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-Hodgkin's lymphoma B cells in patients, although the mechanism of action is not clear. To explore mec hanism(s), we examined the induction of signal transduction events using an ti-CD20 monoclonal antibodies (mAb) in the human non-Hodgkin's lymphoma Ram os B cell line. We found that while Rituximab (a human-mouse hybrid mAb) al one induced apoptotic cell death, other murine anti-CD20 mAbs induced apopt osis of Ramos B cells only upon clustering with a secondary antibody, CD20 clustering was accompanied by activation of tyrosine protein kinase activit y, PLC gamma 2 phosphorylation, influx of Ca2+, and activation of caspase 3 , All signaling events, as well as the subsequent apoptosis, were blocked b y PP2, a selective inhibitor of Src-family kinases, Treatment of Ramos with EGTA and BAPTA to block changes in cytoplasmic Ca2+ likewise prevented CD2 0-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 PLC gamma 2 and increased cytoplasmic Ca2+. These early signal transduction events activate caspase 3 to promote apoptotic cell de ath of NHL B cells, (C) 2000 Academic Press.