Pivotal role of a DEVD‐sensitive step in etoposide‐induced and Fas‐mediated apoptotic pathways.

We investigated the role of proteases in the pathway that leads from specific DNA damage induced by etoposide (VP‐16), a topoisomerase II inhibitor, to apoptotic DNA fragmentation in the U937 human leukemic cell line. In a reconstituted cell‐free system, Triton‐soluble extracts from VP‐16‐treated cells induced internucleosomal DNA fragmentation in nuclei from untreated cells. This effect was inhibited by the tetrapeptide Ac‐DEVD‐CHO, a competitive inhibitor of the interleukin‐1 beta‐converting enzyme (ICE)‐related protease CPP32, but was not influenced by Ac‐YVAD‐CHO and Ac‐YVAD‐CMK, two specific inhibitors of ICE. The three tetrapeptides inhibited Fas‐mediated apoptotic DNA fragmentation in the cell‐free system. Internucleosomal DNA fragmentation, triggered by either VP‐16 or an anti‐Fas antibody, was associated with proteolytic cleavage of the poly(ADP‐ribose)polymerase (PARP), a decrease in the level of 32 kDa CPP32 proenzyme and the appearance of the CPP32 p17 active subunit. Conversely, the expression of Ich‐1L, another ICE‐like protease, remained stable in apoptotic U937 cells. Several cysteine and serine protease inhibitors prevented apoptotic DNA fragmentation by acting either upstream or downstream of the DEVD‐sensitive protease(s) activation and PARP cleavage. We conclude that a DEVD‐sensitive step, which could involve CPP32, plays a central role in the proteolytic pathway that mediates apoptotic DNA fragmentation in VP‐16‐treated leukemic cells at the crossing with Fas‐mediated pathway.