Figure 2.

A mitotic entry delay triggers cell death in p53-deficient cells. Hela or HCT116 cells were synchronized with a double thymidine block protocol, released and pulsed with either DMSO, a combination of S1451 (300 nM; AURKA inhibitor) and BI2536 (0.8 nM; PLK1 inhibitor), or RO3306 (10 μM; CDK1 inhibitor) for 4 hours beginning 6 hours after the second release. Cell viability was measured by morphological changes recorded from phase contrast images or trypan blue exclusion. (A) Representative phase contrast images of a cell undergoing apoptosis following a mitotic entry delay. Time, in minutes, is given in each frame from an arbitrary point prior to cell retraction. Scale bar is 10 μm. (B) Cells were followed by phase contrast imaging for 48–72 hrs after the 4 hr drug inhibitor pulse and cell death measured by morphological changes as shown in (A). The mitotic entry delay induced by either a combination of 300 nM S1451 and 0.8 nM BI2536 or 10 μM RO33306 significantly increased the percentage of cells that died within 72 hours after the drug pulse. (C) Asynchronously growing Hela or HCT116 p53^−/− cells were treated with a 4 hour pulse of inhibitors and followed by live cell recordings as in (A, B). Treatment with the combination of 300 nM S1451 and 0.8 nM BI2536 or with 10 μM RO33306 in asynchronously growing cell populations did not decrease cell viability, demonstrating that the drugs are not simply toxic throughout the cell cycle. (D) Synchronized HCT116 p53^+/+ and p53^−/− cell lines were pulsed with 10 μM RO3306 as in (B). Viability was assayed 48 hours post inhibitor treatment via trypan blue exclusion. A mitotic entry delay via CDK1 inhibition decreased cell viability only in the p53 knockout cell line. Graphs are representative of at least 3 independent experiments with >300 cells/experiment. ** denotes P < 0.01, *** denotes P < 0.001.