ag-213 and herbimycin

Bradykinin (1 microM) and histamine (100 microM) evoked an initial transient increase and a subsequent sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded human gingival fibroblasts, which may be attributed to Ca(2+) release from intracellular stores and Ca(2+) entry from extracellular sites, respectively. In fibroblasts pretreated with tyrosine kinase inhibitors such as herbimycin A (1 microM) and tyrphostin 47 (20 microM), the sustained level of [Ca(2+)](i) induced by bradykinin and histamine increased, but not the initial peak level. In the absence of external Ca(2+), bradykinin and histamine induced only the transient increase in [Ca(2+)](i), but a subsequent addition of Ca(2+) to the medium resulted in a sustained increase in [Ca(2+)](i) caused by Ca(2+)entry. Thapsigargin, an inhibitor of Ca(2+)-ATPase in inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores, mimicked the effect of bradykinin and histamine. In the fibroblasts pretreated with tyrosine kinase inhibitors, the bradykinin-, histamine- and thapsigargin-induced Ca(2+) entry was clearly enhanced, but not the transient [Ca(2+)](i) increase. Tyrosine phosphatase inhibitor benzylphosphonic acid (200 microM) had no effect on Ca(2+)entry or transient [Ca(2+)](i) increase. These results suggest that tyrosine phosphorylation is involved in Ca(2+) entry in human gingival fibroblasts.

Topics: Benzoquinones; Bradykinin; Calcium; Calcium Channels; Enzyme Inhibitors; Fibroblasts; Gingiva; Histamine; Humans; Lactams, Macrocyclic; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Receptors, Histamine H1; Rifabutin; Thapsigargin; Time Factors; Tyrosine; Tyrphostins

Ultraviolet (UV) light is a strong apoptotic trigger that can induce a caspase-dependent biochemical change in cells. We previously showed that UV irradiation can elicit caspase-3 activation and the subsequent cleavage and activation of p21-activated kinase 2 (PAK2) in human epidermal carcinoma A431 cells. We report that genistein, an isoflavone compound with known inhibitory activities to protein tyrosine kinases (PTKs) and topoisomerase-II (topo-II), can prevent UV irradiation-induced apoptotic biochemical changes (DNA fragmentation, caspase-3 activation, and cleavage/activation of PAK2) in A431 cells. Surprisingly, two typical PTK inhibitors (tyrphostin A47 and herbimycin A) and three known topo-II inhibitors (etoposide, daunorubicin, and novomycin) had no effect on UV irradiation-induced apoptotic biochemical changes, suggesting that the inhibitory effect of genistein is not dependent on its property as a PTK/topo-II inhibitor. In contrast, azide, a reactive oxygen species (ROS) scavenger, could effectively block the UV irradiation-induced apoptotic cell responses. Flow cytometric analysis using the cell-permeable dye 2',7'-dichlorofluorescin diacetate as an indicator of the generation of ROS showed that UV irradiation caused increase of the intracellular oxidative stress and that this increase could be abolished by azide, suggesting that oxidative stress plays an important role in mediating the apoptotic effect of UV irradiation. Importantly, the UV irradiation-induced oxidative stress in cells could be significantly attenuated by genistein, suggesting that impairment of ROS formation during UV irradiation is responsible for the antiapoptotic effect of genistein. Collectively, our results demonstrate the involvement of oxidative stress in the UV irradiation-induced caspase activation and the subsequent apoptotic biochemical changes and show that genistein is a potent inhibitor for this process.

Topics: Antineoplastic Agents; Apoptosis; Benzoquinones; Carcinoma, Squamous Cell; Enzyme Inhibitors; Genistein; Humans; Lactams, Macrocyclic; Oxidative Stress; p21-Activated Kinases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Quinones; Reactive Oxygen Species; Rifabutin; Sodium Azide; Topoisomerase II Inhibitors; Tumor Cells, Cultured; Tyrphostins; Ultraviolet Rays

The effect of protein tyrosine kinase (PTK) inhibitors on the replication of herpes simplex virus (HSV) was examined. Tyrphostins AG17, AG213, AG490, and AG555, and herbimycin A all inhibited the plaque formation of HSV type 1 (HSV-1) in Vero cells, but AG17, AG490, and AG555 exhibited a more selective antiviral effect. In the presence of 0.4 microM AG17, the virus production 24 h after infection was decreased to 7.7% of the untreated control level. Even if the treatment was started 12 h after the initiation of infection, the viral titer was reduced by 82.4%, compared with the untreated control level. In HSV-1-infected cells ICPs 6, 17/18, 19/20, and 25 were tyrosine-phosphorylated proteins. The synthesis and phosphorylation of these proteins were inhibited by AG17, and suppression of ICP 19/20, which were identified as the UL47 gene products, was the greatest. In contrast, the in vitro autophosphorylation of viral proteins was not affected by this PTK inhibitor. These results indicate that tyrphostin may represent a novel class of inhibitors of HSV-1, and that the viral proteins which have phosphorylated tyrosine residues and are suppressed by AG17 most significantly are the products of the UL47 gene, the tegument proteins VP13/14.

Topics: Amino Acid Sequence; Animals; Antiviral Agents; Benzoquinones; Catechols; Chlorocebus aethiops; Enzyme Inhibitors; Lactams, Macrocyclic; Molecular Sequence Data; Nitriles; Phenols; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Rifabutin; Simplexvirus; Tyrphostins; Vero Cells; Viral Fusion Proteins; Viral Proteins; Virus Replication

We compared the effects of the tyrosine kinase inhibitor genistein, a naturally occurring isoflavone, to those of tyrphostin A25, tyrphostin A47, and herbimycin on avian osteoclasts in vitro. Inactive analogs daidzein and tyrphostin A1 were used to control for nonspecific effects. None of the tyrosine kinase inhibitors inhibited bone attachment. However, bone resorption was inhibited by genistein and herbimycin with ID50s of 3 microM and 0.1 microM, respectively; tyrphostins and daidzein were inactive at concentrations below 30 microM, where nonspecific effects were noted. Genistein and herbimycin thus inhibit osteoclastic activity via a mechanism independent of cellular attachment, and at doses approximating those inhibiting tyrosine kinase autophosphorylation in vitro; the tyrphostins were inactive at meaningful doses. Because tyrosine kinase inhibitors vary widely in activity spectrum, effects of genistein on cellular metabolic processes were compared to herbimycin. Unlike previously reported osteoclast metabolic inhibitors which achieve a measure of selectivity by concentrating on bone, neither genistein nor herbimycin bound significantly to bone. Osteoclastic protein synthesis, measured as incorporation of 3H-leucine, was significantly inhibited at 10 microM genistein, a concentration greater than that inhibiting bone degradation, while herbimycin reduced protein synthesis at 10 nM. These data suggested that genistein may reduce osteoclastic activity at pharmacologically attainable levels, and that toxic potential was lower than that of herbimycin. To test this hypothesis in a mammalian system, bone mass was measured in 200 g ovariectomized rats treated with 44 mumol/day genistein, relative to untreated controls. During 30 d of treatment, weights of treated and control group animals were indistinguishable, indicating no toxicity, but femoral weight in the treated group was 12% greater than controls (P < 0.05). Our data indicate that the isoflavone inhibitor genistein suppresses osteoclastic activity in vitro and in vivo at concentrations consistent with its ID50s on tyrosine kinases, with a low potential for toxicity.

Topics: Animals; Benzoquinones; Bone Resorption; Caffeic Acids; Cells, Cultured; Chickens; Enzyme Inhibitors; Female; Femur; Genistein; Isoflavones; Lactams, Macrocyclic; Nitriles; Osteoclasts; Ovariectomy; Protein Biosynthesis; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Tyrphostins

The induction of a unique macrophage procoagulant molecule by murine hepatitis virus strain 3 correlates with the severity of viral hepatitis. The role of tyrosine phosphorylation in the signalling pathway leading to procoagulant expression was studied. Murine hepatitis virus strain 3 initiated a rapid increase in phosphotyrosine accumulation. Tyrosine kinase inhibition precluded this increase and abrogated expression of the virus-induced procoagulant mouse fibrinogen-like protein (musfiblp) gene. These findings suggest that manipulation of this signalling pathway in vivo might represent a novel approach to treating this disease.

Topics: Animals; Benzoquinones; Catechols; Cells, Cultured; Female; Genistein; Hepatitis, Viral, Animal; Isoflavones; Kinetics; Lactams, Macrocyclic; Macrophages, Peritoneal; Mice; Murine hepatitis virus; Nitriles; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tyrosine; Tyrphostins

We studied whether the stimulatory effect of human serum basic protein I (BP I) on the formation of cell triacylglycerols and cholesterol may be mediated through protein tyrosine kinase in normal fibroblasts, and whether there was a deficiency in such a process in cells from subjects with hyperapobetalipoproteinemia (hyperapoB). Genistein, a highly specific inhibitor of tyrosine kinase phosphorylation, was used as a probe. When BP I (428.0 nmol/L) alone was added to F-12 medium without genistein, the mean mass of cell triacylglycerols doubled in six normal cell lines from healthy subjects, an effect that was decreased by 50% in six cell lines from subjects with hyperapoB (P = .007). The addition of genistein with BP I to normal cells decreased the stimulation of triacylglycerol formation by BP I by about 50% (P = .008), whereas genistein had little effect in the BP I-treated hyperapoB cells. The effect of genistein on the stimulation of triglyceride and cholesterol production by BP I was shown to be both time and concentration (92.5 nmol/mL medium nadir) dependent. In normal fibroblasts. BP I stimulated the rate of incorporation of both [14C]acetate (P = .0001) and [3H]mevalonolactone (P = .002) into unesterified cholesterol, an effect that was markedly deficient in the hyperapoB cells (P = .0001 for [14C]acetate and P = .0002 for [3H]mevalonolactone). In normal but not hyper-apoB cells, genistein inhibited the significant stimulation by BP I of the rates of both [14C]acetate (P = .0001) and [3H]mevalonolactone (P = .04) incorporation into unesterified cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Apolipoproteins B; Benzoquinones; Cholesterol; Cholesterol Esters; Fibroblasts; Genistein; Humans; Hyperlipoproteinemias; Isoflavones; Lactams, Macrocyclic; Mevalonic Acid; Nitriles; Phenols; Protein-Tyrosine Kinases; Proteins; Quinones; Rifabutin; Triglycerides; Tyrphostins

1. Islets from normal mice were used to test the acute effects of genistein, a potent tyrosine kinase inhibitor, on stimulus-secretion coupling in pancreatic beta-cells. 2. Genistein produced a concentration-dependent (10-100 microM), reversible, increase of insulin release. This effect was marginal on basal release or in the presence of non-metabolized secretagogues, and much larger in the presence of glucose or other nutrients. The increase in insulin release caused by 100 microM genistein was abolished by adrenaline or omission of extracellular Ca2+. It was not accompanied by any rise of cyclic AMP, inositol phosphate or adenine nucleotide levels. 3. Although genistein slightly inhibited ATP-sensitive K+ channels, as shown by 86Rb efflux and patch-clamp experiments, this effect could not explain the action of the drug on insulin release because the latter persisted when ATP-sensitive K+ channels were all blocked by maximally effective concentrations of glucose and tolbutamide. Genistein was also effective when ATP-sensitive K+ channels were opened by diazoxide and the beta-cell membrane depolarized by 30 mM K, but ineffective in the presence of diazoxide and normal extracellular K. 4. Genistein paradoxically decreased Ca2+ influx in beta-cells, as shown by the inhibition of glucose-induced electrical activity, by the inhibition of Ca2+ currents (perforated patches) and by the lowering of cytosolic [Ca2+]i (fura-2 technique). Genistein thus increases insulin release in spite of a lowering of [Ca2+]i in beta-cells. 5. Daidzein, an analogue of genistein reported not to affect tyrosine kinases, was slightly less potent than genistein on K+ and Ca2+ channels, but increased insulin secretion in a similar way. Three other tyrosine kinase inhibitors, tyrphostin A47, herbimycin A and an analogue of erbstatin variably affected insulin secretion.6. Genistein exerts a number of heretofore unrecognized effects. The unusual mechanisms, by which genistein increases insulin release in spite of a decrease in beta-cell [Ca2+]i and without activating known signalling pathways, do not seem to result from an inhibition of tyrosine kinases.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Analysis of Variance; Animals; Benzoquinones; Calcium; Catechols; Cyclic AMP; Cytosol; Dose-Response Relationship, Drug; Epinephrine; Female; Genistein; Hydroquinones; Inositol Phosphates; Insulin; Insulin Secretion; Islets of Langerhans; Isoflavones; Lactams, Macrocyclic; Mice; Nitriles; Patch-Clamp Techniques; Potassium Channels; Protein-Tyrosine Kinases; Quinones; Rifabutin; Rubidium; Tyrphostins

In addition to a constitutive cyclo-oxygenase (Cox-1), human endothelial cells also possess an inducible cyclo-oxygenase (Cox-2) which plays an important role in the regulation of the synthesis of prostacyclin (prostaglandin I2). Cox-2 is regulated and expressed in large quantities upon activation of the cells by inducers such as phorbol myristate acetate (PMA), an activator of protein kinase C (PKC), or interleukin-1 alpha. We have investigated the involvement of protein tyrosine kinases in Cox-2 expression by human endothelial cells upon activation by these inducers. PMA or interleukin-1 alpha provoke an increase in the phosphorylation of substrates of 110 and 120 kDa and additional phosphorylations for a broad band of multiple substrates in the 70 kDa range. This stimulation was accompanied by the induction of Cox-2 protein, detectable after stimulation for 1 h, which is consistent with an increase in activity reflected by prostacyclin synthesis; no variation in the expression of Cox-1 could be observed. Three distinct inhibitors of protein tyrosine kinases, genistein, herbimycin or AG-213, reduced tyrosine phosphorylation of cell substrates, consistently with their pharmacological effects. Under these conditions, there was selective reduction of Cox-2 expression without modification of Cox-1. Regulation of Cox-2 induction is also dependent on the activation of PKC since Ro 31-8220 or PKC depletion by PMA prevented its induction. Our results suggest that within the time-frame of our experiments these effects on kinases are specific for Cox-2 rather than Cox-1.

Topics: Benzoquinones; Blotting, Western; Catechols; Cells, Cultured; Endothelium, Vascular; Enzyme Induction; Enzyme Inhibitors; Genistein; Humans; Indoles; Interleukin-1; Isoenzymes; Isoflavones; Kinetics; Lactams, Macrocyclic; Nitriles; Prostaglandin-Endoperoxide Synthases; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Recombinant Proteins; Rifabutin; Tyrphostins; Umbilical Veins

Mast cells, mastocytoma cells and basophil leukaemia cells are well-established producers of leukotrienes when grown and stimulated appropriately. I report that the cells' ability to produce leukotrienes is dependent on the cells' proliferative status or their provision with growth factors. Proliferating MC/9 and subconfluent RBL2H3 cells respond maximally to stimulation by 1 microM ionomycin with the production of 56 and 32 pmol of cysteinyl-leukotrienes/10(6) cells respectively. In contrast, confluent RBL2H3 or growth-arrested MC/9 cells lose their ability to generate leukotrienes in response to ionomycin treatment. This rapid down-regulation of leukotriene synthesis is also observed when proliferating RBL2H3 cells are transferred to growth-factor-free medium, wherein cellular leukotriene-synthesis capacity has an apparent half-lifetime of 60 min. Transfer back into growth medium results in the regeneration of leukotriene synthesis capacity within 6 h. In growth-arrested MC/9 cells, leukotriene production ability can at least partially be restored by priming the cells with interleukin 3, but not with interleukin 4. In RBL2H3 cells, pretreatment with protein tyrosine kinase inhibitors such as genistein (5 min, 37 microM), herbimycin A (6 h, 3 microM) or tyrphostin 25 (16 h, 100 microM) completely inhibits leukotriene generation, whereas okadaic acid (15 min, 0.5 microM) has no effect. Under these conditions, both genistein and herbimycin A strongly impair ionomycin-induced protein tyrosine phosphorylation. Our study indicates that leukotriene generation in these tumour cells is tightly regulated by their proliferation status and supply with growth factors, and cell stimulation towards leukotriene synthesis appears to involve protein tyrosine kinase activity.

Topics: Animals; Benzoquinones; Catechols; Cell Division; Cell Line; Genistein; Interleukin-3; Interleukin-4; Ionomycin; Isoflavones; Kinetics; Lactams, Macrocyclic; Leukemia, Basophilic, Acute; Leukotrienes; Mast-Cell Sarcoma; Mice; Nitriles; Phosphoproteins; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Tumor Cells, Cultured; Tyrosine; Tyrphostins

DNA fragmentation and cell death in rat thymocytes induced by dexamethasone were inhibited by herbimycin A but not by the other inhibitors of tyrosine kinase including genistein and tyrphostin. Herbimycin A also prevented the inter-nucleosomal DNA fragmentation induced by dexamethasone. On the contrary, apoptosis induced by DNA topoisomerase inhibitors such as camptothecin and etoposide were not affected by herbimycin A. These results demonstrate that dexamethasone-induced apoptosis is specifically inhibited by herbimycin A.

Topics: Animals; Apoptosis; Benzoquinones; Camptothecin; Catechols; Cells, Cultured; Dexamethasone; DNA; DNA Damage; Dose-Response Relationship, Drug; Etoposide; Genistein; Isoflavones; Kinetics; L-Lactate Dehydrogenase; Lactams, Macrocyclic; Nitriles; Nucleosomes; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Thymus Gland; Topoisomerase I Inhibitors; Tyrphostins

Cytosolic free calcium ions concentration ([Ca2+]i) was measured in cell suspensions of cultured human IM-9 lymphocytes by dual wavelength fluorescence spectrometry using the calcium probe fura-2. Human GH (0.2-50 nM) induced a slow, progressive and sustained increase in [Ca2+]i. The GH effect was specific and exhibited a biphasic pattern, presumably reflecting GH receptor dimerization, typical of some other GH actions. The hGH effect depended on extracellular calcium, suggesting that at least part of the [Ca2+]i increase was due to a stimulation of calcium influx. GH did not increase IP3. Somatostatin-14 in the range 10(-10) to 10(-8) M, while having no effect of its own on [Ca2+]i, inhibited the effect of hGH. This inhibition by somatostatin was prevented by pretreatment of the cells with pertussis toxin. The hGH-induced [Ca2+]i increase was not related to either protein tyrosine phosphorylation or protein kinase C activation, thus suggesting a novel mechanism of GH transmembrane signalling.

Topics: Alkaloids; Benzoquinones; Calcium; Catechols; Cell Line; Cell Membrane; Cyclic AMP; Cytosol; Dose-Response Relationship, Drug; Growth Hormone; Humans; Inositol 1,4,5-Trisphosphate; Insulin; Insulin-Like Growth Factor I; Kinetics; Lactams, Macrocyclic; Lymphocytes; Nitriles; Protein-Tyrosine Kinases; Quinones; Rifabutin; Signal Transduction; Staurosporine; Tyrphostins

We report on the potency of two Tyrphostin tyrosine kinase blockers, AG 1112 and AG 568, to inhibit p210bcr-abl tyrosine kinase activity in K562 cells, concomitant with the induction of erythroid differentiation. AG 568 and especially AG 1112 represent a specific group of nontoxic protein tyrosine kinase blockers among more than 1,400 tested. These compounds possess therapeutic potential for purging Philadelphia chromosome-positive cells in preparation for autologous bone marrow transplantation in chronic myelogenous leukemia.

Topics: 3T3 Cells; Animals; Benzoquinones; Catechols; Cell Differentiation; Cell Division; Cell Line; ErbB Receptors; Fusion Proteins, bcr-abl; Humans; Kinetics; Lactams, Macrocyclic; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Molecular Structure; Nitriles; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Receptors, Platelet-Derived Growth Factor; Rifabutin; Time Factors; Tumor Cells, Cultured; Tyrosine; Tyrphostins

Accumulating evidence indicates that the activation of cellular oncogenes is a cause of some human cancers. ErbB-1, erbB-2 and abl oncogenes encoding tyrosine kinases, ras oncogenes encoding GTP binding proteins and myc oncogenes whose functions are not well understood are some examples. Therefore, agents which inhibit the activity of these oncogene products may provide new means to overcome certain human tumors. Herbimycin A and tyrphostins have been found and developed as inhibitors of tyrosine kinases and the effectiveness of these agents against tumors of Ph1-positive leukemia (CML, ALL) or squamous cell carcinomas has been reported. Although specific inhibitors of ras or myc oncogene products have not yet been described, recent studies on the processing of Ras proteins toward the cell membrane provide a strategy to search for inhibitors of ras functions.

Topics: Antibiotics, Antineoplastic; Benzoquinones; Carcinoma, Squamous Cell; Catechols; Cyclin D1; Female; Genes, ras; Humans; Lactams, Macrocyclic; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Neoplasms; Nitriles; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Quinones; Rifabutin; Tyrphostins

Resting memory B lymphocytes specific for the model protein Ag cytochrome c have been shown to be susceptible to tolerance induction in in vitro splenic fragment cultures. This induction of nonresponsiveness is dependent upon the strength of the interaction between surface Ig and specific Ag, where concentration, valency, affinity, and time of exposure all appear to be important factors, as is the case for tolerance induction in immature or primary B cells. The induction of nonresponsivenes in greater than 80% of Ag-specific memory B cells was achieved by incubation with 1 microM cytochrome polymer for 24 h in the absence of T cell help. Not only were memory B cells unresponsive to specific Ag, they were also unable to become activated through nonspecific uptake and presentation of an Ag to which T cells have been primed, demonstrating that the induction of nonresponsiveness involves more than a modulation or blockade of surface Ig receptors. Although soluble factors collected from activated T cells failed to prevent memory B cells from becoming nonresponsive after surface Ig cross-linking, the direct activation of T cells within splenic fragment cultures did partially inhibit tolerance induction in splenic fragment memory B cells. In addition, the induction of tolerance was partially blocked by protein tyrosine kinase inhibitors, suggesting a physiologic change within the B cells associated with the state of nonresponsiveness and resulting from tyrosine-specific phosphorylation.

Topics: Animals; B-Lymphocytes; Benzoquinones; Catechols; Cytochrome c Group; Dose-Response Relationship, Immunologic; Enzyme-Linked Immunosorbent Assay; Female; Genistein; Immunoglobulins; Immunosuppression Therapy; In Vitro Techniques; Isoflavones; Lactams, Macrocyclic; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Nitriles; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tyrphostins