Figure 6. PARP inhibition sensitizes MMR-deficient cells to FdUrd. (A) HT29 cells transfected with control (Luc), MSH2, or MLH1 sRNAs were plated as single cells, exposed to the indicated concentrations of 5-FU or FdUrd for 24 h, washed, cultured for 10 d, and stained with Coomassie Blue. Transfected cells were also sequentially immunoblotted for MSH2 or MLH1 and beta-actin (a loading control). (B) HCT116.ch2 and HCT116.ch3 cells were exposed to the indicated concentrations of FdUrd along with vehicle or 300 nM AZD2281 for 24 h. Following washing, AZD2281 was re-added and the cells were cultured for 8 d to allow colony formation. doi:10.1371/journal.pone.0028862.g006 of all chemotherapy regimes used to treat colon cancer [1], the third leading cause of cancer-related death in the United States [76]. Despite its widespread use in the treatment of colon cancer, it remains unclear how this agent kills colon tumor cells. Similarly, FdUrd, which is often considered to have a similar mechanism of action to 5-FU, is also used to treat colon tumors that have metastasized to the liver. To gain insight into how these agents affect colon cancer cells we first carried out comprehensive analyses of the roles of the ATM and ATR checkpoint signaling pathways in colon cancer cells exposed to 5-FU and FdUrd, and then analyzed the role of the BER pathway, a repair pathway that removes uracil and uracil analogs that are incorporated into the genome. We previously compared the mechanisms by which 5-FU and FdUrd kill ovarian cancer cells. Notably, however, 5-FU has very limited clinical activity against ovarian cancer [49,50], and the DNA repair pathways that are disrupted in ovarian cancer differ from those disrupted in colon cancer. Specifically, ovarian cancers frequently exhibit ‘‘BRCAness’’ due to defects in BRCA1 or BRCA2, or other ill-defined changes that disrupt the homologous recombination DNA repair pathway [46]. In contrast, in colon cancers the mismatch repair pathway is frequently mutated or silenced [39,48], and the MMR pathway has been reported to affect cell killing by 5-FU and FdUrd [36– PARP Inhibitors Sensitize to Floxuridine 38,77,78]. Therefore, in the present report, we have performed head-to-head comparison of these agents in MMR-proficient and - deficient colon cancer cells that have been depleted of key checkpoint signaling and BER pathway intermediates. Important- ly, these mechanistic studies have uncovered novel insights into how these agents kill colon cancer cells and identified a potential therapeutic strategy against colon cancer. First, our studies demonstrated the ATR—but not the ATM— checkpoint signaling pathway plays a critical role facilitating the survival of cells treated with FdUrd. Although previous studies documented that FdUrd activates the ATM- and ATR-dependent checkpoints [10,13,79], these studies did not compare the effects of ATM and ATR depletions on the survival of tumor cells exposed to both agents. Here we have addressed that question. Surpris- ingly, we found that even though FdUrd has been reported to cause double-stranded DNA breaks [20,21], ATM has only a minor role in FdUrd-induced killing. In contrast, ATR depletion severely sensitized to FdUrd, demonstrating that ATR plays a critical role in stabilizing stalled replication forks and preventing their collapse, thus promoting cell survival when cells are treated with replication inhibitors such as the nucleoside analog gemcitabine [60]. Therefore, the present studies suggest that the disruption of DNA replication that occurs when TS is inhibited and the subsequent disruption of dNTP levels is likely a major mechanism by which FdUrd causes cytotoxicity. Second, the present results help clarify the role of BER in colon cancer cells exposed to 5-FU and FdUrd. Previous studies examining the role of the BER pathway have found disparate results, with increased, decreased, or unaltered sensitivity to 5-FU or FdUrd in a variety of experimental systems [17,23,25–35]. In contrast, the present results show that XRCC1 depletion sensitizes to FdUrd but not 5-FU. This finding, along with our published studies showing that an intact BER pathway protects ovarian cancer cells treated with FdUrd [17], indicates that FdUrd inflicts lesions that are cytotoxic to some human cancer cells. Consistent with these findings, two potent and highly specific small molecule inhibitors of PARP also sensitized to FdUrd. These results are similar to what was observed in ovarian cancer cells [17]. However, given that ovarian cancer cells often exhibit BRCAness (due to defects in homologous recombination) [46,80], a phenotype that renders cells exquisitely sensitive to PARP inhibitors [81], it remained an unanswered question whether PARP inhibitors would also sensitize to FdUrd in colon cancer cells, which do not have defects in homologous recombination. It should be noted, however, that although our XRCC1 findings strongly support a protective role for BER, the effects of the PARP inhibitors may be more complicated. PARP not only plays an important role in BER but also participates in other DNA repair pathways and cell signaling pathways, raising the possibility that the tremendous sensitization seen with the PARP inhibitors may stem from effects on BER as well as other cellular pathways. Third, the present studies show that depleting the apical regulators of checkpoint signaling (ATR and ATM) or disabling key BER pathway members (with XRCC1 and APE1 siRNAs or PARP inhibitors) did not sensitize to 5-FU. Such results strongly suggest that 5-FU is exerting its cytotoxic effects independently of its effects on DNA replication or integrity. Notably, this result is consistent with a number of studies showing that 5-FU mediates cell killing by incorporating into RNA and interfering with RNA metabolism [82–89]. In contrast, the finding that disabling the ATR and BER pathways strongly sensitizes to FdUrd, indicates that this agent kills colon tumor cells primarily by affecting DNA metabolism, thus demonstrating that 5-FU and FdUrd have very different mechanisms of action. PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e28862