The novel poly(ADP-ribose) polymerase inhibitor, AG14361, sensitizes cells to topoisomerase I poisons by increasing the persistence of DNA strand breaks

Poly (ADP-ribose) polymerase (PARP) inhibitors enhance DNA topoisomerase I (topo I) poison-induced cytotoxicity and antitumor activity in vitro and in vivo, but the mechanism has not been defined. We investigated the role of PARP-1 in the response to topo I poisons using PARP-1(-/-) and PARP-1(+/+) mouse embryonic fibroblasts and the potent PARP-1 inhibitor, AG14361 (K-i < 5 nmol/L). PARP-1(-/-) mouse embryonic fibroblasts were 3 -fold more sensitive to topotecan than PARP-1(+/+) mouse embryonic fibroblasts (GI(50), 21 and 65 nmol/L, respectively). AG14361 caused a > 3-fold sensitization of PARP-1(+/+) cells to topotecan compared with a < 0.4-fold sensitization in PARP-1(-/-) cells. In human leukemia K562 cells, AG14361 caused a 2-fold sensitization to camptothecin-induced cytotoxicity. AG14361 did not affect the cellular activity of topo I as determined by measurement of cleavable complexes and topo I relaxation activity, showing that sensitization was not due to topo I activation. In contrast, repair of DNA following camptothecin removal, normally very rapid, was significantly retarded by AG14361, resulting in a 62% inhibition of repair 10 minutes after camptothecin removal. This led to a 20% increase in the net accumulation of camptothecin-induced DNA strand break levels in cells coexposed to AG14361 for 16 hours. We investigated the DNA repair mechanism involved using a panel of DNA repair-deficient Chinese hamster ovary cells. AG14361 significantly potentiated camptothecin-mediated cytotoxicity in all cells, except the base excision repair-deficient EM9 cells. Therefore, the most likely mechanism for the potentiation of topo I poison-mediated cytotoxicity by AG14361 is via PARP-1-dependent base excision repair.