CD40-triggered protein tyrosine phosphorylation on Vav and on phosphatidylinositol 3-kinase correlates with survival of the Ramos-Burkitt lymphoma B cell line.

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.

Wortmannin-mediated inhibition of phosphatidylinositol 3-kinase activity triggers apoptosis in normal and neoplastic B lymphocytes which are in cell cycle.

The B cell functional response following ligation of surface (s) IgM is dependent upon the differentiation stage of the population studied: cross-linking sIgM promotes proliferation of resting tonsillar follicular mantle (FM) B lymphocytes but induces apoptosis in the susceptible Epstein-Barr virus genome-negative Burkitt lymphoma (BL) cell line Ramos (Ramos-BL). This study investigates whether phosphatidylinositol-3-kinase (Pl3-kinase), which has been reported to be intimately involved in the regulation of cellular growth, plays a role in the regulation of these sigpromoted B cell responses, and uses the selective and irreversible inhibitor of Pl3-kinase activity, wortmannin (Wm). In Ramos-BL B cells, at 8 h post-treatment, Wm triggers a transient increase in apoptosis of 16 +/- 6.9% with a concomitant cellular loss of 16 +/- 6.1% from the G1 phase of cell cycle; [3H]thymidine incorporation also decreases by 33 +/- 5.0%, from 37,274 c.p.m. +/- 10% to 25,127 c.p.m. +/- 4.0%. Moreover, at 72 h culture, Wm inhibits anti-IgM-induced FM B lymphocyte levels of [3H]thymidine incorporation typically by 47% and triggers 80% apoptosis from the G0G1 phase of cell cycle. Ramos-BL B cells exhibit high basal levels of Pl3-kinase activity, as determined by immunoprecipitation with antibody to the p85 regulatory subunit of Pl3-kinase and 32P incorporation into phosphatidylinositol, which is not significantly affected by anti-IgM stimulation; by contrast, anti-IgM stimulates significant Pl3-kinase activity over negligible basal levels in FM B lymphocytes. Pre-treatment with Wm inhibits Pl3-kinase activity in both cell types. Taken together these data indicate that in Ramos-BL B cells sigM-triggered growth arrest and apoptosis is Pl3-kinase independent, whereas Pl3-kinase activity is critical for sIgM-triggered mitogenesis of FM B lymphocytes. Thus Pl3-kinase plays a pivotal role in the regulation of both normal and neoplastic B lymphocyte progression through the cell cycle, such that if this Pl3-kinase-dependent pathway is inhibited these cells default to apoptosis.