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D Marmé

Publications and source records attributed to D Marmé.

At least 55 records · Page 3Linked to original sources
PubMed1995

Human chorionic gonadotropin-dependent expression of vascular endothelial growth factor/vascular permeability factor in human granulosa cells: importance in ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome (OHSS) is a severe complication arising from controlled ovarian stimulation treatment. This iatrogenic condition is potentially lethal and occurs in 0.3-5% of stimulated ovarian cycles. hCG exacerbates OHSS. The pathophysiology of OHSS is still unknown; therefore, treatment regimens are aimed at ameliorating symptoms. Prominent features of OHSS are an elevated risk of thromboembolism due to enhanced production of von Willebrand factor by endothelial cells and ascites, or pulmonary edema due to increased vascular permeability followed by third space fluid accumulation. Both of these sequelae can be evoked by vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF). High concentrations of VEGF/VPF have been demonstrated in ascitic fluid from patients with OHSS, but the source of VEGF/VPF in these patients remained unidentified. Here we report that the messenger ribonucleic acid expression of VEGF/VPF in human luteinized granulosa cells (GCs) is dose and time dependently enhanced by hCG in vitro. Furthermore, VEGF/VPF proteins are produced by GCs. Our results suggest that the effects of hCG on the development and course of OHSS may be mediated by the production of VEGF/VPF by GCs.

Adult↗
PubMed1994

Differential expression of the two VEGF receptors flt and KDR in placenta and vascular endothelial cells.

Vascular endothelial growth factor (VEGF) is a newly identified growth and permeability factor with a unique specificity for endothelial cells. Recently the flt-encoded tyrosine kinase was characterized as a receptor for VEGF. A novel tyrosine kinase receptor encoded by the KDR gene was also found to bind VEGF with high affinity when expressed in CMT-3 cells. Screening for flt and KDR expression in a variety of species and tissue-derived endothelial cells demonstrates that flt is predominantly expressed in human placenta and human vascular endothelial cells. Placenta growth factor (PIGF), a growth factor significantly related to VEGF, is coexpressed with flt in placenta and human vascular endothelial cells. KDR is more widely distributed and expressed in all vessel-derived endothelial cells. These data demonstrate that cultured human endothelial cells isolated from different tissues express both VEGF receptors in relative high levels and, additionally, that all investigated nonhuman endothelial cells in culture are also positive for KDR gene expression.

Animals↗
PubMed1994

Detection and quantification of vascular endothelial growth factor/vascular permeability factor in brain tumor tissue and cyst fluid: the key to angiogenesis?

In primary malignant brain tumors increased vascularity and marked edema strongly suggest a possible role of the vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). This was confirmed by earlier in situ hybridization studies, by analysis of the expression of the mitogen in different subsets of glioblastoma cells, and by the fact that the VEGF/VPF receptor flt-1 (fms-like tyrosine kinase) is up-regulated in tumor cells in vivo. To assess and quantify the expression of the VEGF/VPF gene and of the receptor gene, 26 surgical specimens of brain tumor tissue from 24 patients were analyzed. In most malignant gliomas, the expression level of the VEGF/VPF gene is elevated and can be increased up to 20- to 50-fold in comparison with low-grade tumors. Using polymerase chain reaction-based amplification, it could be shown that the messenger RNAs of three different VEGF/VPF forms are synthesized in tumor tissue samples. Northern blot studies revealed that in some samples a significant expression of the gene coding for placenta growth factor, a growth factor closely related to VEGF/VPF, was observed. In addition, using a radioreceptor assay it was possible to detect high VEGF/VPF-like activity in the cyst fluids of brain tumors, indicating the accumulation of the mitogen and permeability factor in brain tumor cysts. Further investigations revealed that astrocytoma and glioblastoma cells in culture express the VEGF/VPF gene and secrete the VEGF/VPF protein, whereas gene expression of the two known VEGF/VPF receptors, kinase insert domain-containing receptor and flt-1, could not be detected. These data support previous reports, which stated that VEGF/VPF acts as a paracrine growth and permeability factor in brain tumors and may contribute to tumor growth by initiating tumor angiogenesis.

Animals↗
PubMed1994

Mutant p53 potentiates protein kinase C induction of vascular endothelial growth factor expression.

Many tumor cells produce vascular endothelial growth factor (VEGF), which is thought to be a pivotal mediator of tumor neoangiogenesis. Expression of the VEGF gene can be induced by tumor promoting phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), which activate protein kinase C (PKC). Here we show that in transient transfection assays a mutated form of the murine p53 tumor suppressor gene (ala135-->val) induces expression of VEGF mRNA and potentiates TPA stimulated VEGF mRNA expression. In NIH 3T3 cells which stably overexpress the temperature sensitive p53 (ala135-->val), displaying mutant phenotype at 37 degrees C and wildtype phenotype at 32.5 degrees C, induction of VEGF mRNA and protein by activated PKC is strongly synergistic with mutant, but not wildtype p53. Mutant p53 specifically increases TPA induction of VEGF without affecting the expression of other TPA inducible genes. TPA dependent VEGF expression is also enhanced by human p53 mutated at amino acid 175. Thus, our data link PKC and p53, the gene most frequently altered in human tumors, with the regulation of tumor angiogenesis.

3T3 Cells↗
PubMed1993

Angiotensin II mediates intracellular signalling in vascular smooth muscle cells by activation of tyrosine-specific protein kinases and c-raf-1.

Angiotensin II induces protein tyrosine kinase activation and apparent decreased electrophoretic mobility of the c-raf-1 serine/threonine protein kinase in cultured rat vascular smooth muscle cells. Tyrosine phosphorylation of at least 9 cellular proteins with molecular weights of 151, 131, 116, 110, 90, 65, 62, 60, 52 kd was induced in a time- and concentration-dependent manner, and included a serine/threonine protein kinase. The phosphotyrosine containing proteins differed from those induced by PDGF BB or AB. Angiotensin II by itself was shown not to act as a mitogen in cultured smooth muscle cells.

Angiotensin II↗
PubMed1993

Activation and substrate specificity of the human protein kinase C alpha and zeta isoenzymes.

Protein kinase C (PKC), a class of serine/threonine kinases activated by Ca2+ and/or phospholipids, is involved in a variety of cellular processes such as proliferation, differentiation and secretion. Nine members of the PKC gene family are known; these are differentially expressed in eukaryotic cells and can be divided into two sub-groups: the Ca(2+)-dependent (classical) PKC isoenzymes alpha, beta I, beta II and gamma, and the Ca(2+)-independent neoPKC isoenzymes delta, epsilon, zeta, eta and theta. A detailed biochemical characterisation of these PKC isoenzymes is one prerequisite for the elucidation of their distinct roles within cellular signal transduction. In this study, we report the cloning of a human PKC-zeta cDNA, its expression in recombinant baculovirus-infected insect cells and the partial purification of the PKC-zeta isoenzyme. In comparison to highly purified human PKC alpha, a representative of the classical PKC subgroup, purified PKC zeta was characterised with respect to activator requirement, substrate specificity, proteolytic activation and sensitivity towards PKC inhibitors. In contrast to PKC alpha, PKC zeta exhibits a constitutive kinase activity which is independent of Ca2+, phosphatidylserine and diacylglycerol. Arachidonic acid alone or a combination of gamma-linolenic acid and phosphatidylserine slightly enhance PKC zeta activity. In the presence of the classical PKC activators phosphatidylserine/diacylglycerol, PKC alpha phosphorylates a PKC-alpha pseudosubstrate-derived peptide, an epidermal-growth-factor-receptor-derived peptide, histone III-S and myelin basic protein to an equal extent, whilst PKC zeta phosphorylates only the PKC-alpha-derived peptide. However, arachidonic acid greatly diminishes PKC-alpha activity towards the epidermal-growth-factor-receptor-derived peptide, histone III-S and myelin basic protein, but enhances PKC-zeta activity towards the PKC-alpha-derived peptide. These results indicate a possible modulation of substrate specificity of these two PKC isoenzymes by (the binding of) different activators (to their regulatory domains). In the case of PKC zeta, this finding is strengthened by the fact that the epidermal growth factor receptor-derived peptide, which is not a substrate for the holoenzyme, is significantly phosphorylated by a protein fragment generated by limited proteolysis and comprising only the kinase domain. Furthermore, PKC zeta, in contrast to PKC alpha, is insensitive to PKC inhibitors known to interfere either with the regulatory or the catalytic domain and cannot be activated by phorbol ester treatment of NIH 3T3 cells or insect cells, overexpressing the respective PKC isoenzyme. The potential implications of these findings on the mechanism(s) of activation and the substrate specificity of PKC zeta are discussed.

Amino Acid Sequence↗
PubMed1993

Protein kinase C alpha activates RAF-1 by direct phosphorylation.

The kinase Raf-1 can be activated by treatment of cells with mitogens and by the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (reviewed in refs 1,2). Activated Raf-1 triggers a protein kinase cascade by direct phosphorylation of MAP kinase kinase, resulting in phosphorylation of ternary complex factor and Jun by MAP kinase. Here we investigate the molecular mechanism and biological consequences of PKC alpha-mediated Raf-1 activation in NIH3T3 fibroblasts. PKC alpha directly phosphorylates and activates Raf-1 both in vitro and in vivo. PKC alpha induces Raf-1 phosphorylation at several sites, including a serine residue at position 499. Mutation of serine at this position or at residue 259 does not abrogate Raf-1 stimulation by a combination of Ras plus the src tyrosine kinase Lck, but severely impedes Raf-1 activation by PKC alpha. Consistent with such a direct interaction is the observation that Raf-1 and PKC alpha cooperate in the transformation of NIH3T3 cells. The Ser499 phosphorylation site is necessary for this synergism.

3T3 Cells↗
PubMed1993

Gö 6976, a selective inhibitor of protein kinase C, is a potent antagonist of human immunodeficiency virus 1 induction from latent/low-level-producing reservoir cells in vitro.

Human immunodeficiency virus (HIV-1) infection is followed by a period of latency or a low-level-persistent (LLP) state that results in an asymptomatic infection of the host. Productive viral expression may be triggered by a variety of activators including mitogens, antigens, and cytokines. Protein kinase C (PKC) has been shown to be important in the intracellular cascade of signals induced by such activators. With U1 and ACH-2 cell lines representative of an HIV-1 postintegration state, the effect of Gö 6976, a synthetic inhibitor of PKC was tested. Gö 6976 is a nonglycosidic indolocarbazole found to potently inhibit HIV-1 induction by Bryostatin 1, tumor necrosis factor alpha, and interleukin 6. Gö 6976 effectively blocks viral transcription induced by Bryostatin 1 or tumor necrosis factor alpha that leads to the inhibition of intracellular viral protein synthesis and viral shedding. Gö 6976 also blocks interleukin 6-mediated posttranscriptional induction of viral proteins. The IC50 of Gö 6976 shows a 12- to 60-fold more potent effect than for H-7, another PKC inhibitor with a similar mechanism. The inhibitory effect is reduced when Gö 6976 is not added before or within 1 hr of induction by the potent PKC activator Bryostatin 1. However, U1 cells can be grown for long periods in a nontoxic concentration of Gö 6976 (300 nM), which confers virtual inhibition of HIV-1 induction without the development of resistance. Results indicate that inhibition of HIV-1 proviral induction from latent/low-level-producing infectious states with potent PKC inhibitors like Gö 6976 may represent an additional and promising antiviral approach.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

Selective inhibition of protein kinase C isozymes by the indolocarbazole Gö 6976.

Indolocarbazoles have been identified as novel inhibitors of protein kinase C (PKC), with Gö 6976 as one of its most potent and selective representatives. Recombinant PKC isozymes alpha, beta 1, delta, epsilon, and zeta were used in in vitro kinase assays to investigate Gö 6976 with respect to isozyme-specific PKC inhibition. Gö 6850, identical with GF 109203X, another PKC-specific kinase inhibitor, was included in this study as a reference compound. Nanomolar concentrations of the indolocarbazole Gö 6976 inhibited the Ca(2+)-dependent isozymes alpha and beta 1, whereas even micromolar concentration of Gö 6976 had no effect on the kinase activity of the Ca(2+)-independent PKC subtypes delta, epsilon, and zeta. In contrast, the bisindolymaleimide Gö 6850 inhibited all PKC isozymes, however, with a ranked order of potency (alpha > beta 1 > epsilon > delta > zeta). Kinetic analysis revealed that PKC inhibition by Gö 6976 was competitive with respect to ATP, non-competitive with respect to the protein substrate, and mixed type with respect to phosphatidylserine. Further experiments in the presence of different amounts of free Ca2+ indicated that interference with Ca2+ or its binding site is not responsible for the differential inhibition of PKC isozymes by Gö 6976.

Animals↗
PubMed1993

Over-expression of protein kinase C-alpha enhances platelet-derived growth factor- and phorbol ester- but not calcium ionophore-induced formation of prostaglandins in NIH 3T3 fibroblasts.

Over-expression of human protein kinase C-alpha in murine NIH 3T3 fibroblasts is associated with an increased platelet-derived growth factor- and phorbol ester-mediated formation of prostaglandins, whereas the calcium ionophore-induced release of arachidonic acid metabolites is unaffected; however, the differences of arachidonic acid and prostaglandin formation are much more pronounced with platelet-derived growth factor than with phorbol ester. Platelet-derived growth factor induces an identical elevation of intracellular free calcium in control and protein kinase C-alpha over-expressing cells: the phorbol ester has no effect on intracellular free calcium in both cell lines. These results demonstrate that protein kinase C-alpha may couple to arachidonic acid cascade in NIH 3T3 fibroblasts.

3T3 Cells↗
PubMed1993

Activation of purified human protein kinase C alpha and beta I isoenzymes in vitro by Ca2+, phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate.

The increasing number of eukaryotic protein kinase C (PKC) isoenzymes which have been described has raised great interest in potential differences in the cellular expression, the mode of activation and the substrate specificity of these isoenzymes. The last two aspects have mostly been studied with isoenzymes purified from rat or bovine brain or from recombinant-baculovirus-infected insect cells. In this study, we have expressed the human PKC isoenzymes alpha and beta I in recombinant-baculovirus-infected insect cells. The isoenzymes were purified to homogeneity by a four-step procedure which included a reversible Ca(2+)-dependent association/dissociation to and from the endogenous membranes of the lysed insect cells. Characterization of the purified enzymes with respect to ATP requirement and substrate specificity, using the epidermal-growth-factor receptor peptide and histone III-S respectively, revealed no isoenzyme-specific differences. Activation by trypsin or Ca2+ and a variety of different phospholipids and phosphoinositides (in a mixed-micellar assay) gave the following results. Proteolytic cleavage of the PKC isoenzymes by trypsin generated fully activated phospholipid-independent PKC beta I, whereas PKC alpha reached only 50% of the activity obtained in the presence of phospholipids. PKC alpha and beta I showed no difference in their dependence on Ca2+, diacylglycerol (DAG) and phosphatidylserine (PS). Replacement of either DAG or PS by phosphatidylglycerol, cardiolipin, phosphatidylcholine and several phosphoinositides revealed that PtdIns(4,5)P2 can act as a PKC activator similar to DAG, whereas PtdIns can substitute for PS as a cofactor of activation. Thus, at least for the PKC isoenzymes alpha and beta I, a combination of PtdIns and PtdIns(4,5)P2 can fully replace PS and DAG in vitro as the classical activators of PKC.

Adenosine Triphosphate↗
PubMed1993

Synthesis and assembly of functionally active human vascular endothelial growth factor homodimers in insect cells.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor with a target-cell specificity highly restricted to vascular endothelial cells. Recombinant baculovirus were constructed for the production of two different forms of the human VEGF protein in insect cells. VEGF165 and VEGF121 proteins produced by Sf158 cells underwent a similar processing compared with mammalian cells, including efficient glycosylation, formation of a disulfide-linked dimer and secretion into the media. Only one of these forms, VEGF165 had a high affinity for heparin and this characteristic was used to purify this form to homogenicity by a two-step heparin-affinity chromatography. The biological activity of the purified 43-kDa homodimer was demonstrated by high-affinity binding to VEGF receptors, and by the induction of DNA synthesis in vascular endothelial cells. A positive angiogenic activity in vivo was demonstrated by the day-13 chorioallantoic-membrane assay. The mitogenic potency of VEGF121 for human umbilical vein endothelial cells was very similar compared to VEGF165. These results demonstrate that an angiogenic growth factor whose normal processing requires glycosylation and disulfide-bridge formation can be efficiently expressed in high concentration (up to 5 micrograms/ml) in insects cells.

Amino Acid Sequence↗
PubMed1993

Novel indolocarbazole protein kinase C inhibitors prevent reactivation of HIV-1 in latently infected cells.

Suppression of human immunodeficiency virus-1 (HIV-1) reactivation in latently infected cells by protein kinase C (PKC) inhibitors has been described. Based on an initial finding with the indolocarbazole inhibitor Gö 6976 we have examined several members of this new class of potent and specific PKC inhibitors with respect to their ability to prevent the PKC-mediated induction of HIV-1 replication in the latently infected U1 cell line. Two of these compounds strongly inhibited not only PMA-induced release of p24-antigen and infectious virus particles into the supernatant (50% inhibition at 0.04-0.35 microM) but also TNF-alpha-mediated HIV-1 reactivation in the same concentration range. Significant lower toxicities compared to Gö 6976 were observed for the new compounds, with 50% cytotoxic concentrations at 5.2 microM for Gö 7775 and 3.4 microM for Gö 7716. This resulted in selectivity indices which were 10-20-times higher compared to the reference compound Gö 6976 and were comparable to those of registered anti-AIDS drugs. No anti-HIV-1 activity was observed for a closely related indolocarbazole analogue with no inhibitory activity in the PKC in vitro enzyme assay. This study demonstrates the important role of PKC in reactivation of HIV-1 in latently infected cells and points to the potential of indolocarbazoles to preserve the latent state of HIV-1 infection.

Antineoplastic Agents↗
PubMed1992

Basic fibroblast growth factor modulates the mitogenic potency of the platelet-derived growth factor (PDGF) isoforms by specific upregulation of the PDGF alpha receptor in vascular smooth muscle cells.

Platelet-derived growth factor AA (PDGF AA), in contrast to PDGF AB and BB, is a poor mitogen for smooth muscle cells (SMC). However, together with basic fibroblast growth factor (bFGF) it acts synergistically on DNA synthesis of these cells. Northern blot analysis revealed that bFGF selectively increases the PDGF-receptor alpha subtype (PDGF-R alpha) mRNA level without a significant effect on the PDGF-R beta mRNA level. The amount of PDGF-R alpha protein is also selectively increased after stimulating SMC with bFGF as shown by immunoprecipitation of lysates from SMC with anti-PDGF-R alpha antibodies. The number of binding sites for 125I-PDGF AA is more than doubled after bFGF-treatment, whereas the specific binding for PDGF AB and BB increased only by approximately 30 and 20%, respectively. The increase in the number of PDGF-R alpha renders the SMC responsive for PDGF AA as demonstrated by the induction of the proto-oncogene c-fos as well as by an increased cell proliferation. The enhanced PDGF binding after bFGF treatment may in fact explain the observed synergistic behavior. These data are discussed with regard to a possible role of growth factor-induced transmodulation of receptor expression during atherogenesis.

Animals↗
PubMed1992

Platelet-derived growth factor-induced transcription of the vascular endothelial growth factor gene is mediated by protein kinase C.

Platelet-derived growth factor and phorbol ester cause an increase in vascular endothelial growth factor (VEGF) mRNA expression in control NIH 3T3 fibroblasts and NIH 3T3 fibroblasts overexpressing human protein kinase C (PKC) alpha. In the case of phorbol ester-induced VEGF expression, the VEGF mRNA levels were significantly higher in cells overexpressing human PKC alpha as compared to control cells. In cells stimulated with platelet-derived growth factor or phorbol ester, induction of expression was lost after down-regulation of PKC. This indicates that PKC is involved in the signal transduction leading to VEGF expression.

3T3 Cells↗
PubMed1992

Over-expression of human phospholipase C-gamma 2 enhances platelet-derived growth factor-induced mobilization of intracellular Ca2+ and the release of arachidonic acid and prostaglandins in NIH 3T3 fibroblasts.

Over-expression of human phospholipase C-gamma 2 in murine NIH 3T3 fibroblasts has been shown to result in an increased platelet-derived growth factor-mediated formation of inositol phosphates. Here we show that phospholipase C-gamma 2 over-expression is associated with an increased platelet-derived growth factor-mediated release of arachidonic acid, prostaglandin E2, 6-keto prostaglandin F1 alpha and prostaglandin F2 alpha. The phorbol ester, calcium ionophore- and fluoride-induced release of arachidonate and its metabolites is not affected by phospholipase C-gamma 2 over-expression. Over-expression of phospholipase C-gamma 2 is also associated with an enhancement of platelet-derived growth factor-induced change in intracellular Ca2+. These results demonstrate that stimulation of recombinant human phospholipase C-gamma 2 induces a change in the intracellular Ca2+ concentration, a release of arachidonic acid and formation of prostaglandins in NIH 3T3 cells. In control cells platelet-derived growth factor-induced activation of arachidonic acid cascade is rate-limited by the endogenous phospholipase C.

3T3 Cells↗
PubMed1992

An endogenous membrane-bound protein inhibits the phosphorylation of the L-type calcium channel by the cAMP-dependent protein kinase.

An endogenous protein inhibits the PKA-phosphorylation of the DHP-binding calcium channel complex in vitro. The inhibitory activity could be reduced by a treatment with detergents or dithiothreitol. Further purification separates the inhibitory activity from the dihydropyridine-binding calcium channel complex. Both activities are localized in the plasma membrane indicating that this protein kinase-inhibitor could interfere with the phosphorylation of the calcium channel by the cAMP-dependent protein kinase. The inhibitory activity may therefore take part in the regulation of the calcium channel current.

Animals↗