PARP Inhibitors Sensitize to Floxuridine Figure 3. ATR depletion sensitizes HCT-8 and HCT-116 to FdUrd but not 5-FU. (A, B) HCT-8 (A) or HCT-116 (B) cells transfected with control (Luc) or ATR 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 for ATR and HSP90 (a loading control). *, non-specific band. doi:10.1371/journal.pone.0028862.g003 Because combining FdUrd with a PARP inhibitor may be a potential therapeutic strategy, we reasoned that it would be important to determine whether tumor cells with defects in MMR, which occur in 15–20% of colon cancers [39], were sensitized to FdUrd by a PARP inhibitor. To assess how MMR status affects the sensitivity of colon cancer cells to FdUrd alone and to the combination of FdUrd plus AZD2281 we used two model systems. For the first model system, we used siRNAs to deplete MSH2 and MLH1. Both siRNAs were highly effective, causing near-complete loss of MLH1 and MSH2 (Fig. 6A) and disrupting MNNG (N- methyl-n9-nitro-n-nitrosoguanidine)-induced G2/M arrest (Fig. S3), which requires a functional MMR pathway [51]. Notably, Figure 4. The BER pathway protects cells from FdUrd but not 5-FU. HT29 cells transfected with control (Luc) or XRCC1 siRNA 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 XRCC1 and beta-actin (a loading control). doi:10.1371/journal.pone.0028862.g004 PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28862