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

Authors' Affiliations: ¹ Northern Institute for Cancer Research and ² Institute for Cell and Molecular Biosciences, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom

Requests for reprints: Nicola J. Curtin, Northern Institute for Cancer Research, University of Newcastle upon Tyne, Paul O'Gorman Building, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom. Phone: 44-191-246-4415; Fax: 44-191-246-4301; E-mail: n.j.curtin{at}ncl.ac.uk.

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 (GI₅₀, 21 and 65 nmol/L, respectively). AG14361 caused a >3-fold sensitization of PARP-1^+/+ cells to topotecan compared with a <1.4-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.

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