Volume 27, Issue 4, Pages 647-659 (August 2007) p73 Suppresses Polyploidy and Aneuploidy in the Absence of Functional p53  Flaminia Talos, Alice Nemajerova, Elsa R. Flores, Oleksi Petrenko, Ute M. Moll  Molecular Cell  Volume 27, Issue 4, Pages 647-659 (August 2007) DOI: 10.1016/j.molcel.2007.06.036 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Isolated p73 Loss Induces p53-Mediated Premature Senescence and Transformation Resistance (A) Cell-cycle profiles of MEFs of the indicated genotypes at passage 3. (B) Bromodeoxyuridine incorporation by MEFs of the indicated genotypes after 2 hr and 12 hr pulses. (C) Growth curves of MEFs of the indicated genotypes passaged on a 3T3 protocol. The results are representative of four embryos each. (D) Focus formation assays of E1A and H-RasV12-expressing MEFs of the indicated genotypes. Error bars represent the standard deviation (n = 3). (E) Immunoblots of p53 and p21 expression by MEFs of the indicated genotypes incubated in the absence or presence of adriamycin for 24 hr. Cdk4 is the loading control. (F) Cell-cycle profiles of passage 3 WT and p53−/− MEFs after retroviral transduction with empty vector, ΔNp73α, or p73DD. For comparison, a DNA histogram of DKO MEFs at passage 3 is shown. Inset, immunoblot of ΔNp73α and p73DD expression in WT MEFs. Molecular Cell 2007 27, 647-659DOI: (10.1016/j.molcel.2007.06.036) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Combined Loss of p53 and p73 Results in Increased Genomic Instability (A) DNA histograms of WT and p73−/− MEFs passaged on the 3T3 protocol. Number of passages in culture is indicated on the right. (B) DNA histograms of p53−/− and DKO MEFs passaged on the 3T3 protocol as in (A). Molecular Cell 2007 27, 647-659DOI: (10.1016/j.molcel.2007.06.036) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Combined Loss of p53 and p73 Leads to Excess Polyploidy and Aneuploidy (A) Karyotype analysis of representative metaphases from DKO MEFs at passage 7. Most cells contain unequal chromosome numbers, indicating aneuploidy. (B) Polyploidization of DKO cells at passage 7, as shown by spectral karyotyping. Note lack of diploidy (2n) and high degree of polyploidy (4n and >4n) of DKO cells. (C) Individual chromosome contents of WT, p53−/−, and DKO MEFs at passage 7. Over 100 metaphases were analyzed for each genotype. Error bars represent the standard deviation. To allow precise chromosome identification, only metaphases containing less than 100 chromosomes were quantified. (D) Representative DNA histograms of thymocytes from premalignant p53−/− and p53−/−p73−/− mice. Normal DKO thymus contains a triploid cohort that is absent in the p53−/− animal. Molecular Cell 2007 27, 647-659DOI: (10.1016/j.molcel.2007.06.036) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Primary Mitotic Defects Are Not the Cause of Excess Polyploidy and Aneuploidy in DKO Cells (A) Total number of centrosomes (determined by γ-tubulin staining) and nuclei (determined by Hoechst staining) per cell in WT, p53−/−, and DKO cells at passage 7. More than 500 random cells were counted for each genotype. Note that DKO cells have an improved centrosomal phenotype and are predominantly mononucleated. (B) DNA histograms of p53−/− and DKO MEFs treated with nocodazole for 12 hr, washed out, and then released into media containing the G1/S blocker L-mimosine. Both genotypes recover their original ploidy within 4 hr (i.e., 2n for p53−/− MEFs and 4n for DKO cells), indicating that they had passed through a proper mitosis. (C) Real-time analysis of mitotic progression of GFP-histone H2B-expressing p53−/− and DKO MEFs treated as in (B). Error bars represent the standard deviation (n = 3). At least 500 cells for each time point were counted. Molecular Cell 2007 27, 647-659DOI: (10.1016/j.molcel.2007.06.036) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Dysregulated Activity and Dynamics of Cdk2 and Cdk1 in Unstressed DKO Cells (A) In vitro kinase assays of immunoprecipitated cyclin E-, cyclin A-, and cyclin B-associated Cdk2 and Cdk1 activity in synchronized p53−/− and DKO MEFs. Cells were analyzed at the indicated times after release from G1/S phase block with HU. Error bars represent the standard deviation (n = 3). (B) Immunoblot analysis of p53−/− and DKO MEFs synchronized at the G1/S border by HU for 12 hr and then released for the indicated time. Continuously growing (Asyn) MEFs are also shown. (C) Immunoprecipitation of Cdk1-cyclin B1-p27 complexes in p53−/− and DKO MEFs. Cells were synchronized with aphidicolin in mid-S phase and then released to follow progression through S phase and G2M. Cyclin B1-associated Cdk1 is shown from two independent experiments. Input is shown below. (D) Immunoprecipitation of p27-Cdk4 complexes in p53−/− and DKO MEFs released into G1 after serum-free synchronization. See also Figure S4A. (E) Immunoblot analysis of p57Kip2 of p53−/− and DKO MEFs released into G1 after nocodazole synchronization in late M phase. Molecular Cell 2007 27, 647-659DOI: (10.1016/j.molcel.2007.06.036) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 p73 Activates the G2M Damage Checkpoint (A) DNA contents of p53−/− and DKO MEFs incubated for 20 hr in the absence or presence of VM26. Note the baseline difference in ploidy between untreated p53−/− and DKO MEFs (see also Figure 2B). (B) More DKO than p53−/− cells enter mitosis despite DNA damage. Mitotic indices were determined by real-time quantitative chromosome evaluation of H2B-GFP-expressing p53−/− and DKO cells treated with VM26 for up to 24 hr. At least 500 random cells were scored for each time point. Error bars represent the standard deviation. (C) Immunoblot analysis of histone H2AX phosphorylation in p53−/− and DKO MEFs incubated with 10 μM VM26 for the indicated hours. Cdk1 is shown as control. (D) Cell-cycle profiles of DKO MEFs expressing ectopic TAp73α in the absence or presence of VM26-induced DNA damage. TAp73 reestablishes an efficient S/G2 arrest and rescues the G2M checkpoint defect of DKO cells. (E) In vitro kinase assays of immunoprecipitated cyclin B-associated Cdk1 activity of p53−/− and DKO MEFs. Cells were analyzed at the indicated hours after DNA damage by 10 μM VM26. Error bars represent the standard deviation (n = 3). (F) DKO cells exhibit increased chromatin binding of components of the prereplicative and elongation complexes. p53−/− and DKO MEFs after DNA damage induced by 10 μM VM26 for the indicated hours were trypsinized and counted. Chromosomes were isolated from equal cell numbers and loaded proportionately. A nonspecific 60 kDa band (NSLC) crossreacting with an Rb monoclonal antibody is used as a loading control. (G) DNA histograms of DKO MEFs incubated for 20 hr in the absence or presence of 10 μM VM26, or transduced with retrovirus expressing dominant-negative Cdk2 (Cdk2D145N) prior to VM26 treatment. DKO cells lose their 4n peak upon VM26 treatment and massively shift to the right, while transduced DKO cells maintain their 4n peak. (H) Ectopic expression of Cdk2 increases the rate of polyploidization in p53−/− MEFs. Cells incubated for 20 hr in the presence of 10 μM VM26 or transduced with retroviruses expressing wild-type Cdk2 prior to VM26 treatment are shown. Molecular Cell 2007 27, 647-659DOI: (10.1016/j.molcel.2007.06.036) Copyright © 2007 Elsevier Inc. Terms and Conditions