monensin and Lymphoma

Topics: Amiloride; Animals; Calcimycin; Calcium; Carrier Proteins; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Dimethyl Sulfoxide; Electrolytes; Erythropoiesis; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Lipopolysaccharides; Lymphocytes; Lymphoma; Mice; Monensin; Ouabain; Protons; Sodium; Sodium-Hydrogen Exchangers; Sodium-Potassium-Exchanging ATPase

Monensin, a Na+ ionophore, regulates many cellular functions, including apoptosis. We investigated the in vitro antiproliferative effect of monensin on nine human lymphoma cell lines. Monensin significantly inhibited the proliferation of all the lymphoma cell lines examined with a 50% inhibition concentration of about 0.5 micromol/l, and induced a G1 and/or a G2-M phase arrest in these cell lines. To address the antiproliferative mechanism of monensin, we examined the effect of this drug on cell-cycle-related proteins in CA46 cells (both G1 and G2 arrest) and Molt-4 cells (G2 arrest). Treatment with monensin for 72 h decreased CDK4 and cyclin A levels in CA46 cells, and cdc2 levels in Molt-4 cells. In monensin-treated CA46 cells, increased p21-CDK2, p27-CDK2 and p27-CDK4 complex forms were observed. And, in monensin-treated Molt-4 cells, increased p21-cdc2 complex form was detected. Furthermore, the activities of CDK2- and CDK4-associated kinases were reduced in association with Rb hypophosphorylation in monensin-treated CA46 cells. The activity of cdc2-associated kinase was decreased in both cell lines, which was accompanied by induction of Wee1. Also, monensin induced apoptosis in these cell lines, as evidenced by annexin V binding assay and flow cytometric detection of sub-G1 DNA content. This apoptotic process was associated with loss of mitochondria transmembrane potential (Delta(psi)m). Taken together, these results demonstrated for the first time that monensin potently inhibits the proliferation of human lymphoma cell lines via cell cycle arrest and apoptosis.

Topics: Apoptosis; Blotting, Western; CDC2-CDC28 Kinases; Cell Division; Cyclin A; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Humans; Interphase; Ionophores; Lymphoma; Membrane Potentials; Monensin; Tumor Cells, Cultured

Proprotein convertases are responsible for the endoproteolytic activation of proproteins in the secretory pathway. The most recently discovered member of this family, lymphoma proprotein convertase (LPC), is a type-I transmembrane protein. Previously, we have demonstrated that its cytoplasmic tail is palmitoylated. In this study, we have identified the two most proximal cysteine residues in the cytoplasmic tail as palmitoylation sites. Substitution of either cysteine residue by alanine interfered with palmitoylation of the other. Palmitoylation of LPC was found to be sensitive to the protein palmitoyltransferase inhibitor tunicamycin but not cerulenin. It was also insensitive to the drugs brefeldin A, monensin and cycloheximide, indicating that the modification occurs in a late exocytic or endocytic compartment. Turnover of palmitoylated LPC is significantly faster (t(1/2) approximately 50 min) than that of the LPC polypeptide backbone (t(1/2) approximately 3 h), suggesting that palmitoylation is reversible. Abrogation of palmitoylation reduced the half-life of the LPC protein, but did not affect steady-state localization of LPC in the trans-Golgi network. Finally, LPC could not be detected in detergent-resistant membrane rafts. Taken together, these results suggest that dynamic palmitoylation of LPC is important for stability, but does not function as a dominant trafficking signal.

Topics: Amino Acid Substitution; Brefeldin A; Cerulenin; Cycloheximide; Cysteine; Cytosol; Enzyme Stability; Exocytosis; Fluorescent Antibody Technique, Indirect; Half-Life; Lymphoma; Membrane Microdomains; Monensin; Mutation; Palmitic Acid; Protein Processing, Post-Translational; Protein Sorting Signals; Protein Structure, Tertiary; Protein Transport; Serine Endopeptidases; Subtilisins; trans-Golgi Network; Tunicamycin

Rat neutrophils stimulated with phorbol 12-myristate 13-acetate, Bacillus Calmette-Guérin, zymosan A, and beta-1,3-D-glucan from Alcaligenes faecalis showed cytotoxicity to various tumor cells. Hydrogen peroxide was shown to be an effector molecule in tumor cytotoxicity by inhibition using various active oxygen scavengers. The following findings suggest that tumor cytotoxicity by rat neutrophils stimulated with the four reagents mentioned above is regulated by Na+/H+ exchange: (a) an increase in extracellular pH (pHo) from 6.5 to 8.0 resulted in enhancement of both tumor cytotoxicity and H2O2 production; (b) amiloride and its derivatives, inhibitors of Na+/H+ exchange, inhibited both functions of neutrophils mentioned above; (c) amiloride reduced intracellular pH (pHi) of neutrophils stimulated with the four reagents; (d) a decrease in the extracellular concentration of Na+ [( Na+]o) inhibited H2O2 production; (e) monensin, a Na+/H+ exchange ionophore, enhanced tumor cytotoxicity by neutrophils.

Topics: Amiloride; Animals; Carrier Proteins; Cell Line; Cell Survival; Glucans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Kinetics; Lymphoma; Mice; Mice, Inbred BALB C; Monensin; Mycobacterium bovis; Neutrophils; Rats; Rats, Inbred Strains; Sodium; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate; Zymosan

Rat Nb2 node lymphoma cells proliferate in response to lactogens, but the signal transduction mechanism involved remains unclear. Specific binding, internalization, and degradation of ovine PRL (oPRL) were examined under a variety of experimental conditions to characterize the metabolism of receptor-bound hormone by these cells. Stationary-phase cells were incubated with [125I]oPRL in Fischer's medium containing horse serum. Cell suspensions were centrifuged, and the cell pellets were assayed to determine specific cell-associated radioactivity. Internalized ligand was measured by exposing the cells to an acidic buffer before centrifugation to dissociate hormone from plasma membrane receptors, and cell-surface ligand was calculated by subtracting internalized hormone from the total [125I]oPRL bound by the cells. Hormone degradation was assessed by measuring the radioactivity in an acid-soluble fraction prepared from the incubation medium. Endocytosis of [125I]oPRL was observed within 30 min at 37 C, and the internalized component accounted for approximately 50% of the bound hormone under steady-state conditions. Hormone degradation was detectable within 1 h at 37 C and continued at a relatively linear rate thereafter; by 4 h, 8% of the added [125I]oPRL was acid soluble. Chloroquine (0.2 mM), methylamine (20 mM) and monensin (20 microM) prevented [125I]oPRL degradation and elevated both cell-surface and intracellular hormone 2-fold during a 4-h incubation. Leupeptin (0.2 mM) decreased degradation by only 15% under the same conditions. Phorbol 12-myristate 13-acetate (PMA; 20 nM), a comitogen for lactogen-stimulated Nb2 cells, increased cell-surface hormone by 20% and decreased intracellular hormone by a corresponding amount 1 h after administration. Calcium ionophore A23187 (1 microM) produced similar changes, and a synergistic effect was noted when cells were exposed to both agents for 4 h. Amiloride (125 microM), an inhibitor of Nb2 cell mitogenesis, decreased [125I]oPRL degradation by 25% during a 4-h incubation. This response was abolished when the cells were exposed simultaneously to PMA. These experiments demonstrate that receptor-bound oPRL is rapidly internalized and extensively degraded via the endosome-lysosome pathway when Nb2 cells are maintained at 37 C. The inhibitory effect of PMA on oPRL internalization may help to explain the comitogenic action of this phorbol on Nb2 cells. Since amiloride also produced major changes in oPRL metabolism, pos

Topics: Amiloride; Animals; Calcimycin; Cell Division; Cell Membrane; Chloroquine; Endocytosis; Kinetics; Leupeptins; Lymphoma; Methylamines; Mitosis; Monensin; Prolactin; Rats; Receptors, Prolactin; Sheep; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Murine natural killer (NK) cell-mediated inhibition of growth of a yeast-like target cell, Cryptococcus neoformans, was completely abrogated by blocking the effector cell secretory process with monensin. Therefore, further studies were performed to determine the ability of various cytoplasmic fractions of NK cells to mediate inhibition of cryptococcal growth. Percoll-fractionated homogenates of rat LGL tumor cells demonstrated that the granule-containing fractions plus three additional sets of less dense cytoplasmic fractions displayed anti-cryptococcal activity; whereas only the cytoplasmic granule-containing fractions had cytotoxic activity against YAC-1 tumor cell and sheep erythrocyte targets. Maximal cryptococcal growth inhibition induced by LGL granules occurred after a 1 h incubation, required the presence of Ca2+ (1.0 mM) or Mg2+ (0.5 mM or 5.0 mM), and was completely abrogated in the presence of rabbit anti-LGL granule IgG. Cytolysin, the granule component which mediates tumor cell and sheep erythrocyte lysis, effectively limited the growth of cryptococci. Since Percoll gradient fractionation of the LGL homogenates demonstrated three separate peaks of anti-cryptococcal activity other than the granule peak, it is possible that the cytolysin-containing granules are not the only subcellular component of NK cells playing a role in inhibition of C. neoformans growth.

Topics: Animals; Calcium; Colony Count, Microbial; Cryptococcus; Cryptococcus neoformans; Cytoplasm; Cytoplasmic Granules; Cytotoxins; Immunoglobulin G; Killer Cells, Natural; Lymphoma; Mice; Monensin; Rats; Rats, Inbred F344; Splenic Neoplasms; Tumor Cells, Cultured

Topics: Animals; Biological Transport; Cells, Cultured; Glycoproteins; Golgi Apparatus; H-2 Antigens; Immunoglobulin Heavy Chains; Immunoglobulin M; Lymphocytes; Lymphoma; Membrane Proteins; Mice; Mice, Inbred BALB C; Monensin