guanosine-triphosphate and Brain-Neoplasms

PIKE (PI 3-Kinase Enhancer) is a recently identified brain specific nuclear GTPase, which binds PI 3-kinase and stimulates its lipid kinase activity. Nerve growth factor treatment leads to PIKE activation by triggering the nuclear translocation of phospholipase C-gamma1 (PLC-gamma1), which acts as a physiologic guanine nucleotide exchange factor (GEF) for PIKE through its SH3 domain. To date, three forms of PIKE have been characterized: PIKE-S, PIKE-L and PIKE-A. PIKE-S is initially identified shorter isoform. PIKE-L, a longer isoform of PIKE gene, differs from PIKE-S by C-terminal extension containing Arf-GAP (ADP ribosylation factor-GTPase Activating Protein) and two ankyrin repeats domains. In contrast to the exclusive nuclear localization of PIKE-S, PIKE-L occurs in both the nucleus and the cytoplasm. PIKE-L physiologically associates with Homer 1, an mGluR I binding adaptor protein. The Homer/PIKE-L complex couples PI 3-kinase to mGluR I and regulates a major action of group I mGluRs, prevention of neuronal apoptosis. More recently, a third PIKE isoform, PIKE-A was identified in human glioblastoma multiforme brain cancers. Unlike the brain specific PIKE-L and -S isoforms, PIKE-A distributes in various tissues. PIKE-A contains the same domains present in PIKE-L but lacks N-terminal proline-rich domain (PRD), which binds PI 3-kinase and PLC-gamma1. Instead, PIKE-A specifically binds to active Akt and upregulates its activity in a GTP-dependent manner, mediating human cancer cell invasion and preventing apoptosis. Thus, PIKE extends its roles from the nucleus to the cytoplasm, mediating cellular processes from cell invasion to programmed cell death.

Topics: Animals; Apoptosis; Brain Neoplasms; Carrier Proteins; Glioblastoma; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine Triphosphate; Homer Scaffolding Proteins; Humans; Isoenzymes; Mice; Neoplasm Proteins; Nerve Tissue Proteins; Neurons; Phosphatidylinositol 3-Kinases; Phospholipase C gamma; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Structure-Activity Relationship

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder whose major feature is the occurrence of multiple neurofibromas, which are benign tumors of the nerve sheath. It affects an estimated one in 3000 to 4000 individuals. In addition to neurofibromas, there are many other clinical manifestations, including malignant tumors such as gliomas or malignant peripheral nerve sheath tumors, and nontumor effects such as skeletal dysplasia and learning disability. Diagnosis is established on the basis of clinical criteria. Molecular genetic testing is feasible, but the large size of the gene and wide range of pathogenic mutations have so far impeded the development of a clinical diagnostic test. Insights into pathogenesis have followed from identification of the NF1 gene and the development of animal models. The major function of the gene product appears to be regulation of the ras protein. Tumors are believed to arise by the loss of function of the NF1 protein, suggesting that NF1 behaves as a tumor suppressor gene. Heterozygous effects on some cell types are also likely, however. The role of ras in the pathogenesis of tumors in NF1 has suggested an approach to treatment using ras inhibitors, some of which are likely to begin in clinical trials in NF1 patients in the near future.

Topics: Animals; Brain Neoplasms; Cafe-au-Lait Spots; Cell Transformation, Neoplastic; Female; Genes, Dominant; Genes, Neurofibromatosis 1; Glioma; Guanosine Triphosphate; Humans; Hypertension; Learning Disabilities; Leukemia; Male; Mice; Mice, Knockout; Neurofibroma; Neurofibromatosis 1; Neurofibromin 1; Protein Structure, Tertiary; ras Proteins; Rhabdomyosarcoma; Scoliosis

Mutant p53 (mtp53) is an oncogene that drives cancer cell proliferation. Here we report that mtp53 associates with the promoters of numerous nucleotide metabolism genes (NMG). Mtp53 knockdown reduces NMG expression and substantially depletes nucleotide pools, which attenuates GTP-dependent protein activity and cell invasion. Addition of exogenous guanosine or GTP restores the invasiveness of mtp53 knockdown cells, suggesting that mtp53 promotes invasion by increasing GTP. In addition, mtp53 creates a dependency on the nucleoside salvage pathway enzyme deoxycytidine kinase for the maintenance of a proper balance in dNTP pools required for proliferation. These data indicate that mtp53-harbouring cells have acquired a synthetic sick or lethal phenotype relationship with the nucleoside salvage pathway. Finally, elevated expression of NMG correlates with mutant p53 status and poor prognosis in breast cancer patients. Thus, mtp53's control of nucleotide biosynthesis has both a driving and sustaining role in cancer development.

Topics: Animals; Blotting, Western; Brain Neoplasms; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Deoxycytidine Kinase; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Guanosine Triphosphate; Humans; Immunoprecipitation; Kaplan-Meier Estimate; Mice; Mutation; Neoplasm Invasiveness; Neoplasm Transplantation; Nucleosides; Nucleotides; Prognosis; Promoter Regions, Genetic; Proportional Hazards Models; Tumor Stem Cell Assay; Tumor Suppressor Protein p53

The pseudopeptide [Nphe(1)]N/OFQ(1-13)NH(2) (Nphe) has been shown to act as a pure, selective and competitive antagonist of nociceptin/orphanin FQ (N/OFQ) receptors in different tissues. However, Nphe displayed a highly variable potency, with pA(2) values ranging from 5.96 to 8.45. In the present study, we show that sodium ions and GTP markedly affect the potency of Nphe in blocking N/OFQ receptors coupled to cyclic AMP inhibition in different cellular systems. In intact N1E-115 neuroblastoma cells, the pA(2) value of Nphe increased from 7.13 to 8.02 when the extracellular sodium concentration was reduced from 138 to 2.5 mM. When N/OFQ inhibition of adenylyl cyclase activity was assayed in cell membranes, 100 mM NaCl decreased the pK(i) value of Nphe from 8.38 to 7.32, but increased that of the nonpeptide N/OFQ receptor antagonist CompB from 8.61 to 8.92. Similar effects of sodium ions on the potencies of Nphe and CompB were observed when the compounds were used to antagonize the N/OFQ inhibition of adenylyl cyclase activity in membranes of the external plexiform layer of the rat olfactory bulb. In the same assay, the increase of GTP concentration from 0.1 to 200 micro M decreased Nphe potency by 8-fold. These data demonstrate that sodium ions and GTP affect the potency of Nphe in a manner similar to that of agonists but not of pure antagonists and suggest that these factors may contribute to the reported variability of Nphe affinity constant.

Topics: Adenylyl Cyclases; Animals; Brain Neoplasms; Cell Membrane; Cyclic AMP; Guanosine Triphosphate; Male; Mice; Narcotic Antagonists; Neuroblastoma; Nociceptin Receptor; Olfactory Bulb; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Sodium; Somatostatin; Tumor Cells, Cultured

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

We showed recently that human O(6)-alkylguanine-DNA alkyltransferase (AGT), an important target for improving cancer chemotherapy, is a phosphoprotein and that phosphorylation inhibits its activity [Srivenugopal, Mullapudi, Shou, Hazra and Ali-Osman (2000) Cancer Res. 60, 282-287]. In the present study we characterized the cellular kinases that phosphorylate AGT in the human medulloblastoma cell line HBT228. Crude cell extracts used Mg(2+) more efficiently than Mn(2+) for phosphorylating human recombinant AGT (rAGT) protein. Both [gamma-(32)P]ATP and [gamma-(32)P]GTP served as phosphate donors, with the former being twice as efficient. Specific components known to activate protein kinase A, protein kinase C and calmodulin-dependent kinases did not stimulate the phosphorylation of rAGT. Phosphoaminoacid analysis after reaction in vitro with ATP or GTP showed that AGT was modified at the same amino acids (serine, threonine and tyrosine) as in intact HBT228 cells. Although some of these properties pointed to casein kinase II as a candidate enzyme, known inhibitors and activators of casein kinase II did not affect rAGT phosphorylation. Fractionation of the cell extracts on poly(Glu/Tyr)-Sepharose resulted in the adsorption of an AGT kinase that modified the tyrosine residues and the exclusion of a fraction that phosphorylated AGT on serine and threonine residues. In-gel kinase assays after SDS/PAGE and non-denaturing PAGE revealed the presence of two AGT kinases of 75 and 130 kDa in HBT228 cells. The partly purified tyrosine kinase, identified as the 130 kDa enzyme by the same assays, was strongly inhibited by tyrphostin 25 but not by genestein. The tyrosine kinase used ATP or GTP to phosphorylate the AGT protein; this reaction inhibited the DNA repair activity of AGT. Evidence that the kinases might physically associate with AGT in cells was also provided. These results demonstrate that two novel cellular protein kinases, a tyrosine kinase and a serine/threonine kinase, both capable of using GTP as a donor, phosphorylate the AGT protein and affect its function. The new kinases might serve as potential targets for strengthening the biochemical modulation of AGT in human tumours.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Brain Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Casein Kinase II; Cations; Chromatography, Agarose; Cricetinae; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Genistein; Guanosine Triphosphate; Humans; Magnesium; Manganese; Medulloblastoma; Mice; Molecular Sequence Data; O(6)-Methylguanine-DNA Methyltransferase; Phosphates; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Rats; Recombinant Proteins; Sequence Homology, Amino Acid; Serine; Threonine; Tumor Cells, Cultured; Tyrosine; Tyrphostins

Preliminary studies have demonstrated that the Ras family and related guanosine triphosphate-dependent proteins are overactivated in malignant gliomas and that inhibition of the activation of such proteins, by blockade of their post-translational processing, reduces tumor cell growth in vitro. The current study evaluates the utility of this therapeutic strategy in vivo, using preclinical glioma model systems.. We examined the efficacy against U-87 human malignant glioma cells, in both subcutaneous and intracranial nude mouse models, of selective peptidomimetic inhibitors of farnesyltransferase (FTI-276) and geranylgeranyltransferase (GGTI-297), which are involved in critical steps in the post-translational processing of Ras and related guanosine triphosphate-dependent proteins. For the subcutaneous model, 2 x 10(5) U-87 cells were implanted; after measurable tumors were detected on Day 7, animals were treated with either FTI-276, GGTI-297, or vehicle, administered by continuous infusion for 7 days. Differences in tumor volumes among the treatment groups were examined for significance using a Student's t test. For the intracranial model, 2 x 10(5) U-87 cells were implanted in the right frontal lobe and treatment was initiated on Day 7. In initial studies, animals received a 7-day course of either FTI-276, GGTI-297, or vehicle. In subsequent studies, a 28-day treatment period was used. Comparisons of survival times among treatment groups were performed using a rank-sum test.. Although the two agents exhibited comparable antiproliferative activities in previous in vitro studies, an obvious difference in efficacy was apparent in this study. Whereas the geranylgeranyltransferase inhibitor failed to improve survival rates, compared with those observed for control animals, in either the subcutaneous or intracranial model, the farnesyltransferase inhibitor produced objective regression of tumor growth in the subcutaneous model and significant prolongation of survival times in the intracranial model, without apparent toxicity. In the subcutaneous model, tumor volumes for the control, GGTI-297-treated, and FTI-276-treated animals on Day 28 after implantation were 621+/-420, 107+/-104, and 18.5+/-12.7 mm3, respectively (P < 0.05). In the 7-day-treated intracranial model, survival times for the control, GGTI-297-treated, and FTI-276-treated groups were 27.7+/-2.9, 29.8+/-2.1, and 43.6+/-2.7 days, respectively (P < 0.001). In the 28-day-treated intracranial model, survival times for the control, GGTI-297-treated, and FTI-276-treated groups were 29.2+/-3.7, 28.3+/-3.9, and 58.7+/-6.2 days, respectively, with five of six animals in the latter group surviving more than 55 days after tumor implantation (P < 0.001).. These studies demonstrate that farnesyltransferase inhibition is effective in diminishing the growth of human glioma cells in vivo. Evaluation of this treatment approach in clinical trials is warranted.

Topics: Alkyl and Aryl Transferases; Animals; Benzamides; Brain Neoplasms; Cell Division; Drug Evaluation, Preclinical; Enzyme Inhibitors; Farnesyltranstransferase; Geranyltranstransferase; Glioma; Guanosine Triphosphate; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Proto-Oncogene Proteins p21(ras); Treatment Outcome; Tumor Cells, Cultured

1. We have used somatostatin (SRIF) receptor subtype-selective ligands to determine some of the operational characteristics of somatostatin receptors in Neuro2A mouse neuroblastoma cells. The potent SRIF1-receptor selective ligand, BIM-23027, was able to displace completely the specific binding of radioiodinated somatostatin, [125I]-Tyr11-SRIF-14, with a pIC50 of 10.3, suggesting that Neuro2A cells contain predominantly receptors of the SRIF1 receptor group. The rank order of affinities for several somatostatin analogues tested in competition studies, together with the high affinity of BIM-23027, indicate that the majority of receptors in Neuro2A cells are of the sst2 subtype. 2. The stable radioligand, [125I]-BIM-23027, bound with high affinity (Kd = 13 pM, Bmax = 0.2 pmol mg-1 protein) to Neuro2A cell membranes, but its binding was only partially reversible at room temperature and below. Thus at 4 degrees C, only 36% of the bound ligand dissociated within 2 h. In contrast, 60% of the ligand dissociated at 15 degrees C and 89% of the ligand dissociated at 37 degrees C. 3. Equilibrium binding of [125I]-BIM-23027 was partially (25%) inhibited by 10 microM GTP, and by 120 mM NaCl (42% inhibition) but this inhibition was increased to 75% when sodium chloride and GTP were added together. This effect of GTP and sodium chloride was also seen in dissociation experiments. After incubation to equilibrium with [125I]-BIM-23027, dissociation was initiated with excess unlabelled ligand in the presence of GTP (10 microM) and sodium chloride (120 mM). Under these conditions 67% of the ligand dissociated at 4 degrees C, 81% at 15 degrees C and 93% at 37 degrees C. Binding was totally inhibited by pretreatment of cells with pertussis toxin. 4. Functionally, BIM-23027 inhibited forskolin-stimulated cyclic AMP accumulation in a concentration-dependent manner with an IC50 of 1.0 nM and a maximal inhibition of 37%. This effect was abolished by pretreatment of the cells with pertussis toxin. However, unlike in studies reported with the recombinant sst2 receptor, no rise in intracellular calcium concentration was observed with SRIF-14. 5. We conclude that Neuro2A cells provide a stable neuronal cell line for the study of functionally coupled endogenous somatostatin receptors of the sst2 type. In addition, we have found that activation of the receptor is associated with ligand-receptor internalisation.

Topics: Adenosine Triphosphate; Animals; Brain Neoplasms; Calcium; Cell Membrane; Cyclic AMP; Guanosine Triphosphate; Kinetics; Ligands; Mice; Neuroblastoma; Peptides, Cyclic; Receptors, Somatostatin; Second Messenger Systems; Somatostatin; Tumor Cells, Cultured

Nuclear RNA polymerase activity was studied in homotransplanted rat glial tumors where the primary tumor was produced by transplacental injection of ethylnitrosourea. Alpha amanitin, cycloheximide, and rifampicin were tested as inhibitors of this activity. Alpha amanitin significantly inhibited RNA polymerase activity in all tumors. This indicated that the major nuclear RNA polymerase activity seen in vitro in the tumor nuclei was RNA polymerase II. This is similar to the activity seen in normal glial nuclei. Cycloheximide and rifampicin which have no effect on RNA polymerase activity in normal glial nuclei inhibited about 20% of the polymerase activity in three of the tumors. The size and multiplicity of the nucleoli in these tumor cells suggests that RNA polymerase I could account for the activity which is inhibited by cycloheximide.

Topics: Amanitins; Animals; Autoradiography; Brain Neoplasms; Cell Nucleolus; Cell Nucleus; Cycloheximide; DNA-Directed RNA Polymerases; DNA, Neoplasm; Endoplasmic Reticulum; Ethylnitrosourea; Fibrosarcoma; Glioma; Guanosine Triphosphate; Neoplasm Transplantation; Neoplasms, Experimental; Neurofibroma; Rats; Rifampin; Transplantation, Homologous