TRANSIENT POLY(ADP-RIBOSYL)ATION OF NUCLEAR PROTEINS AND ROLE OF POLY(ADP-RIBOSE) POLYMERASE IN THE EARLY STAGES OF APOPTOSIS

A transient burst of poly(ADP-ribosyl)ation of nuclear proteins occurs early, prior to commitment to death, in human osteosarcoma cells unde rgoing apoptosis, followed by caspase-3-mediated cleavage of poly(ADP- ribose) polymerase (PARP). The generality of this early burst of poly( ADP-ribosyl)ation has now been investigated with human HL-60 cells, mo use 3T3-L1, and immortalized fibroblasts derived from wild-type mice. The effects of eliminating this early transient modification of nuclea r proteins by depletion of PARP protein either by antisense RNA expres sion or by gene disruption on various morphological and biochemical ma rkers of apoptosis were then examined. Marked caspase-3-like PARP clea vage activity, proteolytic processing of CPP32 to its active form, int ernucleosomal DNA fragmentation and nuclear morphological changes asso ciated with apoptosis were induced in control 3T3-L1 cells treated for 24 h with anti-Fas and cycloheximide but not in PARP-depleted 3T3-L1 antisense cells exposed to these inducers. Similar results were obtain ed with control and PARP-depleted human Jurkat T cells. Whereas immort alized PARP +/+ fibroblasts showed the early burst of poly(ADP-ribosyl )ation and a rapid apoptotic response when exposed to anti-Fas and cyc loheximide, PARP -/- fibroblasts exhibited neither the early poly (ADP -ribosyl)ation nor any of the biochemical or morphological changes cha racteristic of apoptosis when similarly treated. Stable transfection o f PARP -/- fibroblasts with wild-type PARP rendered the cells sensitiv e to Fas-mediated apoptosis, These results suggest that PARP and poly( ADP-ribosyl)ation may trigger key steps in the apoptotic program. Subs equent degradation of PARP by caspase-3-like proteases may prevent dep letion of NAD and ATP or release certain nuclear proteins from poly(AD P-ribosyl)ation-induced inhibition, both of which might be required fo r late stages of apoptosis.