calcimycin and Lymphoma--Large-B-Cell--Diffuse

In the U937 histiocytic cell line, we investigated the effect of calcium-mobilizing agents with or without tumor necrosis factor-alpha (TNF) on the regulation of the synthesis of plasminogen activator inhibitor-type 1 (PAI-1). Cultured U937 cells were stimulated with ionophore A23187 and thapsigargin with or without TNF. The response was analyzed in terms of cytosolic calcium mobilization, PAI-1 accumulation in the medium, and PAI-1 mRNA expression. The study was extended to urokinase (uPA) secretion and surface expression of its receptor (uPAR). Using Fluo-3 as a calcium-indicator dye to measure cytosolic calcium mobilization, we showed by flow cytometry that both agents mobilized calcium in a dose-dependent manner. TNF provoked a slight calcium mobilization that was also observed by digital imaging microscopy. Association of TNF with the calcium-mobilizing agents potentiated the calcium mobilization. Both calcium-mobilizing agents induced at 18 hours a dose-dependent accumulation of PAI-1 in culture medium, whereas uPA was not affected. TNF alone induced a more marked accumulation of PAI-1 than of uPA. Association of TNF with the agents induced a PAI-1 response that was more than additive of the two, whereas the secretion of uPA was not enhanced. Membrane expression of uPAR, measured by flow cytometry, tended to be slightly augmented by the calcium-mobilizing agents only. All the treatments resulted in a significant increase in PAI-1 mRNA level at 3 hours after the stimulation, which was very marked when calcium-mobilizing agents were present. Incubation of U937 cells in a calcium-free medium totally prevented both the mRNA expression and accumulation of PAI-1 induced by calcium-mobilizing agents and, to lesser extent, that induced by TNF. The increase in PAI-1 mRNA expression did not require de novo protein synthesis, as cycloheximide did not suppress the increase in PAI-1 mRNA induced by calcium-mobilizing agents. It is concluded that, in U937 cells, calcium triggers a pathway that upregulates PAI-1 synthesis and positively interacts with the TNF-induced pathway that stimulates PAI-1 synthesis. As uPA and uPAR were differently affected, it is suggested that an increase in cytosolic calcium leads to a reduced pericellular proteolysis.

Topics: Calcimycin; Calcium; Cytosol; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Histiocytes; Humans; Interleukin-1; Lymphoma, Large B-Cell, Diffuse; Neoplasm Proteins; Plasminogen Activator Inhibitor 1; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA, Messenger; Signal Transduction; Terpenes; Thapsigargin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Urokinase-Type Plasminogen Activator

We have previously reported that dimethylsulfoxide-differentiation of U937 cells induced significant A23187-stimulatable arachidonate mobilization, consistent with characteristics of cytosolic phospholipase A2 (Rzigalinski, B.A. and Rosenthal, M.D. (1994) Biochim. Biophys. Acta 1223, 219-225). The present report demonstrates that differentiated cells attained higher elevations of intracellular free calcium in response to A23187 and thapsigargin, consistent with enhancement of the capacitative calcium influx pathway. Differentiation induced as significant increase in the size of the intracellular calcium stores, as well as in the capacity for store-activated calcium influx. Alterations in the capacitative calcium influx pathway were coupled to differentiation-induced activation of cPLA2 and mobilization of arachidonate in response to thapsigargin and fMLP stimulation. Although cPLA2 activity is often associated with influx of extracellular calcium, arachidonate mobilization in response to thapsigargin or fMPL was not simply a consequence of calcium influx. Assessment of intracellular free calcium elevations during thapsigargin or fMPL-induced stimulation suggest that a low level of arachidonic acid release was initiated upon release of intracellular store calcium. This initial release of arachidonate was unaffected by inhibition of calcium influx with nickel, EGTA, or SKF96365. Arachidonate release was observed when extracellular calcium was replaced with extracellular strontium, suggesting activation of the cytosolic PLA2 rather than secretory PLA2. Inhibition of PLA2 with prostaglandin B oligomer prevented both thapsigargin and fMLP-stimulated influx of extracellular calcium. Furthermore, exogenous free arachidonate stimulated influx of extracellular calcium in differentiated U937 cells. These results suggest that cPLA2-mediated release of free arachidonate may participate in the formation of a calcium influx factor which controls influx of extracellular calcium through store-controlled channels in the plasma membrane.

Topics: Arachidonic Acid; Biological Transport; Calcimycin; Calcium; Cell Differentiation; Dose-Response Relationship, Drug; Lymphoma, Large B-Cell, Diffuse; N-Formylmethionine Leucyl-Phenylalanine; Strontium; Terpenes; Thapsigargin; Tumor Cells, Cultured

Calpain secreted by lymphoid (MOLT-3, M.R.) or monocytic (U-937, THP-1) cell lines activated with PMA and A23187 degraded myelin antigens. The degradative effect of enzymes released in the extracellular medium was tested on purified myelin basic protein and rat central nervous system myelin in vitro. The extent of protein degradation was determined by SDS-PAGE and densitometric analysis. Various proteinase inhibitors were used to determine to what extent protein degradation was mediated by calpain and/or other enzymes. Lysosomal and serine proteinase inhibitors inhibited 20-40% of the myelin-degradative activity found in the incubation media of cell lines, whereas the calcium chelator (EGTA), the calpain-specific inhibitor (calpastatin), and a monoclonal antibody to m calpain blocked myelin degradation by 60-80%. Since breakdown products of MBP generated by calpain may include fragments with antigenic epitopes, this enzyme may play an important role in the initiation of immune-mediated demyelination.

Topics: Animals; Antibodies, Monoclonal; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Chelating Agents; Culture Media, Conditioned; Demyelinating Diseases; Egtazic Acid; Humans; Leukemia-Lymphoma, Adult T-Cell; Lymphoma, Large B-Cell, Diffuse; Monocytes; Myelin Basic Protein; Myelin Sheath; Neoplasm Proteins; Protease Inhibitors; Rabbits; Rats; T-Lymphocytes; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Differentiation of U937 cells with phorbol myristate acetate (PMA) induces high stimulation by concanavalin A of the respiratory burst as well as an increase in concanavalin A-binding cell capacity. New concanavalin A-binding proteins are detected as differentiated U937 cells acquire their capacity to be activated by concanavalin A. We identified several concanavalin A-binding proteins, of molecular mass 30-200 kDa, in PMA-differentiated cells, but only some of them seem to be directly related to the concanavalin A effect on the respiratory burst. One of these candidates could be a glycoprotein with an apparent molecular mass of 140 kDa which behaved as a major concanavalin A-binding protein and is expressed on differentiated cells at the time these cells respond maximally to concanavalin A.

Topics: Calcimycin; Cell Differentiation; Cell Line; Concanavalin A; Humans; Kinetics; Lymphoma, Large B-Cell, Diffuse; N-Formylmethionine Leucyl-Phenylalanine; Receptors, Concanavalin A; Superoxides; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Zymosan