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. 2015 Jul 30;34(31):4118-29.
doi: 10.1038/onc.2014.342. Epub 2014 Oct 27.

Apc and p53 Interaction in DNA Damage and Genomic Instability in Hepatocytes

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Free PMC article

Apc and p53 Interaction in DNA Damage and Genomic Instability in Hepatocytes

V Méniel et al. Oncogene. .
Free PMC article

Abstract

Disruption of Apc (adenomatous polyposis coli) within hepatocytes activates Wnt signalling, perturbs differentiation and ultimately leads to neoplasia. Apc negatively regulates Wnt signalling but is also involved in organizing the cytoskeleton and may have a role in chromosome segregation. In vitro studies have implicated Apc in the control of genomic stability. However, the relevance of this data has been questioned in vivo as Apc is lost earlier than the onset of genomic instability. Here we analyse the relationship between immediate loss of Apc and the acquisition of genomic instability in hepatocytes. We used Cre-lox technology to inactivate Apc and in combination with p53 in vivo, to define the consequences of gene loss on cell cycle regulation, proliferation, death and aneuploidy. We show that, although Apc loss leads to increased proliferation, it also leads to increased apoptosis, the accumulation of p53, p21 and markers of double-strand breaks and DNA repair. Flow cytometry revealed an increased 4N DNA content, consistent with a G2 arrest. Levels of anaphase bridges were also elevated, implicating failed chromosome segregation. This was accompanied by an increase in centrosome number, which demonstrates a role for Apc in maintaining euploidy. To address the role of p53 in these processes, we analysed combined loss of Apc and p53, which led to a further increase in proliferation, cell death, DNA damages and repair and a bypass of G2 arrest than was observed with Apc loss. However, we observed only a marginal effect on anaphase bridges and centrosome number, which could be due to increased cell death. Our data therefore establishes, in an in vivo setting, that APC loss leads to a DNA damage signature and genomic instability in the liver and that additional loss of p53 leads to an increase in the DNA damage signal but not to an immediate increase in the genomic instability phenotype.

Figures

Fig.1
Fig.1. Loss of Apc in the liver leads to DNA damages, DNA repair and proliferation
(A) Increased %Ki67 labelling at D4 and D6 in APC versus Wt. (B) Increased %BrdU labelling after 2hrs BrdU in APC (D4 and D6) versus Wt. Similar %BrdU labelling from 2hrs to 24hrs in Wt or APC. (C) p53 and p21 increased levels in APC versus Wt. (D) Increased %Caspase3 at D6 or D4 in APC versus Wt. (E,F) γH2AX staining and scoring (G,H) Rad51 staining and scoring in APC versus Wt at D4 and D6. Increased %γH2AX or %Rad51 labelling in APC versus Wt. Bars indicate standard-error. * indicates P<0.05 MW (N=3 mice). Arrows indicate Positive cells (Scale=10μm). Wt, AhCre+Apcfl/fl (APC)
Fig.2
Fig.2. Apc deficiency leads to increased apoptosis, nuclear area, 4N DNA content, cell cycle perturbation and anaphase bridges and decreased of BrdU+P-histone H3+ cells
Increase in nuclear area (μm2) (%=A+B; Cumulative-Frequency=C+D) at D4 (A+C) and D6 (B+D) in APC versus Wt (P=0.01 Kolmogorov-Sminov). (E-F) DNA content by Flow cytometry of hepatocytes at D4 (E) or D6 (F). Decrease in % of cells with 2N DNA content and increase in 4N DNA content in APC versus Wt. (G) Increased anaphase bridges index in APC versus Wt. (H) Anaphase bridge in hepatocyte. (I) Dual immunostaining with P-histone H3 and BrdU 24hrs after injection of BrdU was compared to Brdu positive cells. Decrease in % of cells with double labelling in APC versus Wt. Bars indicate standard-error. * indicates P<0.05 MW. (N≥3 mice) (Scale=50μm). Wt, AhCre+Apcfl/fl (APC)
Fig.3
Fig.3. P53 deficiency enhances DNA damage, DNA repair, proliferation and apoptosis induced by Apc loss
(A) Increased p21 levels in APCP53 versus P53. (B) γH2AX staining. (C) Rad51 staining. (D) Scoring of γH2AX positive cells. (E) Scoring of Rad51 positive cells. Increased %γH2AX or %Rad51 labelling in APCP53. (F) Increased %Ki67 labelling at D4 and D6 in APCP53 (G) Increased %Caspase3 at D6 or D4 in APCP53. (H-I) Increased %Brdu labelling after 2hrs in APCP53 at D4 (H) and D6 (I). No increase is seen in %BrdU labelling from 2hrs to 24hrs in APCP53.; Bars indicate standard-error. * indicates P<0.05 MW, (N=3 mice). Arrows indicate positive cells, (Scale=10μm). Wt, AhCre+Apcfl/fl (APC), AhCre+P53−/−(P53), AhCre+Apcfl/flp53−/−(APCP53)
Fig.4
Fig.4. P53 deficiency enhances increased nuclear area, 4N DNA content, cell cycle perturbation induced after Apc loss
Increase in nuclei area (μm2) (%=A+B; Cumulative-frequency=C+D) at D4 (A+C) and D6 (B+D) in APCP53 versus P53 and APC (P=0.01 Kolmogorov-Smirnov). (E-F) DNA content by Flow cytometry of hepatocytes at D4 (E) or D6 (F) in APC, P53 and APCP53. Increase in cells with DNA content greater than 4N in APCP53. Bars indicate standard-error. * indicates P<0.05 MW, (N≥3 mice). Wt, AhCre+Apcfl/fl (APC), AhCre+P53−/−(P53), AhCre+Apcfl/flp53−/−(APCP53)
Fig.5
Fig.5. P53 deficiency does not enhance anaphases bridges after Apc loss but leads to partial rescue of BrdU+P-histoneH3+ cells number
(A) Dual immunostaining with P-histone H3 and BrdU 24hrs (BrdU+P-histone H3+) was compared to BrdU positive cells (BrdU+). (i) shows a P-histone H3+ positive cell but negative for BrdU labelling; (ii) shows a BrdU+ cell but negative for P-histone H3 labelling, White arrows indicate positive cells.. (B) Increased number of BrdU+P-histone H3+ in APCP53 compared to APC at D4. (C) Increased number of BrdU+P-histone H3+ in APCP53 compared to APC at D6. (D) Increased anaphase bridges index in APCP53. Bars indicate standard-error. * indicates P<0.05 MW, (N≥3 mice). Wt, AhCre+Apcfl/fl (APC), AhCre+P53−/−(P53), AhCre+Apcfl/flp53−/−(APCP53)
Fig.6
Fig.6. Apc loss leads to aneuploidy and increase in centrosomes number which is not significantly enhanced by additional P53 deficiency
(A) Chromosome numbers per hepatocytes in Wt, APC, APCP53 versus P53 with abnormal numbers in APC and APCP53 (N≤2). Bars indicate standard-error. * indicates P<0.05. (B) centrosomes staining (red) representative pictures. cen = centrosome. (Scale=50μm) (C) % nuclei with 1 cen (cen=centrosome), or 2 cen or more than 1 cen. Increase in nuclei with more than 2 centrosomes in APC versus Wt at D4 and D6. This Increase is not significantly altered in APCP53 compared to APC at D6 and D4. Bars indicate standard-error. * indicates P<0.05 MW, (N=3-5 mice). Wt, AhCre+Apcfl/fl (APC), AhCre+P53−/−(P53), AhCre+Apcfl/flp53−/−(APCP53)

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References

    1. Fearon ER. Molecular genetics of colorectal cancer. Annu Rev Pathol. 2011;6:479–507. - PubMed
    1. Clevers H. Wnt/β-Catenin Signaling in Development and Disease. Cell. 2006;127:469–480. - PubMed
    1. Fodde R, Kuipers J, Rosenberg C, Smits R, Kielman M, Gaspar C, et al. Mutations in the APC tumor suppressor gene cause chromosomal instability. Nat Cell Biol. 2001;3:433–438. - PubMed
    1. Kaplan KB, Burds AA, Swedlow JR, Bekir SS, Sorger PK, Näthke IS. A role for the Adenomatous Polyposis Coli protein in chromosome segregation. Nat Cell Biol. 2001;3:429–432. - PubMed
    1. Dikovskaya D, Schiffmann D, Newton IP, Oakley A, Kroboth K, Sansom O, et al. Loss of APC induces polyploidy as a result of a combination of defects in mitosis and apoptosis. The Journal of Cell Biology. 2007;176:183–195. - PMC - PubMed

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