Clustered CD20 Induced Apoptosis: Src-Family Kinase, the Proximal Regulator of Tyrosine Phosphorylation, Calcium Influx, and Caspase 3-Dependent Apoptosis,,

Anti-CD20 antibodies may reduce or eliminate non-Hodgkinʹs lymphoma B cells in patients, although the mechanism of action is not clear. To explore mechanism(s), we examined the induction of signal transduction events using anti-CD20 monoclonal antibodies (mAb) in the human non-Hodgkinʹs lymphoma Ramos B cell line. We found that while Rituximab (a human–mouse hybrid mAb) alone induced apoptotic cell death, other murine anti-CD20 mAbs induced apoptosis of Ramos B cells only upon clustering with a secondary antibody. CD20 clustering was accompanied by activation of tyrosine protein kinase activity, PLCγ2 phosphorylation, influx of Ca2+, and activation of caspase 3. All signaling events, as well as the subsequent apoptosis, were blocked by PP2, a selective inhibitor of Src-family kinases. Treatment of Ramos with EGTA and BAPTA to block changes in cytoplasmic Ca2+ likewise prevented CD20-induced apoptosis. Our findings support a model in which CD20 clustering activates members of the Src family of protein tyrosine kinases, leading to phosphorylation of PLCγ2 and increased cytoplasmic Ca2+. These early signal transduction events activate caspase 3 to promote apoptotic cell death of NHL B cells.