A CYTOSOLIC FACTOR IS REQUIRED FOR MITOCHONDRIAL CYTOCHROME-C EFFLUX DURING APOPTOSIS

Treatment of HL-60 cells with staurosporine (STS) induced mitochondria l cytochrome c efflux into the cytosol, which was followed by caspase- 3 activation and apoptosis, Consistent with these observations, in vit ro experiments demonstrated that, except for cytochrome c, the cytosol of HL-60 cells contained sufficient amounts of all factors required f or caspase-3 activation. In contrast, treatment of HCW-2 cells (an apo ptotic-resistant HL-60 subclone) with STS failed to induce significant amounts of mitochondrial cytochrome c efflux, caspase-3 activation, a nd apoptosis, In vitro assays strongly suggested that a lack of cytoch rome c in the cytosol was the primary limiting factor for caspase-3 ac tivation in HCW-2 cells. To explore the mechanism which regulates mito chondrial cytochrome c efflux, we developed an in vitro assay which sh owed that cytosolic extracts from STS-treated, but not untreated, HL-6 0 cells contained an activity, which we designated 'CIF' (cytochrome c -efflux Inducing factor), which rapidly induced cytochrome c efflux fr om HL-60 mitochondria, In contrast, there was no detectable CIF activi ty in STS-treated HCW-2 cells although the mitochondria from HCW-2 cel ls were responsive to the CIF activity from STS-treated HL-60 cells. T hese experiments have identified a novel activity, CIF, which is requi red for cytochrome c efflux and they indicate that the absence of CIF is the biochemical explanation for the impaired ability of HCW-2 cells to activate caspase-3 and undergo apoptosis.