Mechanisms of proteasome inhibitor-induced apoptosis of B-cell chronic lymphocytic leukaemia (B-CLL) cells

Proteasome inhibitors, including lactacystin, LLnL (N-acetyl-N-leucinyl-L-leucinyl-L- norleucinal) and MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), potently induce apoptosis in leukaemic B-cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL). This pro-apoptotic effect occurs in cells from patients at all stages of the disease, including those resistant to conventional chemotherapy, suggesting that proteasome inhibitors may be useful for treatment of B-CLL. Following initial inhibition of proteasomal activity and an increase in ubiquitinated proteins, these agents induce mitochondrial cytochrome c release and caspase-dependent apoptosis, involving cleavage/activation of caspases -2, -3, -7, -8 and - 9. Pre-treatment with the cell permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe)fluoromethyl ketone (Z-VAD.fmk), does not prevent the release of cytochrome c or partial processing of caspase-9 but prevents activation of effector caspases and induction of apoptosis. These results suggest that the release of cytochrome c is caspase independent and that caspase-9 is the initiator caspase in proteasome inhibitor-induced apoptosis of B-CLL cells. Activation of B-CLL lysates with dATP results in the formation of an -700 kDa caspase-activating apoptosome complex containing Apaf-1. A similar complex is formed in B-CLL cells induced to undergo apoptosis by proteasome inhibitors. Mechanisms of proteasome inhibitor-induced apoptosis vary between cell types, but often involve accumulation of short-lived proteins such as p53, p27 and pro-apoptotic Bcl-2 family members, activation of the stress kinase JNK, or inhibition of NF-kB transcriptional activity. Proteasome inhibitor-induced apoptosis in B-CLL cells is not triggered by alterations in the Bcl-2:Bax ratio, increase in pro-apoptotic t-Bid, or alterations in p27, XIAP, cIAPl or cIAP2. There is also no accumulation of IkB proteins that would inhibit NFkB survival signalling. Although proteasome inhibitors cause an accumulation of p53 and p21, this is not sufficient to induce apoptosis, as etoposide causes more pronounced increases in these molecules but is a less potent inducer of apoptosis. Activation of the stress kinases c-Jun N- terminal kinase (JNK) and p38 also does not appear to be involved. Therefore proteasome inhibitors induce mitochondrial perturbation in B-CLL cells by an apparently novel mechanism