Two Waves of Mitochondrion Disruption in Apoptosis: Implications for the Design of Anti-Cancer Drugs.

Apoptosis plays an important role in preventing cancer development and promoting anti-cancer therapy. During mitochondria-mediated apoptosis, the disruption of mitochondria leads to the release of cytochrome c to form apoptosome with Apaf-1 and caspase-9, resulting in caspase-9 activation. However, the precise mechanisms controlling mitochondrion disruption remain elusive. Ablation of caspase-9 or other apoptosome components causes abnormal brain development and perinatal lethality in mice, making it difficult to assess the in vivo functions of caspase-9 signaling in many cell types and the role of caspase-9 in regulating mitochondria disruption. Through conditional knockout and inducible activation of caspase-9, we show that the loss of mitochondrion membrane potential (Δψm) during apoptosis proceeds in distinct caspase-9-dependent and independent steps. The initial loss of cells with high Δψm (Δψm^h) was independent of caspase-9, but required BH3-only proteins of the Bcl-2 family, such as Bim and BNIP3L. Caspase-9 deficiency arrested the B cells at a stage with intermediate levels of Δψm (Δψm^int). Apoptosis stimuli induced activation of effector caspases and cleavage of anti-apoptotic myeloid cell leukemia-1 (Mcl-1) in wild type but not caspase-9−/− B cells. Specific activation of caspase-9 by chemical-induced dimerization in human leukemia H9 cells also resulted in the loss of Δψm and cleavage of Mcl-1, but silencing of downstream effector caspases inhibited Mcl-1 cleavage and sequestered the cells at the Δψm^int stage. Moreover, a cleavage-resistant Mcl-1 inhibited caspase-9-dependent mitochondrion disruption and apoptosis, indicating that caspase-9-mediated mitochondrion destruction involves Mcl-1 processing. Our data suggest that BH3-only proteins initiate the first wave of mitochondrion disruption during apoptosis with Δψm^h to Δψm^int transition, while caspase-9 signaling orchestrated the second wave of Δψm^int to Δψm⁻ transition through the cleavage of Mcl-1. Defects in mitochondria-mediated apoptosis pathway are associated with the development and treatment failures of many hematological malignancies. Identification of stepwise mitochondrial disruption may allow specific targeting of apoptotic machineries in various therapeutic settings. Chemical-induced dimerization of caspase-9 may be particularly useful for the design of anti-cancer drugs to treat those malignancies involving Mcl-1 over-expression.