Abstract
Senescence is a state of growth arrest resulting mainly from telomere attrition and oxidative stress. It ultimately leads to cell death. We have previously shown that, in keratinocytes, senescence is induced by NF-kappaB activation, MnSOD upregulation and H2O2 overproduction. We have also shown that senescent keratinocytes do not die by apoptosis but as a result of high macroautophagic activity that targets the primary vital cell components. Here, we investigated the mechanisms that activate this autophagic cell death program. We show that corpses occurring at the senescence plateau display oxidatively-damaged mitochondria and nucleus that colocalize with autophagic vacuoles. The occurrence of such corpses was decreased by specifically reducing the H2O2 level with catalase, and, conversely, reproduced by overexpressing MnSOD or applying subtoxic doses of H2O2. This H2O2-induced cell death did occur through autophagy since it was accompanied by an accumulation of autophagic vesicles as evidenced by Lysotracker staining, LC3 vesiculation and transmission electron microscopy. Most importantly, it was partly abolished by 3-methyladenine, the specific inhibitor of autophagosome formation, and by anti-Atg5 siRNAs. Taken together these results suggest that autophagic cell death is activated in senescent keratinocytes because of the upregulation of MnSOD and the resulting accumulation of oxidative damages to nucleus and mitochondria.
Highlights
In vivo as in vitro, normal human cells have a limited lifespan
Senescent keratinocytes and corpses at the senescent plateau display altered mitochondria and nuclei that colocalize with autophagic vacuoles
As we have shown in previous studies, normal human epidermal senescent keratinocytes (NHEKs) cultured in vitro reach a senescence growth plateau after 15–25 population doublings (PDs)
Summary
In vivo as in vitro, normal human cells have a limited lifespan. After having performed a certain number of divisions, they enter a special state termed senescence [1] which is the consequence of both the telomere erosion occurring at each replication cycle and of oxidative damages which increase with time. Senescent cells are cell-cycle arrested and display numerous morphological, metabolic and genetic changes [2,3]. It was shown by us and others that senescence is associated with an increase in macroautophagic activity [4,5,6]. The macroautophagic process, here referred as autophagy, starts by encircling a damaged cell component inside a double membrane. The autophagosome resulting from the closure of this membrane fuses with endosomes and lysosomes to form an autophagolysosome, inside which the sequestered material is submitted to an acidic pH and to the activity of various hydrolytic enzymes. The recognition of the altered material, and the formation, migration and fusion of the autophagic vacuoles at different stages involve about thirty Atg genes [7,8,9]
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