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¹ Evanston Northwestern Healthcare Research Institute, Feinberg School of Medicine, Northwestern University, Evanston, IL 60201
² Department of Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15260

Address correspondence to Evanston Northwestern Healthcare Research Institute, 1001 University Pl., Evanston, IL 60201. Tel.: (847) 570-7660. Fax: (847) 570-8025. E-mail: c-froelich{at}northwestern.edu

Granzyme B (GrB), acting similar to an apical caspase, efficiently activates a proteolytic cascade after intracellular delivery by perforin. Studies here were designed to learn whether the physiologic effector, GrB–serglycin, initiates apoptosis primarily through caspase-3 or through BH3-only proteins with subsequent mitochondrial permeabilization and apoptosis. Using four separate cell lines that were either genetically lacking the zymogen or rendered deficient in active caspase-3, we measured apoptotic indices within whole cells (active caspase-3, mitochondrial depolarization [m] and TUNEL). Adhering to these conditions, the following were observed in targets after GrB delivery: (a) procaspase-3–deficient cells fail to display a reduced m and DNA fragmentation; (b) Bax/Bak is required for optimal m reduction, caspase-3 activation, and DNA fragmentation, whereas BID cleavage is undetected by immunoblot; (c) Bcl-2 inhibits GrB-mediated apoptosis (reduced m and TUNEL reactivity) by blocking oligomerization of caspase-3; and (d) in procaspase-3–deficient cells a mitochondrial-independent pathway was identified which involved procaspase-7 activation, PARP cleavage, and nuclear condensation. The data therefore support the existence of a fully implemented apoptotic pathway initiated by GrB, propagated by caspase-3, and perpetuated by a mitochondrial amplification loop but also emphasize the presence of an ancillary caspase-dependent, mitochondria-independent pathway.

Key Words: granzyme B; apoptosis; caspase-3; mitochondria; mechanism

^* Abbreviations used in this paper: AD, adenovirus; cyt c, cytochrome c; GrB, granzyme B; ICAD, inhibitor of caspase-activated deoxyribonuclease; MEF, murine embryonic fibroblast; NK, natural killer; PFN, perforin; PFU, plaque-forming unit; SG, serglycin; WT, wild type.

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