Checkpoint Signaling, Base Excision Repair, and PARP Promote Survival of Colon Cancer Cells Treated with 5- Fluorodeoxyuridine but Not 5-Fluorouracil Liyi Geng 1 , Amelia M. Huehls1,2, Jill M. Wagner1, Catherine J. Huntoon1, Larry M. Karnitz1,2,3* 1 Division of Oncology Research, Mayo Clinic, College of Medicine, Rochester, Minnesota, United States of America, 2 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, College of Medicine, Rochester, Minnesota, United States of America, 3 Department of Radiation Oncology, Mayo Clinic, College of Medicine, Rochester, Minnesota, United States of America Abstract The fluoropyrimidines 5-fluorouracil (5-FU) and FdUrd (5-fluorodeoxyuridine; floxuridine) are the backbone of chemotherapy regimens for colon cancer and other tumors. Despite their widespread use, it remains unclear how these agents kill tumor cells. Here, we have analyzed the checkpoint and DNA repair pathways that affect colon tumor responses to 5-FU and FdUrd. These studies demonstrate that both FdUrd and 5-FU activate the ATR and ATM checkpoint signaling pathways, indicating that they cause genotoxic damage. Notably, however, depletion of ATM or ATR does not sensitize colon cancer cells to 5-FU, whereas these checkpoint pathways promote the survival of cells treated with FdUrd, suggesting that FdUrd exerts cytotoxicity by disrupting DNA replication and/or inducing DNA damage, whereas 5-FU does not. We also found that disabling the base excision (BER) repair pathway by depleting XRCC1 or APE1 sensitized colon cancer cells to FdUrd but not 5-FU. Consistent with a role for the BER pathway, we show that small molecule poly(ADP-ribose) polymerase 1/2 (PARP) inhibitors, AZD2281 and ABT-888, remarkably sensitized both mismatch repair (MMR)-proficient and -deficient colon cancer cell lines to FdUrd but not to 5-FU. Taken together, these studies demonstrate that the roles of genotoxin- induced checkpoint signaling and DNA repair differ significantly for these agents and also suggest a novel approach to colon cancer therapy in which FdUrd is combined with a small molecule PARP inhibitor. Citation: Geng L, Huehls AM, Wagner JM, Huntoon CJ, Karnitz LM (2011) Checkpoint Signaling, Base Excision Repair, and PARP Promote Survival of Colon Cancer Cells Treated with 5-Fluorodeoxyuridine but Not 5-Fluorouracil. PLoS ONE 6(12): e28862. doi:10.1371/journal.pone.0028862 Editor: Abbes Belkhiri, Vanderbilt University Medical Center, United States of America Received August 2, 2011; Accepted November 16, 2011; Published December 15, 2011 Copyright: 2011 Geng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health Grants R01-CA084321 (LK), P50-CA136393 (LK), GT32-M072474 (AH), a Mayo Clinic Eagles Pilot Project Award, and the Mayo Clinic. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: karnitz.larry@mayo.edu Introduction 5-fluorouracil has activity in multiple cancers and is one of the most widely prescribed anticancer agents, but its most frequent use is in colon cancer, where it is the basis for all modern colon cancer therapies. After uptake into cells, 5-FU undergoes complex metabolic reactions (Fig. 1A; rev. in [1]) to produce 3 known cytotoxic metabolites: FUTP (5-fluorouridine triphosphate), FdUMP (5-deoxyuridine monophosphate), and FdUTP (5-deox- yuridine triphosphate). The FUTP affects RNA metabolism following its incorporation into cellular RNA, where it disrupts snRNA, tRNA, and rRNA processing as well as the ribonucleolytic activity of the exosome and pseudouridylation of RNA [2–8]. In contrast, FdUMP and FdUTP disrupt DNA metabolism. These metabolites are produced following the conversion of 5-FU to FdUrd (5-fluorodeoxyuridine; floxuridine), which is also an FDA- approved chemotherapy agent for the treatment of colon cancer [9] and is often considered to have a similar mechanism of action as 5- FU. FdUrd is then phosphorylated to FdUMP and further phosphorylated to FdUTP [1]. The FdUMP inhibits thymidylate synthase (TS), which prevents the conversion of dUMP to dTMP, ultimately causing the depletion of dTTP, the accumulation of dUTP, and the disruption of dNTP ratios. In contrast, FdUTP, as well as the accumulated dUTP, are incorporated into DNA. Consistent with their abilities to disrupt dNTP levels, both FdUrd and 5-FU activate the ATR checkpoint signaling pathway [10–17], a pathway that is triggered when DNA replication is inhibited and that also plays critical roles in promoting survival of cells experiencing replication stress produced by dNTP disruption and/or DNA lesions [18]. Once activated, ATR phosphorylates multiple substrates, including Chk1. The collective kinase activities of ATR and Chk1 orchestrate the S phase checkpoint and regulate DNA repair to promote cell viability and recovery [19]. Additionally, 5-FU and FdUrd also cause double strand DNA breaks [20,21], which in turn activate the ATM checkpoint signaling pathway. The ATM pathway also regulates cell survival by either inducing apoptosis or preventing cell cycle progression and activating DNA repair, both of which promote survival [22]. Notably, however, it remains unclear whether the ATR and/or ATM checkpoint pathways play important roles in determining the survival of human colon cancer cells, the cells that are targeted by 5-FU and FdUrd in patients, when they are treated with these agents. The uracil and 5-FU that are incorporated into the genome are also recognized by 2 DNA repair pathways that may play roles in the survival of cells treated with 5-FU and FdUrd. One pathway is the base excision repair (BER) pathway [1,23]. In this pathway, genomically incorporated uracil and 5-FU are first recognized by PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28862