p16INK4a protects against dysfunctional telomere–induced ATR-dependent DNA damage responses
Yang Wang, … , Norman Sharpless, Sandy Chang
Yang Wang, … , Norman Sharpless, Sandy Chang
Published October 1, 2013; First published September 16, 2013
Citation Information: J Clin Invest. 2013;123(10):4489-4501. https://doi.org/10.1172/JCI69574.
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Research Article Cell biology

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 1

Loss of p16INK4a accelerates premature aging phenotypes in Pot1bΔ/Δ;mTR+/– and Pot1bΔ/Δ mice.

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Loss of p16INK4a accelerates premature aging phenotypes in Pot1bΔ/Δ;mTR+...
(A) Real-time PCR quantification of p16INK4a mRNA expression levels in young (10-week-old) and old (60-week-old) WT and Pot1b-null mouse spleens. Three individual samples were analyzed per genotype, and each experiment was repeated in triplicate. Error bars represent the SEM. A two-tailed Student’s t test was used to calculate statistical significance. (B) Kaplan-Meier survival analysis of Pot1bΔ/Δ;mTR+/– and Pot1bΔ/Δ;mTR+/;p16–/– mice. A log-rank test was used to calculate statistical significance. (C) Kaplan-Meier survival analysis showing the survival percentage of mice of the indicated genotypes as a function of age. All groups of mice were monitored for a minimum of 65 weeks and sacrificed when moribund. A log-rank test was used to calculate statistical significance. (D) Testicular weights from mice of the indicated genotypes. Each genotype includes testes from a minimum of 4 mice. Error bars represent the SEM. A two-tailed Student’s t test was used to calculate statistical significance. (E) Quantification of the number of apoptotic cells in basal crypts of small intestines isolated from WT, p16–/–, Pot1bΔ/Δ, and DK mice. Error bars represent the SEM. A two-tailed Student’s t test was used to calculate statistical significance. Representative H&E-stained histological sections of testes (F), small intestine (G), and BM (H) isolated from 40- to 45-week-old mice of the indicated genotypes. Original magnification, ×4 for testes and ×40 for intestines and BM. Arrowheads point to apoptotic intestinal cells.