Senescence In Mammalian Cells Research Articles

Hog1 acts in a Mec1-independent manner to counteract oxidative stress following telomerase inactivation in Saccharomyces cerevisiae

Replicative senescence is triggered when telomeres reach critically short length and activate permanent DNA damage checkpoint-dependent cell cycle arrest. Mitochondrial dysfunction and increase in oxidative stress are both features of replicative senescence in mammalian cells. However, how reactive oxygen species levels are controlled during senescence is elusive. Here, we show that reactive oxygen species levels increase in the telomerase-negative cells of Saccharomyces cerevisiae during replicative senescence, and that this coincides with the activation of Hog1, a mammalian p38 MAPK ortholog. Hog1 counteracts increased ROS levels during replicative senescence. While Hog1 deletion accelerates replicative senescence, we found this could stem from a reduced cell viability prior to telomerase inactivation. ROS levels also increase upon telomerase inactivation when Mec1, the yeast ortholog of ATR, is mutated, suggesting that oxidative stress is not simply a consequence of DNA damage checkpoint activation in budding yeast. We speculate that oxidative stress is a conserved hallmark of telomerase-negative eukaryote cells, and that its sources and consequences can be dissected in S. cerevisiae.

Typhoid toxin hijacks Wnt5a to establish host senescence and Salmonella infection

Damage to our genome causes acute senescence in mammalian cells, which undergo growth arrest and release a senescence-associated secretory phenotype (SASP) that propagates the stress response to bystander cells. Thus, acute senescence is a powerful tumor suppressor. Salmonella enterica hijacks senescence through its typhoid toxin, which usurps unidentified factors in the stress secretome of senescent cells to mediate intracellular infections. Here, transcriptomics of toxin-induced senescent cells (TxSCs) and proteomics of their secretome identify the factors as Wnt5a, INHBA, and GDF15. Wnt5a establishes a positive feedback loop, driving INHBA and GDF15 expression. In fibroblasts, Wnt5a and INHBA mediate autocrine senescence in TxSCs and paracrine senescence in naive cells. Wnt5a synergizes with GDF15 to increase Salmonella invasion. Intestinal TxSCs undergo apoptosis without Wnt5a, which is required for establishing intestinal TxSCs. The study reveals how an innate defense against cancer is co-opted by a bacterial pathogen to cause widespread damage and mediate infections.

An antioxidant suppressed lung cellular senescence and enhanced pulmonary function in aged mice

Oxidative stress is one of the major causes of cellular senescence in mammalian cells. The excess amount of reactive oxygen species generated by oxygen metabolism is pathogenic and facilitates tissue aging. Lung tissue is more susceptible to oxidative stress than other organs because it is directly exposed to environmental stresses. The aging of lung tissues increases the risk of chronic diseases. Senescent cells accumulate in tissues during aging and contribute to aging-associated morbidity; however, the roles of cellular senescence in lung aging and diseases have not yet been elucidated in detail.To clarify the physiological role of oxidative stress-induced cellular senescence in aging-associated declines in pulmonary function, we herein investigated the effects of the antioxidant N-acetyl-L-cysteine (NAC) on lung cellular senescence and aging in mice. The administration of NAC to 1-year-old mice reduced the expression of senescence-associated genes in lung tissue. Pulmonary function and lung morphology were partly restored in mice administered NAC. Collectively, these results suggest that oxidative stress is a major inducer of cellular senescence in vivo and that the control of oxidative stress may prevent lung aging and diseases.

Histone variant H2A.J accumulates in senescent cells and promotes inflammatory gene expression

The senescence of mammalian cells is characterized by a proliferative arrest in response to stress and the expression of an inflammatory phenotype. Here we show that histone H2A.J, a poorly studied H2A variant found only in mammals, accumulates in human fibroblasts in senescence with persistent DNA damage. H2A.J also accumulates in mice with aging in a tissue-specific manner and in human skin. Knock-down of H2A.J inhibits the expression of inflammatory genes that contribute to the senescent-associated secretory phenotype (SASP), and over expression of H2A.J increases the expression of some of these genes in proliferating cells. H2A.J accumulation may thus promote the signalling of senescent cells to the immune system, and it may contribute to chronic inflammation and the development of aging-associated diseases.

Aberrant localization of lamin B receptor (LBR) in cellular senescence in human cells

5-Bromodeoxyuridine (BrdU), a thymidine analogue, induces cellular senescence in mammalian cells. BrdU induces cellular senescence probably through the regulation of chromatin because BrdU destabilizes or disrupts nucleosome positioning and decondenses heterochromatin. Since heterochromatin is tethered to the nuclear periphery through the interaction with the nuclear envelope proteins, we examined the localization of the several nuclear envelope proteins such as lamins, lamin-interacting proteins, nuclear pore complex proteins, and nuclear transport proteins in senescent cells. We have shown here that lamin B receptor (LBR) showed a change in localization in both BrdU-induced and replicative senescent cells.

Electrophoretically mediated microanalysis for in-capillaryelectrical cell lysis and fast enzyme quantification by capillary electrophoresis

In this study, a novel capillary electrophoresis (CE)-based enzymatic assay was developed to evaluate enzymatic activity in whole cells. β-Galactosidase expression was used as an example, as it is a biomarker for assessing replicative senescence in mammalian cells. It catalyzes the hydrolysis of para-nitrophenyl-β-D-galactopyranoside (PNPG) into para-nitrophenol (PNP). The CE-based assay consisted of four main steps: (1) hydrodynamic injection of whole intact cells into the capillary, (2) in-capillary lysis of these cells by using pulses of electric field (electroporation), (3) in-capillary hydrolysis of PNPG by the β-galactosidase--released from the lysed cells--by the electrophoretically mediated microanalysis (EMMA) approach, and (4) on-line detection and quantification of the PNP formed. The developed method was applied to Escherichia coli as well as to human keratinocyte cells at different replicative stages. Results obtained by CE were in excellent agreement with those obtained from off-line cell lysates which proves the efficiency of the in-capillary approach developed. This work shows for the first time that cell membranes can be disrupted in-capillary by electroporation and that the released enzyme can be subsequently quantified in the same capillary. Enzyme quantification in cells after their in-capillary lysis has never been conducted by CE. The developed CE approach is automated, economic, eco-friendly, and simple to conduct. It has attractive applications in bacteria or human cells for early disease diagnostics or insights for development in biology.

Conserved role of medium acidification in chronological senescence of yeast and mammalian cells

The yeast chronological life span (CLS) model has led to the identification of the pro-aging effects of the TOR-Sch9 /S6K and Ras-Adenylate cyclase-PKA pathways, components of which play conserved role in nutrient sensing and aging in mammals [1-4]. One of the early changes that occurs in yeast cells grown in media containing 2% glucose and excess amino acids is the production of acetic acid and acidification of the medium to below pH 4. This acidification has been shown to accelerate yeast aging [5-9]. However, it is clear that it does not explain the effect of the TOR-Sch9/S6K and Ras-AC-PKA pathways on aging since their inhibition extends chronological life span in media that is not acidified and that does not contain acetic acid [10]. The assumption that acetic acid is an organic toxin, which is the key mediator of chronological aging under standard conditions, is probably not true for most genetic backgrounds, since under physiological conditions acetic acid is generated at low levels compared to another metabolite, ethanol [6-7, 11]. Additionally, acetic acid, in spite of its potential toxicity, represents one among several carbon sources that can be utilized by Saccharomyces cerevisiae for growth and metabolism [12-15]. In previous issue of Aging, Leontieva and Blagosklonny describe a yeast-like chronological senescence (CS) model in mammalian cells (Leontieva and Blagosklonny). They show that human tumor cells maintained in stationary culture lose their viability (colony forming units) and that this process is accelerated by medium acidification caused in part by lactate accumulation, which mirrors the accumulation of ethanol and some acetic acid, and the acidification of the medium in S. cerevisiae [5-7, 9]. In yeast, the ethanol accumulated during the growth phase can be used as carbon source during the diauxic shift and the post-diauxic phase, when cells stop dividing and switch from a fermentation- to a respiration-based metabolism [5, 16-17]. Long-lived mutants with deficiencies in the TOR- Sch9/S6K and Ras-AC-PKA pathways deplete ethanol, show a reduced accumulation of extracellular acetic acid [6, 11](M. Wei unpublished results) as well as activate glycerol biosynthesis [11]. As opposed to glucose and ethanol and, possibly, acetic acid, glycerol does not elicit adverse effects on cellular protection and life span suggesting that the Tor1/Sch9-regulated glycerol biosynthesis results in the removal of pro-aging carbon sources [11]. Leontieva and Blagosklonny show that the “yeast-like” chronological senescence in mammalian cells is delayed and attenuated by the inhibition of the mTOR and PI3K signaling pathways, both of which have been implicated in longevity regulation in organisms ranging from yeast to mice. Conditioned medium produced by rapamycin-treated cells was less toxic in inducing CS. However, the addition of rapamycin did not protect fibrosarcoma cells from high concentration of lactate suggesting that rapamycin did not protect cells from CS per se. Rather, inhibition of mTOR affected cellular metabolism and inhibited lactate production during the early phase of stationary survival, which led to a reduced initial lactate accumulation and delayed CS (Leontieva and Blagosklonny). Interestingly, mTOR was spontaneously inactivated after one day in culture, possibly a protective response to lactate accumulation and medium acidification. These results suggest that mTOR promotes CS by favoring lactate production and medium acidification in agreement with the role for TOR-Sch9/S6K in promoting ethanol and acetic acid accumulation in yeast [5, 11, 18]. By contrast, the deletion of either TOR1 or SCH9/S6K are known to extend yeast chronological life span in part by depleting ethanol and acetic acid but largely by mechanisms that are cell autonomous [10-11, 19-21]. It has been argued that acidification of the culture medium and the accumulation of non-fermentable carbon sources such as ethanol and acetic acid render the CLS a paradigm for the identification of “private” mechanisms specific for yeast chronological aging [7, 22-23]. However, not only the yeast CLS method has been remarkably effective in discovering genes later shown to promote aging in mammals [4], it has also revealed the multi-factorial nature of yeast chronological senescence and points to the involvement of diverse cellular processes, such as mitochondrial respiration, reactive oxygen species signaling [1, 19, 24-27], stress response [3, 10, 28], autophagy [29-30], and genome maintenance, in the regulation of life span [31-35]. Although, accumulation of toxic metabolic byproducts may not represent a mechanism of aging in yeast [5-8] or mammalian cells (Leontieva and Blagosklonny [36]), chronological senescence provides a simple model for probing the roles of genes and signaling pathways that affect aging and a powerful platform for high-throughput screening of agents that modulate aging and age-related disease progression.

Yeast-like chronological senescence in mammalian cells: phenomenon, mechanism and pharmacological suppression

In yeast, chronological senescence (CS) is defined as loss of viability in stationary culture. Although its relevance to the organismal aging remained unclear, yeast CS was one of the most fruitful models in aging research. Here we described a mammalian replica of yeast CS: loss of viability of overgrown "yellow" cancer cell culture. In a density and time (chronological)-dependent manner, cell culture loses the ability to re-grow in fresh medium. Rapamycin dramatically decelerated CS. Loss of viability was caused by acidification of the medium by lactic acid (lactate). Rapamycin decreased production of lactate, making conditioned medium (CM) less deadly. Both deadly CM and lactate caused loss of viability in low cell density, not preventable by either rapamycin or additional glucose. Also, NAC, LY294002, U0126, GSK733, which all indirectly inhibit mTOR and have been shown to suppress the senescent phenotype in traditional models of mammalian cell senescence, also decreased lactate production and decelerated CS. We discuss that although CS does not mimic organismal aging, the same signal transduction pathways that drive CS also drive aging.

SIRT1 controls circadian clock circuitry and promotes cell survival: a connection with age‐related neoplasms

Aging is believed to be a primary risk factor for cancer. Interestingly, the sirtuin family of class III histone deacetylases (HDACs) has been implicated in the regulation of longevity and may be a lost link between aging and cancer. SIRT1, a nicotinamide adenine dinucleotide (NAD(+))-dependent sirtuin, has been shown to promote cell survival by inhibiting apoptosis or cellular senescence in mammalian cells. Recent studies have provided a link between the cellular metabolic function of SIRT1 and the circadian rhythm (controlled by a clock machinery), which, if deregulated, may lead to an increased risk for some cancers. Interestingly, the loss of the pineal hormone melatonin, a known regulator of circadian rhythm, has been shown to cause deregulation in the circadian rhythm machinery and an increase in susceptibility to cancer. On the basis of scientific evidence, we propose a hypothesis that SIRT1 inhibition will impart an antiproliferative response in age-related cancers via resynchronization of deregulated core clock circuitry at the cellular level. If this hypothesis is found valid, it may ultimately lead to the development of novel approaches toward management of age-related malignancies and possibly other diseases.

Pseudomonas pyocyanin inhibits wound repair by inducing premature cellular senescence: Role for p38 mitogen-activated protein kinase

Pseudomonas aeruginosa is an important nosocomial pathogen of burn wounds. Pyocyanin, a virulence factor produced by the bacterium, induces persistent intracellular oxidative stress and premature senescence in mammalian cells. Our aims were to evaluate pyocyanin levels in infected wound dressings and the potential of the toxin to influence wound repair. Surgical dressings from infected burn patients were examined for pyocyanin and normal primary human diploid fibroblasts (HDFs) were treated with comparable concentrations and their replicative ability examined. Pyocyanin was detected in the exudate of infected wound dressings in amounts up to 5.3 μg/g (mean: 2.0 ± 2.3 μg/g). HDFs exposed to pyocyanin (1–50 μM; 0.2–10.5 μg/ml) underwent growth arrest at all concentrations and developed morphological characteristics associated with cellular senescence, including expression of senescence-associated β-galactosidase. Using an in vitro wound repair model, a single exposure to pyocyanin inhibited wound repair in a concentration-dependent manner. Prior treatment with a specific p38 MAPK inhibitor allowed cells to maintain their replicative ability and pre-senescent morphology indicating pyocyanin operates through the Erk/p38 MAPK senescence pathway. These data support the hypothesis that bacterial virulence factors capable of inducing persistent low-level oxidative stress play a pivotal role in modulating the tissue repair response to infection by inducing premature cellular senescence.

Uncoupling of Longevity and Telomere Length in C. elegans

The nematode Caenorhabditis elegans, after completing its developmental stages and a brief reproductive period, spends the remainder of its adult life as an organism consisting exclusively of post-mitotic cells. Here we show that telomere length varies considerably in clonal populations of wild-type worms, and that these length differences are conserved over at least ten generations, suggesting a length regulation mechanism in cis. This observation is strengthened by the finding that the bulk telomere length in different worm strains varies considerably. Despite the close correlation of telomere length and clonal cellular senescence in mammalian cells, nematodes with long telomeres were neither long lived, nor did worm populations with comparably short telomeres exhibit a shorter life span. Conversely, long-lived daf-2 and short-lived daf-16 mutant animals can have either long or short telomeres. Telomere length of post-mitotic cells did not change during the aging process, and the response of animals to stress was found independent of telomere length. Collectively, our data indicate that telomere length and life span can be uncoupled in a post-mitotic setting, suggesting separate pathways for replication-dependent and -independent aging.

Quantitative assay of senescence-associated β-galactosidase activity in mammalian cell extracts

Senescence-associated β-galactosidase activity is a widely used biomarker for assessing replicative senescence in mammalian cells. This enzymatic activity has generally been measured by staining cells with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β- d-galactopyranoside (X-gal) at pH 6.0, a reaction condition that suppresses lysosomal β-galactosidase activity sufficiently to ensure that most nonsenescent cells will appear unstained. This article describes a quantitative method for measuring this activity and characterizes the method using extracts from senescent, quiescent, and presenescent human fibroblasts. The assay is capable of detecting relatively subtle changes in activity and confirms previous indications based on staining that confluency and contact inhibition of growth can cause a small increase in the expression of this biomarker. Investigation of the pH dependence of the activity in the cell extracts suggests that the senescent phenotype is correlated with an increase in total β-galactosidase rather than with a shift in the pH optimum of the enzyme. This assay for measuring senescence-associated changes in β-galactosidase is suitable for mechanistic studies of senescence regulation in which graduated changes in biomarker expression may be anticipated.

Amplification of nuclear aldolase A in mouse cell mutants resistant to Hoechst 33342

5-Bromodeoxyuridine (BrdU) induces a phenomenon similar to cellular senescence in mammalian cells. AT-binding ligands such as Hoechst 33258 synergistically potentiate the effect of BrdU. We isolated mouse FM3A cell mutants resistant to Hoechst 33342 and characterized two highly resistant mutants. Two-dimensional gel electrophoresis followed by peptides sequence tags revealed that nuclear aldolase A was markedly increased in both mutants. Western blot analysis confirmed that nuclear aldolase A was increased leaving cytosolic aldolase A unaltered. Its mRNA levels were also increased in the mutants. Enforced expression of aldolase A conferred resistance to Hoechst 33342 on wild-type cells. Taken together, nuclear aldolase A was shown to somehow protect the cytotoxic effect of Hoechst 33342.

AT-hook proteins stimulate induction of senescence markers triggered by 5-bromodeoxyuridine in mammalian cells

5-Bromodeoxyuridine (BrdU) induces a phenomenon similar to cellular senescence in mammalian cells. To address an underlying molecular mechanism in this phenomenon, we assessed the role of AT-hook proteins that bind to the minor grooves of specific AT-rich sequences. We expressed DsRed-tagged HMGI, MATH2, and MATH20 proteins in HeLa cells in a doxycycline dependent manner. Modest expression of these proteins revealed no apparent effect on the cells although high levels of expression were toxic to the cells. In contrast, their modest expression in the presence of low concentrations of BrdU similarly and dose-dependently induced senescence markers examined, although the same concentrations of BrdU alone showed no obvious effect. In both cases, DsRed fluorescence was mainly observed as foci or intense dots on Hoechst 33342-staining regions. These distribution patterns were not changed by addition of BrdU. Since AT-hook domains can displace chromatin compacting proteins pre-bound on AT-rich sequences, these results suggest that chromatin unpacking is one of the factors stimulating expression of the senescence markers in human cells.

5-Halogenated thymidine analogues induce a senescence-like phenomenon in HeLa cells.

We tested various thymidine analogues for induction of a senescence-like phenomenon in HeLa cells. CldU, BrdU, and IdU similarly induced the morphology of senescent cells and typical senescence markers. Thymidine analogues other than 5-halogenated forms caused only cell death. BrdU efficiently killed the cells in cooperation with irradiation with light and a brief treatment with Hoechst 33258, but CldU did not at all. 5-Halogenated thymidine analogues were thus shown to be specific inducers of cellular senescence in mammalian cells.

Specific association of a small protein with the telomeric DNA-protein complex during the onset of leaf senescence in Arabidopsis thaliana.

Telomeres and their changes in length throughout the life span of cells have been intensively investigated in different organisms. Telomere length is assumed to control replicative senescence in mammalian cells. However, only very few data are available on the developmental dynamics of plant telomeres. Here, changes of telomere length and DNA-protein structure of Arabidopsis thaliana telomeres were analysed in different stages of development, with the main focus resting on the transition from pre-senescent to senescent leaves. The lengths of the telomeres, ranging from ca. 2.0 to 6.5 kb, do not significantly change during plant development indicating that telomere length is not involved in differentiation and replicative senescence nor in post-mitotic senescence of A. thaliana. In dedifferentiated cultured cells a slight increase in length can be determined. The nucleoprotein structure of the telomeric DNA was investigated by gel mobility shift assays, with synthetic oligonucleotides and nuclear protein extracts derived from four defined stages of post-mitotic leaf senescence. In all four stages, a highly salt-resistant DNA-protein complex was formed with the double-stranded as well as with the single-stranded G-rich telomeric DNA. An additional DNA-protein complex was identified in nuclear protein extracts isolated from plants in the transition stage from pre-senescence to senescence. The protein components of the DNA-protein complexes were analysed on native PAGE and SDS-PAGE gels. A protein of 67 kDa (ATBP1) bound to the telomeric DNA in all developmental stages. An additional protein of merely 22 kDa (ATBP2) was associated via protein-protein interaction with ATBP to form a higher-order complex exclusively during the onset of senescence. DNA interaction of this higher-order protein complex seems to be restricted to double-stranded telomeric DNA. The defined period of ATBP1/ATBP2 complex formation with the telomeric DNA probably indicates that ATBP2 is involved in the onset of post-mitotic leaf senescence by either disturbing an established or establishing an additional function exhibited by the telomeres in the interphase nuclei.

Cell immortalization as a key, rate-limiting event in malignant transformation: approaches toward a molecular genetic analysis

Recent advances using somatic cell genetic approaches have provided a convincing body of evidence that the senescence of mammalian cells in culture is controlled by a small group of genes, one or more of which are functionally deleted in the process of immortalization. Microcell-mediated mono-chromosomal transfer methods should permit precise mapping of these genes to specific chromosomal regions. Cloning of senescence genes, using either conventional ‘positional cloning’ techniques or retroviral insertion mutagenesis, is now a realistic possibility. The leap in our understanding of the molecular genetic events driving the alternative cellular states of limited proliferative capacity and immortality, which such advances should precipitate, will finally permit the question of the role of cell immortalization in cancer to be addressed, and may open the door to the design of new modes of cancer therapy. In addition, the precise mechanism underlying the wide difference in transformability between human and rodent cells, which should also emerge from these investigations, is likely to make a significant contribution towards resolving the key issue of the relevance of rodent tumour induction assays in assessing the potential carcinogenicity of environmental chemicals.

Diphenylhydantoin intoxication attendant to slow inactivation of isoniazid.

Damage to our genome causes acute senescence in mammalian cells, which undergo growth arrest and release a secretome that elicits cell cycle arrest in bystander cells through the senescence-associated secretory phenotype (SASP). Thus, acute senescence is a powerful tumour suppressor. <i>Salmonella enterica</i> hijacks senescence through its typhoid toxin, which usurps unidentified factors in the stress secretome of senescent cells to mediate intracellular infections. Here, transcriptomics of toxin-induced senescent cells (txSCs) and proteomics of their secretome identified secreted ligands that activate the TGFβ pathway through SMAD transcription factors. The ligand Wnt5a established a self-amplifying positive feedback loop driving TGFβ signalling, which enforced autocrine senescence in txSCs and paracrine senescence in naive bystander cells by activation of DDRs. Wnt5a and GDF15 increased host cell susceptibility to infection. The study reveals how an innate defence against cancer is co-opted by a bacterial pathogen to cause widespread damage and mediate infections.