Cellular senescence and organismal ageing in the absence of p21(CIP1/WAF1) in ku80(-/-) mice

Abstract

Ku80 is important in the repair of DNA double-strand breaks by its essential function in non-homologous end-joining. The absence of Ku80 causes the accumulation of DNA damage and leads to premature ageing in mice. We showed that mouse embryonic fibroblasts (MEFs) from ku80(-/-) mice senesced rapidly with elevated levels of p53 and p21. Deletion of p21 delayed the early senescence phenotype in ku80(-/-) MEFs, despite an otherwise intact response of p53. In contrast to ku80(-/-)p53(-/-) mice, which die rapidly primarily from lymphomas, there was no significant increase in tumorigenesis in ku80(-/-)p21(-/-) mice. However, ku80(-/-)p21(-/-) mice showed no improvement with respect to rough fur coat or osteopaenia, and even showed a shortened lifespan compared with ku80(-/-) mice. These results show that the increased lifespan of ku80(-/-) MEFs owing to the loss of p21 is not associated with an improvement of the premature ageing phenotypes of ku80(-/-) mice observed at the organismal level.

Figure 1

Early senescence of ku80^−/− mouse embryonic fibroblasts correlates with elevated levels of p53 and p21. (A) Growth curves of MEFs starting at PD8 (which reflects time zero on the x axis), with genotypes as indicated. (B) Quantification of BrdU/SA-β-gal of WT and ku80^−/− MEFs at PD8. Mean±s.d. reflects the results of three separate experiments from individual isolates. (C) Western blot showing levels of p53 and p21 in WT, ku80^−/−, p53^−/− (PD12) and p21^−/− (PD6) MEFs either untreated (−) or treated (+) with doxorubicin (Doxo; 100 ng/ml) for 20 h. (D) Quantifications of the levels of p53 and p21 were normalized to β-actin, and to basal levels of p53 and p21 in WT MEFs, respectively. Mean±s.d. reflects the results of three separate experiments from individual isolates. For all the experiments, at least two sets of isolates for each genotype were assayed and similar results were obtained. BrdU, 5-bromodeoxyuridine; MEF, mouse embryonic fibroblast; PD, population doubling; SA-β-gal, senescence-associated β-galactosidase; WT, wild type.

Figure 2

Effects of p21 deletion on growth properties of ku80^−/− mouse embryonic fibroblasts. (A) Growth curves of MEFs starting at PD8, with genotypes as indicated. Of note is that p21^−/−, WT and ku80^−/−p21^−/− MEFs showed sub-populations associated with a loss of p53, which bypassed senescence and eventually took over the culture, as reflected by the accelerated phase on their growth curves. (B) Saturation densities of MEFs. MEFs (1 × 10⁴) at PD4 were plated in six 60-mm petri dishes and the medium was changed every 3 days. At 1 week, three dishes were stained with crystal violet, and representative images are shown on the left. Cell numbers from parallel dishes are shown on the right. Mean±s.d. reflects results of 3 separate experiments from individual MEFs. (C) Representative images of BrdU (brown)/SA-β-gal (blue) double staining and cell morphology after 22 days in culture; PDs are extrapolated from growth curves. Quantifications for BrdU or SA-β-gal staining are shown on the right. Mean±s.d. reflects the results of three separate experiments from individual isolates. For all the experiments, at least two sets of isolates for each genotype were assayed and similar results were obtained. BrdU, 5-bromodeoxyuridine; MEF, mouse embryonic fibroblast; PD, population doubling; SA-β-gal, senescence-associated β-galactosidase; WT, wild type.

Figure 3

Cell-cycle profiles and p53 responses in ku80^−/−p21^−/− mouse embryonic fibroblasts. Representative cell-cycle profiles of (A) Ku80^+/+ and ku80^−/− MEFs with or without p21 at PD10, and (B) in WT and ku80^−/−p21^−/− MEFs at PD26. Mean±s.d. reflects the results of three separate experiments from individual MEFs. Two sets of MEFs for each genotype were assayed with similar results (^*P<0.05). (C) Western blot analysis of the response of p53 in ku80^−/−p21^−/− MEFs with increasing PDs. Cells were either untreated (−) or treated (+) with doxorubicin (Doxo; 300 ng/ml) for 20 h, and levels of p53 and p19 are shown; β-actin was used as a loading control. MEF, mouse embryonic fibroblast; PD, population doubling; WT, wild type.

Figure 4

Effects of p21 deletion on ageing phenotypes of ku80^−/− mice. (A) Survival curves (100% × number of mice alive after each week/total number of mice at 2 weeks) for mice with each genotype. (B) Comparison of the appearance of 32-week-old mice with each genotype; note the rough fur coat and kyphosis in ku80^−/− and ku80^−/−p21^−/− mice. (C) Comparison of the distal femoral epiphysis in mice with each genotype. The cortical wall and part of the diaphysis are also shown. Chondrocyte numbers in the growth plates from two mice between 32 and 34 weeks old for each genotype were counted, and the quantification is shown on the right. Scale bar 200μm.

Figure 5

Effects of p21 deletion on lymphoid tissue in ku80^−/− mice. (A) Spleen/body weight ratio from mice with various genetic backgrounds at 16 weeks. (B) HE staining of spleen tissue from each group; note the loss of splenic follicles in ku80^−/− compared with Ku80^+/+ mice. (C) TUNEL staining of spleen tissue was performed on paraffin sections (three mice for each genotype). Quantification of three high-power fields (HP) per spleen is shown on the right. HE, haematoxylin and eosin; TUNEL, TdT-mediated dUTP nick-end labelling.