p16INK4a protects against dysfunctional telomere–induced ATR-dependent DNA damage responses
Yang Wang, … , Norman Sharpless, Sandy Chang
Yang Wang, … , Norman Sharpless, Sandy Chang
Published September 16, 2013
Citation Information: J Clin Invest. 2013;123(10):4489-4501. https://doi.org/10.1172/JCI69574.
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p16INK4a protects against dysfunctional telomere–induced ATR-dependent DNA damage responses

Abstract

Dysfunctional telomeres limit cellular proliferative capacity by activating the p53-p21– and p16INK4a-Rb–dependent DNA damage responses (DDRs). The p16INK4a tumor suppressor accumulates in aging tissues, is a biomarker for cellular senescence, and limits stem cell function in vivo. While the activation of a p53-dependent DDR by dysfunctional telomeres has been well documented in human cells and mouse models, the role for p16INK4a in response to telomere dysfunction remains unclear. Here, we generated protection of telomeres 1b p16–/– mice (Pot1bΔ/Δ;p16–/–) to address the function of p16INK4a in the setting of telomere dysfunction in vivo. We found that deletion of p16INK4a accelerated organ impairment and observed functional defects in highly proliferative organs, including the hematopoietic system, small intestine, and testes. Pot1bΔ/Δ;p16–/– hematopoietic cells exhibited increased telomere loss, increased chromosomal fusions, and telomere replication defects. p16INK4a deletion enhanced the activation of the ATR-dependent DDR in Pot1bΔ/Δ hematopoietic cells, leading to p53 stabilization, increased p21-dependent cell cycle arrest, and elevated p53-dependent apoptosis. In contrast to p16INK4a, deletion of p21 did not activate ATR, rescued proliferative defects in Pot1bΔ/Δ hematopoietic cells, and significantly increased organismal lifespan. Our results provide experimental evidence that p16INK4a exerts protective functions in proliferative cells bearing dysfunctional telomeres.

Authors

Yang Wang, Norman Sharpless, Sandy Chang

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Figure 4

Severe telomere dysfunction in Pot1bΔ/Δ;p16–/– hematopoietic cells.

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Severe telomere dysfunction in Pot1bΔ/Δ;p16–/– hematopoietic cells. (A...
(A) Telomere FISH analysis on metaphase chromosome spreads of BM cells of the indicated genotypes using Tam-OO-(CCCTAA)4 telomere PNA (red) and DAPI (blue). A minimum of 50 metaphases were analyzed per genotype. Arrows indicate fused chromosomes; red arrowheads indicate signal-free ends. Bottom panels show examples of the fragile telomere phenotype observed in 40- to 45-week-old Pot1bΔ/Δ and DK mouse BM cells. White arrowheads point to fragile telomeres. (B) Quantification of the number of telomere signal–free ends found in BM metaphases isolated from mice of the indicated genotypes. n = 4 for each genotype examined. A two-tailed Student’s t test was used to calculate statistical significance. (C) Quantification of the number of chromosome fusions in BM metaphases isolated from mice of the indicated genotypes. n = 4 for each genotype examined. A two-tailed Student’s t test was used to calculate statistical significance. (D) Telomere length determination in 40- to 45-week-old mouse spleen cells. HinfI/RsaI digested splenocyte genomic DNA of the indicated genotypes was hybridized under denatured conditions with a 32P-labeled [CCCTAA]4-oligo to detect total telomere DNA. Telomere signal intensity (percentage) was quantified by setting WT total telomere DNA as 100%. A two-tailed Student’s t test was used to calculate statistical significance. (E) Quantification of fragile telomeres observed in BM cells of the indicated genotypes. A total of 50 metaphases were scored for each mouse, and quantification of MTSs from metaphases isolated from individual animals is shown. A two-tailed Student’s t test was used to calculate statistical significance.