Human Astroglial Cell Research Articles

Mushrooms as potential therapeutic agents in the treatment of cancer: Evaluation of anti-glioma effects of Coprinus comatus, Cantharellus cibarius, Lycoperdon perlatum and Lactarius deliciosus extracts

Cancer incidence rates are on the increase worldwide. The most common brain cancer in adults is glioblastoma. Currently available treatment modalities are limited and natural products such as mushrooms could enhance them. Apart from nutritional value, mushrooms are an excellent source of bioactive compounds and therefore could be used to treat various disorders. The aim of the study was to assess the anti-glioma potential of selected mushrooms on U87MG, LN-18 glioblastoma and SVGp12 normal human astroglial cell lines. The materials were Cantharellus cibarius, Coprinus comatus, Lycoperdon perlatum and Lactarius delicious. Aqueous, 70 % ethanol or 95 % ethanol extracts from mushrooms were used for analysis including assessment of antioxidant activity by DPPH assay, cell viability by MTT assay, DNA biosynthesis by thymidine incorporation assay, activity of metalloproteinase by gelatin zymography and cell cycle assay by flow cytometry. Mushroom extracts influenced the viability and DNA biosynthesis of cancer cells. Activity of ethanol mushroom extracts was stronger than that of aqueous extracts. Anti-glioma mechanism consisted in inhibition of cancer cell proliferation and induction of apoptosis associated with arrest of cells in subG1 or G2/M phase of cell cycle, and inhibition of metalloproteinases activity. Among investigated mushrooms, L. deliciosus and C. comatus showed the greatest anti-glioma potential.

Celastrol enhances TRAIL-induced apoptosis in human glioblastoma via the death receptor pathway.

Glioblastoma is the most common, malignant and devastating type of primary brain tumor. Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is characterized by its lethality to precancerous and cancerous cells. However, many kinds of tumor cells, including most glioma cells, tend to evade TRAIL-induced apoptosis. Celastrol is a pleiotropic compound from a traditional Chinese medicine that has proven to be useful as a sensitizer for TRAIL treatment. However, the underlying mechanism and role of celastrol in the sensitization of glioma cells remain to be elucidated. The viability of glioma cell lines was examined by the CCK-8 assay. The expression of DR5 was detected by reverse transcriptase quantitative real-time PCR. The protein expression of DR5, cleaved caspase-8, cleaved caspase-3 and PARP were measured by western blot. The apoptosis rates and the sub-G1 population were detected by flow cytometry. The cellular morphological changes were assessed by TUNEL apoptosis and Hoechst 33258 staining assays. The knockdown of DR5 expression was conducted by siRNA. In this study, we observed that celastrol treatment inhibited cell viability in a dose-dependent manner, while glioma and normal human astroglial cell lines were resistant to TRAIL treatment. We also observed that the antiproliferative effects of TRAIL in combination with a noncytotoxic concentration of celastrol were significantly greater than those of celastrol or TRAIL alone. In addition, cell death induced by the combination treatment was apoptotic and occurred through the death receptor pathway via activation of caspase-8, caspase-3, and PARP. Furthermore, celastrol upregulated death receptor 5 (DR5) at the mRNA and protein levels, and siRNA-mediated DR5 knockdown reduced the killing effect of the combination drug treatment on glioma cells and reduced the activation of caspase-3, caspase-8 and PARP. Taken together, the results of our study demonstrate that celastrol sensitizes glioma cells to TRAIL via the death receptor pathway and that DR5 plays an important role in the effects of this cotreatment. The results indicate that this cotreatment is a promising tumor-killing therapeutic strategy with high efficacy and low toxicity.

Abstract 531: Heterozygous IDH1 R132H/WT created by “single base editing” inhibits human astroglial cell growth by downregulating YAP

Abstract Mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been identified in a number of cancer types, including brain cancer. The Cancer Genome Atlas project has revealed that IDH1 mutations occur in 70-80% of grade II and grade III gliomas. Until recently, most of the functional studies of IDH1 mutations in cellular models have been conducted in overexpression systems with the IDH1 wild type background. In this study, we employed a modified CRISPR/Cas9 genome editing technique called “single base editing”, and efficiently introduced heterozygous IDH1 R132H mutation in human astroglial cells (~20%). Compared with astroglial cells with IDH1 wild type, monoallelic IDH1 R132H mutation (IDH1R132H/WT) inhibited cell proliferation and promoted cell migration. Global gene expression analysis identified molecular targets and pathways altered by IDH1R132H/WT, including cell proliferation, extracellular matrix, and cell migration, consistent with our phenotype characterization. Global DNA methylation profiling showed hypermethylation as well as hypomethylation induced by IDH1R132H/WT. Using our mutated IDH1 models generated by genome editing, we identified novel molecular targets of IDH1R132H/WT, namely Yes-associated protein (YAP) and its downstream signaling pathway Notch, to mediate the cell growth-inhibiting effect of IDH1R132H/WT. Our findings are in line with the clinical observations that brain tumor patients with mutated IDH1 have a better prognosis. In summary, the “single base editing” strategy has successfully created heterozygous IDH1 R132H mutation that recapitulate the naturally occurring IDH1 mutant. Our isogenic cellular systems that differ in a single nucleotide in one allele of the IDH1 gene provide a valuable model for novel discoveries of IDH1R132H/WT-driven biological events. Citation Format: Shuang Wei, Jie Wang, Olutobi Oyinlade, Ding Ma Ma, Shuyan Wang, Shuli Xia. Heterozygous IDH1R132H/WT created by “single base editing” inhibits human astroglial cell growth by downregulating YAP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 531.

Heterozygous IDH1R132H/WT created by "single base editing" inhibits human astroglial cell growth by downregulating YAP.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been identified in a number of cancer types, including brain cancer. The Cancer Genome Atlas project has revealed that IDH1 mutations occur in 70–80% of grade II and grade III gliomas. Until recently, most of the functional studies of IDH1 mutations in cellular models have been conducted in overexpression systems with the IDH1 wild type background. In this study, we employed a modified CRISPR/Cas9 genome editing technique called “single base editing”, and efficiently introduced heterozygous IDH1 R132H mutation (IDH1R132H/WT) in human astroglial cells. Global DNA methylation profiling revealed hypermethylation as well as hypomethylation induced by IDH1R132H/WT. Global gene expression analysis identified molecular targets and pathways altered by IDH1R132H/WT, including cell proliferation, extracellular matrix, and cell migration. Our phenotype analysis indicated that compared with IDH1 wild type cells, IDH1R132H/WT promoted cell migration by upregulating integrin β4 (ITGB4), and significantly inhibited cell proliferation. Using our mutated IDH1 models generated by “single base editing”, we identified novel molecular targets of IDH1R132H/WT, namely Yes-associated protein (YAP) and its downstream signaling pathway Notch, to mediate the cell growth-inhibiting effect of IDH1R132H/WT. In summary, the “single base editing” strategy has successfully created heterozygous IDH1 R132H mutation that recapitulates the naturally occurring IDH1 mutation. Our isogenic cellular systems that differ in a single nucleotide in one allele of the IDH1 gene provide a valuable model for novel discoveries of IDH1R132H/WT-driven biological events.

Interplay between Endothelin and Erythropoietin in Astroglia: The Role in Protection against Hypoxia

We show that, under in vitro conditions, the vulnerability of astroglia to hypoxia is reflected by alterations in endothelin (ET)-1 release and capacity of erythropoietin (EPO) to regulate ET-1 levels. Exposure of cells to 24 h hypoxia did not induce changes in ET-1 release, while 48–72 h hypoxia resulted in increase of ET-1 release from astrocytes that could be abolished by EPO. The endothelin receptor type A (ETA) antagonist BQ123 increased extracellular levels of ET-1 in human fetal astroglial cell line (SV-FHAS). The survival and proliferation of rat primary astrocytes, neural precursors, and neurons upon hypoxic conditions were increased upon administration of BQ123. Hypoxic injury and aging affected the interaction between the EPO and ET systems. Under hypoxia EPO decreased ET-1 release from astrocytes, while ETA receptor blockade enhanced the expression of EPO mRNA and EPO receptor in culture-aged rat astroglia. The blockade of ETA receptor can increase the availability of ET-1 to the ETB receptor and can potentiate the neuroprotective effects of EPO. Thus, the new therapeutic use of combined administration of EPO and ETA receptor antagonists during hypoxia-associated neurodegenerative disorders of the central nervous system (CNS) can be suggested.

Differential expression of miRNA-146a-regulated inflammatory genes in human primary neural, astroglial and microglial cells

Micro RNA-146a (miRNA-146a) is an inducible, 22 nucleotide, small RNA over-expressed in Alzheimer's disease (AD) brain. Up-regulated miRNA-146a targets several inflammation-related and membrane-associated messenger RNAs (mRNAs), including those encoding complement factor-H (CFH) and the interleukin-1 receptor associated kinase-1 (IRAK-1), resulting in significant decreases in their expression (p<0.05, ANOVA). In this study we assayed miRNA-146a, CFH, IRAK-1 and tetraspanin-12 (TSPAN12), abundances in primary human neuronal-glial (HNG) co-cultures, in human astroglial (HAG) and microglial (HMG) cells stressed with Aβ42 peptide and tumor necrosis factor alpha (TNFα). The results indicate a consistent inverse relationship between miRNA-146a and CFH, IRAK-1 and TSPAN12 expression levels, and indicate that HNG, HAG and HMG cell types each respond differently to Aβ42-peptide+TNFα-triggered stress. While the strongest miRNA-146a-IRAK-1 response was found in HAG cells, the largest miRNA-146a-TSPAN12 response was found in HNG cells, and the most significant miRNA-146a-CFH changes were found in HMG cells, the ‘resident scavenging macrophages’ of the brain.

Role of C/EBP‐β, p38 MAPK, and MKK6 in IL‐1β‐mediated C3 gene regulation in astrocytes

Complement component C3, the central player in the complement cascade and the pro-inflammatory cytokine IL-1β is expressed by activated glial cells and may contribute to neurodegeneration. This study examines the regulation of the expression of C3 by IL-1β in astroglial cells focusing on the role of the upstream kinase MKK6, p38-α MAPK, and C/EBP-β isoforms (LAP1, LAP2, or LIP) in astroglial cells. Activation of human astroglial cell line, U373 with IL-1β, led to the induction of C3 mRNA and protein expression as determined by real-time RT-PCR and Western blot analysis, respectively. This induction was suppressed by the pharmacological inhibitor of p38 MAPK (i.e., SB202190-HCl), suggesting the involvement of p38 MAPK in C3 gene expression. IL-1β also induced C3 promoter activity in U373 cells in a MAP kinase- and C/EBP-β-dependent manner. Cotransfection of C3 luciferase reporter construct with constitutively active form of the upstream kinase in the MAP kinase cascade, that is, MKK6 (the immediate upstream activator of p38 kinase) resulted in marked stimulation of the promoter activity, whereas overexpression of a dominant negative forms of MKK6 and p38α MAPK inhibited C3 promoter activity. Furthermore, a mutant form of C/EBP-β, LAP(T235A) showed reduction in IL-1β-mediated C3 promoter activation. These results suggest that the p38α, MAPK, and MKK6 play prominent roles in IL-1β and C/EBP-β-mediated C3 gene expression in astrocytes.

H02 Low levels of transforming growth factor β1 in Huntington's disease peripheral macrophages and brain glial cells

BackgroundHuntington's disease (HD) is a neurodegenerative disorder caused by a CAG expansion mutation in huntingtin (HTT). Mutant HTT induces cell death modulating several molecular pathways, including cell signalling, transcriptional machineries,...

Similar protective effects of BQ-123 and erythropoietin on survival of neural cells and generation of neurons upon hypoxic injury

Our recent study [Danielyan et al., 2005. Eur. J. Cell Biol. 84, 567–579] showed an additive protective effect of endothelin (ET) receptor A (ETA-R) blockade and erythropoietin (EPO) on the survival and rejuvenation of rat astroglial cells exposed to hypoxia. Whether the effects observed with rodent astroglial cells can be reproduced in human astrocytes and whether these effects of ETA-R blockade and EPO on astrocytes are associated with neuronal survival remained open. Therefore, in the present study, the effects of the ETA-R antagonist BQ-123 and EPO on the maintenance of the neuronal population and survival of the human fetal astroglial cell line (SV-FHAS) under normoxic and hypoxic conditions (NC and HC, respectively) were investigated. Rat brain primary cultures exposed to BQ-123 and/or EPO revealed an increase in the number of β-III tubulin-positive neurons under NC. The hypoxia-caused loss of neurons was abolished by administration of BQ-123 or EPO. Simultaneous application of EPO and BQ-123 led to an additive protective effect on the generation of neurons under NC only. By contrast, BQ-788, the selective ETB-R antagonist, diminished the neuronal population both in NC and HC. Both under NC and HC the number of non-differentiated nestin +/GFAP − neural cells increased upon application of EPO or BQ-123. SV-FHAS responded to BQ-123 or EPO by a decrease in LDH activity in the culture medium under NC (35%) and HC (26% LDH decrease). Concomitant effects of EPO and BQ-123 were illustrated in an additional increase in the survival of human astrocytes (33% under NC and 17% under HC). These data hint at a neuroprotective therapeutic potency of ETA-R blockade, which either alone or in combination with EPO may improve the survival of astroglial and neuronal cells upon hypoxic injury.

The Amyloid-β Peptide Suppresses Transforming Growth Factor-β1-induced Matrix Metalloproteinase-2 Production via Smad7 Expression in Human Monocytic THP-1 Cells

Accumulation of the amyloid-beta (Abeta) peptide in the brain is a crucial factor in the development of Alzheimer disease. Expression of transforming growth factor-beta1 (TGF-beta1), an immunosuppressive cytokine, has been associated in vivo with Abeta accumulation in transgenic mice and recently with Abeta clearance by activated microglia, suggesting its deleterious and beneficial effects in neuronal cells. In this study, we demonstrated that TGF-beta1 stimulated the production of matrix metalloproteinase-2 (MMP-2) in a time- and dose-dependent manner in a human monocytic THP-1 cell line. Notably, we found that Abeta1-42 consistently inhibited the TGF-beta1-induced production of MMP-2, the endogenous gene containing Smad response elements, whereas the reverse peptide, Abeta42-1, evidenced little effect. Additionally, Abeta1-42 reduced TGF-beta1-induced increase in plasminogen activator inhibitor-1 (PAI-1). This inhibitory effect of Abeta1-42 was also seen in human astroglial T98G cell line. Furthermore, Abeta1-42 significantly induced the expression of Smad7, which appears in turn to mediate the Abeta suppression of the TGF-beta1-induced MMP-2 production. Indeed, Smad7 overexpression mimicked the inhibitory effect of Abeta1-42 on TGF-beta1-induced MMP-2 production. Importantly, Abeta1-42 markedly suppressed the transactivation of the transfected reporter construct, p3TP-Lux, which contains TGF-beta1-inducible Smad response elements. This was concomitant with a decreased MMP-2 production in TGF-beta1-treated cells. Inhibition of cellular Smad7 levels via the small interference RNA method significantly ameliorated the Abeta1-42-mediated suppression of TGF-beta1-inducible transcription reporter activity, thereby restoring MMP-2 induction, whereas Smad7 transfection down-regulated TGF-beta1-inducible transcription reporter activity. Collectively, these data suggest that Abeta1-42 may play an important role in the negative regulation of TGF-beta1-induced MMP-2 production via Smad7 expression.

Selective decrease of membrane-associated PKC-α and PKC-ε in response to elevated intracellular O-GlcNAc levels in transformed human glial cells

Increased flux through the hexosamine biosynthetic pathway (HBP) has been shown to affect the activity and translocation of certain protein kinase C (PKC) isoforms. It has been suggested that this effect is due to increases in the β- O-linked N-acetylglucosamine ( O-GlcNAc) modification. Herein, we demonstrate the effect of increasing the O-GlcNAc modification on the translocation of select PKC isozymes in a human astroglial cell line. Treating cells with either 8 mM d-glucosamine (GlcN), 5 mM streptozotocin (STZ), or 80 μM O-(2-acetamido-2-deoxy- d-glucopyranosylidene)amino- N-phenylcarbamate (PUGNAc) produced a significant increase in the O-GlcNAc modification on both cytosolic and membrane proteins; however, both the level and rate of O-GlcNAc increase varied with the compound. GlcN treatment resulted in a rapid, transient translocation of PKC-βII that was maximal after 3 h (73±8%) and also produced a 48±15% decrease in membrane-associated PKC-ε after 9 h of treatment. Similar to GlcN treatment, STZ and PUGNAc treatment also resulted in decreased levels of PKC-ε in the membrane fraction. Significant decreases were seen as early as 5 h and, by 9 h of treatment, had decreased by 87±6% with STZ and 73±7% with PUGNAc. Unlike GlcN, both STZ and PUGNAc produced a decrease in PKC-α membrane levels by 9 h posttreatment (78±10% with STZ and 66±8% with PUGNAc) while neither compound produced any changes in PKC-βII translocation. In addition, none of the three compounds affected membrane levels of PKC-ι. Altogether, these results demonstrate a novel link between increased levels of the O-GlcNAc modification and the regulation of specific PKC isoforms.

An anti-inflammatory role for N-acetyl aspartate in stimulated human astroglial cells

Although N-acetyl-l-aspartate (NAA) has been shown to be important to myelin synthesis and osmotic regulation, the biological rationale for the high levels of NAA found in the brain remains unknown. Here, a human astroglial cell line (STTG) was treated with NAA and stimulated with ionomycin, ionomycin/PMA, or IL-1β. PGE2 levels in ionomycin-stimulated STTG cells decreased by 76% and >95% at NAA concentrations of 10 and 20mM, respectively. NAA also decreased the levels of COX-2 protein and activated NF-κB in IL-1β-stimulated STTG cells but had little effect on unstimulated cells. Also, NAA significantly decreased intracellular calcium levels in ionomycin/PMA-stimulated cells. NAA had no effect on total COX-2 activity or COX-2 mRNA. Acetylation of IκBα kinase, an acetylation target of aspirin, was not observed when NAA was present. These results demonstrate that NAA appears to be important in the modulation of inflammation in the human STTG astroglial cell line. The results of these findings are discussed in relation to neuronal pathologies that exhibit abnormal NAA levels within the brain.

Acute ethanol exposure modulates expression of inducible nitric-oxide synthase in human astroglia: evidence for a transcriptional mechanism

Astroglia are important in immunocompetence and response to injury within the CNS. Activated astroglia respond, in part, by expressing inducible nitric-oxide synthase (iNOS) and subsequent catalytic production of nitric oxide. Results from a previous study in our laboratory, in the human A172 astroglial cell line, revealed that induction of iNOS activity by tumor necrosis factor-α + interferon-γ + interleukin-1β was inhibited by 24-h exposure to a high ethanol concentration (200 mM), but enhanced by 50 mM ethanol. In the work reported in this article, we tested the working hypothesis that ethanol acts transcriptionally to modulate cytokine-induced expression of the iNOS gene, NOS2A, in human astroglia. Ethanol, 50 or 200 mM, did not directly alter in vitro catalytic activity of the iNOS enzyme, indicating that ethanol does not affect the enzyme directly. Likewise, ethanol exposure after a 12-h cytokine-stimulation period had no effect on in vivo iNOS activity. However, when cells were simultaneously exposed to ethanol and cytokines for 12 h, in vivo iNOS activity was altered. That ethanol must be present during cytokine stimulation to influence iNOS activity is consistent with a transcriptional mechanism of action. In addition, steady-state expression of iNOS protein and NOS2A mRNA levels were modulated in a biphasic manner by ethanol similar to that noted previously for iNOS activity. These findings strongly support the suggestion that ethanol modulates cytokine-induced iNOS expression in A172 cells at a pretranslational site. These findings should be instrumental in the identification of the critical ethanol-sensitive elements involved in the regulation of NOS2A in human astroglia.

NF-κB mediates IL-1β-induced synthesis/release of α 2-macroglobulin in a human glial cell line

Increasingly, inflammatory responses in localized areas of brain parenchyma in response to the extracellular deposition of the Aβ peptides are thought to play a causative role in Alzheimer’s disease (AD) progression. Anti-inflammatory agents, in particular non-steroid anti-inflammatory drugs (NSAIDs), such as aspirin and ibuprofen, have been shown to be associated with a delayed onset or slowed rate of disease progression in several epidemiological studies. Activation of glial cells and the subsequent expression of a number of proteins including α 2-macroglobulin (α 2M) are associated with the induction of brain tissue inflammation. Additionally cytokines, such as interleukin-1α (IL-1α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) are co-localized to senile plaques, a neuropathological hallmark of AD. α 2M binds various cytokines, including IL-1β, as well as Aβ. In this study, we demonstrate that IL-1β induces α 2M synthesis/release from a human astroglial cell line (U373) via the activation of the nuclear transcription factor NF-κB. Our data suggest that attenuation of IL-1β-induced α 2M synthesis/release by blocking NF-κB activation may potentially be ‘protective’ against the development of late-onset AD.

Ultrastructural Analysis of Beta-Amyloid Production in Monocytes, Astrocytes, Epithelial, and Endothelial Cells Infected with Chlamydia Pneumoniae Isolated from Alzheimer Brains

Abstract Previously, we uncovered evidence of an infectious agent, Chlamydia pneumoniae, associated with sporadic Alzheimer's disease (AD). We demonstrated that C. pneumoniae was present in 90% of brain materials examined from AD patients, compared with 5% in age-matched, non-AD patients. We further showed that microglia and astroglia in the CNS are host cells for the bacterium. RT-PCR analyses indicated that the bacterium is metabolically-active in the brain and could be cultured in a human monocyte cell line (THP-1). Importantly, we showed that the organism is concentrated in areas of AD-neuropathology also containing tau and beta-amyloid. The present study was designed to investigate the effects of C. pneumoniae infection on beta amyloid production in human monocytes (THP-1), astroglial (CCF-STTG1), epithelial (Hep 2), and endothelial (HBMEC) cell lines. We investigated the host pathogen relationship with C. pneumoniae and whether entities characteristic of AD neuropathology such as betaamyloid are associated directly with this infectious agent.

Expression of complement inhibitor protein CD59 in human neuronal and glial cell lines treated with HIV-1 gp41 peptides.

In attempts to elucidate the pathogenic mechanisms involved in neurodegeneration in AIDS patients with cognitive deficits, the possible effect of HIV-1 transmembrane envelope protein gp41 on expression of the membrane inhibitor of complement mediated cytolysis (CD59) was assessed in human neuronal (SK-N-SH) and astroglial (T98G) cell lines. Western blotting analyses demonstrated that an immunodominant (ID, aa 598-613) gp41 peptide as well as the recombinant gp41 protein encompassing this domain markedly reduced CD59 level in a dose dependent manner whereas p24 and control peptide had little effect. RT-PCR showed that ID peptide also elicited a reduction in the expressed CD59 mRNA level. This gp41 peptide apparently down-regulated phorbol 12,13-dibutyrate induced elevation of CD59 at the protein and mRNA levels in a manner similar to that conferred by protein kinase C inhibitor, H-7 or staurosporine in SK-N-SH. Interestingly, proinflammatory cytokines such as IL-1beta or IFN-gamma as well as LPS greatly decreased CD59 in SK-N-SH and to a lesser extent in T98G whereas TNF-alpha did not significantly alter it. In contrast, antioxidants and anti-inflammatory agents enhanced CD59 expression reversing gp41 peptide mediated inhibitory effect in SK-N-SH. Our data suggest that high level of gp41 or its metabolites as well as impaired protein kinase response, chronic inflammation or antioxidant depletion within HIV-1 infected brains may be associated with a diminished expression of CD59 which would render neuronal cells to susceptible to indirect bystander lysis in the presence of autologous complement.

Overexpression of protein kinase C epsilon in astroglial brain tumor derived cell lines and primary tumor samples.

Prognosis for astroglial brain tumors that are not amenable to surgical resection remains poor. Consequently, a need to identify new cellular targets and chemotherapeutics for the treatment of astroglial tumors remains. Important reports indicate that human astroglial cell lines express higher protein kinase C (PKC) activity in comparison to normal astrocytes. PKC designates a family of kinases that regulate many cellular functions including cell growth and differentiation. The tight regulation of PKC activity is crucial for maintaining normal cellular proliferation since excessive activity leads to uncontrolled growth and cellular transformation. PKCepsilon, one of the 11 known PKC isozymes, has been shown to function as an oncogene in rodent fibroblasts by enhancing c-Raf-1 kinase activity leading to the stimulation of mitogen-activated protein (MAP) kinase pathway. We recently demonstrated that the ability of substance P (SP) neuropeptide to activate MAP kinase pathway in U-373MG astrocytoma cells correlates with its ability to selectively translocate PKCepsilon from cytosolic to membrane fraction, and that PKC inhibitors (e.g. CGP 41251) inhibit the activation of this pathway by SP or the PKC activator 12-O-tetradecanoyl phorbol 13-acetate (TPA). In this study, we demonstrated that PKCepsilon is overexpressed in many astroglial cell lines (n=27 lines), thus providing new evidence as to the possible involvement of this isozyme in the pathology of astroglial tumors. Consistently, we demonstrated that PKCepsilon is overexpressed in primary pediatric anaplastic astrocytoma (grade III) tumor samples as well as in cell lines derived from them, and that glioblastoma multiforme (grade IV) and gliosarcoma tumor samples, but not pilocytic astrocytomas (grade I), also express high levels of PKCepsilon. Therefore, the reported increase in PKC activity in brain tumor derived cell lines may be, in part, attributed to the overexpression of PKCepsilon and possibly other PKC isozymes. Consequently, we propose that the use of PKCepsilon selective inhibitors may be beneficial in the treatment of astroglial brain tumors.

Effect of HIV-1 gp41 peptides on secretion of beta-amyloid precursor protein in human astroglial cell line, T98G.

To understand the mechanism underlying cognitive deficits in AIDS patients, we examined the influence of gp41 peptides on the expression and the secretion of Alzheimer's amyloid precursor protein (APP) in human astroglial cell line T98G. Western blotting analyses demonstrated that treatment of glial cells with a putative immunosuppressive domain (aa 583-599) of gp41 remarkably downregulated the interleukin 1beta- (IL-1beta) induced elevation of the secreted form of APP (sAPP alpha) containing Kunitz-type protease inhibitor (KPI) domain without significant changes of the expression pattern of APP mRNAs as revealed by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Recombinant gp41 protein encoding for ectodomain, including aa 583-599 residues, also elicited a similar dose-dependent inhibitory effect, whereas the control peptides resulted in little change. The molecular mechanism underlying this gp41-mediated reduction of sAPP alpha secretion appears not to be owing to the difference in the function of extracellular proteases based on the finding of similar proteolytic activities responsible for APP metabolism in vitro present in the conditioned media from the cultures treated with or without gp41 peptide. However, the known PKC inhibitors such as H-7 or staurosporine, partially inhibited the elevation of sAPP alpha secretion in response to protein kinase C (PKC) agonist phorbol 12,13-dibutyrate (PdBu) as well as to IL-1beta, mimicking the immunosuppressive gp41 peptide. These observations implicate that part of the neurodegenerative cascade in AIDS brains may involve the inhibitory effect of gp41 on secretion of sAPP alpha, a potent glial neurotrophic factor, through impaired PKC response.

Expression of a NOS-III-like Protein in Human Astroglial Cell Culture

Evidence for the presence of a type-III nitric oxide synthase-like protein (NOS-III-like protein) in astroglial cells is reported. The mRNA of a NOS-III-like protein is constitutively expressed in human astrocytoma T67 cells, taken as an astroglial model. The nucleotide sequence of the PCR product (422 bp) shares more than 99% identity with the cDNA (from 1588 to 2009) of the human endothelial nitric oxide synthase (NOS-III). The molecular mass of the astroglial NOS-III-like protein is about 140 kDa, as observed for human NOS-III. Moreover, the astroglial NOS-III-like protein is constitutively tyrosine-phosphorylated and associated with caveolin-1. The astroglial NOS-III-like protein is apparently inactive, as reported for phosphorylated human NOS-III associated with caveolin-1.

Expression of tumour necrosis factor-α, -β and interferon-γ genes within human neuroglial tumour cells and brain specimens

Expression of cytokine genes, TNF-α, TNF-β and IFN-γ, in human astroglial cell lines and in fresh brain specimens was studied by PCR. mRNA transcripts of TNF-α could be detected in three out of five astrocytomas and neuroblastoma cell lines, and after stimulation with IL-1β/IFN-γ or LPS/IFN-γ all these cell lines expressed TNF-α genes. TNF-β genes could not be detected in these cell lines. We were able to detect expression of IFN-γ genes within two astrocytoma cell lines, which interestingly did not show TNF-α activity. In addition to the cultured cells, we also examined gene expression of these cytokines within four human malignant astrocytoma specimens, two peritumoral brain and two autopsied normal brains. The results show that tumour and surrounding reactive lesions express TNF-α genes (four of six) but not normal brains. The concentration of these cytokines in the supernatant of cultured cells was measured quantitatively by TNF-α, -β or IFN-γ ELISA. The combined stimulation of these neuroglial cell lines with IL-1β and LPS or IFN-γ, revealed a high level of TNF-α activity. This was especially evident with a neuroblastoma cell line. The concentration of TNF-α in the supernatant of the IMR32 neuroblastoma cell line increased markedly upon stimulation with IL-1β in both a time- and dose-dependent fashion in the presence of LPS or IFN-γ. Next, we examined expression of IL-1β and IFN-γ genes in the brain specimens. The result shows that four in six tumour and peritumoral regions expressed IFN-γ genes and one specimen showed IL-β gene by PCR. From these experiments it is suspected that neuroglial cell-derived TNF-α induced by IL-1β of IFN-γ may participate in local immune reactions of the brain in an autocrine and paracrine fashion.