Suppression of Glial Tumor Growth by Expression of Glial Fibrillary Acidic Protein

We examined the effect of expression of glial fibrillary acidic protein (GFAP) on the tumor growth of astrocytoma in vivo. When rat astrocytoma C6 cells were injected subcutaneously in athymic mice, the cells produced tumors that grew rapidly. The tumor growth of C6 cells transfected with GFAP cDNA was significantly reduced compared to that of control NeoC6 cells transfected only with the neomycin resistant gene. After implantation of C6 cells transfected with mutated GFAP cDNA at the phosphorylation sites, the tumor growth was suppressed similar to that of the wild GFAP transfectants. To determine whether the cell growth suppression by GFAP is specific for astroglial cells, we assessed the effect of GFAP on the cell growth of an L cell of fibroblast origin in vitro. By GFAP cDNA transfection, L cells showed morphological changes, but the cell growth was not reduced. These results suggest that GFAP is a critical regulator of the tumor growth of astrocytoma.     

Suppression by antisense mRNA demonstrates a requirement for the glial fibrillary acidic protein in the formation of stable astrocytic processes in response to neurons

The glial fibrillary acidic protein (GFAP) is a glial-specific intermediate filament protein, which is expressed in astrocytes in the central nervous system, as well as in astrocytoma cell lines. To investigate the function of GFAP, we have studied the human astrocytoma cell line, U251, which constitutively expresses GFAP and vimentin in the same 10-nm filaments. These cells respond to neurons in vitro in the same way as primary astrocytes: they withdraw from the cell cycle, support neuronal cell survival and neurite outgrowth, and they extend complex, GFAP-positive processes. To determine the role of GFAP in these responses, we have specifically suppressed its expression by stably transfecting the U251 cells with an antisense GFAP construct. Two stable antisense cell lines from separate transfections were isolated and were shown to be GFAP negative by Northern and Western blot analyses, and by immunofluorescence studies. The antisense cell lines were inhibited in their ability to extend significant glial processes in response to neurons. In culture with primary neurons, the average increase in process length of the U251 cells was nearly 400%, as compared to only 14% for the antisense transfectants. The other neuron induced responses of astrocytes, i.e., proliferative arrest and neuronal support, were not affected in these cell lines. These data support the conclusion that the glial-specific intermediate filament protein, GFAP, is required for the formation of stable astrocytic processes in response to neurons.     

Redistribution of GFAP and αB-crystallin after thermal stress in C6 glioma cell line

Summary Some intermediate filament (IF) proteins expressed in the development of glia include nestin, vimentin, and glial fibrillary acidic protein (GFAP). However, GFAP is the major intermediate filament protein of mature astrocytes. To determine the organization of GFAP in glial cells, rat GFAP cDNA tagged with enhanced green fluorescent protein (EGFP) was transfected into the rat C6 glioma cell line. After selection, two stable C6-EGFP-GFAP cell lines were established. Stable C6-EGFP-GFAP cell lines with or without heat shock treatment were analyzed by immunocytochemistry, electron microscopy, and Western blot analysis. In the transient transfection study, EGFP-GFAP transiently expressed in C6 cells formed punctate aggregations in the cytoplasm right after transfection, but gradually a filamentous structure of EGFP-GFAP was observed. The protein level of nestin in the C6-EGFP-GFAP stable clone was similar to that in the pEGFP-C1 transfected C6 stable clones and non-transfected C6 cells, whereas the level of vimentin was reduced in Western blotting. Interestingly, the expression level of small heat shock protein αB-crystallin in C6-EGFP-GFAP cells was also enhanced after transfection. Immunostaining patterns of C6-EGFP-GFAP cells showed that GFAP was dispersed as a fine filamentous structure. However, after heat shock treatment, GFAP formed IF bundles in C6-EGFP-GFAP cells. In the meantime, αB-crystallin also colocalized with IF bundles of GFAP in C6-EGFP-GFAP cells. The heat-induced GFAP reorganization we found suggested that small heat shock protein αB-crystallin may play a functional role regulating the cytoarchitecture of GFAP.     

Phosphatidylinositol 3-kinase activity is required for the expression of glial fibrillary acidic protein upon cAMP-dependent induction of differentiation in rat C6 glioma

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein expressed upon maturation of astrocytes and upregulated during reactive astrogliosis. Its expression is modulated by several growth factors and hormones. Although an upregulation of intracellular cAMP is required for the induction of GFAP expression in astrocytes, little information is available on other downstream factors of the signal transduction pathways involved in the regulation of its expression. In this communication, we identified phosphatidylinositol 3-kinase (PI 3-K) as a necessary enzyme for GFAP expression in rat C6 glioma cells. Use of the specific PI 3-K inhibitors wortmannin and LY294002 and transfection of C6 cells with a dominant negative PI 3-K construct, resulting in a decrease of the enzymatic activity of PI 3-K, inhibited the cAMP-dependent expression of GFAP. Furthermore, confocal laser scanning microscopy demonstrated that inhibition of the PI 3-K activity by LY294002 or wortmannin concomitant with induction of differentiation changes the cellular distribution leading to a pericentrosomal localization of GFAP and an altered cell shape lacking process formation. We conclude that the expression and cellular distribution of GFAP is mediated through a PI 3-K-dependent mechanism.     

Ghrelin and the growth hormone secretagogue receptor constitute a novel autocrine pathway in astrocytoma motility

Originally thought of as a stomach-derived endocrine peptide acting via its receptors in the central nervous system to stimulate food intake and growth hormone expression, ghrelin and its receptor (growth hormone secretagogue receptor (GHS-R)) are widely expressed in a number of organ systems, including cancer cells. However, the direct functional role of ghrelin and its receptor in tumors of central nervous system origin remains to be defined. Here, we demonstrate that the human astrocytoma cell lines U-118, U-87, CCF-STTG1, and SW1088 express 6-, 11-, 15-, and 29-fold higher levels of GHS-R compared with primary normal human astrocytes. The ligation of GHS-R by ghrelin on these cells resulted in an increase in intracellular calcium mobilization, protein kinase C activation, actin polymerization, matrix metalloproteinase-2 activity, and astrocytoma motility. In addition, ghrelin led to actin polymerization and membrane ruffling on cells, with the specific co-localization of the small GTPase Rac1 with GHS-R on the leading edge of the astrocytoma cells and imparting the tumor cells with a motile phenotype. Disruption of the endogenous ghrelin/GHS-R pathway by RNA interference resulted in diminished motility, matrix metalloproteinase activity, and Rac expression, whereas tumor cells stably overexpressing GHS-R exhibited increased cell motility. The relevance of ghrelin and GHS-R expression was verified in clinically relevant tissues from 20 patients with oligodendrogliomas and grade II-IV astrocytomas. Analysis of a central nervous system tumor tissue microarray revealed that strong GHS-R and ghrelin expression was significantly more common in high grade tumors compared with low grade ones. Together, these findings suggest a novel role for the ghrelin/GHS-R axis in astrocytoma cell migration and invasiveness of cancers of central nervous system origin.     

Necrotic tumor cells oppositely affect nitric oxide production in tumor cell lines and macrophages

Nitric oxide (NO) is a highly reactive free radical with profound tumoricidal activity, produced by both macrophages and tumor cells. While it has been postulated that necrotic tumor cells can augment macrophage anti-tumor action, we investigated the effect of tumor cell necrosis on NO synthesis and viability of L929 fibrosarcoma and C6 astrocytoma cell lines. The presence of necrotic tumor cells dose-dependently reduced NO production in IFN-gamma stimulated L929 cells, and rescued them from NO-dependent autotoxicity. This effect was mediated through soluble products, since it was completely preserved after blocking the contact between the necrotic and live cells. On the other hand, apoptotic tumor cells were unable to suppress IFN-gamma-triggered NO release and subsequent decrease of cell respiration in L929 cultures. Similar results were obtained with C6 astrocytoma cell line. This down-regulation of NO synthesis in response to necrotic cell products was not specific for tumor cell lines, since necrotic tumor cells markedly suppressed NO production in cytokine-stimulated primary fibroblasts and astrocytes. In contrast, both murine and rat peritoneal macrophages readily increased their basal or IFN-gamma-induced NO production when incubated with necrotic tumor cells. Taken together, these results suggest that tumor cell necrosis might promote or restrict tumor growth through suppression or enhancement of NO synthesis in tumor cells and macrophages, respectively, with net effect presumably depending on the extent of macrophage infiltration.     

Differential inducibilities of GFAP expression, cytostasis and apoptosis in primary cultures of human astrocytic tumours

Glial fibrillary acidic protein (GFAP) is an astrocytic lineage-specific intermediate filament protein, and its expression or non-expression is inversely correlated with the tumourigenecity of astrocytoma cells. To estimate the GFAP levels of astrocytes in intracranial tumour tissues, we established primary cultures from six astrocytic tumour specimens and used a double-staining flow cytometric method to detect the different levels of GFAP among these primary cultures. Although these primary cultures exhibited the same Matrigel invasiveness, their GFAP expression is inversely related to the rate of cell growth and the histologic grade of the original tumour. Phenylacetate, 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, which are potent inducers of differentiation in various cancer cells, have been examined for their effects on these primary cultures. Cytostasis was more or less caused by these compounds in all six primary cultures, but induction of GFAP was observed only in the primary culture derived from a less malignant astrocytoma specimen having the highest intrinsic GFAP level. Interestingly, this primary culture, but not others, also exhibited increased HRG-α expression after phenylacetate or sodium butyrate treatment. Loss of the inducibility of differentiation-related gene expression could be one of the events involved in the malignant progression of astrocytomas. In addition, the chemotherapeutic agent BiCNU has a killing effect on all six primary culture cells, with LD50 less than 60nM. The underlying mechanism was through the induction of apoptosis in these primary culture cells regardless of their varying malignancies of original tumours. However, unlike colon cancer and leukaemia cells, sodium butyrate could not induce apoptosis within 4 days in these astrocytic tumour cells, indicating that the cell context of different cell types indeed determined the ability of sodium butyrate to induce apoptosis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Protein tyrosine kinase-dependent regulation of adenylate cyclase and phosphatidylinositol 3-kinase activates the expression of glial fibrillary acidic protein upon induction of differentiation in rat c6 glioma

Glial fibrillary acidic protein (GFAP) is expressed upon cAMP-mediated induction of differentiation of glial progenitor cells into type II astrocytes. The protein is regulated by hormones, growth factors and cytokines but the signal transduction pathways involved in the regulation of GFAP expression are largely unknown. Specific protein kinase inhibitors were used to study their effect on the expression of GFAP in rat C6 glioma cells. Herbimycin A, a selective protein tyrosine kinase inhibitor, reduced GFAP mRNA and protein expression upon cAMP analog or beta-adrenergic receptor-mediated induction of differentiation. The latter inhibitor attenuated the elevation of cAMP by adenylate cyclase and abolished the activity of phosphatidylinositol 3-kinase (PI 3-K). These data indicate that GFAP expression is regulated by protein tyrosine phosphorylations, modulating the cAMP concentration and PI 3-K activity in C6 glioma cells.     

Growth inhibition and differentiation of C6 glioma cells on treatment with hmba.

HMBA, a differentiation inducer belonging to the class of hybrid polar compounds, is known to induce terminal differentiation of a number of leukemic and solid tumour cell lines. In this report we have shown that HMBA markedly inhibits growth of C6 glioma cells at non-cytotoxic concentrations ranging from 2.5 m m to 10 m m in a dose-dependent manner. The growth inhibitory effect can be detected as early as 18--24 h. By the sixth day the growth inhibition decreases at all the concentrations tested. Treatment with HMBA results in an accumulation of C6 cells in G0/G1 phase along with a decrease in the number of cells in S phase. HMBA induces morphological differentiation of C6 cells and increases expression of glial fibriliary acidic protein (GFAP), a marker for mature astrocytes. HMBA induces c-fos and represses cycloheximide-induced c-jun and fra-1 expression. HMBA-induced growth inhibition of C6 cells is accompanied by a decrease in Cdk4 protein levels. However, HMBA fails to sustain low Cdk4 levels, which may be responsible for HMBA's failure to sustain the growth inhibitory effect.     

Endothelins Stimulate c-fos and Nerve Growth Factor Expression in Astrocytes and Astrocytoma

Abstract: Endothelin receptors have been identified on astrocytes and astrocytoma, but their physiological significance has remained elusive. It is shown here that endothelins induce c- fos in primary cultures of mouse embryo astrocytes, as well as in two subclones of rat astrocytoma C6 cells, although with different kinetics. In addition, nerve growth factor expression is stimulated, as seen by mRNA accumulation and protein secretion, in primary astrocytes and one of the two C6 subclones, with an apparent correlation with the transience of c- fos induction. The activation of protein kinase C appears as an obligatory step during these processes, because (a) inhibition of protein kinase C by staurosporine blocks the induction by endothelin or phorbol esters of both c- fos and nerve growth factor, and (b) phorbol esterevoked down-regulation of protein kinase C completely abolishes the c- fos induction by endothelin, but not that by the β-adrenergic agonist isoproterenol, a known activator of the cyclic AMP-dependent pathway. Our results support the hypothesis that c- fos product might be implicated in nerve growth factor expression by astrocytes, and also suggest that endothelins may participate in vivo in the modulation of the glial neurotrophic activity during brain development or wound healing.     

Differentiation of embryonic stem cell to astrocytes visualized by green fluorescent protein

Green fluorescent protein (GFP) gene was transfected and expressed in murine embryonic stem (ES) cells under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Stably transfected cells were characterized by immunohistochemistry and by fluorescence microscopy. Cells containing GFP were differentiated to Type I and Type II astrocytes after induction by all-trans retinoic acid. Differentiated cells were expressed GFP and visualized by fluorescence microscopy. Differentiated cells expressed GFP were correlated with the expression of GFAP and morphological change. It demonstrates that the cell line expressed GFP can be used to trace the morphological changes of astrocytes during differentiation, and further for the isolation of astrocytes from the mixed cells differentiated from ES cell.     

A2B5+/GFAP+ Cells of Rat Spinal Cord Share a Similar Lipid Profile with Progenitor Cells: A Comparative Lipidomic Study

The central nervous system (CNS) harbors multiple glial fibrillary acidic protein (GFAP) expressing cell types. In addition to the most abundant cell type of the CNS, the astrocytes, various stem cells and progenitor cells also contain GFAP+ populations. Here, in order to distinguish between two types of GFAP expressing cells with or without the expression of the A2B5 antigens, we performed lipidomic analyses on A2B5+/GFAP+ and A2B5?/GFAP+ cells from rat spinal cord. First, A2B5+/GFAP? progenitors were exposed to the leukemia inhibitory factor (LIF) or bone morphogenetic protein (BMP) to induce their differentiation to A2B5+/GFAP+ cells or A2B5?/GFAP+ astrocytes, respectively. The cells were then analyzed for changes in their phospholipid, sphingolipid or acyl chain profiles by mass spectrometry and gas chromatography. Compared to A2B5+/GFAP? progenitors, A2B5?/GFAP+ astrocytes contained higher amounts of ether phospholipids (especially the species containing arachidonic acid) and sphingomyelin, which may indicate characteristics of cellular differentiation and inability for multipotency. In comparison, principal component analyses revealed that the lipid composition of A2B5+/GFAP+ cells retained many of the characteristics of A2B5+/GFAP? progenitors, but their lipid profile was different from that of A2B5?/GFAP+ astrocytes. Thus, our study demonstrated that two GFAP+ cell populations have distinct lipid profiles with the A2B5+/GFAP+ cells sharing a phospholipid profile with progenitors rather than astrocytes. The progenitor cells may require regulated low levels of lipids known to mediate signaling functions in differentiated cells, and the precursor lipid profiles may serve as one measure of the differentiation capacity of a cell population.     

Cocaine induces astrocytosis through ER stress-mediated activation of autophagy

Cocaine is known to induce inflammation, thereby contributing in part, to the pathogenesis of neurodegeneration. A recent study from our lab has revealed a link between macroautophagy/autophagy and microglial activation. The current study was aimed at investigating whether cocaine could also mediate activation of astrocytes and, whether this process involved induction of autophagy. Our findings demonstrated that cocaine mediated the activation of astrocytes by altering the levels of autophagy markers, such as BECN1, ATG5, MAP1LC3B-II, and SQSTM1 in both human A172 astrocytoma cells and primary human astrocytes. Furthermore, cocaine treatment resulted in increased formation of endogenous MAP1LC3B puncta in human astrocytes. Additionally, astrocytes transfected with the GFP-MAP1LC3B plasmid also demonstrated cocaine-mediated upregulation of the green fluorescent MAP1LC3B puncta. Cocaine-mediated induction of autophagy involved upstream activation of ER stress proteins such as EIF2AK3, ERN1, ATF6 since blockage of autophagy using either pharmacological or gene-silencing approaches, had no effect on cocaine-mediated induction of ER stress. Using both pharmacological and gene-silencing approaches to block either ER stress or autophagy, our findings demonstrated that cocaine-induced activation of astrocytes (measured by increased levels of GFAP) involved sequential activation of ER stress and autophagy. Cocaine-mediated-increased upregulation of GFAP correlated with increased expression of proinflammatory mediators such as TNF, IL1B, and IL6. In conclusion, these findings reveal an association between ER stress-mediated autophagy and astrogliosis in cocaine-treated astrocytes. Intervention of ER stress and/or autophagy signaling would thus be promising therapeutic targets for abrogating cocaine-mediated neuroinflammation.     

Effect of reactive cell density on net [2-14C]acetate uptake into rat brain: labeling of clusters containing GFAP+- and lectin+-immunoreactive cells

Astrocytic proliferation is a hallmark of brain injury, but the biological functions and metabolic activities of reactive astrocytes in vivo are poorly understood. [2-14C]Acetate, which is preferentially transported into and, therefore, metabolized by astrocytes, was used to assess injury- and trophic factor-induced changes in astrocyte metabolic activity. Local rates of net [2-14C]acetate uptake and glucose utilization (CMR(glc)), determined with [14C]deoxyglucose to assay overall metabolic activity of all brain cells, were assayed 7 days after a cannula placement; adjacent brain sections were immunostained to identify glial fibrillary acidic protein-positive (GFAP(+)) astrocytes and microglia plus macrophages (lectin-positive cells). GFAP(+) cells were abundant in tissue surrounding the cannula compared to the contralateral hemisphere, whereas lectin(+) cells were restricted to the wound boundary. CMR(glc) fell 25% in regions enriched in reactive astrocytes compared to the homologous contralateral hemisphere, whereas [14C]acetate uptake increased slightly (6%) but statistically significantly; metabolism of both tracers in 13 other brain structures was unchanged. Injection of basic fibroblast growth factor (b-FGF) into cerebral cortex or superior colliculus produced fiber-rich cell clusters containing both GFAP(+) and lectin(+) cells that had a 37% increase in [14C]acetate uptake; GFAP(+)-cell density rose in the nearby neuropil but the corresponding change in [14C]acetate uptake was small (6-8%). Sensory stimulation did not alter [14C]acetate uptake into the clusters. Thus, [14C]acetate uptake was relatively stable with respect to changes in the density of reactive astrocytes that are dispersed throughout the neuropil and to changes in cellular activity arising from sensory stimulation. In contrast, b-FGF-induced cell clusters that contain mixed cell types and numerous fibers accumulated higher levels of [14C]acetate, raising the possibility that increased uptake might be due to high numbers of activated astrocytes and, perhaps, acetate metabolism by other cell types.     

Modulation of lysine transport in cultured rat astrocytes and astrocytoma cells

In the previous paper, it was shown that the transport of lysine into astrocytes and astrocytoma cells obeys the classical enzyme kinetics. Although unmodulated lysine transport into both normal rat astrocytes and rat astrocytoma cells is somewhat slower than needed for observed growth in the culture, it is capable of a large degree of enhancement. Insulin increases the Vmax for lysine influx in astrocytes tenfold and in astrocytoma cells fivefold. Glutathione produces a Vmax enhancement of 80% for astrocytes and 70% for astrocytoma cells. gamma-Glutamyl hydrazide is a weak inhibitor of lysine transport. Diethyl maleate appears to break down the regulation of lysine transport and allows a large increase in lysine influx in both cell types studied. Basic amino acid analogues canaline and S-aminoethylcysteine are not potent inhibitors of lysine transport. Lysine efflux kinetics are slower for C6 cells than for astrocytes; this difference is abolished by diethyl maleate and by dithiothreitol.     

Responses in Primary Astrocytes and C6-Glioma Cells to Ammonium Chloride and Dibutyryl Cyclic-AMP

Elevated brain ammonia levels are a major factor in the genesis of hepatic encephalopathy (HE). The mechanism of ammonium chloride (NH₄Cl) neurotoxicity involves interruption of oxidative metabolism. This leads to decreased levels of ATP concentration and subsequent glial fibrillary acidic protein (GFAP) degradation of astrocytes and fibrous C6-glioma cells. Our study investigates NH₄Cl toxicity by evaluating changes in ATP concentration and mitochondrial function as well as by evaluating alterations in GFAP expression. NH₄Cl induced decreases in ATP were detected after 15 minutes in C6-glioma cells and 24 hours in both cell types. Mitochondrial function, assessed by MTT (2–4,5-dimethylthiazol A-yl)-2, 5-diphenyltetrazolium bromide) assay, was impaired in both cell types at 24 hours following NH₄Cl treatment. GFAP was also significantly decreased in both cell types. Morphologic and metabolic toxicities were greater in C6-glioma cells. The data clearly indicate that NH₄Cl interrupts oxidative metabolism. The greater toxicity seen in C6-glioma cells may be due to their greater dependence on oxidative metabolism. Lastly, the decrease in GFAP is probably a consequence of diminished ATP.     

Spontaneous differentiation of porcine and bovine embryonic stem cells (epiblast) into astrocytes or neurons

The culture of porcine or bovine epiblasts, i.e., embryonic stem cells, on STO feeder cells resulted in their spontaneous differentiation into multiple cell types that were subsequently isolated as separate cell lines. Some of these cell lines were "neuron-like" in morphology. Immunofluorescent analysis of two porcine epiblast-derived cell lines demonstrated that the cells were positive for the expression of vimentin and the glial fibrillary acidic protein (GFAP). Because of their stellate morphology and lack of neurofilament expression, it is possible that the cells are type 2 astrocytes. Similar analysis of a bovine epiblast-derived cell line showed that the cells were positive for vimentin but that they did not express GFAP. However, a few cells within the population expressed neurofilaments and alpha-internexin. It is possible that the bovine cells are neural precursor cells. The results confirm and extend the demonstrated in vitro pluripotency of porcine and bovine epiblast cultures and provide evidence for an in vitro model of embryonic neuroectoderm development.     

Differential expression of gap junctions in neurons and astrocytes derived from P19 embryonal carcinoma cells

The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Exposure to retinoic acid induces P19 cells to differentiate into neurons and astrocytes that express similar developmental markers as their embryonic counterparts. We examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes. Untreated P19 cells express at least two gap junction proteins, connexins 26 and 43. Connexin32 could not be detected in these cells. Treatment for 96 hr with 0.3 mM retinoic acid induced the P19 cells to differentiate first into neurons followed by astrocytes. Retinoic acid produced a decrease in connexin43 mRNA, protein, and functional gap junctions. Connexin26 message was not affected by retinoic acid treatment. The neurons that developed consisted of small round cell bodies extending two to three neurites and expressed MAP2. Connexin26 was detected at sites of cell–cell and cell–neurite contact within 3 days following differentiation with retinoic acid. The astrocytes were examined for production of their intermediate filament marker, glial fibrillary acidic protein (GFAP). GFAP was first detected at 8 days by Western blotting. In culture, astrocytes co-expressed GFAP and connexin43 similar to primary cultures of mouse brain astrocytes. These results suggest that differentiation of neurons and glial cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model with which to examine the role gap junctions play during differentiation events of developing neurons and astrocytes. Dev. Genet. 21:187–200, 1997. © 1997 Wiley-Liss, Inc.