PARP Inhibitors Sensitize to Floxuridine Figure 1. 5-FU and FdUrd activate the ATR and ATM checkpoint signaling pathways. (A) Metabolism of 5-FU and FdUrd. (B, C) HT29 (B) and HCT-8 (C) cells were treated with 5-FU (80 mM, HT29 cells; 200 mM HCT-8 cells), FdUrd (40 mM for both cell lines), or 10 mM hydroxyurea (HU) for the indicated times. Cell extracts were blotted for phospho-Ser317-Chk1 (P-Chk1), phospho-Thr68-Chk2 (P-Chk2), Chk1, or Chk2. TS, thymidylate synthase; TP, thymidine phosphorylase; UP, uridine phosphorylase; UK, uridine kinase; OPRT, orotate phosphoribosyltransferase; RR, ribonucleotide reductase; FUR, 5-fluorouridine; FUMP, 5-fluorouridine monophosphate; FUDP, 5-fluorouridine diphosphate; FUTP, 5-fluorouridine triphosphate; FdUMP, 5-fluorodeoxyuridine monophosphate; FdUDP, 5-fluorodeoxyuridine diphosphate; FdUTP, 5-fluorodeoxyuridine triphosphate. doi:10.1371/journal.pone.0028862.g001 uracil glycosylases, which excise the lesion, leaving an abasic site. The abasic site is further processed by an endonuclease (e.g., APE1), creating a single-stranded DNA break that is recognized by poly(ADP-ribose) polymerase, which poly(ADPribosyl)ates itself as well as other repair proteins, recruiting XRCC1 and other proteins that complete repair [24]. Investigations into the role of BER in cells treated with 5-FU or FdUrd have reached disparate conclusions using a wide variety of model systems. Given that these studies have shown that disabling BER protects, sensitizes, or has no effect on the cytotoxicity induced by 5-FU and FdUrd in these varied systems, including mouse [17,23,25–35], it remains unclear what, if any, role BER plays in the survival of colon cancer cells exposed to 5-FU or FdUrd. The other implicated repair pathway is the mismatch repair (MMR) system. In vitro studies have found that the MMR pathway can remove 5-FU from artificial substrates containing 5-FU:G mispairs. Notably, however, studies in cells suggest that MMR plays only a minor role in the excision of 5-FU lesions in the genome [35]. Analyses of the effects of the MMR pathway on cell viability following treatment with 5-FU and FdUrd have generally indicated that cells with defective MMR are more resistant to 5- FU and FdUrd [10,36–38], a result consistent with the finding that MMR-defective colon cancer patients do not benefit from 5-FU therapy [39]. Given the multiple mechanisms of action of 5-FU and FdUrd and the wide range of experimental systems used in these studies (including studies in mouse cell lines and in human cell lines derived from tumors that are not typically treated with 5-FU or FdUrd), we have initiated studies to address the roles of individual checkpoint signaling and DNA repair pathways in human cells derived from human tumors. In our first analysis, we found that 5- FU and FdUrd have very different mechanisms of action in ovarian cancer cells [17]. Disabling ATR or BER sensitized these cells to FdUrd, but not 5-FU, indicating that FdUrd kills cells by causing DNA damage and that 5-FU exerts its cytotoxicity by another mechanism, possibly by disrupting RNA metabolism. Notably, we also found that PARP inhibitors, which have shown unprecedented activity against ovarian tumors and other tumors that have mutated BRCA1 and BRCA2 [40–43], remarkably sensitized ovarian cancer cells to FdUrd but not 5-FU [17]. Given that FdUrd is active in ovarian cancer [44,45], that defects in BRCA1 and BRCA2 (or other genes in the homologous repair pathway) are common in ovarian cancer [46], and that PARP inhibitors will likely have a role in the treatment of ovarian cancers [24], these findings suggested a novel therapeutic strategy in ovarian cancer. Notably, however, the biology of ovarian cancer is very different from the biology of colon cancer. The oncogenes and tumor suppressor genes commonly mutated in colon cancers (APC, p53, PI3K, KRAS) differ from the genes commonly mutated in ovarian cancers (NF1, RB1, CDK12, CCNE1, NOTCH) [46,47]. Furthermore, the DNA repair pathways that are disrupted in colon cancer cells are vastly different from those disrupted in PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28862