h-89 and Brain-Neoplasms

Mannose-binding lectin (MBL)-Associated Serine Proteases (MASP)-1 and 3, key enzymes in the lectin complement pathway of innate immune response, are also expressed in glioma cell lines. We investigated MASP-1 and MASP-3 expression during dibutyryl cyclic AMP (dbcAMP)- or Interleukin-6 (rIL-6)-induced astrocytic differentiation of C6 glioma cells. Our results demonstrate that C6 cells express basal levels of MASP-1 and MASP-3 and following exposure to dbcAMP or IL-6, a consistent MASP-1 and MASP-3 mRNA up-regulation was found, with a behavior similar to that showed by the fibrillary acidic protein (GFAP). Furthermore, in cell conditioned media, rIL-6 stimulated MASP-3 secretion which reached levels similar to those obtained by dbcAMP treatment. Moreover, the detection of a 46-kDa MASP-3 suggested its processing to the mature form in the extracellular cell medium. Interestingly, the H89 PKA inhibitor, mostly affected dbcAMP-induced MASP-1 and MASP-3 mRNA levels, compared to that of rIL-6, suggesting that cAMP/PKA pathway contributes to MASP-1 and MASP-3 up-regulation. MASP-1 and MASP-3 expression increase was concomitant with dbcAMP- or rIL-6-induced phosphorylation of STAT3. Our findings suggest that the increase in intracellular cAMP concentration or rIL-6 stimulation can play a role in innate immunity enhancing MASP-1 and MASP-3 expression level in C6 glioma cells.

Topics: Animals; Astrocytes; Brain Neoplasms; Bucladesine; Cell Differentiation; Cell Line, Tumor; Glioma; Immunity, Innate; Interleukin-6; Isoquinolines; Mannose-Binding Protein-Associated Serine Proteases; Phosphorylation; Protein Kinase Inhibitors; Rats; Recombinant Proteins; RNA, Messenger; STAT3 Transcription Factor; Sulfonamides

Dedicator of cytokinesis 1 (Dock1 or Dock180), a bipartite guanine nucleotide exchange factor for Rac1, plays critical roles in receptor tyrosine kinase-stimulated cancer growth and invasion. Dock180 activity is required in cell migration cancer tumorigenesis promoted by platelet derived growth factor receptor (PDGFR) and epidermal growth factor receptor.. To demonstrate whether PDGFRα promotes tumor malignant behavior through protein kinase A (PKA)-dependent serine phosphorylation of Dock180, we performed cell proliferation, viability, migration, immunoprecipitation, immunoblotting, colony formation, and in vivo tumorigenesis assays using established and short-term explant cultures of glioblastoma cell lines.. Stimulation of PDGFRα results in phosphorylation of Dock180 at serine residue 1250 (S1250), whereas PKA inhibitors H-89 and KT5720 oppose this phosphorylation. S1250 locates within the Rac1-binding Dock homology region 2 domain of Dock180, and its phosphorylation activates Rac1, p-Akt, and phosphorylated extracellular signal-regulated kinase 1/2, while promoting cell migration, in vitro. By expressing RNA interference (RNAi)-resistant wild-type Dock180, but not mutant Dock180 S1250L, we were able to rescue PDGFRα-associated signaling and biological activities in cultured glioblastoma multiforme (GBM) cells that had been treated with RNAi for suppression of endogenous Dock180. In addition, expression of the same RNAi-resistant Dock180 rescued an invasive phenotype of GBM cells following intracranial engraftment in immunocompromised mice.. These data describe an important mechanism by which PDGFRα promotes glioma malignant phenotypes through PKA-dependent serine phosphorylation of Dock180, and the data thereby support targeting the PDGFRα-PKA-Dock180-Rac1 axis for treating GBM with molecular profiles indicating PDGFRα signaling dependency.

Topics: Animals; Brain Neoplasms; Carbazoles; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclic AMP-Dependent Protein Kinases; Female; Glioblastoma; HEK293 Cells; Humans; Isoquinolines; Mice; Phosphorylation; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Pyrroles; rac GTP-Binding Proteins; Receptor, Platelet-Derived Growth Factor alpha; Signal Transduction; Sulfonamides

We investigated whether cAMP-mediated protein kinase A(PKA) and Epac1/Rap1 pathways differentially affect brain tumor cell death using 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone(rolipram), specific phosphodiesterase type IV(PDE IV) inhibitor.. A172 and U87MG human glioblastoma cells were used. Percentage of cell survival was determined by MTT assay. PKA and Epac1/Rap1 activation was determined by western blotting and pull-down assay, respectively. Cell cycle and hypodiploid cell formation were assessed by flow cytometry analysis.. Non-specific PDE inhibitors, isobutylmethylxanthine(IBMX) and theophylline reduce survival percentage of A172 and U87MG cells. The expression of PDE4A and PDE4B was detected in A172 and U87MG cells. Rolipram-treated A172 or U87MG cell survival was lower in the presence of forskolin, adenylate cyclase activator, than that in its absence. Co-treatment with rolipram and forskolin also enhanced CREB phosphorylation on serine 133 that was inhibited by H-89, PKA inhibitor and cAMP-responsive guanine nucleotide exchange factor 1(Epac1), a Rap GDP exchange factor-mediated Rap1 activity in A172 cells. When A172 cells were treated with cell-permeable dibutyryl-cAMP(dbcAMP), PKA activator or 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate(CPT), Epac1 activator, basal level of cell death was increased and cell cycle was arrested at the phase of G2/M. Rolipram-induced A172 cell death was also increased by the co-treatment with dbcAMP or CPT, but it was inhibited by the pre-treatment with H-89.. These findings demonstrate that PKA and Epac1/Rap1 pathways could cooperatively play a role in rolipram-induced brain tumor cell death. It suggests that rolipram might regulate glioblastoma cell density through dual pathways of PKA- and Epac1/Rap1-mediated cell death and cell cycle arrest.

Topics: 1-Methyl-3-isobutylxanthine; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Brain Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 4; Dose-Response Relationship, Drug; Glioblastoma; Guanine Nucleotide Exchange Factors; Humans; Isoquinolines; Phosphodiesterase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Rolipram; Shelterin Complex; Signal Transduction; Sulfonamides; Telomere-Binding Proteins; Theophylline

Elevation in the level of intracellular cAMP is known to induce the astrocytic differentiation of C6 glioma cells by unknown mechanisms. In this report, we show that cAMP-induced autocrine interleukin 6 (IL-6) promoted astrocytic differentiation of C6 cells. Treatment of cells with N(6),2'-O-dibutyryl cAMP (Bt(2)AMP) and theophylline caused the delayed phosphorylation of signal transducer and activator of transcription 3 (STAT3), as well as the expression of an astrocyte marker, glial fibrillary acidic protein (GFAP). Overexpression of the dominant-negative form of STAT3 leads to the suppression of GFAP promoter activity, suggesting that STAT3 activity was essential for cAMP-induced GFAP promoter activation. On the other hand, the IL-6 gene was quickly induced by Bt(2)AMP/theophylline, and subsequent IL-6 protein secretion was stimulated. In addition, recombinant IL-6 induced GFAP expression and STAT3 phosphorylation. Most importantly, treatment with IL-6-neutralizing antibody dramatically reduced the cAMP-induced GFAP expression and STAT3 phosphorylation and reversed the cellular morphological changes that had been caused by Bt(2)AMP/theophylline. Taken together, these results indicated that Bt(2)AMP/theophylline lead to delayed STAT3 activation via autocrine IL-6. These processes subsequently led to the induction of GFAP. IL-6 secretion is thus thought to be a key event in controlling the astrocytic differentiation of C6 cells.

Topics: Animals; Astrocytes; Blotting, Western; Brain Neoplasms; Bucladesine; Cell Differentiation; Cell Line, Tumor; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Genes, Reporter; Glial Fibrillary Acidic Protein; Glioma; Interleukin-6; Isoquinolines; Microscopy, Fluorescence; Models, Biological; Phosphorylation; Plasmids; Promoter Regions, Genetic; Rats; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; STAT3 Transcription Factor; Sulfonamides; Theophylline; Time Factors; Trans-Activators; Transcription, Genetic

We have demonstrated that treatment with 200 nM okadaic acid (OA) for 1 h followed by a 15-min heat shock (HS) at 45 degrees C (termed OA-->HS treatment) leads to a rapid transactivation of grp78, the gene for the 78-kDa glucose-regulated protein, in 9L rat brain tumor cells. The level of Grp78 mRNA rose 15-fold in 60 min after the combined treatment. Nuclear extracts from cells subjected to OA-->HS treatment, compared to those of treatment with OA or HS alone, exhibited an increased binding activity toward an oligonucleotide probe containing the cAMP-responsive element-like (CRE-like, TGACGTGA) regulatory element in electrophoretic mobility shift assays (EMSA). The binding resulted in the formation of two protein-EMSA probe complexes exhibiting different association and dissociation rates in kinetic studies. The protein factors in the upper band (complex I) and lower band (complex II) were identified as the activating transcription factor-2 (ATF-2) and the CRE binding factor 1 (CREB-1), respectively, by antibody interference assays. In addition, the identity of CREB-1 was confirmed by supershift analysis. The binding activity, as well as the transactivation of the grp78 gene, can be abolished by a 1-h treatment with the cAMP-dependent protein kinase (PKA) inhibitor but not with protein kinase C or Ca2+/calmodulin-dependent protein kinase II inhibitors. Accumulation of steady-state level of ATF-2 was observed and was also modulated by treatment with H-89, a PKA inhibitor. From these results, we conclude that the CRE-like element plays an important role in the rapid transactivation of the grp78 gene and that the PKA signaling pathway is involved. In addition, PKA-mediated transcriptional regulation of grp78 in OA-->HS treatment is through regulation of protein phosphorylation as well as de novo synthesis of ATF-2.

Topics: Activating Transcription Factor 1; Activating Transcription Factor 2; Animals; Base Sequence; Brain Neoplasms; Carrier Proteins; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; DNA Primers; DNA-Binding Proteins; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Heat-Shock Proteins; Hot Temperature; Isoquinolines; Molecular Chaperones; Okadaic Acid; Phosphorylation; Polymerase Chain Reaction; Promoter Regions, Genetic; Rats; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Sulfonamides; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured

The brain creatine kinase (CKB) gene is expressed in a variety of tissues with highest expression seen in the brain. We have previously shown in primary rat brain cell cultures that CKB mRNA levels are high in oligodendrocytes and astrocytes and low in neurons (Molloy et al.: J Neurochem 59:1925-1932, 1992). In this report we show that treatment of human U87 glioblastoma cells with forskolin and IBMX, to elevate intracellular cAMP, induces expression of CKB mRNA from the transiently transfected rat CKB gene by 14-fold and also increases expression from the endogenous human CKB gene. This induction of CKB mRNA i) is due to increased transcription; ii) occurs rapidly (with maximal induction after 6 hr; iii) requires the activity of protein kinase A (PKA), but iv) does not require de novo protein synthesis and, in fact, is superinduced in the presence of cycloheximide. Given the role of oligodendrocytes in the energy-demanding process of myelination and of astrocytes in ion transport, these results have physiological significance, since they suggest that changes in cellular energy requirements in the brain during events, such as glial cell differentiation and increased neuronal activity, may in part be met by a cAMP-mediated modulation of CKB gene expression. Of particular importance is the possible modulation of CKB gene expression during myelinogenesis, since oligodendrocyte differentiation has been shown previously to be stimulated by increases in cAMP.

Topics: 1-Methyl-3-isobutylxanthine; Adenylyl Cyclases; Animals; Brain; Brain Neoplasms; Cloning, Molecular; Colforsin; Creatine Kinase; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Energy Metabolism; Enzyme Activation; Enzyme Induction; Glioblastoma; Glioma; HeLa Cells; Humans; Isoenzymes; Isoquinolines; Myelin Sheath; Nerve Tissue Proteins; Neuroglia; Protein Synthesis Inhibitors; Rats; Recombinant Fusion Proteins; RNA, Antisense; Sulfonamides; Transcription, Genetic; Transfection; Tumor Cells, Cultured