INTACT CELL EVIDENCE FOR THE EARLY SYNTHESIS, AND SUBSEQUENT LATE APOPAIN-MEDIATED SUPPRESSION, OF POLY(ADP-RIBOSE) DURING APOPTOSIS

Poly(ADP-ribose) polymerase (PARP), which is catalytically activated b y DNA strand breaks, has been implicated in apoptosis, or programmed c ell death. A protease (CPP32) responsible for the cleavage of PARP and necessary for apoptosis was recently purified and characterized. The coordinated sequence of events related to PARP activation and cleavage in apoptosis has now been examined in individual cells. Apoptosis was studied in a human osteosarcoma cell. line that undergoes a slow (8 t o 10 days), spontaneous, and reproducible death program in culture. Ch anges in the abundance of intact PARP, poly(ADP-ribose) (PAR), and a p roteolytic cleavage product of PARP that contains the DNA-binding doma in were examined during apoptosis in the context of individual, whole cells by immunofluorescence with specific antibodies. The synthesis of PAR from NAD increased early, within 2 days of cell plating for apopt osis, prior to the appearance of internucleosomal DNA cleavage and bef ore the cells become irreversibly committed to apoptosis, since replat ing yields viable, nonapoptotic cells. Strong expression of full-lengt h PARP was also detected, by immunofluorescence as well as by Western analysis, during this same time period. However, after similar to 4 da ys in culture, the abundance of both full-length PARP and PAR decrease d markedly. After 6 days, a proteolytic cleavage product containing th e DNA-binding domain of PARP was detected immunocytochemically and con firmed by Western analysis, both in the nuclei and in the cytoplasm of cells. A recombinant peptide spanning the DNA-binding domain of PARP was expressed, purified, and biotinylated, and was then used as a prob e for DNA strand breaks. Fluorescence microscopy with this probe revea led extensive DNA fragmentation during the later stages of apoptosis. This is the first report, using individual, intact cells, demonstratin g that poly(ADP-ribosyl)ation of nuclear proteins occurs prior to the commitment to apoptosis, that inactivation and cleavage of PARP bean s hortly thereafter, and that very little PAR per se is present during t he later stages of apoptosis, despite the presence of a very large num ber of DNA strand breaks. These results suggest a negative regulatory role for PARP during apoptosis, which in turn may reflect the requirem ent for adequate NAD and ATP during the later stages of programmed cel l death. (C) 1997 Academic Press.