A Lethal Injection

The stealthy assassin creeps up to the victim and quickly injects a dose of deadly poison.No, it's not a scene from a spy novel, but one of the ways in which the immune system's natural assassins -cytotoxic T lymphocytes and natural killer cells -can kill their target cells.The deadly poison in this case is a serine protease called granzyme B (GrB), and the killing mechanism is discussed by Christopher Froelich and colleagues in The Journal of Cell Biology.Much is known about GrB -there's evidence that it kills with or without caspase activation, and that it causes mitochondrial permeabilization with or without members of the Bcl-2 family.So Froelich and co-workers set out to assess the relative contributions of these pathways, to come up with a probable scheme for how GrB normally acts.To do this, they measured several different hallmarks of apoptosis -loss of mitochondrial membrane potential (ψ m ), DNA fragmentation and activation of procaspase precursors.Most previous studies have been done on cell lysates, after GrB has been delivered to its target.By contrast, Froelich and colleagues studied whole cells after delivery of the physiological form of GrB, a GrB-Serglycin (SG) complex.Previous studies with lysates had indicated that GrB-treated cells rapidly acquire cleaved (and presumably active) procaspase-3.Mature caspase-3 and GrB then process caspase-7 to complete the initial phase of apoptosis.The authors therefore asked how the absence of procaspase-3 might alter the pattern of apoptosis in whole cells after delivery of GrB-SG.The authors compared MCF-7 cells that lacked procaspase-3 (MCF-7 vec ) with cells that contained a stable transfectant that expressed this zymogen (MCF-7 casp-3 ), after delivery of GrB-SG.They saw DNA fragmentation and a loss of ψ m in the MCF-7 casp-3 cells, but not in MCF-7 vec cells, indicating that caspase-3 is essential for starting apoptosis.The authors also showed that the requirement for caspase-3 was not due simply to the fact that caspase-7 could no longer be activated in the caspase-3-deficient cells.If caspase-3 is the crucial initiator, what's the contribution of the Bcl-2-family members?First, the