Loss of p53-mediated cell-cycle arrest, senescence and apoptosis promotes genomic instability and premature aging

Abstract

Although p53-mediated cell cycle arrest, senescence and apoptosis are well accepted as major tumor suppression mechanisms, the loss of these functions does not directly lead to tumorigenesis, suggesting that the precise roles of these canonical activities of p53 need to be redefined. Here, we report that the cells derived from the mutant mice expressing p533KR, an acetylation-defective mutant that fails to induce cell-cycle arrest, senescence and apoptosis, exhibit high levels of aneuploidy upon DNA damage. Moreover, the embryonic lethality caused by the deficiency of XRCC4, a key DNA double strand break repair factor, can be fully rescued in the p533KR/3KR background. Notably, despite high levels of genomic instability, p533KR/3KRXRCC4-/- mice, unlike p53-/- XRCC4-/- mice, are not succumbed to pro-B-cell lymphomas. Nevertheless, p533KR/3KR XRCC4-/- mice display aging-like phenotypes including testicular atrophy, kyphosis, and premature death. Further analyses demonstrate that SLC7A11 is downregulated and that p53-mediated ferroptosis is significantly induced in spleens and testis of p533KR/3KRXRCC4-/- mice. These results demonstrate that the direct role of p53-mediated cell cycle arrest, senescence and apoptosis is to control genomic stability in vivo. Our study not only validates the importance of ferroptosis in p53-mediated tumor suppression in vivo but also reveals that the combination of genomic instability and activation of ferroptosis may promote aging-associated phenotypes.

Keywords: acetylation; ferroptosis; genomic instability; p53; tumor suppression.

Figure 1. Loss of p53-mediated acute DNA damage response causes genomic instability

A. Flow cytometric analysis of cell cycle distribution in p53^+/+, p53^3KR/3KR, and p53^−/− MEFs. MEFs were either left untreated or exposed to 10 Gy of γ-irradiation; 24 hours later, MEFs were collected and fixed with 70% ethanol for 1hour at 4°C, then subjected to FACS after propidium iodide (PI) staining. B. Quantification of the percentage of MEFs with aneuploidy. Error bars represent averages ± SD from at least three independent MEF lines for each genotype. C. Western blot analysis of p53^+/+, p53^3KR/3KR and p53^−/− MEFs. Cells were either untreated or exposed to 10 Gy of γ-irradiation, then lyzed and analyzed for the expression of p53, p21, and Puma. β-actin was used as a loading control. D. Table showing the expected and observed frequency from the intercross of p53^3KR/3KRXrcc4^+/− mice. E. Representative pictures of p53^3KR/3KRXRCC4^−/− mice and p53^3KR/3KR littermates at 2 days of age. F. The percentage of aneuploidy by FACS analysis of cell cycle distribution in p53^+/+, p53^3KR/3KRXRCC4^−/−, and p53^−/−XRCC4^−/− MEFs. Cells were either left untreated or exposed to 10 Gy of γ-irradiation. 24 hours post-radiation, MEFs were collected, fixed with 70% ethanol for 1hour at 4°C, and then subjected to FACS after propidium iodide (PI) staining. Data are shown as averages ± SD from three independent MEF lines for indicated genotypes.

Figure 2. Spontaneous genomic instability in p^533KR/3KRXRCC4^−/− mice was augmented with age

A. Percentage of abnormal metaphase spreads obtained from early passage p53^+/+, p53^3KR/3KR, p53^−/−, p53^3KR/3KRXRCC4^−/−, and p53^−/−XRCC4^−/− MEFs. Metaphases were prepared from MEFs for the indicated phenotypes after 3 hours treatment with 0.1μg/ml colcemid and analyzed using telomere-FISH assay. At least 100 metaphases were counted. Results are shown as averages ± SD from three different MEF lines of each genotype. B. Frequency of cytogenetic abnormalities in three categories: chromosomal breaks, chromatid breaks and chromosomal fusions, in MEFs with the indicated genotypes. Values shown are the averages ± SD from three independent MEF lines. C. Representative images of abnormalities observed in p53^3KR/3KRXRCC4^−/− MEF metaphases. Chromosomes were stained with telomere specific probes (red) and counterstained with DAPI (blue).D. Percentage of metaphases with abnormalities in bone marrows from 4-week-old and 22-week-old p53^+/+ and p53^3KR/3KRXRCC4^−/− mice. Metaphase spreads were obtained from mice bone marrows with the indicated genotypes after with incubation with 0.1μg/ml colcemid for 6 hours and a minimum of 100 metaphases was analyzed for each sample using telomere-FISH. Results are reported as averages ± SD from three mice for each genotype. E. Immunoblot assays of p53, phopho-p53, Kap1, phopho-Kap1, γ-H2ax and β-actin proteins in the lysates prepared from the spleens of p53^+/+, p53^3KR/3KR, and p53^−/− p53^3KR/3KRXRCC4^−/− mice at the indicated ages. *; The 3-month-old p53^−/− mice (lane 7) was used for control. β-actin was used as a loading control. F. Representative immunohistochemical staining of spleens from 4 week- and 22 week-old p53^+/+ and p53^3KR/3KRXRCC4^−/− mice for γ-H2ax.

Figure 3. p^533KR/3KRXRCC4^−/− mice bearing high levels of genomic instability are resistant to cancer but display premature aging phenotypes

A. Kaplan-Meier overall survival curves of p53^3KR/3KR and p53^3KR/3KRXRCC4^−/− mice. B. Images of spleens of p53^3KR/3KRXRCC4^−/− mouse and p53^3KR/3KR littermate at the age of 22 weeks.C. Hematoxylin and eosin (H&E) staining of spleens from 22-week-old p53^3KR/3KR and p53^3KR/3KRXRCC4^−/− mice.D. Average body weight of p53^+/+, p53^3KR/3KR, and p53^3KR/3KRXRCC4^−/− mice at different ages. Error bars means averages ± SD. E. p53^3KR/3KRXRCC4^−/− mouse and p53^3KR/3KR littermate at 22 weeks of age.F. Representative whole-body radiographs of 22-week-old p53^3KR/3KRXRCC4^−/− mice with p53^3KR/3KR littermates. G. H&E staining of testis from 22-week-old p53^3KR/3KR and p53^3KR/3KRXRCC4^−/− mice.

Figure 4. The roles of p53-mediated effects on Slc7a11 and ferroptosis in tumor suppression and aging associated testicular atrophy in p^533KR/3KRXRCC4^−/− mice

A. qRT-PCR analysis of Slc7a11 mRNA levels in p53^3KR/3KR, p53^3KR/3KRXRCC4^−/− and p53^−/−XRCC4^−/− MEFs. Data were shown as average ± SEM from three independent MEF lines.B. p53^3KR/3KRXRCC4^−/− and p53^−/−XRCC4^−/− MEFs were either left untreated or exposed to ROS (Tert-butyl-hydroperoxide; 400 μM) and ferroptosis inhibitor, ferrostatin-1 (ferr-1, 2 μM) for 8 hours and then representative phase-contrast images were taken.C. Quantification of cell death of MEFs after the treatment with ROS and ferr-1. Data were shown as average ±SD from three independent MEF lines.D. and F. Western blot analysis of p53, Slc7a11 and β-actin proteins in the lysates prepared from the spleens (D) or testis (F) of p53^+/+, p53^3KR/3KR, p53^3KR/3KRXRCC4^−/−, and p53^−/− mice at the age of 22 weeks. *; The 3-month-old tumor-free p53^−/− mice were used for control.E. and G. qRT-PCR analysis of Ptgs2 mRNA levels in spleens (E) or in testis (G) of 22-week-old p53^+/+, p53^3KR/3KR, p53^3KR/3KRXRCC4^−/− and 3-month-old p53^−/− mice. Error bars represent averages ± SEM of three mice for each genotype.