The Novel Poly(ADP-Ribose) Polymerase Inhibitor, AG14361, Sensitizes Cells to Topoisomerase I Poisons by Increasing the Persistence of DNA Strand Breaks

Abstract

Poly(ADP-ribose) polymerase (PARP) inhibitors enhance DNA topoisomerase I (topo I) poisoninduced 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 À/À and PARP-1 +/+ mouse embryonic fibroblasts and the potent PARP-1 inhibitor, AG14361 (K i < 5 nmol/L). PARP-1 À/À mouse embryonic fibroblasts were 3-fold more sensitive to topotecan than PARP-1 +/+ mouse embryonic fibroblasts (GI 50 , 21 and 65 nmol/L, respectively). AG14361caused a >3-fold sensitization of PARP-1 +/+ cells to topotecan compared with a <1.4-fold sensitization in PARP-1 À/À 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.

Enhanced Killing of Cancer Cells by Poly(ADP-ribose) Polymerase Inhibitors and Topoisomerase I Inhibitors Reflects Poisoning of Both Enzymes

“…studies demonstrated that a variety of PARP inhibitors enhance the antiproliferative and cytotoxic effects of topo I poisons (23)(24)(25)(26)(27)(28)(29)(30)(31). Consistent with those earlier studies, we observed that treating various solid tumor cell lines, including A2780, SKOV3, Ovcar5, and A549 cells, with the PARP inhibitor veliparib (78) sensitized cells to the topo I poison topotecan (Fig.…”

“…Our observation that Parp1 Ϫ/Ϫ and Parp1 ϩ/ϩ fibroblasts exhibit similar camptothecin sensitivity appears to be at odds with a prior report that Parp1 Ϫ/Ϫ fibroblasts are hypersensitive to camptothecin (30). In that study Smith et al (30) of control cells (Fig.…”

“…A number of previous studies have demonstrated that small molecule PARP inhibitors selectively sensitize cells to topo I poisons in vitro and in vivo (23)(24)(25)(26)(27)(28)(29)(30)(31)(32). At least three explanations have been advanced to explain these observations.…”

“…TDP1 interacts with several components of the base excision repair pathway, including XRCC1, polynucleotide kinase phosphatase, and DNA ligase III (48,49). Other studies have shown that cells lacking functional base excision repair components such as XRCC1 are also hypersensitive to topo I poisons (30,50,51). Moreover, XRCC1 and DNA ligase III are typically recruited to sites of DNA damage by PARP1 and pADPr (52,53).…”

A Ubiquitin-Proteasome Pathway for the Repair of Topoisomerase I-DNA Covalent Complexes

“…PARP1, known to be involved in base excision repair, has also been shown to participate in the repair of CPT-induced DNA damage (27,28). To test whether CPT-induced SSBs are the substrates for PARP1-mediated repair, PARP1 inhibitors , and Top1 down-regulation assay were performed as described under "Experimental Procedures."…”