Immunofluorescence and biochemical studies of the type VI collagen expression by human glioblastoma cells in vitro

Abstract

The human glioblastoma cell line U-87 MG was found to express a 140 kD polypeptide which was recognized on immunoblot analysis by a monoclonal antibody to type VI collagen. This polypeptide was digestible by a highly purified bacterial collagenase. After treatment of U-87 MG cells by pepsin, the protein profile revealed the two major pepsin-resistant fragments identical in Mr to those of collagen VI extracted from human placenta. The respective peptide maps from V8 protease one-dimensional gels of these two fragments were identical to those obtained with human collagen VI. Immunofluorescent staining by antibodies to type VI collagen was observed in the extracellular matrix. Moreover; U-87 MG cells were found to be positive for A2B5, a cell surface marker specific for 0-2A type glial precursor cells. These data indicate that the human glioblastoma cell line U-87 MG exhibits the properties of glial precursor cells and expresses collagen type VI in vitro. This cell line therefore may prove valuable for comparative investigations of the regulation of type VI collagen synthesis, and may be useful as a model to study the function and pathological importance of type VI collagen in human brain tumours, both in vitro and in vivo. [Neurol Res 1994; 16: 370-375]     

Functional activation of EphA5 receptor does not promote cell proliferation in the aberrant EphA5 expressing human glioblastoma U-118 MG cell line

Eph receptors are a subfamily of receptor tyrosine kinases (RTKs), that are activated by ephrin ligands and appear to play important roles in axon guidance and cell migration during development of the nervous system. Over-expression or constitutive activation of Eph receptors has been linked with increased proliferation in various tumours. We have recently described lineage aberrant expression of EphA5 in primary human astrocytomas, glioblastomas and in the human glioblastoma U-118 MG cell line. A role for EphA5 expression in these tumours is not apparent, and we have investigated the cellular effects of EphA5 activation using the human glioblastoma U-118 MG cell line as a model. Immunofluorescent staining demonstrated cell surface expression of EphA5. Activation of the EphA5 receptor using an ephrin-A1 recombinant fusion protein resulted in tyrosine phosphorylation of EphA5 in a time-dependent manner. Exposure of U-118 MG glioblastoma cells to ephrin-A1 did not result in significant spontaneous or FCS-stimulated cell proliferation, though a marginal decrease was observed. This is in converse to the effects of Eph activation in other tumour cell lines, and is the first study to investigate EphA5 in glioblastoma cell lines.     

Differential regulation of glial cell line–derived neurotrophic factor (GDNF) expression in human neuroblastoma and glioblastoma cell lines

Human SK‐N‐AS neuroblastoma and U‐87MG glioblastoma cell lines were found to secrete relatively high levels of glial cell line–derived neurotrophic factor (GDNF). In response to growth factors, cytokines, and pharmacophores, the two cell lines differentially regulated GDNF release. A 24‐hr exposure to tumor necrosis factor‐α (TNFα; 10 ng/ml) or interleukin‐1β (IL‐1β; 10 ng/ml) induced GDNF release in U‐87MG cells, but repressed GDNF release from SK‐N‐AS cells. Fibroblast growth factors (FGF)‐1, ‐2, and ‐9 (50 ng/ml), the prostaglandins PGA₂, PGE₂, and PGI₂ (10 μM), phorbol 12,13‐didecanoate (PDD; 10 nM), okadaic acid (10 nM), dexamethasone (1 μM), and vitamin D₃ (1 μm) also differentially effected GDNF release from U‐87MG and SK‐N‐AS cells. A result shared by both cell lines, was a two‐ to threefold increase in GDNF release by db‐cAMP (1 mM), or forskolin (10 μM). In general, analysis of steady‐state GDNF mRNA levels correlated with changes in extracellular GDNF levels in U‐87MG cells but remained static in SK‐N‐AS cells. The data suggest that human GDNF synthesis/release can be regulated by numerous facto, signaling through multiple and diverse secondary messenger systems. Furthermore, we provide evidence of differential regulation of human GDNF synthesis/release in cells of glial (U‐87MG) and neuronal (SK‐N‐AS) origin. J. Neurosci. Res. 55:187–197, 1999. © 1999 Wiley‐Liss, Inc.     

Interleukin 1 and gliomas: immunohistochemical and immunocytochemical examinations

Interleukin 1 is a monokine produced by macrophage and has an ability to activate thymocytes. In addition to the immunological regulatory effect, interleukin 1 has attracted a great deal of investigators as a new peptide hormone that was secreted by many cells and has a various physiological activities. In central nervous systems, interleukin 1 promotes the glial cells proliferations on the injured brain and the fetal brain. The cell sources of interleukin 1 in central nervous systems are considered to the microglial cells. On gliomas, Lachmann and Dinarello reported growth promoting effect of IL-1 on U-373 MG human glioblastoma cell. The authors investigated the roles and effects of IL-1 on the growth of gliomas using recombinant human IL-1 beta and anti-HuIL-1 beta monoclonal antibody. On Immunohistochemistry, paraffin sections of 10 cases of gliomas were stained with immunoperoxidase method using anti-human IL-1 beta and anti-GFAP mouse monoclonal antibody. All astrocytomas examined and 2 of 4 glioblastomas were stained by anti-IL-1 beta. The origin of IL-1 that was stained by immunoperoxidase staining is unknown. The authors think it that IL-1 existed in glioma cells were secreted by microglial cells or that the glioma cells themselves secreted IL-1. In either case, IL-1 must be related to the growth of glioma in situ. On immunocytochemistry, U-373 MG human glioblastoma cells purchased from ATCC were incubated on cover-slip with 0 and 10 U/ml of rHuIL-1 beta for 3 or 7 days. The cells were stained with immunoperoxidase method using anti-GFAP monoclonal antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sphingosine kinase-1 expression correlates with poor survival of patients with glioblastoma multiforme: roles of sphingosine kinase isoforms in growth of glioblastoma cell lines

Sphingosine-1-phosphate is a bioactive lipid that is mitogenic for human glioma cell lines by signaling through its G protein-coupled receptors. We investigated the role of sphingosine-1-phosphate receptors and the enzymes that form sphingosine-1-phosphate, sphingosine kinase (SphK)-1, and -2 in human astrocytomas. Astrocytomas of various histologic grades expressed three types of sphingosine-1-phosphate receptors, S1P1, S1P2, and S1P3; however, no significant correlation with histologic grade or patient survival was detected. Expression of SphK1, but not SphK2, in human astrocytoma grade 4 (glioblastoma multiforme) tissue correlated with short patient survival. Patients whose tumors had low SphK1 expression survived a median 357 days, whereas those with high levels of SphK1 survived a median 102 days. Decreasing SphK1 expression using RNA interference or pharmacologic inhibition of SphK significantly decreased the rate of proliferation of U-1242 MG and U-87 MG glioblastoma cell lines. Surprisingly, RNA interference to knockdown SphK2 expression inhibited glioblastoma cell proliferation more potently than did SphK1 knockdown. SphK knockdown also prevented cells from exiting G1 phase of the cell cycle and marginally increased apoptosis. Thus, SphK isoforms may be major contributors to growth of glioblastoma cells in vitro and to aggressive behavior of glioblastoma multiforme.     

Growth effects of epidermal growth factor (EGF) and a monoclonal antibody against the EGF receptor on four glioma cell lines

Summary Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis and cell division in normal glia. At least half of malignant human gliomas (MHG) express high levels of the EGF receptor (EGFR), which are above those detected in normal brain. The demonstration that antibodies against the EGFR inhibit the growth of squamous cell carcinoma line A-431, with large numbers of EGFR, in vitro and in vivo raises the possibility that these agents could be used therapeutically against malignant human gliomas either alone or conjugated to other agents. We have measured the growth effects of EGF and an anti-EGFR monoclonal antibody, 528 (Ab-528), on four well-characterized human malignant glioma cell lines, D-263 MG, D-247 MG, U-343 MGa Cl 26, and D-37 MG, with 2.9×10⁴, 1.5×10⁵, 8.6×10⁵ and 1.59×10⁶ EGFRs per cell, respectively. EGF significantly increased cell number in D-263 MG and D-37 MG by 65% and 74%, respectively, had no effect on D-247 MG, and significantly decreased cell number in U-343 MGa Cl 26 by 39%. U-343 MGa Cl 26 growth was inhibited 19% by Ab-528, but Ab-528 had no effect on growth of the other MHG lines. Ab-528 significantly inhibited all EGF-mediated growth effects. These studies demonstrate that, although Ab-528 alone has little antiproliferative activity on MGH, it successfully competes with EGF to reduce the biological effects of EGF-EGFR binding. Therefore, this antibody could potentially be used to target radioisotopes to MHG via the EGFR for diagnosis and therapy.Supported by Grants CA-11898, NS-20023, CA-43722, and the Association for Brain Tumor Research (MHW, PAH)     

Human glioblastoma cell line 86HG39 activates T cells in an antigen specific major histocompatibility complex class II-dependent manner.

The capacity of three different human glioblastoma cell lines to activate human T cells was analysed by measuring major histocompatibility complex (MHC) antigen expression, monokine secretion and lectin, mAb OKT3 and antigen-driven T cell proliferation. All glioblastoma cells tested were able to induce PHA and concanavalin A (ConA)-driven T cell proliferation in a dose-dependent fashion, while all failed to induce T cell activation with mAb OKT3. In addition, the glioblastoma cell line 86HG39 was able to induce tetanus toxoid and toxoplasma lysate antigen-specific T cell proliferation. The responding T cell lines originated from only one out of five different donors. This foreign antigen-specific T cell proliferation induced by 86HG39 cells could be inhibited with mAb L243 directed against HLA-DR molecules. The study of monokine secretion by 86HG39 cells showed a strong interleukin (IL)-6 secretion after lipopolysaccharide (LPS) treatment, whilst no IL-1 secretion was observed. Furthermore, only 86HG39 cells were positive for HLA-DR molecules, whereas interferon (IFN) gamma treatment of 87HG28 and 87HG31 cells was necessary for the induction of class II antigen expression. Thus, cell line 86HG39 shows many features of an antigen presenting cell and the interaction of these cells with MHC compatible human T cells might be a useful model to study cellular immune reactions within the central nervous system.     

Physiology of transformed glial cells

Much of our present knowledge of glial cell function stems from studies of glioma cell lines, both rodent (C6, C6 polyploid, and TR33B) and human (1321N1, 138MG, D384, R-111, T67, Tp-301MG, Tp-483MG, Tp-378MG, U-118MG, U-251MG, U-373MG, U-787MG, U-1242MG, and UC-11MG). New methods such as patch clamp and Ca²⁺ imaging have lead to rapid progress the last few years in our knowledge about glial cells, where an unexpected presence and diversity of receptors and ion channels have emerged. Basic mechanisms related to membrane potential and K⁺ transport and the presence of voltage gated ion channels (Na⁺, inwardly rectifying K⁺, Ca²⁺ activated K⁺, Ca²⁺, and Cl^? channels) have been identified. Receptor function and intracellular signaling for glutamate, acetylcholine, histamine, serotonin, cathecolamines, and a large number of neuropeptides (bradykinin, cholecystokinin, endothelin, opioids, and tachykinins) have been characterized. Such studies are facilitated in cell lines which offer a more homogenous material than primary cultures. Although the expression of ion channels and receptors vary considerably between different cell lines and comparative studies are rare, a few differences (compared to astrocytes in primary culture) have been identified which may turn out to be characteristic for glioma cells. Future identification of specific markers for receptors on glial and glioma cells related to cell type and growth properties may have great potential in clinical diagnosis and therapy. © 1995 Wiley-Liss, Inc.     

Invasiveness of human glioma cell lines in vitro: Relation to tumorigenicity in athymic mice

Summary Five established cell lines derived from human anaplastic astrocytomas or glioblastoma multiforme were tested for invasiveness into precultured chick heart fragments in vitro. Four of the cell lines (U118 MG, D54 MG, U373 MG and A172) were strongly invasive into the heart tissue. A fifth cell line, U251 MG sp, which was only tumorigenic at doses of greater than 1×10⁸ cells in athymic mice, was non-invasive in vitro. One line, A172, was invasive but not tumorigenic in athymic mice, although a related invasive subline, D54 MG, at later passage levels was tumorigenic even at low cell doses. Invasion of the glioma cells was characterized by progressive and irreversible replacement of the precultured chick heart tissue. Both by light and transmission electron microscopy, a similar pattern of invasion was observed as earlier found with experimental rat glioma cells in the same system. Some human cell lines established from human gliomas retain invasive properties after a prolonged culture period in vitro.Supported by the Norwegian Cancer Society (LIdeR), and by grants CA 11898, CA32672, and NS 20023 (DDB)     

Synthesis of New Boron Derived Compounds; Anticancer,Antioxidant and Antimicrobial Effect in Vitro Glioblastoma Tumor Model

ObjectiveThe aim of our study is to investigate the cytotoxic, antioxidant, and antimicrobial effects of newly synthesized boron compounds in U87MG glioblastoma cell treatment. MethodsWe synthesized boron glycine monoester (BGM) and boron glycine diester (BGD) structures containing boron atoms and determined their cytotoxic activities on glioblastoma by the MTT method. The inhibitory concentration 50 (IC₅₀) value was calculated with GraphPad Prism 5.0 program. The IC₅₀ values were administered 48 hours on U87MG glioblastoma cell. Catalase (CAT), acid phosphatase (ACP) and alkaline phosphatase (ALP) enzyme activity, malondialdehyde (MDA), total glutathione (GSH), and total protein levels were detected using spectrophotometric methods. We determined the antimicrobial activities of BGM and BGD with the disc diffusion method.ResultsAfter 48 hours of BGM and BGD application to U87MG glioblastoma cells, we found the IC₅₀ value as 6.6 mM and 26 mM, respectively. CAT and ACP enzyme activities were decreased in BGM and BGD groups. MDA which is a metabolite of lipid peroxidation was increased in both boron compounds groups. GSH level was reduced especially in BGD group. BGM and BGD have been found to be antimicrobial effects. ConclusionBoron compounds, especially the BGM, can provide a new therapeutic approach for the treatment of glioblastoma with their anticancer, antioxidant, and antimicrobial effects.     

Role of high molecular weight extracellular matrix proteins in glioma cell migration

Malignant human gliomas are characterized by an uncontrolled cell proliferation and infiltrative growth within the brain. Complete surgical removal is difficult due to disseminated tumour cells, and the fundamental mechanisms responsible for this spread are poorly understood. An extensive tumour cell movement along blood vessels is frequently observed and this may be due to specific interactions between tumour cell surface receptors and specific extracellular matrix (ECM) components present in conjunction with vascular elements. In order to investigate the influence of ECM on glioma cell migration, three different human glioma cell lines (U-373 MG, A-172 MG and HF-66) were exposed to known ECM components of the basement membrane (laminin, fibronectin and collagen type IV). Cell migration from multicellular spheroids was studied, using a custom-made medium which was prepared by removing the high molecular weight protein fraction (>100 kDa) from newborn calf serum by ultrafiltration. To this medium, the specific ECM components were added. For two of the cell lines (A-172 MG and U-373 MG), laminin was the most potent stimulator of glioma cell migration; the effect of laminin exceeded that evoked by ordinary serum-supplemented medium. For the HF-66 cell line, fibronectin was the most potent stimulator of migration. Western blot analysis showed that the A-172 MG and HF-66 cell lines expressed low amounts of laminin compared with U-373 MG, which showed extensive intrinsic synthesis of this ligand. U-373 MG was the only cell line that migrated in pure filtered medium. The cells stimulated by fibronectin expressed a different morphology from those stimulated by laminin suggesting that specific ECM-receptor binding may activate different cytoskeletal components within the cells. Furthermore, it was shown that there was no difference in the amount of protein synthesis between cells grown in filtered medium and in filtered medium supplemented with different ECM components. This suggests that ECM-induced cell migration is not dependent on a high level of protein synthesis. It is also shown that α3 integrin, which is a receptor-subunit for laminin, fibronectin and collagen type IV, was highly expressed in all cell lines. This study indicates that glioma cells need serum proteins with a molecular weight >100 kDa to migrate in vitro, and that laminin and fibronectin play an important role in this process.     

Expression of extracellular matrix components in a highly infiltrative in vivo glioma model

This work demonstrates the expression of extracellular matrix (ECM) components in a highly infiltrative brain tumor model developed by simple inoculation of spheroids from five human glioma biopsy tissues directly into the brains of immunodeficient rats. Non-invasive tumors derived from one glioblastoma biopsy specimen and two glioma cell lines (D-54MG and U-251MG) were also included in this study. The extent of tumor cell infiltration was studied using a pan-human monoclonal anti-vimentin antibody. The cellular origin for several of these ECM components was identified using human-specific monoclonal antibodies and polyclonal antibodies detecting epitopes from both species. Immunostaining revealed a diffuse parenchymal staining of glioma-produced tenascin, whereas vitronectin was produced mainly by the invading glioma cells. ECM components such as laminin, fibronectin and collagen type IV were most probably produced by the host and were mainly associated with the blood vessels in the tumors. However, some parenchymal staining with regional variations was observed. The expression pattern of these components was different in cell lines tumors as compared to the biopsy specimen tumors. The alpha3 and beta1 integrin subunits were mainly observed in areas of tumor cell invasion in the invasive tumors. In conclusion, the observed staining patterns clarify the cellular origin and indicate the possible biological function of tenascin, vitronectin, laminin, fibronectin and collagen type IV in these highly invasive malignant tumors of glial origin.     

表达VASH1基因的人脑胶质瘤U-87MG细胞对化疗药物敏感性的变化

目的 探讨表达人血管生成抑制因子１（VASH1）的人脑胶质瘤U-87MG细胞对化疗药物的敏感性变化。方法 构建针对VASH1的慢病毒载体pGCL-GFP-VASH1,经测序鉴定后转染293T细胞,筛选出适合浓度的慢病毒转染人脑胶质瘤U-87MG细胞,荧光显微镜下检测转染效率;通过RT-PCR和Western blot分析U-87MG细胞VASH1 mRNA和蛋白表达水平;用CCK-8法检测U-87MG细胞在化疗药物顺铂和替莫唑胺作用下的存活率。流式细胞仪检测U-87MG细胞凋亡。结果 成功构建pGCL-GFP-VASH1慢病毒载体,并成功转染U-87MG细胞,转染率达70％以上;RT-PCR和Western blot结果证实转染VASH1慢病毒载体的U-87MG细胞表达VASH1 mRNA和蛋白。在顺铂或替莫唑胺作用下,表达VASH1的U-87MG细胞存活率均较未表达VASH1的U-87MG细胞明显降低（P<0.01）,而且U-87MG细胞凋亡率明显增加（P<0.01）。结论 VASH1慢病毒载体转染U-87MG细胞可使其稳定表达VASH1,并提高人脑胶质瘤U-87MG细胞对化疗药物敏感性、增加细胞凋亡率。     

Identification of differentially expressed proteins in human glioblastoma cell lines and tumors

An in-frame deletion of 801 bp in exons 2-7 (type III mutation) of the epidermal growth factor receptor (EGFR) is detected at high incidence in primary glioblastoma tumors. A proteomic approach was used to generate differential protein expression maps of fetal human astrocytes (FHA), human glioblastoma cell lines U87MG and U87MG expressing type III EGFR deletion (U87MGdeltaEGFR) that confers high malignancy to tumor cells. Two-dimensional gel electrophoresis followed by in-gel digestion of separated spots and protein identification by LC-MS-MS and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identified 23 proteins expressed at higher levels or exclusively in FHA and 29 proteins expressed at higher levels or exclusively in U87MG cells. Three proteins, ubiquitin, cystatin B, and tissue transglutaminase (TTG), were upregulated in U87MGdeltaEGFR relative to U87MG. Four proteins highly expressed by U87MG cells, Hsp27, major vault protein, TTG, and cystatin B, were analyzed by Western blot, ELISA, or RT-PCR in cell extracts and in tissue samples of glioblastoma multiforme (GBM; grade IV), low-grade astrocytomas (grades I and II), and nonmalignant brain lesions. All four proteins were highly expressed in GBM tissues compared to nonmalignant brain. These proteins may be used as diagnostic or functional (e.g., multiple drug resistance, invasiveness) markers for glioblastoma tumors.     

Presence of a novel epithelial antigen on rat cerebellar cell lines as detected by a monoclonal antibody

We have derived a monoclonal antibody, MCAb 51, following immunization of BALB/c mice with a Rous sarcoma virus-transformed rat cerebellar cell line. When assayed by immunofluorescence on primary rat cerebellar cultures MCAb 51 recognizes only islands of cells with an epitheloid morphology. Double-label immunofluorescence experiments with MCAb 51 and antisera to tetanus toxin, glial fibrillary acidic protein, galactocerebroside and fibronectin reveal that these cells do not appear to be neurons, astrocytes, oligodendrocytes, or fibroblasts, respectively. In contrast, cells from kidney, liver, tongue and choroid plexus epithelium are positive for the antigen. Of 12 Rous sarcoma virus-transformed cell lines, in contrast to 2 out of 9 chemically transformed lines, 11 exhibit the MCAb 51 antigen. These findings demonstrate that MCAb 51 recognizes an epithelial cell surface marker. Possible explanations for the difference in the expression of the antigen on Rous sarcoma virus and chemically transformed neural lines are discussed.     

Differential control of VEGF synthesis and secretion in human glioma cells by IL-1 and EGF.

VEGF (vascular endothelial growth factor), one of the most potent angiogenic factors, has recently been identified as an inducer of neoangiogenesis in many tumors including gliomas. VEGF itself appears to be regulated through different pathways. Since malignant gliomas frequently show EGF receptor amplification and express IL-1, a pivotal regulatory cytokine involved in angiogenesis, we analyzed interactions between EGF/EGF receptor and IL-1/IL-1 receptor and VEGF in the established glioblastoma cell lines U-87 MG and A-172. Basal VEGF expression was an order of magnitude higher in U-87 MG compared to A-172. IL-1 caused a fast and strong increase of VEGF secretion in U-87 MG which appeared to harbor an intracellular VEGF pool for enhanced exocytosis. The IL-1 receptor antagonist (IL-1-ra) reversed this effect suggesting an IL-1 receptor-associated mechanism. In contrast, VEGF secretion could not be increased by exogenous IL-1 exposure in A-172, which apparently lacked an intracellular VEGF pool for augmented exocytosis. However, IL-1-ra treatment alone caused a significant reduction of basal VEGF secretion in both U-87 MG and A-172. This suggests that baseline secretion of VEGF involves IL-1 receptor activation by endogenously produced IL-1. EGF also stimulated the secretion of VEGF into the cell supernatant. However, this effect, observed in both U-87 MG and A-172, was delayed and only occurred following replenishment of the intracellular VEGF pool. EGF upregulated the amount of VEGF mRNA. In general, the effects of IL-1 and EGF on VEGF were additive, suggesting independent mechanisms. Since IL-1 appears to be involved in VEGF secretion in glial tumors through an autocrine/paracrine mechanism, recombinant human IL-1-ra may evolve as a new agent for anti-angiogenic glioma therapy.     

Microtubule-severing ATPase spastin in glioblastoma: increased expression in human glioblastoma cell lines and inverse roles in cell motility and proliferation

We studied the expression and distribution of the microtubule-severing enzyme spastin in 3 human glioblastoma cell lines (U87MG, U138MG, and T98G) and in clinical tissue samples representative of all grades of diffuse astrocytic gliomas (n = 45). In adult human brains, spastin was distributed predominantly in neuronsand neuropil puncta and, to a lesser extent, in glia. Compared with normal mature brain tissues, spastin expression and cellular distribution were increased in neoplastic glial phenotypes, especiallyin glioblastoma (p < 0.05 vs low-grade diffuse astrocytomas). Overlapping punctate and diffuse patterns of localization wereidentified in tumor cells in tissues and in interphase and mitotic cells ofglioblastoma cell lines. There was enrichment of spastin in the leading edges of cells in T98G glioblastoma cell cultures and in neoplastic cell populations in tumor specimens. Real-time polymerase chain reaction and immunoblotting experiments revealed greater levels of spastin messenger RNA and protein expression in theglioblastoma cell lines versus normal human astrocytes. Functional experiments indicated that spastin depletion resulted in reduced cell motility and higher cell proliferation of T98G cells. Toour knowledge, this is the first report of spastin involvement incellmotility. Collectively, our results indicate that spastinexpression in glioblastomas might be linked to tumor cell motility, migration, and invasion.