Bcl-2 acts upstream of the PARP protease and prevents its activation

Apoptosis has recently been extensively studied and multiple factors have been implicated in its regulation. It remains unclear how these factors are ordered in the cell death pathway. Here we investigate the relationship between the inhibitor of apoptosis, bcl-2, and the PARP protease, prlCE/CPP32, recently implicated in apoptosis. Using PARP proteolysis as an indicator of the activation of the PARP protease, we find that the chemotherapeutic agent, etoposide, induces apoptosis and PARP proteolysis in Molt4 cells as early as 4 h with cell death lagging behind this event. In contrast, Molt4 cells that over-express bcl-2 show no PARP proteolysis or cell death. In order to determine if bcl-2 inhibits the PARP protease or its activation, we developed a cell-free system. Using this system with extracts from etoposide-treated cells and purified bovine PARP, we demonstrate that extracts from bcl-2 over-expressing cells cause little or no PARP proteolysis. Whereas, extracts from control vector cells contain an active PARP protease. This protease is inhibited by the tetrapeptide ICE-like protease inhibitor, YVAD-chloromethylketone. Interestingly, this protease is not inhibited by the addition of purified bcl-2 protein. These results rule out that bcl-2 directly inhibits the active protease or that it has an effect downstream of prlCE/CPP32 such as preventing access to the PARP substrate. These results also demonstrate a role of bcl-2 in interfering with an upstream signal required to activate the PARP protease and allow us to begin to order the components in the apoptotic pathway.