The role of PARP-1 in the cellular response to topoisomerase I poisons

Abstract

P ARP-l inhibitors enhance DNA topoisomerase I (Topo I) poison-induced cytotoxicity and anti-tumour activity in vitro and in vivo but the underlying mechanism has not been defined. Two hypotheses have been proposed to explain this a) PARP-I modulates topo I activity via poly(ADP-ribosylation), or b) P ARP-l participates in the repair of topo 1- induced DNA lesions. To explore these mechanisms we have investigated the cellular effects of a novel potent P ARP-l inhibitor, AG 14361 (Ki < 5 nM), in combination with the topo I poisons, camptothecin and topotecan. PARP-l null mouse embryonic fibroblasts (MEFs) were 3-fold more sensitive to topotecan than P ARP-l wild type MEFs. AG 14361 significantly enhanced topotecaninduced growth inhibition by 3-fold in PARP-l wild-type cells but not PARP-l null cells. This confirms that P ARP-l activity promotes survival after topo I poison-induced cytotoxicity and the cellular effects of AG 14361 are due to P ARP-l inhibition. AG 14316 also increased camptothecin-induced growth inhibition in human K562 cells ~2-fold. AG14361 did not affect topo I-DNA cleavable complexes or topo I relaxation activity. In contrast, AG 14361 increased camptothecin-induced DNA strand breaks by 20% and significantly retarded DNA repair following camptothecin removal, (620/0 inhibition of repair 10 mins). These data indicate a role for PARP-l in the repair of topo I poison-mediated damage. The repair pathways by which P ARP-l acts to repair this damage was investigated using repair-deficient cells. AG 14361 significantly potentiated the cytotoxicity of camptothecin in AA8 repair-proficient and V3 nonhomologous end joining-deficient cells, but not the irs 1 SF homologous recombinationdeficient or EM9 base excision repair-deficient cells. AG 14361 also failed to retard the repair of camptothecin-induced DNA damage in EM9 cells. This suggests that P ARP-l may act via BER and possibly HR to repair topo I poison-mediated DNA damage.