Effects Of Tyrosine Kinase Inhibitors Research Articles

The role of tyrosine phosphorylation in lipopolysaccharide- and zymosan-induced procoagulant activity and tissue factor expression in macrophages.

The expression of surface procoagulants by exudative macrophages represents an important mechanism underlying local fibrin deposition at sites of extravascular inflammation. The present studies investigated the contribution of tyrosine phosphorylation to the generation of macrophage procoagulant activity (PCA) and tissue factor expression in response to proinflammatory stimuli. Both lipopolysaccharide (LPS) and zymosan rapidly stimulated tyrosine phosphorylation in elicited murine peritoneal macrophages. This effect was prevented by the tyrosine kinase inhibitors genistein and herbimycin and augmented by the addition of the phosphotyrosine phosphatase inhibitor vanadate. The vanadate-mediated rise in phosphotyrosine accumulation was abrogated by the use of diphenylene iodonium, an inhibitor of the respiratory burst oxidase, suggesting a role for peroxides of vanadate as contributors to the tyrosine phosphorylation. This notion was supported by the finding that vanadyl hydroperoxide markedly increased the accumulation of phosphotyrosine residues. To define the role of tyrosine phosphorylation in the induction of macrophage PCA by LPS, the effects of tyrosine kinase inhibition by genistein and herbimycin were investigated. Both agents inhibited the expression of macrophage PCA. Further, Northern blot analysis with the cDNA probe for murine tissue factor indicated that the inhibition occurred at the mRNA level or earlier. Since vanadate augmented phosphotyrosine accumulation, it was hypothesized that it might enhance generation of macrophage products. However, vanadate reduced induction of PCA in response to LPS. By contrast, vanadate augmented basal prostaglandin E2 (PGE2) release and stimulated PGE2 release by macrophages. Indomethacin prevented the increase in PGE2 but only partially restored normal levels of PCA. The effect of vanadate on tissue factor expression appeared to be posttranscriptional. These studies thus demonstrate, by functional Western blotting and Northern blotting techniques, that tyrosine phosphorylation plays a role in the regulation of macrophage PCA and tissue factor expression in response to proinflammatory stimuli.

Protein tyrosine kinase inhibitors synergize with nerve growth factor in embryonic chick sensory neuronal cell survival

The survival of developing sensory neurons is dependent upon target-derived growth factors, in their absence neurons undergo programmed cell death. The molecular mechanisms underpinning neuronal cell survival and death are poorly understood. Tyrosine kinases are important signalling proteins that have been implicated in both cell survival and death. The aim of this study was to examine the effects of tyrosine kinase inhibition on embryonic chick sensory neuronal survival using the tyrosine kinase inhibitors, herbimycin, genistein and tyrphostin. In low concentrations of nerve growth factor, NGF (100 fg/ml), the majority of neurons die, however neuronal survival was significantly potentiated in the presence of each of the tyrosine kinase inhibitors, herbimycin (40 ng/ml), genistein (2.5 μM) and tyrphostin (8 μM). In the presence of each of these inhibitors, sensory neurons exhibited typical phase bright morphology and fibre outgrowth was increased. These results demonstrate that the tyrosine kinase inhibitors support the survival of neurons in the presence of low concentrations of NGF. Herbimycin was used at lower concentrations than previously reported, and at this concentration it has been shown to be non-cytotoxic in animals. Therefore it will be important to determine if herbimycin can be used as a therapeutic agent for enhancing nerve regeneration following injury.

Ca2+ channel activation by platelet-derived growth factor-induced tyrosine phosphorylation and Ras guanine triphosphate-binding proteins in rat glomerular mesangial cells.

We investigated the signaling pathways mediating 1-pS Ca2+ channel activation by PDGF in cultured rat mesangial cells. In cell-attached patches, intrapipette PDGF-BB (PDGF B chain homodimer isoform) (50 ng/ml) dramatically stimulates channel activity (P < 0.003, n = 6). Tyrosine kinase inhibition (100 microM genistein or 10 microM tryphostin 9) abolished PDGF-induced channel activation (P < 0.02, n = 6). In excised patches, the effect of tyrosine kinase inhibition could be reversed by 200 microM GTPgammaS (P < 0.02, n = 4). In contrast, 200 microM GDPbetaS inhibited PDGF-induced channel activity (P < 0.04, n = 6). Pertussis toxin (250 ng/ml) had no effect on PDGF-induced channel activity (P = 0.45, n = 6). When excised patches were exposed to anti-Ras antibody (5 microg/ml), PDGF-induced channel activity was abolished (P < 0.002, n = 11). Western immunoblots revealed that PDGF-BB binding stimulates the formation of a membrane-bound complex consisting of growth factor receptor-binding protein 2, son of sevenless, and the PDGF-beta receptor. Complex formation was abolished by genistein. In mesangial cells, the intrinsic tyrosine kinase activity of the PDGF-beta receptor stimulates the formation of a membrane-bound growth factor receptor-binding protein 2/son of sevenless/PDGF-beta receptor complex and activation of the pertussis toxin-insensitive GTP-binding protein, p21-Ras, which leads to the opening of 1-pS Ca2+ channels.

The effect of tyrosine kinase inhibition on phorbol 12,13-dibutyrate- and ionomycin-induced LH and GH release.

The effect of tyrosine kinase inhibition on phorbol 12,13-dibutyrate- and ionomycin-induced LH and GH release.

Effect of tyrosine kinase inhibition on basal and epidermal growth factor‐stimulated human Caco‐2 enterocyte sheet migration and proliferation

Mucosal healing requires enterocyte migration (restitution) supplemented by proliferation. Proliferation and migration may be studied independently by thymidine uptake and proliferation-blocked cell migration using human Caco-2 enterocyte monolayers in culture. Since epidermal growth factor (EGF) promotes mucosal healing and the EGF receptor is a tyrosine kinase, we hypothesized that tyrosine kinases might therefore modulate enterocyte migration and proliferation. The tyrosine kinase inhibitors genistein and 2,5-dihydroxymethylcinnamate, which block kinase ATP-binding and substrate-binding sites, respectively, were studied alone and with EGF. Proliferation was blocked with mitomycin. Although each inhibitor decreased basal and EGF-stimulated monolayer expansion when cell proliferation occurred, neither genistein nor 2,5-dihydroxymethylcinnamate decreased migration when proliferation was blocked. However, each inhibitor prevented EGF stimulation of proliferation-blocked migration and thymidine uptake. More substantial inhibition of basal proliferation by genistein correlated with increased protein-linked DNA breaks, which may reflect nonspecific inhibition of DNA topoisomerase activity by genistein. The more specific 2,5-dihydroxymethylcinnamate blocked changes in the alpha 2 integrin subunit organization which may modulate EGF-stimulated migration. Antiproliferative effects of tyrosine kinase inhibitors decrease basal monolayer expansion but true basal enterocyte migration appears independent of tyrosine kinase regulation. However, a specific tyrosine kinase-dependent modulation of cell-matrix interaction inhibits EGF-stimulated migration.

Differential Effects of Tyrosine Kinase Inhibition in CD69 Antigen Expression and Lytic Activity Induced by rIL-2, rIL-12, and rIFN-α in Human NK Cells

The effect of rIL-12 on induction of CD69 antigen expression and cytolytic activity in purified human NK cells was evaluated in comparison to the effects of rIL-2 and rIFN-α. It was found that rIL-12 directly induced CD69 antigen expression in NK cells, although the period of incubation required by rIL-12 was longer than the period required by rIL-2 or by rIFN-α. Similarly, the cytolytic activity induced by rIL-12 in NK cells against the NK-resistant target cell line Raji was consistently lower than the cytolytic activity induced by rIL-2 or rIFN-α when measured after 6 hr of incubation, and increased during the following 18 hr of incubation. To compare the involvement of tyrosin kinuses in activation of NK cells induced by rIL-2, rIL-12, and rIFN-α, the effect of the specific inhibitor of tyrosin kinases, genistein, was evaluated on induction of CD69 antigen expression and lyric function mediated by the three cytokines. It was found that gentstein inhibited CD69 antigen expression induced by rIL-2 and by rIL-12, but not that induced by rIFN-α. Unlike the effect on CD69 antigen expression, the cytolytic activity induced by all three cytokines was inhibited by genistein. These results, together with the finding that CD69 antigen expression induced by rIL-2 but not by rIL-12 or rIFN-α was inhibited by addition of rIL-4, strongly suggest that IL-2, IL-12, and IFN-α mediate their effects, leading to induction of CD69 antigen expression through different activation pathways.