Cytoskeletal disruption accelerates caspase-3 activation and alters the intracellular membrane reorganization in DNA damage-induced apoptosis

In actinomycin D (AD)-induced apoptosis, caspase-3 activation and DNA cleavage in human megakaryoblastic leukemia CMK-7 cells were greatly accelerated by tubulin and actin polymerization inhibitors [e.g., colcemid (CL) and cytochalasin D (CD), respectively], but the acceleration was not found with Taxol or phalloidin. A decrease in mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of procaspase-9 to its active form preceded the activation of caspase-3 and, moreover, all of these events began earlier and/or proceeded faster in cells treated with AD plus CL or CD than in cells treated with AD only. These results suggest that cytoskeletal disruption in the apoptotic cells promotes damage of the mitochondrial membrane, resulting in the enhanced release of cytochrome c necessary for the activation of caspase-9 that initiates the caspase cascade. On the other hand, apoptotic bodies were rapidly formed from cells treated with AD and CL, but were suppressed when treated with AD and CD. Intracellular membranes and the actin system were reorganized to surround the nuclear fragments in the AD-and CL-treated cells, but such a membrane system was not formed in the presence of CD, implying that the apoptotic bodies are formed via reorganization of intracellular membranes under regulation by actin polymerization. Thus, the cytoskeletal change in CMK-7 cells has a strong effect on the early biochemical process as well as on the later morphologic process in AD-induced apoptosis. (C) 2000 Academic Press.