Finally, and most importantly, these studies, which were initiated to identify the checkpoint and DNA repair pathways that regulate colon tumor responses to FdUrd and 5-FU, demonstrated that BER was a critical repair pathway when these cells were exposed to FdUrd (but not 5-FU). Based on these findings, and the fact that PARP inhibitors disrupt BER, we then discovered that small molecule PARP inhibitors robustly sensitized MMR-deficient and –proficient colon cancer cells to FdUrd (but not 5-FU). These findings may be of particular importance in tumors with defects in MMR, which account for 15–20% of all colon cancers [39]. Previous studies found that MMR-deficient cell lines are less sensitive to 5-FU and FdUrd. Consistent with this result, clinical studies have shown that 5-FU has limited activity against MMR-deficient colon cancers compared to MMR- proficient tumors [39]. Given that 1) FdUrd is approved for the treatment of colon cancer; and 2) there are limited therapeutic options for these tumors because tumors with defects in MMR are commonly considered to be unresponsive to 5-FU-based therapies, our finding that PARP inhibitors robustly sensitize MMR-deficient cells to FdUrd raises the possibility that therapies that combine FdUrd with a PARP inhibitor may have activity against these tumors. Similarly, because PARP inhibitors also sensitize mis- match proficient tumors to FdUrd, this drug combination may also be useful in the treatment of these tumors. Further preclinical and clinical development of this combination is warranted. Supporting Information Figure S1 Effects of ATR and ATM disruptions on sensitivity to gemcitabine and ionizing radiation. (A) ATR depletion sensitizes to gemcitabine. HT29 cells transfected with control (Luc) or ATR siRNAs from experiment shown in Fig. 2B were plated as single cells, exposed to the indicated concentrations of gemcitabine for 24 h, washed, and cultured for 10 d to allow colony formation. (B) ATM depletions sensitize to ionizing radiation (IR). HT29 cells transfected with control (Luc) References 1. Longley DB, Harkin DP, Johnston PG (2003) 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 3: 330–338. 2. Samuelsson T (1991) Interactions of transfer RNA pseudouridine synthases with RNAs substituted with fluorouracil. Nucleic Acids Res 19: 6139–6144. 3. Sun XX, Dai MS, Lu H (2007) 5-fluorouracil activation of p53 involves an MDM2-ribosomal protein interaction. J Biol Chem 282: 8052–8059. 4. Patton JR (1993) Ribonucleoprotein particle assembly and modification of U2 small nuclear RNA containing 5-fluorouridine. Biochemistry 32: 8939–8944. 5. 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HT29 cells were plated as single cells and allowed to adhere for 4 h. For the FdUrd experiment (C), the cells were first exposed to the indicated concentrations of KU-55933 for 15 min and then FdUrd was added. Cells were then incubated for 24 h, washed, and cultured for 10 d to allow colony formation. For the IR experiment (D), the cells were exposed to the indicated concentrations of KU-55933 for 15 min, irradiated, washed after 24 h to remove the KU- 55933, and cultured for 10 d to allow colony formation. (TIF) Figure S2 APE1 depletion sensitizes HT29 cells to FdUrd. Cells were transfected with control (Luc) or APE1 siRNAs. 48 h later, the cells were plated as single cells, treated with the indicated concentrations of FdUrd for 24 h, washed, and cultured for 10 d to allow colony formation. (TIF) Figure S3 Depletion of MSH2 and MLH1 disrupts MNNG-induced G2/M cell cycle arrest. 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