Executioner caspase-3,-6, and-7 perform distinct, non-redundant roles during the demolition phase of apoptosis

Apoptosis is orchestrated by a family of cysteine proteases known: as the c aspases. Fourteen mammalian caspases have been identified, three of which ( caspase-3, -6, and -7) are thought to coordinate the execution phase of apo ptosis by cleaving multiple structural and repair proteins. However, the re lative contributions that the "executioner" caspases make to the demolition of the cell: remains speculative. Here we have used cell-free extracts imm une-depleted of either caspase-3, -6, or -7 to examine the caspase requirem ents for apoptosis-associated proteolysis of 14 caspase substrates as well as nuclear condensation, chromatin margination, and DNA fragmentation. We s how that caspase-3 is the primary executioner caspase in this system, neces sary for cytochrome c/dATP-inducible cleavage of fodrin, gelsolin, U1 small nuclear ribonucleopretein, DNA fragmentation factor 45 (DFF45)/inhibitor o f caspase-activated DNase (ICAD), receptor-interacting protein (RIP), X-lin ked inhibitor of apoptosis protein (X-IAP), signal transducer and activator of transcription-1 (STAT1), topoisomerase I, vimentin, Rb, and lamin B but not for cleavage of poly(ADP-ribose) polymerase (PARP) or lamin A. In addi tion, caspase-3 was also essential for apoptosis-associated: chromatin marg ination, DNA fragmentation, and nuclear collapse in this system. Surprising ly, although caspase-6 and -7 are considered to be important downstream-eff ector caspases, depletion of either caspase had minimal impact on any of th e parameters investigated, calling into question their precise role during the execution phase of apoptosis.