PARP Inhibitors Sensitize to Floxuridine Figure 2. Depletion of ATR sensitizes to FdUrd but not 5-FU, whereas ATM depletion does not affect sensitivity to either agent. (A, B) HT29 cells, transfected with control (Luc), ATM (A), or ATR (B) siRNAs, 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 (insets) to detect ATM, ATR, and HSP90 (as a loading control). *, non-specific band. doi:10.1371/journal.pone.0028862.g002 and 5-fluorouracil substituents are substrates for the BER machinery [35,63–70]. Furthermore, a recent report demonstrat- ed that in intact cells, uracil glycosylases remove 5-FU from the genomes of colon cancer cells exposed to FdUrd [35]; notably, however, in these studies, depletion of the glycosylases did not affect the sensitivity to FdUrd. Therefore, to examine whether disabling BER affected the sensitivity of HT29 cells to FdUrd, we used siRNAs to deplete XRCC1 and APE1, two downstream key participants in the BER pathway, and examined their sensitivity to FdUrd. Significantly, depletion of XRCC1 (Fig. 4) and APE1 (Fig. S2) sensitized cells to FdUrd. In contrast, XRCC1 depletion did not sensitize these cells to 5-FU (Fig. 4), thus indicating that BER does not play a role in promoting the survival of cells treated with 5-FU and further suggesting that 5-FU exerts its cytotoxic effects independently of DNA replication or damage. Small molecule PARP inhibitors sensitize colon cancer cells to FdUrd but not 5-FU Given that XRCC1 and APE1 depletion sensitized colon cancer cells to FdUrd, and that PARP plays a key role in BER, we reasoned that PARP inhibitors may sensitize colon cancer cells to FdUrd. We therefore exposed HCT-8 and HT29 cells to graded concentrations of FdUrd or 5-FU along with 3 mM ABT-888 (veliparib [71]), a concentration that was reported previously to sensitize multiple tumor cell lines to a variety of chemotherapy agents [17,72]. As shown in Fig. 5, ABT-888 robustly sensitized HCT-8 and HT29 cells to FdUrd, whereas ABT-888 did not alter the antiproliferative effects of 5-FU. To further demonstrate that PARP inhibitors sensitize these cells to FdUrd, we also tested the PARP inhibitor AZD2281 (olaparib [73]), which has shown unprecedented activity in heavily pretreated patients with BRCA1- and BRCA2-deficient tumors [40–43]. Similar to the results seen with ABT-888, AZD2281 robustly sensitized both cell lines to FdUrd (Fig. 5), further supporting the idea that PARP inhibition sensitizes colon tumor cells to FdUrd. Small molecule PARP inhibitor sensitization to FdUrd is independent of MMR status Previous reports demonstrated that cells with defects in MMR are more resistant to FdUrd [10,36–38]. Similarly, patients treated with 5-FU do not benefit from 5-FU-based chemotherapies [39], suggesting that an intact MMR pathway promotes killing by 5-FU. PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e28862