CHARACTERIZATION OF ANTIBODIES SPECIFIC FOR THE CASPASE CLEAVAGE SITEON POLY(ADP-RIBOSE) POLYMERASE - SPECIFIC DETECTION OF APOPTOTIC FRAGMENTS AND MAPPING OF THE NECROTIC FRAGMENTS OF POLY(ADP-RIBOSE)POLYMERASE

Intracellular cysteine proteases are important mediators of apoptosis. Indeed, some nuclear proteins and enzymes are cleaved during apoptosi s. in particular poly(ADP-ribose) polymerase (PARP), which is activate d by DNA strand interruptions and is involved in DNA repair. PARP is c leaved into two fragments of 29 and 85 kDa (apparent molecular mass) i n human promyelomonocytic leukemia cells, HL-60, treated with etoposid e to induce apoptosis. These cells possess protease activities, caspas es, that share many features with the ICE/CED-3 family. The cleavage o ccurs between Asp-214 and Gly-215, a site that is conserved in human, bovine, and chicken PARP. This cleavage has been shown to be an early marker of apoptosis. To monitor apoptosis, to understand the role of P ARP cleavage by caspases, and to study the role of the two fragments i n DNA repair, members of our laboratory have developed two polyclonal antipeptide antibodies directed against the two human PARP sequences: [196-214] for LP96-22 and [215-228] for LP96-24. Moreover, these antib odies will be useful to map the necrotic cleavage of PARP, which gener ates fragments different from those obtained during apoptosis, and thu s to discriminate between apoptotic and necrotic cell death.