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

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RecQ-like helicase, presumed to function in DNA replication, recombination, or repair. BLM localizes to promyelocytic leukemia protein (PML) nuclear bodies and is expressed during late S and G2. We show, in normal human cells, that the recombination/repair proteins hRAD51 and replication protein (RP)-A assembled with BLM into a fraction of PML bodies during late S/G2. Biochemical experiments suggested that BLM resides in a nuclear matrix–bound complex in which association with hRAD51 may be direct. DNA-damaging agents that cause double strand breaks and a G2 delay induced BLM by a p53- and ataxia-telangiectasia mutated independent mechanism. This induction depended on the G2 delay, because it failed to occur when G2 was prevented or bypassed. It coincided with the appearance of foci containing BLM, PML, hRAD51 and RP-A, which resembled ionizing radiation-induced foci. After radiation, foci containing BLM and PML formed at sites of single-stranded DNA and presumptive repair in normal cells, but not in cells with defective PML. Our findings suggest that BLM is part of a dynamic nuclear matrix–based complex that requires PML and functions during G2 in undamaged cells and recombinational repair after DNA damage.

Primary liver cancer represents a major health problem. It comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), which differ markedly with regards to their morphology, metastatic potential and responses to therapy. However, the regulatory molecules and tissue context that commit transformed hepatic cells towards HCC or ICC are largely unknown. Here we show that the hepatic microenvironment epigenetically shapes lineage commitment in mosaic mouse models of liver tumorigenesis. Whereas a necroptosis-associated hepatic cytokine microenvironment determines ICC outgrowth from oncogenically transformed hepatocytes, hepatocytes containing identical oncogenic drivers give rise to HCC if they are surrounded by apoptotic hepatocytes. Epigenome and transcriptome profiling of mouse HCC and ICC singled out Tbx3 and Prdm5 as major microenvironment-dependent and epigenetically regulated lineage-commitment factors, a function that is conserved in humans. Together, our results provide insight into lineage commitment in liver tumorigenesis, and explain molecularly why common liver-damaging risk factors can lead to either HCC or ICC.

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In this study, we investigated the subcellular and molecular mechanisms underlying promyelocytic leukemia (PML)-induced premature senescence. We demonstrate that intact PML nuclear bodies are not required for the induction of senescence. We have determined further that of seven known PML isoforms, only PML IV is capable of causing premature senescence, providing the ®rst evidence for functional differences among these isoforms. Of interest is the fact that in contrast to PML +/+ ®broblasts, PML ±/± cells are resistant to PML IV-induced senescence. This suggests that although PML IV is necessary for this process to occur, it is not suf®cient and requires other components for activity. Finally, we provide evidence that PML IV-induced senescence involves stabilization and activation of p53 through phosphorylation at Ser46 and acetylation at Lys382, and that it occurs independently of telomerase and differs from that elicited by oncogenic Ras. Taken together, our data assign a speci®c pro-senescent activity to an individual PML isoform that involves p53 activation and is independent from PML nuclear bodies.

p53 functions to prevent malignant progression, in part by inhibiting proliferation or inducing the death of potential tumour cells. One of the most important regulators of p53 is MDM2, a RING domain E3 ligase that ubiquitinates p53, leading to both proteasomal degradation and relocation of p53 from the nucleus to the cytoplasm. Previous studies have suggested that although polyubiquitination is required for degradation, monoubiquitination of p53 is sufficient for nuclear export. Using a p53-ubiquitin fusion protein we show that ubiquitination contributes to two steps before export: exposure of a carboxy-terminal nuclear export sequence (NES), and dissociation of MDM2. Monoubiquitination can directly promote further modifications of p53 with ubiquitin-like proteins and MDM2 promotes the interaction of the SUMO E3 ligase PIASy with p53, enhancing both sumoylation and nuclear export. Our results suggest that modifications such as sumoylation can regulate the strength of the p53-MDM2 interaction and participate in driving the export of p53.

Cellular senescence is a tumour suppressor mechanism that is triggered by cancer-initiating or promoting events in mammalian cells. The molecular underpinnings for this stable arrest involve transcriptional repression of proliferation-promoting genes regulated by the retinoblastoma (Rb)/E2F repressor complex. Here, we demonstrate that AGO2, Rb and microRNAs (miRs), as exemplified here by let-7, physically and functionally interact to repress Rb/E2F target genes in senescence, a process that we refer to as senescence-associated transcriptional gene silencing (SA-TGS). Herein, AGO2 acts as the effector protein for miR-let7-directed implementation of silent state chromatin modifications at target promoters and inhibition of the let-7-AGO2 effector complex perturbs the timely execution of senescence. Thus, we identify cellular senescence as the an endogenous signal of miR-AGO2-mediated TGS in human cells. Our results suggest that miR-AGO2-mediated SA-TGS may contribute to tumour suppression by stably repressing proliferation-promoting genes in pre-malignant cancer cells.

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