herbimycin and Burkitt-Lymphoma

Signals transduced through CD40 rescue cells of the Ramos-Burkitt lymphoma (Ramos-BL) B cell line from surface immunoglobulin M (sIgM)-triggered growth arrest and apoptosis. This study investigates whether protein tyrosine kinase (PTK) activity and tyrosine phosphorylation on p95(vav) and on the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3 kinase) play a role in the regulation of Ramos-BL B cell survival. The PTK inhibitor herbimycin A (HA) triggers significant growth arrest prior to apoptosis from the G1-phase of the cell cycle, indicating that tyrosine phosphorylation of key proteins is critical for Ramos-BL cell cycle progression and survival. Indeed, signals transduced through CD40 fail to rescue Ramos-BL B cells from HA-triggered growth arrest and apoptosis. Since Vav and PI3 kinase are intimately involved in the regulation of cellular growth, their tyrosine phosphorylation status was determined in unstimulated and anti-IgM- and anti-CD40-treated Ramos-BL B cells: Vav and p85 are devoid of tyrosine-phosphorylated epitopes in control cells whereas p85, but not Vav, is significantly phosphorylated following ligation of sIgM and anti-CD40 triggers tyrosine phosphorylation on both proteins. Thus, tyrosine-phosphorylated Vav may be a critical effector of CD40-mediated survival. As tyrosine-phosphorylated PI3 kinase is common to both sIgM-triggered death and CD40-triggered survival pathways, its lipid kinase activity was correlated with tyrosine phosphorylation on p85: Ramos-BL B cells exhibit high basal levels of PI3 kinase activity, determined by immunoprecipitation with anti-p85 and 32P incorporation into phosphatidylinositol, which is not significantly affected by stimulation with anti-IgM but which is elevated by 36 +/- 2.9% following ligation of CD40. Thus, tyrosine phosphorylation on p85 correlates with the CD40-triggered increase in PI3 kinase activity but not with basal levels nor with sIgM-triggered levels of enzymatic activity: these data suggest the presence of different PI3 kinase isoforms or the existence of multiple regulatory pathways for the same PI3 kinase isotype in Ramos-BL B cells.

Topics: B-Lymphocytes; Benzoquinones; Burkitt Lymphoma; CD40 Antigens; Cell Cycle Proteins; Child, Preschool; Enzyme Inhibitors; G1 Phase; Humans; Immunoglobulin M; Lactams, Macrocyclic; Male; Neoplasm Proteins; Palatine Tonsil; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Processing, Post-Translational; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-vav; Quinones; Rifabutin; Tumor Cells, Cultured

The CD40 receptor is an important molecule regulating B lymphocyte proliferation, maturation, Ab class switching, and cell survival. In the present study, we identified signal transduction events triggered by cross-linking the CD40 receptor. Stimulation of Daudi B cells with anti-CD40 resulted in activation of p21ras, an important switch point in the regulation of cell growth and differentiation. Ras activation correlated with a stimulation of Rac1 and MEK-1 as well as tyrosine phosphorylation of phosphatidylinositol 3-kinase. Inhibition of endogenous Ras by transfection of transdominant inhibitory Ras prevented tyrosine phosphorylation or stimulation of phosphatidylinositol 3-kinase, Rac1, or MEK-1 upon CD40 receptor triggering, proving an activation of the Ras pathway by CD40. Ras activation was partially inhibited by either herbimycin A or calphostin pretreatment and completely inhibited by preincubation with a combination of both inhibitors, indicating a synergistic role for protein tyrosine kinases and diglycerides in Ras activation after CD40 stimulation. In support of a role for diglycerides, we detected a 30 +/- 5% decrease of cellular phosphatidylcholine content, correlating with a threefold increase of diacylglycerol synthesis induced by CD40. Supporting a role for protein tyrosine kinase, we measured a five- to eightfold stimulation of p56lyn and p58blk kinase activity. These results suggest the activation of the Ras pathway via an additive function of src kinases and phospholipases that may be important in the mediation of biologic effects after CD40 receptor engagement.

Topics: Antibodies, Monoclonal; B-Lymphocytes; Benzoquinones; Burkitt Lymphoma; CD40 Antigens; Diglycerides; Enzyme Activation; Enzyme Inhibitors; GTP-Binding Proteins; Humans; Lactams, Macrocyclic; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase Kinases; Naphthalenes; Neoplasm Proteins; Phosphatidylcholines; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Quinones; rac GTP-Binding Proteins; Rifabutin; Signal Transduction; src-Family Kinases; Tumor Cells, Cultured

CD40 plays an important role in B cell activation, proliferation, and Ig class switching. The signal transduction pathway mediated by CD40 was studied using monoclonal antibody (mAb) 626.1 to CD40. Burkitt's lymphoma and Epstein-Barr virus-transformed B cell lines and tonsilar B lymphocytes were treated with the anti-CD40 mAb for various lengths of time. The early events triggered by CD40 were examined by monitoring the changes in tyrosine phosphorylation of cellular proteins with anti-phosphotyrosine mAb. Dephosphorylation of specific proteins ranging between 50-110 kD and the appearance of a 28-kD tyrosine phosphorylated protein were seen within 30 s in human B cell lines. The dephosphorylation was reversed and the 28-kD protein was dephosphorylated in cells stimulated for 1 min. In resting B cells, the appearance of the 28-kD phosphoprotein was observed in 30 s after the addition of the anti-CD40 mAb. The tyrosine phosphorylation of this protein persisted. The patterns of protein tyrosine phosphorylation differed from those induced by an anti-immunoglobulin M mAb. The changes in the state of tyrosine phosphorylation induced by the anti-CD40 mAb were obviated by mAb to CD45, a protein tyrosine phosphatase (PTP) or by the addition of sodium orthovanadate, a broad PTP inhibitor. They were also blocked by protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, and PKC and protein serine/threonine kinase inhibitors, H7 and HA1004. In addition, the alteration in the tyrosine phosphorylation of PTKs Lyn, Fyn, and Syk was directly demonstrated. Engagement of CD40 for 30 s induced a transient decrease in tyrosine phosphorylation of these PTKs. These results indicate that the early events in CD40 signaling involve the complex interaction between PTP and protein kinases.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; Benzoquinones; Burkitt Lymphoma; CD40 Antigens; Cell Line; Cell Line, Transformed; Enzyme Precursors; Genistein; Herpesvirus 4, Human; Humans; Intracellular Signaling Peptides and Proteins; Isoflavones; Isoquinolines; Lactams, Macrocyclic; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Molecular Weight; Palatine Tonsil; Phosphoproteins; Phosphorylation; Phosphotyrosine; Piperazines; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fyn; Quinones; Rifabutin; Sulfonamides; Syk Kinase; Tumor Cells, Cultured; Tyrosine