Normal Glial Cell Research Articles

Histone deacetylase 1 promotes glioblastoma cell proliferation and invasion via activation of PI3K/AKT and MEK/ERK signaling pathways

Histone deacetylase 1 (HDAC1) plays a crucial role in cancer progression and development. This enzyme has been confirmed to be a key regulator of tumor biology functions, such as tumor cell proliferation, migration and invasion. However, HDAC1 expression in glioma remains controversial, and its specific function and molecular mechanism in glioblastoma is poorly understood. In this study, our findings demonstrated that protein and mRNA levels of HDAC1 were increased in glioma cell lines and glioma tissues compared to normal glial cell lines and non-neoplastic brain tissues, respectively. Furthermore, HDAC1 knockdown cells displayed decreased proliferation and invasion capabilities, whereas HDAC1 overexpressing glioblastoma cells displayed more proliferation and invasion capabilities in vitro. These novel outcomes suggested that knockdown of HDAC1 possibly suppressed the expression of phosphorylated AKT (p-AKT) and phosphorylated ERK (p-ERK) proteins, while overexpression of HDAC1 significantly increased p-AKT and p-ERK protein in glioblastoma cells. In addition, knockdown of HDAC1 repressed subcutaneous tumor growth in vivo, and led to down-regulation of p-AKT and p-ERK protein in U87 MG xenograft tumors. For the first time, we have demonstrated that HDAC1 promotes proliferation and invasion in glioblastoma cells by activating PI3K/AKT and MEK/ERK signaling pathways in vitro and in vivo. These results suggest that HDAC1 may be a novel biomarker and potential therapeutic target in glioblastoma.

GLUT5 increases fructose utilization and promotes tumor progression in glioma

Fructose is now such an important component of human diets, and several studies have found that some cancer cells could utilize fructose to overcome low glucose micro-environment, but the study on the role of fructose in glioma is rare. To explore the role of fructose in glioma, we detected the proliferation and colony formation ability of glioma cells in fructose medium, and found that the abilities of proliferation and colony formation of glioma cells in fructose medium were similar with abilities in glucose medium, however, fructose just partly restored proliferation ability of normal glial cells. To explore the mechanism, we compared the expression level of GLUT5 (Glucose transporter type 5) in these cell lines, and the results showed that glioma cell lines had higher GLUT5 expression than normal glial cell lines. And knockdown of GLUT5 could significantly inhabit cell proliferation of glioma cells in fructose medium. Furthermore, we found that GLUT5 was also higher expressed in glioma tissues, and GLUT5 expression correlated significantly with glioma malignancy and poor survival of glioma patients (p < 0.01). In addition, we also demonstrated that knockdown of GLUT5 could significantly inhabit tumor proliferation in vivo, and intake fructose could increase tumor volume prominently. Taken together, our data show that fructose can be used by glioma cells, and restrict the fructose intake or targeting GLUT5 could be efficacious strategies in glioma.

Identification of Driver Genes and Key Pathways of Glioblastoma Shows JNJ-7706621 as a Novel Antiglioblastoma Drug.

The aim of this study is to identify novel targets of diagnosis, therapy, and prognosis for glioblastoma, as well as to verify the therapeutic effect of JNJ-7706621 regarding glioblastoma. The gene expression profiles of GSE42656, GSE50161, and GSE86574 were obtained respectively from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified with comparison between gene expression profiles of the glioblastoma tissues and normal tissues. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and protein-protein interaction (PPI) network analyses were performed. Quantitative reverse transcription polymerase chain reaction and survival curve analysis were also conducted to verify the correlation between expression of hub genes and prognosis. Moreover, invitro, MTT assay, colony-forming assay, the scratch assay, and flow cytometry were performed to verify the therapeutic effect of JNJ-7706621. AURKA, NDC80, KIF4A, and NUSAP1 were identified as hub genes after PPI network analysis. Differential expression of those genes was detected between human normal glial cells and glioblastoma cells by quantitative reverse transcription polymerase chain reaction (P< 0.05), and the survival curve analysis showed that the patients with low expression of gene AURKA, NDC80, KIF4A, and NUSAP1 had a significant favorable prognosis (P < 0.05). Invitro assays showed that JNJ-7706621 inhibited glioblastoma cellular viability, proliferation, and migration via inducing glioblastoma cells apoptosis. AURKA, NDC80, KIF4A, and NUSAP1 were significantly more highly expressed in glioblastoma cells than in human normal glial cell. Patients with low expression of those 4 genes had a favorable prognosis. JNJ-7706621 was a potential drug in treatment of patients with glioblastoma.

MiR-330-5p suppresses glioblastoma cell proliferation and invasiveness through targeting ITGA5.

The present study intended to investigate the biological effects of miR-330-5p on glioblastoma (GBM) cell proliferation and invasiveness by targeting integrin α5 (ITGA5). The expressions of miR-330-5p and ITGA5 mRNA in GBM cell lines (U87, U251, and U373) and normal brain glial cell line (HEB) were detected using RT-qPCR. Protein expression of ITGA5 was examined using Western blot. The present study used MTT assay, colony formation assay, Transwell assay, wound healing assay, and flow cytometry analysis in order to determine the biological functions of GBM cells (including cell proliferation, invasion, migration, apoptosis, and cell cycle). The present study applied dual-luciferase reporter gene assay to identify the target relationship between miR-330-5p and ITGA5. miR-330-5p was low-expressed in GBM cell lines while ITGA5 was high-expressed compared with HEB. miR-330-5p could directly target ITGA5 as well as suppress its expression in GBM cells. Up-regulation of miR-330-5p and down-regulation of ITGA5 both have an inhibitory effect on cell proliferation, invasion, and migration. Meanwhile, they could also promote GBM cell apoptosis. miR-330-5p could suppress proliferation and invasion of GBM cells through targeting ITGA5.

MicroRNA‑205 acts as a tumor suppressor and directly targets YAP1 in glioma.

Glioma is the most common form of primary malignant tumor that occurs in the central nervous system. The underlying molecular mechanism of the carcinogenesis and progression of glioma remains to be elucidated. It is well‑established that microRNAs (miRs) are associated with the regulation of glioma initiation and progression, and may represent a novel effective therapeutic strategy for the treatment of glioma. In the present study, the expression, roles and molecular mechanisms of miR‑205 in glioma were investigated. The expression levels of miR‑205 in glioma tissues, normal brain tissues, human glioma and normal HEB glial cell lines were determined using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). To explore the functional roles of miR‑205 in glioma cells, a Cell Counting kit 8 assay, and Transwell migration and invasion assays were employed. The molecular mechanisms underlying the roles of miR‑205 in glioma cells were investigated using bioinformatics analysis, a luciferase reporter assay, RT‑qPCR and western blot analysis. The results of the present study demonstrated that miR‑205 expression was markedly low in glioma tissues and cell lines compared with normal brain tissue and a glial cell line. Upregulation of miR‑205 invitro decreased cell viability, migration and invasion in glioma. Further investigation of the potential molecular mechanism demonstrated that the tumor suppressive functions of miR‑205 in regulating the proliferation, migration and invasion of glioma cells were mediated by a direct target gene, yes associated protein 1 (YAP1). The results of the present study suggested that miR‑205 inhibited glioma growth and metastasis by directly targeting YAP1, and that miR‑205 should be investigated as a novel therapeutic target for anti‑cancer treatment.

Inference of Low and High-Grade Glioma Gene Regulatory Networks Delineates the Role of Rnd3 in Establishing Multiple Hallmarks of Cancer.

Gliomas are a highly heterogeneous group of brain tumours that are refractory to treatment, highly invasive and pro-angiogenic. Glioblastoma patients have an average survival time of less than 15 months. Understanding the molecular basis of different grades of glioma, from well differentiated, low-grade tumours to high-grade tumours, is a key step in defining new therapeutic targets. Here we use a data-driven approach to learn the structure of gene regulatory networks from observational data and use the resulting models to formulate hypothesis on the molecular determinants of glioma stage. Remarkably, integration of available knowledge with functional genomics datasets representing clinical and pre-clinical studies reveals important properties within the regulatory circuits controlling low and high-grade glioma. Our analyses first show that low and high-grade gliomas are characterised by a switch in activity of two subsets of Rho GTPases. The first one is involved in maintaining normal glial cell function, while the second is linked to the establishment of multiple hallmarks of cancer. Next, the development and application of a novel data integration methodology reveals novel functions of RND3 in controlling glioma cell migration, invasion, proliferation, angiogenesis and clinical outcome.

Differential effects of miR-34c-3p and miR-34c-5p on the proliferation, apoptosis and invasion of glioma cells

Glioblastoma is the most malignant and common intrinsic brain tumor, but the molecular mechanism of glioma pathophysiology is poorly understood. Recent data have shown that microRNAs regulate the expression of several genes associated with human cancer. In the present study, the function of miR-34c in glioma cells was analyzed. It was demonstrated that miR-34c-3p and miR-34c-5p were downregulated in gliomas, by performing qPCR on tumor tissues from glioma patients and glioma cell lines, compared with normal brain tissues and a normal glial cell line. Furthermore, the miR-34c expression was found to be inversely correlated with glioma WHO grades. Overexpression of miR-34c-3p inhibited U251 and U87 cell proliferation; however, miR-34c-5p only had an effect on U251 cells. Transfection with miR-34c-3p or miR-34c-5p in U251 cells and with miR-34c-3p in U87 cells produced S-phase arrest with G0/G1 reduction and induced cell apoptosis, but no significant changes were observed with miR-34c-5p transfection in U87 cells, normal or negative control groups. However, significant inhibition of glioma cell invasion was observed following transfection with miR-34c-3p and miR-34c-5p. Moreover, it was identified that miR-34c-3p overexpression reduced the expression of Notch pathway members, but miR-34c-5p overexpression did not. Therefore, these results suggest differential tumor suppressor roles for miR-34c-3p and miR-34c-5p and provide new insights into the role of miR-34c in glioma, which includes tumor-suppressing effects on proliferation, apoptosis and invasiveness.

Abstract A21: High-throughput small molecule screening of genetically diverse glioblastoma stem cells

Abstract Survival statistics for glioblastoma multiforme (GBM) have remained static despite years of research. Mounting evidence suggests that glioblastoma stem cells (GSCs) are a primary cause of GBM growth, spread, vascularity, and chemotherapeutic resistance. Increasing survival, therefore, demands a therapeutic strategy with efficacy against GSCs. To address this, we have conducted a high-throughput screen of over 3,000 small molecule compounds for activity against three independently derived GSC lines that vary in their expression of TCGA subtype-related genes. Our fluorescence-based assay is sensitive, reliable, and largely free of row- and column-based systematic errors. We identify novel compounds that impair the viability of all three of these genetically diverse GSC lines but have comparably limited toxicity against a normal human glial cell line. Many of these compounds are small and lipophilic, as well, making them ideal candidates for follow-up in vivo evaluation either through intravenous delivery or through encapsulation in polymeric nanoparticles. Citation Format: Garth W. Strohbehn, Jiangbing Zhou, Michael Fu, Toral R. Patel, Joseph M. Piepmeier, W. Mark Saltzman. High-throughput small molecule screening of genetically diverse glioblastoma stem cells [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr A21.

Bantam Is Required for Optic Lobe Development and Glial Cell Proliferation

microRNAs (miRNAs) are small, conserved, non-coding RNAs that contribute to the control of many different cellular processes, including cell fate specification and growth control. Drosophila bantam, a conserved miRNA, is involved in several functions, such as stimulating proliferation and inhibiting apoptosis in the wing disc. Here, we reported the detailed expression pattern of bantam in the developing optic lobe, and demonstrated a new, essential role in promoting proliferation of mitotic cells in the optic lobe, including stem cells and differentiated glial cells. Changes in bantam levels autonomously affected glial cell number and distribution, and non-autonomously affected photoreceptor neuron axon projection patterns. Furthermore, we showed that bantam promotes the proliferation of mitotically active glial cells and affects their distribution, largely through down regulation of the T-box transcription factor, optomotor-blind (omb, Flybase, bifid). Expression of omb can rescue the bantam phenotype, and restore the normal glial cell number and proper glial cell positioning in most Drosophila brains. These results suggest that bantam is critical for maintaining the stem cell pools in the outer proliferation center and glial precursor cell regions of the optic lobe, and that its expression in glial cells is crucial for their proliferation and distribution.

SMN deficiency attenuates migration of U87MG astroglioma cells through the activation of RhoA

Spinal muscular atrophy (SMA) is a neurodegenerative disease that affects alpha motoneurons in the spinal cord caused by homozygous deletion or specific mutations in the survival motoneuron-1 (SMN1) gene. Cell migration is critical at many stages of nervous system development; to investigate the role of SMN in cell migration, U87MG astroglioma cells were transduced with shSMN lentivectors and about 60% reduction in SMN expression was achieved. In a monolayer wound-healing assay, U87MG SMN-depleted cells exhibit reduced cell migration. In these cells, RhoA was activated and phosphorylated levels of myosin regulatory light chain (MLC), a substrate of the Rho kinase (ROCK), were found increased. The decrease in cell motility was related to activation of RhoA/Rho kinase (ROCK) signaling pathway as treatment with the ROCK inhibitor Y-27632 abrogated both the motility defects and MLC phosphorylation in SMN-depleted cells. As cell migration is regulated by continuous remodeling of the actin cytoskeleton, the actin distribution was studied in SMN-depleted cells. A shift from filamentous to monomeric (globular) actin, involving the disappearance of stress fibers, was observed. In addition, profilin I, an actin-sequestering protein showed an increased expression in SMN-depleted cells. SMN is known to physically interact with profilin, reducing its actin-sequestering activity. The present results suggest that in SMN-depleted cells, the increase in profilin I expression and the reduction in SMN inhibitory action on profilin could lead to reduced filamentous actin polymerization, thus decreasing cell motility. We propose that the alterations reported here in migratory activity in SMN-depleted cells, related to abnormal activation of RhoA/ROCK pathway and increased profilin I expression could have a role in developing nervous system by impairing normal neuron and glial cell migration and thus contributing to disease pathogenesis in SMA.

Regulation ofDrosophilaglial cell proliferation by Merlin-Hippo signaling

Glia perform diverse and essential roles in the nervous system, but the mechanisms that regulate glial cell numbers are not well understood. Here, we identify and characterize a requirement for the Hippo pathway and its transcriptional co-activator Yorkie in controlling Drosophila glial proliferation. We find that Yorkie is both necessary for normal glial cell numbers and, when activated, sufficient to drive glial over-proliferation. Yorkie activity in glial cells is controlled by a Merlin-Hippo signaling pathway, whereas the upstream Hippo pathway regulators Fat, Expanded, Crumbs and Lethal giant larvae have no detectable role. We extend functional characterization of Merlin-Hippo signaling by showing that Merlin and Hippo can be physically linked by the Salvador tumor suppressor. Yorkie promotes expression of the microRNA gene bantam in glia, and bantam promotes expression of Myc, which is required for Yorkie and bantam-induced glial proliferation. Our results provide new insights into the control of glial growth, and establish glia as a model for Merlin-specific Hippo signaling. Moreover, as several of the genes we studied have been linked to human gliomas, our results suggest that this linkage could reflect their organization into a conserved pathway for the control of glial cell proliferation.

Analysis of Gene Expression in a Human-derived Glial Cell Line Exposed to 2.45 GHz Continuous Radiofrequency Electromagnetic Fields

The increasing use of mobile phones has aroused public concern regarding the potential health risks of radiofrequency (RF) fields. We investigated the effects of exposure to RF fields (2.45 GHz, continuous wave) at specific absorption rate (SAR) of 1, 5, and 10 W/kg for 1, 4, and 24 h on gene expression in a normal human glial cell line, SVGp12, using DNA microarray. Microarray analysis revealed 23 assigned gene spots and 5 non-assigned gene spots as prospective altered gene spots. Twenty-two genes out of the 23 assigned gene spots were further analyzed by reverse transcription-polymerase chain reaction to validate the results of microarray, and no significant alterations in gene expression were observed. Under the experimental conditions used in this study, we found no evidence that exposure to RF fields affected gene expression in SVGp12 cells.

Roles of Specific Membrane Lipid Domains in EGF Receptor Activation and Cell Adhesion Molecule Stabilization in a Developing Olfactory System

BackgroundReciprocal interactions between glial cells and olfactory receptor neurons (ORNs) cause ORN axons entering the brain to sort, to fasciculate into bundles destined for specific glomeruli, and to form stable protoglomeruli in the developing olfactory system of an experimentally advantageous animal species, the moth Manduca sexta. Epidermal growth factor receptors (EGFRs) and the cell adhesion molecules (IgCAMs) neuroglian and fasciclin II are known to be important players in these processes.Methodology/Principal FindingsWe report in situ and cell-culture studies that suggest a role for glycosphingolipid-rich membrane subdomains in neuron-glia interactions. Disruption of these subdomains by the use of methyl-β-cyclodextrin results in loss of EGFR activation, depletion of fasciclin II in ORN axons, and loss of neuroglian stabilization in the membrane. At the cellular level, disruption leads to aberrant ORN axon trajectories, small antennal lobes, abnormal arrays of olfactory glomerul, and loss of normal glial cell migration.Conclusions/SignificanceWe propose that glycosphingolipid-rich membrane subdomains (possible membrane rafts or platforms) are essential for IgCAM-mediated EGFR activation and for anchoring of neuroglian to the cytoskeleton, both required for normal extension and sorting of ORN axons.

Essential Role for Epidermal Growth Factor Receptor in Glutamate Receptor Signaling to NF-κB

Glutamate is a critical neurotransmitter of the central nervous system (CNS) and also an important regulator of cell survival and proliferation. The binding of glutamate to metabotropic glutamate receptors induces signal transduction cascades that lead to gene-specific transcription. The transcription factor NF-kappaB, which regulates cell proliferation and survival, is activated by glutamate; however, the glutamate receptor-induced signaling pathways that lead to this activation are not clearly defined. Here we investigate the glutamate-induced activation of NF-kappaB in glial cells of the CNS, including primary astrocytes. We show that glutamate induces phosphorylation, nuclear accumulation, DNA binding, and transcriptional activation function of glial p65. The glutamate-induced activation of NF-kappaB requires calcium-dependent IkappaB kinase alpha (IKKalpha) and IKKbeta activation and induces p65-IkappaBalpha dissociation in the absence of IkappaBalpha phosphorylation or degradation. Moreover, glutamate-induced IKK preferentially targets the phosphorylation of p65 but not IkappaBalpha. Finally, we show that the ability of glutamate to activate NF-kappaB requires cross-coupled signaling with the epidermal growth factor receptor. Our results provide insight into a glutamate-induced regulatory pathway distinct from that described for cytokine-induced NF-kappaB activation and have important implications with regard to both normal glial cell physiology and pathogenesis.

Control of glial precursor cell development in the mouse optic nerve by sonic hedgehog from retinal ganglion cells

The development of glial precursor cells in the mammalian optic nerve depends on retinal ganglion cell (RGC) axons, but the signals that mediate this neuron-to-glia interaction have not been fully characterized. Sonic hedgehog (Shh) is expressed by RGCs, and we showed previously that it is required for the specification of astrocyte lineage cells at the optic disc. To study the role of RGC-derived Shh on astrocyte development at later developmental stages, we generated mice with a conditional ablation of Shh in the peripheral retina and analyzed gene expression and glial cell development in the optic nerve. Astrocyte development was initiated in the optic nerves of these mutant mice; however, the expression of Hedgehog (Hh) target genes, Gli1 and Ptch1 and cell cycle genes, Ccnd1 and Cdc25b in the optic nerves were downregulated. Astrocyte proliferation was markedly reduced. Oligodendrocyte precursor cells were fewer in the optic nerves of mutant mice, possibly as a consequence of reduced secretion of growth factors by astrocytes. At a later developmental stage, optic nerve axons displayed signs of Wallerian degeneration, including reduction of astrocyte processes, degenerating glial cells and formation of distended axons. We also demonstrate that the Hh pathway can be activated in optic nerve-derived astrocytes in vitro, but fails to induce cell cycle gene expression and proliferation. RGC-derived Shh signalling isthus necessary in vivo for maintenance of astrocyte proliferation, affecting both axo-glial and normal glial cell development in the optic nerve.

Identification of two novel glial-restricted cell populations in the embryonic telencephalon arising from unique origins

BackgroundConsiderably less attention has been given to understanding the cellular components of gliogenesis in the telencephalon when compared to neuronogenesis, despite the necessity of normal glial cell formation for neurological function. Early proposals of exclusive ventral oligodendrocyte precursor cell (OPC) generation have been challenged recently with studies revealing the potential of the dorsal telencephalon to also generate oligodendrocytes. The identification of OPCs generated from multiple regions of the developing telencephalon, together with the need of the embryonic telencephalon to provide precursor cells for oligodendrocytes as well as astrocytes in ventral and dorsal areas, raises questions concerning the identity of the precursor cell populations capable of generating macroglial subtypes during multiple developmental windows and in differing locations.ResultsWe have identified progenitor populations in the ventral and dorsal telencephalon restricted to the generation of astrocytes and oligodendrocytes. We further demonstrate that the dorsal glial progenitor cells can be generated de novo from the dorsal telencephalon and we demonstrate their capacity for in vivo production of both myelin-forming oligodendrocytes and astrocytes upon transplantation.ConclusionBased on our results we offer a unifying model of telencephalic gliogenesis, with the generation of both oligodendrocytes and astrocytes from spatially separate, but functionally similar, glial restricted populations at different developmental times in the dorsal and ventral CNS.

Induction of transforming growth factor‐β1 by basic fibroblast growth factor in rat C6 glioma cells and astrocytes is mediated by MEK/ERK signaling and AP‐1 activation

Basic fibroblast growth factor (bFGF) and transforming growth factor-beta1 (TGF-beta1) play an important role in proliferation, differentiation, and survival of malignant gliomas and in normal glial cell biology. Because of these critical roles, potential interactions between these key growth factors were investigated. We previously demonstrated that bFGF potently stimulates TGF-beta1 release from rat glioma cells. The purpose of the present study was to elucidate the mechanism(s) of this regulatory effect, establish its functional importance, and examine whether it extends to nontransformed rat hypothalamic astrocytes (RHA). The results revealed that RHA express the high-affinity FGF(1-4) receptors, and similarly to glioma cells, bFGF stimulated TGF-beta1 release in an isoform-specific manner. A mediatory role for ERK signaling in bFGF-induced TGF-beta release was suggested by the fact that MEK1 inhibition prevented this effect. Additionally, bFGF enhanced MEK1/2 phosphorylation and ERK activation/nuclear translocation, which culminated in increased activity of AP-1-mediated gene transcription. bFGF markedly induced TGF-beta1 mRNA levels in an isoform-specific manner, an effect that was dependent on MEK/ERK/AP-1 signaling. Functionally, bFGF-induced proliferation of glioma cells was attenuated by MEK/ERK inhibition or immunoneutralization of TGF-beta1, suggesting that this pathway may have important implications for brain tumor progression.

GDNF family members and their receptors: expression and functions in two oligodendroglial cell lines representing distinct stages of oligodendroglial development.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), and persephin (PSP) constitute a subfamily of transforming growth factor-betas (TGF-betas) with prominent roles in the regulation of neuron survival and differentiation. Although numerous members of the TGF-beta superfamily are important regulators of glial cell functions in health and disease, it is unknown whether any member of the GDNF subfamily may have functions in normal or pathological glial cell performances. To begin to address this issue, we have studied expression and putative functions of GDNF, NTN, PSP, and their receptors in two cell lines representing models for oligodendrocyte progenitor cells (OLI-neu) and immature oligodendrocytes (OLN-93), respectively. RT-PCR analysis revealed expression of all three growth factor mRNAs in OLI-neu and OLN-93 cells. Expression was weak in OLI-neu cells, while both NTN and PSP mRNAs were strongly expressed in OLN-93 cells. Furthermore, OLI-neu and OLN-93 cells expressed transcripts encoding the GDNF receptors Ret and GFRalpha-1. The two splice variants for GFRalpha-2 were exclusively synthesized in OLI-neu cells. Similarly, primary O-2A progenitor cells and enriched mature oligodendrocytes expressed Ret, GFRalpha-1 and GFRalpha-2 mRNAs. Both GDNF and NTN stimulated DNA synthesis monitored by BrdU incorporation of OLI-neu cells in a dose-dependent fashion. Co-administration of TGF-beta significantly reduced this effect. Similarly, PDGF co-applied with GDNF or NTN down-regulated proliferation in OLI-neu cells. In contrast, OLN-93 cells did not respond to GDNF or NTN with increased incorporation of BrdU. Expression of GDNF, NTN, and their receptors and distinct effects in two model cell lines of oligodendrocyte development suggest that functions of members of the GDNF family and their receptors may not be restricted to neurons and may be implicated in oligodendrocyte development.

The identification of four protein kinase C isoforms in human glioblastoma cell lines: PKC alpha, gamma, epsilon, and zeta

Levels of protein kinase C (PKC) isoforms in eight human glioblastoma cell lines and two normal human glial cell cultures were determined. Earlier studies identified PKC-alpha and PKC-gamma in these cell lines but PKC-beta was not present. In this study, PKC-epsilon and PKC-zeta are demonstrated immunologically in these cell lines and also in two normal human glial cell cultures. Protein kinase C-delta was not present. When levels of the four isoforms in the tumor cells were compared to levels in the normal cells, no increase was observed in PKC-alpha or PKC-gamma, but PKC-epsilon was elevated three to 30 times in six of the eight tumors, and PKC-zeta was elevated approximately two times in all of the tumors. Incubation of cell line A172 with phorbol ester for 6 hours resulted in a 48-fold maximum increase in the nuclear PKC-epsilon and a sevenfold increase in the plasma membrane fraction with no change in the cytoplasmic fraction. A similar incubation for 4 hours produced a 0.5- to onefold increase of PKC-zeta in cytoplasmic, nuclear, and plasma membrane fractions. Other researchers have shown that overexpression of PKC-epsilon in fibroblasts results in tumorigenesis, and that blocking PKC-zeta function inhibits deoxyribonucleic acid synthesis. These data suggest that alteration in the expression of PKC-epsilon and PKC-zeta could be a factor in the conversion of normal glial cells to glioblastomas.

High-resolution one- and two-dimensional 1H MRS of human brain tumor and normal glial cells.

Astrocytoma (WHO grade II, III), glioblastoma, malignant melanoma, and normal glial cell cultures, established from biopsies, were investigated by 1H MRS. At a 1H resonance frequency of 500 MHz (11.75 T) a high spectral resolution was achieved in 1D 1H spectra; in conjunction with 2D shift-correlated (COSY) MRS, resonances of alanine, aspartate, choline, creatine, glutamate, glutamine, hypotaurine, myo-inositol, phosphocreatine, phosphoryl-ethanolamine, phosphoryl-choline, lactate, lysine, N-acetylaspartate, taurine, threonine and valine could be identified. T1 relaxation times for the most prominent compounds are presented. T1 values of lactate ranged between 450 ms and 850 ms. The intensity of the lactate signal revealed differences between individual spectra, but exhibited no correlation between different tumor specimens or degree of malignancy. It was shown that the lactate signal at 1.3 ppm is covered by peaks arising from threonine and fatty acids. The choline signal level varied among spectra of different tumors, among tumors with similar degree of malignancy, and within the same tumor. Further preliminary differences due to aspartate, inositol and glutamine/glutamate were found in 1D and 2D COSY spectra between normal glial cells as well as different tumors. These results indicate that some differences observed in in vivo spectra may be attributable to secondary macroscopic structural changes (hypoxia, necrosis) and not to tumor inherent characteristics. Further correlation between in vivo and in vitro spectroscopy is therefore required.