotic pathway clearly indicate that transferrin-associated lipoplexes are internalized into primary neurons by endocytosis. Promising results were obtained in terms of the levels and duration of gene expression, particularly in cortical neurons when transfected with the Tf-associated lipoplexes, this finding suggesting the usefulness of these lipid-based carriers to deliver genes within the CNS.     

抑胶素对C6胶质瘤细胞p16蛋白表达的影响

目的探讨不同浓度的抑胶素对C6胶质瘤细胞p16蛋白表达的影响。方法体外培养C6胶质瘤细胞,应用顺铂作为对照,通过免疫细胞化学染色法检测不同浓度的抑胶素作用后细胞周期负性调控基因p16在不同时问点的蛋白表达。结果 C6胶质瘤细胞经过抑胶素处理后,随着抑胶素浓度的增加,p16蛋白的表达也明显增强。结论抑胶素能够抑制C6胶质瘤细胞的增殖,并且具有改变肿瘤细胞增殖周期的作用。     

Adaptation of C6 glioma cells to serum-free conditions leads to the expression of a mixed astrocyte-oligodendrocyte phenotype and increased production of neurite-promoting activity

C6 cells were adapted to proliferate in defined culture medium to allow the study of long-term effects of serum-free growth conditions on their phenotypic antigen expression and production of neurite promoting factors (NPFs). Cultures were grown in either Ham's F-12 or supplemented Opti-MEM-I containing 15% heat-inactivated horse serum and 2.5% fetal calf serum (serum-containing) or in supplemented OptiMEM-I alone (serum-free). Immunocytochemical and immunofluorescence techniques were used to determine the antigenic expression of A2B5, galactocerebroside (Ga1C), and glial fibrillary acidic protein (GFAP) in passage matched and sister cultures of serum and serum-free grown C6 cells. When C6 cells were grown under serum-containing conditions, two populations of cells were seen: young oligodendrocytes (A2B5+, GFAP-, GalC+ ), and mixed astrocyte-oligodendrocyte phenotype (A2B5+, GFAP-, Ga1C+). After adaptation of the C6 cells to serumfree conditions over 2–3 passages, only one population of cells was observed, the mixed astrocyte-oligodendrocyte phenotype. The serum-free conditions also resulted in, greater staining of the C6 cells. Conditioned media from the two growth conditions were fractionated by ultrafiltration into two fractions: components >50 kDa and components of 10–50 kDa. The amount of neurite promoting activity seen between the two culture conditions resulted in a 3-fold increase in NPF activity under serum-free conditions in the >50 kDa fraction. The 10–50 kDa fraction only expressed NPF activity if obtained from the serumgrown C6 cells. This alteration in NPF activity appears to be the result of the phenotypical alteration of the C6 cells, and may suggest that the NPF activities from the two culture conditions may not be identical. © 1995 Wiley-Liss, Inc.     

Identification of a collagen potentiated neurite promoting factor isolated from C6 glioma cells

The C6 glial cell line has been used as a model cell system for the investigation of new glial produced neurotrophic and neurotropic molecules. By using the C6 cell line grown in a defined medium on collagen, this laboratory has isolated a distinct neurite promoting factor (NPF) that is potentiated by the presence of collagen (CPNPF). We have observed that C6 cells cultured in a defined medium on collagen (rat type-I) slowed their growth rate and expressed an astrocytic- or oligodendrocytic-like morphology. CPNPF, at this state of purity, appears to be a distinct NPF which induces neurite outgrowth (neurites of 1 or more somal diameters) in PC12 cells. These neurite promotion effects, however, appear to support the neuron morphology for only a short period (4 days) of time without the presence of neurotrophic factor (NTF). The neurite promoting activity is ineffective in inducing neurite outgrowth using mouse neuroblastoma cells (neuro-2a). CPNPF appears to be a heat stable protein whose activity does not depend on the presence of intact collagen, heparin sulfate proteoglycan (HSPG), or chondroitin sulfate proteoglycan (CSPG). Exposure to dissociative conditions results in a loss of neurite promoting activity. CPNPF is not a glycoprotein that contains an accessible α-D-mannopyranosyl, α-D-glucopyranosyl, or a sterically related residue (hydroxyl groups in the C-3,4, and 5 positions). Although these residues are not present on all glycoproteins, it does indicate that CPNPF is most likely not a glycoprotein. CPNPF activity is not blocked by neutralizing antibodies directed toward NGF, β-FGF, IL-1β, IL-6, TGF-β2, TGF-β1.2, TGF-β3, TGF-β5, or EGF. CPNPF appears to either be oligomeric protein or a complex of proteins. On the basis of indirect evidence, it does not appear to be glial derived protease nexin-I. The alteration in morphology of the C6 glial cell line by serum-free conditions in the presence of collagen may have induced the production of a potentially new NPF not seen by previous investigators. © 1993 Wiley-Liss, Inc.     

Induction of blood-brain barrier properties in cultured brain capillary endothelial cells: comparison between primary glial cells and C6 cell line

The communication between glial cells and brain capillary endothelial cells is crucial for a well-differentiated blood-brain barrier (BBB). It has been suggested that in vitro primary glial cells (GCs) be replaced by the glial C6 cell line to standardise the model further. This study compares directly the structural and functional differentiation of bovine brain capillary endothelial cells (BBCECs) induced by co-culture with rat primary GCs or C6 cells, for the first time. Trans-endothelial electrical resistance (TEER) measurements showed that under no condition were C6 cells able to reproduce TEER values as high as in the presence of GCs. At the same time, permeability of the BBCECs to both radioactive sucrose and FITC-inulin was 2.5-fold higher when cells were co-cultured with C6 than with GCs. Furthermore, immunocytochemistry studies showed different cell morphology and less developed tight junction pattern of BBCECs co-cultured with C6 toward GCs. Additionally, studies on P-glycoprotein (P-gp) showed much lower P-gp presence and activity in BBCECs co-cultured with C6 than GCs. Both VEGF mRNA expression and protein content were dramatically increased when compared with GCs, suggesting that VEGF could be one of the factors responsible for higher permeability of BBB. Our results clearly indicate that, in the presence of the glial C6 cell line, BBCECs did not differentiate as well as in the co-culture with primary GCs at both structural and functional levels.     

Signaling effects of α-thrombin and SFLLRN in rat glioma C6 cells

Effects of thrombin on brain cells, including change of neurite outgrowth and astrocyte shape, are described, but the molecular mechanisms are unclear. We investigated the effects of human α-thrombin and a six amino acid thrombin receptor activating peptide (TRAP-6, SFLLRN) on [Ca²⁺]₁, phosphoinositide hydrolysis, and protein kinase C in rat glioma C₆ cells. Stimulation of C₆ cells with both α-thrombin and TRAP-6 resulted in [Ca²⁺]₁ mobilization, [³H]Inositol phosphate response, and enhanced immunoreactivity of the protein kinase C (PKC) isoenzymes α, β, γ, δ, and ϵ. Results suggest that α-thrombin and TRAP-6 activate at least partially the same intracellular signaling pathways in rat glioma C₆ cells, which is evidence for involvement of “tethered ligand” receptor in thrombin induced signaling in glioma C₆ cells. © 1996 Wiley-Liss, Inc.     

Effect of growth factors on the in vitro growth and differentiation of early and late passage C6 glioma cells

The effect of different hormones and growth factors was assayed on the in vitro growth and enzymatic activities of 2′,3′-cyclic nucleotide 3′phosphohydrolase (CNP) and glutamine synthetase (GS) of rat glioma C6 cells at two different passages in culture. Young cultures (passage 26), mainly oligodendrocytic, and older cultures (passage 134), predominantly astrocytic, were treated with 10 μM dexamethasone, 20 ng/ml transforming growth factor alpha (TGFα), 10 ng/ml insulin, 20 ng/ml platelet-derived growth factor (PDGF), and 20 ng/ml, epidermal growth factor (EGF) in serum-free chemically defined media. In vitro growth rate was measured in terms of DNA content, by a fluorometric method of diaminobenzoic acid, and rate of DNA synthesis by ³H-thymidine incorporation. CNP activity (marker for in vitro oligodendrocytes) and GS activity (marker for astrocytes) were determined spectrophotometrically. Dexamethasone reversibly and significantly inhibited growth of C6 glioma in early and late passages. PDGF and insulin promoted in vitro growth only in late passage but not in early passage cells, whereas EGF and TGFα did not significantly affect growth. An increase in CNP activity was observed in early passage cells under the effect of PDGF and insulin. The increase in GS activity induced by insulin and dexamethasone suggests a differentiating role for these factors in C6 glioma cells. These results further present the C6 glioma cell line as a useful model for studies on glial cell properties and responsiveness in culture and support its use in experimental aging in vitro.     

Src抑制的蛋白激酶C底物在细胞因子诱导的C6胶质瘤细胞中表达的改变

目的研究肿瘤坏死因子（TNF-α）对培养的C6胶质瘤细胞中Src抑制的蛋白激酶C底物（Src-suppressed C Kinase Substrate,SSeCKS）表达的影响。方法根据TNF-α刺激时间与浓度的差异,将培养的C6胶质瘤细胞随机分为TNF-α时间刺激组与浓度刺激组,运用实时荧光定量PCR（Realtime PCR）、免疫印迹和免疫细胞化学法分析SSeCKS的表达变化和亚细胞定位。结果细胞因子TNF-α可引起C6胶质瘤细胞中蛋白激酶C（Protein kinase C,PKC）底物的广泛磷酸化,并以时间及浓度依赖的方式上调PKC底物SSeCKS的表达。免疫细胞化学分析显示,正常情况下,SSeCKS散在分布于细胞质,浓集于细胞伸长的足突中。TNF-α刺激后,SSeCKS向核周迁移。这些改变可被PKC的抑制剂Ro-31-8220部分抑制。结论TNF-α可诱导C6胶质瘤细胞中PKC的活性,上调SSeCKS表达,这些改变与PKC的活性相关,提示SSeCKS可能参与胶质细胞中炎症信号的转导。     

2-甲氧基雌二醇对C6脑胶质瘤细胞增殖与凋亡的作用

目的 探讨2-甲氧基雌二醇对C6脑胶质瘤细胞增殖及凋亡的影响。方法 以不同浓度的2-甲氧基雌二醇分别作用C6胶质瘤细胞不同时间,采用甲基噻唑蓝比色法（MTT）检测细胞活性,流式细胞仪检测细胞凋亡和细胞周期,并用光镜及电镜观察细胞形态学变化。结果 经2-甲氧基雌二醇作用后,C6胶质瘤细胞活性受抑制,且呈时间依赖性,作用组与对照组之间的差异具有统计学意义（P〈0.05）。此外,2-甲氧基雌二醇还可诱导C6胶质瘤细胞凋亡,凋亡率在作用组与对照组之间存在显著差异（P〈0.05）;细胞周期检测发现作用组G1及S期细胞减少,G2期细胞增多,与对照组存在显著差异（P〈0.05）。结论 2-甲氧基雌二醇可抑制C6胶质瘤细胞生长,诱导C6胶质瘤细胞凋亡。     

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.     

砷剂对胶质瘤细胞的促凋亡作用

目的 研究三氧化二砷(As2O3)对不同胶质瘤细胞系的作用以及机制.方法 应用MTT法观察As2O3对细胞生长的影响,并用透射电镜、Hoechst 33342/PI双染荧光和TUNEL观察C6胶质瘤细胞与9L胶质肉瘤细胞凋亡的形态变化;Annexin-v-FITC/PI双标记法检测细胞凋亡率.结果 MTF法发现As2O3在0.5-8.0μmol/L的浓度均可显著抑制C6与9L胶质瘤细胞株的生长;透射电镜、Hoechst 33342/PI双染荧光和TUNEL观察均显示两种胶质瘤细胞发生了显著的凋亡形态改变;Annexin-v-FITC/PI法检测显示随As2O3浓度的增大和时间的延长,C6与9L胶质瘤细胞株的凋亡率明显上升,而在相同时间及浓度下9L胶质瘤细胞株凋亡率要小于C6胶质瘤细胞株.结论 As3O3可诱导C6和9L胶质瘤细胞株产生凋亡,并且其作用具有选择性.