concanavalin-a and Glioma

Matrix metalloproteinase-2 (MMP-2) has been suggested to play a crucial role in tumor invasion and angiogenesis. In order to understand the mechanisms underlying proMMP-2 activation, we compared the biochemical and cellular events triggered by two potent MMP-2 activators, the lectin concanavalin A (ConA) and the cytoskeleton disrupting agent cytochalasin D (CytoD). Incubation of U87 human glioma cells for 24 h in the presence of ConA or CytoD induced a marked activation of proMMP-2 and this activation was correlated in both cases with an increase in the mRNA levels of MT1-MMP. At the protein level, proMMP-2 activation induced by CytoD or ConA strongly correlated with the appearance of a 43-kDa MT1-MMP proteolytic breakdown product in cell lysates. Interestingly, CytoD also induced a very rapid (2 h) activation of proMMP-2 that was independent of protein synthesis. Under these conditions, CytoD also promoted the rapid proteolytic breakdown of the 63 kDa pro form of MT1-MMP, resulting in the appearance of the 43 kDa MT1-MMP processed form. Overexpression of a recombinant full-length MT1-MMP protein in glioma cells resulted in the activation of proMMP-2 that was correlated with the generation of the 43 kDa fragment of the protein. By contrast, overexpression of the protein in COS-7 cells promoted proMMP-2 activation without inducing the production of the 43 kDa fragment. These results thus suggest that activation of proMMP-2 occurs through both translational and post-translational mechanisms, both involving proteolytic processing of membrane-associated MT1-MMP. This processing of MT1-MMP is, however, not essential to proMMP-2 activation but may represent a regulatory mechanism to control the activity of MT1-MMP.

Topics: Concanavalin A; Culture Media, Conditioned; Cytochalasin D; Enzyme Activation; Enzyme Inhibitors; Extracellular Matrix Proteins; Gene Expression Regulation, Enzymologic; Glioma; Humans; Immunoblotting; Matrix Metalloproteinase 2; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Protein Precursors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

Maitotoxin (MTX) at 0.3 nM elicited a 10-20-fold increase in the level of Ca(2+) influx in rat glioma C6 cells. At higher doses (3-30 nM), MTX induced marked Ca(2+) influx in human erythrocyte ghosts when monitored with the fluorescent dye Fura-2. Although the ghosts were not as susceptible to MTX as intact erythrocytes or other cell lines, Fura-2 experiments under various conditions suggested that the MTX-induced entry of ions into the ghosts was mediated by a mechanism similar to that reported for cells or tissues. These ghosts are the simplest system known to be sensitive to MTX and thus may be suitable for research on the direct action of MTX. Gangliosides GM1 and GM3, glycosphingolipids which have a sialic acid residue, strongly inhibited MTX-induced Ca(2+) influx in C6 cells, while the inhibitory action by asialo-GM1, which lacks a sialic acid residue, was somewhat weaker. Their inhibitory potencies were in the following order: GM1 (IC(50) approximately 2 microM) > GM3 (IC(50) approximately 5 microM) > asialo-GM1 (IC(50) approximately 20 microM). GM1 (3 microM) completely blocked MTX (30 nM)-induced Ca(2+) influx in human erythrocyte ghosts. When C6 cells were pretreated with tunicamycin, an antibiotic which inhibits N-linked glycosylation, or concanavalin A, a lectin which exhibits a high affinity for cell-surface oligosaccharides, MTX-induced Ca(2+) influx was significantly potentiated. This suggests that removal of oligosaccharides from the cell surface by tunicamycin or capping of sugar chains on plasma membranes by concanavalin A can potentiate the action of MTX.

Topics: Animals; Anti-Bacterial Agents; Brain Neoplasms; Calcium; Calcium Radioisotopes; Concanavalin A; Erythrocyte Membrane; Fluorescent Dyes; Fura-2; Gangliosides; Glioma; Marine Toxins; Membrane Lipids; Membrane Potentials; Models, Molecular; Molecular Conformation; Oxocins; Rats; Tumor Cells, Cultured; Tunicamycin

18 cases of low-graded mixed gliomas were studied using the two lectins Concanavalin A (Con A) and Peanut lectin (PNA). Con A stained cytoplasm and processes of tumoral astrocytes, whereas PNA stained cell membranes of tumoral oligodendrocytes. Con A and PNA are reliable markers for astrocyte and oligodendrocyte areas of mixed gliomas, respectively. A part of cells were overlappingly positive for both lectins. They expressed an oligosaccharide pattern of both glioma types and represented a third, intermediate cell type of mixed gliomas. The existence of intermediate cells close to astrocytic and oligodendroglial cell types in mixed gliomas could result from transformation processes of neoplastic glial cells or from the malignant transformation of a common glial precursor cell.

Topics: Adult; Aged; Astrocytes; Brain Neoplasms; Concanavalin A; Female; Glial Fibrillary Acidic Protein; Glioma; Glucose; Humans; Male; Mannose; Middle Aged; Oligodendroglia; Peanut Agglutinin

In this study, we investigated the expression of activated gelatinase A and membrane-type metalloproteinase (MT-MMP) induced by concanavalin A (ConA) in four highly invasive glioma cell lines (UWR2, UWR3, U251MG, and SNB-19). We also examined gelatinase A and MT-MMP expression in human brain tumor tissues in vivo. Gelatin zymography showed that all four cell lines expressed latent progelatinase A (M(r) 66,000). Activated gelatinase A (M(r) 62,000) was induced by ConA in only UWR2 or UWR3 cells. MT-MMP mRNA was present in all four cell lines prior to ConA treatment, and the relative hybridization signals were 1, 0.80, 0.25, and 0.15 in UWR2, UWR3, U251MG, and SNB-19 cells, respectively. These mRNA signals were dramatically increased (2,8-, 5.4-, and 2.2-fold in UWR2, UWR3, and U251MG cells, respectively) following ConA treatment; however, MT-MMP mRNA expression was unchanged in SNB-19 cells. MT-MMP protein was detected in various amounts in the four cell lines, but only after ConA pretreatment. The amount of MT-MMP mRNA was unchanged in SNB-19 after ConA treatment, and the MT-MMP mRNA level in ConA-treated U251MG was lower than in UWR2 and UWR3 without ConA treatment. MT-MMP protein was detected in SNB-19 and U251 cell lines only after ConA treatment. Gelatin zymography of human brain tumor tissues revealed that almost all samples examined contained a latent form of gelatinase A, whereas the activated form of gelatinase A was only seen in metastatic lung adenocarcinomas and malignant astrocytomas, and especially in glioblastomas. MT-MMP mRNA levels were significantly higher in malignant astrocytomas than in low-grade gliomas and normal brain tissues. These results were confirmed by PCR analysis, which showed that MT-MMP mRNA was absent or barely detectable in normal brain white matter but was easily detectable in malignant astrocytomas. Immunohistochemistry of MT-MMP in frozen sections showed that MT-MMP was localized in neoplastic astrocytes of malignant astrocytomas but was undetectable in normal white brain matter. The data indicate that MT-MMP is present in malignant human glial tumors and that MT-MMP expression correlates with expression and activation of gelatinase A during malignant progression in vivo. A direct correlation between the levels of MT-MMP protein and its transcripts was not found in vitro, suggesting that MT-MMP expression in glioma cell lines might be regulated either at the level of transcription message stability or at posttranscrip

Topics: Astrocytoma; Base Sequence; Blotting, Northern; Blotting, Western; Brain; Brain Neoplasms; Concanavalin A; Disease Progression; Enzyme Activation; Enzyme Precursors; Fluorescent Antibody Technique; Gelatinases; Glioma; Humans; Immunohistochemistry; Matrix Metalloproteinase 2; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Molecular Sequence Data; Polymerase Chain Reaction; Proteins; Receptors, Cell Surface; Reference Values; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-2; Tumor Cells, Cultured

According to current concepts, agonists can effect the down-regulation of cell surface receptors, whereas antagonists can cause their up-regulation. We have discovered that the opioid antagonists naltrexone, naloxone, and ICI174864 induce a transient down-regulation of delta-opioid receptors before up-regulation, in NG108-15 cells. The possibility of an apparent loss of sites due to blockade by residual antagonist was ruled out by several lines of evidence. The reduction in delta receptors was time, temperature, and antagonist concentration dependent. This down-regulation could not be induced by either the highly mu-selective opioid antagonist cyclic D-Phe-Cys-Try-D-Trp-Arg-Thr-Pen-Thr-amide or the muscarinic antagonist atropine. In the same neurohybrid cells, the opioid agonist [D-Ala2,D-Leu5]enkephalin (0.1 microM, 60 min) effected a greater down-regulation of delta-opioid receptors. Similar qualitative changes in opioid binding of subcellular fractions were elicited with [D-Ala2,D-Leu5]enkephalin and naltrexone. However, the agonist was 2-fold more effective in reducing the heavy membrane population of receptors and 4-fold more potent in increasing the light membrane sites. Because heavy membranes are enriched in plasma membrane, whereas light membranes contain intracellular sites, these findings indicate that internalization occurs in both instances. Naltrexone and the delta-specific antagonists ICI174864 and naltrindole also diminished specific activities of two lysosomal enzymes, whereas opioid agonist-induced down-regulation was accompanied by an increase in their specific activities. Pretreatment of cell cultures with concanavalin A blocked both down-regulation and alterations in the lysosomal enzyme activities elicited by agonists and antagonists, suggesting that the latter is an opioid receptor-mediated process. The up-regulation of delta-opioid receptors by antagonists appears, then, to entail down-regulation that differs from that of agonists.

Topics: Amino Acid Sequence; Binding Sites; Binding, Competitive; Concanavalin A; Down-Regulation; Enkephalin, Leucine-2-Alanine; Glioma; Kinetics; Lysosomes; Molecular Sequence Data; Naloxone; Naltrexone; Narcotic Antagonists; Neuroblastoma; Receptors, Opioid; Receptors, Opioid, delta; Subcellular Fractions; Tritium; Tumor Cells, Cultured

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.

Topics: Animals; Antigen-Antibody Reactions; Antigen-Presenting Cells; Cell Line; Concanavalin A; Cytotoxicity Tests, Immunologic; Dose-Response Relationship, Immunologic; Glioma; HLA-D Antigens; Humans; Immunity, Cellular; Interferon-gamma; Interleukin-1; Interleukin-6; Lectins; Lipopolysaccharides; Lymphocyte Activation; Monokines; Phytohemagglutinins; T-Lymphocytes; Tetanus Toxoid; Toxoplasma

Anticonvulsant-induced alteration in C6 glioma cell adhesivity has been evaluated in two independent in vitro assay systems. A centrifugal shear assay was employed to determine drug-induced change in cell-substratum adhesivity. Valproate and clonazepam were found to significantly increase cell-substratum adhesivity when cells were cultured at concentrations which were within twice their therapeutic plasma level. A second assay evaluated change in affinity for concanavalin A lectin coated surfaces to determine change in cell surface glycoconjugate expression. Valproate and clonazepam and, to a lesser extent, diazepam significantly decreased drug-exposed C6 glioma cell affinity for concanavalin A lectin coated surfaces. Valproate and clonazepam had approximate IC50 values of 0.75 mM and 75 microM, respectively. These findings are compared and discussed in relation to those obtained with an anti-proliferative assay which has been suggested to predict teratogen potential.

Topics: Animals; Anticonvulsants; Cell Adhesion; Cell Division; Concanavalin A; Glioma; Rats; Teratogens; Tumor Cells, Cultured

The lectins Concanavalin A (Con A), Ricinus communis agglutinin (RCA-I), Peanut agglutinin (PNA) and Wheat germ agglutinin (WGA) as well as the immunomarkers for glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were used in a series of 21 glial tumors (4 pylocytic astrocytomas, 5 grade II astrocytomas, 3 anaplastic astrocytomas, 4 glioblastomas and 5 oligodendrogliomas). ConA binds to all tumoral astrocytes in low grade astrocytomas, as well as to well differentiated tumoral astrocytes in anaplastic astrocytomas and glioblastomas. RCA-I has a similar behaviour. PNA, and to a lesser degree WGA, binds selectively to the oligodendroglial plasma membrane in well differentiated oligodendrogliomas. The results suggest that these lectins are markers of differentiation in gliomas rather than of malignancy.

Topics: Astrocytoma; Cell Differentiation; Central Nervous System Neoplasms; Concanavalin A; Glioma; Humans; Lectins; Peanut Agglutinin; Ricin; Wheat Germ Agglutinins

We have studied the Gal beta 1-3GalNAc-R alpha 2,3 sialyltransferase from C6 glioma cells transferring Neu5Ac from CMP-Neu5Ac onto O-glycans of glycoproteins. Using synchronized C6 glioma cells, we showed that the alpha 2,3 sialyltransferase activity was inhibited by tunicamycin to a greater extend than DNA and protein biosynthesis suggesting inhibition of N-glycosylation of this enzyme. Additional demonstration of N-glycosylation of the alpha 2,3 sialytransferase was provided through ConA-Sepharose binding. Treatment of partially purified alpha 2,3 sialytransferase by peptide-N-glycosidase F showed a significative inhibition demonstrating that N-glycan moiety is required for complete activity of the C6 glioma cell alpha 2,3 sialyltransferase.

Topics: Animals; beta-Galactoside alpha-2,3-Sialyltransferase; Cell Line; Chromatography, Affinity; Concanavalin A; DNA Replication; Glioma; Glycoproteins; Glycosylation; Kinetics; Protein Processing, Post-Translational; Rats; Sialyltransferases; Tunicamycin

These experiments set out to assess the role of NK and B cells in the resistance of nude mice to human tumor xenotransplantation. The transplantability of 9 fresh and 8 cultured human tumors was compared in 2 strains of mice with different genetic immune deficiencies: athymic NCr/Sed (nu/nu) nude mice, and nude-beige-xid (N:NIH-nu-bg-xid/Sed mice). Flow cytometric studies showed both strains to be deficient in Thy. 1.2 (T) cells and unresponsive to stimulation by Concanavalin A (Con A) or direct T-cell-receptor triggering with anti-CD3. The number of B cells was similar in the 2 strains, but the response to lipopolysaccharide (LPS) was markedly reduced in the nude-beige-xid animals. The number of asialoGM1-positive cells (predominantly NK) detected by flow cytometry was also reduced in the nude-beige-xid mice. The transplantability of the human tumors was found to be equivalent in the 2 strains. Quantitative cell-transplantation assays performed for 2 of the tumor cell lines did not reveal any subtle transplantation advantage for the more broadly immune-deficient animals. No evidence could, therefore, be found to suggest that NK or B cells were major determinants of human tumor xenotransplantability in these strains of mice.

Topics: Animals; B-Lymphocytes; Cell Line; Concanavalin A; Flow Cytometry; Glioma; Humans; Killer Cells, Natural; Lipopolysaccharides; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Transplantation, Heterologous

An in vitro technique was developed to generate activated rat T cells, with antitumor activity. Splenic mononuclear cells (SMC) from outbred Wistar and inbred Wistar-Munich rats were stimulated with Concanavalin A and recombinant human interleukin-2 (rIL-2) in vitro for 48 h. After 2 days, the nonadherent cells began proliferating and were maintained in rIL-2 for up to 18 days in vitro. FACScan analysis revealed that SMC was a mixture of cell types; however, CD5+ T cells rapidly increased and became the predominant cell type after 5 days in culture. SMC induced cytolysis of YAC-1, but not C6 glioma cells in 4 h 51Cr release assays. In contrast, 5- and 9-day T cells lysed C6 glioma and YAC-1 cells. The C6 cells were admixed with cultured effector cells at various effector-to-target (E:T) ratios and were injected into the right cerebral hemisphere of Wistar and Wistar-Munich rats for a Winn assay. Histopathologic evaluations revealed that a) SMC had no effect; b) 2- and 5-day T cells, injected at E:T ratios greater than 5:1, caused significant reduction in tumor size; and c) 2- or 5-day T cells, at a 40:1 E:T ratio, resulted in little or no histologic evidence of tumor. Eighty-three percent of animals receiving C6 and 5-day mitogen-stimulated lymphokine activated killer cells at an E:T ratio of 40:1 were alive 120 days postinjection (p less than 0.05).

Topics: Animals; Brain Neoplasms; Cell Division; Cell Survival; Cells, Cultured; Concanavalin A; Glioma; Immunophenotyping; Immunotherapy, Adoptive; Interleukin-2; Killer Cells, Lymphokine-Activated; Lymphoma; Male; Neoplasm Transplantation; Rats; Rats, Inbred Strains; Recombinant Proteins; Spleen; Tumor Cells, Cultured

Mononuclear cells infiltrating human brain tumors were isolated from seven of nine surgical biopsy specimens. These cells were small T11+, T3+ lymphocytes that did not express DR antigens or the receptor for interleukin-2. In addition, large granular lymphocytes were recovered from two of these tumors. The clonogenic potential of tumor-infiltrating lymphocytes (TIL's) was assessed by limiting-dilution analysis (LDA) using a microculture system that permits proliferation of virtually 100% of normal peripheral blood T lymphocytes (PBL-T's). In comparison to normal and autologous PBL-T's, TIL's had a strikingly reduced proliferative potential revealed by a decrease in the frequency of proliferating T lymphocyte precursors calculated by LDA. On average, only one of every 100 T cells from TIL's was able to proliferate, as compared to one of every two or all of the T cells from the patient's peripheral blood or from normal donors. Furthermore, the TIL populations showed depressed proliferative responses to the lectins phytohemagglutinin and concanavalin A and to the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate. Clonal analysis performed on the proliferating microcultures from three tumors demonstrated that the majority of these clones possessed cytolytic activity against various tumor cell targets. Among clones tested for cytolytic activities with glioma cells, four lysed cultured autologous tumor cells, and the specific lysis was greater than 50% in all cases. Numerous clones with natural killer (NK)-like activity were obtained from two TIL preparations, and the frequency of cytolytic T lymphocyte precursors with NK-like activity was determined for one of these preparations and was found to be higher than that in the patient's peripheral blood. Glioma cells grown in culture and then mixed with normal peripheral blood lymphocytes (PBL's) were capable of inhibiting the PBL's response to lectins. This inhibitory property may account in part for the observed poor clonogenicity of TIL's from brain tumors. Nevertheless, nearly all proliferating clones displayed cytotoxicity against either autologous or allogeneic tumor cell targets and may imply selective accumulation of cytolytic effector cells at the tumor site.

Topics: Adult; Aged; Antibodies, Monoclonal; Brain Neoplasms; Cell Division; Child; Clone Cells; Concanavalin A; Cytotoxicity, Immunologic; Female; Glioma; Humans; Immunohistochemistry; Male; Middle Aged; Phytohemagglutinins; T-Lymphocytes; Tumor Cells, Cultured

Exposure of rat glioma C6 cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused an activation of protein kinase C wherein the enzyme rapidly became membrane-bound (T 1/2 of 15 min). This translocation of protein kinase C from cytosol to membrane was followed by a sequestration of cell surface beta-adrenergic receptors and a loss of isoproterenol-stimulated adenylate cyclase activity. We had reported previously that prior exposure of rat glioma cells to concanavalin A prevents the TPA-mediated sequestration of receptors and desensitization of adenylate cyclase (Kassis et al., 1985). We now show that the concanavalin A treatment also prevents the translocation and activation of protein kinase C. These results are further evidence that in the TPA-treated cells, sequestration of beta-adrenergic receptors is mediated by membrane-bound protein kinase C.

Topics: Animals; Cell Line; Cell Membrane; Concanavalin A; Cytosol; Glioma; Kinetics; Protein Kinase C; Rats; Receptors, Adrenergic, beta; Tetradecanoylphorbol Acetate

Human glioblastoma cell lines showed profound suppression of both DNA and RNA synthesis when exposed to supernatants (SNs) of mitogen-activated blood mononuclear cells. Cloning efficiency of these glioma cells also decreased 10- to 500-fold. In monolayer cultures, growth inhibition was evident within 12 h of adding SN and peaked at 24 h. A decrease in absolute cell number was evident by 72 h. The inhibitory effect of SNs, however, was not permanent as more cells entered S-phase when SN-treated cultures were refed with fresh medium (without SN). The factor(s) responsible for this inhibitory activity was a product of lymphocytes and was produced in comparable amounts by cells of normal blood donors and patients with glioma. The compromised immunological status of glioblastoma patients did not influence their capacity to produce cytostatic lymphokines.

Topics: Adult; Aged; Animals; Cell Line; Colorimetry; Concanavalin A; DNA; Female; Glioblastoma; Glioma; Humans; In Vitro Techniques; Lymphocyte Activation; Lymphocytes; Lymphokines; Male; Middle Aged; Phytohemagglutinins; Rats; RNA

The distribution of beta-adrenergic receptors in lysates from several mammalian cell lines was analyzed on nonlinear sucrose density gradients before and after desensitization by isoproterenol. On the nonlinear gradients, the receptors in lysates from untreated HeLa, A431, S49 cyc- and C6 cells were well resolved into light and heavy density membrane fractions. In contrast, with the former three cell lines, there was very poor or no separation of the two peaks of receptors on linear sucrose gradients. With C6 cells, resolution was better on the nonlinear than on the linear gradient. In all cases, successful separation of the two density fractions of the receptor was achieved only when cells had been treated with concanavalin A prior to lysis. Adenylate cyclase activity cosedimented with the heavy membrane fraction of the receptor, and no activity was detected with the light fraction. After desensitization of adenylate cyclase by isoproterenol, there was a redistribution of the receptors to the light density fraction. This shift of receptors, but not desensitization, was prevented when cells were pretreated at 37 degrees C with concanavalin A prior to exposure to isoproterenol. Thus, sequestration of beta-adrenergic receptors away from the plasma membrane and adenylate cyclase to a lighter density membrane fraction appears to accompany, but may not be a prerequisite for desensitization in mammalian cells. This receptor redistribution, however, can be readily detected on nonlinear sucrose gradients.

Topics: Adenylyl Cyclases; Cell Line; Cells, Cultured; Centrifugation, Density Gradient; Concanavalin A; Glioma; HeLa Cells; Humans; Receptors, Adrenergic, beta; Sucrose

A crucial manifestation of malignant gliomas is the regrowth of already-invaded neoplastic cells after surgical intervention. One possible approach for inhibiting such tumor growth is to utilize the tumoricidal potential of macrophages. In order to investigate the clinical application of this concept, peritoneal exudate cells (PEC) activated in vitro and in vivo by immunomodulating agents were tested for cytotoxic activity against murine glioma (203-glioma) cells. As immunomodulating agents, heat-killed Propionibacterium acnes (P. acnes), OK-432 and Concanavilin A supernatant (Con A sup) were used in these experiments. P. acnes was provided by Kowa Pharmaceutical Co., Tokyo, and OK-432 by Chugai Pharmaceutical Co., Ltd., Tokyo. Klinische Einheit (KE) units were used to express the strength of the preparation, with 1 KE equal to 0.1 mg of dried streptococci. Con A sup was produced by Con A pulsing of BALB/c splenocytes resuspended in complete medium. PEC harvested from mice to which 5% glycogen in saline had been inoculated intraperitoneally 6 d previously were activated in vitro by P. acnes (P. acnes-PEC), OK-432 (OK-432-PEC) and Con A sup (Con A-PEC). The cytotoxic activities of P. acnes-PEC, OK-432-PEC and Con A-PEC were approximately 25%, 65% and 60%, respectively. PEC were then collected from mice into which either 100 micrograms of P. acnes or 1 KE of OK-432 had been injected intraperitoneally several times. The antitumor effects of P. acnes-PEC and OK-432-PEC were about 35% and 50%, respectively. These activated PEC demonstrated cytotoxic activity against murine glioma in the tumor neutralization assay (Winn assay). Also, the antitumor efficacy of OK-432-PEC belonged mainly to adherent cells. Meningeal gliomatosis (MG) models were prepared for clinical studies. Viable 203-glioma cells (5 X 10(6) were injected percutaneously into the cisterna magna of C57BL/6 mice. The median survival time (MST) of the untreated group was 8.5 days. The MST of the groups treated by intraperitoneal and intracisternal administration of P. acnes were 26 and 33 days. This therapy significantly prolonged the survival time of these models, particularly by the intracisternal treatment. The differential cell count by Giemsa staining and latexphagocytic cell findings revealed that macrophages accounted for more than 90% of the P. acnes-PEC. These results may indicate that activated (PEC) macrophages were induced intracisternally by P. acnes and that activated macrophages induc

Topics: Animals; Concanavalin A; Cytotoxicity, Immunologic; Female; Glioma; Macrophage Activation; Macrophages; Mice; Mice, Inbred C57BL; Picibanil; Propionibacterium acnes

Exposure of rat glioma C6 cells to either isoproterenol or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in desensitization of isoproterenol-stimulated adenylate cyclase activity. After either treatment, the affinity of beta-receptors for isoproterenol was reduced. Thus, desensitization by TPA or isoproterenol appeared to involve an "uncoupling" of the beta-receptor from the stimulatory regulatory component (Ns) of adenylate cyclase. The activity of Ns, assayed by reconstitution of S49 cyc- adenylate cyclase activity, was found to be unchanged after desensitization. The activity of beta-receptors was measured by inactivating Ns and the catalytic component of adenylate cyclase in C6 membranes and fusing them with membranes lacking beta-receptors. Receptors from isoproterenol-treated C6 cells were less active in "coupling" to the foreign adenylate cyclase than receptors from untreated cells, whereas receptors from TPA-treated cells were fully active. This unexpected latter result was explored further. Lysates from C6 cells were centrifuged on linear sucrose density gradients and the gradient fractions assayed for beta-receptor binding activity. Most of the receptors were recovered in a "heavy" plasma membrane peak but some receptors also appeared in a "light" membrane peak. After treatment of the cells with isoproterenol or TPA, the proportion of receptors in the light peak increased. Prior treatment of the cells with concanavalin A prevented the increase in light receptors caused by isoproterenol or TPA. In addition, the concanavalin A treatment prevented the desensitization of adenylate cyclase caused by TPA but not that caused by isoproterenol. Finally, desensitization of adenylate cyclase was reversed by polyethylene glycol-induced fusion of membranes from cells treated with TPA but not isoproterenol. We conclude that beta-agonists and phorbol esters desensitize adenylate cyclase by distinct mechanisms. Agonists cause a reduction in the functional activity of the beta-receptors followed by a segregation of the receptors into a light membrane fraction devoid of Ns. Phorbol esters do not alter the activity of the receptors but do cause their segregation.

Topics: Adenylyl Cyclases; Adrenergic beta-Agonists; Animals; Caenorhabditis elegans Proteins; Carrier Proteins; Cells, Cultured; Concanavalin A; Enzyme Activation; Glioma; Isoproterenol; Phorbols; Phosphorylation; Protein Kinase C; Protein Kinases; Rats; Receptors, Adrenergic, beta; Receptors, Drug; Receptors, Immunologic; Tetradecanoylphorbol Acetate

Immunosuppressive mechanisms in patients with malignant brain tumors were studied with the use of a nylon wool column and monoclonal antibodies. Peripheral blood lymphocytes (P.B.L.) from the patients (82 malignant gliomas, 65 metastatic brain tumors) were tested for their ability to inhibit lymphocytoblastogenesis, and reacted with monoclonal anti Leu 1, 2a and 3a antibodies to identify the subsets of T lymphocytes. Depression of the lymphocytoblastogenesis was detected significantly by the patients' P.B.L. passed through the nylon-wool column, but not detected by that adhering to the column. This suppressor cell activity was shown to do its work over the barrier of major histocompatibility complex, and seemed to be associated with Con. A-induced suppressor cell activity. Especially, in the patients with malignant gliomas, the suppressor T cells seemed to be induced by tumor cells, and mediate the noted immunodepression. Furthermore, analysis of T cell subsets using monoclonal antibodies showed that the suppressor cell activity in the patients with malignant gliomas seemed to be closely correlated with Leu 2a+ cells, and the Leu 3a+/Leu 2a+ ratio decreased with tumor loads suggesting that the suppressor T cells are more dominant than the helper T cells. These immunological studies help to advance therapeutic protocols of the patients, because suppressor cells may be related to the escape mechanism of malignant brain tumors.

Topics: Antibodies, Monoclonal; Brain Neoplasms; Concanavalin A; Female; Glioma; Humans; Immune Tolerance; Lymphocyte Activation; Male; Middle Aged; T-Lymphocytes

Topics: Adolescent; Adult; Brain Neoplasms; Child; Concanavalin A; Dose-Response Relationship, Drug; Female; Glioma; Humans; Male; Middle Aged; T-Lymphocytes, Regulatory

Using a lectin-peroxidase method, Concanavalin A binding was examined on formalin-fixed paraffin-embedded biopsy specimens (n = 143) of the most frequent central nervous system tumours. The brain tumours included oligodendrogliomas, astrocytomas, glioblastomas, ependymomas, neurinomas, meningiomas, medulloblastomas and plexus papillomas. In oligodendroglioma cells, only a weak granular intracytoplasmic staining was observed. The astrocytomas showed a strong reaction in fibrillary astrocytes and in tumour areas undergoing small cystic degeneration. Staining of protoplasmic astrocytes was weaker; pilocytic astrocytes demonstrated poor perinuclear staining. Intracytoplasmic Con A binding in gemistocytic astrocytes was distinct but inconstant and rather diffuse. In the glioblastomas the lymphocyte-like small astrocytes were negative. Giant multinucleated astrocytes stained strongly. In ependymomas no or at most a weak perinuclear reaction was observed, whereas the acceptor density was as high as in the normal ependymocytes in areas where the tumour was capable of producing organotypical structures. Plexus papillomas showed a strong intracytoplasmic staining comparable to the normal plexus epithelial cell. This feature was preserved in the malignant variants. In general, meningiomas and neurinomas were negative. Xanthomatous-degenerated meningioma cells, however, showed a distinct to strong intracytoplasmic staining. This feature was characteristic for the xanthomatous subtype of meningiomas. Granular cells with strong intracytoplasmic Con A staining often occurred at the border of fibrillary to reticular differentiated areas of neurinomas. Medulloblastomas were completely negative. Our results indicate that Con A binding to human brain tumours is specific and rather cytotypical than histotypical . The Con A acceptor density is probably related to the grade of differentiation. Lectin mapping of tumours leads to cytotypical binding patterns which may contribute to the differential diagnosis of neoplasias.

Topics: Astrocytoma; Brain; Brain Neoplasms; Cerebellar Neoplasms; Concanavalin A; Ependymoma; Glioma; Humans; Immunoenzyme Techniques; Medulloblastoma; Meningeal Neoplasms; Meningioma; Neurilemmoma; Oligodendroglioma; Papilloma; Receptors, Concanavalin A

Plasma membranes were isolated from C6 glioblastoma cells by two methods. In the first method cells were treated with concanavalin A and lysed in hypotonic medium. After partial separation of plasma membranes from other cell material, the lectin was displaced with alpha-methyl-D-mannoside. In the second method untreated cells or cells iodinated in a lactoperoxidase-catalyzed reaction were homogenized in isotonic medium. Membrane fractions obtained by either homogenization procedure were further purified by rate zonal and equilibrium centrifugations into linear density gradients. Disruption of the glioblastoma cell membrane gives rise to heterogeneous assemblies of membrane fragments. Two populations of plasma membranes were isolated from untreated and from iodinated cells: a "lighter" membrane fraction characterized by relatively lower sedimentation velocity and buoyant density, and a "heavier" membrane fraction of relatively faster sedimentation velocity and higher buoyant density. Both fractions showed electrophoretic patterns similar to those of 125I-labeled cell surface proteins. Their specific (Na+ + K+)-ATPase activity was seven- to eightfold the homogenate activity (recovery, 13.1%). Both fractions were, however, still contaminated by smooth endoplasmic reticulum, as judged from the activity of NADPH-dependent cytochrome c reductase (recovery, 2.4%). It is suggested that plasma membrane fragments present in the two fractions might differ in the organization of their structures, e.g., membrane vesicle intactness and membrane orientation.

Topics: Animals; Cell Fractionation; Cell Membrane; Cells, Cultured; Centrifugation, Density Gradient; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Glioma; Molecular Weight; Rats; Sodium-Potassium-Exchanging ATPase

The intracellular content of the nervous system specific protein S-100 began to increase with 4 days latency following the morphological differentiation of cultured rat glioma cells (C-6) with 1 mM dibutyryl cyclic AMP (dbcAMP), rising to approximately 10-fold over the control level at 15 days after the treatment. The concanavalin A (Con A) binding pattern on the external cell surface of C-6 cells exposed to dbcAMP appeared as a smooth layer of 40-60 nm thickness whereas that of control cells was irregularly thick and patchy. The correlation between morphological and biochemical changes of C-6 cells after dbcAMP treatment is discussed in relation to the mechanism controlling the differentiation of glioma cells.

Topics: Animals; Binding Sites; Bucladesine; Cell Differentiation; Concanavalin A; DNA, Neoplasm; Glioma; Kinetics; Neoplasms, Experimental; Nerve Tissue Proteins; Rats; S100 Proteins

The concentration of glycerol-3-phosphate dehydrogenase (GPDH; sn-glycerol-3-phosphate:NAD(+) 2-oxidoreductase, EC 1.1.1.8) had previously been determined to be regulated by glucocorticoids in rat brain cells in vivo and in cell culture. We now demonstrate that concanavalin A (Con A) can inhibit the induction of GPDH in dose-dependent manner in C6 rat glioma cells and in primary cultures of rat brain oligodendrocytes. Con A is not cytotoxic, because its effect can be prevented or reversed by alpha-methyl mannoside. The inhibition specifically prevents the appearance of new molecules of GPDH, although Con A does not significantly inhibit protein synthesis in these cells, nor does it affect the activity of another soluble enzyme, lactate dehydrogenase. The ability to block enzyme induction is not limited to Con A, because other lectins also inhibit induction, with Ricinus communis agglutinin 60 being the most potent (50% inhibition of induction at 0.0083 muM) and wheat germ agglutinin being the least potent (50% inhibition of induction at 1.2 muM). The molecular mechanism by which Con A inhibits GPDH induction appears to be by the "down regulation" of the cytoplasmic glucocorticoid receptors, because exposure to Con A results in the loss of more than 90% of the receptor activity. Con A does not inhibit the receptor assay and no direct interaction between the receptor and Con A could be demonstrated. This down regulation is not tumor cell specific and appears to be a general phenomenon, because it occurs in normal oligodendrocytes and even in normal astrocytes (a cell type in which the gene for GPDH is not expressed). The down regulation of glucocorticoid receptors in normal brain cells suggests two important corollaries. First, it demonstrates the existence of a rate-limiting step controlling the glucocorticoid-dependent gene expression in brain cells and possibly represents a regulatory site common to all glucocorticoid target cells. Second, it suggests that the response to glucocorticoids of oligodendrocytes and astrocytes can be regulated in vivo by cell surface contact with endogenous lectins, neighboring cells, or both.

Topics: Animals; Astrocytes; Brain; Cell Line; Concanavalin A; Dexamethasone; Enzyme Induction; Glioma; Glyceraldehyde-3-Phosphate Dehydrogenases; Oligodendroglia; Rats; Receptors, Glucocorticoid; Receptors, Steroid

Topics: Amphotericin B; Animals; Cell Line; Concanavalin A; Glioma; Mice; Models, Biological; Neuroblastoma; Rats; Receptors, Concanavalin A; Receptors, Mitogen; Ricin

The effect of ethanol on an enzyme system within an intact functional plasma membrane has been studied using neural cells grown in culture. Rat C6 glioma cells in mono-layer culture were treated acutely or chronically with 100 mM ethanol and the effect of this exposure on the activity of ecto-5'-nucleotidase was determined. Acute exposure led to an increase in enzyme activity with maximum stimulation occurring at concentrations of 100 - 400 mM ethanol. Chronic treatment of cells with 100 mM ethanol for 4 - 8 days also caused an increase in ecto-5'-nucleotidase activity. Both the acute and chronic ethanol-induced stimulation of enzyme activity was completely reversible by removing the ethanol; the acute effects reversed immediately, whereas the chronic effects required several hours. The addition of Concanavalin A demonstrated that the effects on enzyme activity of both chronic and acute exposure to ethanol were blocked by modification of the external cell surface. The effect of chronic exposure to 100 mM ethanol was further localized to an action on the plasma membrane by studies which showed chronic exposure to have no effect on the intracellular 5'-nucleotidase activity. Furthermore, the occurrence of pharmacological tolerance to acute ethanol was observed in this plasma membrane system following chronic treatment of C6 cells with 100 mM ethanol. These findings are consistent with the hypothesis that mammalian neural cells can adapt to the chronic presence of ethanol through changes in their plasma membrane.

Topics: 5'-Nucleotidase; Animals; Cell Line; Cell Membrane; Concanavalin A; Ethanol; Glioma; Kinetics; Nucleotidases; Protein Binding; Rats; Substrate Specificity

Topics: 5'-Nucleotidase; Animals; Cells, Cultured; Concanavalin A; Ethanol; Glioma; Hydrogen-Ion Concentration; Kinetics; Neoplasms, Experimental; Nucleotidases; Rats; Substrate Specificity

Plastic dishes were coated with an agarose layer. The layer was modified by covalently binding proteins to it, using the CNBr-method. Cells were seeded on the dishes and the number of attached cells was evaluated. The specificity of the attachment was demonstrated by showing that cells, carrying specific membrane-bound immunoglobulins, attached only to the corresponding anti-immunoglobulins. This indicated that the method could be used for cell sorting. The attachment of cells to proteins was influenced by the amount of bound protein, incubation time, temperature and the degree of trypsinization. Most attached cells were viable for several days and when dying they detached. Detailed morphological and cytochemical analyses of the dynamics of attachment and cytoplasmic spreading on the chemically well-defined surfaces were possible using the new method.

Topics: Burkitt Lymphoma; Cell Adhesion; Cell Line; Concanavalin A; Glioma; Humans; Leukemia; Lymphoma, Non-Hodgkin; Neuroglia; Polylysine; Polysaccharides; Protamines; Protein Binding; Proteins; Sepharose

The mitogenic responsiveness of spleen cells obtained from avian sarcoma virus-inoculated Fischer 344 rats was studied. Sixty % of the rats had astrocytomas, 13% had sarcomas, 7% had mixed gliosarcomas, and 20% had no evidence of tumors. Only spleen cells from rats bearing astrocytomas had significantly diminished responses to phytohemagglutinin and concanavalin A (Con A) when compared to control responses. The decreased responsiveness observed with phytohemagglutinin was limited to the optimal concentration range (10 and 20 microgram) while a broader concentration of Con A (0.01 to 50 microgram) induced significant suppression. Moreover, a more profound immunosuppression was observed with Con A. The results also demonstrated that spleen cells from rats with the largest astrocytomas exhibited the greatest suppression. From the results of this study, it appears the avian sarcoma virus-induced astrocytoma in rats is an immunological parallel of the human disease based on the loss of general immunological competence as assessed by responsiveness of lymphocytes to phytohemagglutinin and Con A.

Topics: Alpharetrovirus; Animals; Astrocytoma; Brain Neoplasms; Concanavalin A; Glioma; Immunity; Lectins; Lymphocyte Activation; Male; Neoplasms, Experimental; Rats; Rats, Inbred F344; Sarcoma, Avian; Spleen

Topics: Agglutination Tests; Animals; Cell Aggregation; Cell Membrane; Concanavalin A; Glioma; Microscopy, Electron; Neoplasms, Experimental; Neuroglia; Rats; Receptors, Concanavalin A; Receptors, Drug

Topics: Astrocytoma; Brain Neoplasms; Concanavalin A; Glioma; Humans; Lectins; Lymphocyte Activation

Topics: Agglutination; Animals; Cell Aggregation; Cell Count; Cell Division; Cell Line; Concanavalin A; Dose-Response Relationship, Drug; Edetic Acid; Extracellular Space; Glioma; Kinetics; Methylmannosides; Rats; Trypsin

Topics: Adult; Agglutination; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Concanavalin A; Depression, Chemical; Fibroblasts; Glioma; Glucose; Glutaral; Humans; Hyaluronoglucosaminidase; In Vitro Techniques; Lactose; Lectins; Lung; Methods; Neuroglia; Plant Lectins; Plants, Toxic; Ricinus; Sarcoma; Stimulation, Chemical; Temperature; Trypsin