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.

Reversion of deregulated expression of vascular endothelial growth factor in human renal carcinoma cells by von Hippel-Lindau tumor suppressor protein.

Mutations or loss of both alleles of the von Hippel-Lindau (VHL) tumor suppressor gene has been documented in sporadic renal cell carcinomas and neoplasms that arise in individuals having the VHL syndrome. The well-vascularized phenotype of tumors that form in VHL disease let us consider vascular endothelial growth factor (VEGF) as a mediator of tumor growth in VHL disease. Human renal carcinoma cells that either lacked endogenous wild-type VHL or were transfected with an inactive mutant VHL showed deregulated expression of VEGF on the mRNA and protein level that was reverted by introduction of wild-type VHL. Stimulation of proliferation of endothelial cells by conditioned medium of cells expressing mutant VHL was almost abolished by neutralizing the VEGF. In contrast, expression of basic fibroblast growth factor and of c-myc proto-oncogene was not affected by VHL. Our data suggest VEGF as the key tumor angiogenesis factor in VHL disease.

Vascular endothelial growth factor up-regulates its receptor fms-like tyrosine kinase 1 (FLT-1) and a soluble variant of FLT-1 in human vascular endothelial cells.

The growth of solid tumors and the formation of metastases are dependent on neoangiogenesis. One of the most important factors in inducing the formation of new blood vessels is the vascular endothelial growth factor (VEGF), which acts specifically on endothelial cells. VEGF is expressed and secreted by almost all solid tumors. The molecular mechanisms leading to enhanced production of this angiogenic mitogen are manyfold and have been elucidated to some degree. Two VEGF receptors, fms-like tyrosine kinase 1 (FLT-1) and KDR, have been identified almost specifically on human endothelial cells. They are expressed preferentially in the proliferating endothelium of vessels lining and/or penetrating solid tumors, whereas they are almost undetectable by convenient methods in vessels of healthy tissue. However, the underlying mechanisms are not understood. We could show that media conditioned by various cancer cell lines grown under hypoxic conditions were able to up-regulate expression of FLT-1 mRNA and protein but not of KDR mRNA. Furthermore, up-regulation of a shorter mRNA species was observed that most probably codes for the soluble variant of FLT-1. These effects were completely inhibited by VEGF-neutralizing extracellular VEGF receptor domains. The effect could be mimicked by adding recombinant VEGF instead of conditioned cancer cell medium to the endothelial cell cultures. Both mutant VEGF, which activates only KDR, and placenta growth factor, which activates only FLT-1, were able to enhance FLT-1 expression. VEGF-stimulated FLT-1 mRNA expression was inhibited by actinomycin D. These data suggest that VEGF itself is the main factor secreted by tumor cells that is able to enhance the expression of its receptor FLT-1 and of a soluble variant of FLT-1 in endothelial cells.

Two independent mechanisms essential for tumor angiogenesis: inhibition of human melanoma xenograft growth by interfering with either the vascular endothelial growth factor receptor pathway or the Tie-2 pathway.

Protein ligands and receptor tyrosine kinases that specifically regulate endothelial cell function are mainly involved in physiological as well as in disease-related angiogenesis. These ligand/receptor systems include the vascular endothelial growth factor (VEGF) and the angiopoietin (Ang) families, and their receptors, the VEGF receptor family and the tyrosine kinase with immunoglobulin-like and epidermal growth factor homology domains (Tie) family. In the present study, the contribution of these endothelium-specific ligand/receptor systems to tumor angiogenesis was evaluated. A375v human melanoma cells, which express at least the angiogenic growth factors VEGF, VEGF-C, and Ang-1, were stably transfected to overexpress the extracellular ligand-binding domains of the endothelium-specific receptor tyrosine kinases fms-like tyrosine kinase-1 (Flt-1), Flt-4, Tie-1, and Tie-2, respectively. In vitro proliferation and colony formation assays confirmed that expression of the extracellular receptor domains inhibited neither tumor cell mitogenesis nor the ability to produce anchorage-independent growth. Nude mouse xenografts revealed that interference with either the VEGF receptor pathway or the Tie-2 pathway resulted in a significant inhibition of tumor growth and tumor angiogenesis. In contrast, interference with the Flt-4 pathway or the Tie-1 pathway was without significant effect. Our results show that both the VEGF receptor pathway and the Tie-2 pathway are essential for A375v melanoma xenograft growth. The inhibition of the VEGF receptor pathway cannot be compensated by the Tie-2 pathway, nor vice versa. These findings suggest that the VEGF receptor pathway and the Tie-2 pathway have to be considered as two independent mediators essential for the process of in vivo angiogenesis.

Effects of PTK787/ZK 222584, a specific inhibitor of vascular endothelial growth factor receptor tyrosine kinases, on primary tumor, metastasis, vessel density, and blood flow in a murine renal cell carcinoma model.

Antiangiogenic therapy is a promising new strategy to inhibit tumor growth and formation of metastases. Vascular endothelial growth factor (VEGF) and its receptors, VEGF-receptor 1 (VEGF-R1; FLT-1) and VEGF-R2 (KDR), have been shown to play a major role in tumor angiogenesis. PTK787/ZK 222584, a specific inhibitor of both VEGF-receptor tyrosine kinases, was investigated for its antitumoral and antiangiogenic activity in a murine renal cell carcinoma model. After intrarenal application of the renal carcinoma cells, mice develop a primary tumor and metastases to the lung and to the abdominal lymph nodes. Daily oral therapy with PTK787/ZK 222584 at a dose of 50 mg/kg resulted in a significant decrease of 61 and 67% in primary tumors after 14 and 21 days, respectively. The occurrence of lung metastases was significantly inhibited at both time points (98% reduction and 78% reduction, respectively). After 14 days, no lymph node metastases developed in the PTK787/ZK 222584-treated group, whereas after 21 days of treatment, the lymph node metastases were reduced by 87%. Vessel density in tumor tissues, detected by immunohistochemistry with an anti-CD31 antibody, was significantly decreased by PTK787/ZK 222584. Using color Doppler imaging ultrasound, significant changes in blood flow in the tumor feeding renal artery were found under treatment with PTK787/ZK 222584. Blood flow changes correlated with changes in vessel density but not with tumor volume. The compound was well tolerated in all in vivo experiments and had no significant effects on body weight or general well-being of the animals. This was in contrast to the animals treated with the antiangiogenic agent TNP-470. s.c. therapy with 30 mg/kg TNP-470 every other day had to be discontinued after 13 days because of animal weight loss (>20%) and ataxia. These results demonstrate that PTK787/ZK 222584 is a potent inhibitor of tumor growth, metastases formation, and tumor vascularization in murine renal cell carcinoma. Furthermore, we have been able to demonstrate that color Doppler imaging ultrasound can be used to measure blood flow to a tumor and that flow correlates with vessel density. Thus, this may be a valuable noninvasive method for monitoring the effects of antiangiogenic agents such as PTK787/ZK 222584 on tumor vasculature.

Expression of the vascular endothelial growth factor gene is inhibited by p73.

Recently, p73, a new member of the p53 family, has been cloned and mapped to chromosome 1p36, a region that is frequently deleted in a variety of human cancers. p73 can activate p53-responsive promoters and induce apoptosis when overexpressed in certain p53-deficient tumor cells. In contrast to p53, analysis of the p73 gene in several human solid tumors did not reveal loss of p73 expression or mutations in the p73 gene. However, transcriptional silencing of the p73 gene by hypermethylation of a CpG island was observed in several leukemias and lymphomas. These lymphoid neoplasms also show increased expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a key mediator of angiogenesis. To evaluate a possible relationship between p73 status and VEGF expression, we have studied the effect of ectopically expressed p73 on the regulation of the VEGF gene. Our results demonstrate that p73 can down-regulate endogenous VEGF gene expression on mRNA and protein level. This effect is mediated by transcriptional repression of the VEGF promoter and involves the promoter region -85 to -50 bp, containing a cluster of Sp 1 binding sites. Our results suggest a regulatory role for p73 in tumor angiogenesis. Oncogene (2000) 19, 3470 - 3476

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.

Sp1 recognition sites in the proximal promoter of the human vascular endothelial growth factor gene are essential for platelet-derived growth factor-induced gene expression.

Stimulation of NIH3T3 cells with platelet-derived growth factor (PDGF)-BB enhances expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a key mediator of tumor angiogenesis. Here, we identified cis-acting VEGF promoter elements and trans-acting factors which are involved in PDGF-stimulated VEGF expression. By 5'-deletion and transient transfection analysis, a G + C-rich region at -85 to -50 of the human VEGF promoter was shown to be necessary and sufficient for both PDGF inducible and basal expression. The region contains three potential recognition sites for Sp1 transcription factors, which overlap with two Egr-1 sites. Mutations that abolish the ability of Sp1 to interact with the VEGF promoter element also abrogate expression induced by PDGF. Mutations of the potential Egr-1 binding sites did not affect PDGF responsiveness. Gel shift and antibody supershift analyses showed that Sp1 and Sp3 interact constitutively with the VEGF promoter element. Our data strongly suggest that enhanced VEGF gene expression in PDGF-induced NIH3T3 cells is mediated by Sp1 and/or Sp3 transcription factors bound to the -85 to -50 promoter region of the VEGF gene.

Calmodulin-stimulated cyclic nucleotide phosphodiesterase from Neurospora crassa.

Cyclic nucleotide phosphodiesterase has been partially purified by calmodulin-Sepharose affinity chromatography from a soluble extract of Neurospora crassa. The phosphodiesterase activity remained bound to the affinity column even in the presence of 6 M urea and could only be eluted by calcium chelation. The enzyme exhibits cAMP and cGMP phosphodiesterase activities. Both activities can be enhanced by calmodulin in a Ca2+-dependent manner. Stimulation of cyclic nucleotide phosphodiesterase by calmodulin can be inhibited by calmodulin antagonists such as pimozide, trifluoperazine and chlorpromazine.

Characterisation of calmodulin from Drosophila heads.

Calmodulin from Drosophila heads has been purified to apparent electrophoretic homogeneity. It has the same characteristics as bovine brain calmodulin with respect to the migration upon polyacrylamide gel electrophoresis and maximal activation of a calmodulin-deficient cAMP phosphodiesterase. The amino acid composition resembles bovine brain calmodulin with the exception that trimethyllysine is absent and that it contains only one tyrosine. The tryptic peptide map of Drosophila calmodulin suggests some differences in the amino acid sequence as compared to bovine brain calmodulin. These proposed differences in the primary structure may explain why Drosophila calmodulin is less potent than bovine brain calmodulin in the activation of a cAMP phosphodiesterase from bovine brain.

Targeting of endothelial KDR receptors with 3G2 immunoliposomes in vitro.

Immunoliposomes (IL) containing anti-angiogenic drugs directed selectively to the easily accessible kinase insert domain containing receptor (KDR) vascular endothelial growth factor (VEGF), which is predominantly expressed on tumour vessels are a promising tool to inhibit tumour angiogenesis. To explore this strategy, we have prepared fluorescent-labelled IL presenting antibodies against the KDR receptor (3G2) on their surface. 3G2-IL were composed of egg phosphatidylcholine and cholesterol (6:4), containing 2 mol% of the new thiol reactive linker lipid O-(3-cholesteryloxycarbonyl)propionyl-O'-m-maleimido-benzoyl tetraethylene glycol. Specific binding of 3G2-IL to immobilised recombinant KDR was used to show the maintenance of sufficient immunoreactivity of 3G2 antibodies upon the coupling procedure. 3G2-IL bound to Chinese hamster ovarian (CHO) cells stably transfected to overexpress KDR to a five times higher amount as compared to mock-transfected CHO cells. Subsequently, specific binding of 3G2-IL to KDR could also be demonstrated on KDR expressing cells, human umbilical vein endothelial cells and human microvascular endothelial cells, whereas only low binding of 3G2-IL to NIH-3T3 mouse fibroblast cells, which do not express KDR, was found. The binding of 3G2-IL to KDR receptors could not be blocked by VEGF, suggesting that the binding site for VEGF is not identical with the epitope recognised by 3G2. We could demonstrate that 3G2-IL is able to bind in vitro even in the presence of high levels of VEGF.

Dihydropyridine binding of the calcium channel complex from skeletal muscle is modulated by subunit interaction.

The dihydropyridine-binding subunit alpha 1 of the calcium channel complex from rabbit skeletal muscle can be partially depleted from the alpha 2 delta beta-complex using wheat germ agglutinin-affinity chromatography. This depletion of the alpha 1 from the other subunits leads to a loss of dihydropyridine-binding, which can be fully reconstituted by repletion of the alpha 1 with the other subunits. Reassembly of these subunits results in an increase in the Kd and Bmax of the dihydropyridine-binding indicating that the non-dihydropyridine-binding subunits influence dihydropyridine-binding. The affinity of the alpha 1 subunit for the other subunits was determined to be approximately 35 nM. Since the free alpha 1 subunit will not bind to the beta subunit alone, there is evidence, given the selective partitioning of the beta subunit to the lectin-bound subunit pool, that either beta binds with higher affinity to the alpha 2 delta-complex than to the free alpha 1 subunit or that the bound alpha 1 creates or modulates beta-binding. This indicates a functional high affinity interaction between the dihydropyridine-binding alpha 1 subunit and the alpha 2 delta beta-complex.

Inducible overexpression of human protein kinase C alpha in NIH 3T3 fibroblasts results in growth abnormalities.

We have stably overexpressed the human protein kinase C alpha (hPKC alpha) in NIH 3T3 fibroblasts under the control of the interferon (IFN) type I inducible murine Mx promoter. These cells showed a 10-fold increase in the transcription of hPKC alpha mRNA after induction with interferon alpha. The increase in the amount and activity of protein kinase C (PKC)-protein in these cells was only about 3-fold after induction with interferon alpha. Compared to control cells which were transfected with the vector only, the NIH 3T3 fibroblasts transfected with the hPKC alpha cDNA showed already a slightly increased PKC-activity and amount of PKC-protein in the absence of interferon alpha. The hPKC alpha overexpressing cells had an altered, "transformed-like" morphology, which was reversed by staurosporine, an increased growth rate and a higher saturation density. The growth rate was further increased by treating the cells with interferon alpha. The hPKC alpha overexpressing cells were able to grow in soft agarose after treatment with phorbol ester such as TPA (12-O-tetradecanoylphorbol 13-acetate). After phorbol ester and interferon treatment a stronger expression of the protooncogene c-jun was detectable in the hPKC alpha overexpressing cells, whereas expression of c-fos and c-myc was not affected. Since these cells show a specific response pattern due to induced PKC alpha expression they might be useful as an assay system for the development of PKC isozyme-specific inhibitors and activators.

VEGF-mediated tumour angiogenesis: a new target for cancer therapy.

Considerable evidence is gathering for the involvement of vascular endothelial growth factor (VEGF) in the vascularization and growth of primary tumours as well as in the formation of metastases. The expression of VEGF depends on activated oncogenes and inactivated tumour suppressor genes as well as several other factors (e.g. growth factors, tumour promoters and hypoxia). Substantial expression of the receptors for VEGF is restricted mainly to the tumour blood vessels. The causal involvement of this angiogenic factor in the progression of disease has been successfully evaluated by means of monoclonal antibodies against VEGF, dominant-negative receptor mutants and the use of antisense oligonucleotides against the VEGF mRNA. Thus, the VEGF signalling system seems to be an appropriate target to inhibit tumour angiogenesis and metastases formation.

Soluble Tie2 and Flt1 extracellular domains in serum of patients with renal cancer and response to antiangiogenic therapy.

Antiangiogenesis drugs can be difficult to evaluate because they produce disease stabilization rather than tumor regression. Markers of endothelial mass in tumors may be of value to monitor therapy and evaluate such drugs. Soluble domains of the endothelial receptor tyrosine kinases, sTie2 (angiopoietin receptor) and sFlt1 (vascular endothelial growth factor receptor-1) were analyzed by sandwich ELISA in serum samples from 43 patients with advanced renal cancer before and 1 month after antiangiogenic therapy with razoxane. Pretreatment sFlt1 levels were 0.77 ng/ml +/- 0.48 (SD) and sTie2 74.3 ng/ml +/- 15 (SD). Pretreatment sFlt1 levels above the median were associated with a lesser chance of stable disease (P = 0.04) and poorer survival (P = 0.01). Fall of sTie2 on treatment was associated with stable disease (P = 0.05) and improved survival (P = 0.04). The soluble receptors measured weeks before response were assessed and correlated with response and survival, showing they may be useful to monitor and develop antiangiogenic therapy.

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.

Pertussis toxin inhibits the angiotensin II and serotonin-induced rise of free cytoplasmic calcium in cultured smooth muscle cells from rat aorta.

Angiotensin II, serotonin and K+-depolarization cause an increase in free cytoplasmic Ca2+ in cultured smooth muscle cells. The involvement of a guanine nucleotide-binding protein has been investigated by using pertussis toxin. When smooth muscle cells were pretreated with pertussis toxin angiotensin II and serotonin-induced rise of cytosolic Ca2+ was found to be significantly reduced whereas the Ca2+ influx mediated by K+-depolarization remained unchanged. These results suggest the participation of a guanine nucleotide-binding protein in the receptor-mediated rise of intracellular Ca2+.

Synthetic diacylglycerols induce a rise of quin2-detectable free intracellular calcium in human platelets.

The two activators of protein kinase C, oleoylacetylglycerol (OAG) and dioctanoylglycerol (DOG), are able to induce a concentration-dependent rise in cytoplasmic free Ca2+ concentration in gel-filtered human platelets, detected as an increase in quin2 fluorescence. The phorbol ester phorbol-12-myristate-13-acetate (PMA) has no effect. The OAG-induced increase of intracellular Ca2+ is not influenced by forskolin, in contrast to the effect of the diterpene on the thrombin-stimulated increase in cytoplasmic Ca2+. It is concluded that the increase in intracellular free Ca2+ concentration induced by synthetic diacylglycerols and their activation of protein kinase C are two different and independent processes.

High-efficiency expression of rat protein kinase C-gamma in baculovirus-infected insect cells.

We have expressed rat protein kinase C-gamma in insect cells using a baculovirus vector. The yield of expressed protein kinase C-gamma is about 4% of total protein. The recombinant protein shows a prominent band at about 80 kDa on SDS-polyacrylamide gels, which can be identified as protein kinase C-gamma by Western blotting using monoclonal antibodies against protein kinase C-gamma. Upon incubation with [gamma-32P]ATP and in the presence of Ca2+, phosphatidylserine and diacylglycerol this protein autophosphorylates. Its enzyme activity shows the characteristic properties of mammalian protein kinase C.

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.