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

https://doi.org/10.1006/bcmd.2000.0287Get rights and content

Abstract

ABSTRACT

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 mechanism(s), we examined the induction of signal transduction events using anti-CD20 monoclonal antibodies (mAb) in the human non-Hodgkin's 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.

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    Abbreviations used: BAPTA-AM, acetoxymethyl ester of 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetracetic acid; BSA, bovine serum albumin; DEVD, peptide composed of aspartic acid–glutamic acid–valine–aspartic acid; DMSO, dimethyl sulfoxide; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetracetic acid; FcR, Fc receptor; FITC, fluorescein isothiocyanate; GAM, goat anti-mouse IgG; ITAM, immunoreceptor tyrosine-based activation motif; NHL, non-Hodgkin's lymphoma; PLC, phospholipase C; PI, propidium iodide; PTK, protein tyrosine kinase.

    ☆☆

    This work was supported by NIH Grants AI41447 and CA64268. Dr. Coggeshall is a scholar of the Leukemia Society of America, and Dr. Hofmeister is supported by a Fellowship from the Lymphoma Research Foundation of America, Inc.

    Communicated by Alvin, Mauer, M.D., 03/16/00

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    Correspondence and reprint requests to: Dr. K. M. Coggeshall, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK. 73104. Fax: (405) 271-8568. E-mail: [email protected].

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