In this paper the specific mitochondrial respiratory chain inhibitors rotenone and antimycm A and the highly specific mitochondrial ATP-synthase inhibitor oligomycin are shown to induce an apoptotic suicide response in cultured human lymphoblastoid and other mammalian cells wlthin 12-18 h. The mitochondrial inhibitors do not induce apoptosis in cells depleted of mitochondrial DNA and thus lacking an intact mitochondrial respiratory chain. Apoptosis induced by respiratory chain inhibitors is not inhibited by the presence of Bcl-2. We discuss the possible role of mitochondrial induced apoptosis in the ageing process and age-associated diseases.

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In the present paper we show that the immunosuppressant rapamycin inhibits the induction of apoptosis by didemnin B in human promyeloid HL‐60 cells. The mechanism of this inhibition is investigated using FK506, which competes with rapamycin for binding to their common target FK506‐binding protein (FKBP)12. The lack of competition for rapamycin‐mediated inhibition of didemnin B‐induced apoptosis by FK506 suggests that rapamycin inhibits apoptosis through some mechanism other than inhibition of p70 S6 kinase activation. The lack of inhibition of didemnin B‐induced apoptosis by inhibitors of phosphatidylinositol 3‐kinase and mitogen‐activated protein (MAP) kinase kinase further supports the conclusion that rapamycin does not inhibit didemnin B‐induced apoptosis through inhibition of the MAP kinase pathway. Furthermore, didemnin B‐induced apoptosis is not inhibited by the inhibitors of cyclin‐dependent kinase, roscovitine and olomoucine. This indicates that rapamycin does not act through inhibition of cyclin‐dependent kinases. Together with the lack of competition for the effect of rapamycin by FK506, our data suggest the possible involvement of the FK506‐binding protein, FKBP25, which is localized in the nucleus. This interpretation of our data gains support from the fact that didemnin B does not induce apoptosis in enucleated HL‐60 cells, which supports the possible involvement of FKBP25 in the inhibition of apoptosis by rapamycin.

Caspases have been implicated in the induction of apoptosis in most systems studied. The importance of caspases for apoptosis was further investigated using the system of didemnin B-induced apoptosis. We found that benzyloxycarbonyl-VAD-fluoromethylketone, a general caspase inhibitor, inhibits didemnin B-induced apoptosis in HL-60 and Daudi cells. Acetyl-YVAD-chloromethylketone, a caspase-1-like activity inhibitor, inhibits didemnin B-induced apoptosis in Daudi cells, whereas the caspase-3-like activity inhibitor, acetyl-DEVD-aldehyde, has no effect. Using immunoblots to investigate cleavage of caspases-1 and -3, we found that both caspases are activated in both cell lines. We showed that the caspase substrate poly(ADP-ribose)polymerase is cleaved in these cells after didemnin B treatment. In both cell lines, poly(ADP-ribose)polymerase cleavage is inhibited by benzyloxycarbonyl-VAD-fluoromethylketone and also by acetyl-YVAD-chloromethylketone in Daudi cells. These results indicate that a caspase(s) other than caspase-3 is required for didemnin B-induced apoptosis. We show that caspases may be activated during apoptosis that are not required for the progression of apoptosis.