Chemoresistance in B cell lymphoma: Role of apoptosome activation

Abstract

Chemoresistance is a major clinical problem in the treatment of cancer, and is thoughtto be related to an underlying resistance to apoptosis (programmed cell death) in cancercells. In the present study, Burkitta??s lymphoma (BL)-derived B cell lines were used toinvestigate the intracellular signaling pathways involved in apoptosis induction bychemotherapeutic agents; in particular, we focused on post-mitochondrial signalingand its implications for chemoresistance.Apaf-1 (apoptotic protease-activating factor-1) is the main component of a proteincomplex termed the apoptosome, which is required for caspase activation downstreamof mitochondria. Upon release from these organelles, cytochrome c binds to Apaf-1thus driving its oligomerization; this results in the activation of caspase-9 and caspase-3, and the rapid dismantling of the cell. In the B cell lines utilized herein, cytochrome cfailed to stimulate apoptosome formation and caspase activation, and this was shown tobe due to insufficient levels of Apaf-1 in the cytosolic compartment. However, thelevel of Apaf-1 in total cellular extracts was normal. Enforced expression of Apaf-1increased its concentration in the cytosol, restored cytochrome c-dependent caspaseactivation, and rendered the prototypic BL cell line Raji sensitive to etoposide- andstaurosporine-induced apoptosis. Subsequent investigations revealed that, in Raji BLcells, the bulk of Apaf-1 was sequestered in the plasma membrane. Moreover,liberation of Apaf-1 using lipid raft-disrupting agents served to sensitize BL cells tochemotherapeutic drugs. Our studies also show that IAPs (inhibitor of apoptosisproteins) are highly expressed in BL cell lines. The combination of Smac peptides,which serve to antagonize IAPs, thereby alleviating caspase inhibition, andstaurosporine triggered apoptosis in Apaf-1-overexpressing BL cells, but failed to doso in mock transfectants. Similar results were obtained when Smac peptides wereadded together with the proteasome inhibitor, lactacytsin. Hence, Smac-mediatedpotentiation of apoptosis was shown to be Apaf-1-dependent, and IAPs wereconcluded to modulate the sensitivity to apoptosis in this model.These studies are the first to show that plasma membrane sequestration of Apaf-1, a key component of the apoptosome, may render cancer cells resistant to treatment.From the point of view of clinical utilization of apoptosis-targeted therapies, our resultssuggest that IAP modulation using, for instance, Smac peptides or Smac mimetics, maybe beneficial only if sufficient amounts of Apaf-1 are present. Our results also suggestthat proteasome inhibition-triggered apoptosis is dependent on apoptosome formation.The latter findings may be of potential importance since clinical trials usingproteasome inhibitors are currently ongoing in several hematological malignancies.