guanosine-triphosphate and Astrocytoma

Focal adhesion kinase (FAK) signaling may be mediated through the modulation of Ras activity. We have shown previously that grade III malignant astrocytoma biopsy samples exhibit elevated levels of FAK, and that overexpression of FAK in U-251MG malignant astrocytoma cells promotes the phosphorylation of Shc, a potential upstream mediator of Ras activity. Here, we report that overexpression of FAK promotes Ras activity in U-251MG malignant astrocytoma cells cultured in aggregate suspension or as monolayers adherent to vitronectin. The overexpression of FAK also promoted the association of FAK with p120RasGAP, which is a negative regulator of Ras activity, in the U-251MG cells cultured in aggregate suspension, with this association being abrogated upon plating of the cells onto vitronectin. An association of FAK with p120RasGAP also was observed in malignant astrocytoma biopsy samples, but not in normal brain samples. As overexpression of FAK in U-251MG cells in aggregate suspension culture reduced the amount of p120RasGAP complexed with active Ras, we hypothesize that the association of FAK with p120 RasGAP may facilitate Ras activity. The overexpression of a mutated FAK in which the Y397 had been mutated to F did not result in the formation of the FAK/p120RasGAP complex and did not promote Ras activity, indicating that the Y397 residue of FAK plays a role in the formation of this complex and in the activation of Ras. Moreover, the overexpression of mutated FAK (397F) was found to inhibit anchorage-independent growth. These data provide the basis for a previously undescribed mechanism in which the elevated expression of FAK can promote Ras activity through its competitive recruitment of p120RasGAP, thereby diminishing the association of p120RasGAP with active Ras.

Topics: Animals; Astrocytoma; Cell Division; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Guanosine Triphosphate; Humans; p120 GTPase Activating Protein; Protein-Tyrosine Kinases; ras Proteins; Rats

Increased small GTPase protein mitogenic signaling is common in tumors. We have previously demonstrated that sporadic astrocytomas exhibit high levels of activated Ras important for tumor growth. Individuals with tuberous sclerosis complex (TSC) develop astrocytoma-like tumors resulting from mutations in the TSC2 protein, tuberin, which is hypothesized to function as a Rap1 GTPase activating protein (GAP). Since we have previously reported that high-grade astrocytomas frequently exhibit loss of tuberin expression or increased Rap1 levels, we sought to determine whether there is a correlation between decreased tuberin Rap1-GAP function or Rap1 overexpression and tumor Rap1 activity. In this study, we compared levels of Rap1-GTP, Rap1 and tuberin levels in normal brain tissue and 24 grade II-IV astrocytoma specimens. Whereas Rap1 overexpression was observed in astrocytomas of all malignancy grades, tuberin loss was seen most frequently in the higher-grade astrocytomas. In the grade IV glioblastoma multiforme tumors, Rap1 activity was 2-3-fold higher than in lower grade or non-neoplastic brain. However, there was no correlation between tuberin expression or Rap1 overexpression and the levels of Rap1 activity in the tumors studied, suggesting that the increased Rap1 activation is not the direct result of reduced tuberin Rap1-GAP function or elevated Rap1 protein expression.

Topics: Astrocytoma; Gene Expression; Guanosine Triphosphate; Humans; rap1 GTP-Binding Proteins; Repressor Proteins; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Previous studies have demonstrated that astrocytomas express elevated levels of activated Ras.GTP despite the absence of activating Ras mutations. Farnesyl transferase inhibitors (FTIs) exert their antitumor effect in part through inhibition of Ras-mediated signaling. SCH66336 is a potent FTI presently undergoing clinical trials in patients with solid tumors. We evaluated the efficacy of SCH66336 against a panel of eight human astrocytoma cell lines and three human astrocytoma explant xenograft models in NOD-SCID mice. SCH66336 demonstrated variable antiproliferative effects against the cell lines, with IC(50) ranging from 0.6 microM to 32.3 microM. Two of the three human glioblastoma multiforme (GBM) xenografts demonstrated substantial growth inhibition in response to SCH66336, with up to 69% growth inhibition after 21 days of treatment. Drug efficacy could be accurately predicted using a combination of the H-, K-, and N-isotype-specific Ras.GTP levels. These data indicate that the absence of Ras mutations does not preclude chemotherapeutic efficacy by FTIs, that Ras is likely a major target of FTIs regardless of Ras mutational status, and that isotype-specific Ras.GTP levels are a promising marker of drug efficacy.

Topics: Alkyl and Aryl Transferases; Animals; Astrocytoma; Brain Neoplasms; Cell Division; Enzyme Inhibitors; Farnesyltranstransferase; Genes, ras; Glioblastoma; Guanosine Triphosphate; Humans; Immunohistochemistry; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Monomeric GTP-Binding Proteins; Mutation; Piperidines; Pyridines; ras Proteins; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Overexpression and activation of receptor tyrosine kinases, such as platelet derived growth factor receptors (PDGFRs) and epidermal growth factor receptor (EGFR), leads to proliferation of human malignant astrocytoma cells. Although oncogenic mutations affecting Ras are not prevalent in human malignant astrocytomas, we have investigated whether levels of activated Ras.GTP might be elevated in these tumors secondary to the mitogenic signals originating from activated receptor tyrosine kinases. In support of this hypothesis high levels of Ras.GTP, similar to those found in oncogenic Ras transformed fibroblasts, were present in four established human malignant astrocytoma cell lines which express PDGFRs and EGFR, and 20 operative malignant astrocytoma specimens. Stimulation of PDGFR's and EGFR's induced tyrosine phosphorylation of the Shc adaptor protein and its association with Grb2, suggesting a mechanism by which Ras may be activated in human malignant astrocytoma cells. Furthermore, blocking Ras activation by expression of the Ha-Ras-Asn17 dominant-negative mutant, or by farnesyl transferase inhibitors, decreased in vitro proliferation of the human astrocytoma cell lines. These results support the hypothesis that proliferative signals from receptor tyrosine kinases expressed by human malignant astrocytoma cells utilize the Ras mitogenic pathway. Pharmacological inhibitors of the Ras pathway may therefore be of therapeutic value in these presently terminal tumors.

Topics: Alkyl and Aryl Transferases; Astrocytoma; Brain; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; ErbB Receptors; Farnesyltranstransferase; Genes, ras; Guanosine Triphosphate; Humans; Mutation; ras Proteins; Receptors, Platelet-Derived Growth Factor; Signal Transduction; Tumor Cells, Cultured; Up-Regulation

To investigate substance P (SP) receptors on an established human astrocytoma cell line (U-87 MG), [3H][Sar9,Met(O2)11]-SP, a selective SP receptor agonist, was used to identify and characterize the cell membrane binding sites for SP. SP receptor mRNA was examined by solution hybridization analysis, and the existence of SP binding protein on the surface of membranes was evaluated by flow cytometry using an anti-SP binding protein antibody. In U-87 MG and U-373 MG RNA preparations, transcripts were identified that corresponded to both mature and partially spliced receptor forms. In U-87 MG cell membrane-enriched preparations, the binding of [3H][Sar9,Met(O2)11]-SP was found to be time and cell number dependent, specific, saturable, and of high affinity. Equilibrium binding analysis revealed a single class of binding sites with an apparent KD of 1.15 +/- 0.15 nM and a Bmax of 108 +/- 9.8 fmol/mg of protein. [3H][Sar9, Met(O2)11]-SP binding was basically not influenced by addition of mono (Na+, Li+) or divalent (Mg2+, Mn2+, Ca2+) cations; only high doses of divalent cations decreased the binding. GTP and guanylyl-5'-imidodiphosphate, but not GDP and GMP, reduced the Bmax without changing the affinity of [3H][Sar9,Met(O2)11]-SP. We also examined the effects of pretreatment with three lectins [concanavalin A (con A), wheat germ agglutinin (WGA), and Lens culinaris agglutinin (LCA)] to determine the nature of carbohydrate chains on the U-87 MG cell. Of three lectins analyzed for effects on agonist binding, WGA and LCA had an inhibitory effect, whereas con A was ineffective. These results suggest that SP receptors on the human astrocytoma cell line U-87 MG have either a biantennary complex-type or a high mannose-type of carbohydrate chain and may be regulated by GTP-binding protein(s).

Topics: Astrocytoma; Binding Sites; Binding, Competitive; Cations, Monovalent; Cell Line; Exons; Glioblastoma; Guanine Nucleotides; Guanosine Triphosphate; Humans; Kinetics; Lectins; Receptors, Neurokinin-1; RNA, Antisense; RNA, Complementary; RNA, Messenger; Substance P; Tritium; Tumor Cells, Cultured

In 1321N1 astrocytoma cells, thrombin, but not carbachol, induces AP-1-mediated gene expression and DNA synthesis. To understand the divergent effects of these G protein-coupled receptor agonists on cellular responses, we examined Gq-dependent signaling events induced by thrombin receptor and muscarinic acetylcholine receptor stimulation. Thrombin and carbachol induce comparable changes in phosphoinositide and phosphatidylcholine hydrolysis, mobilization of intracellular Ca2+, diglyceride generation, and redistribution of protein kinase C; thus, activation of these Gq-signaling pathways appears to be insufficient for gene expression and mitogenesis. Thrombin increases Ras and mitogen-activated protein kinase activation to a greater extent than carbachol in 1321N1 cells. The effects of thrombin are not mediated through Gi, since ribosylation of Gi/Go proteins by pertussis toxin does not prevent thrombin-induced gene expression or thrombin-stimulated DNA synthesis. We recently reported that the pertussis toxin-insensitive G12 protein is required for thrombin-induced DNA synthesis. We demonstrate here, using transfection of receptors and G proteins in COS-7 cells, that G alpha 12 selectively couples the thrombin receptor to AP-1-mediated gene expression. This does not appear to result from increased mitogen-activated protein kinase activity but may reflect activation of a tyrosine kinase pathway. We suggest that preferential coupling of the thrombin receptor to G12 accounts for the selective ability of thrombin to stimulate Ras, mitogen-activated protein kinase, gene expression, and mitogenesis in 1321N1 cells.

Topics: Animals; Astrocytes; Astrocytoma; Calcium-Calmodulin-Dependent Protein Kinases; Carbachol; COS Cells; DNA; Gene Expression Regulation; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Muscarinic Agonists; Pertussis Toxin; Protein Kinase C; ras Proteins; Receptors, Muscarinic; Receptors, Thrombin; Signal Transduction; Thrombin; Transcription Factor AP-1; Transfection; Tumor Cells, Cultured; Virulence Factors, Bordetella

Thrombin stimulation of 1321N1 astrocytoma cells results in polyphosphoinositide hydrolysis, Ca2+ mobilization, AP-1-mediated transcriptional activation, and DNA replication. Thrombin stimulation also activates Ras as assessed by an increase in the proportion of Ras in a GTP bound state. We examined the functional requirement for endogenous Ras protein in mediating thrombin-induced responses. Microinjection of a dominant interfering mutant of H-Ras into 1321N1 cells inhibited DNA synthesis in response to thrombin as did microinjection of an inhibitory antibody to Ras. Stimulation of AP-1-mediated transcriptional activity was also reduced by the expression of interfering Ras mutants. However, neither the stimulation of polyphosphoinositide hydrolysis nor the mobilization of intracellular Ca2+ was dependent on endogenous Ras function. These observations indicate that thrombin stimulation of mitogenesis requires Ras protein function. Our data suggest that the G-protein-coupled thrombin receptor stimulates pathways, which in part are convergent with those stimulated by tyrosine kinase growth factor receptors.

Topics: Astrocytoma; Cell Division; DNA Replication; Genes, ras; Growth Substances; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Humans; Kinetics; Mutagenesis, Site-Directed; Phosphatidylinositols; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins p21(ras); Thrombin; Transfection; Tumor Cells, Cultured

Mastoparan inhibited [3H]inositol phosphate accumulation induced by carbachol as well as cyclic AMP accumulation induced by isoproterenol in 1321N1 human astrocytoma cells. Mastoparan inhibited GTP gamma S-induced, but not Ca2(+)-induced, [3H]inositol phosphate accumulation in membrane preparations with an IC50 of approximately 10 microM. The inhibitory effect of mastoparan on carbachol-induced [3H]inositol phosphate accumulation was resistant to pertussis toxin (IAP) treatment in intact cells. These results suggest that mastoparan inhibits phospholipase C in human astrocytoma cells via a GTP binding protein, which is not a substrate for IAP.

Topics: Astrocytoma; Bee Venoms; Calcium; Cyclic AMP; Dose-Response Relationship, Drug; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Hydrolysis; Inositol Phosphates; Intercellular Signaling Peptides and Proteins; Peptides; Pertussis Toxin; Phosphatidylinositols; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella; Wasp Venoms

Carbachol, histamine and bradykinin activate phospholipase C in pertussis toxin-insensitive manner in human astrocytoma cells. Pretreatments of the cells with these agonists resulted in the reduction of GTP gamma S-induced accumulation of inositol phosphates in membrane preparations. Treatment of cells with carbachol mobilized GTP gamma S binding activities as well as muscarinic receptors from heavy membrane fraction to light fraction, reflecting from an agonist-induced desensitization. The treatment of the cells with agonists reduced a 32 kDa GTP binding protein in heavy membrane fraction, determined by a photoaffinity labeling with [35S]GTP gamma S. The data suggest that the 32 kDa GTP binding protein is involved in desensitization by agonists which activate phospholipase C in human astrocytoma cells.

Topics: Affinity Labels; Astrocytoma; Calcium Channel Agonists; Cells, Cultured; Centrifugation, Density Gradient; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Membranes; Neoplasms, Experimental; Protein Binding; Receptors, Muscarinic; Thionucleotides

Digitonin-permeabilized monolayers of C6-2B rat astrocytoma cells exhibit adenylate cyclase activity in the presence of exogenously added ATP. The adenylate cyclase retains the qualitative and quantitative characteristics of hormone stimulated cyclic AMP accumulation in whole cells including GTP dependency and 100 fold stimulation by isoproterenol. Forskolin increased enzymatic activity in the absence of added GTP, however forskolin efficacy and potency was enhanced by GTP. Low non-efficacious concentrations of forskolin, without added GTP, supported isoproterenol-stimulated cyclase activity. The GTP-stimulated isoproterenol response was potentiated by forskolin. Forskolin support of isoproterenol stimulated cyclase in the absence of GTP raises the possibility that forskolin can act independently of GTP in coupling receptors to cyclase catalytic units and/or that forskolin could increase the efficacy and potency of GTP in the coupling reaction. Permeabilization of C6-2B and other cultured cells yields a preparation of adenylate cyclase which retains the enzyme in a state which closely approximates its activity in the native membrane--a system which could prove useful in studies of the regulation of adenylate cyclase in vivo.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Animals; Astrocytoma; Cell Line; Cell Membrane Permeability; Colforsin; Digitonin; Guanosine Triphosphate; Isoproterenol; Kinetics; Rats

The efficacies of a series of six muscarinic cholinergic receptor agonists for stimulation of phosphoinositide breakdown and unidirectional efflux of 45Ca2+ in 1321N1 human astrocytoma cells were compared with the relative capacity of these agonists for formation of a GTP-sensitive high-affinity binding state in washed membranes. Carbachol and methacholine were 'full' agonists as regards phosphoinositide breakdown and Ca2+ mobilization, whereas bethanechol, arecoline and oxotremorine were 'partial' agonists for these two responses. Pilocarpine was the least efficacious of the six drugs tested. Except for pilocarpine, competition curves generated with the agonists and [3H]quinuclidinyl benzilate did not follow the Law of Mass Action for ligand interaction at a single site. Non-linear regression analyses of these data indicated that the data significantly better fit a two-, rather than a single-, site model with a high- and a low-affinity binding component. Competition curves generated in the presence of GTP were shifted to the right, and the extent of receptors in the high-affinity agonist-binding state was decreased. The relative efficacies of the six agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization were significantly correlated with the difference in affinities (KL/KH) between the two affinity states for each agonist. The relative efficacy of the agonists for stimulation of Ca2+ mobilization also was significantly correlated with the extent of receptors in the high-affinity state (%H) for each agonist. The results suggest that interaction with an as-yet unidentified guanine nucleotide regulatory protein is important in the mechanism whereby muscarinic receptors stimulate phosphoinositide breakdown in 1321N1 astrocytoma cells.

Topics: Astrocytoma; Binding Sites; Calcium; Cell Line; Guanosine Triphosphate; Humans; Parasympathomimetics; Phosphatidylinositols; Receptors, Muscarinic

Incubation of 1321N1 human astrocytoma cells with carbachol resulted in a rapid loss of binding of [3H]N-methylscopolamine ([3H]NMS) to muscarinic cholinergic receptors measured at 4 degrees C on intact cells; loss of muscarinic receptors in lysates from the same cells measured with [3H]quinuclidinyl benzilate [( 3H]QNB) at 37 degrees C occurred at a slower rate. Upon removal of agonist from the medium, the lost [3H]NMS binding sites measured on intact cells recovered with a t1/2 of approximately 20 min, but only to the level to which [3H]QNB binding sites had been lost; no recovery of "lost" [3H]QNB binding sites occurred over the same period. Based on these data and the arguments of Galper et al. (Galper, J. B., Dziekan, L. C., O'Hara, D. S., and Smith, T. W. (1982) J. Biol. Chem. 257, 10344-10356) regarding the relative hydrophilicity of [3H]NMS versus [3H]QNB, it is proposed that carbachol induces a rapid sequestration of muscarinic receptors that is followed by a loss of these receptors from the cell. These carbachol-induced changes are accompanied by a change in the membrane form of the muscarinic receptor. Although essentially all of the muscarinic receptors from control cells co-purified with the plasma membrane fraction on sucrose density gradients, 20-35% of the muscarinic receptors from cells treated for 30 min with 100 microM carbachol migrated to a much lower sucrose density. This conversion of muscarinic receptors to a "light vesicle" form occurred with a t1/2 approximately 10 min, and reversed with a t1/2 approximately 20 min. In contrast to previous results in this cell line regarding beta-adrenergic receptors (Harden, T. K., Cotton, C. U., Waldo, G. L., Lutton, J. K., and Perkins, J. P. (1980) Science 210, 441-443), agonist binding to muscarinic receptors in the light vesicle fraction obtained from carbachol-treated cells was still regulated by GTP. One interpretation of these data is that agonists induce an internalization of muscarinic receptors with the retention of their functional interaction with a guanine nucleotide regulatory protein.

Topics: Astrocytoma; Atropine; Carbachol; Cell Fractionation; Cell Line; Cell Membrane; Centrifugation, Density Gradient; Guanosine Triphosphate; Humans; Kinetics; N-Methylscopolamine; Quinuclidinyl Benzilate; Receptors, Muscarinic; Scopolamine Derivatives

Incubation of 1321N1 astrocytoma cells for 15 min with 1 microM (-)-isoproterenol resulted in a 50-65% loss of isoproterenol-stimulated adenylate cyclase activity. No decrease occurred in basal adenylate cyclase activity or in the density of beta-adrenergic receptors as assessed by (125I)-hydroxybenzylpindolol binding. Concentration-effect studies indicated that the apparent affinity of isoproterenol for inhibition of (125I)-hydroxbenzylpindolol binding was decreased by approximately 10-fold in membranes prepared from cells that had been exposed to 1 umM isoproterenol for 15 min. In the presence of GTP there was a shift to the right of the concentration-effect curve for isoproterenol in control membranes. GTP had little effect on the apparent affinity of isoproterenol in desensitized membranes. In desensitized cells that were subsequently washed free of catecholamine, the decrement in isoproterenol-stimulated adenylate cyclase activity and the decrease in the capacity of isoproterenol to inhibit 125IHYP binding returned to control levels within 15 min. These data are consistent with the hypothesis that an early event in the process of desensitization in 1321N1 cells involves a reversible uncoupling of beta-adrenergic receptors and adenylate cyclase.

Topics: Adenylyl Cyclases; Astrocytoma; Benzyl Alcohols; Binding Sites; Cell Line; Dose-Response Relationship, Drug; Guanosine Triphosphate; Isoproterenol; Membranes; Pindolol; Protein Binding; Receptors, Adrenergic; Receptors, Adrenergic, beta

Epinephrine increased adenylate cyclase activity 10 to 15 fold in lysates of the cultured human astrocytoma cell line 132-1N1. GTP had little effect on adenylate cyclase activity of lysed cell preparations either with or without added epinephrine. However, the epinephrine stimulation of adenylate cyclase was essentially lost (less than 90%) when a washed nuclei-free membrane preparation of the cyclase was assayed. A 10 to 15 fold epinephrine stimulation of the membrane adenylate cyclase could be demonstrated if cytosol of GTP were added to the assay with the hormone. The criteria of anion exchange, cation exchange, gel exclusion and paper chromatography indicated that the cytosolic agents which acted synergistically with hormones were GTP and GDP. The apparent Kact's for the synergistic action of GDP and GTP were essentially identical (1.0 muM) and of all the other nucleotides examined only GDP had a potency similar to GTP. However, the effect of GDP was apparently due to its rapid conversion to GTP even in the absence of a regenerating system. With epinephrine pretreatment of the intact 132-1N1 cells there was a specific loss of epinephrine stimulation of adenylate cyclase activity. The hormone pretreatment did not alter the capacity of the cytosol from these desensitized cells to potentiate epinephrine stimulation of the cyclase. Rather, the alteration was in the particulate fraction of the lysate. The desensitization of the membranous cyclase was stable and not reversed by GTP.

Topics: Adenylyl Cyclases; Astrocytoma; Cell Membrane; Cells, Cultured; Cytosol; Epinephrine; Guanosine Diphosphate; Guanosine Monophosphate; Guanosine Triphosphate; Humans; Prostaglandins E