A possible intermediate step during apoptotic execution.

Many proteases are known to be involved in apoptosis. Among them, interleukin-1beta converting enzyme (ICE) and its family proteases, which are called caspases, play critical roles in the execution stage of apoptosis. We previously reported that a proteasome-inhibitor, benzyloxycarbonyl Leu-Leu-leucinal (ZLLLal), induced apoptosis in MOLT-4 cells. In the present study, in order to analyze the detailed mechanism of ZLLLal-induced apoptosis, we examined the effect of a caspase-inhibitor, acetyl(Ac)-Tyr-Val-Ala-Asp-chloromethyl ketone (AcYVADcmk), on ZLLLal-induced apoptosis in the cells. Agarose gel electrophoresis revealed that low concentrations of AcYVADcmk efficiently suppressed apoptotic DNA fragmentation. However, the cells presented morphology different from normal, apoptotic or necrotic cells, although DNA fragmentation was suppressed. The same examination was performed on the cells with anti-Fas antibody-induced apoptosis, and the same results were obtained. Some cells with a similar morphology were found even without the caspase-inhibitor in the early stage of anti-Fas antibody-induced physiological apoptosis. In addition, apoptotic cascade was reactivated by washing out the caspase inhibitor from the DNA degradation-suppressed cells. Therefore, this newly found morphological feature shows the presence of a step prior to caspase activation in the cells, and this is the first report presenting the pre-caspase-activated step in the apoptotic cascade.