BCL-2 ACTS UPSTREAM OF THE PARP PROTEASE AND PREVENTS ITS ACTIVATION

Apoptosis has recently been extensively studied and multiple factors h ave been implicated in its regulation, It remains unclear how these fa ctors are ordered in the cell death pathway. Here we investigate the r elationship between the inhibitor of apoptosis, bcl-2, and the PARP pr otease, prICE/CPP32, recently implicated in apoptosis, Using PARP prot eolysis as an indicator of the activation of the PARP protease, we fin d that the chemotherapeutic agent, etoposide, induces apoptosis and PA RP proteolysis in Molt4 cells as early as 4h with cell death lagging b ehind this event, In contrast, Molt4 cells that over-express bcl-2 sho w no PARP proteolysis or cell death. In order to determine if bcl-2 in hibits the PARP protease or its activation, we developed a cell-free s ystem. Using this system with extracts from etoposide-treated cells an d purified bovine PARP, we demonstrate that extracts from bcl-2 over-e xpressing cells cause little or no PARP proteolysis. Whereas, extracts from control vector cells contain an active PARP protease. This prote ase 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 bc l-2 directly inhibits the active protease or that it has an effect dow nstream of prICE/CPP32 such as preventing access to the PARP substrate . These results also demonstrate a role of bcl-2 in interfering with a n upstream signal required to activate the PARP protease and allow us to begin to order the components in the apoptotic pathway.