Figure 2.

A, Radiosensitization of A549 cells by AZD6738 demonstrated by a clonogenic assay. Number of colonies expressed as a fraction of the number on untreated plates. Surviving fractions normalized to the cell kill by AZD6738 alone, expressed in the box on each graph. B, Cal27cells, as per (A); C, FaDu cells, as per (A). D, clonogenic assay demonstrating radiosensitization of HCT116 wild-type and p53-null cells by AZD6738. Clonogenic data were analyzed by ANOVA with multiple comparisons; *, P < 0.05. A minimum of 3 replicates; error bars represent SEM. SER, sensitizer enhancement ratio obtained by fitting a linear quadratic curve and obtaining the ratio for sensitized and unsensitized conditions to achieve survival of 0.37. E, Western blot for ATM, Chk1, Chk2 function 1 hour after 4 Gy in the presence or absence of AZD6738. F, Cell-cycle distribution effect of AZD6738 and radiation, propidium iodide flow cytometry. Cells exposed to 0.5 µmol/L AZD6738 or DMSO control, 1 hour prior to 4 Gy or mock irradiation, harvested 16 or 24 hours later. Percentage cell-cycle distributions are displayed. G, Cal27 cells were stained for mitotic fraction using phospho-histone-H3. AZD6738 was added 1 hour prior to irradiation and cells harvested 6 hours after. Percentage of cells staining positive for phospho-histone-H3 are displayed. Error bars, SEM. Minimum of 3 replicates. H, Analysis of tumor spheroid growth for FaDu and SCC7 cells treated with AZD6738 (1 µmol/L), fractionated radiation (20 Gy in 10 fractions over 2 weeks), or the combination. Drug was added 1 hour prior to radiation and removed after the final fraction of radiation. Spheroids were imaged weekly. Error bars: SEM, 3 replicates; *, P < 0.05 between RT-only and RT-ATRi curves.