Centrosome amplification and proliferation of untransformed cells after cleavage failure. (A) Centrosome amplification after repeated cleavage failure in RPE1 cell populations. Asynchronous cultures were treated with 0.5 µM cytochalasin D for 10 h (green bars) and split 100 h later. One culture was passaged seven times; the other was treated again with cytochalasin. This was repeated four more times. The percentages of binuclear cells after cytochalasin treatments are shown in blue. The percentages of centrosome amplification (more than four centrioles) are shown in bold with the number of cells counted shown above or below. (B) Proliferative capacity of binuclear cells and their progeny (yellow bars) and same-preparation control cells (green bars) after cytochalasin-induced cleavage failure. Bars show the percentage of cells that enter mitosis after completing the prior mitosis for each cell cycle; cells arresting in a previous cell cycle are not included. Percentages were calculated from lineages of multiple individual cells followed in one experiment. (C) Proliferative capacity of binuclear cells (yellow bars) and same-preparation control cells (green bars) after blebbistatin-induced cleavage failure. One experiment is shown on multiple cells.

Expression of p53 and p21 in untransformed cells that stop cycling after cleavage failure. (A) Binuclear cell arrested after cleavage failure expressing p53 (red), p21 (green), and DNA (blue). (B) Daughters of a binuclear cell fixed 15 h after first tetraploid mitosis showing p53 (red) and p21 (green). (C) Percentages of cells expressing only p53, only p21, or both. The first column shows binucleates that arrested in first interphase, the second column shows daughters of binucleates, and the third column shows same-preparation control cells. Fluorescence micrographs are shown. Bar, 50 µm.

Centrosome amplification and proliferation of HCT116 p53^−/− cells after cleavage failure. (A) Centrosome amplification in proliferative HCT116 p53^−/− cell populations after repeated cleavage failure. The experimental protocol and display of the results are the same as those shown in Fig. 1 A. Large multinucleated cells are not scored because they do not proliferate. The percentages of binuclear cells after cytochalasin treatments are shown in blue. The percentages of centrosome amplification (more than four centrioles) are shown in bold with the number of cells counted shown above or below. (B) Proliferative capacity of binuclear cells (yellow bars) and same-preparation control cells (green bars). Orange portions of the bars indicate the percentages of cells that spontaneously fail cleavage at mitosis. Such cells cycle but repeatedly fail cleavage and cease proliferating. One experiment is shown on multiple cells. (C, top) Binuclear cell (asterisks) exhibiting multiple rounds of cleavage failure, resulting in a large multinucleated cell. (bottom) Death of another large multinuclear cell. Times are in hours and minutes. Phase-contrast microscopy is shown. Bar, 50 µm.

Centrosome amplification and proliferation of HeLa and CHO cells after cleavage failure. (A) Proliferative capacity of binuclear HeLa cells (yellow bars) and same preparation control cells (green bars). Black portions of bars indicate the percentage of the cells that die during or just after that mitosis. (B) Proliferative capacity of binuclear CHO cells (yellow bars) and same-preparation control cells (green bars). The darker portions of the yellow bars denote the percentage of cells that fail cleavage at each mitosis. (A and B) One experiment is shown on multiple cells. (C) Centrosome amplification after repeated cleavage failure in CHO cell populations. The experimental protocol and display of the results are the same as those shown in Fig. 1 A. The percentages of binuclear cells after cytochalasin treatments are shown in blue. The percentages of centrosome amplification (more than four centrioles) are shown in bold with the number of cells counted shown above or below.