Involvement of PARP and poly(ADP-ribosyl)ation in the early stages of apoptosis and DNA replication

We have focused on the roles of PARP and poly(ADP-ribosyl)ation early in ap optosis, as well as during the early stages of differentiation-linked DNA r eplication. In both nuclear processes, a transient burst of PAR synthesis a nd PARP expression occurs early, prior to internucleosomal DNA cleavage bef ore commitment to apoptosis as well as at the round of DNA replication prio r to the onset of terminal differentiation. In intact human osteosarcoma ce lls undergoing spontaneous apoptosis, both PARP and PAR decreased after thi s early peak, concomitant with the inactivation and cleavage of PARP by cas pase-3 and the onset of substantial DNA and nuclear fragmentation. Whereas 3T3-L1, osteosarcoma cells, and immortalized PARP +/+ fibroblasts exhibited this early burst of PAR synthesis during Fas-mediated apoptosis, neither P ARP-depleted 3T3-L1 PARP-antisense cells nor PARP -/- fibroblasts showed th is response. Consequently, whereas control cells progressed into apoptosis, as indicated by induction of caspase-3-like PARP-cleavage activity, PARP-a ntisense cells and PARP -/- fibroblasts did not, indicating a requirement f or PARP and poly(ADP-ribosyl)ation of nuclear proteins at an early reversib le stage of apoptosis. In parallel experiments, a transient increase in PAR P expression and activity were also noted in 3T3-L1 preadipocytes 24 h afte r induction of differentiation,, a stage at which similar to 95% of the cel ls were in S-phase, but not in PARP-depleted antisense cells, which were co nsequently unable to complete the round of DNA replication required for dif ferentiation. PARR a component of the multiprotein DNA replication complex (MRC) that catalyzes viral DNA replication in vitro, poly(ADP-ribosyl)ates 15 of similar to 40 MRC proteins, including DNA pol alpha, DNA topo I, and PCNA. Depletion of endogenous PARP by antisense RNA expression in 3T3-L1 ce lls results in MRCs devoid of any DNA pol alpha and DNA pol delta activitie s. Surprisingly, there was no new expression of PCNA and DNA pol alpha, as well as the transcription factor E2F-1 in PARP-antisense cells during entry into S-phase, suggesting that PARP may play a role in the expression of th ese proteins, perhaps by interacting with a site in the promoters for these genes.