Accumulation of DNA Damage-Induced Chromatin Alterations in Tissue-Specific Stem Cells: The Driving Force of Aging?

<div><p>Accumulation of DNA damage leading to stem cell exhaustion has been proposed to be a principal mechanism of aging. Using 53BP1-foci as a marker for DNA double-strand breaks (DSBs), hair follicle stem cells (HFSCs) in mouse epidermis were analyzed for age-related DNA damage response (DDR). We observed increasing amounts of 53BP1-foci during the natural aging process independent of telomere shortening and after protracted low-dose radiation, suggesting substantial accumulation of DSBs in HFSCs. Electron microscopy combined with immunogold-labeling showed multiple small 53BP1 clusters diffusely distributed throughout the highly compacted heterochromatin of aged HFSCs, but single large 53BP1 clusters in irradiated HFSCs. These remaining 53BP1 clusters did not colocalize with core components of non-homologous end-joining, but with heterochromatic histone modifications. Based on these results we hypothesize that these lesions were not persistently unrepaired DSBs, but may reflect chromatin rearrangements caused by the repair or misrepair of DSBs. Flow cytometry showed increased activation of repair proteins and damage-induced chromatin modifications, triggering apoptosis and cellular senescence in irradiated, but not in aged HFSCs. These results suggest that accumulation of DNA damage-induced chromatin alterations, whose structural dimensions reflect the complexity of the initial genotoxic insult, may lead to different DDR events, ultimately determining the biological outcome of HFSCs. Collectively, our findings support the hypothesis that aging might be largely the remit of structural changes to chromatin potentially leading to epigenetically induced transcriptional deregulation.</p></div

Quantification of gold-labeled pKu70 and 53BP1 in cortical neurons analyzed by TEM. Induction.

<p>Quantification of pKu70 and 53BP1 cluster in euchromatic and heterochromatic domains at 5<b> </b>min and 40<b> </b>min after irradiation with doses ranging from 1 to 10 Gy. The induction of pKu70 (<b>A</b>) and 53BP1 cluster (<b>B</b>) was clearly dependent on the radiation dose, with a linear correlation in the dose range of 1 to 10 Gy. <b>Repair.</b> Quantification of pKu70 (<b>C</b>) and 53BP1 cluster (<b>D</b>) per nucleus at 5<b> </b>min, 20<b> </b>min, 40<b> </b>min, 5 h, 24 h, 48 h, and 72 h after irradiation (6Gy). 53BP1 clusters disappeared with kinetics similar to those observed with heterochromatin-associated pKu70 clusters.</p

Quantification of the different pKu70 and 53BP1 clusters in euchromatic and heterochromatic compartments of cortical neurons analyzed by TEM.

<p>(<b>A</b>) Quantification of pKu70 clusters consisting of 1, 2, 4, or ≥6 beads, analyzed separately in euchromatic and heterochromatic compartments at the different time-points after irradiation (6 Gy). We observed solely pKu70 clusters of 1 and 2 beads in euchromatin, but increasingly complex pKu70 clusters with 4 or ≥6 beads in heterochromatin. (<b>B</b>) Quantification of 53BP1 clusters <10 and ≥10 beads, analyzed in the heterochromatic compartment at different time-points after irradiation (6Gy). We observed huge 53BP1 clusters at late repair-times.</p

Gold-labeled pKu70 and 53BP1 in mouse tissues and human fibroblasts analyzed 40 min after irradiation with 6Gy.

<p>TEM micrographs of double-labeling of pKu70 (10-nm beads) and p53BP1 (6-nm beads) at different magnifications. In enterocytes of small intestine, keratinocytes of skin as well as in human fibroblast, radiation-induced pKu70 clusters (consisting of 2 gold beads, red arrows) co-localize with 53BP1 only in heterochromatic regions.</p

Gold-labeled pKu70 and 53BP1 clusters in cortical neurons analyzed at different time-points after irradiation.

<p>TEM micrographs of double-labeling of pKu70 (10-nm beads) and pDNA-PKcs or 53BP1 (6-nm beads) at different magnifications. (<b>A</b>) All pKu70 clusters consisting of 2 (upper panel) or more gold beads (lower panel) co-localize with pDNA-PKcs, suggesting that these lesions are actively processed DSBs (analyzed at 20<b> </b>min and 24 h post-irradiation). (<b>B</b>) In contrast, huge 53BP1 clusters in heterochromatin at late repair-times (72 h post-irradiation) do not co-localize with pKu70, suggesting that these lesions do not reflect persistently unrepaired DSBs. (pKu70 beads are marked by red arrows).</p

Gold-labeled pKu70 and 53BP1 in cortical neurons of brain analyzed 40 min after irradiation with 6Gy.

<p>TEM micrographs of double-labeling of pKu70 (10-nm beads) and p53BP1 (6-nm beads) at different magnifications (boxed regions are shown at higher magnifications in the following images). Complex pKu70 clusters (consisting of 4 gold beads) co-localizing with 53BP1 were observed in heterochromatic regions, but only isolated pKu70 clusters without any p53BP1 binding were observed in euchromatic regions (pKu70 beads are marked by red arrows).</p

Correlation between the pre-RT IL-6 and TGF-β1 plasma levels, respectively, and the IL-6 and TGF-β1 staining intensity of the corresponding tumour biopsies (grade 1–4) (IL-6: no grade 4 samples).

<p>For both cytokines, statistically significant correlations were found between the amount of pre-RT plasma levels and the staining intensity of the corresponding tumour biopsies.</p

TNF-α, IL-1β, IL-6 and TGF-β1 plasma levels at the onset of radiation pneumonitis (mean values), depicted separately for patients with moderate (grade I/II) (n = 14) and severe lung toxicity (grade III/IV) (n = 7).

<p>Error bars represent SEs of the means. Gray-shaded area indicates the normal range of the cytokine plasma levels.</p

Correlation between the tumour response (CR = complete remission; PR = partial remission; NC = no change; PD = progressive disease) and the IL-6 and TGF-β1 plasma levels assessed at the same time-points during follow-up.

<p>For both cytokines, statistically significant correlations were observed between the tumour response categories and IL-6 and TGF-β1 plasma levels.</p

Time courses of TNF-α, IL-1β, IL-6 and TGF-β1 plasma levels (mean values) before (0), during (1–6 weeks) and after radiotherapy (1–9 months), depicted separately for patients <i>with</i> (n = 21) and <i>without</i> radiation pneumonitis (n = 31).

<p>Error bars represent SEs of the means. Gray-shaded area indicates the normal range of the cytokine plasma levels.</p