NEW INSIGHTS INTO THE REGULATION OF MITOCHONDRIAL OUTER MEMBRANE PERMEABILIZATION DURING APOPTOSIS

Abstract

Disruption of normal apoptosis can contribute to the onset of cancer. Additionally, many cancer drugs are effective for their ability to initiate the apoptotic process. Often this involves the activation of the mitochondria-mediated pathway. The existing paradigm of mitochondria-mediated apoptosis, which can be activated by DNA damage, indicates this pathway proceeds in a linear fashion. Mitochondria outer membrane permeabilization (MOMP) and the release of intermembrane space proteins (e.g., cytochrome c) are early events during mitochondria-mediated apoptotic signaling. In addition, Apaf-1 is a critical component of the mitochondrial pathway and is generally thought to reside downstream of MOMP. My dissertation investigates the molecular requirements essential for MOMP during stress-induced and receptor-mediated apoptosis. Distinct clones of Jurkat T-lymphocytes were used in which the mitochondria-mediated pathway had been inhibited at three different steps. The first aim investigated the molecular requirements necessary for Bak activation. Apaf-1-deficient cells and cells overexpressing full-length XIAP or the BIR1/BIR2 domains of XIAP were refractory mitochondrial apoptotic events. These data suggest that caspase-mediated positive amplification of initial mitochondrial changes can determine the threshold for irreversible activation of the intrinsic apoptotic pathway. In so-called types II cells, the mitochondria-mediated pathway is required for cell death upon stimulation of the receptor-mediated pathway. In the second aim, I investigated the molecular requirements necessary for Fas-mediated apoptosis. Interestingly, Apaf-1-deficient type II Jurkat cells were sensitive to anti-Fas. Inhibiting downstream caspases decreased all anti-Fas-induced apoptotic changes. Combined, my findings strongly suggest that Fas-mediated activation of executioner caspases and induction of apoptosis does not depend on apoptosome-mediated caspase-9 activation in prototypical type II cells. In the third aim, mitochondrial apoptotic events were examined after prolonged treatment with etoposide ( 6 h). Total cellular cytochrome c and Smac were decreased after 24 h of incubation. Interestingly, inhibition of the 26S proteasome by co-treatment of Apaf-1-deficient cells with bortezomib or MG132 led to the robust retention of total cellular cytochrome c and Smac. Combined, the major findings suggest that proteasomal degradation is largely responsible for the loss of intracellular cytochrome c and Smac in Apaf-1-deficient cells incubated with etoposide over extended time periods.