X-linked-inhibitor-of-apoptosis-protein (XIAP) is the most potent intracellular inhibitor of caspases-9, -3 and -7. While highly elevated XIAP levels reduce the apoptotic response to various stimuli, the potency of physiological XIAP expression to control caspase activation and the consequences of XIAP deficiency on apoptosis execution remain controversial. We therefore analyzed parental and XIAP-deficient DLD-1 and HCT-116 colon cancer cells by employing fluorescence-based single-cell imaging of mitochondrial permeabilisation and effector caspase activation. Our results demonstrate that physiological XIAP expression maintains a transient "off"-state for effector caspase activation following mitochondrial permeabilisation. Loss of XIAP expression instead accelerated the caspase activation response, but did not enhance the measured caspase activity. Apoptosis execution kinetics were independent of activating the intrinsic or extrinsic pathway by either staurosporine or TRAIL, and corresponded to computational systems analyses of caspase activation dynamics. We confirmed a protective role of XIAP upstream of mitochondrial permeabilisation during TRAIL-induced apoptosis, however, once mitochondria permeabilised ultimately no cell could escape effector caspase activation, regardless of potential cell-to-cell variability within the populations or the presence of XIAP. Our study provides comprehensive kinetic and mechanistic insight into the rapid molecular dynamics during apoptosis execution in the presence or absence of physiological XIAP expression.
Royal College of Surgeons in Ireland ->