Figure 3

AZD1152-HQPA induces apoptosis and reduces clonogenic potential in breast cancer cells. (A) HER18 and MDA-MB-231 cells were exposed to AZD1152-HQPA (100 nM and 105 nM) for up to 72 hours and cells were stained with Annexin V-FITC plus propidium iodide. Samples were analyzed by flow cytometry for PI (y-axis) and Annexin V (x-axis) at 3 time points. Representative scattergrams are shown with percentages of cells displayed for each quadrant: Lower Left - live cells, Lower Right - early apoptosis, Upper Right - late apoptosis and Upper Left - necrotic cells. Overall percent apoptosis was calculated for each time point by adding Lower Right and Upper Right quadrants. (B) Apoptosis of HER18 and MDA-MB-231 cells was analyzed by Western blotting with anti-PARP antibody. PARP appears at 115 KD while the apoptotic indicator, cleaved PARP, appears at 85 KD. (C) HER18 cells were plated in triplicate at 5000 cells/plate in 6 cm tissue culture dishes and treated with either vehicle or 40 nM AZD1152-HQPA. Representative plates are shown after incubation for 12 days and staining with crystal violet. (D) Mean colony numbers from C are plotted for AZD1152-HQPA versus control. (E) Soft agar clonogenic assay was performed using HER18 cells. Mean soft agar colony numbers from triplicate plates are plotted for AZD1152-HQPA versus control. All Error bars represent 95% confidence intervals.