Showing papers on "Tyrosine-kinase inhibitor published in 1998"

Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain.

Abstract: Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is an essential physiological process in development, yet also plays a major role in the progression of human diseases such as diabetic retinopathy, atherosclerosis and cancer. The effects of the most potent angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin and fibroblast growth factor (FGF) are mediated through cell surface receptors that possess intrinsic protein tyrosine kinase activity. In this report, we describe a synthetic compound of the pyrido[2,3-d]pyrimidine class, designated PD 173074, that selectively inhibits the tyrosine kinase activities of the FGF and VEGF receptors. We show that systemic administration of PD 173074 in mice can effectively block angiogenesis induced by either FGF or VEGF with no apparent toxicity. To elucidate the determinants of selectivity, we have determined the crystal structure of PD 173074 in complex with the tyrosine kinase domain of FGF receptor 1 at 2.5 A resolution. A high degree of surface complementarity between PD 173074 and the hydrophobic, ATP-binding pocket of FGF receptor 1 underlies the potency and selectivity of this inhibitor. PD 173074 is thus a promising candidate for a therapeutic angiogenesis inhibitor to be used in the treatment of cancer and other diseases whose progression is dependent upon new blood vessel formation.

Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor

Abstract: A class of high-affinity inhibitors is disclosed that selectively target and irreversibly inactivate the epidermal growth factor receptor tyrosine kinase through specific, covalent modification of a cysteine residue present in the ATP binding pocket. A series of experiments employing MS, molecular modeling, site-directed mutagenesis, and 14C-labeling studies in viable cells unequivocally demonstrate that these compounds selectively bind to the catalytic domain of the epidermal growth factor receptor with a 1:1 stoichiometry and alkylate Cys-773. While the compounds are essentially nonreactive in solution, they are subject to rapid nucleophilic attack by this particular amino acid when bound in the ATP pocket. The molecular orientation and positioning of the acrylamide group in these inhibitors in relation to Cys-773 entirely support these results as determined from docking experiments in a homology-built molecular model of the ATP site. Evidence is also presented to indicate that the compounds interact in an analogous fashion with erbB2 but have no activity against the other receptor tyrosine kinases or intracellular tyrosine kinases that were tested in this study. Finally, a direct comparison between 6-acrylamido-4-anilinoquinazoline and an equally potent but reversible analog shows that the irreversible inhibitor has far superior in vivo antitumor activity in a human epidermoid carcinoma xenograft model with no overt toxicity at therapeutically active doses. The activity profile for this compound is prototypical of a generation of tyrosine kinase inhibitors with great promise for therapeutic significance in the treatment of proliferative disease.

Ca2+-Independent Activation of the Endothelial Nitric Oxide Synthase in Response to Tyrosine Phosphatase Inhibitors and Fluid Shear Stress

Abstract: Fluid shear stress enhances NO formation via a Ca2+-independent tyrosine kinase inhibitor-sensitive pathway. In the present study, we investigated the effects of the protein tyrosine phosphatase inhibitor phenylarsine oxide and of fluid shear stress on endothelial NO production as well as on the membrane association and phosphorylation of the NO synthase (NOS) III. Phenylarsine oxide (10 micromol/L) induced an immediate and maintained NO-mediated relaxation of isolated rabbit carotid arteries, which was insensitive to the removal of extracellular Ca2+ and the calmodulin antagonist calmidazolium. This phenylarsine oxide-induced vasodilatation was unaffected by genistein but abrogated by the tyrosine kinase inhibitor erbstatin A. Incubation of native or cultured endothelial cells with phenylarsine oxide resulted in a time-dependent tyrosine phosphorylation of mainly Triton X-100-insoluble (cytoskeletal) proteins, along with a parallel change in the detergent solubility of NOS III, such that the enzyme was recovered in the cytoskeletal fraction. A similar, though slightly delayed, phenomenon was also observed after the application of fluid shear stress but not in response to any receptor-dependent agonist. Although Ca2+-independent NO formation was sensitive to erbstatin A, phenylarsine oxide treatment was associated with the tyrosine dephosphorylation of NOS III rather than its hyperphosphorylation. Proteins that also underwent redistribution in response to the tyrosine phosphatase inhibitor included paxillin, phospholipase C-gamma1, mitogen-activated protein kinase, and the tyrosine kinases Src and Fyn. We envisage that fluid shear stress and tyrosine phosphatase inhibitors may alter the conformation and/or protein coupling of NOS III, facilitating its interaction with specific phospholipids, proteins, and/or protein kinases that enhance/maintain its Ca2+-independent activation.

Tyrosine Kinase-Dependent Activation of a Chloride Channel in CD95-Induced Apoptosis in T Lymphocytes

Abstract: CD95/Fas/APO-1 mediated apoptosis is an important mechanism in the regulation of the immune response. Here, we show that CD95 receptor triggering activates an outwardly rectifying chloride channel (ORCC) in Jurkat T lymphocytes. Ceramide, a lipid metabolite synthesized upon CD95 receptor triggering, also induces activation of ORCC in cell-attached patch clamp experiments. Activation is mediated by Src-like tyrosine kinases, because it is abolished by the tyrosine kinase inhibitor herbimycin A or by genetic deficiency of p56lck. In vitro incubation of excised patches with purified p56lck results in activation of ORCC, which is partially reversed upon addition of anti-phosphotyrosine antibody. Inhibition of ORCC by four different drugs correlates with a 30–65% inhibition of apoptosis. Intracellular acidification observed upon CD95 triggering is abolished by inhibition of either ORCC or p56lck. The results suggest that tyrosine kinase-mediated activation of ORCC may play a role in CD95-induced cell death in T lymphocytes.

Selective inhibition of cell proliferation and BCR-ABL phosphorylation in acute lymphoblastic leukemia cells expressing Mr 190,000 BCR-ABL protein by a tyrosine kinase inhibitor (CGP-57148).

Abstract: The excessive proliferation of the myeloid marrow compartment in Philadelphia chromosome (Ph)-positive acute and chronic leukemias has been largely attributed to a hyperactive and autonomously acting hybrid tyrosine kinase BCR-ABL, a product of the fusion between the second exon of the c-ABL proto-oncogene and 5' portions of the BCR gene on chromosome 22. This specific molecular event, amenable to attack with specifically designed inhibitors, has recently been successfully influenced by the drug CGP-57148 in mammalian cells transfected with full-length BCR-ABL gene and expressing full-length p210Bcr-Abl protein, as well as in primary human leukemic cells expressing p210Bcr-Abl fusion protein. In view of the heterogeneity of BCR-ABL transcripts associated with various phenotypes, we investigated the effect of CGP-57148 on p190Bcr-Abl- and p210Bcr-Abl-expressing, patient-derived cell lines and primary intact blast cells. In particular, we were interested in whether the variations in molecular events and/or the phenotype of Ph-positive cells would affect their susceptibility to the specific tyrosine kinase inhibitor CGP-57148. We have demonstrated that the sensitivity of human cells with lymphoblastic immunophenotype expressing p190Bcr-Abl protein is comparable to that for leukemic myeloid cells expressing p210Bcr-Abl protein. After documenting profound and phenotype-independent suppression of both autophosphorylation and cell growth, we explored the importance of time and dose of exposure on the manifestation and stability of the induced events. Although there were variations between target cells, in vitro exposure for 24-48 h induced extensive and apparently irreversible apoptosis in BCR-ABL-expressing but not other normal or BCR-ABL-negative leukemic cells. These findings support the potential use of CGP-57148 to purge Ph-positive cells from autologous bone marrow in vitro. Another important finding was the comparable suppressive effect of temporary CGP-57148 exposure on both clonogenic KBM-5 cells and the whole cell population. Exposure time and dose appeared to be important variables among various cell types. Moreover, effective doses appeared uniformly harmless to cells lacking BCR-ABL protein functioning as tyrosine kinase. Thus, the continuous exposure of target cells, at least during the initial period of 24-48 h, may prove to be an important variable in the design of in vitro and in vivo therapy using tyrosine kinase inhibitors.

Inhibition of JAK3 and STAT6 Tyrosine Phosphorylation by the Immunosuppressive Drug Leflunomide Leads to a Block in IgG1 Production

Abstract: Leflunomide is an immunosuppressive drug capable of inhibiting T and B cell responses in vivo. A number of studies demonstrate that leflunomide functions both as a pyrimidine synthesis inhibitor and as a tyrosine kinase inhibitor. We previously reported that leflunomide inhibits LPS-stimulated B cell proliferation, cell cycle progression, and IgM secretion. This inhibition can be reversed by the addition of exogenous uridine, suggesting that leflunomide functions as a pyrimidine synthesis inhibitor in B cells. We report here that while the addition of uridine restored proliferation and IgM secretion to leflunomide-treated LPS-stimulated B cells, as determined by metabolic labeling and immunoprecipitation, it did not completely restore secretion of IgG Ab. We hypothesized that leflunomide inhibits LPS-induced IgG secretion by inhibiting tyrosine kinase activity required for isotype switch. We tested this hypothesis in a well-defined model of isotype switch, LPS plus IL-4 induction of IgG1. Leflunomide inhibited IgG1 secretion in this model in a dose-dependent manner. The signal transduction pathway utilized by IL-4 to induce IgG1 involves tyrosine phosphorylation of the IL-4 receptor, JAK1, JAK3, and STAT6 proteins induced by IL-4 binding to the IL-4R. Leflunomide diminished the tyrosine phosphorylation of JAK3 and STAT6 in the absence or presence of uridine. In gel mobility shift studies, STAT6 binding to the STAT6 DNA binding site in the IgG1 promoter decreased in the presence of leflunomide or leflunomide plus uridine. Taken together, these data suggest that leflunomide acts as a tyrosine kinase inhibitor to block IgG1 production.

Activation of mitogen-activated protein kinase cascades during priming of human neutrophils by TNF-alpha and GM-CSF.

Abstract: The signal transduction pathways activated by tumor necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) that lead to priming of polymorphonuclear leukocytes (PMNs) are unknown. The hypotheses that these cytokines stimulate multiple mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), and p38 MAPK, and that these MAPKs participate in priming of human PMNs were examined. TNF-alpha stimulated a dose-dependent increase in ERK and p38 MAPK activities that was maximal at 10 min. JNKs were not stimulated by TNF-alpha or GM-CSF. GM-CSF stimulated ERK activity comparable to that of TNF-alpha, but GM-CSF was a less potent stimulus of p38 MAPK activity. The tyrosine kinase inhibitor, genistein, inhibited ERK and p38 MAPK stimulation by both cytokines. The phosphatidylinositol 3-kinase inhibitor, wortmannin, attenuated stimulation of ERKs and p38 MAPK by GM-CSF, but not TNF-alpha. GM-CSF, but not TNF-alpha, stimulated wortmannin-sensitive activation of Raf-1. TNF-alpha and GM-CSF priming of superoxide release stimulated by N-formyl-methionyl-leucyl-phenylalanine was significantly attenuated by the MEK inhibitor, PD098059, and the p38 MAPK inhibitor, SB203580. Incubation with both MAPK inhibitors produced an additive effect. Our data suggest that TNF-alpha and GM-CSF activate ERKs and p38 MAPK by different signal transduction pathways. Both ERK and p38 MAPK cascades contribute to the ability of TNF-alpha and GM-CSF to prime the respiratory burst response in human PMNs.

Cell cycle-independent death of prostate adenocarcinoma is induced by the trk tyrosine kinase inhibitor CEP-751 (KT6587).

Abstract: Advanced prostate cancer remains largely incurable, primarily because the very low growth fraction present in these tumors makes them generally resistant to treatment with standard chemotherapeutic agents that target cell division. Effective therapies should therefore induce death of prostate cancer cells, independent of their growth rate. trkA, the high-affinity tyrosine kinase-linked receptor for nerve growth factor, has been implicated in prostatic cancer growth and may represent a molecular target for therapeutic agents. At low mg/kg doses, the trk tyrosine kinase inhibitor CEP-751 (KT6587) inhibits prostatic cancer growth in nine different animal models independent of the tumor growth rate, androgen sensitivity, metastatic ability, or state of tumor differentiation. CEP-751 is selective for cancerous versus normal prostate cells and affects the growth of only a limited number of nonprostate tumors. Importantly, CEP-751 induces cell death of prostate cancer cells in a cell cycle-independent fashion and, therefore, represents a novel therapeutic approach to the management of both hormone-dependent and hormone-independent prostate cancer.

EGFR blockade by tyrosine kinase inhibitor or monoclonal antibody inhibits growth, directs terminal differentiation and induces apoptosis in the human squamous cell carcinoma HN5.

Abstract: Human squamous cell carcinomas frequently overexpress the epidermal growth factor receptor (EGFR) and this is often associated with poor prognosis in patients with these cancers. The high level of expression of the EGFR provides an important target for therapy and we and others have shown that monoclonal antibodies (mAbs) which block the activation of the receptor by the EGF family of ligands inhibit the growth of EGFR overexpressing tumours in vitro and induce the regression of established tumours grown as xenografts in athymic mice. Inhibitors of the tyrosine kinase associated with the EGFR have also been shown to block receptor activation and prevent tumour cell proliferation. Using the EGFR-overexpressing head and neck carcinoma cell line HN5, we have compared the biological consequences of treatment with an inhibitor of EGFR tyrosine kinase (PD153035) with anti-EGFR monoclonal antibodies (mAbs) ICR63 or ICR80. We found that both the anti-EGFR mAbs and the TK inhibitor produce similar biological changes namely, they inhibit the EGF and TGFa-induced tyrosine phosphorylation of the receptor and the growth in culture of HN5 cells. At concentrations above 100 nM, the TK inhibitor prevented the growth in culture of HN5 cells completely with an IC50 of 40 nM. With the anti-EGFR mAbs, growth of HN5 cells was inhibited completely at concentrations above 4 nM with an IC50 of 1 nM. More importantly we found that, like the anti-EGFR mAbs, treatment with the TK inhibitor directs HN5 cells to undergo terminal differentiation as monitored by the expression of cytokeratin 10. In addition, our results indicate that the growth inhibitory effects of the anti-EGFR agents also lead to induction of apoptosis as determined by 7-amino actinomycin D staining (7-AAD). We conclude that EGFR blockade by anti-EGFR mAbs or TK inhibitor influences the growth in culture of EGFR overexpressing tumours by directing terminal differentiation and inducing apoptosis.

Identification of the FcϵRI-activated tyrosine kinases Lyn, Syk, and Zap-70 in human basophils

Abstract: Background: In human blood basophils, cross-linking the high-affinity IgE receptor FcϵRI with multivalent antigen activates a signaling pathway leading to Ca 2+ mobilization, actin polymerization, shape changes, secretion, and cytokine production. Methods and Results: The role of tyrosine kinases in human FcϵRI signaling was explored by using human basophils isolated by Percoll gradient centrifugation followed by negative and/or positive selection with antibody-coated magnetic beads. FcϵRI cross-linking of more than 95% pure basophil preparations activates the protein-tyrosine kinases Lyn and Syk, previously linked to FcϵRI-coupled rodent mast cell activation, as well as Zap-70, previously implicated in T-cell receptor signaling, and causes the tyrosine phosphorylation of multiple proteins. The presence of Lyn, Syk, and Zap-70 in basophils was confirmed by Western blotting in lysates of highly purified basophils and independently by confocal fluorescence microscopy in cells labeled simultaneously with kinase-specific antibodies and with the basophil-specific antibody 2D7. Comparable amounts of Lyn and Syk were found in basophils and B cells, whereas T cells appear to have greater amounts of Zap-70 than basophils. The tyrosine kinase inhibitor piceatannol spares IgE-mediated Lyn activation but inhibits IgE-induced Syk and Zap-70 activation as well as overall protein tyrosine phosphorylation and secretion. Overall protein-tyrosine phosphorylation increases steadily over a range of anti-IgE concentrations that are low to optimal for secretion. However, tyrosine phosphorylation continues to increase at high anti-IgE concentrations that elicit very little secretion (the characteristic high-dose inhibition of secretion). Conclusions: Our data demonstrate the association of anti-IgE–stimulated, protein-tyrosine phosphorylation by a cascade of tyrosine kinases, including Zap-70 as well as Lyn and Syk, with the initiation of FcϵRI-mediated signaling in human basophils. (J Allergy Clin Immunol 1998;102:304-15.)

Induction of tyrosine phosphorylation in human MHC class II-positive antigen-presenting cells by stimulation with contact sensitizers.

Abstract: To investigate the intracellular signaling mechanisms involved in the activation of APC by contact sensitizers, we studied the induction of tyrosine phosphorylation by these agents. Selective analysis of phosphotyrosine (p-tyr) in human Langerhans cells and different mononuclear cell types was achieved using a multicolor flow-cytometric technique. Stimulation with contact sensitizers revealed a distinct increase in p-tyr exclusively for MHC class II-positive cells. For different haptens, irritants, as well as activators of distinct signal transduction pathways, it was demonstrated that only strong sensitizers or the protein tyrosine phosphatase inhibitor sodium orthovanadate or cross-linking of MHC class II molecules were able to induce formation of p-tyr in human blood-derived dendritic cells serving as model for the dendritic cell family. This event required physiologic cell culture conditions and was blocked by specific inhibitors of protein tyrosine kinases. No evidence for the inhibition of protein tyrosine phosphatases by haptens was found. Western blot analysis of monocyte-enriched populations revealed an augmented phosphorylation of distinct proteins after hapten stimulation partly resembling the pattern noticed after cross-linking of HLA-DR molecules. In dendritic cells generated from mononuclear progenitors, the protein tyrosine kinase inhibitor genistein was able to block tyrosine phosphorylation as well as production of IL-1β mRNA transcripts. Our data underline the unique capacity of haptens to activate APC and the important role of tyrosine phosphorylation for this process.

Mechanisms of Macrophage Stimulation Through CD8: Macrophage CD8α and CD8β Induce Nitric Oxide Production and Associated Killing of the Parasite Leishmania major

Abstract: Prior studies demonstrated that rat macrophages express CD8, which differs from T lymphocyte CD8 within the ligand binding domain. We investigated whether stimulation of macrophage CD8 could induce mediator release and regulate host defense. Cross-linking either CD8α (OX8, 5 μg/ml) or CD8β (341, 10 μg/ml) stimulated nitric oxide (NO) production, which correlated with an up-regulation of inducible NO synthase protein. Cell signaling inhibitors were used to elucidate the pathways of CD8α and CD8β stimulation. Genistein (broad spectrum protein tyrosine kinase inhibitor, 10 μg/ml), PP1 ( src family kinase inhibitor, 5 μg/ml), polymyxin B (protein kinase C (PKC) inhibitor, 100 μg/ml), and Ro 31-8220 (PKC inhibitor, 1 μM) significantly inhibited anti-CD8α- and anti-CD8β-stimulated NO production and inducible NO synthase up-regulation, suggesting that tyrosine kinase(s) ( src family) and PKC are involved in CD8 signaling. In addition, cross-linking CD8α stimulated NO-dependent macrophage killing of the parasite Leishmania major . For the first time, this work demonstrates that the β-chain of macrophage CD8, in addition to the α-chain, can regulate mediator release. These results further illustrate the importance of this molecule and support our previous data demonstrating differences between macrophage and T lymphocyte CD8. Additional studies on the signaling mechanisms and possible ligand(s) for macrophage CD8 will lead to a greater understanding of inflammation and host defense.

Tyrphostin AG490, a tyrosine kinase inhibitor, blocks actively induced experimental autoimmune encephalomyelitis

Abstract: Migration of lymphocytes from blood into the brain is a critical event in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Previous observations made in our laboratory showed that protein tyrosine kinase inhibitors were able to block lymphocyte adhesion to brain endothelium and prevent the entry of encephalitogenic T cell lines into the brain of SJL/J mice. Here we show that systemic administration of the protein tyrosine kinase inhibitor, tyrphostin AG490, blocks the development of actively induced EAE in a dose-dependent manner. Administration of 1 mg of drug daily significantly decreased the severity of the disease, while 3 mg of AG490 daily totally blocked the disease in 62% of treated animals, and in those that developed the disease, paralysis was delayed and clinical score was significantly reduced. Blood leukocytes isolated from mice treated with tyrphostin AG490 were less adhesive on VCAM-1 and fibronectin, when compared with control animals. AG490 treatment had no effect on the proliferation by antigen-stimulated peripheral lymph nodes cells. Interestingly, cells obtained from draining lymph nodes in AG490-treated animals and stimulated with antigen secreted two times more IFN-gamma and four times more IL-10, when compared with control animals, whereas no difference was observed in TNF-alpha production. Our results suggest that tyrphostin AG490 may have therapeutic potential by blocking tyrosine kinase activities involved in key mechanisms leading to demyelinating diseases of the central nervous system.

IkappaBalpha degradation is not a requirement for the X-ray-induced activation of nuclear factor kappaB in normal rat astrocytes and human brain tumour cells.

Abstract: Purpose: To investigate the mechanism of NF kappa B activation by X-rays in normal primary rat astrocytes. Materials and methods: Primary cultures of type I astrocytes generated from the cortex of neonatal rats were exposed to X-rays with and without various kinase inhibitors and a protease inhibitor. The nuclear or cytoplasmic protein extracts were collected at specified times after treatment and analysed for NF kappa B-DNA binding activity and I kappa B protein levels. Results: The NF kappa B-DNA binding activity was induced by X-rays in a dose- and time-dependent manner in the absence of I kappa B protein degradation in astrocytes as well as in the human glioma cell line U-373MG. Whereas a protease inhibitor (calpain inhibitor 1) and a protein kinase C inhibitor (CGP-41251) did not affect X-ray-induced NF kappa B-DNA binding, treatment of astrocytes with the tyrosine kinase inhibitor (erbstatin) completely prevented the increase in NF kappa B activity after irradiation. Erbstatin also reduced the phosp...

Combination of tyrosine kinase inhibitor and chemical castration to treat prostate cancer

Abstract: A method of treating prostate cancer by coadministering a tyrosine kinase inhibitor such as an indolocarbazole and a chemical castration agent is disclosed. A composition containing a tyrosine kinase inhibitor and a chemical castration agent is also disclosed.

Tyrphostin AG957, a tyrosine kinase inhibitor with anti-BCR/ABL tyrosine kinase activity restores beta1 integrin-mediated adhesion and inhibitory signaling in chronic myelogenous leukemia hematopoietic progenitors.

Abstract: Abnormal beta1 integrin receptor function may contribute to the continuous proliferation and abnormal circulation of malignant hematopoietic progenitors in chronic myelogenous leukemia (CML). Previous studies suggest that abnormal integrin function in CML progenitors is related to the presence of the BCR/ABL oncogene. BCR/ABL may alter integrin function in CML by phosphorylating cytoskeletal and/or signaling proteins important for normal integrin function. We evaluated the effect of Tyrphostin AG957, a protein tyrosine kinase (PTK) inhibitor which has activity against the p210BCR/ABL kinase, on beta1 integrin function in CML progenitors. Incubation of CML marrow CD34+HLA-DR+ cells with Tyrphostin AG957 at concentrations that did not affect colony-forming cells (CFC) viability, but which partly inhibited p210BCR/ABL kinase activity, significantly increased CML CFC adhesion to stroma and alpha4beta1 and alpha5beta1 integrin binding fragments of fibronectin (FN). CML CFC proliferation, unlike that of normal CFC, is not inhibited following integrin receptor engagement with FN or anti-integrin antibodies. AG957 did not alter CML CFC proliferation by itself, but resulted in significant inhibition of CML CFC proliferation following integrin engagement. Another PTK inhibitor, Tyrphostin AG555, which does not have anti-p210BCR/ABL kinase activity, did not affect CML CFC adhesion or proliferation. Neither AG957 nor AG555 affected normal CFC adhesion or proliferation. In BCR/ABL expressing cells, AG957 partially inhibited phosphorylation of several proteins that are BCR/ABL PTK substrates and are involved in normal integrin signaling. These studies suggest that abnormal tyrosine phosphorylation may play an important role in defective integrin function in CML progenitors.

Priming of Platelet αIIbβ3 by Oxidants Is Associated With Tyrosine Phosphorylation of β3

Abstract: Reactive oxygen species play an important role at the site of vascular injuries and arterial thromboses. We studied the mechanism mediating platelet aggregation induced by H2O2, a major cellular oxidant. Exposure to H2O2 triggered platelet aggregation, but only when the platelets were stirred. Strong platelet aggregation induced99032416 required the presence of the tyrosine phosphatase inhibitor sodium orthovanadate (NaVO4) and was dependent on the participation of integrin alphaIIbbeta3 (glycoprotein IIb-IIIa). A specific inhibitor of alphaIIbbeta3 blocked platelet aggregation induced by H2O2 and NaVO4, thus confirming that aggregation requires this receptor. In the presence of H2O2 and NaVO4, multiple platelet substrates were phosphorylated on tyrosine. Such tyrosine kinase response was necessary but not sufficient to activate alphaIIbbeta3, as detected by binding of soluble fibrinogen to platelets. Stirring of the platelets exposed to H2O2 and NaVO4 was also needed to allow for binding of fibrinogen to alphaIIbbeta3. The tyrosine kinase inhibitor genistein was able to block platelet aggregation induced by H2O2 and NaVO4, thus confirming that tyrosine kinase activity was needed to trigger alphaIIbbeta3 activation on stirring. N-Acetyl-L-cysteine, a cell-permeant antioxidant, blocked the tyrosine phosphorylation of platelet substrates and also the platelet aggregation induced by H2O2 and NaVO4. We found that beta3 was phosphorylated on tyrosine in platelets exposed to H2O2 and NaVO4, even in the absence of aggregation. Hence, tyrosine phosphorylation of beta3 might contribute to the "priming" of alphaIIbbeta3 induced by H2O2 and NaVO4, whereby the receptor can become activated on stirring of the platelets.

Constitutively tyrosine phosphorylated p52 Shc in breast cancer cells: correlation with ErbB2 and p66 Shc expression

Abstract: Breast cancer cell lines display a wide variety of growth factor receptors, and considerable evidence implicates signaling from these receptors, especially ErbB2, in the important early stages of this disease, contributing to malignant progression. If this is true, then we would hypothesize that a useful prognostic indicator would be the level of activity of a second messenger protein used in common by these receptors. One such second messenger is the Shc adapter protein, which is activated when tyrosine phosphorylated by receptors. Therefore, one prediction from the hypothesis is that the level of tyrosine-phosphorylated Shc (PY-Shc) in breast cancer cell lines would correlate with total receptor tyrosine kinase activity. To begin to test this prediction, we examined Shc tyrosine phosphorylation in a diverse group of breast cancer cell lines that display varied levels of ErbB2. Using Shc immunoprecipitation and anti-phosphotyrosine immunoblotting analysis, we found a strong correlation between the level of ErbB2 overexpression (r = 0.91, p < 0.0002) and PY-ErbB2 levels (r = 0.89, p = 0.0005) compared with the level of tyrosine phosphorylation of the p52 and p46 Shc isoforms. Consistent with Shc tyrosine phosphorylation being driven by ErbB2, an ErbB2-specific tyrosine kinase inhibitor markedly reduced Shc tyrosine phosphorylation. Unexpectedly, although all cell lines had comparable total amounts of p52 and p46 Shc, the amount of an inhibitory Shc isoform, p66, was inversely related to the level of ErbB2 expression (r = -0.86, p = 0.0013). This suggests that reduced p66 Shc expression may play a role in ErbB2-positive breast cancer. In summary, these data are consistent with our prediction that the cellular level of PY-Shc would correlate with the levels of activated ErbB2 displayed by cell lines derived from breast cancers.

Lipopolysaccharide induction of monocyte matrix metalloproteinases is regulated by the tyrosine phosphorylation of cytosolic phospholipase A2.

Abstract: Activation of human monocytes with lipopolysaccharide (LPS) results in the production of matrix metalloproteinases (MMPs) through a prostaglandin E2 (PGE2)-cAMP-dependent pathway. In this study, the early signaling events involved in this signal transduction pathway were evaluated. Pretreatment of human peripheral blood monocytes with herbimycin A, a tyrosine kinase inhibitor, or arachidonyl trifluoromethyl ketone (AACOCF3), a specific inhibitor of cytosolic phospholipase A2 (cPLA2) inhibited the induction of PGE2 by LPS. This resulted in the inhibition of protein expression of gelatinase B (MMP-9) and interstitial collagenase (MMP-1), two major MMPs secreted by activated monocytes. Addition of arachidonic acid (AA) reversed the inhibitory effect of herbimycin A or AACOCF3 on monocyte MMP production, indicating the importance of tyrosine phosphorylation and the involvement of cPLA2 at an early stage in the signal transduction pathway of MMPs. This finding was further supported by LPS-induced shift in cPLA2 migration and tyrosine phosphorylation based on immunoblotting and immunoprecipitation studies. These results provide evidence that tyrosine phosphorylation of cPLA2 is one of the initial steps needed for the LPS induced MMP production in human monocytes.

EGF-isoflavone conjugates for the prevention of restenosis

Abstract: A protein conjugate containing EGF coupled to a tyrosine kinase inhibitor such as Genistein, for inhibiting or preventing restenosis following vascular injury.

A potent protein-tyrosine kinase inhibitor which selectively blocks proliferation of epidermal growth factor receptor-expressing tumor cells in vitro and in vivo.

Abstract: A calculated 3-D model of the kinase domain of the epidermal growth factor receptor (EGF-R) protein-tyrosine kinase (PTK) was used to develop a pharmacophore model for ATP-competitive inhibitors and, subsequently, a new class of selective EGF-R kinase inhibitors. CGP 59326A, a highly selective and potent inhibitor of the EGF-R in vitro, inhibited the proliferation of EGF-R-expressing epithelial lines, while having little anti-proliferative activity against EGF-R-negative lines. In contrast to previously described inhibitors, CGP 59326A had potent and selective in vivo anti-tumor activity at well-tolerated doses against EGF-R-expressing tumors (e.g., ED50 of 0.78 to 1.5 mg/kg for inhibition of A431 tumor growth). CGP 59326A inhibited growth of human tumor xenografts expressing the EGF-R but showed little activity against EGF-R-negative xenografts. Combination of CGP 59326A with cytotoxic agents resulted in tumor regression and cures. The high selectivity and attractive biological profile of CGP 59326A suggest that it could have therapeutic value in the treatment of proliferative diseases which involve mitogenic signaling from the EGF-R.

Endogenous insulin-like growth factor-I is obligatory for stimulation of rat inhibin alpha-subunit expression by follicle-stimulating hormone.

Abstract: Insulin-like growth factor-I (IGF-I) is essential for FSH-dependent steroidogenesis by rat granulosa cells (GC), but whether IGF-I is required for other FSH-dependent functions is unknown. To investigate the role of IGF-I in the mechanisms of FSH-stimulated inhibin alpha-subunit (Inh-alpha) production, rat GC were cultured with FSH, IGF-I, insulin-like growth factor-binding protein (IGFBP)-4, IGFBP-5, and/or anti-IGF-I antibody. Inh-alpha protein and mRNA levels were measured in conditioned medium and cells by Western immunoblotting and Northern analysis, respectively. Inh-alpha expression was increased by FSH (3.5-fold) and IGF-I (2.5-fold), and the effects were dose and time dependent. FSH stimulation of Inh-alpha was attenuated by IGFBP-4 or -5 in a dose-dependent fashion, and the effects were reversed by IGF-I. Anti-IGF-I antibody mimicked the inhibitory effects of IGFBP-4 and -5. Forskolin, cholera toxin, and 8-bromo-cAMP increased Inh-alpha production approximately 3.5-fold, and the effects were blocked by IGFBP-4 or -5. Increases in Inh-alpha by FSH, IGF-I, forskolin, cholera toxin, and 8-bromo-cAMP were totally blocked by the protein tyrosine kinase inhibitor, tyrphostin A23. In summary, these results suggest that the stimulation of Inh-alpha expression by FSH requires activation of protein tyrosine kinases by endogenously produced IGF-I. We propose that the IGF-I signaling is obligatory for FSH stimulation of Inh-alpha expression in rat GC.

Internalization of Aeromonas hydrophila by fish epithelial cells can be inhibited with a tyrosine kinase inhibitor.

Abstract: Aeromonas hydrophila is a Gram-negative bacterium that is pathogenic in fish, causing motile aeromonad septicaemia. It can enter (invade) fish cells, and survive as an intracellular parasite. The host-pathogen interaction and signal transduction pathway were studied by screening signal transduction inhibitors using carp epithelial cells and a virulent strain of the bacterium, PPD134/91. Genistein, a tyrosine kinase inhibitor, postponed internalization of A. hydrophila into host cells, suggesting that tyrosine phosphorylation plays a role in internalization. In contrast, staurosporine, a protein kinase C inhibitor, and sodium orthovanadate, a protein tyrosine phosphatase inhibitor, accelerated internalization of PPD134/91. Other virulent strains of A. hydrophila were also examined and it is likely that all strains, irrespective of serogroup, use the same signalling pathway to facilitate bacterial uptake.

High cell density induces vascular endothelial growth factor expression via protein tyrosine phosphorylation.

Abstract: Expression of vascular endothelial growth factor (VEGF), an angiogenic factor and endothelial cell-specific mitogen, is induced by hypoxia in various cell lines as well as in solid tumors. In this study, we report that cell density has a profound effect on the expression of VEGF in human glioblastoma cells (U87) and human fibrosarcoma cells (HT1080), an effect that is independent of hypoxia. Northern blot analysis revealed that VEGF mRNA levels were four- to eightfold higher in cells seeded at high density compared to cells seeded at low density. This upregulation of VEGF message in response to seeding at high density was not seen with other mRNAs such as those for TGF-beta1 or GAPDH. Conditioned medium switch experiments between sparse and dense cells suggested that soluble factor(s) may not account for the observed changes in VEGF expression. Incubation with genistein, a protein tyrosine kinase inhibitor, for 3 h following seeding resulted in the reduction of the VEGF mRNA levels in highly confluent cultures but not in sparse cultures. To identify protein tyrosine kinases involved in the upregulation of the steady-state levels of VEGF mRNA in highly dense cultures, we analyzed the phosphorylation state of the c-src tyrosine kinase, in high versus low confluency cultures of U87 and HT1080 cells. Interestingly, an increased phosphorylation at Tyr416 of c-src was noted in high compared to low confluency, suggesting the activation of c-src in highly confluent cultures. Because extracellular signal-regulated kinases (ERKs) such as MAP kinase have been shown to be activated by extracellular stimuli and act downstream of c-src, we examined their possible involvement in this process. We found that the tyrosine phosphorylation level of MAP kinase is higher in dense compared to sparse cultures and, moreover, 6-thioguanine (6-TG), a potent inhibitor of ERKs, reduced VEGF mRNA levels in high but not low confluency. Furthermore, reintroduction of wild-type, but not mutant, von Hippel-Lindau (VHL) gene product in 786-O cells (a renal carcinoma cell line) specifically abrogated the induction of VEGF mRNA due to high cell density. Taken together, these data suggest that VEGF gene expression is regulated by cell density, and the protooncogene c-src and the tumor-suppressor VHL are modulators of this regulation.