Lack of release of cytochrome C from mitochondria into cytosol early in the course of Fas-mediated apoptosis of Jurkat cells.

Several groups have reported that during apoptosis, cytochrome c is released from the mitochondria into the cytosol, but we have found that in apoptotic cells the cytochrome appears to remain with the mitochondria. In hopes of reconciling these findings, we compared the results obtained from cells disrupted by our method (nitrogen cavitation) with those obtained using the cell-disruption method employed by others (homogenization). We observed that at 2 h, cytochrome c levels in apoptotic cytosols from homogenized cells exceeded control levels, whereas cytochrome c levels in apoptotic cytosols from cavitated cells were similar to control. Outer membranes of homogenized mitochondria appeared damaged because the mitochondria had become permeable to cytochrome c, whereas outer membranes of cavitated mitochondria excluded the cytochrome. 4 h after Fas ligation, both cavitated and homogenized mitochondria had released small amounts of cytochrome c into the cytosol, whereas after 6 h the cytochrome had disappeared from the cell lysate. We believe that the differences between our results and those reported by others were due to 1) our examining the cells after a short (2 h) incubation with the anti-Fas antibody, and 2) our use of nitrogen cavitation instead of homogenization to disrupt the cells.