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Identification and isolation of senescent cells is challenging, rendering their detailed analysis an unmet need. We describe a precise one-step protocol to fluorescently label senescent cells, for flow cytometry and fluorescence microscopy, implementing a fluorophore-conjugated Sudan Black-B analog, GLF16. Also, a micelle-based approach allows identification of senescent cells in vivo and in vitro, enabling live-cell sorting for downstream analyses and live in vivo tracking. Our protocols are applicable to cellular systems, tissues, or animal models where senescence is present. For complete details on the use and execution of this protocol, please refer to Magkouta et al. 1 ., Competing Interests: Declaration of interests Pending patents: (1) Greek Patent Application 20230100019 and (2) University of Dundee 20230100019, regarding SBB analogs hydrophilic compounds on chemical synthesis, method(s), and application(s) use., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Activating mutations in the BRAF-MAPK pathway have been reported in histiocytoses, hematological inflammatory neoplasms characterized by multi-organ dissemination of pro-inflammatory myeloid cells. Here, we generate a humanized mouse model of transplantation of human hematopoietic stem and progenitor cells (HSPCs) expressing the activated form of BRAF (BRAF V600E ). All mice transplanted with BRAF V600E -expressing HSPCs succumb to bone marrow failure, displaying myeloid-restricted hematopoiesis and multi-organ dissemination of aberrant mononuclear phagocytes. At the basis of this aggressive phenotype, we uncover the engagement of a senescence program, characterized by DNA damage response activation and a senescence-associated secretory phenotype, which affects also non-mutated bystander cells. Mechanistically, we identify TNFα as a key determinant of paracrine senescence and myeloid-restricted hematopoiesis and show that its inhibition dampens inflammation, delays disease onset and rescues hematopoietic defects in bystander cells. Our work establishes that senescence in the human hematopoietic system links oncogene-activation to the systemic inflammation observed in histiocytic neoplasms., (© 2021. The Author(s).)

Down syndrome (DS) patients prematurely show clinical manifestations usually associated with aging. Their immune system declines earlier than healthy individuals, leading to increased susceptibility to infections and higher incidence of autoimmune phenomena. Clinical features of accelerated aging indicate that trisomy 21 increases the biological age of tissues. Based on previous studies suggesting immune senescence in DS, we hypothesized that induction of cellular senescence may contribute to early thymic involution and immune dysregulation. Immunohistochemical analysis of thymic tissue showed signs of accelerated thymic aging in DS patients, normally seen in older healthy subjects. Moreover, our whole transcriptomic analysis on human Epcam-enriched thymic epithelial cells (hTEC), isolated from three DS children, which revealed disease-specific transcriptomic alterations. Gene set enrichment analysis (GSEA) of DS TEC revealed an enrichment in genes involved in cellular response to stress, epigenetic histone DNA modifications and senescence. Analysis of senescent markers and oxidative stress in hTEC and thymocytes confirmed these findings. We detected senescence features in DS TEC, thymocytes and peripheral T cells, such as increased β-galactosidase activity, increased levels of the cell cycle inhibitor p16, telomere length and integrity markers and increased levels of reactive oxygen species (ROS), all factors contributing to cellular damage. In conclusion, our findings support the key role of cellular senescence in the pathogenesis of immune defect in DS while adding new players, such as epigenetic regulation and increased oxidative stress, to the pathogenesis of immune dysregulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Marcovecchio, Ferrua, Fontana, Beretta, Genua, Bortolomai, Conti, Montin, Cascarano, Bergante, D’Oria, Giamberti, Amodio, Cancrini, Carotti, Di Micco, Merelli, Bosticardo and Villa.)

Cellular senescence, first described in vitro in 1961, has become a focus for biotech companies that target it to ameliorate a variety of human conditions. Eminently characterized by a permanent proliferation arrest, cellular senescence occurs in response to endogenous and exogenous stresses, including telomere dysfunction, oncogene activation and persistent DNA damage. Cellular senescence can also be a controlled programme occurring in diverse biological processes, including embryonic development. Senescent cell extrinsic activities, broadly related to the activation of a senescence-associated secretory phenotype, amplify the impact of cell-intrinsic proliferative arrest and contribute to impaired tissue regeneration, chronic age-associated diseases and organismal ageing. This Review discusses the mechanisms and modulators of cellular senescence establishment and induction of a senescence-associated secretory phenotype, and provides an overview of cellular senescence as an emerging opportunity to intervene through senolytic and senomorphic therapies in ageing and ageing-associated diseases.

Hematopoietic stem and progenitor cells (HSPC) reside in the bone marrow (BM) niche and serve as a reservoir for mature blood cells throughout life. Aging in the BM is characterized by low-grade chronic inflammation that could contribute to the reduced functionality of aged HSPC. Mesenchymal stromal cells (MSC) in the BM support HSPC self-renewal. However, changes in MSC function with age and the crosstalk between MSC and HSPC remain understudied. Here, we conducted an extensive characterization of senescence features in BM-derived MSC from young and aged healthy donors. Aged MSC displayed an enlarged senescent-like morphology, a delayed clonogenic potential and reduced proliferation ability when compared to younger counterparts. Of note, the observed proliferation delay was associated with increased levels of SA-β-galactosidase (SA-β-Gal) and lipofuscin in aged MSC at early passages and a modest but consistent accumulation of physical DNA damage and DNA damage response (DDR) activation. Consistent with the establishment of a senescence-like state in aged MSC, we detected an increase in pro-inflammatory senescence-associated secretory phenotype (SASP) factors, both at the transcript and protein levels. Conversely, the immunomodulatory properties of aged MSC were significantly reduced. Importantly, exposure of young HSPC to factors secreted by aged MSC induced pro-inflammatory genes in HSPC and impaired HSPC clonogenic potential in a SASP-dependent manner. Altogether, our results reveal that BM-derived MSC from aged healthy donors display features of senescence and that, during aging, MSC-associated secretomes contribute to activate an inflammatory transcriptional program in HSPC that may ultimately impair their functionality., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Cellular senescence constitutes a stable growth arrest characterized by DNA damage response (DDR) activation and by the senescence-associated secretory phenotype (SASP). SASP, through its paracrine effects, stimulates the immune system for senescence eradication. Similarly, chemotherapy-treated cancers activate an interferon-mediated response to induce anti-tumor immunity. Recent studies now uncover a new role for the innate DNA sensing pathway in the recognition of cytosolic chromatin in senescence and cancer., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The generation of DNA lesions and the resulting activation of DNA damage response (DDR) pathways are both affected by the chromatin status at the site of damaged DNA. In turn, DDR activation affects the chromatin at both the damaged site and across the whole genome. Cellular senescence and cancer are associated with the engagement of the DDR pathways and with profound chromatin changes. In this Opinion article, we discuss the interplay between chromatin and DDR factors in the context of cellular senescence that is induced by oncogenes and in cancer.

In normal human somatic cells, telomere dysfunction causes cellular senescence, a stable proliferative arrest with tumour suppressing properties. Whether telomere dysfunction-induced senescence (TDIS) suppresses cancer growth in humans, however, is unknown. Here, we demonstrate that multiple and distinct human cancer precursor lesions, but not corresponding malignant cancers, are comprised of cells that display hallmarks of TDIS. Furthermore, we demonstrate that oncogenic signalling, frequently associated with initiating cancer growth in humans, dramatically affected telomere structure and function by causing telomeric replication stress, rapid and stochastic telomere attrition, and consequently telomere dysfunction in cells that lack hTERT activity. DNA replication stress induced by drugs also resulted in telomere dysfunction and cellular senescence in normal human cells, demonstrating that telomeric repeats indeed are hypersensitive to DNA replication stress. Our data reveal that TDIS, accelerated by oncogene-induced DNA replication stress, is a biological response of cells in human cancer precursor lesions and provide strong evidence that TDIS is a critical tumour suppressing mechanism in humans.

Two major mechanisms have been causally implicated in the establishment of cellular senescence: the activation of the DNA damage response (DDR) pathway and the formation of senescence-associated heterochromatic foci (SAHF). Here we show that in human fibroblasts resistant to premature p16(INK4a) induction, SAHF are preferentially formed following oncogene activation but are not detected during replicative cellular senescence or on exposure to a variety of senescence-inducing stimuli. Oncogene-induced SAHF formation depends on DNA replication and ATR (ataxia telangiectasia and Rad3-related). Inactivation of ATM (ataxia telangiectasia mutated) or p53 allows the proliferation of oncogene-expressing cells that retain increased heterochromatin induction. In human cancers, levels of heterochromatin markers are higher than in normal tissues, and are independent of the proliferative index or stage of the tumours. Pharmacological and genetic perturbation of heterochromatin in oncogene-expressing cells increase DDR signalling and lead to apoptosis. In vivo, a histone deacetylase inhibitor (HDACi) causes heterochromatin relaxation, increased DDR, apoptosis and tumour regression. These results indicate that heterochromatin induced by oncogenic stress restrains DDR and suggest that the use of chromatin-modifying drugs in cancer therapies may benefit from the study of chromatin and DDR status of tumours., (© 2011 Macmillan Publishers Limited. All rights reserved)

Primary mouse embryonic fibroblasts (MEFs) are a popular tool for molecular and cell biology studies. However, when MEFs are grown in vitro under standard tissue culture conditions, they proliferate only for a limited number of population doublings (PD) and eventually undergo cellular senescence. Presently, the molecular mechanisms halting cell cycle progression and establishing cellular senescence under these conditions are unclear. Here, we show that a robust DNA damage response (DDR) is activated when MEFs undergo replicative cellular senescence. Senescent cells accumulate senescence-associated DDR foci (SDFs) containing the activated form of ATM, its phosphorylated substrates and gammaH2AX. In senescent MEFs, DDR markers do not preferentially accumulate at telomeres, the end of linear chromosomes. It has been observed that proliferation of MEFs is extended if they are cultured at low oxygen tension (3% O(2)). We observed that under these conditions, DDR is not observed and senescence is not established. Importantly, inactivation of ATM in senescent MEFs allows escape from senescence and progression through the S-phase. Therefore, MEFs undergoing cellular senescence arrest their proliferation due to the activation of a DNA damage checkpoint mediated by ATM kinase. Finally, we observed that spontaneously immortalized proliferating MEFs display markers of an activated DDR, indicating the presence of chromosomal DNA damage in these established cell lines.

Oncogene activation in normal cells induces a permanent proliferative arrest known as cellular senescence. This phenomenon restrains the expansion of cells that bear an activated oncogene and acts as a powerful tumor-suppressive process. Although the full molecular mechanisms are still being elucidated, it has been observed recently that some oncogenes alter the DNA-replication process and cause DNA-damage accumulation. DNA-damage checkpoint-response activation together with the increased appearance of heterochromatin formation that leads to transcriptional silencing of proliferative genes are, presently, the two main mechanisms known that establish and maintain oncogene-induced senescence. Here, we discuss the most recent advancements in understanding the molecular and cellular mechanisms that control cellular senescence caused by oncogene activation and their impact on cancer studies.

Early tumorigenesis is associated with the engagement of the DNA-damage checkpoint response (DDR). Cell proliferation and transformation induced by oncogene activation are restrained by cellular senescence. It is unclear whether DDR activation and oncogene-induced senescence (OIS) are causally linked. Here we show that senescence, triggered by the expression of an activated oncogene (H-RasV12) in normal human cells, is a consequence of the activation of a robust DDR. Experimental inactivation of DDR abrogates OIS and promotes cell transformation. DDR and OIS are established after a hyper-replicative phase occurring immediately after oncogene expression. Senescent cells arrest with partly replicated DNA and with DNA replication origins having fired multiple times. In vivo DNA labelling and molecular DNA combing reveal that oncogene activation leads to augmented numbers of active replicons and to alterations in DNA replication fork progression. We also show that oncogene expression does not trigger a DDR in the absence of DNA replication. Last, we show that oncogene activation is associated with DDR activation in a mouse model in vivo. We propose that OIS results from the enforcement of a DDR triggered by oncogene-induced DNA hyper-replication.