herbimycin and Lymphoma--Large-B-Cell--Diffuse

Anaplastic large cell lymphoma (ALCL) exhibiting the t(2;5) translocation is characterized by the resulting expression of the oncogenic fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) gene product. The ALK domain of NPM-ALK contains kinase activity, which is responsible for the autophosphorylation of tyrosine residues of the oncogenic protein and phosphorylation of SH2-protein substrates. Herbimycin A is a general protein tyrosine kinase inhibitor active as an antiproliferative compound against different types of mammalian cells. Herbimycin A inhibited the NPM-ALK-associated autophosphorylating activity in an in vitro cell-free kinase assay. The inhibition was specific when tested against other kinase inhibitors and extended to other cell lines derived from t(2;5)-ALCL. SUDHL-1 cells showed increasing percentage of cells in G(1) after 18 h of incubation with a dose of herbimycin A. NPM-ALK, Akt, and pAkt were down-regulated after 24 h of incubation with herbimycin A. Apoptosis was observed only if the dose of inhibitor was given every 12 h for prolonged time. Our results show that herbimycin A interferes with NPM-ALK and Akt pathways in SUDHL-1 cells. It seems that prolonged inhibition of these biochemical pathways may lead to cell cycle arrest and apoptosis. This study supports the idea of investigating protein kinase inhibitors as therapeutic compounds for t(2;5)-ALCL.

Topics: Anaplastic Lymphoma Kinase; Apoptosis; Benzoquinones; Blotting, Western; Cell Cycle; Enzyme Inhibitors; Humans; Indoles; Lactams, Macrocyclic; Lymphoma, Large B-Cell, Diffuse; Oncogene Proteins, Fusion; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein-Tyrosine Kinases; Quinones; Receptor Protein-Tyrosine Kinases; Rifabutin; Tumor Cells, Cultured

The t(2;5)(p23;q35) translocation creates a fusion gene NPM-ALK (p80) that encodes a product with tyrosine kinase activity believed to play an important role in development of anaplastic large cell lymphoma (ALCL). Our study was aimed to analyze tyrosine kinase activity and phosphotyrosine in ALCLs. We were also interested in determining the effect of tyrosine kinase inhibitors on survival of ALCL.. Eleven cases of ALCL and three ALCL cell lines with t(2;5)(Karpas-299, SUPM2, SU-DHL-1) and 10 Hodgkin's disease (HD) samples were stained with anti-phosphotyrosine antibody. The tyrosine kinase activity, p80 phosphorylation, and the apoptotic effects of two tyrosine kinase inhibitors, herbimycin A and STI-571, were determined on ALCL cell lines.. Herbimycin A had showed both a time- and dose-dependent apoptotic effect on all three cell lines, while STI-571 demonstrated a minimal effect. Following herbimycin A treatment, a decrease in tyrosine kinase activity in the ALCL cell lines and inhibition in NPM-ALK (p80) autophosphorylation was demonstrated by immunoprecipitation and Western blotting. Herbimycin A-induced apoptosis was accompanied by caspase-3 activation. Furthermore, apoptosis induced by herbimycin A was blocked by both z-VAD-FMK and z-DEVD-FMK, suggesting a critical role of caspases.. These findings indicate that tyrosine kinase activity is a common characteristic of ALCLs and necessary for ALCL cell survival. These findings further suggest that therapies targeting tyrosine kinases, including p80, may have clinical utility.

Topics: Anaplastic Lymphoma Kinase; Antineoplastic Agents; Apoptosis; Benzamides; Benzoquinones; Caspases; Chromosomes, Human, Pair 2; Chromosomes, Human, Pair 5; Enzyme Inhibitors; Hodgkin Disease; Humans; Imatinib Mesylate; Immunohistochemistry; Lactams, Macrocyclic; Lymphoma, Large B-Cell, Diffuse; Oncogene Proteins, Fusion; Phosphorylation; Piperazines; Protein-Tyrosine Kinases; Pyrimidines; Quinones; Receptor Protein-Tyrosine Kinases; Rifabutin; Translocation, Genetic; Tumor Cells, Cultured

This study describes the activation conditions for tumor necrosis factor-alpha (TNF alpha) production in myelomonocytic U937 cells and human primary peripheral blood monocytes in response to lipopolysaccharide (LPS) and/or phorbol 12-myristate 13-acetate (PMA). PMA itself induced only low levels of TNF alpha production with delayed kinetics (e.g. 0.758 +/- 0.128 ng/ml from U937 cells after 48 h) while LPS induced greater levels of TNF alpha production in less time (e.g. 2.083 +/- 0.96 ng/ml from monocytes in 24 h). Pharmacological agents with various molecular sites of action were used to validate the two systems, with the protein serine-threonine kinase inhibitors staurosporine and Ro-31-8220, the protein tyrosine kinase inhibitor herbimycin A (HBA) and dexamethasone exhibiting the greatest potency (IC50S 5-350 nM). In contrast to the effect on TNF alpha production, PMA induced strong phosphorylation/activation of p42/p44mapk in monocytes by 10 min determined in a mobility shift assay, while LPS was a weaker inducer. Additionally, staurosporine (to LPS and PMA) and HBA (to LPS only) inhibited the activation of these mitogen-activated protein kinase (MAPK) isoforms at doses 10-100 fold higher than those required to inhibit maximal TNF alpha production. These data indicate the involvement of the p42/p44mapk signalling pathway in LPS-induced pro-inflammatory cytokine production but suggest that other signalling pathways are also implicated in this phenomenon.

Topics: Benzoquinones; Binding Sites; Calcium-Calmodulin-Dependent Protein Kinases; Dexamethasone; Enzyme Activation; Enzyme Inhibitors; Humans; Interleukin-10; Interleukin-4; Lactams, Macrocyclic; Lipopolysaccharides; Lymphoma, Large B-Cell, Diffuse; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Monocytes; Quinones; Rifabutin; Signal Transduction; Staurosporine; Structure-Activity Relationship; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha