Cellular senescence is a cell state implicated in various physiological processes and a wide spectrum of age-related diseases. Recently, interest in therapeutically targeting senescence to improve healthy aging and age-related disease, otherwise known as senotherapy, has been growing rapidly. Thus, the accurate detection of senescent cells, especially in vivo, is essential. Here, we present a consensus from the International Cell Senescence Association (ICSA), defining and discussing key cellular and molecular features of senescence and offering recommendations on how to use them as biomarkers. We also present a resource tool to facilitate the identification of genes linked with senescence, SeneQuest (available at http://Senequest.net). Lastly, we propose an algorithm to accurately assess and quantify senescence, both in cultured cells and in vivo. Cellular Senescence: Walking a Line between Life and Death Cell states link both physiological and stress signals to tissue homeostasis and organismal health. In both cases, the outcomes vary and are determined by the signal characteristics (type, magnitude, and duration), spatiotemporal parameters (where and when), and cellular capacity to respond (Gorgoulis et al., 2018). In the case of potentially damaging stress, damage is reversed and the structural and functional integrity of cells restored. Alternatively, damage can be irreversible, and cells activate death mechanisms mainly to restrict the impact on tissue degeneration. Between these extremes, cells can acquire other states, often associated with survival but also with permanent structural and functional changes. An example is the non-proliferative but viable state, distinct from G0 quiescence and terminal differentiation, termed cellular senescence (Rodier and Campisi, 2011). Formally described in 1961 by Hayflick and colleagues, cellular senescence, derived from the latin word senex meaning ''old'' (Hayflick and Moorhead, 1961), was originally observed in normal diploid cells that

XPF-ERCC1 endonuclease is required for repair of helix-distorting DNA lesions and cytotoxic DNA interstrand crosslinks. Mild mutations in XPF cause the cancer-prone syndrome xeroderma pigmentosum. A patient presented with a severe XPF mutation leading to profound crosslink sensitivity and dramatic progeroid symptoms. It is not known how unrepaired DNA damage accelerates ageing or its relevance to natural ageing. Here we show a highly significant correlation between the liver transcriptome of old mice and a mouse model of this progeroid syndrome. Expression data from XPF-ERCC1-deficient mice indicate increased cell death and anti-oxidant defences, a shift towards anabolism and reduced growth hormone/insulin-like growth factor 1 (IGF1) signalling, a known regulator of lifespan. Similar changes are seen in wild-type mice in response to chronic genotoxic stress, caloric restriction, or with ageing. We conclude that unrepaired cytotoxic DNA damage induces a highly conserved metabolic response mediated by the IGF1/insulin pathway, which re-allocates resources from growth to somatic preservation and life extension. This highlights a causal contribution of DNA damage to ageing and demonstrates that ageing and end-of-life fitness are determined both by stochastic damage, which is the cause of functional decline, and genetics, which determines the rates of damage accumulation and decline.

Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (␥-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced ␥-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, ␥-H2AX foci were also induced in Ercc1 MMC-induced ␥-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1؊/؊ and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.Interstrand cross-links (ICLs) comprise a unique class of DNA lesions that have a potent biological effect. By definition, ICLs involve covalent modification of both strands of DNA. Therefore, these adducts prevent DNA strand separation and block DNA metabolism, such as transcription and replication (31). DNA-damaging agents that cause ICLs are extremely cytotoxic, and their utility as anticancer chemotherapeutics likely stems from their selective toxicity to proliferating cells. ICLs occur via a two-step reaction mechanism in which first a monoadduct involving one strand of DNA is formed (24). Although cross-linking agents induce a variety of DNA adducts, the relative cytotoxicity of each agent correlates with its ability to form ICLs (43, 44).The repair of DNA ICLs presents a unique challenge to cells. Since both strands of DNA are covalently modified, simple excision of the lesion followed by template-driven DNA resynthesis is precluded. In Escherichia coli, two solutions to this problem have been identified (reviewed in reference 19). In both these repair mechanisms, the ICL is excised from one strand. In error-free repair, an undamaged chromosome is then utilized as a template for gap-filling DNA polymerization (55). ICL repair also occurs in recombination-deficient E. coli, likely via translesional DNA polymerization of the second damaged strand (5). Similarly, genetic analysis of Saccharomyces cerevisiae (23) and mammalian DNA repair mutants (reviewed in reference 19) indicates the involvement of proteins from multiple DNA repair pathways in ICL repair: nucleotide excision repair (NER), homologous recombination, and postreplication...

Senolytic drugs are agents that selectively induce apoptosis of senescent cells. These cells accumulate in many tissues with aging and at sites of pathology in multiple chronic diseases. In studies in animals, targeting senescent cells using genetic or pharmacological approaches delays, prevents, or alleviates multiple age-related phenotypes, chronic diseases, geriatric syndromes, and loss of physiological resilience. Among the chronic conditions successfully treated by depleting senescent cells in pre-clinical studies are frailty, cardiac dysfunction, vascular hyporeactivity and calcification, diabetes, liver steatosis, osteoporosis, vertebral disk degeneration, pulmonary fibrosis, and radiation-induced damage. Senolytic agents are at the point of being tested in proof-of-concept clinical trials. To do so, new clinical trials paradigms for testing senolytics and other agents that target fundamental aging mechanisms are being developed, since use of long-term endpoints such as life- or healthspan is not feasible. These strategies include testing effects on multi-morbidity, accelerated aging-like conditions, diseases with localized accumulation of senescent cells, potentially fatal diseases associated with senescent cell accumulation, age-related loss of physiological resilience, and frailty. If senolytics or other interventions that target fundamental aging processes prove to be effective and safe in clinical trials, they could transform geriatric medicine by enabling prevention or treatment of multiple diseases and functional deficits in parallel, instead of one-at-a-time.

No abstract

The accumulation of cellular damage, including DNA damage, is thought to contribute to aging-related degenerative changes, but how damage drives aging is unknown. XFE progeroid syndrome is a disease of accelerated aging caused by a defect in DNA repair. NF-κB, a transcription factor activated by cellular damage and stress, has increased activity with aging and aging-related chronic diseases. To determine whether NF-κB drives aging in response to the accumulation of spontaneous, endogenous DNA damage, we measured the activation of NF-κB in WT and progeroid model mice. As both WT and progeroid mice aged, NF-κB was activated stochastically in a variety of cell types. Genetic depletion of one allele of the p65 subunit of NF-κB or treatment with a pharmacological inhibitor of the NF-κB-activating kinase, IKK, delayed the age-related symptoms and pathologies of progeroid mice. Additionally, inhibition of NF-κB reduced oxidative DNA damage and stress and delayed cellular senescence. These results indicate that the mechanism by which DNA damage drives aging is due in part to NF-κB activation. IKK/NF-κB inhibitors are sufficient to attenuate this damage and could provide clinical benefit for degenerative changes associated with accelerated aging disorders and normal aging.

Human telomeres are protected by TRF2. Inhibition of this telomeric protein results in partial loss of the telomeric 3' overhang and chromosome end fusions formed through nonhomologous end-joining (NHEJ). Here we report that ERCC1/XPF-deficient cells retained the telomeric overhang after TRF2 inhibition, identifying this nucleotide excision repair endonuclease as the culprit in overhang removal. Furthermore, these cells did not accumulate telomere fusions, suggesting that overhang processing is a prerequisite for NHEJ of telomeres. ERCC1/XPF was also identified as a component of the telomeric TRF2 complex. ERCC1/XPF-deficient mouse cells had a novel telomere phenotype, characterized by Telomeric DNA-containing Double Minute chromosomes (TDMs). We speculate that TDMs are formed through the recombination of telomeres with interstitial telomere-related sequences and that ERCC1/XPF functions to repress this process. Collectively, these data reveal an unanticipated involvement of the ERCC1/XPF NER endonuclease in the regulation of telomere integrity and establish that TRF2 prevents NHEJ at telomeres through protection of the telomeric overhang from ERCC1/XPF.

DNA interstrand crosslinks (ICLs) are lesions caused by a variety of endogenous metabolites, environmental exposures, and cancer chemotherapeutic agents that have two reactive groups. The common feature of these diverse lesions is that two nucleotides on opposite strands are covalently joined. ICLs prevent the separation of two DNA strands and therefore essential cellular processes including DNA replication and transcription. ICLs are mainly detected in S phase when a replication fork stalls at an ICL. Damage signaling and repair of ICLs are promoted by the Fanconi anemia pathway and numerous posttranslational modifications of DNA repair and chromatin structural proteins. ICLs are also detected and repaired in non-replicating cells, although the mechanism is less clear. A unique feature of ICL repair is that both strands of DNA must be incised to completely remove the lesion. This is accomplished in sequential steps to prevent creating multiple double-strand breaks. Unhooking of an ICL from one strand is followed by translesion synthesis to fill the gap and create an intact duplex DNA, harboring a remnant of the ICL. Removal of the lesion from the second strand is likely accomplished by nucleotide excision repair. Inadequate repair of ICLs is particularly detrimental to rapidly dividing cells, explaining the bone marrow failure characteristic of Fanconi anemia and why cross-linking agents are efficacious in cancer therapy. Herein, recent advances in our understanding of ICLs and the biological responses they trigger are discussed.