Multipolar mitosis of tetraploid cells: inhibition by p53 and dependency on Mos Effect of p53 on the survival and on the genomic stability of tetraploid.

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Multipolar mitosis of tetraploid cells: inhibition by p53 and dependency on Mos Effect of p53 on the survival and on the genomic stability of tetraploid HCT 116 cells. (A, B) The absence of p53 increases the clonogenic survival of freshly generated polyploid cells. Wild type (WT), p53−/−, Bax−/− and p21−/− diploid human colon carcinoma HCT 116 cells and HCT 116 cells stably transfected with a plasmid encoding the baculoviral inhibitor of caspases p35 (p35) were left untreated or treated with 100 nM nocodazole (Noco) for 48 h. After washing, cells were cultured for additional 48 h in drug‐free culture medium, then stained with Hoechst 33342, followed by fluorescence‐activated cell sorter (FACS) purification of diploid (2n, white and grey symbols for untreated and nocodazole‐treated cells, respectively) or polyploid (>4n, black symbols) cell populations. In A, representative cell cycle distributions of WT and p53−/− cells from one out of three independent experiments are shown. X‐axis = Hoechst 33342 fluorescence (DNA content); Y‐axis = cell number per channel (counts). In B, the results of clonogenic survival assays carried out on such FACS‐purified cells are reported. The upper part shows representative pictures of colonies formed by WT and p53−/− cells as observed upon crystal violet staining 10 days after FACS purification. Columns depict the survival fraction (mean±s.e.m., n=3 parallel wells, normalized for plating efficiency and to control diploid cells) of the diploid and polyploid cell populations with the indicated genotype and corresponding to the FACS‐purified populations represented in A. Asterisks indicate statistically significant differences as compared to WT tetraploid cells (Student's t‐test, P<0.05). (C, D) Genomic instability of viable polyploid cells. Diploid HCT 116 cells with the indicated genotype were treated for 48 h with nocodazole and tetraploid populations were FACS‐purified as in A (>4n, black symbols). Cells then were cultured for the indicated number of days and analyzed for DNA content. Representative cell cycle profiles of WT and p53−/− tetraploid cells are shown in C, whereas quantitative data are reported in D (mean±s.e.m., n=6 independent determinations). In C, the percentages of cells with a sub‐tetraploid DNA content (indicating chromosomal loss) are indicated. Statistical significance resulting from the comparison to WT cells is highlighted by asterisks (Student's t‐test, P<0.05). IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 29, Issue: 7, Pages: 1272-1284, First published: 25 February 2010, DOI: (10.1038/emboj.2010.11)