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1.
Analysis of the calcium-modulated proteins, S100 and calmodulin, and their target proteins during C6 glioma cell differentiation 总被引:4,自引:2,他引:2
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We have analyzed the levels, subcellular distribution, and target proteins of two calcium-modulated proteins, S100 and calmodulin, in differentiated and undifferentiated rat C6 glioma cells. Undifferentiated and differentiated C6 cells express primarily the S100 beta polypeptide, and the S100 beta levels are four-fold higher in differentiated compared to undifferentiated cells. Double fluorescent labeling studies of undifferentiated cells demonstrated that S100 beta staining localized to a small region of the perinuclear cytoplasm and colocalized with the microtubule organizing center and Golgi apparatus. Analysis of differentiated C6 cells demonstrated that S100 beta distribution and S100 beta-binding protein profile changed significantly upon differentiation. In addition, the brain-specific isozyme of one S100-binding protein, fructose-1,6-bisphosphate aldolase C, can be detected in differentiated but not undifferentiated C6 cells. While changes in the subcellular distribution of calmodulin were not observed during differentiation, calmodulin levels and calmodulin-binding protein profiles did change. Altogether these data suggest that S100 beta and calmodulin regulate different processes in glial cells and that the regulation of the expression, subcellular distribution, and target proteins of S100 beta and calmodulin during differentiation is a complex process which involves multiple mechanisms. 相似文献
2.
Nardin P Tramontina F Leite MC Tramontina AC Quincozes-Santos A de Almeida LM Battastini AM Gottfried C Gonçalves CA 《Neurochemistry international》2007,50(5):774-782
S100B is an astrocyte calcium-binding protein that plays a regulatory role in the cytoskeleton and cell cycle. Moreover, extracellular S100B, a marker of glial activation in several conditions of brain injury, has a trophic or apoptotic effect on neurons, depending on its concentration. Hyperglycemic rats show changes in glial parameters, including S100B expression. Here, we investigated cell density, morphological and biochemical alterations in primary cortical astrocytes from rats and C6 glioma cells cultured in high-glucose medium. Astrocytes and C6 glioma cells have a reduced content of S100B and glial fibrillary acidic protein when cultured in a high-glucose environment, as well as a reduced content of glutathione and cell proliferation rate. Although these cells have been used indistinctly to study S100B secretion, we observed a contrasting profile of S100B secretion in a high-glucose medium: a decrease in primary astrocytes and an increase in C6 glioma cells. Based on the in vitro neurotrophic effects of the S100B protein, our data suggest that chronic elevated glucose levels affect astrocyte activity, reducing extracellular secretion of S100B and that this, in turn, could affect neuronal activity and survival. Such astrocyte alterations could contribute to cognitive deficit and other impairments observed in diabetic patients. 相似文献
3.
Meroni SB Pellizzari EH Cánepa DF Cigorraga SB 《The Journal of steroid biochemistry and molecular biology》2000,75(4-5):307-313
In the present study, a possible role of a ceramide-dependent pathway in the regulation of Leydig cell function was investigated. Intracellular ceramide levels were increased by: (a) adding ceramide analogs; (b) inhibiting ceramidase activity; and (c) adding sphingomyelinase (SMase). The cell-permeable ceramide analogs N-acetyl-, N-hexanoyl- and N-octanoylsphingosine (C2, C6 and C8) were used. As inhibitor of ceramidase activity 1S,2R-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (MAPP) was used. Sphingomyelinase from S. aureus origin was utilized. Leydig cells were cultured for 3 or 24 h with or without the different drugs (10 microM) and SMase (0.3 U/ml) in the presence or absence of hCG (10 ng/ml). Basal testosterone production was not modified under any of the experimental conditions. A decrease in hCG-stimulated testosterone production was observed at 3 and 24 h in all cases. The inactive analog (N-hexanoyl dihydrosphingosine) did not produce inhibition in hCG-stimulated testosterone production. TNFalpha and IL1beta, two possible inducers of sphingomyelin hydrolysis, produced similar effects on hCG-stimulated testosterone production. In experiments performed in the presence of C6, inhibition in hCG-stimulated cAMP production was observed. The inhibitory effect of ceramide was also observed in dbcAMP-stimulated cultures indicating that this pathway inhibits post-cAMP formation events. To study possible loci for the action of ceramide on the steroidogenic pathway, cells were incubated with C6 and MAPP in the presence of different testosterone precursors. The drugs inhibited testosterone produced from 22(R)-hydroxycholesterol (22R-OHChol), pregnenolone and 17alpha-hydroxyprogesterone (17OHP4) but not from androstenedione (Delta4). These results suggest that a ceramide-dependent pathway regulates hCG-stimulated Leydig cell steroidogenesis at the level of cAMP production and at post-cAMP events. 相似文献
4.
Sense and antisense modification of glial alpha B-crystallin production results in alterations of stress fiber formation and thermoresistance 总被引:1,自引:0,他引:1
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《The Journal of cell biology》1994,125(6):1385-1393
The phenotypic effects of selectively altering the levels of alpha B- crystallin in cultured glial cells were analyzed using sense and antisense approaches. Rat C6 glioma cells and human U-373MG glioma cells were transfected with a rat alpha B-crystallin sense cDNA or an antisense cDNA regulated by a Rous sarcoma virus promoter to alter cellular levels of alpha B-crystallin. The antisense strategy resulted in decreased alpha B-crystallin levels, as revealed by Western blot and immunocytochemical analyses. The reduced alpha B-crystallin expression was accompanied by alterations in cellular phenotype: (a) a reduction of cell size and/or a slender cell morphology; (b) a disorganized microfilament network; and (c) a reduction of cell adhesiveness. Like HSP27, the presence of additional alpha B-crystallin protein confers a thermoresistant phenotype to stable transfectants. Thus, alpha B- crystallin in glioma cells plays a role in their thermal resistance and may contribute to the stability of cytoskeletal organization. 相似文献
5.
Striking paucity of HLA-A, B, C and beta 2-microglobulin on human neuroblastoma cell lines 总被引:11,自引:0,他引:11
L A Lampson C A Fisher J P Whelan 《Journal of immunology (Baltimore, Md. : 1950)》1983,130(5):2471-2478
Monoclonal antibodies to beta 2-microglobulin (beta 2m), and to the native two-chain molecule, were used to assess the expression of the HLA-A, B, C molecules on human neuroblastoma-derived cell lines. In radioimmuno-, cytotoxic, and microscopic assays, employing fresh and fixed cells, neuroblastoma cells show at best weak activity as compared to glial or lymphoid cells. In binding inhibition assays, neuroblastoma extracts were 200- to 1800-fold less efficient in inhibiting the antibodies than were glial or lymphoid extracts. Immunoprecipitation and SDS-PAGE analysis confirmed that a beta m-like chain is synthesized by the neuroblastoma cells, but the HLA chain could not be visualized by this technique. HLA-A, B, C and beta 2m levels are known to vary among tissues and cell lines. Yet the magnitude of the differences between the neuroblastoma and lymphoid lines is much greater than the reported differences in expression between some of these same lymphoid lines and many other nonlymphoid malignant or nonmalignant cell types. Metastatic neuroblastoma tumor in bone marrow also showed weak HLA-A, B, C activity, with the cells appearing negative in microscopic assays. Possible clinical implications are discussed. 相似文献
6.
Levels and Distribution of the Calcium-Modulated Proteins S100 and Calmodulin in Rat C6 Glioma Cells 总被引:5,自引:3,他引:2
To understand better the mechanisms involved in the transduction of a calcium signal into an intracellular response via multiple calcium-modulated proteins, we have examined the calcium-modulated proteins, S100 and calmodulin, and their intracellular targets in rat C6 glioma cells. Subconfluent, confluent, and postconfluent C6 cells contain predominantly, if not exclusively, the S100 beta polypeptide. The level of S100 beta in C6 cells increases approximately 20-fold from subconfluency to postconfluency whereas the level of calmodulin increases only about two-fold. The subcellular distribution of S100 beta and calmodulin in mitotic cells is similar. However, the subcellular distribution of these proteins in interphase cells is different and appears to change with cell density. Gel overlay analysis demonstrated that the S100- and calmodulin-binding protein profiles are significantly different and that some of the binding proteins appear to change in intensity with cell density. These data demonstrate that S100 beta is the predominant S100 polypeptide in C6 cells and suggest that changes in S100 beta and S100 beta-binding proteins may be involved in regulating S100-mediated intracellular processes in C6 cells. Our studies also suggest that the levels of S100 and calmodulin may be differentially regulated in C6 cells. 相似文献
7.
8.
Isolated sensory neurons in vitro do not contain or synthesize S100, whereas glial cell precursor populations do. These precursor cells, when isolated from other cell types, produce low levels of S100 but never undergo the developmental transition to produce high levels of S100. When glial cell precursors are combined with isolated, live or paraformaldehyde-fixed sensory neurons, the precursor cells do undergo the second transition, and accumulate high levels of S100. Peroxidase-anti-peroxidase immunohistochemical staining for S100 confirms previous conclusions (B. Holton and J. A. Weston, 1982, Develop. Biol.89, 64–71) that only those glial cells which are closely apposed to neurons contain augmented levels of S100. This stimulation appears to be specific to neuronal/glial interactions since live or fixed fibroblasts, when cocultured with glial precursor cells, do not promote accumulation of S100 by the glial cells. 相似文献
9.
10.
We have isolated two colcemid-resistant mutant sublines, CMR (7A) and CMR (7B), from rat glial cells, C6, using multiple consecutive selections with increasing concentrations of colcemid. The mutant sublines show a decreased uptake of [3H]colchicine but have no apparent defect in the cytoplasmic binding of the drug. The synthesis of the brain-specific S100 protein is less sensitive to colcemid inhibition in the mutant cell lines than in parental C6 cells, suggesting that colcemid must enter the cell to inhibit S100 protein synthesis. 相似文献
11.
Previous studies from this laboratory (Schroy, P., Rifkin, J., Coffey, R.J., Winawer, S., and Friedman, E. (Cancer Res., 50: 261-265, 1990; Schroy, P.C., Winawer, S., and Friedman, E. Cancer Lett., 48: 53-58, 1989) found that a 7-day treatment of the human colon carcinoma cell line HT29 with the differentiation agent hexamethylene bisacetamide (HMBA) induces both a 4-5-fold increase in transforming growth factor beta 1 (TGF beta 1) mRNA levels and reduced tumorigenicity in vivo. A series of 15 cloned lines with different commitments to differentiation has been isolated from 20-day HMBA-treated HT29 cells, maintained without HMBA, and utilized to study the role of TGF beta 1 in colon carcinoma differentiation. Two such lines, HD6 and HD8, differentiate to 97 and 76% mucus-secreting goblet cells, respectively, in columnar monolayers in postconfluent culture. Both HD6 and HD8 cells exhibit low TGF beta 1 mRNA levels, little different from the undifferentiated HT29 parental line, and exhibit no growth modulation in response to exogenous TGF beta 1. In contrast, two other lines, HD3 and HD4, differentiate to fluid-transporting enterocytic cells with functional brush borders and exhibit autocrine-negative growth response to TGF beta 1. Both lines express TGF beta 1 mRNA at levels 11-12-fold higher than the parental line and respond to exogenous TGF beta 1 by growth inhibition. HD3 cells secrete biologically active TGF beta 1 into conditioned media, which inhibited growth of a TGF beta 1-sensitive mink cell line. This inhibition was blocked by antisera to TGF beta 1, proving the specificity of the inhibition. A range of concentrations of this TGF beta 1 antiserum stimulated HD3 cell growth in a dose-dependent manner, further documenting the autocrine-negative response of the cells to TGF beta 1. Another cell line, HI1, was blocked in enterocytic differentiation. HI1 cells synthesized as much TGF beta 1 mRNA as HD3 and HD4 cells, yet they responded to exogenous TGF beta 1 with less growth inhibition, suggesting some impairment in their response to TGF beta 1. A third class of response to TGF beta 1 was exhibited by the HP1 cell line, which was resistant to HMBA-induced differentiation, remaining undifferentiated with a multilayered growth pattern. HP1 cells synthesized TGF beta 1 mRNA at levels over 20 times the parental level but were stimulated to divide by TGF beta 1, exhibiting autocrine-positive response to this growth factor.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
12.
Growth factors play an important role in proliferation and differentiation of malignant brain gliomas in humans. Glial cell line-derived neurotrophic factor (GDNF) has been shown recently to be highly expressed in human glioblastomas and in rat glial cell lines B49 and C6. The aim of the present study was to knockdown GDNF, its receptor GFR-alpha1, and the related family member persephin by using antisense oligonucleotides and to observe the effects on cell proliferation. To enhance cellular uptake into C6 glioma cells, 15-mer phosphorothioate oligonucleotides were complexed with the cationic lipid Lipofectamine. The complex was applied for 3 x 12 hours to C6 glioma cells, and cells were allowed to recover for 24 hours after each transfection and then analyzed. This protocol markedly reduced GDNF and GFR-alpha1 protein levels in C6 glioma cells compared with control oligonucleotides. Knockdown of C6 cells with GDNF and GFR-alpha1 but not with persephin antisense oligonucleotides significantly decreased the number of C6 glioma cells and also inhibited the incorporation of bromodeoxyuridine as a sign of reduced DNA synthesis. In conclusion, it is shown that GDNF but not persephin is a potent proliferation factor for rat glioma cells. Knockdown of GDNF using antisense oligonucleotides complexed with lipids as carriers may be useful in gene therapeutic approaches in vitro and possibly also in vivo. 相似文献
13.
Quincozes-Santos A Abib RT Leite MC Bobermin D Bambini-Junior V Gonçalves CA Riesgo R Gottfried C 《Molecular and cellular biochemistry》2008,314(1-2):59-63
We investigated the effect of the atypical neuroleptic risperidone on morphology and S100B secretion in C6 glioma cells, considering the putative involvement of astroglial cells in neuropsychiatric disorders. In the presence of high experimental doses of risperidone, C6 cells become stellate, with process-bearing cells and partial retraction of the cell body followed by detachment from the adhesion surface with practically no cell death. These results indicate that risperidone is able to interfere with C6 cell adhesion without toxic effects. RhoA activator LPA prevented the effects of risperidone on cell morphology. From 6 h risperidone induced a statistically significant increment of about 80% in S100B secretion. These data contribute to the proposal that glial cells are targets of risperidone, which could be involved in the therapeutic response of risperidone to improve autism symptoms. 相似文献
14.
Sadok A Bourgarel-Rey V Gattacceca F Penel C Lehmann M Kovacic H 《Biochimica et biophysica acta》2008,1783(1):23-33
Reactive oxygen species are well-known mediators of various biological responses. Recently, new homologues of the catalytic subunit of NADPH oxidase have been discovered in non-phagocytic cells. These new homologues (Nox1-Nox5) produce low levels of superoxides compared to the phagocytic homologue Nox2/gp91phox. Using Nox1 siRNA, we show that Nox1-dependent superoxide production affects the migration of HT29-D4 colonic adenocarcinoma cells on collagen-I. Nox1 inhibition or down-regulation led to a decrease of superoxide production and alpha 2 beta 1 integrin membrane availability. An addition of arachidonic acid stimulated Nox1-dependent superoxide production and HT29-D4 cell migration. Pharmacological evidences using phospholipase A2, lipoxygenases and protein kinase C inhibitors show that upstream regulation of Nox1 relies on arachidonic acid metabolism. Inhibition of 12-lipoxygenase decreased basal and arachidonic acid induced Nox1-dependent superoxide production and cell migration. Migration and ROS production inhibited by a 12-lipoxygenase inhibitor were restored by the addition of 12(S)-HETE, a downstream product of 12-lipoxygenase. Protein kinase C delta inhibition by rottlerin (and also GO6983) prevented Nox1-dependent superoxide production and inhibited cell migration, while other protein kinase C inhibitors were ineffective. We conclude that Nox1 activation by arachidonic acid metabolism occurs through 12-lipoxygenase and protein kinase C delta, and controls cell migration by affecting integrin alpha 2 subunit turn-over. 相似文献
15.
S100 beta stimulates calcium fluxes in glial and neuronal cells. 总被引:7,自引:0,他引:7
The glial-derived protein S100 beta can act as a mitogen or a neurotrophic factor, stimulating proliferation of glial cells or differentiation of immature neurons. We report here that dimeric S100 beta evokes increases in intracellular free calcium concentrations ([Ca2+]i) in both glial cells and neuronal cells. The [Ca2+]i increase exhibited a rapid transient component which was not affected by removal of extracellular calcium and a sustained component which appeared to require influx of extracellular calcium through Ni(2+)-sensitive channels. S100 beta also stimulated hydrolysis of phosphoinositides, suggesting a mobilization of calcium from intracellular stores. These data suggest that although the final biological responses of neuronal and glial cells to S100 beta are different, transduction of the S100 beta signal in both cell types involves changes in [Ca2+]i. 相似文献
16.
The influence of prostaglandins on glial functions and, more specifically, on glial activation is not well understood. We report here that prostaglandin E(2) (PGE(2)), one of the major prostaglandins produced in the brain, acts as a potent and selective inhibitor of tumor necrosis factor alpha (TNF-alpha) production in lipopolysaccharide-stimulated primary microglia and the microglial cell line BV-2. The IC(50) for this effect is 1 nM, and 100 nM PGE(2) suppresses TNF-alpha production by >95%. More detailed studies of BV-2 cells show that PGE(2) also prevents the secretion of interleukin (IL)-6 but does not significantly modify lipopolysaccharide-stimulated expression of cyclooxygenase-2, pro-IL-1beta, or inducible nitric oxide synthase. PGE(2) appears to act primarily at the level of translation or protein stability, because TNF-alpha and IL-6 mRNA levels were only modestly decreased at high PGE(2) concentrations; concomitantly with this inhibition, PGE(2) up-regulated the levels of IL-1beta mRNA. The effects of PGE(2) could be largely mimicked by 8-bromo-cAMP, suggesting that, as in other cell types, PGE(2) action is mediated at least in part by a rise in intracellular cyclic AMP. However, the protein kinase A inhibitor H89 only partially reversed the inhibition of TNF-alpha production by PGE(2), implying that the PGE(2) effect in BV-2 cells is mediated through both protein kinase A-dependent and -independent pathways. 相似文献
17.
In rat pancreatic islets and insulin-producing cell lines, IL-1beta induces expression of inducible nitric oxide synthase and NO production leading to impairment of glucose-stimulated insulin release and decreased cell survival. NADPH is an obligatory cosubstrate for iNOS synthesis of NO. We hypothesized that IL-1beta stimulates an increase in activity of NADPH-producing enzyme(s) prior to NO production and that this increase is necessary for NO production. Using rat insulin-secreting RINm5F cells, we found that (1) IL-1beta caused a biphasic change in the NADPH level (increased by 6 h and decreased after prolonged incubation in the presence of 2 ng/mL IL-1beta); (2) IL-1beta stimulated increased activity of glucose-6-phosphate dehydrogenase (G6PD) in a time- and dose-dependent manner, and G6PD expression was increased by about 80% after exposure to 2 ng/mL IL-1beta for 18 h: (3) IL-1beta-stimulated NO production was positively correlated with increased G6PD activity; (4) IL-1beta did not cause any significant change in enzyme activity of another NADPH-producing enzyme, malic enzyme; (5) IL-1beta-induced NO production was significantly reduced either by inhibiting G6PD activity using an inhibitor of G6PD (dehydroepiandrosterone) or by inhibiting G6PD expression using an antisense oligonucleotide to G6PD mRNA; and (6) IL-1beta stimulated a decrease in the cAMP level. 8-Bromo-cAMP caused decreased G6PD activity, and the protein kinase A inhibitor H89 led to a increase in G6PD activity in RINm5F cells. In conclusion, our data show that IL-1beta stimulated G6PD activity and expression level, providing NADPH that is required by iNOS for NO production in RINm5F cells. Also, inhibition of the cAMP-dependent PKA signal pathway is involved in an IL-1beta-stimulated increase in G6PD activity. 相似文献
18.
S Dedhar 《Journal of cellular physiology》1989,138(2):291-299
Recombinant human interleukin-1 beta, a mediator of osteoblastic cell function, was found to regulate the expression of the cell adhesion receptors, integrins, on human osteosarcoma cells. Interleukin-1 beta (IL-1 beta) at picomolar concentrations, specifically elevated approximately six- to tenfold the expression of the beta 1 subunit and its associated alpha subunits, but not the related vitronectin receptor, within 20 hours. Integrin beta 1 messenger RNA levels were elevated within 6 hours and peaked to tenfold higher levels after 20 hours exposure to IL-1 beta in two human osteosarcoma cell lines. The increase in the cell-surface beta 1 integrins resulted in a stronger binding of the IL-1 beta-treated cells to fibronectin. Cell growth was also inhibited by IL-1 beta, cell morphology was altered, and IL-1 beta-treated cells expressed an approximately two- to threefold higher alkaline phosphatase. This increase in alkaline phosphatase activity was found to be independent of the inhibition of cell proliferation. These data indicate that the beta 1 integrin family of cell surface receptors is a target for regulation by IL-1 beta, which also regulates cell proliferation and the expression of the osteoblastic phenotype in human osteosarcoma cells. 相似文献
19.
The synthesis of S100 protein increases toward the end of the exponential phase of growth of clonal rat glial cells C6 in monolayer culture. Moreover the synthesis of this protein can be increased by treatment of C6 cells with the lectin succinylated concanavalin A (succinyl ConA). In order to study the relationship between these two inductions of S100 protein we have isolated a cell line resistant to ConA from a population of C6 cells. The resistant cells (C6-ConAR) have less succinyl ConA receptors than C6 cells. In contrast to C6 cells, the synthesis of S100 protein does not increase in C6-ConAR cells after treatment with succinyl ConA. However in both cell types the synthesis of S100 protein increases toward the end of the exponential phase of growth. These results suggest firstly that the induction of S100 protein in C6 cells by succinyl ConA is mediated by an interaction of the lectin with its membrane receptors and secondly that the initial steps in the induction of S100 protein by the lectin are different from the initial steps in the induction of this protein which occurs toward the end of the exponential phase of growth in monolayer culture. 相似文献
20.
The synthesis of S100 protein increases toward the end of the exponential phase of growth of clonal rat glial cells C6 in monolayer culture. Moreover the synthesis of this protein can be increased by treatment of C6 cells with the lectin succinylated concanavalin A (succinyl ConA). In order to study the relationship between these two inductions of S100 protein we have isolated a cell line resistant to ConA from a population of C6 cells. The resistant cells (C6-ConAsuitr) have less succinyl ConA receptors than C6 cells.In contrast to C6 cells, the synthesis of S100 protein does not increase in C6-ConAsuitr cells after treatment with succinyl ConA. However in both cell types the synthesis of S100 protein increases toward the end of the exponential phase of growth.These results suggest firstly that the induction of S100 protein in C6 cells by succinyl ConA is mediated by an interaction of the lectin with its membrane receptors and secondly that the initial steps in the induction of S100 protein by the lectin are different from the initial steps in the induction of this protein which occurs toward the end of the exponential phase of growth in monolayer culture. 相似文献