herbimycin and Lymphoma--B-Cell

The effect of lauryl gallate (antioxidant E-312) has been studied on the mouse B-cell lymphoma line Wehi 231. This compound is able to inhibit protein tyrosine kinases (PTKs) in whole cells and in crude extracts with a better efficiency than other well-known PTK inhibitors such as herbimycin or genistein. Initial events triggered upon the incubation of cells with lauryl gallate in phosphate-buffered saline (up to 1 h) include the inhibition of tyrosine phosphorylation, discharge of the mitochondrial transmembrane potential, and induction of mRNA for Bcl-2. Long-term cultures in complete medium supplemented with fetal calf serum (up to 24 h) in the presence of this compound exhibit clear apoptotic features such as increase in phosphatidylserine in the cell surface, decrease in the functionality of mitochondria, cytochrome c release to the cytosol, activation of caspases, hypodiploidy, and oligonucleosomal breakdown of DNA. Comparison between Wehi cells overexpressing Bcl-2 (Wehi-bcl-2) with Wehi-neo cells shows a delay in the manifestations of the apoptotic signs, indicating that Bcl-2 has a partial protective effect on the apoptosis induced by lauryl gallate. The proapoptotic effect of lauryl gallate is not dependent on DNA or protein synthesis, is not blocked by the chelation of calcium, and is not reverted by N-acetylcysteine.

Topics: Acetylcysteine; Adenosine Triphosphate; Animals; Antioxidants; Apoptosis; Benzoquinones; Blotting, Western; Calcium; Caspase 3; Caspases; Cattle; Cell Membrane; Cell Separation; Cytochrome c Group; Cytosol; Diploidy; DNA; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Flow Cytometry; Food Preservatives; Gallic Acid; Glutathione; Herbicides; Kinetics; Lactams, Macrocyclic; Lymphoma, B-Cell; Membrane Potentials; Mice; Mice, Inbred BALB C; Mitochondria; Phosphatidylserines; Poly(ADP-ribose) Polymerases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-2; Quinones; Reverse Transcriptase Polymerase Chain Reaction; Rifabutin; RNA, Messenger; Sodium Chloride; Time Factors; Transfection; Tumor Cells, Cultured

In addition to providing the framework for peptide presentation, major histocompatibility complex class I (MHC-I) molecules can act as signal transducing molecules in lymphoid cells. Here we show that the mobilization of intracellular calcium, which follows crosslinking of MHC-I molecules on human B lymphoma cells, is dependent on protein tyrosine kinases and the phosphatidylinositol 3 (PI-3) kinase. Functional studies showed that MHC-I crosslinking induced almost complete inhibition of the spontaneous proliferation of the B lymphoma cells as early as 6 h post-crosslinking and apoptosis 24 h post-crosslinking. Preincubation with either protein tyrosine kinase or protein serine/threonine kinase inhibitors reduced the MHC-I-induced apoptosis to background levels, whereas inhibition of PI-3 kinase had no effect. These data demonstrate a pivotal role for protein tyrosine and serine/threonine kinases in MHC-I-mediated apoptosis in human B-cells and suggest the presence of several MHC-I signaling pathways leading to diverse effects in these cells.

Topics: Androstadienes; Apoptosis; Benzoquinones; beta 2-Microglobulin; Calcium; Cell Division; Chromones; Enzyme Activation; Histocompatibility Antigens Class I; Humans; Lactams, Macrocyclic; Lymphoma, B-Cell; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-2; Quinones; Receptor Aggregation; Rifabutin; Signal Transduction; Time Factors; Tumor Cells, Cultured; Wortmannin

BAL17 cells pulsed with goat anti-IgM or anti-IgD as antigens stimulated a goat IgG specific T cell clone in terms of inositol phosphate production. The antigen-presenting capacity of BAL17 cells was inhibited by pretreatment with the tyrosine kinase inhibitors herbimycin A or genistein. Furthermore, ligand-induced capping and endocytosis of membrane immunoglobulin, monitored at the single cell level, was also blocked by herbimycin A. These results indicate that tyrosine phosphorylation plays an important role in receptor-mediated antigen presentation by B cells.

Topics: Antibiotics, Antineoplastic; Antigen-Presenting Cells; Benzoquinones; Endocytosis; Enzyme Activation; Immunologic Capping; Lactams, Macrocyclic; Lymphoma, B-Cell; Protein-Tyrosine Kinases; Quinones; Receptors, Antigen, B-Cell; Rifabutin; Tumor Cells, Cultured

BAL17 B lymphoma cells, representing mature B lymphocytes, were used to analyze the role of tyrosine kinase in B cell activation. Anti-IgM-induced tyrosine phosphorylation was inhibited by preincubation of cells with tyrosine kinase inhibitor herbimycin A. Enzymatic activity of lyn protein was also inhibited by this drug, accompanied by down-regulation of p53lyn and p56lyn. However, a protein kinase C-mediated event was intact in the herbimycin A-pretreated cells, suggesting that the inhibitor acts selectively on tyrosine kinase. Anti-IgM failed to stimulate herbimycin A-pretreated cells to induce increases in inositol phospholipid metabolism or increased [Ca2+]i, whereas aluminum fluoride-induced metabolism was not altered. Moreover, membrane IgM density as revealed by flow cytometry was not changed by herbimycin A. These results indicate that tyrosine kinase(s) participates in the coupling of an Ag receptor cross-linkage to phospholipase C activation through a phosphorylation event in B lymphoma cells.

Topics: Animals; B-Lymphocytes; Benzoquinones; Calcium; Down-Regulation; Immunoglobulin M; In Vitro Techniques; Inositol Phosphates; Lactams, Macrocyclic; Lymphocyte Activation; Lymphoma, B-Cell; Mice; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Receptors, Antigen, B-Cell; Rifabutin; Signal Transduction; src-Family Kinases; Tumor Cells, Cultured; Type C Phospholipases