Caspase-independent commitment phase to apoptosis in activated blood T lymphocytes: reversibility at low apoptotic insult.

Little is known about the mechanisms of programmed death triggered in T lymphocytes by stimuli that can bypass caspase activation. Anti-CD2 monoclonal antibody and staurosporine are such apoptosis inducers because they operate in the presence of broad-spectrum caspase inhibitors BOC-D.fmk and Z-VAD.fmk. A system was devised, based on the isolation according to density of activated blood T cells progressively engaged in the apoptotic process. This allowed definition of a sequence of caspase-dependent and caspase-independent apoptogenic events that are triggered by anti-CD2 and staurosporine. Thus, a commitment phase to apoptosis was defined that is entirely caspase independent and that is characterized by cell volume loss, partial chromatin condensation, and release into the cytosol and the nucleus of mitochondrial "apoptosis-inducing factor " (AIF). Committed cells were viable, displayed a high mitochondrial inner transmembrane potential (triangle upPsim), and lacked large-scale and oligonucleosomal DNA fragmentation. Mitochondrial release of AIF was selective because cytochrome c was retained in mitochondria of the very same cells. Mitochondrial release of cytochrome c occurred later, at the onset of the execution phase of apoptosis, concurrently with triangle upPsim collapse, poly (ADP-ribose) polymerase cleavage, and DNA fragmentation. The apoptogenic events of this commitment phase are reversible if the strength of the stimulus is low and of short duration.