p21Waf1/Cip1 plays a critical role in modulating senescence through changes of DNA methylation

It has been reported that genomic DNA methylation decreases gradually during cell culture and an organism's aging. However, less is known about the methylation changes of age-related specific genes in aging. p21(Waf1/Cip1) and p16(INK4a) are cyclin-dependent kinase (Cdk) inhibitors that are critical for the replicative senescence of normal cells. In this study, we show that p21(Waf1/Cip1) and p16(INK4a) have different methylation patterns during the aging process of normal human 2BS and WI-38 fibroblasts. p21(Waf1/Cip1) promoter is gradually methylated up into middle-aged fibroblasts but not with senescent fibroblasts, whereas p16(INK4a) is always unmethylated in the aging process. Correspondently, the protein levels of DNA methyltransferase 1 (DNMT1) and DNMT3a increase from young to middle-aged fibroblasts but decrease in the senescent fibroblasts, while DNMT3b decreases stably from young to senescent fibroblasts. p21(Waf1/Cip1) promoter methylation directly represses its expression and blocks the radiation-induced DNA damage-signaling pathway by p53 in middle-aged fibroblasts. More importantly, demethylation by 5-aza-CdR or DNMT1 RNA interference (RNAi) resulted in an increased p21(Waf1/Cip1) level and premature senescence of middle-aged fibroblasts demonstrated by cell growth arrest and high beta-Galactosidase expression. Our results suggest that p21(Waf1/Cip1) but not p16(INK4a) is involved in the DNA methylation mediated aging process. p21(Waf1/Cip1) promoter methylation may be a critical biological barrier to postpone the aging process.     

Regulation of Survival Networks in Senescent Cells: From Mechanisms to Interventions

Cellular senescence is a state of stable cell cycle arrest arising in response to DNA and mitochondrial damages. Senescent cells undergo morphological, structural and functional changes that are influenced by a number of variables, including time, stress, tissue, and cell type. The heterogeneity of the senescent phenotype is exemplified by the many biological properties that senescent cells can cover. The advent of innovative model organisms has demonstrated a functional role of senescent cells during embryogenesis, tissue remodeling, tumorigenesis and aging. Importantly, prolonged and aberrant persistence of senescent cells is often associated with tissue dysfunction and pathology, and is partially the consequence of mechanisms that enhance survival and resistance to cell death. Here, we describe the main molecular players involved in promoting survival of senescent cells, with particular emphasis on the regulation of senescence-associated anti-apoptotic pathways. We discuss the consequences these pathways have in providing resistance to intrinsic and extrinsic pro-apoptotic signals. Finally, we highlight the importance of these pathways in the development of targets for senolytic interventions.     

Postmitotic neurons develop a p21‐dependent senescence‐like phenotype driven by a DNA damage response

In senescent cells, a DNA damage response drives not only irreversible loss of replicative capacity but also production and secretion of reactive oxygen species (ROS) and bioactive peptides including pro‐inflammatory cytokines. This makes senescent cells a potential cause of tissue functional decline in aging. To our knowledge, we show here for the first time evidence suggesting that DNA damage induces a senescence‐like state in mature postmitotic neurons in vivo. About 40–80% of Purkinje neurons and 20–40% of cortical, hippocampal and peripheral neurons in the myenteric plexus from old C57Bl/6 mice showed severe DNA damage, activated p38MAPkinase, high ROS production and oxidative damage, interleukin IL‐6 production, heterochromatinization and senescence‐associated β‐galactosidase activity. Frequencies of these senescence‐like neurons increased with age. Short‐term caloric restriction tended to decrease frequencies of positive cells. The phenotype was aggravated in brains of late‐generation TERC?/? mice with dysfunctional telomeres. It was fully rescued by loss of p21(CDKN1A) function in late‐generation TERC?/?CDKN1A?/? mice, indicating p21 as the necessary signal transducer between DNA damage response and senescence‐like phenotype in neurons, as in senescing fibroblasts and other proliferation‐competent cells. We conclude that a senescence‐like phenotype is possibly not restricted to proliferation‐competent cells. Rather, dysfunctional telomeres and/or accumulated DNA damage can induce a DNA damage response leading to a phenotype in postmitotic neurons that resembles cell senescence in multiple features. Senescence‐like neurons might be a source of oxidative and inflammatory stress and a contributor to brain aging.     

A newly adapted pulsed-field gel electrophoresis technique allows to detect distinct types of DNA damage at low frequencies in human dermal fibroblasts upon exposure to non-toxic H2O2 concentrations

Reactive oxygen species (ROS) comprise several oxygen containing compounds, among them hydrogen peroxide (H₂O₂), which are generated by internal and external sources and play pleiotropic roles in physiological and pathological states. Skin cells as well as cells from other tissues have developed antioxidant defense mechanisms to protect themselves from high concentrations of ROS. Although biological and pathological roles of ROS have previously been elucidated, so far only limited knowledge exists regarding ROS-mediated generation of DNA breaks and base lesions occurring at low frequency in intact skin cells. This study was therefore designed to probe a newly adapted pulsed-field gel electrophoresis technique for the adequate measurement of high molecular weight DNA fragments as well as to investigate the protective role of the antioxidant enzyme catalase against H₂O₂-mediated damage in human dermal fibroblasts. We stably transfected and overexpressed the full-length catalase cDNA in the human dermal fibroblast cell line 1306 in culture and found that these cells are significantly more protected from cytotoxicity, overall DNA strand breaks, and 8-oxodeoxyguanine base lesions resulting from H₂O₂-triggered oxidative stress compared to vector-transfected 1306 cells or secondary dermal fibroblasts. This work has outlined the importance of catalase in the protection from H₂O₂-mediated cytotoxicity and DNA damage which — if unbalanced — even when occurring at low frequency are known to lead to genomic instability, a hallmark in carcinogenesis and premature aging.     

A plant type 2 metallothionein (MT) from cork tissue responds to oxidative stress

Expression of plant metallothionein genes has been reported in a variety of senescing tissues, such as leaves and stems, ripening fruits, and wounded tissues, and has been proposed to function in both metal chaperoning and scavenging of reactive oxygen species. In this work, it is shown that MT is also associated with suberization, after identifying a gene actively transcribed in Quercus suber cork cells as a novel MT. This cDNA, isolated from a phellem cDNA library, encodes a MT that belongs to type 2 plant MTs (QsMT). Expression of the QsMT cDNA in E. coli grown in media supplemented with Zn, Cd, or Cu has yielded recombinant QsMT. Characterization of the respective metal aggregates agrees well with a copper-related biological role, consistent with the capacity of QsMT to restore copper tolerance to a MT-deficient, copper-sensitive yeast mutant. Furthermore, in situ hybridization results demonstrate that RNA expression of QsMT is mainly observed under conditions related to oxidative stress, either endogenous, as found in cork or in actively proliferating tissues, or exogenous, for example, in response to H(2)O(2) or paraquat treatments. The putative role of QsMT in oxidative stress, both as a free radical scavenger via its sulphydryl groups or as a copper chelator is discussed.     

p21 maintains senescent cell viability under persistent DNA damage response by restraining JNK and caspase signaling

Cellular senescence is a permanent state of cell cycle arrest that protects the organism from tumorigenesis and regulates tissue integrity upon damage and during tissue remodeling. However, accumulation of senescent cells in tissues during aging contributes to age‐related pathologies. A deeper understanding of the mechanisms regulating the viability of senescent cells is therefore required. Here, we show that the CDK inhibitor p21 (CDKN1A) maintains the viability of DNA damage‐induced senescent cells. Upon p21 knockdown, senescent cells acquired multiple DNA lesions that activated ataxia telangiectasia mutated (ATM) and nuclear factor (NF)‐κB kinase, leading to decreased cell survival. NF‐κB activation induced TNF‐α secretion and JNK activation to mediate death of senescent cells in a caspase‐ and JNK‐dependent manner. Notably, p21 knockout in mice eliminated liver senescent stellate cells and alleviated liver fibrosis and collagen production. These findings define a novel pathway that regulates senescent cell viability and fibrosis.     

Endogenously generated DNA nucleobase modifications source,and significance as possible biomarkers of malignant transformation risk,and role in anticancer therapy

The DNA of all living cells undergoes continuous structural and chemical alteration, which may be derived from exogenous sources, or endogenous, metabolic pathways, such as cellular respiration, replication and DNA demethylation. It has been estimated that approximately 70,000 DNA lesions may be generated per day in a single cell, and this has been linked to a wide variety of diseases, including cancer. However, it is puzzling why potentially mutagenic DNA modifications, occurring at a similar level in different organs/tissue, may lead to organ/tissue specific cancers, or indeed non-malignant disease – what is the basis for this differential response? We suggest that it is perhaps the precise location of damage, within the genome, that is a key factor. Finally, we draw attention to the requirement for reliable methods for identification and quantification of DNA adducts/modifications, and stress the need for these assays to be fully validated. Once these prerequisites are satisfied, measurement of DNA modifications may be helpful as a clinical parameter for treatment monitoring, risk group identification and development of prevention strategies.     

Relationship between antioxidant potential and oxidative damage to lipids, proteins and DNA in aged rats

Oxidative stress has been implicated to play a major role in aging and age-related diseases. In the present study, we investigated the effects of aging on the total antioxidant capacity, uric acid, lipid peroxidation, total sulfhydryl group content and damage to DNA in adult (6 months), old (15 months) and senescent (26 months) male Wistar rats. The antioxidant capacity, determined by phycoerythrin-based TRAP method (total peroxyl radical-trapping potential) was significantly decreased in the plasma and myocardium of old and senescent rats, whereas plasma level of uric acid was elevated in 26-month-old rats. Age-related decline in plasma and heart antioxidant capacity was accompanied by a significant loss in total sulfhydryl group content, increased lipid peroxidation and higher DNA damage in lymphocytes. Correlations between TRAP and oxidative damage to lipids, proteins and DNA suggest that the decline in antioxidant status may play an important role in age-related accumulation of cell damage caused by reactive oxygen species.     

Aging alters the force-frequency relationship and toxicity of oxidative stress in rabbit heart

Adult (6 months) and senescent (greater than 5 years) rabbit atria were studied under conditions known to increase cytoplasmic calcium (increased frequency of contraction and oxidative stress). At a contraction frequency of 1/sec, cardiac relaxation (90% relaxation time) was similar in senescent and adult atria but at a frequency of 2 or 3/sec, relaxation was significantly slower in senescent preparations (P less than 0.05). Additional experiments indicated that H2O2 (500 microM), a powerful oxidant, increased resting force and decreased developed force (DF) much more rapidly in senescent than adult atria; the maximum decrease in DF, however, was less in senescent preparations (adult = 81 +/- 6% and senescent = 42 +/- 27% of pre-H2O2 values; P less than 0.05). Age-related differences in effects of H2O2 did not result simply from a decreased ability of senescent hearts to detoxify an oxidative stress by the glutathione pathway. Both basal glutathione (GSH) concentrations and the H2O2-mediated decreases in GSH were similar in adult and senescent ventricular preparations, as were activities of glutathione peroxidase and glutathione reductase. These observations suggest that interventions known to increase cytoplasmic calcium can amplify age-related impairments of cardiac relaxation through mechanisms that may be independent of the glutathione pathway.     

Environmental-induced oxidative stress in neurodegenerative disorders and aging

The aetiology of most neurodegenerative disorders is multifactorial and consists of an interaction between environmental factors and genetic predisposition. Free radicals derived primarily from molecular oxygen have been implicated and considered as associated risk factors for a variety of human disorders including neurodegenerative diseases and aging. Damage to tissue biomolecules, including lipids, proteins and DNA, by free radicals is postulated to contribute importantly to the pathophysiology of oxidative stress. The potential of environmental exposure to metals, air pollution and pesticides as well as diet as risk factors via the induction of oxidative stress for neurodegenerative diseases and aging is discussed. The role of genetic background is discussed on the light of the oxidative stress implication, focusing on both complex neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis) and monogenic neurological disorders (Huntington's disease, Ataxia telangiectasia, Friedreich Ataxia and others). Emphasis is given to role of the repair mechanisms of oxidative DNA damage in delaying aging and protecting against neurodegeneration. The emerging interplay between environmental-induced oxidative stress and epigenetic modifications of critical genes for neurodegeneration is also discussed.     

Mitochondrial DNA repair of oxidative damage in mammalian cells

Nuclear and mitochondrial DNA are constantly being exposed to damaging agents, from endogenous and exogenous sources. In particular, reactive oxygen species (ROS) are formed at high levels as by-products of the normal metabolism. Upon oxidative attack of DNA many DNA lesions are formed and oxidized bases are generated with high frequency. Mitochondrial DNA has been shown to accumulate high levels of 8-hydroxy-2'-deoxyguanosine, the product of hydroxylation of guanine at carbon 8, which is a mutagenic lesion. Most of these small base modifications are repaired by the base excision repair (BER) pathway. Despite the initial concept that mitochondria lack DNA repair, experimental evidences now show that mitochondria are very proficient in BER of oxidative DNA damage, and proteins necessary for this pathway have been isolated from mammalian mitochondria. Here, we examine the BER pathway with an emphasis on mtDNA repair. The molecular mechanisms involved in the formation and removal of oxidative damage from mitochondria are discussed. The pivotal role of the OGG1 glycosylase in removal of oxidized guanines from mtDNA will also be examined. Lastly, changes in mtDNA repair during the aging process and possible biological implications are discussed.     

Emerging role of NF-κB signaling in the induction of senescence-associated secretory phenotype (SASP)

The major hallmark of cellular senescence is an irreversible cell cycle arrest and thus it is a potent tumor suppressor mechanism. Genotoxic insults, e.g. oxidative stress, are important inducers of the senescent phenotype which is characterized by an accumulation of senescence-associated heterochromatic foci (SAHF) and DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS). Interestingly, senescent cells secrete pro-inflammatory factors and thus the condition has been called the senescence-associated secretory phenotype (SASP). Emerging data has revealed that NF-κB signaling is the major signaling pathway which stimulates the appearance of SASP. It is known that DNA damage provokes NF-κB signaling via a variety of signaling complexes containing NEMO protein, an NF-κB essential modifier, as well as via the activation of signaling pathways of p38MAPK and RIG-1, retinoic acid inducible gene-1. Genomic instability evoked by cellular stress triggers epigenetic changes, e.g. release of HMGB1 proteins which are also potent enhancers of inflammatory responses. Moreover, environmental stress and chronic inflammation can stimulate p38MAPK and ceramide signaling and induce cellular senescence with pro-inflammatory responses. On the other hand, two cyclin-dependent kinase inhibitors, p16INK4a and p14ARF, are effective inhibitors of NF-κB signaling. We will review in detail the signaling pathways which activate NF-κB signaling and trigger SASP in senescent cells.     

Increased Arf/p53 activity in stem cells,aging and cancer

Arf/p53 pathway protects the cells against DNA damage induced by acute stress. This characteristic is the responsible for its tumor suppressor activity. Moreover, it regulates the chronic type of stress associated with aging. This is the basis of its anti‐aging activity. Indeed, increased gene dosage of Arf/p53 displays elongated longevity and delayed aging. At a cellular level, it has been recently shown that increased dosage of Arf/p53 delays age‐associated stem cell exhaustion and the subsequent decline in tissue homeostasis and regeneration. However, p53 can also promote aging if constitutively activated. In this context, p53 reduces tissue regeneration, which correlates with premature exhaustion of stem cells. We discuss here the current evidence linking the Arf/p53 pathway to the processes of aging and cancer through stem cell regulation.     

Mitochondrial DNA damage induces apoptosis in senescent cells

Senescence is a cellular response to damage and stress. The senescence response prevents cancer by suppressing the proliferation of cells with a compromised genome and contributes to optimal wound healing in normal tissues. Persistent senescent cells are also thought to drive aging and age-associated pathologies through their secretion of inflammatory factors that modify the tissue microenvironment and alter the function of nearby normal or transformed cells. Understanding how senescent cells alter the microenvironment would be aided by the ability to induce or eliminate senescent cells at will in vivo. Here, we combine the use of the synthetic nucleoside analog ganciclovir (GCV) with herpes simplex virus thymidine kinase (HSVtk) activity to create or eliminate senescent human cells. We show that low concentrations of GCV induce senescence through the accumulation of nuclear DNA damage while higher concentrations of GCV, similar to those used in vivo, kill non-dividing senescent cells via mitochondrial DNA (mtDNA) damage and caspase-dependent apoptosis. Using this system, we effectively eliminated xenografted normal human senescent fibroblasts or induced senescence in human breast cancer cells in vivo. Thus, cellular senescence and mtDNA damage are outcomes of synthetic nucleoside analog treatment, indicating that the GCV–HSVtk combination can be used effectively to promote the targeted formation or eradication of senescent cells.     

Emerging roles of extracellular vesicles in cellular senescence and aging

Cellular senescence is a cellular program that prevents the proliferation of cells at risk of neoplastic transformation. On the other hand, age‐related accumulation of senescent cells promotes aging at least partially due to the senescence‐associated secretory phenotype, whereby cells secrete high levels of inflammatory cytokines, chemokines, and matrix metalloproteinases. Emerging evidence, however, indicates that extracellular vesicles (EVs) are important mediators of the effects of senescent cells on their microenvironment. Senescent cells secrete more EphA2 and DNA via EVs, which can promote cancer cell proliferation and inflammation, respectively. Extracellular vesicles secreted from DNA‐damaged cells can also affect telomere regulation. Furthermore, it has now become clear that EVs actually play important roles in many aspects of aging. This review is intended to summarize these recent progresses, with emphasis on relationships between cellular senescence and EVs.