Figure 5.

A, Clonogenic survival assay comparing DLD1 cells with wild-type or absent BRCA2. Surviving fractions normalized to the cell kill by AZD6738 alone, expressed in the inset box. Solid lines: wild-type, interrupted lines: BRCA2 null. B, Analysis of cell-cycle distributions over a 60-hour time period following 4 Gy or mock irradiation in the presence of AZD6738 (0.9 µmol/L, the IC₅₀ value derived from 72-hour MTT assay) or DMSO control. Nuclei were automatically quantified and cell-cycle phase assigned to each nucleus depending upon fluorescence. C, Quantification of micronuclei (expressed as number of micronuclei counted divided by number of nuclei counted) over the time course. Points represent mean of 3 measurements and lines represent the smoothed mean. D, Representative images from time lapse microscopy. Top, a cell going through normal mitosis; middle, the first mitosis yielding micronuclei, dyssynchrony of cell-cycle between the micronucleus and nucleus and subsequent apoptosis at the second mitosis; bottom, a cell undergoing apoptosis at the first mitosis. E, Representative images showing micronuclei with γH2AX staining (top) and representative images showing cell-cycle phase dyssynchrony between the main nuclei and micronuclei. F, Western analysis showing levels of cleaved caspase 3 and cleaved PARP, reflective of apoptotic cell death, 24 hours after mock irradiation or 4 Gy in the presence of 0.25 μmol/L AZD6738 or DMSO control in Cal27. G, Quantification of fixed Cal27 and FaDu cells, 24 hours after mock irradiation or 4 Gy in the presence of 0.25 mmol/L AZD6738 or DMSO control; box plots represent range, with line at median number of micronuclei per nucleus. H, Proportion of micronuclei positive or negative for the kinetochore marker CENPA, treated with 4 Gy or 4 Gy in the presence of 0.25 µmol/L AZD6738 in Cal27 (left) and FaDu (right). Images show representative nuclei and micronuclei showing absence of CENPA in micronuclei. Scale bar, 10 µm. I, Quantification of the average number of micronuclei over the 60-hour time course, in cells treated with AZD6738, the Chk1 inhibitor CCT244747 at isoeffective doses for single-agent growth inhibition (0.9 µmol/L AZD6738, 1.3 µmol/L CCT244747).