Cell Senescence: Methods and Protocols

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One of the most prominent features of cellular senescence, a stress response that prevents the propagation of cells that have accumulated potentially oncogenic alterations, is a permanent loss of proliferative potential.

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Thus, at odds with quiescent cells, which resume proliferation when stimulated to do so, senescent cells cannot proceed through the cell cycle even in the presence of mitogenic factors. Taken together, these methods allow for the identification of bonafide cytostatic effects as well as for a refined characterization of cell cycle distributions, providing information on proliferation, DNA content, as well as the presence of cell cycle phase-specific markers.

At the end of the chapter, a set of guidelines is offered to assist researchers that approach the study of the cell cycle with the interpretation of results.

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Detection of senescence-associated heterochromatin foci SAHF. N2 - One of the most prominent features of cellular senescence, a stress response that prevents the propagation of cells that have accumulated potentially oncogenic alterations, is a permanent loss of proliferative potential. AB - One of the most prominent features of cellular senescence, a stress response that prevents the propagation of cells that have accumulated potentially oncogenic alterations, is a permanent loss of proliferative potential.

Abstract One of the most prominent features of cellular senescence, a stress response that prevents the propagation of cells that have accumulated potentially oncogenic alterations, is a permanent loss of proliferative potential. Fingerprint Heterochromatin. Mutations in ATM, an important molecule involved in activating checkpoint signaling in response to DNA double strand breaks, cause Ataxia-telangiectasia, another rare early-onset neurodegenerative disease in which patients display skin abnormalities including vitiligo, hair graying and telangiectasia in sun-exposed skin Zou, ; Derheimer and Kastan, ; Rothblum-Oviatt et al.

Dyskeratosis congenita DKC is multi-systemic premature aging syndrome resulting in some skin defects. Initial clinical features appear during childhood and include abnormal skin pigmentation and nail dystrophy. During the second and third decade, patients develop bone marrow failure, ultimately resulting in their death. In addition, some patients exhibit an increased risk to develop premalignant and malignant lesions, including AK and SCC Powell et al.

Cell Senescence: Methods and Protocols

These mutations result in impaired telomere maintenance, presumably in adult stem cell compartments, and premature telomere shortening Mitchell et al. A common feature of the abovementioned genetic syndromes is the accumulation of persistent DNA damage, either by impaired DNA replication, repair, or signaling, as well as by DNA damage at unrepairable loci such as telomeres, resulting in premature cellular senescence and accelerated skin aging Carrero et al. These phenotypes can be recapitulated in patient-derived fibroblasts, cell- or iPSC-based disease models and mouse models Crabbe et al.

However, the rarity of these genetic diseases coupled with limited access to tissue biopsies have hampered efforts to test whether senescent cells accumulate within patient tissues in vivo. How are these accelerated aging syndromes relevant to normal aging? During chronological aging, at least the accumulation of genomic alterations correlates with the onset of age-related phenotypes and diseases Chen et al.

Furthermore, an increase susceptibility to DNA damage Xu et al. Chronological aging can be accelerated by extrinsic factors that result in DNA damage, including UV radiation, particles from traffic pollution and cigarette smoke. In conclusion, increased DNA damage, impaired repair or signaling play a key conserved feature of premature aging syndromes and normal aging.

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Aging results in a progressive decline of skin function, leading to fragility, impaired barrier function, increased susceptibility to physical insults, infection, accumulation of age-related pathologies such as impaired wound healing and an increased risk of cancer Lewis et al. Skin wound healing occurs in four stages: hemostasis, inflammation, proliferation and remodeling. Wound closure takes place during the proliferation phase and depends on the formation of granulation tissue and the generation of contractile myofibroblasts.

A ground breaking study by Demaria et al. Elimination of senescent cells reduced the number of myofibroblasts, delayed wound closure and increased fibrosis, all of which could be prevented by ectopic administration of PDGF-AA Jun and Lau, ; Demaria et al. Similarly, senescent cells prevent fibrosis during liver regeneration Krizhanovsky et al.

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These results demonstrate that senescent cells play an important role in tissue repair via cell-autonomous mechanisms: in the skin through the secretion of PDGF-AA; in the regenerating liver by secreting Mmp9, Mmp13 and IL Thus, senescent cells may limit tissue regeneration in order to protect against unrestricted cell proliferation.

On the flip side, a prolonged presence of senescent cells in the elderly prevents re-epithelialization and wound closure resulting in chronic wounds Holt et al. Taken together, cellular senescence plays a complex role during normal wound healing as well as in chronic wounds. Cellular senescence is a double-edged sword: on the one hand, the accumulation of senescent cells may contribute to various age-related complications; on the other hand, RS and OIS suppress uncontrolled proliferation of cells that are at risk for preneoplastic transformation, thereby limiting tumorigenesis.

As a result, cells from premalignant clinical specimens of breast, colon, lung and bladder tumors exhibited persistent DNA damage checkpoint activation and expressed some senescence markers Bartkova et al. In the skin, the vast majority of benign nevi are caused either by activating mutations in N-RAS or BRAF, and are unlikely to progress toward melanoma. Kras mutant mice also expressed increased senescence markers in premalignant lung and pancreatic adenomas, and in chemically induced skin papillomas Collado et al.

Skin cancer represents the most common human cancer type and its incidence increases with age and UV exposure Narayanan et al. Long-term exposure to sunlight results in persistent DNA damage, increased cellular senescence and formation of premalignant lesions such as AK. Although cells within AK lesions exhibit genomic instability and telomere shortening Rehman et al. As senescent cells generally accumulate in premalignant, but not malignant lesions, the identification and quantification of senescent cells within AK lesions may serve as a valuable tool to diagnose and distinguish AK from SCC and predict the course of disease progression.

Numerous studies have aimed at preventing the accumulation of senescent cells within aging tissues. However, strategies that involve blocking p16 INK4a and p53, or activating telomerase to extend the proliferative capacity of cells inevitably lead to an increased cancer risk.

Thus, a safer approach might be to either selectively eliminate senescent cells from tissues or modulate their function particularly the SASP. The use of a progeroid mouse model expressing a mutated form of the mitotic checkpoint BubR1 has proven instrumental in demonstrating that p16 INK4a -positive senescent cells drive age-related pathologies, and that selective elimination of these cells can prevent or delay age-related tissue deterioration Baker et al. Similarly, clearance of p16 INK4a -positive cells in normal-aging mice extended their median lifespan, delayed tumorigenesis, improved healthspan indices including heart and kidney function, and delayed age-related decline in exploratory behavior Baker et al.

Multiple recent studies confirmed that clearing senescent cells improved tissue function in various age-related pathologies including degenerative joint disease, hepatic fat accumulation and liver chemotoxicity Xu et al. These studies established senescent cells as potential therapeutic targets in the treatment of aging and age-related diseases. As a result, compounds that selectively eliminate senescent cells senolytics are under investigation to treat and prevent various age-related diseases Baar et al.

While several molecular pathways that trigger senescence are under consideration for the development of senolytics, the majority are directed at the pathways that confer apoptosis-resistance in senescent cells in particular the anti-apoptotic BCL protein family Chang et al. However, few studies have probed how treatment with senolytics may affect skin function and homeostasis.

In one study involving a skin-specific keratin 5-driven p14 ARF transgenic mouse model, senescent epidermal cells were efficiently eliminated upon delivery of ABT, a BCL-specific senolytic compound Tokarsky-Amiel et al. Although elimination of senescent cells from the epidermis led to increased proliferation within hair follicles, the underlying mechanism or other consequences on skin function were not investigated.

Thus, it remains to be determined whether senolytics offer novel therapeutic approaches to treat chronic wounds, skin inflammation, age-related pigmentation disorders and ECM breakdown, or whether their clearance would impair wound healing or other skin functions in younger individuals. A prerequisite for any such studies is a better understanding as to which skin compartment or cell type becomes senescent in the myriad age-related skin pathologies.

Multiplexing senescence biomarkers with cell-type specific markers will pave the way to address this question. In addition, it is important to better understand how senescence affects or modulates the function of various skin cell types. To date, most SASP studies have focused on dermal fibroblasts and little is known about the SASP of other skin resident cell types, including melanocytes, keratinocytes or cell types from skin appendages, such as sebocytes.

This is particularly relevant in skin as the interplay between these different cell types is essential to regulate skin functions including pigmentation, barrier function, sebum production, wound healing and cancer progression.


An integrated approach for monitoring cell senescence.

The recognition of the role of cellular senescence in tissue aging has progressed tremendously in the past decade and this is in part attributed to better characterization of senescence biomarkers which allows qualitative and quantitative detection of senescent cells in vitro and in situ. Recent studies provided evidence that senescent cells accumulate in aged skin and may be driving the functional deterioration that characterizes aging skin and age-related skin diseases.

Improved methodologies to detect senescent cells and a better understanding how senescence affects cellular function will enable us to determine how they contribute to various age-related skin changes and pathologies, including impaired wound healing and tumorigenesis. Moreover, the careful evaluation of senolytics in animal models and 3D reconstructed skin organotypics will facilitate the successful translation of senolytics as a therapeutic intervention for skin aging. The accessibility of human skin offers advantages to test these hypotheses.

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. We thank Philipp Kaldis, Steven Thng Tien Guan, and the members of the Dreesen lab for discussions and careful reading of the manuscript.

Acosta, J. Chemokine signaling via the CXCR2 receptor reinforces senescence. Cell , — Allsopp, R. Telomere length predicts replicative capacity of human fibroblasts. Baar, M. Targeted apoptosis of senescent cells restores tissue homeostasis in response to chemotoxicity and aging. Cell ,— Bachrati, C. RecQ helicases: suppressors of tumorigenesis and premature aging. Baker, D. Naturally occurring p16 Ink4a-positive cells shorten healthy lifespan.

Nature , — Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency. Cell Biol. Clearance of p16 Ink4a-positive senescent cells delays ageing-associated disorders. Bakkenist, C. Nature ,— Bartkova, J. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis.

Cell Senescence: Methods and Protocols Cell Senescence: Methods and Protocols
Cell Senescence: Methods and Protocols Cell Senescence: Methods and Protocols
Cell Senescence: Methods and Protocols Cell Senescence: Methods and Protocols
Cell Senescence: Methods and Protocols Cell Senescence: Methods and Protocols
Cell Senescence: Methods and Protocols Cell Senescence: Methods and Protocols

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