ovarian cancers. For example, the genes required for mismatch repair pathway (e.g., MLH1 and MSH2) are often mutated or epigenetically silenced in colon cancers [39,48], whereas defects in homologous recombination (e.g., BRCA1 and BRCA2 mutations) occur in ovarian cancers [46,47]. Finally, ovarian and colon tumors have very different responses to 5-FU. Whereas 5-FU has very limited activity in ovarian cancer [49,50], 5-FU is the cornerstone for all chemotherapy regimens used to treat colon cancers due to its high activity in this disease [1]. Therefore, given these biological differences between ovarian and colon cancers, and the fact that 5-FU is universally used in colon cancer chemotherapy regimens, we have now determined how 5-FU and FdUrd kill colon cancer cells to gain important insights underlying the biology of these agents and improve their use in the clinic to treat this disease. To that end, we initiated a systematic analysis of the roles of genotoxin-activated checkpoint signaling, the BER pathway, and the MMR pathway by depleting key signaling intermediates in these pathways using highly effective siRNAs. These findings not only further illuminate our understanding of the signaling and DNA repair pathways that are important in these cells, they also reveal that colon tumor cells are sensitized to FdUrd by small molecule PARP inhibitors that are currently in clinical trials, thus suggesting a novel therapeutic approach that combines FdUrd, a drug approved for the treatment of colon cancer, with a PARP inhibitor, an emerging class of agents with exciting anticancer activity. Materials and Methods Cell lines and culture HT29, HCT-8, and HCT-116 cells were obtained from American Type Culture Collection. HCT-116.ch2 and HCT- 116.ch3 [51] cells were from Scott Kaufmann (Mayo Clinic). Cells were cultured in RPMI-1640 media (Mediatech) supplemented with 10% fetal bovine serum (Atlanta Biologicals) at 37uC in 5% CO2. For clonogenic assays, the medium was supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin (Mediatech). Materials Reagents were from the following suppliers: 5-fluorouracil (APP Pharmaceuticals), FdUrd (Bedford Laboratories), ABT-888 (Sell- eck Chemicals and ChemieTek), AZD2281 (ChemieTek), KU- 55933 (Tocris Bioscience), gemcitabine (Eli Lilly), SuperSignal Pico West (Thermo Scientific). All other materials were from Sigma-Aldrich. Antibodies were as follows: phospho-Ser317-Chk1 (R&D Systems); phospho-Thr68-Chk2, ATR, horseradish peroxidase- linked rabbit IgG, and horseradish peroxidase-linked mouse IgG (Cell Signaling); Chk1 (Santa Cruz Biotechnology); Chk2 and ATM (Epitomics); APE1 (Abcam); XRCC1 (Bethyl Laboratories); beta-actin (Sigma-Aldrich); and HSP90, D. Toft (Mayo Clinic, Rochester, MN). Cell transfections and small interfering (si)RNAs siRNAs were transfected by electroporation as described [17]. The transfected cells were cultured for 48 h before use. Sequences of siRNAs were: ATM-1, 59-GCACCAGUCCAGUAUUGGC-39 [52]; ATR-2, 59-CCUCCGUGAUGUUGCUUGA-39 [53]; XRCC1-2, 59-CUCGACUCACUGUGCAGAA-39 [54]; APE1, 59-GGACAGAGCCAGAGGCCAA-39; MLH1, 59-GGAAGAU- UCUGAUGUGGAA-39; MSH2, 59-GAUCCUAAUCUCAGU- GAAU-39; and luciferase, 59-CUUACGCUGAGUACUUCGA- 39 [55]. PARP Inhibitors Sensitize to Floxuridine Clonogenic assays, cell lysis, and cell irradiation Cell cycle analyses, clonogenic assays, cell lysis, immunoblotting and immunostaining were performed as described [56,57]. For clonogenic assays using non-transfected cells, percent survivals of all individual and combination treatments were normalized to cells treated with vehicle only. For clonogenic assays using cells transfected with siRNA, percent survivals at each drug concen- tration were normalized to the vehicle-treated control for the given siRNA. Cells were irradiated with a RS-2000 Biological Irradiator, Rad Source (Suwanee, GA) 4–6 h after plating. Results 5-FU and FdUrd activate the ATR and ATM checkpoint signaling pathways in colon cancer cells To assess the effects of 5-FU and FdUrd on the ATM and ATR checkpoint signaling pathways, we compared the abilities of these agents to activate checkpoint signaling in two colon cancer cell lines: HT29, which have a functional MMR system, and HCT-8, which have a mutations in MSH6 and are MMR deficient [58]. Cells were treated with concentrations of each agent that inhibit clonogenicity by 90% (IC90) and, as a positive control, the replication inhibitor hydroxyurea. Activation of the ATM and ATR pathways was then assessed by immunoblotting for phosphorylated Chk1 and Chk2, two protein kinases that are phosphorylated and activated by ATR and ATM [59]. These studies revealed that 5-FU and FdUrd strongly activated Chk1 and Chk2 in HT29 cells (Fig. 1B), with 5- FU causing even greater levels of Chk1 phosphorylation than did FdUrd. Similarly, in HCT-8 cells, both agents induced Chk1 phosphorylation (Fig. 1C); however, in these cells 5-FU induced less Chk1 than did FdUrd. Analyses of Chk2 phosphorylation were not possible due to the very low levels of Chk2 in the HCT-8 cells (data not shown). Taken together, these results demonstrate that both agents cause genotoxic damage that activates checkpoint signaling pathways in colon cancer cells. ATR and ATM promote the survival of colon cancer cells treated with FdUrd but not 5-FU ATM and ATR are the two apical kinase regulators of genotoxin-induced checkpoint signaling. To determine if either ATM or ATR activate pathways that affect the survival of cells treated with FdUrd or 5-FU, we transiently depleted ATM and ATR using siRNAs, and then assessed the capacity of cells treated with graded concentrations of FdUrd or 5-FU to proliferate using clonogenic assays. Surprisingly, depletion of ATM or ATR did not sensitize either cell line to 5-FU (Fig. 2A and 2B), even though this agent activated these pathways (see Fig. 1B and 1C). A far different picture emerged when the cells were treated with FdUrd. Depletion of ATR dramatically sensitized HT29 cells to FdUrd (Fig. 2B) and to gemcitabine (Fig. S1A), a nucleoside analog that inhibits ribonucleotide reductase and disrupts DNA replication when incorporated into DNA [60]. In contrast, ATM depletion (Fig. 2A) and the ATM inhibitor KU-55933 [61] (Fig. S1C), both of which sensitized to ionizing radiation (Fig. S1B and Fig S1D), had minimal effects on FdUrd cytotoxicity. Similar results were also seen in HCT-8 and HCT-116 cells, in which ATR depletion sensitized both cell lines to FdUrd but not 5-FU (Fig. 3). Disruption of BER by depleting XRCC1 sensitizes to FdUrd but not 5-FU 5-FU and FdUrd cause the accumulation of uracil and 5- fluorouracil in genomic DNA [23,62]. Studies using purified uracil glycosylases have shown that synthetic substrates bearing uracil PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28862