Active caspases and cleaved cytokeratins are sequestered into cytoplasmic inclusions in TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) -induced ap optosis, in transformed human breast epithelial MCF-7 cells, resulted in a time-dependent activation of the initiator caspases-8 and -9 and the effect or caspase-7, Cleavage of caspase-8 and its preferred substrate, Bid, prece ded processing of caspases-7 and -9, indicating that caspase-8 is the apica l initiator caspase in TRAIL-induced apoptosis. Using transient transfectio n of COOH-terminal-tagged green fluorescent protein fusion constructs, casp ases-3, -7, and -8 were localized throughout the cytoplasm of MCF-7 cells. TRAIL-induced apoptosis resulted in activation of caspases-3 and -7, and th e redistribution of most of their detectable catalytically active small sub units into large spheroidal cytoplasmic inclusions, which lacked a limiting membrane. These inclusions, which were also induced in untransfected cells , contained cytokeratins 8, 18, and 19, together with both a phosphorylated form and a caspase-cleavage fragment of cytokeratin 18. Similarly, in untr ansfected breast HBL100 and lung A549 epithelial cells, TRAIL induced the f ormation of cytoplasmic inclusions that contained cleaved cytokeratin 18 an d colocalized with active endogenous caspase-3,We propose that effector cas pase-mediated cleavage of cytokeratins, resulting in disassembly of the cyt oskeleton and formation of cytoplasmic inclusions, may be a characteristic feature of epithelial cell apoptosis.