ag-879 and staurosporine-aglycone

Neurotrophin receptor signaling has been increasingly recognized as an important factor in the development and progression of a variety of malignancies. In order to analyze the potential contribution of neurotrophin signaling to lymphoma cell survival, we investigated the role of a neurotrophin axis in promoting survival and proliferation of non-Hodgkin lymphoma (NHL) cells.. The role of neurotrophins in the survival and proliferation of NHL cells was determined by exposing cells to the Trk-specific inhibitor, K252a, and then performing (3)H-thymidine incorporation and Annexin-V/propidium iodide staining. The involvement of nuclear factor-kappaB (NF-kappaB) in this process was studied using Western blot, electrophoretic mobility shift assay, and immunofluorescence assays.. Here we demonstrate that both primary NHL cells and diffuse large B-cell lymphoma cell lines express Trk receptors and their neurotrophin ligands. Furthermore, these cells are sensitive to the Trk-specific inhibitor, K252a, as evidenced by the inhibition of proliferation and/or induction of apoptosis. Analysis of the mechanism into the effects of K252a revealed that, in the OCI-LY3 cell line, K252a induced a subnuclear distribution of NF-kappaB resulting in the sequestration of RelA in the nucleolus, thereby inhibiting NF-kappaB-dependent gene transcription. This results in the loss of interleukin-6 production; a known survival-promoting signal for OCI-LY3, as well as many primary diffuse large B-cell lymphomas.. Thus, Trk receptors represent a novel therapeutic target for the treatment of NHL.

Topics: Apoptosis; Autocrine Communication; B-Lymphocytes; Brain-Derived Neurotrophic Factor; Carbazoles; Cell Division; Cell Line, Tumor; Culture Media, Conditioned; DNA Replication; Humans; Indole Alkaloids; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Non-Hodgkin; Neoplasm Proteins; Nerve Growth Factor; Nerve Growth Factors; NF-kappa B; Receptors, Nerve Growth Factor; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Tyrphostins

The first morphological sign of testicular differentiation is the formation of testis cords. Prior to cord formation, newly specified Sertoli cells establish adhesive junctions, and condensation of somatic cells along the surface epithelium of the genital ridge occurs. Here, we show that Sertoli cell aggregation is necessary for subsequent testis cord formation, and that neurotrophic tyrosine kinase receptors (NTRKs) regulate this process. In a three-dimensional cell culture assay, immature rat Sertoli cells aggregate to form large spherical aggregates (81.36+/-7.34 microm in diameter) in a highly organized, hexagonal arrangement (376.95+/-21.93 microm average distance between spherical aggregates). Exposure to NTRK inhibitors K252a and AG879 significantly disrupted Sertoli cell aggregation in a dose-dependent manner. Sertoli cells were prevented from establishing cell-cell contacts and from forming spherical aggregates. In vitro-derived spherical aggregates were xenografted into immunodeficient nude mice to investigate their developmental potential. In controls, seminiferous tubule-like structures showing polarized single-layered Sertoli cell epithelia, basement membranes, peritubular myoid cells surrounding the tubules, and lumen were observed in histological sections. By contrast, grafts from treatment groups were devoid of tubules and only few single Sertoli cells were present in xenografts after 4 weeks. Furthermore, the grafts were significantly smaller when Sertoli cell aggregation was disrupted by K252a in vitro (20.87 vs 6.63 mg; P<0.05). We conclude from these results that NTRK-regulated Sertoli-Sertoli cell contact is essential to the period of extensive growth and remodeling that occurs during testicular tubulogenesis, and our data indicate its potential function in fetal and prepubertal testis differentiation.

Topics: Animals; Carbazoles; Cell Aggregation; Cell Communication; Cell Culture Techniques; Dose-Response Relationship, Drug; Indole Alkaloids; Male; Mice; Mice, Nude; Morphogenesis; Rats; Rats, Inbred Strains; Receptor, trkA; Seminiferous Tubules; Sertoli Cells; Testis; Transplantation, Heterologous; Tyrphostins

Neurotrophin-mediated signalling cascades can be initiated by activation of either the p75 neurotrophin receptor (p75(NTR)) or the more selective tyrosine kinase receptors. Previously, we demonstrated that nerve growth factor (NGF) increased the excitability of sensory neurons through activation of p75(NTR) to liberate sphingosine 1-phosphate. If neurotrophins can modulate the excitability of small diameter sensory neurons through activation of p75(NTR), then brain-derived neurotrophic factor (BDNF) should produce the same sensitizing action as did NGF. In this report, we show that focally applied BDNF increases the number of action potentials (APs) evoked by a ramp of depolarizing current by reducing the rheobase without altering the firing threshold. This increased excitability results, in part, from the capacity of BDNF to enhance a tetrodotoxin-resistant sodium current (TTX-R I(Na)) and to suppress a delayed rectifier-like potassium current (I(K)). The idea that BDNF acts via p75(NTR) is supported by the following observations. The sensitizing action of BDNF is prevented by pretreatment with a blocking antibody to p75(NTR) or an inhibitor of sphingosine kinase (dimethylsphingosine), but not by inhibitors of tyrosine kinase receptors (K252a or AG879). Furthermore, using single-cell RT-PCR, neurons that were sensitized by BDNF expressed the mRNA for p75(NTR) but not TrkB. These results demonstrate that neurotrophins can modulate the excitability of small diameter capsaicin-sensitive sensory neurons through the activation of p75(NTR) and its downstream sphingomyelin signalling cascade. Neurotrophins released upon activation of a variety of immuno-competent cells may be important mediators that give rise to the enhanced neuronal sensitivity associated with the inflammatory response.

Topics: Action Potentials; Animals; Antibodies; Brain-Derived Neurotrophic Factor; Carbazoles; Enzyme Inhibitors; Gene Expression Regulation; Indole Alkaloids; Male; Neurons, Afferent; Rats; Rats, Sprague-Dawley; Receptor, Nerve Growth Factor; Receptor, trkB; RNA, Messenger; Signal Transduction; Sodium Channels; Sphingomyelins; Sphingosine; Tetrodotoxin; Tyrphostins

Nerve growth factor (NGF) is an important substance in the skin, where it modulates nerve maintenance and repair. However, the direct link between NGF and pruritic diseases such as atopic dermatitis is not yet fully understood. Our previous study showed that NGF plays an important role in the pathogenesis of atopic dermatitis-like skin lesions in NC/Nga mice. NGF mediates its effects by binding to two classes of transmembrane receptors, a high-affinity receptor (tropomyosin-related kinase A, TrkA) and a low-affinity receptor (p75).. To determine the significance of NGF receptors in the pathogenesis of atopic dermatitis, the effects of TrkA inhibitors AG879 and K252a on the symptoms of NC/Nga mice were evaluated.. Male NC/Nga mice with severe skin lesions were used. AG879 or K252a was applied to the rostral part of the back of mice five times a week. The dermatitis score for the rostral back was assessed once a week. The scratching behaviour was measured using an apparatus, MicroAct (Neuroscience, Tokyo, Japan). Immunofluorescence examinations were made in the rostral back skin for nerve fibres, NGF and TrkA receptor.. Repeated applications of AG879 or K252a significantly improved the established dermatitis and scratching behaviour, and decreased nerve fibres in the epidermis. NGF was observed more weakly in keratinocytes, and a lower expression of TrkA was observed in stratum germinativum of the epidermis of mice treated with AG879 or K252a compared with those treated with vehicle.. We suggest that NGF plays an important role in the pathogenesis of atopic dermatitis-like skin lesions via the high-affinity NGF receptor. These findings provide a new potential therapeutic approach for the amelioration of symptoms of atopic dermatitis.

Topics: Animals; Carbazoles; Dermatitis, Atopic; Disease Models, Animal; Drug Evaluation, Preclinical; Enzyme Inhibitors; Indole Alkaloids; Male; Mice; Mice, Inbred Strains; Pruritus; Receptors, Nerve Growth Factor; Treatment Outcome; Tyrphostins

The neurotrophin nerve growth factor (NGF) has been implicated as a mediator in allergic asthma. Direct evidence that inhibition of NGF-induced activation of neurotrophin receptors leads to improvement of airway symptoms is lacking. We therefore studied the effects of inhibitors of NGF signal transduction on the development of airway hyper-responsiveness (AHR) and pulmonary inflammation in a guinea-pig model for allergic asthma.. Airway responsiveness to the contractile agonist histamine was measured in vivo in guinea-pigs that were sensitized and challenged with ovalbumin (OVA). Inflammatory cell influx and NGF levels were determined in bronchoalveolar lavage fluid (BALF). Substance P, a key mediator of inflammation, was measured in lung tissue by radioimmunoassay, while substance P immunoreactive neurons in nodose ganglia were measured by immunohistochemistry.. OVA challenge induced an AHR after 24 h in OVA-sensitized guinea-pigs. This coincided with an increase in the amount of NGF in BALF. Simultaneously, an increase in the percentage of substance P immunoreactive neurons in the nodose ganglia and an increase in the amount of substance P in lung tissue were found. We used tyrosine kinase inhibitors to block the signal transduction of the high-affinity NGF receptor, tyrosine kinase A (trkA). Treatment with the tyrosine kinase inhibitors (K252a or tyrphostin AG879) both inhibited the development of AHR, and prevented the increase in substance P in the nodose ganglia and lung tissue completely whereas both inhibitors had no effect on baseline airway resistance. Neither treatment with K252a or tyrphostin AG879 changed the influx of inflammatory cells in the BALF due to allergen challenge.. We conclude that substance P plays a role in the induction of AHR in our model for allergic asthma which is most likely mediated by NGF. As both tyrosine kinase inhibitors AG879 and K252a show a similar inhibitory effect on airway function after allergen challenge, although both tyrosine kinase inhibitors exhibit different non-specific inhibitory effects on targets other than trkA tyrosine kinases, it is likely that the induction of substance P derived from sensory nerves is mediated by NGF via its high-affinity receptor trkA.

Topics: Animals; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Carbazoles; Disease Models, Animal; Enzyme Inhibitors; Female; Guinea Pigs; Immunohistochemistry; Indole Alkaloids; Lung; Male; Nerve Growth Factor; Neurons; Nodose Ganglion; Ovalbumin; Receptor, trkA; Signal Transduction; Substance P; Tyrphostins

The sIgG(+) lymphoblastoid B cell line CESS spontaneously produces a high amount of NGF and expresses both high affinity (p140(Trk-A)) and low affinity (p75(NTR)) NGF receptors. Blocking NGF signals with neutralizing antibodies or specific Trk-A inhibitors induces a rapid phosphorylation of antiapoptotic Bcl-2 protein, followed by caspase activation, and apoptotic death of CESS cells. Bcl-2 phosphorylation in several sites within a approximately 60 aa "loop" domain of protein is known to regulate its antiapoptotic function. Accordingly, CESS cells expressing the loop deletional mutant cDNA constructs Bcl-2 Delta40-91 were completely resistant to apoptosis induced by NGF withdrawal, indicating that Bcl-2 phosphorylation is a critical event. NGF withdrawal induces p38 MAPK, but not JNK, activation in CESS cells, and SB203580, a specific inhibitor of p38 MAPK, is able to prevent both Bcl-2 phosphorylation and apoptosis, indicating that p38 MAPK is the enzyme responsible for these events.

Topics: Apoptosis; B-Lymphocytes; Carbazoles; Cell Division; Enzyme Activation; Enzyme Inhibitors; Genes, bcl-2; Humans; Indole Alkaloids; JNK Mitogen-Activated Protein Kinases; Kinetics; Mitogen-Activated Protein Kinases; Nerve Growth Factor; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Receptor, trkA; Receptors, Nerve Growth Factor; Sequence Deletion; Signal Transduction; Tumor Cells, Cultured; Tyrphostins

A 96-well microtiter enzyme-linked immunosorbent assay was developed to assay the activity of the cytoplasmic domain of trkA tyrosine kinase. The assay involves immobilization of phospholipase C- gamma/glutathione S-transferase fusion protein on a microtiter plate, addition of the kinase reaction mixture, and detection by an antibody to phosphotyrosine followed by an alkaline phosphatase-conjugated second antibody. The substrate used in this system, phospholipase C-gamma, is one of several biologically important substrates for the phosphorylation reaction of receptor-linked tyrosine kinases. The assay was then used to characterize kinase inhibitory activities of various small molecules including analogs of K-252a.

Topics: Adenosine Triphosphate; Benzylidene Compounds; Carbazoles; Cations, Divalent; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Humans; Indole Alkaloids; Kinetics; Nitriles; Receptor, trkA; Recombinant Proteins; Type C Phospholipases; Tyrphostins