Apoptosis in factor-dependent haematopoietic cells is linked to calcium-sensitive mitochondrial rearrangements and cytoskeletal modulation

Apoptosis in murine haematopoietic interleukin (IL)3-dependent cell lines is induced within 6-8 h by IL-3 withdrawal. Direct introduction of cytochrome c by electroporation induces apoptosis within 2 h and was inhibited by caspase inhibitors, such as Z-VADfmk and Z-Dfmk. We report here that apoptosis induced by IL-3 withdrawal was refractory to these inhibitors but was accompanied by striking redistribution of mitochondria, which aggregated into an area associated with centrioles without loss of Delta psi(m). Both mitochondrial redistribution and apoptosis were inhibited by the calcium ionophore, ionomycin. Nocodozole, an inhibitor of microtubule assembly, also induced apoptosis, which was unaffected by caspase inhibitors. Although nocodozole did not alter mitochondrial distribution, it significantly reduced Delta psi(m), and both reduction of Delta psi(m) and apoptosis were inhibited by ionomycin. Oligomycin, which inhibits the mitochondrial FoF1 ATPase, similarly induced apoptosis, which was unaffected by caspase inhibitors but was inhibited by ionomycin. Further, oligomycin stimulated the novel formation and release of surface membrane-derived vesicles containing mitochondria with intact Delta psi(m); ionomycin also inhibited their production. In all these conditions, Bcl-2 protected cells from apoptosis. Our studies show that apoptosis induced by three very different agents shares insensitivity to caspase inhibitors, suppression by ionomycin and effects on mitochondria, which all appear to be linked to cytoskeletal/microtubule activity. They suggest that microtubules and the cytoskeleton play an important role in apoptosis through mechanisms affecting mitochondria but which are independent of cytochrome c release.