CD20-induced B cell death can bypass mitochondria and caspase activation.
The apoptotic pathway activated by chimeric anti-CD20 monoclonal antibodies (rituximab, IDEC.C2B8) was analyzed using the Burkitt lymphoma cell line Ramos. Crosslinking of CD20 (CD20XL) induced apoptosis in Ramos cells, which involved loss of mitochondrial membrane potential (Deltapsi(m)), the release of cytochrome-c (cyt-c), and activation of caspases-9 and -3. Nevertheless, several lines of evidence showed that the apoptotic outcome did not depend on these events. First, under circumstances where Ramos cells display resistance to either CD95- or B cell receptor (BCR)-induced apoptosis, CD20XL-induced apoptosis was not affected, pointing to a distinct pathway. Second, the broad-spectrum caspase inhibitor zVAD-fmk prevented processing of caspase-9, -3 and PARP as well as DNA fragmentation, but did not block apoptosis as measured by annexin V staining, cell size and membrane integrity. Lastly, Bcl-2 overexpression blocked cyt-c release and the decrease in Deltapsi(m), and completely prevented CD95- or BCR-mediated apoptosis; however, it did not affect CD20XL-induced cell death. We conclude that although CD20XL can initiate the mitochondrial apoptosis pathway, CD20-induced apoptosis does not necessarily require active caspases and cannot be blocked by Bcl-2. Since most chemotherapeutic drugs require the activation of caspases to exert their cytotoxicity, these findings provide an important rationale for the use of CD20 mAbs in chemoresistant malignancies.
Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands.
L E
LE
van der Kolk
L M
LM
Evers
C
C
Omene
S M A
SM
Lens
S
S
Lederman
R A W
RA
van Lier
M H J
MH
van Oers
E
E
Eldering
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20 / Q000276:immunology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001402:B-Lymphocytes / Q000187:drug effects* / Q000473:pathology; D002051:Burkitt Lymphoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D008564:Membrane Potentials; D008928:Mitochondria / Q000187:drug effects* / Q000201:enzymology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D000069283:Rituximab; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism / Q000473:pathology
D000590:Amino Acid Chloromethyl Ketones; D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D019253:Proto-Oncogene Proteins c-bcl-2; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D000069283:Rituximab; D020169:Caspases