Proteasome inhibitors induce caspase-dependent apoptosis and accumulation of p21(WAF1/Cip1) in human immature leukemic cells

The 26S proteasome is a non-lysosomal multicatalytic protease complex for d egrading intracellular proteins by ATP/ubiquitin-dependent proteolysis. Tig htly ordered proteasomal degradation of proteins critical for cell cycle co ntrol implies a role of the proteasome in maintaining cell proliferation an d cell survival. In this study, we demonstrate that cell-permeable proteaso me inhibitors, lactacystin, benzyloxycarbonyl(Z)-leucyl-leucyl-leucinal (ZL LLal; MG-132) and 4-hydroxy-5-iodo-3-nitrophenylacetyl-leucyl-leucine vinyl sulfone (NLVS), induce apoptosis abundantly in p53-defective leukemic cell lines CCRF-CEM, U937 and K562 as well as in myelogenic and lymphatic leuke mic cells obtained from adult individuals with relapsed acute leukemias. Le ukemic cell apoptosis induced by the proteasome inhibitors was dependent on activation of caspase-3 and related caspase family pro teases, because cas pase-3 inhibitor N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartal (Ac-DEVD -cho) and, more effectively, the general casp ase-inhibitor N-benzyloxycarb onyl-L-valyl-L-alanyl- L-aspartate fluoromethylketone (Z-VAD-fmk) were capa ble of blocking apoptosis induced by lactacystin, ZLLLal or NLVS. Induction of apoptosis by lactacystin or ZLLLal was accompanied by cell cycle arrest at G2/M phase and by accumulation and stabilization of cyclin-dependent ki nase inhibitor p21(WAF1/Cip) and tumor suppressor protein p53. A role of p5 3 in mediating apoptosis or induction of p21(WAF/Cip1) was ruled out since CCRF-CEM and U937 cells express non-functional mutant p53, and K562 cells l ack expression of p53. Viability and hematopoietic outgrowth of human CD34 + progenitor cells treated with lactacystin were slightly reduced, whereas treatment of CD34 + cells with ZLLLal or the cytostatic drugs doxorubicin a nd gemcitabine resulted in markedly reduced viability and hematopoietic out growth. These results demonstrate a basic role of the proteasome in maintai ning survival of human leukemic cells, and may define cell-permeable protea some inhibitors as potently anti-leukemic agents which exhibit a moderate h ematopoietic toxicity in vitro.