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. 2001 May;12(5):1315–1328. doi: 10.1091/mbc.12.5.1315

Figure 6.

Figure 6

p53 is required for tetraploid arrest whether induced by inhibition of cytokinesis or by exit from mitosis without the formation of a mitotic spindle. p53 was inactivated in REF-52 (p53DD) by expression of a dominant-negative mutant (Shaulian et al., 1992). (A) Inactivation of p53 in cells expressing p53DD resulted in their inability to induce p21WAF1 following exposure to gamma irradiation, as demonstrated by Western blots. By contrast, control REF-52 cells (p53+/+) induced p21WAF1 following irradiation. (B) FACSscan analysis showed that both p53+/+ and p53DD cells had similar cell cycle profiles when untreated and had the 4N DNA content expected of cells following mitotic synchronization (mitotic shakeoff) and subsequent tetraploidization by exposure to 10 μM DCB for 5 h (DCB 5 h). Control p53+/+ cells remained arrested with 4N DNA content following exposure to DCB, whether released into drug-free medium for 19 or 67 h, or released into 0.04 μg/ml nocodazole for 19 h. By contrast, p53DD cells continued cycling when made tetraploid by exposure to DCB. Cells accumulated with 8N DNA content when released from DCB into nocodazole and became aneuploid when released from DCB into drug-free medium for 19 or 67 h. (C) Similarly, p53DD cells continued cycling when made tetraploid by aberrant exit from mitosis in the presence of 0.04 μg/ml nocodazole, whereas control p53+/+ cells arrested with 4N DNA content. p53DD cells accumulated a subpopulation of 8N cells when exposed to 0.04 μg/ml nocodazole for 24 h following mitotic synchronization, and they became aneuploid when mitoticallysynchronized, then treated with 0.04 μg/ml nocodazole for 5 h, and finally released into drug-free medium for either 19 or 67 h. (D) p21WAF1 was induced in a p53-dependent manner in cells made tetraploid with DCB, relative to controls or actively cycling cells 2 d after release from mitotic synchronization. Western blots show p21WAF1 was induced in p53+/+, but not p53DD cells following mitotic synchronization and then treatment with DCB for 5 h to obtain a tetraploid G1 population.