Specific Protein Tyrosine Kinase Inhibitor Research Articles

9(B-5) v-Srcチロシンキナーゼ阻害物質ヒバリマイシンの構造・生合成および活性(口頭発表の部)

9(B-5) v-Srcチロシンキナーゼ阻害物質ヒバリマイシンの構造・生合成および活性(口頭発表の部)

Involvement of tyrosine-specific protein kinase and protein kinase C in J774A.1 macrophage functions activated by Tinospora cordifolia

BackgroundMacrophages are the first line of defense and constitute important participant in the bi-directional interaction between innate and specific immunity. Macrophages are in a quiescent form and get activated when given a stimulus. In our previous studies we have reported that guduchi or LPS treatment of macrophages enhanced production of nitric oxide (NO) and increased tumoricidal activity against L929 fibroblast cells. ObjectiveIn the present study effect of Tinospora cordifolia commonly known as guduchi on macrophage activation and the mechanism of action i.e. involvement of protein kinase C inhibitor and tyrosine-specific protein kinase inhibitor was investigated. Materials and MethodsThe present study was undertaken to determine whether H-7 (inhibitor of protein kinase C) and/or genistein (inhibitor of tyrosine-specific protein kinase) could inhibit guduchi or LPS-induced macrophage NO and TNF-α production or reduce the cytolysis of L929 fibroblast cells. ResultsIt was observed that in vitro incubation with H-7 and/or genistein completely inhibited guduchi or LPS-induced NO and TNF-α production by macrophages (J774A.1). ConclusionThe inhibitory effects of H-7 and/or genistein, suggest that phosphorylation via these kinases may upregulate the NO synthase activity in macrophages.

Platelet-activating factor attenuation of long-term potentiation in rat hippocampal slices via protein tyrosine kinase signaling

It is well established that HIV-1-infected mononuclear phagocytes release platelet activating factor (PAF) and elevated levels of PAF have been detected in blood and in the cerebrospinal fluid (CSF) of acquired immunodeficiency syndrome (AIDS) patients with HIV-associated neurocognitive disorders (HAND). It is our hypothesis that the elevated levels of PAF alter long-term potentiation (LTP) in the hippocampus, leading to neurocognitive dysfunction. To test this hypothesis, we studied the effects of PAF on LTP in the CA1 region of rat hippocampal slices. Our results showed incubation of hippocampal slices with PAF attenuated LTP. The PAF-mediated attenuation was blocked by ginkgolide B, a PAF receptor antagonist, suggesting PAF attenuation of LTP via PAF receptors. Application of lyso-PAF, an inactive PAF analog, had no apparent effect on LTP. Further investigation revealed an involvement of tyrosine kinase in PAF attenuation of LTP, which was demonstrated by lavendustin A (a specific protein tyrosine kinase inhibitor) blockage of PAF attenuation of LTP. As LTP is widely considered as the cellular and synaptic basis for learning and memory, the attenuation of LTP by PAF may contribute at least in part to the HAND pathogenesis.

Combined effects of PI3K and SRC kinase inhibitors with imatinib on intracellular calcium levels, autophagy, and apoptosis in CML-PBL cells

Imatinib induces a complete cytogenetic regression in a large percentage of patients affected by chronic myeloid leukemia (CML) until mutations in the kinase domain of BCR-ABL appear. Alternative strategies for CML patients include the inhibition of phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, which is constitutively activated in leukemia cells and seems important for the regulation of cell proliferation, viability, and autophagy. In this study, we verified the effect of imatinib mesylate (IM), alone or in association with LY294002 (LY) (a specific PI3K protein tyrosine kinase inhibitor) or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1) (a Src tyrosine kinase inhibitor), on viability, intracellular calcium mobilization, apoptosis, and autophagy, in order to verify possible mechanisms of interaction. Our data demonstrated that PP1 and LY interact synergistically with IM by inducing apoptosis and autophagy in Bcr/Abl+ leukemia cells and this mechanism is related to the stress of the endoplasmic reticulum (ER). Our findings suggest a reasonable relationship between apoptotic and autophagic activity of tyrosine kinase inhibitors (TKIs) and the functionality of smooth ER Ca2+-ATPase and inositol triphosphate receptors, independently of intracellular calcium levels. Therapeutic strategies combining imatinib with PI3K and/or Src kinase inhibitors warrant further investigations in Bcr/Abl+ malignancies, particularly in the cases of imatinib mesylate-resistant disease.

Genistein directly inhibits native and recombinant NMDA receptors

The protein tyrosine kinase (PTK) inhibitor genistein has been widely used to examine potential effects of tyrosine phosphorylation on neurotransmitter function. We report here that genistein inhibits N-methyl-d-aspartate (NMDA) receptors through a direct effect. Whole-cell NMDA-activated current was recorded in native receptors from mouse hippocampal slice culture and rat recombinant NR1aNR2A and NR1aNR2B receptors transiently expressed in HEK293 cells. Extracellular application of genistein and NMDA reversibly inhibited NMDA-activated current. The inhibition of NMDA-activated current by genistein applied externally was not affected when genistein was also pre-equilibrated in the intracellular solution. Daidzein, an analog of genistein that does not block PTK, also inhibited NMDA-activated current. Coapplication of lavendustin A, a specific inhibitor of PTK, had no effect on the NMDA response. Moreover, genistein-induced inhibition of NMDA-activated current displayed concentration- and voltage-dependence. Our results demonstrate that genistein has a direct inhibitory effect on NMDA receptors that is not mediated via inhibition of tyrosine kinase. Thus, other PTK inhibitors may be more suitable for studying involvement of PTKs in NMDA receptor-mediated events.

Role of Epidermal Growth Factor Receptor in Ovine Fetal Pulmonary Vascular Remodeling Following Exposure to High Altitude Long-Term Hypoxia

High altitude long-term hypoxia (LTH) in the fetus may result in pulmonary vascular smooth muscle cell (PVSMC) proliferation and pulmonary vascular remodeling. Our objective was to determine if epidermal growth factor receptor (EGFR) is involved in hypoxia induced PVSMC proliferation or in pulmonary vascular remodeling in ovine fetuses exposed to high altitude LTH. Fetuses of pregnant ewes that were held at 3820-m altitude from *30 to 140 days (LTH) gestation and sea level control pregnant ewes were delivered near term. Morphometric analyses and immunohistochemistry were done on fetal lung sections. Pulmonary arteries of LTH fetuses exhibited medial wall thickening and distal muscularization. Western blot analyses done on protein isolated from pulmonary arteries demonstrated an upregulation of EGFR. This upregulation was attributed in part to PVSMC in the medial wall by immunohistochemistry.Proliferation of fetal ovine PVSMC after 24 h of hypoxia (2% O2) was attenuated by inhibition of EGFR with 250 nmol tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), a specific EGFR protein tyrosine kinase inhibitor, when measured by [3H]-thymidine incorporation. Our data indicate that EGFR plays a role in fetal ovine pulmonary vascular remodeling following long-term fetal hypoxia and that inhibition of EGFR signaling may ameliorate hypoxia-induced pulmonary vascular remodeling.

Role of Nonreceptor Protein Tyrosine Kinases During Phospholipase C-γ1-related Uterine Contractions in the Rat

Activated phospholipase C1, produced in response to tyrosine phosphorylation, appears to play an important role during uterine contractions. These studies sought to determine which non-receptor protein tyrosine kinases are involved in the activation of phospholipase C1 in rat uterine tissue. In vitro contraction studies were performed utilizing isoform specific protein tyrosine kinase inhibitors. Western blots were performed utilizing antibodies to phosphotyrosine-phospholipase C1, total phospholipase C1, c-Src kinase and Lck kinase. Spontaneous, stretch-stimulated, and bpV(phen) (tyrosine phosphatase inhibitor) enhanced uterine contractions were significantly suppressed in response to Damnacanthal (Lck kinase inhibitor) and PP1 (c-Src kinase inhibitor). Damnacanthal and PP1 also significantly suppressed bpV(phen)-enhanced tyrosine phosphorylation of phospholipase C1. Western blots confirmed expression of Lck kinase and c-Src kinase in uterine tissue. In conclusion, the Lck and c-Src kinases appear to play an important role in regulating tyrosine phosphorylation of phospholipase C1 and contractile activity in the rat uterus.

High glucose-induced NF-κB activation occurs via tyrosine phosphorylation of IκBα in human glomerular endothelial cells: involvement of Syk tyrosine kinase

Activation of nuclear factor-kappaB (NF-kappaB) occurs by dissociation from IkappaB after serine or tyrosine phosphorylation of IkappaBalpha, but the way of NF-kappaB activation by high glucose has not been defined. High glucose is known to activate NF-kappaB via protein kinase C and reactive oxygen species (ROS). In this study, we investigated how high glucose activates NF-kappaB for CC chemokine ligand 2 production in cultured human glomerular endothelial cells. High glucose increased nuclear translocation of p65 and also increased NF-kappaB DNA binding activity. High glucose-induced NF-kappaB activation occurred without degradation of IkappaBalpha. In agreement with this, there was no increase in serine phosphorylation of IkappaBalpha, while tyrosine phosphorylation of IkappaBalpha was increased by high glucose. High glucose increased the generation of ROS, whereas both alpha-lipoic acid and N-acetylcysteine scavenged the ROS and decreased high glucose-induced tyrosine phosphorylation of IkappaBalpha, nuclear translocation of p65, and NF-kappaB DNA binding activity. Protein kinase C pseudosubstrate inhibited high glucose-induced ROS production, tyrosine phosphorylation of IkappaBalpha, and nuclear translocation of p65. Both BAY 61-3606, a specific inhibitor of Syk protein-tyrosine kinase, and small interfering RNA directed against Syk inhibited high glucose-induced tyrosine phosphorylation of IkappaBalpha as well as p65 nuclear translocation. High glucose increased tyrosine phosphorylation of Syk, while it was inhibited by alpha-lipoic acid and protein kinase C pseudosubstrate. In summary, high glucose-induced NF-kappaB activation occurred not by serine phosphorylation of IkappaBalpha. Our data suggest that ROS-mediated tyrosine phosphorylation of IkappaBalpha is the mechanism for high glucose-induced NF-kappaB activation, and Syk may play a role in tyrosine phosphorylation of IkappaBalpha.

The metabolism of serotonin in neuronal cells in culture and platelets

The aim of this study is to find a relationship between serotonin (5-HT) and its metabolite 5-hydroxy indol acetic acid (5-HIAA) in hippocampus, frontal neocortex and platelets. Serotonin and 5-HIAA were measured in cultured neurons and compared with those produced by human platelets. The cortical neuronal 5-HIAA/serotonin ratio was 4.7 and for hippocampal neurons it was 3.2. In human platelets, this ratio was 1.35 suggesting that the highest serotonin metabolism occurs in the frontal neocortex followed by the hippocampus and platelets. In the presence of 0.3 microM of p-chlorophenylalanine both cultured neurons and platelets exhibited an approximately 50% decrease in serotonin and 5-HIAA concentration suggesting similarities in the metabolic profile in both preparations. In addition, we found that serotonin by itself does not play any role in platelet aggregation but potentiates this phenomenon in the presence of calcium ionophore A23187. This synergistic interaction between serotonin (2-5 microM) and A23187 (0.5-2 microM) was inhibited by serotonin receptor blockers [methysergide (IC50 = 18 microM) and cyproheptadine (IC50, 20 microM)] and calcium channel blockers (verapamil and diltiazem, IC50 = 20 and 40 microM, respectively) that indicate both mechanisms are receptor mediated. Similarly, U73122, an inhibitor of phospholipase C (PLC), blocked the synergistic effect of serotonin and ionophore at an IC50 value of 9.2 microM. Wortmannin, a phosphoinositide 3-kinase (PI 3-K) inhibitor, also blocked the response (IC50 = 2.6 microM) by inhibiting respiratory burst. However, neither genistein, a tyrosine-specific protein kinase inhibitor, nor chelerythrine, a protein kinase C (PKC) inhibitor, affected aggregation. Our results are strongly suggestive of a synergistic interaction between serotonin type-2 and Ca-ionophore via a PLC/Ca signalling pathway.

ABT-737 Neutralizes Resistance to FLT3 Inhibitor Treatment Mediated by Overexpression of BCL2 in Primary AML Blasts.

Mutated FLT3 defines a promising target for the treatment of acute myeloid leukemia (AML) with specific protein tyrosine kinase (PTK) inhibitors. The clinical efficacy of this approach, however, is limited due to molecular mechanisms that remain to be elucidated. As we demonstrated previously, overexpression of antiapoptotic proteins of the BCL2 family lead to resistance against PTK inhibitors in cell lines with activating FLT3 mutations (Bagrintseva, Blood, 2005). In primary AML samples tested so far in our study, we found a correlation of the expression level of BCL-XL, a known downstream target of FLT3, and the presence of activating FLT3 mutations whereas MCL1 protein, another antiapoptotic member of the BCL2 family, showed very low expression. In contrast, we found a high expression level of BCL2 protein in all AML samples whether or not FLT3 mutations were present and this level remained unchanged after dephosphorylation of mutated FLT3. Thus, we speculated that an overexpression of BCL2 independent from FLT3 activation might at least in part explain the limited clinical efficacy of PTK inhibitors in the treatment of FLT3 positive AML. To test this hypothesis, we stably expressed mock, BCL2 or BCL-XL, respectively, in Ba/F3 cell lines carrying constitutively activated FLT3 with internal tandem duplication. The cells overexpressing BCL2 or BCL-XL, respectively, did not respond to treatment with the FLT3 specific inhibitor SU5614 up to high doses. To overcome the observed resistance, we tested the small molecule inhibitor ABT-737 (kindly provided by Abbott Laboratories) that has been described to efficiently disrupt intracellular BCL2 family interactions by binding to the hydrophobic BH3 groove of these proteins (Oltersdorf, Nature, 2005). Surprisingly, treatment of our generated cell lines with ABT-737 alone did not result in increased levels of apoptotic cell death. This finding is in line with previous reports showing that mono-treatment with ABT-737 does not directly activate proapoptotic proteins, but needs activator BH3-only proteins such as BID or BIM. Co-treatment of the cell lines with SU5614 and ABT-737, however, rendered them again susceptible to the PTK inhibitor in a concentration-dependent manner. SU5614 and ABT-737 showed synergism as confirmed by immunoblotting against cleaved and full-length caspase-3. As a negative control to all our experiments, we used the functionally inactive enantiomer of ABT-737 (ABT control) that caused significant cytotoxicity neither alone nor in combination with SU5614 up to high doses.To underline the clinical relevance of these findings, a panel of AML patient samples is currently tested for response to ABT-737 alone or in combination with PTK inhibitors. Two AML samples tested so far showed an IC50 of 10 and 25nM (vs. 300 and 1000 nM, respectively, for ABT control) after 24h of mono-treatment with ABT-737, whereas peripheral blood mononuclear cells of a healthy donor showed an IC50 of 80 nM for ABT-737 and 400nM for ABT control. This might be explained by recent findings indicating that native tumor and leukemia cells are addicted to the expression of antiapoptotic proteins and tonically exposed to proapoptotic stimuli (Certo, Cancer Cell, 2006).Since BCL2 has been reported to be involved in cell cycle regulation by facilitating G0/G1 arrest, we are also going to study the effects of ABT-737 on non-proliferating CD34+ progenitor AML cells that cannot be eliminated by conventional chemotherapy.

Molecular Mechanism of Adaphostin-mediated G1 Arrest in Prostate Cancer (PC-3) Cells

Adaphostin (NSC680410), a small molecule congener of tyrphostin AG957, has been demonstrated previously to have significant anti-proliferative effects in several leukemia models. However, this effect of adaphostin in adherent cells/solid tumor models has not been examined. In this study, we investigated the anti-proliferative effects of adaphostin in the human prostate cancer cell line PC-3. Specifically, we explored the potential molecular mechanism(s) by which adaphostin elicits its anti-proliferative effect(s). We demonstrate that adaphostin inhibits the proliferation of PC-3 cells by inducing a G(1) phase cell cycle arrest. This adaphostin-induced G(1) arrest was associated with an increase in the expression of p21 and p27 and a decrease in the expression of G(1)-specific cyclins (cyclin A, D1, and D3) and cyclin-dependent kinases 4 and 6. Consequently, a dramatic decrease in the phosphorylation of retinoblastoma protein was also observed. Additionally, we found that adaphostin treatment induced a decrease in the phosphorylation of nucleophosmin, a major nuclear phosphoprotein, and that this decreased phosphorylation was a result of the p21- and p27-mediated inactivation of cyclin E-cyclin-dependent kinase 2 complex kinase activity. Furthermore, we have determined that the adaphostin-mediated cell cycle arrest of PC-3 cells is dependent upon activation of the p38 MAPK. We also demonstrate that the hepatocyte growth factor receptor-c-Met is involved in the adaphostin-mediated signaling events that regulate p38 MAPK. Taken together, these results identify for the first time a signaling cascade of adaphostin-mediated G(1) phase-specific cell cycle arrest in PC-3 cells. These findings suggest that the tyrphostin member has a broader spectrum of activity than originally predicted.

Complex formation between tissue transglutaminase II (tTG) and vascular endothelial growth factor receptor 2 (VEGFR-2): Proposed mechanism for modulation of endothelial cell response to VEGF

We have recently demonstrated that thrombin-activated FXIII (FXIIIA-subunit), a plasma transglutaminase, activates VEGFR-2 by crosslinking it with the α vβ 3 integrin on the surface of endothelial cells (EC), thereby stimulating angiogenesis. Tissue transglutaminase (tTG), which is functionally and structurally related to FXIIIA, is expressed by numerous cell types, among them EC. However, its role in EC function has not been fully characterized. In the present study, we investigated the potential involvement of tTG in angiogenesis. Using co-immunoprecipitation and immunofluorescent staining experiments, we observed that tTG forms a complex with VEGFR-2 on the cell surface and within the cytoplasm of EC. Stimulation of EC with VEGF resulted in translocation of the tTG–VEGFR-2 complex from the cytoplasm to the nucleus. In VEGF-treated cells, tTG–VEGFR-2 interaction resulted in incorporation of VEGFR-2 into high molecular weight crosslinked complex (es), as revealed by an antibody against γ-glutamyl-ε-lysine isopeptide bond. tTG –VEGFR-2 association was inhibited by a specific VEGFR-2 protein tyrosine kinase inhibitor (PTKI ), as well as by cystamine, inhibitor of the transglutaminase activity of tTG, but not by bacitracin which inhibits the protein-disulfide isomerase (PDI) activity of tTG. Furthermore, cystamine completely abolished the VEGF-induced nuclear translocation of the tTG–VEGFR-2 complex. Blockade of the crosslinking activity of tTG by cystamine enhanced VEGF-induced migration of EC in Boyden chamber by 31% ( P < 0.02), and prolonged VEGF-induced signaling response, as demonstrated by sustained activation of the MAP kinase ERK. Taken together, our findings suggest that endothelial cell tTG might be involved in modulation of the cellular response to VEGF by forming an intracellular complex with VEGFR-2, and mediating its translocation into the nucleus upon VEGF stimulation.

Tyrphostin‐A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoa

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.

The selective inhibition of nitric oxide production in the avian macrophage cell line HD11

The production of reactive nitrogen, nitric oxide (NO), has previously been demonstrated to be a major mechanism by which the innate immune system defends against microbial invasion. The induction of many antimicrobial mechanisms is regulated by numerous components during the transduction of the signal from the cell surface to the cell nucleus where response genes are upregulated. Toll-like cell surface receptor activation often leads to sequential modulation of protein tyrosine kinases (PTK), mitogen activated protein kinases (MAPK), degradation of I kappa B (IκB) regulatory molecules which, in turn, release the nuclear factor-kappa B (NF-κB) family proteins for translocation into the nucleus and subsequent gene transcription. The purpose of this study was to investigate components of the upstream signal transduction pathway induced by bacterial and viral-like stimulation of NO for antimicrobial defense by the transformed chicken macrophage cell line, HD11. We quantified the production of nitrite by chicken macrophages after exposure to selective pharmacological inhibitors of specific signal transduction components prior to stimulation by polyinosinic polycytidylic acid (poly I:C), formalin-fixed Enterococcus gallinarum (EG) or formalin-fixed Klebsiella pneumoniae (KP). We found that NO production induced by dsRNA or bacteria was reduced in a dose dependent manner by specific inhibitors of PTK, p38 MAPK, IκB, and NF-κB. Inhibition efficacy varied dependent on stimulation by bacterial or viral-like ligands. In general, NO production induced by bacterial stimulation was most effectively reduced by inhibition of p38 MAPK and least effectively reduced by inhibition of IκB. NF-κB and IκB inhibition affected NO production induced by dsRNA more than that induced by bacterial stimulation.

Characterization of dual specificity protein kinase from maize seedlings.

A protein kinase of 57 kDa, able to phosphorylate tyrosine in synthetic substrates pol(Glu4,Tyr1) and a fragment of Src tyrosine kinase, was isolated and partly purified from maize seedlings (Zea mays). The protein kinase was able to phosphorylate exogenous proteins: enolase, caseins, histones and myelin basic protein. Amino acid analysis of phosphorylated casein and enolase, as well as of phosphorylated endogenous proteins, showed that both Tyr and Ser residues were phosphorylated. Phosphotyrosine was also immunodetected in the 57 kDa protein fraction. In the protein fraction there are present 57 kDa protein kinase and enolase. This co-purification suggests that enolase can be an endogenous substrate of the kinase. The two proteins could be resolved by two-dimensional electrophoresis. Specific inhibitors of typical protein-tyrosine kinases had essentially no effect on the activity of the maize enzyme. Staurosporine, a nonspecific inhibitor of protein kinases, effectively inhibited the 57 kDa protein kinase. Also, poly L-lysine and heparin inhibited tyrosine phosphorylation by 57 kDa maize protein kinase. The substrate and inhibitor specificities of the 57 kDa maize protein kinase phosphorylating tyrosine indicate that it is a novel plant dual-specificity protein kinase.

Characterization of dual specificity protein kinase from maize seedlings.

A protein kinase of 57 kDa, able to phosphorylate tyrosine in synthetic substrates pol(Glu4,Tyr1) and a fragment of Src tyrosine kinase, was isolated and partly purified from maize seedlings (Zea mays). The protein kinase was able to phosphorylate exogenous proteins: enolase, caseins, histones and myelin basic protein. Amino acid analysis of phosphorylated casein and enolase, as well as of phosphorylated endogenous proteins, showed that both Tyr and Ser residues were phosphorylated. Phosphotyrosine was also immunodetected in the 57 kDa protein fraction. In the protein fraction there are present 57 kDa protein kinase and enolase. This co-purification suggests that enolase can be an endogenous substrate of the kinase. The two proteins could be resolved by two-dimensional electrophoresis. Specific inhibitors of typical protein-tyrosine kinases had essentially no effect on the activity of the maize enzyme. Staurosporine, a nonspecific inhibitor of protein kinases, effectively inhibited the 57 kDa protein kinase. Also, poly L-lysine and heparin inhibited tyrosine phosphorylation by 57 kDa maize protein kinase. The substrate and inhibitor specificities of the 57 kDa maize protein kinase phosphorylating tyrosine indicate that it is a novel plant dual-specificity protein kinase.

Molecularly targeted treatment for dermatofibrosarcoma protuberans

Traditionally, treatment for dermatofibrosarcoma protuberans (DFSP), a rare cutaneous tumor that is locally aggressive, has been limited to wide surgical excision with negative margins. Although not usually metastatic, DFSP has significant potential for recurrence and interference in local structures. The pathogenesis of DFSP stems from a chromosomal rearrangement involving chromosomes 17 and 22, in which the collagen 1α1 gene is fused to the gene for platelet-derived growth factor (PDGF) B-chain. The resultant deregulated expression of PDGFB leads to continuous activation of the PDGF receptor β (PDGFRβ) protein-tyrosine kinase that promotes DFSP tumor cell growth. Imatinib is a potent and specific inhibitor of several protein-tyrosine kinases, including the PDGFRs. Preclinical investigations and clinical reports have shown the efficacy of imatinib in DFSP. Imatinib may provide an alternative for the treatment of unresectable or partially resectable tumors, thereby possibly improving the effectiveness of surgery.

The role of protein tyrosine kinases in CYP1A1 induction by omeprazole and thiabendazole in rat hepatocytes

Benzimidazoles compounds like omeprazole (OME) and thiabendazole (TBZ) mediate CYP1A1 induction differently from classical aryl hydrocarbon receptor (AhR) ligands, 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). To clarify the involvement of an intracellular signal pathway in CYP1A1 induction by OME and TBZ, the TBZ, OME and 3-MC signal-transducing pathways were compared by using specific protein tyrosine kinase inhibitors in primary culture of rat hepatocytes. The effect of OME and TBZ (75–250 μM) on cytochrome P450 1A1 (CYP1A1) expression was therefore studied in primary cultures of rat hepatocytes after 24 h, 48 h and 72 h of exposure. Both compounds provoked a dose- and time-dependent increase in CYP1A1 (EROD activity, protein and mRNA levels), but OME was less effective at all the concentrations and times tested. The mechanism of benzimidazole-mediated induction of CYP1A1 was investigated by comparison with 3-MC, a prototypical AhR ligand. As expected, OME and TBZ were unable to displace [ 3H]-TCDD from its binding sites to the AhR in competitive binding studies. Moreover, classic tyrosine kinase inhibitor herbimycin A (HA) inhibited the two benzimidazoles-mediated CYP1A1 inductions, but only partially inhibited the 3-MC-mediated one. Another two tyrosine kinase inhibitors, Lavendustin A (LA) and genistein (GEN), had no effect on CYP1A1 induction by benzimidazoles and 3-MC. These results are consistent with the implication of a tyrosine kinase, most probably the Src tyrosine kinase, in the mechanism of CYP1A1 induction in rat hepatocytes.

Galectin-3 expression in macrophages is signaled by Ras/MAP kinase pathway and up-regulated by modified lipoproteins

To study the signaling pathway involved in the regulation of galectin-3 expression we used phorbol ester to stimulate macrophage differentiation of THP-1 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) increased significantly the level of expression of galectin-3 in THP-1 cells. PMA-induced galectin-3 overexpression was blocked by: protein kinase C inhibitors staurosporine, calphostin C, and apigenin; tyrosine-specific protein kinase inhibitors genistein and tyrphostin A25; PD 98059, a selective inhibitor of mitogen-activated protein kinase (MAPK) kinase 1 (MEK1 or MKK1); and SB 203580, a specific inhibitor of p38 MAPK. Galectin-3 up-regulation was not affected by exposure to two inhibitors of cAMP-dependent protein kinase (PKA), H-89 and KT5720. Co-transfection of pPG3.5, a plasmid vector containing the rabbit galectin-3 promoter and the constructs pMCL-MKK1 N3 or pRC-RSV-MKK3Glu that constitutively express MKK1 and MKK3, raised the activity of galectin-3 promoter by 185% and 110%, respectively. Co-transfection with a Ha- Ras expression vector stimulated galectin-3 promoter activity approximately 10-fold. Expression of c- Jun or v- Jun raised the level of galectin-3 promoter activity more the three- and fourfold, respectively. Co-transfection of c- Jun and pPG3.5 5′-upstream deletion mutants resulted in a reduction of the galectin-3 promoter activity by 50% to 80%. Transfection of c- Jun, v- Jun or Ha- Ras increased significantly galectin-3 protein in THP-1 cells. These findings indicated that Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway plays an important role in the expression of galectin-3 in PMA-stimulated macrophages. We further investigated the effect of modified lipoproteins on galectin-3 expression in macrophages. Murine resident peritoneal macrophages loaded with acetylated low-density lipoprotein (AcLDL) or oxidized LDL (OxLDL) showed increased galectin-3 protein and mRNA. These results showed that treatment of macrophages with PMA or modified lipoproteins results in galectin-3 overexpression. These findings may explain the enhanced expression of galectin-3 in atherosclerotic foam cells and suggest that Ras/MAPK signal transduction pathway is involved in controlling this gene.

Protein tyrosine kinases and L-type Ca2+ currents in cells that have survived in epicardial border zone of canine infarcted heart.

Previously a reduction was shown in the density of the L-type Ca currents in cells that have survived in the epicardial border zone of the 5-day infarcted canine heart (IZ). A hyporesponsiveness of I(CaL) to beta-adrenergic stimulation in IZs versus cells from the noninfarcted heart (NZs) was also shown. To determine the role of protein tyrosine kinase (PTK) activity in this altered adrenergic response as well as in the reduced basal current function in IZs, the effects of genistein and T23, specific inhibitors of PTK, on basal I(CaL) in the absence and presence of isoproterenol (5 nM ) were studied using whole-cell patch-clamp techniques. Genistein reduction of I(CaL) was similar in NZs and IZs and was not mimicked by daidzein, an inactive analogue of genistein. Submaximal isoproterenol produced a small response in both cell types that was potentiated in the presence of genistein. T23 also reduced I(CaL) in both NZs and IZs; however, submaximal isoproterenol was not potentiated in its presence. In sum, basal I(CaL) is sensitive to genistein and T23, suggesting that persistent PTK activity contributes to I(CaL) in both NZs and IZs. With genistein but not with T23, there is an enhanced sensitivity of I(CaL) to isoproterenol in both NZs and IZs but peak I(CaL) is not fully restored in IZs. Thus, dysregulation of PTK activity cannot account for the reduced basal Ca currents or hyporesponsiveness of I(CaL) to isoproterenol in the cells that have survived in the infarcted heart.