myelin-basic-protein and Glioma

Topics: Animals; Brain Neoplasms; Genetic Therapy; Genetic Vectors; Glioma; Humans; Myelin Basic Protein; Promoter Regions, Genetic; Retroviridae

Retroviral vector is often used for gene therapy of malignant tumors. The main characteristic of this vector is that it integrates only into the genes of dividing and proliferating cells. Glioma cells proliferate actively, while surrounding normal brain cells rarely divide. Thus, we can expect the recombinant retrovirus modified to express cytotoxic genes to kill glioma cells selectively. However, this characteristic of specific toxicity to the dividing cells is also observed in many chemotherapeutic agents, and it is well known that they cause severe side effects, such as bone marrow suppression or diarrhea caused by simultaneous toxicity of the drugs to proliferating bone marrow cells or intestinal epithelial cells, respectively. We have cloned many genes which are specifically expressed in brain, and identified their promoter regions conferring tissue-specific expression. If we use the brain-specific promoters to regulate the expression of the toxic genes, these genes may not be expressed in the myeloid cells or intestinal epithelial cells, even if they were infected with the retrovirus. Therefore, we searched for brain-specific promoters which are also active in glioma cells to kill glioma cells specifically. Then, MBP promoter showed the strongest promoter activity in mouse glioma cells. These mouse glioma cells transduced with retrovirus containing the MBP promoter directing the herpes simplex virus type 1 thymidine kinase (HTK) gene were extremely sensitive to ganciclovir, even when transduced with the MBP promoter-HTK gene-containing retrovirus. And we could get complete remission in the mouse brain tumor models, which were transfected HTK genes in more than 25% glioma cells, with ganciclovir.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Brain Neoplasms; Genetic Therapy; Genetic Vectors; Glioma; Humans; Mice; Myelin Basic Protein; Promoter Regions, Genetic; Retroviridae

The technical modalities of the immunohistochemical demonstration of antigens of neuroectodermal differentiation and of mesenchymal nature in neural tissue are discussed. On the main antigens (S-100 protein, GFAP, Vimentin, Neuronal specific Enolase, neurofilaments, myelin basic protein, carboanhydrase C, FVIII/RAg, laminin, fibronectin) the most important findings are described and critically considered. They are interpreted in the light of the present knowledge on the problems of differentiation of the oncotypes and on particular aspects of bio-pathology of brain tumors.

Topics: Astrocytes; Fixatives; Glial Fibrillary Acidic Protein; Glioma; Humans; Immunoenzyme Techniques; Laminin; Medulloblastoma; Myelin Basic Protein; Naphthol AS D Esterase; Neoplasms, Nerve Tissue; p-Dimethylaminoazobenzene; Staining and Labeling; Tyrosine 3-Monooxygenase; Vimentin

Lineage progression and diversification is regulated by the coordinated action of unique sets of transcription factors. Oligodendrocytes (OL) and astrocytes (AS) comprise the glial sub-lineages in the CNS, and the manner in which their associated regulatory factors orchestrate lineage diversification during development and disease remains an open question. Sox10 and NFIA are key transcriptional regulators of gliogenesis associated with OL and AS. We found that NFIA inhibited Sox10 induction of OL differentiation through direct association and antagonism of its function. Conversely, we found that Sox10 antagonized NFIA function and suppressed AS differentiation in mouse and chick systems. Using this developmental paradigm as a model for glioma, we found that this relationship similarly regulated the generation of glioma subtypes. Our results describe the antagonistic relationship between Sox10 and NFIA that regulates the balance of OL and AS fate during development and demonstrate for the first time, to the best of our knowledge, that the transcriptional processes governing glial sub-lineage diversification oversee the generation of glioma subtypes.

Topics: Animals; Animals, Newborn; Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; Cells, Cultured; Cerebral Cortex; Chick Embryo; Chromatin Immunoprecipitation; Electroporation; Embryo, Mammalian; Glioma; Green Fluorescent Proteins; Homeodomain Proteins; Mice; Myelin Basic Protein; Nerve Tissue Proteins; Neuroglia; NFI Transcription Factors; Oligodendrocyte Transcription Factor 2; SOXE Transcription Factors; Transfection

Tumor stem cells (TSCs) possess the ability of chemoresistance. This study was to isolate and identify TSCs from human glioma cell line U87.. After U87 cells were grown as monolayer attached at the bottom of flasks in the medium containing serum, neural stem cell culture medium containing 5 ng/ml vincristine was added to obtain the first generation of tumorosphere. Then, the first generation of tumorosphere was dissociated into single cell suspension and seeded in the neural stem cell culture medium to obtain the second generation of tumorosphere. The morphology of the first and the second generations of tumorosphere was observed by light and scanning electron microscopy, respectively. The expression of nestin, glial fibrillary acidic protein (GFAP), beta-tubulin III and myelin basic protein (MBP) in the second generation of tumorosphere and differentiated cells were determined by immunofluorescence assay and confocal laser scanning microscopy.. The first and second generations of tumorosphere were obtained successfully. Cells inside the second generation of tumorosphere with a few protrusions attached with each other tightly. The expression of nestin, a stem cell marker, was detected in the second generation of tumorosphere. The expression of nestin, GFAP, beta-tubulin III, MBP was detected in the differentiated cells derived from the second generation of tumorosphere.. Tumor stem cells with the capacity of self-renewal and multipotency exist in U87 cells. Selective culture of tumor cells with treatment of vincristine might be a simple and practical method for isolation of TSCs.

Topics: Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Separation; Glial Fibrillary Acidic Protein; Glioma; Humans; Intermediate Filament Proteins; Myelin Basic Protein; Neoplastic Stem Cells; Nerve Tissue Proteins; Nestin; Tubulin; Vincristine

Malignant gliomas (MGs), lethal human central nervous system (CNS) neoplasms, contain tumor infiltrating lymphocytes (TIL). Although MHC class II molecules are frequently detected on MG cells, suggesting that they may be capable of antigen (Ag) presentation to CD4(+) T cells, deficiencies in CD4(+) T-cell activation are associated with these nonimmunogenic tumors. We evaluated regulation of the MHC class II transactivator (CIITA), the key intermediate that controls class II expression, in MG cells and tested whether MG cells could process native Ag. After interferon-gamma (IFN-gamma) stimulation, MG cells upregulated CIITA and class II molecules. IFN-gamma-inducible CIITA expression in MG cells, as well as primary human astrocytes, was directed by two CIITA promoters, pIV, the promoter for IFN-gamma-inducible CIITA expression in nonprofessional antigen-presenting cells (APC), and pIII, the promoter that directs constitutive CIITA expression in B cells. Both pIII and pIV directed CIITA transcription in vivo in MGs and ex vivo in IFN-gamma-activated primary MG cultures. We also demonstrate for the first time that MG cells can process native Ag for presentation to CD4(+) MHC class II-restricted Th1 cells, indicating that MG cells can serve as nonprofessional APC. CIITA may be a key target to modulate MHC class II expression, which could augment immunogenicity, Ag presentation, and CD4(+) T-cell activation in MG therapy.

Topics: Adult; Antigen Presentation; Antigen-Presenting Cells; Antigens, Surface; Astrocytes; Autoantigens; Base Sequence; Brain Neoplasms; CD4-Positive T-Lymphocytes; Exons; Female; Gene Expression Regulation, Neoplastic; Glioma; Histocompatibility Antigens Class II; Humans; Immunohistochemistry; Interferon-gamma; Male; Middle Aged; Molecular Sequence Data; Myelin Basic Protein; Nuclear Proteins; Promoter Regions, Genetic; RNA, Messenger; Trans-Activators; Tumor Cells, Cultured

Gliomas express a higher amount of Fas than normal brain tissue. It is of interest to know whether expression of the Fas receptor is unfavorable to the antiapoptotic pathways in gliomas. In this study, we introduced the Fas gene via an adenovirus vector (Adeno-Fas) into the A-172, U251, and U-373 MG glioma cell lines, each of which expresses Fas on the cell surface. Infection of Adeno-Fas induced apoptosis in each glioma cell line. In U251 cells and A-172 cells that express the same level of Fas as a result of infection with Adeno-Fas, a much higher percentage of U251 cells underwent apoptosis than did A-172 cells. This suggests that each glioma cell line has its own threshold of Fas expression, above which apoptosis is induced, and that the constitutive expression of Fas is below the level of this threshold. It was found that the constitutive expression of the anti-apoptotic gene Bcl-X(L) is higher in A-172 cells than in U251 cells. Adenovirus-mediated transduction of the Bcl-X(L) gene into U251 cells effectively suppressed Adeno-Fas-mediated apoptosis. These data indicate that the Bcl-X(L) gene is one of the important determinants of the threshold for Fas-mediated apoptosis. When U251 and U-373 MG cells were transduced with the Fas gene controlled by the myelin basic protein promoter, which had been shown to be active in gliomas but not in neural tissues, the cells underwent markedly enhanced apoptosis. Taken together, these results indicate that the overexpression of Fas alone induced apoptosis in each glioma cell line. The degree of Fas-mediated apoptosis was attenuated by the expression of an anti-apoptotic gene, Bcl-X(L). The adenovirus-mediated induction of Fas gene controlled by a tissue-specific promoter (e.g., myelin basic protein promoter) would be a promising therapeutic approach for malignant glioma.

Topics: Adenoviridae; Apoptosis; bcl-X Protein; fas Receptor; Glioma; Humans; Myelin Basic Protein; Promoter Regions, Genetic; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

The transduction of Bax protein, which is up-regulated in radiation- and chemotherapy-induced apoptosis, augments the cytotoxicity of radiotherapy and chemotherapy for cancers. The cytotoxicity of Bax overexpression is caused primarily by mitochondrial dysfunction, which is also involved in the apoptosis triggered by caspase-8. In this study, we transduced the Bax gene in combination with caspase-8 gene to evaluate whether or not this approach induces effective cytotoxicity in glioma cells. In terms of cancer gene therapy, it is critically important to induce cytotoxic genes in a cancer-specific manner. Therefore, we used the myelin basic protein promoter to drive cytotoxic genes. The expression level controlled by the myelin basic protein promoter was relatively low in gliomas. In U251 and U-373 MG glioma cells, adenovirus-mediated transduction of the Bax gene combined with caspase-8 gene induced enhanced apoptosis and cell death as determined by morphological analysis and assay for dead cells, hypodiploid cells, and DNA fragmentation (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling method). This therapeutic modality would be useful to induce a specific and enhanced cytotoxic effect for gliomas.

Topics: Adenoviridae; bcl-2-Associated X Protein; Brain Neoplasms; Caspase 8; Caspase 9; Caspases; Cell Death; Cell Separation; DNA Fragmentation; DNA, Complementary; Flow Cytometry; Gene Transfer Techniques; Glioma; Humans; Immunoblotting; Lac Operon; Microscopy, Electron; Mitochondria; Myelin Basic Protein; Promoter Regions, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Transduction, Genetic; Tumor Cells, Cultured

Caspase-8 is a member of the family of caspases, which are involved in the execution of apoptosis. To investigate whether caspase-8 can be used for gene therapy of gliomas, we transduced A-172 and U251 glioma cells with the caspase-8 gene via an adenoviral vector (Adv) controlled by the chicken beta-actin (CA) promoter (Advcaspase-8), and found that a similar level of caspase-8 protein induced A-172 cells to undergo necrotic cell death and U251 cells to undergo apoptotic cell death. Neither Bcl-XL nor Bcl-2, which play important roles in antiapoptotic mechanisms in gliomas, protected glioma cells from apoptosis induced by overexpression of caspase-8. Injection of Adv-caspase-8 suppressed the in vivo growth of U251 xenografts, in which apoptotic cell death remarkably increased as revealed by TUNEL analysis. Finally, we assessed whether gene therapy with a tissue-specific promoter, the myelin basic protein (MBP) promoter, is applicable to gliomas. Adv for caspase-8 controlled by the MBP promoter induced drastic apoptosis in U251 and U-373MG glioma cells, whereas it did not induce apoptosis in human endothelial cells, fibroblasts, and nerve growth factor-treated PC12 cells. These results indicate that Adv for caspase-8 effectively induced cell death in gliomas, and that this approach may be a useful modality for gene therapy of gliomas.

Topics: Actins; Adenoviridae; Animals; Apoptosis; Brain Neoplasms; Caspase 8; Caspase 9; Caspases; Cell Separation; Diploidy; DNA Fragmentation; Endothelium; Fibroblasts; Flow Cytometry; Genetic Therapy; Genetic Vectors; Glioma; Humans; In Situ Nick-End Labeling; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Electron; Myelin Basic Protein; Necrosis; Neoplasm Transplantation; Neoplasms, Experimental; Nerve Growth Factor; PC12 Cells; Promoter Regions, Genetic; Rats; Transduction, Genetic

Deletion of T cells due to apoptosis induction is a regulatory mechanism in the human immune system that may be impaired in autoimmune diseases such as multiple sclerosis (MS). Involvement of the apoptosis-mediating CD95/CD95 ligand system in MS has been demonstrated. Here, we report that (auto)antigen-specific human T cells are not killed in vitro by soluble TNF-related apoptosis-inducing ligand (TRAIL) although expressing death-inducing receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2. Apoptosis was assessed by caspase activation and DNA fragmentation, receptor expression was detected by RT - PCR and flow cytometry. The (auto)antigen-specific T cells were also resistant to specific TRAIL-R1/TRAIL-R2-directed induction of apoptosis, indicating that coexpression of the truncated TRAIL-R3 and TRAIL-R4 in these T cells is not responsible for the observed resistance. Upon stimulation, levels of death-inducing TRAIL receptors decreased whereas TRAIL was up-regulated on the cell surface. In contrast to CD95, the role of TRAIL receptors in MS might not involve regulation of T cell vulnerability.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Autoantigens; Caspases; CD4-Positive T-Lymphocytes; Cells, Cultured; DNA Fragmentation; fas Receptor; Glioma; GPI-Linked Proteins; Humans; Jurkat Cells; Membrane Glycoproteins; Multiple Sclerosis; Myelin Basic Protein; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Member 10c; Recombinant Proteins; T-Lymphocytes; Tetanus Toxoid; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor Decoy Receptors; Tumor Necrosis Factor-alpha

The p53 tumor suppressor gene is an important target for the gene therapy of cancers, and clinical trials targeting this gene have been conducted. Some cancers, however, are refractory to p53 gene therapy. Therefore, it has been combined with other therapies, including chemotherapy and radiotherapy, to enhance the cytopathic effect of p53 induction. The p33ING1 gene cooperates with p53 to block cell proliferation. In this study, we investigated whether adenovirus (Adv)-mediated coinduction of p33ING1 and p53 enhances apoptosis in glioma cells (U251 and U-373 MG), which showed no genetic alterations but low expression levels of p33ING1. Although the single infection of Adv for p33ING1 (Adv-p33) at a multiplicity of infection (MOI) of 100, or Adv for p53 controlled by myelin basic protein (MBP) promoter (Adv-MBP-p53), a glioma-specific promoter, at a MOI of 50, did not induce apoptosis in U251 and U-373 MG glioma cells; coinfection of Adv-p33 and Adv-MBP-p53 at the same MOIs induced drastically enhanced apoptosis in both cell lines. Apoptosis was not induced in NGF-treated PC-12 cells infected with a high MOI (300) of Adv-p33 nor in those coinfected with Adv-p33 (100) and Adv-MBP-p53 (50). Coinfection of Adv-p33 and Adv-MBP-p53 demonstrated morphological mitochondrial damage during the initial stage of apoptosis, which likely led to apoptotic cell death. Our results indicate that this coinfection approach can be used as a modality for the gene therapy of gliomas, sparing damage to normal tissues.

Topics: Adenoviridae; Animals; Apoptosis; bcl-2-Associated X Protein; Cell Cycle Proteins; DNA Mutational Analysis; DNA-Binding Proteins; Flow Cytometry; Gene Expression Regulation, Neoplastic; Gene Transfer Techniques; Genes, Tumor Suppressor; Glioma; Humans; Immunoblotting; Inhibitor of Growth Protein 1; Intracellular Signaling Peptides and Proteins; Lac Operon; Mitochondria; Myelin Basic Protein; Nuclear Proteins; Promoter Regions, Genetic; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Up-Regulation

Intracranial nervous-system tumours were diagnosed in three of 1092 bovine necropsy specimens submitted to the Department of Veterinary Pathology, Obihiro University between April 1983 and March 1996. A fourth case was a referral from the Department of Veterinary Pathology, Rakuno Gakuen University. Histopathological examination revealed four types of tumour: intracranial malignant peripheral nerve sheath tumour (MPNST), choroid plexus papilloma, differentiated fibrillary astrocytoma and anaplastic (malignant) astrocytoma. Immunohistochemically, the intracranial MPNST was strongly positive for S-100 protein and vimentin, and in places weakly positive for glial fibrillary acid protein (GFAP). The choroid plexus papilloma was strongly positive for epithelial membrane antigen (EMA), keratin, S-100 protein and vimentin, and positive for GFAP in places. The cytoplasm and fibrous component in the differentiated fibrillary astrocytoma were strongly positive for S-100 protein and GFAP. The anaplastic (malignant) astrocytoma was strongly positive for vimentin, S-100 protein and keratin in the cytoplasm and fibrous processes, and weakly positive for GFAP and EMA in places. Myelin basic protein (MBP) and synaptophysin showed a weak positive reaction in the marginal areas of the tumour.

Topics: Animals; Astrocytoma; Brain Neoplasms; Cattle; Cattle Diseases; Glial Fibrillary Acidic Protein; Glioblastoma; Glioma; Immunohistochemistry; Keratins; Mucin-1; Myelin Basic Protein; Nerve Sheath Neoplasms; S100 Proteins; Vimentin

We examined the expression of glial- and neuronal-specific mRNAs within human gliomas using in situ hybridization. We found that low-grade astrocytomas contained a high number of proteolipid protein (PLP) mRNA-positive cells and that the number of PLP-stained cells decreased markedly with increasing tumor grade. Interestingly, the ratio of PLP mRNA-stained cells:myelin basic protein (MBP) mRNA-stained cells in normal white matter and low-grade astrocytoma was about 2:1 but approached 1:1 with increasing tumor grade. This parameter appeared to be a good indicator of tumor infiltration in astrocytomas, so we tested this in the analysis of other gliomas. Unlike astrocytomas, oligodendrogliomas were found consistently to contain few PLP mRNA- or MBP mRNA-expressing cells. In contrast, gemistocytic astrocytomas, typically highly invasive tumors, contained high numbers of PLP-positive cells and a ratio of PLP mRNA:MBP mRNA-stained cells of about 1.5:1, similar to low-grade astrocytomas. Nonradioactive in situ hybridization also enabled the morphological identification of specific cells. For example, gemistocytic astrocytes, which were found to be strongly vimentin mRNA positive, contained little glial fibrillary acidic protein mRNA and did not stain for PLP or MBP mRNAs. Neuronal mRNAs, such as neurofilament 68, were observed in small numbers of entrapped neurons within gliomas but were uninformative with respect to predicting tumor grade. Our results suggest that oligodendrocytes survive low-grade tumor infiltration and that glial tumor cells, unlike cell lines derived from them, do not express oligodendrocyte or neuronal mRNAs. In addition, the expression of mRNAs for the two major myelin protein genes, PLP and MBP, could be used to predict the grade and extent of tumor infiltration in astrocytomas.

Topics: Astrocytoma; Brain Neoplasms; Gene Expression Regulation, Neoplastic; Glial Fibrillary Acidic Protein; Glioma; Humans; In Situ Hybridization; Keratins; Myelin Basic Protein; Myelin Proteolipid Protein; Neurofilament Proteins; Neuroglia; Neurons; Oligodendroglia; Oligodendroglioma; RNA, Messenger; RNA, Neoplasm; Vimentin

We have searched for suitable promoters to regulate the expression of suicide genes for use in gene therapy. We have shown that the 1.3-kb fragment of the mouse myelin basic protein (MBP) promoter region initiates transcription in mouse glioma cells more efficiently than glial fibrillary acidic protein (GFAP) or myelin proteolipid protein (PLP) promoter. Among three different lengths of the MBP promoter, the shortest (256-bp) core promoter region initiates transcription as efficiently as 650-bp or 1.3-kb MBP promoter lengths in RSV-M glioma cells. To assess the suitability of the MBP promoter for use in clinical trials of malignant glioma gene therapy, we also had to show that it (the 1.3-kb length in this case) is effective in human glioma cells, as well as in murine glioma cells. The activity of the MBP promoter is much higher than that of GFAP or PLP promoter in most human glioma cells, suggesting that the MBP promoter would be best for directing toxic gene expression in gene therapy for patients with malignant glioma. Human glioma cells in which the MBP promoter was strongly active were sensitive to ganciclovir when they were transduced with MBP promoter/herpes simplex virus thymidine kinase gene-bearing retroviruses. In conclusion, retrovirus-targeted gene therapy for malignant glioma using this MBP promoter is a promising candidate for clinical trials.

Topics: 3T3 Cells; Animals; Genetic Therapy; Glial Fibrillary Acidic Protein; Glioma; Herpesvirus 1, Human; Humans; Mice; Myelin Basic Protein; Myelin Proteolipid Protein; Promoter Regions, Genetic; Rats; Retroviridae; Thymidine Kinase; Transcription, Genetic; Transduction, Genetic; Tumor Cells, Cultured

Pathological differentiation of oligodendroglioma and mixed oligoastrocytoma from astrocytoma is difficult, relying on morphological characteristics due to the lack of reliable immunohistochemical stains. Oligodendrocytes, the presumed cell of origin of oligodendrogliomas, highly express the genes encoding myelin basic protein (MBP) and proteolipid protein (PLP). We analyzed the expression of these genes to determine whether they might be useful molecular markers of oligodendrocytic tumors. MBP and PLP were highly expressed in all oligodendrogliomas and minimally expressed in glioblastomas multiforme. MBP was highly expressed in mixed oligoastrocytomas, whereas PLP expression was minimal. The association between tumor classification and expression of the MBP and PLP genes was statistically significant. Expression of these genes may serve as a useful molecular marker for some subtypes of human gliomas.

Topics: Adult; Aged; Biomarkers, Tumor; Brain Neoplasms; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Humans; Male; Middle Aged; Myelin Basic Protein; Myelin Proteolipid Protein; Oligodendroglia; Oligodendroglioma; Reverse Transcriptase Polymerase Chain Reaction; Survival Analysis; Time Factors

A subline of rat C6 glioma cells, C6-10A cells, in which epinephrine can induce nerve growth factor (NGF) synthesis/secretion, was isolated. C6-10A cells have retained their sensitivity to epinephrine for more than 2 years in a medium containing 0.5% fetal calf serum (FCS) but easily lose it in 10% FCS. C6-10A cells are S-100- and glial fibrillary acidic protein-positive, and the doubling time is about 60 h in the medium containing 0.5% FCS and about 20 h in 10% FCS. Epinephrine induced NGF synthesis/secretion prominently in serum-free cultures of C6-10A cells and in cultures with a high cell density, but not in serum-containing cultures. The induction did not occur with parent C6 cells under the appropriate conditions in C6-10A cells. NGF secretion was induced by catecholaminergic compounds in the following order isoproterenol > epinephrine = norepinephrine >> dopamine. The induction caused by epinephrine was blocked by propranolol (beta-blocker) but not by phentolamine (alpha-blocker). Various compounds that activate the adenylate cyclase system also induced NGF synthesis/secretion. These results indicate that C6-10A cells are astrocytes that are highly responsive to beta-adrenergic receptor agonists, which stimulate NGF synthesis/secretion via receptors coupled with adenylate cyclase machinery. These cells may be a useful aid in studying the mechanism of NGF synthesis/secretion.

Topics: 1-Methyl-3-isobutylxanthine; Adrenergic alpha-Antagonists; Adrenergic beta-Antagonists; Animals; Blood; Bucladesine; Cattle; Cell Division; Cell Line; Cholera Toxin; Colforsin; Culture Media; Dose-Response Relationship, Drug; Epinephrine; Fibronectins; Glial Fibrillary Acidic Protein; Glioma; Immunoenzyme Techniques; Immunohistochemistry; Kinetics; Myelin Basic Protein; Nerve Growth Factors; Neurotransmitter Agents; Rats; S100 Proteins; Time Factors; Tumor Cells, Cultured

We have previously demonstrated that retrovirus-mediated genes were transferred to mouse glioma cells in a meningeal gliomatosis model (Yamada et al.: Japanese Journal of Cancer Research 83:1244-1247, 1992). This retrovirus vector contains the Escherichia coli. beta-galactosidase (beta-gal) gene as a marker for integration of the lacZ gene, which is controlled by the SV40 early promoter. We investigated whether lacZ genes could be specifically controlled in mouse glioma cells by glial-specific promoters, including the 2.5 kb 5' flanking region of the mouse glial fibrillary acidic protein (GFAP) gene, the 1.3 kb 5' flanking region of the myelin basic protein (MBP) gene, and the 1.5 kb 5' flanking region of the myelin proteolipid protein (PLP) gene. Psi-2 packaging cells were transfected with each retrovirus vector (GFAP promoter-, MBP promoter-, and PLP promoter-lacZ) and the infectious virus particles were recovered from the supernatants. Blue staining for beta-gal was detected in various fibroblast, myeloma, and glioma cell lines transduced with the retrovirus BAG vector. On the other hand, blue staining was only detected in glioma cells after transduction with the lacZ gene-bearing retrovirus controlled by glial-specific promoters. The strongest promoter activity was detected after transduction with the retrovirus in which the MBP promoter controlled the lacZ gene. Mouse glioma cells transduced with retrovirus containing the MBP promoter directing the herpes simplex virus type 1 thymidine kinase (HTK) gene were extremely sensitive to ganciclovir, while the parental cells and cells transduced with retrovirus containing the lacZ gene were not sensitive to ganciclovir.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3T3 Cells; Animals; beta-Galactosidase; Escherichia coli; Ganciclovir; Gene Expression; Genetic Therapy; Genetic Vectors; Glial Fibrillary Acidic Protein; Glioma; Herpesvirus 1, Human; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Myelin Basic Protein; Myelin Proteins; Myelin Proteolipid Protein; Promoter Regions, Genetic; Retroviridae; Simian virus 40; Thymidine Kinase; Transfection; Tumor Cells, Cultured

Since the regulation of myelin basic protein expression depends primarily on the initiation of transcription, we analyzed the 5' flanking region of the human myelin basic protein gene in transient transfection studies in primary cultures of developing oligodendrocytes. We demonstrated that 149 base pairs 5' of the initiation of transcription was sufficient to direct oligodendrocyte-specific expression of myelin basic protein. The capsite of the fusion transcript was identical with that of the endogenous myelin basic protein transcript, and chloramphenicol acetyl transferase reporter gene expression was restricted to oligodendrocytes in these cultures. Within this 149 base pair region, one distal, negative cis-acting segment, containing a consensus nuclear factor I site, and one proximal, positive cis-acting segment were identified. The distal segment behaved more negatively in Cos-7 cells than in oligodendrocytes, reducing expression to background levels. Furthermore, these functionally important cis-acting segments bound oligodendrocyte nuclear proteins in a pattern differing from other cells, including Cos-7 cells. Interestingly, the distal segment increased heterologous SV40 promoter activity in oligodendrocytes but had no effect on the SV40 promoter in Cos-7 cells. We conclude that the functionally negative distal segment may mediate oligodendrocyte-specific expression of MBP by restricting its expression in other cells. These experiments strongly support using primary cultures of oligodendrocytes for analyzing the myelin-specific promoters.

Topics: Animals; Base Composition; Base Sequence; Cell Line; Cells, Cultured; Chloramphenicol O-Acetyltransferase; Cricetinae; DNA-Binding Proteins; Fluorescent Antibody Technique; Gene Expression Regulation; Glioma; HeLa Cells; Humans; In Situ Hybridization; Molecular Sequence Data; Myelin Basic Protein; Neuroglia; Oligodendroglia; Plasmids; Promoter Regions, Genetic; Rats; Recombinant Fusion Proteins; RNA, Messenger; Transfection

Using stable cell lines containing a series of deletions of the myelin basic protein (MBP) promoter directing the bacterial chloramphenicol acetyltransferase gene in a peripheral neurinoma cell line, we have studied the sequences in the MBP promoter needed for induction by cyclic AMP. Stimulation of expression from the MBP promoter by cyclic AMP is not a rapid response. Expression begins after 24 h and reaches a maximum at approximately 72 h. The results from the stable transformants indicate at least one region that appears to be essential to the induction of transcription directed by the MBP promoter. The region that is necessary for induction does not contain a consensus cyclic AMP response element. A specific binding site involved in the induction by cyclic AMP was localized to an NFI binding site.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Base Sequence; Binding Sites; Bucladesine; CCAAT-Enhancer-Binding Proteins; Chloramphenicol O-Acetyltransferase; Colforsin; Cyclic AMP; DNA; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Glioma; Kinetics; Molecular Sequence Data; Myelin Basic Protein; NFI Transcription Factors; Nuclear Proteins; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Rats; Restriction Mapping; Sequence Deletion; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Y-Box-Binding Protein 1

35 intracranial tumours, 18 gliomas, 12 meningiomas, one neurilemmoma (neurinoma), one malignant melanoma and two metastases were successfully grown in-vitro and were submitted to immunocytochemical reactions, including cytokeratin, glial fibrillary acid protein (GFAP), vimentin, fibronectin, S-100 protein, neurofilament proteins, neuron-specific enolase (NSE) and basic myelin protein (MBP). Cytokeratin in metastases, GFAP and vimentin in gliomas, vimentin in meningiomas were consistently positive. S-100 protein was weakly and partially positive in gliomas, meningiomas, the neurilemmoma and malignant melanoma. Positive demonstration of fibronectin within cells was interpreted as a consequence of phagocytosis, except in meningiomas where fibronectin expression next to cell membranes seemed genuine. All other tested markers proved negative. The most important result seems to be that cells expressed markers irrespective of cellular shape and cytological morphology. It can be concluded that the cellular population as a whole consisted of tumour cells during the short time under observation and that supportive cell contamination during this early growth period was negligible.

Topics: Biomarkers, Tumor; Brain Neoplasms; Fibronectins; Glial Fibrillary Acidic Protein; Glioma; Humans; Immunohistochemistry; Intermediate Filament Proteins; Melanoma; Meningioma; Myelin Basic Protein; Neurilemmoma; Phosphopyruvate Hydratase; S100 Proteins; Vimentin

The present study was undertaken to evaluate the utility of pathologic features and specific immunohistochemical studies in estimating the prognosis of oligodendroglioma. The pathological diagnosis of an oligodendroglioma was made on HE stained-sections according to WHO classification. Sixteen oligodendrogliomas, twelve mixed oligoastrocytomas and ten anaplastic oligodendrogliomas were immunotested by the peroxidase-antiperoxidase (PAP) method with anti-GFAP serum, anti-S-100 serum and anti-MBP (Myelin basic protein) serum and by the avidin biotin peroxidase-complex (ABC) method with anti-vimentin serum and ant-Leu 7 monoclonal antibody. GFAP positive cells were interpreted as reactive astrocytes, neoplastic astrocytes and neoplastic oligodendrocytes, S-100 positive cells were interpreted as reactive astrocytes and neoplastic astrocytes. Leu 7 positive cells were found in only one case of anaplastic oligodendroglioma. Anti-Leu 7 could not be considered as a specific marker for oligodendroglioma. Of the anaplastic oligodendroglioma 60% displayed MBP positively and 70% displayed vimentin positively. NSE positive cells were found in a few anaplastic oligodendrogliomas. The present study has not so far uncovered any marker that is restricted to oligodendrogliomas. However GFAP may be useful to assess the extent of reactive astrocytes and neoplastic astrocytes in the oligodendroglioma or mixed oligoastrocytoma. MBP and vimentin will help to determine the malignancy of oligodendroglioma.

Topics: Adult; Aged; Biomarkers, Tumor; Brain Neoplasms; Child; Female; Glial Fibrillary Acidic Protein; Glioma; Humans; Immunohistochemistry; Male; Middle Aged; Myelin Basic Protein; Oligodendroglioma; Phosphopyruvate Hydratase; Prognosis; S100 Proteins; Vimentin

Studies were undertaken to investigate the regulation of myelin-specific mRNA expression in cultured cells. Three experimental systems were investigated: primary oligodendrocytes grown as enriched cell populations, primary oligodendrocytes grown in the presence of chick spinal cord neurons, and C6 cells. cDNA probes specific for the myelin proteolipid mRNA and the myelin basic protein mRNA were used to quantitate proteolipid and myelin basic protein mRNA levels in cells under different experimental conditions. C6 cells expressed less than 0.2% of the proteolipid mRNA that was expressed in primary oligodendrocytes. Primary oligodendrocytes expressed the myelin-specific mRNAs for at least 104 days in culture, and the level of these mRNAs in cultures was elevated fourfold by coculturing rat oligodendrocytes with chick spinal cord neurons.

Topics: Animals; Cells, Cultured; Chick Embryo; Glioma; Myelin Basic Protein; Nucleic Acid Hybridization; Oligodendroglia; Proteolipids; Rats; RNA, Messenger; Spinal Cord

In 44 patients undergoing neurosurgical procedures for intracranial tumors, subarachnoid hemorrhage, or spinal and peripheral nerve lesions, serum myelin basic protein (MBP) immunoreactivity was measured preoperatively and serially in the first 10 postoperative days. The double-antibody radioimmunoassay method was used, with a detection limit of 2.5 ng/ml in serum. Clinical evaluation was carried out at admission and on successive days during the period of neurosurgical management; outcome was assessed later. In the early postoperative phase, there was a fall in MBP immunoreactivity in all groups of patients. In the groups with intracranial tumor and subarachnoid hemorrhage, there was a subsequent rise in MBP immunoreactivity before the end of the 10-day period, which was not found in the group with spinal and peripheral nerve lesions.

Topics: Adenoma; Brain Neoplasms; Central Nervous System Diseases; Female; Glioma; Humans; Male; Meningioma; Middle Aged; Myelin Basic Protein; Radioimmunoassay; Spinal Cord Diseases; Spinal Diseases; Subarachnoid Hemorrhage

Topics: Brain Neoplasms; Electrophoresis; Glioma; Humans; Lymphocytes; Macrophages; Myelin Basic Protein; Neoplasm Proteins; Stomach Neoplasms

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Cattle; Clone Cells; Enzyme Induction; Galactosylceramides; Glioma; Glycerolphosphate Dehydrogenase; Hybrid Cells; Hydrocortisone; Myelin Basic Protein; Neuroglia; Oligodendroglia; Phosphoric Diester Hydrolases; Rats; Sulfoglycosphingolipids

Myelin basic protein in spinal fluid was measured with a radioimmunoassay method after surgery of brain tumors and posttraumatic brains in thirteen cases. Three cases were studied daily for up to three weeks. Immediately after the operation the values were high but then successively returned to normal. Repeated measurement of the myelin basic protein in spinal fluid seem to be useful for assessing the healing rate of brain tissue after surgery for brain tumors and after other brain damage.

Topics: Adult; Aged; Brain Diseases; Brain Neoplasms; Contusions; Ependymoma; Female; Glioma; Hematoma; Humans; Male; Meningioma; Middle Aged; Myelin Basic Protein; Postoperative Period; Skull Fractures

Topics: Cell Line; Glioma; Myelin Basic Protein; Radioimmunoassay

The effect of low concentrations of bovine encephalitogenic protein on the migration of human peripheral leukocytes in agarose was studied. A concentration of 0.3 mug/ml of the protein stimulated the migration of cells from many donors, including some healthy subjects. An indirect technique suggested that the migration enhancement is due to the production of soluble factor, possibly corresponding to the leukocyte migration enhancement factor described by others. The frequency of subjects whose cells could be stimulated and the recorded degree of stimulation tended to be higher in a group of patients with multiple sclerosis than in a group of healthy subjects. When the effect of some of the main peptide fragments of the protein was studied on cells that were stimulated by the intact protein, one or more of these peptides sometimes induced the opposite effect: a migration inhibition. There is, apparently, a complex balance between enhancing and inhibiting factors acting on leukocyte migration in vitro; and the character of the antigen seems to be one important factor.

Topics: Amyotrophic Lateral Sclerosis; Brain Neoplasms; Cell Migration Inhibition; Cell Movement; Glioma; HLA Antigens; Humans; Infarction; Leukocytes; Lymphocyte Activation; Meningioma; Multiple Sclerosis; Myelin Basic Protein; Peptides; Stimulation, Chemical

Topics: Amyotrophic Lateral Sclerosis; Antigens, Viral; Brain; Brain Neoplasms; Cell Migration Inhibition; Female; Gastrointestinal Neoplasms; Glioma; Humans; Immunity, Cellular; Infarction; Leukocytes; Lung Neoplasms; Meningioma; Methods; Multiple Sclerosis; Myelin Basic Protein; Peptides; Polysaccharides