Treatment of HL-60 cells with staurosporine (STS) induced mitochondrial cytochrome c efflux into the cytosol, which was followed by caspase-3 activation and apoptosis. Consistent with these observations, in vitro experiments demonstrated that, except for cytochrome c, the cytosol of HL-60 cells contained sufficient amounts of all factors required for caspase-3 activation. In contrast, treatment of HCW-2 cells (an apoptotic-resistant HL-60 subclone) with STS failed to induce significant amounts of mitochondrial cytochrome c efflux, caspase-3 activation, and apoptosis. In vitro assays strongly suggested that a lack of cytochrome c in the cytosol was the primary limiting factor for caspase-3 activation in HCW-2 cells. To explore the mechanism which regulates mitochondrial cytochrome c efflux, we developed an in vitro assay which showed that cytosolic extracts from SIS-treated, but not untreated, HL-60 cells contained an activity, which we designated 'CIF' (cytochrome c-efflux inducing factor), which rapidly induced cytochrome c efflux from HL-60 mitochondria. In contrast, there was no detectable CIF activity in STS-treated HCW-2 cells although the mitochondria from HCW-2 cells were responsive to the CIF activity from STS-treated HL-60 cells. These experiments have identified a novel activity, CIF, which is required 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.
Bibliographical noteFunding Information:
This work is supported in part by a National Science Foundation grant MCB-9630362 (to JHW and EAH), and by a National Institute of Health grant AI 35763 (to EAH). EAH is a Leukemia Society of America Scholar. TB is supported by a Visiting Associate Professor Fellowship from the Howard Hughes Medical Institute to Brown University (grant #5-29251). We thank Drs. A.-K. Bielinsky and D. Chatterjee for their comments and helpful discussions.
- Cytochrome c