monensin and Glioma

Topics: Animals; Bacterial Toxins; Brain Neoplasms; Carcinoma; Clinical Trials as Topic; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Diphtheria Toxin; Drug Carriers; Drug Synergism; Glioma; Guinea Pigs; Humans; Immunotoxins; Injections, Intralesional; Injections, Spinal; Ligands; Medulloblastoma; Meningeal Neoplasms; Mice; Monensin; Plant Proteins; Rats; Rats, Nude; Recombinant Fusion Proteins; Tissue Distribution; Transferrin; Treatment Outcome; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is preferentially cytotoxic to cancer cells over normal cells. However, many cancer cells, including malignant glioma cells, tend to be resistant to TRAIL. Monensin (a polyether ionophore antibiotic that is widely used in veterinary medicine) and salinomycin (a compound that is structurally related to monensin and shows cancer stem cell-inhibiting activity) are currently recognized as anticancer drug candidates. In this study, we show that monensin effectively sensitizes various glioma cells, but not normal astrocytes, to TRAIL-mediated apoptosis; this occurs at least partly via monensin-induced endoplasmic reticulum (ER) stress, CHOP-mediated DR5 upregulation and proteasome-mediated downregulation of c-FLIP. Interestingly, other polyether antibiotics, such as salinomycin, nigericin, narasin and lasalocid A, also stimulated TRAIL-mediated apoptosis in glioma cells via ER stress, CHOP-mediated DR5 upregulation and c-FLIP downregulation. Taken together, these results suggest that combined treatment of glioma cells with TRAIL and polyether ionophore antibiotics may offer an effective therapeutic strategy.

Topics: Anti-Bacterial Agents; Antineoplastic Agents; Apoptosis; Astrocytes; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Down-Regulation; Endoplasmic Reticulum Stress; Glioma; Humans; Monensin; Proteasome Endopeptidase Complex; Receptors, TNF-Related Apoptosis-Inducing Ligand; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand; Up-Regulation

Influence of high salt culture conditions on the expression of immediate early gene egr-1 in rat C6 glioma cells was investigated by measuring both Egr-1 mRNA and protein levels in the cells exposed to the medium containing high concentrations of NaCl. The exposure to high salt medium reduced Egr-1 mRNA and protein levels, while Egr-1 mRNA levels were not altered by the medium containing either sucrose or glycerol. Veratridine and monensin also reduced Egr-1 mRNA levels, similar in extent to that induced by high salt medium. Imaging analysis indicated that the exposure to high salt medium induced the elevation of Na+ levels within the cells. These results indicate that neither hyperosmotic pressure nor ionic strength of high salt medium contribute to the reduction of Egr-1 expression, and suggest that the elevation of intracellular Na+ concentration is closely associated with the down-regulation of egr-1 gene expression.

Topics: Animals; Calcium; Cell Line, Tumor; Culture Media; DNA-Binding Proteins; Down-Regulation; Early Growth Response Protein 1; Gene Expression Regulation; Genes, Immediate-Early; Glioma; Immediate-Early Proteins; Monensin; Osmolar Concentration; Rats; Saline Solution, Hypertonic; Sodium; Transcription Factors; Veratridine

Comparative analyses were conducted to determine the effects of Na(+) (monensin, MON), K(+) (nigericin, NIG) and Ca(2+) (A23187) selective carboxylic ionophores on differentiated NG108-15 (neuroblastoma X glioma hybrid) cells. Alterations in membrane potential (V(m)), input resistance (Rin) and electrically induced action potential generation were measured using intracellular microelectrode techniques in cells treated with 0.1-30 microM MON and NIG and 0.1-10 microM A23187. Responses to the ionophores were similar in that membrane hyperpolarization and unchanged R(in) predominated with all three compounds. However, significant differences between the ionophores were also detected. MON- and A23187-induced hyperpolarization was generally maintained throughout the 24-min superfusion whereas that produced by NIG diminished with time or was replaced by depolarization. In addition, action potential generation was blocked by NIG, whereas MON had no effect and action potential alterations were evident only with the highest A23187 concentration (10 microM). This study represents the initial comprehensive analysis of the effects of carboxylic ionophores on membrane electrical characteristics of an intact cell system and forms the basis for subsequent work using NG108-15 cells as a model system to evaluate potential therapeutic treatments against the carboxylic ionophores.

Topics: Calcimycin; Dose-Response Relationship, Drug; Glioma; Ionophores; Membrane Potentials; Monensin; Neuroblastoma; Neurons; Nigericin; Tumor Cells, Cultured

The effects of two different stressors, heat shock (HS; 44 degrees C, 20 min) and ethanol (1.2 M, 60 min), on ion content and membrane potential were investigated in C6 rat glioma cells. Both treatments were previously shown to induce the HS response [26]. Intracellular pH (pH(i)), sodium ion concentration ([NA+]i), potassium ion concentration ([K+]i) and membrane potential were determined by means of continuous 31P and 23Na nuclear magnetic resonance (NMR), continuous fluorescence spectroscopy and 86Rb uptake. Lactate extrusion was determined in addition with respect to pH(i) regulation. The aim of this study was a detailed picture of HS and ethanol-induced ion changes in a single cell type, because stress-induced changes in the intracellular ionic balance may be important factors for determining proliferation, stress response and apoptosis. HS lowered the pH(i) from 7.38 +/- 0.04 to about 7.05 +/- 0.04. [Na+]i decreased during HS to 50% of the control and recovered to normal level 95 min after HS treatment. During HS, [K+]i remained constant but increased after HS. The membrane potential hyperpolarized from -83 mV to -125 mV and returned to initial values during HS treatment. Lactate extrusion increased 3-fold after HS. Ethanol (1.2 M) lowered the pH(i) from pH 7.38 +/- 0.04 to pH 7.0 +/- 0.04, but in contrast to heat strongly increased [Na]i. It hyperpolarized the membrane potential from -83 to -125 mV. Ethanol also increased lactate extrusion similar to HS. Also in contrast to the effect of HS, the potassium concentration decreased during ethanol treatment. The Na(+)-H+ exchanger monensin was used to overcome the apparent inhibition of the cellular Na(+)-H+ exchanger by HS. At normal pH(e) (7.4) monensin increased [Na+]i and pH(i) considerably. A subsequent HS reduced [Na+]i only minimally. Acidification of the cells by low pH(e) (6.2) prior to HS did not abolish the HS-induced drop of pH(i), indicating that the Na(+)-H+ exchanger was also inhibited at low pH(i). At low pH(e), monensin transports H+ into the cell. A subsequent HS decreased pH(i) only little, showing the importance of inhibition of the Na(+)-H+ exchanger for the HS-induced pH(i) decrease. 100 microM amiloride reduced pH(i) and [Na+]i in a similar way as HS, but did not change pH(i) and [Na+]i much during a HS. These results indicate that some of the HS-induced ionic changes are mediated by inhibition of the Na(+)-H+ exchanger, activation of Na(+)-K(+)-ATPase and changes of membrane con

Topics: Animals; Biological Transport; Central Nervous System Depressants; Ethanol; Glioma; Hot Temperature; Hydrogen-Ion Concentration; Ionophores; Kinetics; Lactates; Magnetic Resonance Spectroscopy; Membrane Potentials; Monensin; Potassium; Protons; Rats; Sodium; Sodium-Hydrogen Exchangers; Sodium-Potassium-Exchanging ATPase; Spectrometry, Fluorescence; Tumor Cells, Cultured

The effect of intracellular pH (pHi) on heat shock protein (HSP) synthesis was investigated in C6 rat glioma cells. pHi changes were analysed by means of fluorescence spectroscopy in a perfused monitoring system allowing continuous measurements before, during and after treatments. HSP induction was determined by means of Western blots and autoradiographs. A 20 min heat shock (HS) of 44 degrees C decreased the pHi from 7.36 to 7.05 during exposure [17] and elicited the synthesis of heat shock proteins 2-8 h later. A pHi decrease, brought about by low extracellular pH (pHe) of 4.5 and 5.0 or 5.5, induced HSP synthesis after 1 h or 3 h, respectively. During these treatments, pHi decreased to values significantly lower than that caused by HS. Three h exposure to pHe 6.2, however, was not inductive. These results indicate that the heat shock-induced pHi decrease alone is not sufficient to stimulate HSP synthesis. In order to investigate the effect of alkaline pHi on the induction of HSP by heat, pHi was increased prior to HS treatments. Preincubation of cells at pHe ranging from 6.8 to 8.0 had little effect on pHi and on HSP synthesis. A shift of pHi to more alkaline values was achieved by adding the H+/Na+ exchanger monensin at alkaline pHe. Twenty microM monensin raised the pHi and inhibited the HSP induction depending on the pHe values: as pHe was increased from pH 7.2 to 8.0 HSP synthesis was increasingly inhibited. Monensin also diminished the HS-induced drop of pHi particularly at higher pHe. The result showed that neither a lower pHi nor a drop of pHi during HS is a necessary prerequisite for the induction, whereas alkalosis inhibits the synthesis of HSP.

Topics: Alkalosis; Animals; Autoradiography; Blotting, Western; Fluorescent Dyes; Glioma; Heat-Shock Proteins; Hydrogen-Ion Concentration; Monensin; Neutral Red; Rats; Tumor Cells, Cultured

1. Alzheimer's disease is characterized by the deposition in the brain of extracellular amyloid plaques and vascular deposits consisting mostly of amyloid beta-peptide (A beta). A beta, a polypeptide of 39-43 amino acids (M(r), approximately 4 kDa), is derived proteolytically from a family of proteins of 695-770 amino acids (M(r), approximately 110-140 kDa) called beta-amyloid precursor protein (beta APP). 2. beta APP, an integral membrane glycoprotein, is extensively posttranslationally modified within the endoplasmic reticulum (ER) and various Golgi compartments. beta APP is cleaved by proteases in either the trans-Golgi network or the post-Golgi apparatus and then secreted as a truncated soluble form into the conditioned media of cultured cells and cerebrospinal fluid samples from human subjects. beta APP can be processed either by an antiamyloidogenic secretory pathway or by an endosomal/lysosomal pathway. 3. I studied the effect of two ionophores on the processing of beta APP in cultured cells. Monensin and, in some cases, ammonium chloride increase the intracellular accumulation of beta APP in several cell lines and may alter its processing. Monensin, which had the most consistent effects, also inhibited secretion of beta APP in a differentiated (growth factor mediated) cell line. Nigericin, with greater K+ selectivity, was less able to alter the accumulation and possible processing of the protein. 4. These results suggest that the increase in the accumulation of intracellular beta APP observed after treating cells with ionophores has some specificity. The selective effect of these ionophores on the metabolism of beta APP may provide a model system to analyze the pathways for studying maturation, secretion, and degradation of beta APP.

Topics: Ammonium Chloride; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Cell Line; Endopeptidases; Endoplasmic Reticulum; Fibroblast Growth Factor 2; Glioma; Golgi Apparatus; HeLa Cells; Humans; Ionophores; Monensin; Neoplasm Proteins; Nerve Growth Factors; Neuroblastoma; Nigericin; PC12 Cells; Protein Processing, Post-Translational; Rats; Tumor Cells, Cultured

Cholera toxin (CT) consists of a pentameric B subunit which binds to ganglioside GM1 on the cell surface and an A subunit which activates adenylylcyclase. The latter process involves the reduction of A to the A1 peptide which ADP-ribosylates the stimulatory G protein, Gs of adenylylcyclase. There is a distinct lag phase between toxin binding and activation of adenylylcyclase. Little is known about the events during this lag including where A1 is generated and how it gains access to Gs on the cytoplasmic side of the plasma membrane. We explored the effects of several inhibitors of intracellular trafficking on the response of human SK-N-MC neurotumor and Caco-2 intestinal tumor cells to CT. Whereas chloroquine or monensin had little or no effect on CT stimulation of cyclic AMP accumulation, brefeldin A (BFA) totally inhibited the response to CT in a time- and dose-dependent and reversible manner. BFA was effective when added at the same time as CT and had an IC50 of 30 ng/ml. BFA did not alter cell surface GM1 as cells treated with BFA for 30 min bound as much 125I-CT as control cells. Furthermore, BFA inhibited CT stimulation of GM1-treated rat glioma C6 cells. BFA treatment did not affect beta-adrenergic agonist stimulation of cyclic AMP. In addition, adenylylcyclase was activated by A1 peptide and NAD+ to the same extent in membranes from control and BFA-treated cells, or when BFA was added directly to the assay. Whereas control cells generated small amounts of A1 from bound CT with time, no A1 was detected in BFA-treated cells. BFA treatment did not prevent the internalization of CT but did inhibit its degradation. BFA is known to disrupt the organization of the Golgi complex, resulting in inhibition of protein transport from the endoplasmic reticulum and redistribution of Golgi enzymes to the endoplasmic reticulum. BFA also prevents the formation of non-clathrin-coated vesicles from Golgi membranes and thus vesicular transport between Golgi cisternae. We confirmed that BFA caused the morphological disruption of the Golgi apparatus in Caco-2 cells. The data support a role for a functional Golgi apparatus with its associated vesicular routing in CT action.

Topics: Adenocarcinoma; Adenylyl Cyclases; Animals; Biological Transport; Brefeldin A; Cell Line; Chloroquine; Cholera Toxin; Colonic Neoplasms; Cyclic AMP; Cyclopentanes; Enzyme Activation; Glioma; Golgi Apparatus; Humans; Isoproterenol; Kinetics; Monensin; Mycotoxins; Rats; Time Factors; Tumor Cells, Cultured

Human alpha 2-macroglobulin (alpha 2M) is a high molecular weight plasma proteinase inhibitor exhibiting a broad specificity; in fact it is capable of binding endopeptidases from all known classes of proteases (Barret 1981). Two human glioma cell lines, namely an astrocytoma and a glioblastoma, were found to synthesize and secrete in the culture medium a protein which resembles the serum alpha 2M for immunological, biochemical and biological features. Using polyclonal antibodies to serum alpha 2M, an alpha 2M-like factor could be detected in the cytoplasm and in the culture medium of the tumor cells. Furthermore this factor accumulated in cytoplasmic granules if cells were incubated with monensin and its production was dramatically reduced following a treatment with cycloheximide. This protein behaved like the serum alpha 2M in immunoblotting analysis and exhibited the same antiproteolytic activity. Its role in human brain is unknown at present. Since interactions of proteinases and proteinase-inhibitors appear to influence the host-tumor immune response and to play a crucial role during the migration of metastasizing tumor cells, alpha 2M expression observed in these glioma cells could be involved in tumor cell proliferation and invasion.

Topics: alpha-Macroglobulins; Astrocytoma; Cell Line; Cycloheximide; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Glioma; Humans; Kinetics; Molecular Weight; Monensin; Protease Inhibitors; Time Factors

Treatment of rat brain synaptosomes with 10 microM monensin stimulated activity of the Na/K pump, which enhanced oxygen consumption and lactate production. Glycolytic flux was also increased independently of the pump activation by a fall in [H+]i. Under such conditions, glycolysis provided 26% of ATP for the ouabain-sensitive ATPase, a value substantially greater than the 4% obtained in veratridine-treated preparations (Erecińska and Dagani, 1990). In C6 glioma cells, a glia-derived line endowed with high rates of aerobic lactate synthesis, the cytosolic and mitochondrial ATP generation contributed 50% each for the support of the pump in the presence of 10 microM monensin. The fraction of energy utilized by the pump was greater in synaptosomes than in C6 cells. Enhancement of ion movements was accompanied by changes in the levels of high-energy phosphate compounds. Measurements with ion-sensitive microelectrodes in C6 cells and cultured neurons showed that monensin caused an increase in pHi by 0.4-0.5 unit and a parallel rise in [Na+]i. The increases in [Na+]i were about twofold in both types of cells, but the absolute values attained were much higher in neurons (40-50 mM) than in C6 cells (10-12 mM). Membrane potentials transiently declined by less than 10 mV and returned to their original values after 20 min of treatment. Rises in [Ca2+]i were small in neurons as well as in C6 cells. These changes could be explained by the known mechanism and/or consequences of monensin action. In contrast, in synaptosomes monensin caused an internal alkalinization of 0.1-0.15 pH unit, a large depolarization of the plasma membrane, and massive leakage of potassium into the external medium. The decrease in plasma membrane potential was accompanied by an increase in [Ca/+]i and release of the neurotransmitter amino acids GABA, aspartate, and glutamate. The depolarization and loss of K+ were unaffected by calcium withdrawal, replacement of chloride with gluconate, and addition of 1 mM 4-acetamido-4'-isothiocyanostilebene-2,2'-disulfonic acid (SITS), but was markedly attenuated by elimination of Na+. It is proposed that in synaptosomes monensin and/or the consequences of its action open a nonspecific cation channel that allows Na+ entry and K+ exit, with a consequent decrease in membrane potential.

Topics: Animals; Energy Metabolism; Glioma; Intracellular Membranes; Ions; Monensin; Neurons; Synaptosomes; Tumor Cells, Cultured

Tritium-labeled neoglycolipids consisting of the oligosaccharide of ganglioside GM1 attached to cholesterol (GM1OSNH-X-CHOL), phosphatidylethanolamine (GM1OS-PE) and stearylamine (GM1OSNHC18) were synthesized and their uptake and metabolism by GM1-deficient rat glioma C6 cells were determined. When the neoglycolipids were added to serum-free culture medium, all three were rapidly taken up by the cells and initially inserted into the plasma membrane based on their resistance to trypsin and their ability to bind cholera toxin. With time, the neoglycolipids underwent internalization as the ratio of cell-associated radioactivity to cell surface toxin binding increased; this process was slow for GM1OSNH-X-CHOL and GM1OS-PE and rapid for GM1OSNHC18. Analysis of lipids extracted from the cells indicated that the neoglycolipids also underwent metabolism to GD1aOS-based analogues. In addition, GM1OSNH-X-CHOL and GM1OSNHC18 were degraded to their GM2OS-based analogues, whereas GM2OS-PE was not detected. In contrast, large amounts of 3H were recovered in the medium from cells treated with GM1OS-PE and the label was associated with material that behaved neither as an oligosaccharide or a neoglycolipid. In the presence of monensin or chloroquine, metabolism of the three neoglycolipids was inhibited. Thus, GM1OS-based neoglycolipids were taken up by the cells, internalized and sorted both to the Golgi apparatus (sialylated to GD1aOS-based analogues) and to lysosomes (hydrolyzed to GM2OS-based analogues). The rate and extent of these processes, however, were strongly influenced by the nature of lipid moiety.

Topics: Amines; Animals; Carbohydrate Sequence; Chloroquine; Cholesterol; G(M1) Ganglioside; Glioma; Molecular Sequence Data; Monensin; Phosphatidylethanolamines; Rats; Tumor Cells, Cultured

The cytotoxic effects of an antihuman transferrin receptor monoclonal antibody-ricin A-chain conjugate (anti-TfR-A) immunotoxin on glioma cells were assessed in vitro. Five human glioma cell lines were studied; three were derived from surgical explants (MG-1, MG-2, MG-3) and two were well characterized established glioma cells (U-87 MG, U-373 MG). The C6 rat glioma line served as a nonhuman control. One of six lines (U-373) expressed glial fibrillary acidic protein, as assessed by immunohistochemistry. All five human lines expressed human transferrin receptor, as assessed by flow cytometry; no human transferrin receptor was demonstrable on rat C6 cells. Potent inhibition of protein synthesis was found after an 18-h incubation with anti-TfR-A. Fifty % inhibitory concentration (IC50) values for human glioma cells ranged from 1.9 X 10(-9) to 1.8 X 10(-8) M. In contrast, no significant inhibition of leucine incorporation was observed when anti-TfR-A was tested on rat cells (IC50 greater than 10(-7) M) or when a control immunotoxin directed against carcinoembryonic antigen was substituted for anti-TfR-A on human glioma cells (IC50 greater than 10(-7) M). Coincubation with the carboxylic ionophore monensin (10(-7) M) decreased the IC50 of anti-TfR-A against human glioma lines from 16- to 842-fold (range, 7.0 X 10(-12) to 1.5 X 10(-10) M). In contrast, an IC50 of greater than 10(-7) M was obtained when C6 cells were incubated with anti-TfR-A and monensin. Anti-TfR-A immunotoxins potentiated by monensin are extremely potent in vitro cytotoxins for human glioma cells.

Topics: Antibodies, Monoclonal; Drug Synergism; Glioma; Humans; Immunotoxins; Male; Middle Aged; Monensin; Receptors, Transferrin; Ricin; Tumor Cells, Cultured

Chemotaxis is an important step in monocyte recruitment in inflammation, wound healing, and tumor growth. We reported previously that monocyte chemotactic activity secreted by malignant cells and normal smooth muscle cells is associated with a protein or family of proteins that are related to the monocyte-specific smooth muscle cell-derived chemotactic factor (SMC-CF) (Graves, D. T., Jiang, Y. L., Williamson, M. J., and Valente, A. J. (1989) Science 245, 1490-1493). Similar monocyte chemotactic proteins (MCP-1) produced by U-105MG human glioma cells have also been identified (Yoshimura, T., Robinson, E. A., Tanaka, S., Appella, E., Kuratsu, J., and Leonard, E. J. (1989) J. Exp. Med. 169, 1449-1459). We now report that the MCP-1 gene is expressed in MG-63 human osteosarcoma and vascular smooth muscle cells and that SMC-CF antiserum specifically immunoprecipitates proteins synthesized by U-105MG glioma cells. Experiments were undertaken to elucidate the processing pathway of MCP-1/SMC-CF-like proteins in each of these cell types. These experiments demonstrate that larger MCP-1/SMC-CF-like proteins are derived from a Mr = 9000 precursor. Post-translational modification involves the addition of O-linked carbohydrates and sialic acid residues. Differences in carbohydrate processing account for the heterogeneity in MCP-1/SMC-CF-like proteins produced by different cell types. Secretion of these proteins occurs rapidly following processing events in the endoplasmic reticulum-Golgi compartment.

Topics: Blotting, Northern; Chemokine CCL2; Chemotactic Factors; Glioma; Glycosylation; Humans; Molecular Weight; Monensin; Muscle, Smooth, Vascular; Osteosarcoma; Precipitin Tests; Protein Processing, Post-Translational; RNA, Messenger; Tumor Cells, Cultured; Tunicamycin

The clonal analysis in the ultrastructure of tumor-lymphocyte interaction was carried out in order to investigate the precise mechanism responsible for CTL-mediated cytolysis of tumor cells. A glioma-derived cell line (GI-1) and autologous tumor-specific cytotoxic T lymphocyte (CTL) clones were established. The CTL lines were composed of the morphologically homogeneous lymphocytes with intracytoplasmic electron-dense secretory granules. After the stimulation by GI-1, the size of the CTLs increased, and the intracytoplasmic organellas were developed. It was noted that the intracytoplasmic secretory granules markedly increased in number and size, and many of them exhibited an "immature" appearance. On the other hand, the tumor cells underwent a progressive degeneration. In contrast, the stimulation by other antigens caused only small morphological changes in the CTLs. It is suggested, therefore, that the secretory function of tumor-specific CTLs is activated by the stimulation of the specific antigen, and that soluble factors in the secretory granules in the CTLs may be closely associated with the mechanism of target cell lysis.

Topics: Cell Communication; Clone Cells; Cytoplasmic Granules; Cytotoxicity, Immunologic; Glioma; Humans; Microscopy, Electron; Monensin; Phenotype; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Cathepsin D was assessed in C6 glioma cells grown in medium with an intermediate- or low-percent composition of serum. The amount, form, and subcellular location of cathepsin D differed after treatment with cyanate or monensin in cells grown in a low-serum, growth-factor-supplemented medium. Immunoblotting showed that cathepsin D in the lysosomal fraction of the C6 cell line had a molecular weight (Mr) of 42 kD, whereas that in the microsomal fraction had Mr's of 42, 47, and 78 kD. After treatment for 1 to 16 hr with 4 mmol/L cyanate and subcellular fractionation, the molecular weight of lysosomal cathepsin D was the same in treated and untreated cells, but more enzyme was found in lysosomes of treated cells at 8 and 16 hr. In the microsomal fraction, the amounts of both the 42 and 47 kD forms were increased after 1 to 16 hr of treatment. When exposed to 20 mmol/L cyanate, C6 cells remained viable, but compared with untreated cells, they showed 25% less lysosomal cathepsin D, with increased amounts found in the microsomal fraction. The 78 kD protein detected by immunoblotting was present in both the lysosomal and microsomal fractions but was predominant in the latter. The apparent molecular weight of this protein was the same after cyanate but differed with monensin, where Mr's of 39, 42, and 73 kD were found. Monensin-treated cells had less lysosomal cathepsin D and relatively more microsomal enzyme. The differing molecular weights of cathepsin D from cyanate- and monensin-treated cells suggest that their inhibitions occur at different processing loci in distal elements of the Golgi stacks. The differences in the pI of cathepsin D and the number of its forms from cyanate- and monensin-treated cells are also consistent with interference in the late stages of glycoprotein maturation. In this paper we show that the amount, molecular form, and consequent intracellular location of cathepsin D in cells of the C6 line can be affected by agents that selectively disrupt stages in Golgi-related protein modification and transport.

Topics: Animals; Cathepsin D; Cell Line; Cyanates; Fluorescent Antibody Technique; Glioma; Lysosomes; Microsomes; Mitochondria; Molecular Weight; Monensin; Protein Processing, Post-Translational

We have investigated the phenotype of seven human glioma cell lines established in vitro from primary tumour explants. Indirect immunofluorescence and flow cytofluorimetry revealed a heterogeneous distribution of surface GE 2 and CG 12 Tumour Associated Antigens (TAA). In one group of cell lines TAA were detected both at the cell surface and in the cytosol, whereas in a second group of glioma cell lines TAA were found only in the cytosol. We have also investigated the sensitivity of glioma-derived cell lines to antibody-toxin and ligand-toxin conjugates (Immunotoxins). Monoclonal antibodies anti GE 2 antigen linked to ricin toxin A subunit (RTA) showed poor cytotoxicity, which increased about 50 fold when the whole toxin was linked to anti GE 2 monoclonals. Treatment with human recombinant interferon gamma (IFN-gamma) greatly augmented the percentage of HLA-DR+ cells and the amount of HLA-DR antigens per cell. IFN-gamma treatment resulted in a net increase of sensitivity to anti HLA-DR Immunotoxins (IT). Human diferric transferrin linked to RTA exhibited a potent cytotoxic effect against human glioma-derived cells when used in the presence of the lysosomotropic carboxylic ionophore monensin.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Drug Synergism; Glioma; Humans; Immunotoxins; Monensin; Protein Biosynthesis; Proteins; Ricin; Transferrin

The effects of varying the sodium concentration (at constant ionic strength) on opioid binding at mu- and delta-opioid receptors in 7315c and NG108-15 cells has been examined. The binding of [3H]etorphine to mu-receptors on 7315c cells was increased by replacing the sodium in the incubation medium with potassium or N-methyl-D-glucamine. This effect was shown to be attributable to an increase in affinity, with no change in the maximum number of binding sites, both in cell membrane suspensions and in intact 7315c cells. Replacement of sodium with potassium or N-methyl-D-glucamine in NG108-15 membrane or intact cell suspensions also resulted in an increase in [3H]etorphine binding, but in these cells the effect was associated with an increase in the number of binding sites measurable under these experimental conditions. The effects of sodium on opioid inhibition of adenylate cyclase in membrane preparations from 7315c and NG108-15 cells also differed. Sodium reduced apparent agonist affinity in 7315c membranes. In NG108-15 cell membranes, sodium was essential for the demonstration of opioid inhibition of cyclase activity. Increasing the sodium concentration above 0.5 mM resulted in an increase in the fraction of total enzyme activity inhibited by opioid, but the opioid IC50 did not change. In the companion paper, it is shown that the effects of sodium removal on mu- and delta-receptor binding in guinea pig brain neural membranes were similar to those observed in the cell preparations. An increase in intracellular sodium concentration without change in extracellular concentration was effected by incubation of 7315c and NG108-15 cells with the sodium-selective ionophore, monensin. When sodium was present in the extracellular medium, monensin reduced [3H]etorphine binding by 50% or more, both at mu-receptors in 7315c cells and at delta-receptors in NG108-15 cells. In the absence of sodium, however, monensin treatment produced only a small inhibition of binding. These results suggest that sodium acts at an intracellular site to regulate opioid agonist binding at both mu- and delta-receptors, but that the mode of regulation is not identical at each site. Since a reduction in intracellular sodium concentration by removal of extracellular sodium increases agonist binding, and an increase in intracellular sodium following monensin treatment reduces agonist binding, it is probable that the intracellular sodium concentration is a critical regulator of opioid agonist b

Topics: Adenylyl Cyclase Inhibitors; Animals; Cell Line; Cell Membrane; Cyclazocine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylketocyclazocine; Etorphine; Glioma; Guinea Pigs; Monensin; Naloxone; Neuroblastoma; Pituitary Neoplasms; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Sodium

We have found that several types of cultured mammalian cells, including both normal and transformed human, rat, and mouse cell lines, have an active transport system for a diverse group of structurally related compounds possessing an amine group and various types of aromatic ring structures. Ligands such as the beta-adrenergic antagonists (-)-[3H] dihydroalprenolol (DHA), (-)-[3H]propranolol, and (-)-[125I] iodocyanopindolol, and the tricyclic antidepressant [3H]imipramine, which are used to assess cell surface receptors for catecholamines and serotonin, appear to be actively transported into cells rather than simply bound to cell surface sites or accumulated by passive diffusion. DHA transport was competed by many structurally related amines including imipramine and certain alpha-and beta-adrenergic ligands, but not by catecholamines or serotonin. Ligand transport in HeLa cells was saturable at micromolar levels, selective, nonstereospecific, temperature- and pH-dependent, and sensitive to the ionophore monensin and the amine transport inhibitor reserpine, thus indicating dependence on a carrier system driven by a transmembrane proton gradient. In C6 glioma cells, amine transport was clearly distinguishable from beta-adrenergic receptor binding which could be measured with the recently developed hydrophilic beta-blocker (+/-)-[3H] 4-(3-tertiarybutylamino-2-hydroxy-propoxy)-benzimidazole-2-on hydrochloride (CGP-12177); binding of this ligand met rigorous pharmacological criteria, was not influenced by monensin or reserpine, and, therefore, did not appear to be transported. Membrane vesicles from HeLa and C6 cells transported DHA but not CGP-12177 via a MgATP-dependent mechanism which was inhibited by N,N'-dicyclohexylcarbodiimide, monensin, and reserpine, indicating a carrier system driven by a proton gradient maintained by a proton-pumping ATPase.

Topics: Adenosine Triphosphate; Amines; Binding, Competitive; Biological Transport, Active; Carrier Proteins; Cell Line; Diffusion; Dihydroalprenolol; Glioma; HeLa Cells; Humans; Hydrogen-Ion Concentration; Ligands; Magnesium; Membrane Potentials; Monensin; Receptors, Adrenergic, beta; Reserpine; Solubility; Temperature

Neuroblastoma and glioma cells were grown in the presence of [3H]galactose, and the incorporation of 3H into gangliosides and the transport of newly synthesized gangliosides to the cell surface were examined under different experimental conditions. A variety of drugs, including inhibitors of protein synthesis and energy metabolism, modulators of the cytoskeleton and the ionophore monensin, had no effect on the transport of newly synthesized GD1a in neuroblastoma cells. Only low temperature effectively blocked translocation to the plasma membrane. Monensin, however, had marked effects on the biosynthesis of gangliosides and neutral glycosphingolipids. Whereas incorporation of 3H into complex glycosphingolipids was reduced, labeling of glucosylceramide was increased in cells exposed to monensin. In addition, biosynthesis of the latter glycolipid was less susceptible to low temperatures than that of more complex ones. Previous studies have implicated the Golgi apparatus as the predominant site of glycosylation of gangliosides. As monensin has been reported to interfere with the Golgi apparatus, our results indicate that glucosylceramide may be synthesized at a site that is separate from the site where further glycosylation occurs. Once synthesis of a ganglioside is completed, transport of the molecule to the cell surface proceeds under conditions of cytoskeletal disruption, energy depletion and ionic inbalance , but not low temperature.

Topics: Animals; Biological Transport; Cells, Cultured; Colchicine; Cycloheximide; Cytochalasin B; Glioma; Glycosphingolipids; Golgi Apparatus; Ionophores; Mice; Monensin; Neuroblastoma; Puromycin; Rats; Temperature; Vinblastine

Addition of the ionophore monensin to mouse neuroblastoma-rat glioma hybrid NG108-15 cells leads to a 20 to 30-mV increase in the electrical potential across the plasma membrane as shown by direct intracellular recording techniques and by distribution studies with the lipophilic cation [3H]-tetraphenylphosphonium+ (TPP+) [Lichtshtein, D., Kaback, H.R. & Blume, A.J. (1979) Proc. Natl. Acad. Sci. USA 76, 650-654]. The effect is not observed with cells suspended in high K+ medium, is dependent upon the presence of Na+ externally, and the concentration of monensin that induces half-maximal stimulation of TPP+ accumulation is approximately 1 microM. The ionophore also causes rapid influx of Na+, a transient increase in intracellular pH, and a decrease in extracellular pH, all of which are consistent with the known ability of monensin to catalyze the transmembrane exchange of H+ for Na+. Although ouabain has no immediate effect on the membrane potential, the cardiac glycoside completely blocks the increase in TPP+ accumulation observed in the presence of monensin. Thus, the hyperpolarizing effect of monensin is mediated apparently by an increase in intracellular Na+ that acts to stimulate the electrogenic activity of the Na+,K+-ATPase. Because monensin stimulates TPP+ accumulation in a number of other cultured cell lines in addition to NG108-15, the techniques described may be of general use for studying the Na+,K+ pump and its regulation in situ.

Topics: Animals; Biological Transport; Cell Line; Furans; Glioma; Hybrid Cells; Kinetics; Membrane Potentials; Mice; Monensin; Neuroblastoma; Onium Compounds; Organophosphorus Compounds; Ouabain; Potassium; Rats; Sodium