Telomere dysfunction suppresses spontaneous tumorigenesis in vivo by initiating p53-dependent cellular senescence

Abstract

Dysfunctional telomeres induce p53-dependent cellular senescence and apoptosis, but it is not known which function is more important for tumour suppression in vivo. We used the p53 ( R172P ) knock-in mouse, which is unable to induce apoptosis but retains intact cell-cycle arrest and cellular senescence pathways, to show that spontaneous tumorigenesis is potently repressed in Terc -/- p53 ( R172P ) mice. Tumour suppression is accompanied by global induction of p53, p21 and the senescence marker senescence-associated-beta-galactosidase. By contrast, cellular senescence was unable to suppress chemically induced skin carcinomas. These results indicate that suppression of spontaneous tumorigenesis by dysfunctional telomeres requires the activation of the p53-dependent cellular senescence pathway.

Figure 1

TRF2 ^ΔBΔM induces telomere dysfunction and cellular senescence in p53 ^P/P primary mouse embryonic fibroblasts. (A) BrdU incorporation of p53 ^+/+, p53 ^P/P and p53 ^−/− MEFs at day 4 after infection with H-Ras^V12, TRF2^ΔBΔM or empty (vector) retrovirus. The percentage of BrdU positive cells relative to controls is indicated. Error bars represent standard error of the mean (s.e.m.). (B) Prominent SA-β-gal staining was observed only in p53 ^P/P but not in p53 ^−/− MEFs infected with Myc-tagged TRF2 ^ΔBΔM or H-Ras^V12. (C) Quantitation of the percentage of SA-β-gal positive cells. Error bars represent s.e.m. (D) Immunoblots of lysates from p53 ^+/+, p53 ^−/− and p53 ^P/P MEFs. Cells were infected with either Myc-TRF2 ^ΔBΔM , H-Ras^V12 or vector and proteins were detected with the indicated antibodies. (E) Colocalization of DDR foci to dysfunctional telomeres (arrowheads) in p53 ^P/P MEFs 48 h after infection with TRF2 ^ΔBΔM : telomeres (TRITC, red; left), γ-H2AX (FITC, green; middle) and merged (right). Arrowheads indicate telomere dysfunction induced foci. (F) End-to-end chromosomal fusions were detected after infection with TRF2 ^ΔBΔM , but not with vector, in p53 ^P/P and p53 ^−/− MEFs. (E,F) Arrowheads point to sites of fusions. BrdU, bromodeoxyuridine; DDR, DNA damage response; FITC, fluorescein isothiocyanate; MEF, mouse embryonic fibroblast; SA-β-gal, senescence-associated-β-galactosidase; TRF2, telomere repeat-binding factor 2; TRITC, tetramethyl rhodamine isothiocyanate.

Figure 2

Dysfunctional telomeres do not initiate an apoptotic response in iG1 Terc ^−/− p53 ^P/P mice. (A) Bone marrow metaphases isolated from iG1 Terc ^−/− p53 ^P/P mice show chromosome end-to-end fusions (arrow), whereas Terc ^+/− p53 ^P/P metaphases showed minimal abnormalities. (B) Quantitation of cytogenetic aberrations in bone marrow from mice of the indicated genotypes. Error bars represent s.e.m. (C) Anaphase bridges (indicated by arrows) in intestines of iG1 Terc ^−/− p53 ^P/+ and iG1 Terc ^−/− p53 ^P/P mice. (D) Quantitation of anaphase bridges in intestines of mice of the indicated genotypes. Error bars represent s.e.m. (E) TUNEL staining of testes of the indicated genotypes. (F) TUNEL staining of intestines of the indicated genotypes. (G) Quantitation of the number of apoptotic cells present in intestinal crypts of the indicated genotypes. Error bars represent s.e.m. iG1, first intergenerational mating; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling.

Figure 3

Kaplan–Meier analysis of mouse cohorts. Tumour incidence (A,C) and overall survival (B,D) of mice of the indicated genotypes are shown. The number of tumours identified (t), the total number of mice (n) and the P-values (calculated using the log-rank test) are indicated. Note the near-complete tumour suppression in iG1 p53 ^P/+, iG1 p53 ^P/P and iG1 p53 ^+/+ mice. Deletion of p53 reduced the lifespan of iG1 mice owing to increased tumour formation. iG1, first intergenerational mating; NS, statistically not significant.

Figure 4

Telomere dysfunction induces cellular senescence in vivo. (A) Immunostaining with p53 antibody detected staining in intestinal crypts of iG1 iG1 Terc ^−/− p53 ^P/P and Terc ^−/− p53 ^P/+ mice, but not in Terc ^+/− p53 ^P/P and Terc ^+/− p53 ^P/+ mice. (B) Immunostaining with p21 antibody detected staining in intestinal crypts of mice of the indicated genotypes. (C) Robust SA-β-gal activity detected in tissues from iG1 Terc ^−/− p53 ^P/+ mice (top) but not in tissues from iG1 Terc ^−/− p53 ^+/+ mice (bottom). (D) End-to-end chromosomal fusions (indicated by arrows) and telomere signal-free ends in an iG1 Terc ^−/− p53 ^P/P lymphoma. iG1, first intergenerational mating.

Figure 5

Skin papillomas in mice of the indicated genotypes. (A) Total number of papillomas of the indicated sizes plotted against the number of weeks after carcinogen treatment. The genotypes of treated mice are indicated. Asterisks indicate the week when a mouse died. n: total number of treated mice; t: number of mice with tumours. (B) Increased anaphase bridge index in iG1 Terc ^−/− p53 ^P/P papillomas. (C) Immunohistochemistry of carcinogen-treated iG1 Terc ^−/− p53 ^P/P mouse (left) show high p21, p53 and Ki-67 positivity in the nuclei of keratinocytes. Upper panel: squamous papilloma. Lower panel: invasive SCC. iG1, first intergenerational mating; SCC; squamous-cell carcinomas.