ING1a expression increases during replicative senescence and induces a senescent phenotype

The ING family of tumor suppressor proteins affects cell growth, apoptosis and response to DNA damage by modulating chromatin structure through association with different HAT and HDAC complexes. The major splicing isoforms of the ING1 locus are ING1a and INGlb. While INGlb plays a role in inducing apoptosis, the function of ING1a is currently unknown. Here we show that alternative splicing of the ING1 message alters the INGla:INGlb ratio by approximately 30-fold in senescent compared to low passage primary fibroblasts. INGla antagonizes INGlb function in apoptosis, induces the formation of structures resembling senescence-associated heterochromatic foci containing heterochromatin protein 1 gamma, the accumulation of senescence-associated beta-galactosidase activity and promotes senescent cell morphology and cell cycle arrest. Phenotypic effects may result from differential effects on gene expression since ING1a increases levels of both retinoblastoma and the p16 cyclin-dependent kinase inhibitor and ING1a and ING1b have opposite effects on the expression of proliferating nuclear cell antigen (PCNA), which is required for cell growth. Gene expression appears to be altered by targeting of HDAC complexes to gene promoters since INGla associates with several-fold higher levels of HDAC1 in senescent, compared to replication-competent cells and ING1 is found on the PCNA promoter by chromatin immunoprecipitation analysis. These data demonstrate a novel role for the ING1 proteins in differentially regulating senescence-associated chromatin remodeling vs. apoptosis and support the idea that altered ratios of the ING1 splicing isoforms may contribute to establishing the senescent phenotype through HDAC and HAT complex-mediated effects on chromatin structure.     

The p33ING1b tumor suppressor cooperates with p53 to induce apoptosis in response to etoposide in human osteosarcoma cells

p33ING1b induces cell cycle arrest and stimulates DNA repair, apoptosis and chemosensitivity. The magnitude of some p33ING1b effects may be due to activation of the tumor suppressor p53. To investigate if the p33ING1b protein affected chemosensitivity of osteosarcoma cells, we overexpressed p33ING1b in p53+/+ U2OS cells or in p53-mutant MG63 cells, and then assessed for growth arrest and apoptosis after treatment with etoposide. p33ING1b increased etoposide-induced growth inhibition and apoptosis to a much greater degree in p53+/+ U2OS cells than in p53-mutant MG63 cells. Moreover, ectopic expression of p33ING1b markedly upregulated p53, p21WAF1 and bax protein levels and activated caspase-3 protein kinase in etoposide-treated U2OS cells. Together, our data indicate that p33ING1b prominently enhances etoposide-induced apoptosis through p53-dependent pathways in human osteosarcoma cells. p33ING1b may be an important marker and/or therapeutic target in the prevention and treatment of metastatic osteosarcoma.     

miR-138转录可下调p33ING1在衰老细胞中的表达

p33ING1参与了多种生物学过程,包括细胞生长抑制、凋亡、DNA损伤修复、染色质重塑等．近来研究显示,p33在细胞衰老过程中表达降低,这可能与衰老细胞的抗凋亡有关．但p33在衰老细胞中表达下调的分子机理仍不清楚．我们发现,在衰老细胞中miR-138表达升高与p33基因的表达降低密切相关．以下实验结果支持如此结论：(1)与年轻细胞相比,带p33ING1 3′UTR 报告载体荧光素酶活性在衰老细胞中降低;突变3′UTR上的miR-138结合位点可升高报告载体荧光素酶在衰老细胞中的活性;(2)在衰老细胞中miR-138的表达升高;(3)在年轻细胞中,过表达miR-138不仅可抑制带p33ING1 3′UTR 报告载体荧光素酶活性,而且下调细胞内p33ING1基因mRNA和蛋白水平.与此相反,抑制miR-138活性可升高带p33ING1 3′UTR 报告载体荧光素酶活性,并且上调细胞内p33ING1基因mRNA和蛋白水平．这些结果表明,p33ING1基因是miR-138的靶基因;在衰老过程中,miR-138表达升高, 由此导致该基因的表达降低．     

Wig1 prevents cellular senescence by regulating p21 mRNA decay through control of RISC recruitment

Premature senescence, a key strategy used to suppress carcinogenesis, can be driven by p53/p21 proteins in response to various stresses. Here, we demonstrate that Wig1 plays a critical role in this process through regulation of p21 mRNA stability. Wig1 controls the association of Argonaute2 (Ago2), a central component of the RNA‐induced silencing complex (RISC), with target p21 mRNA via binding of the stem‐loop structure near the microRNA (miRNA) target site. Depletion of Wig1 prohibited miRNA‐mediated p21 mRNA decay and resulted in premature senescence. Wig1 plays an essential role in cell proliferation, as demonstrated in tumour xenografts in mice, and Wig1 and p21 mRNA levels are inversely correlated in human normal and cancer tissues. Together, our data indicate a novel role of Wig1 in RISC target accessibility, which is a key step in RNA‐mediated gene silencing. In addition, these findings indicate that fine‐tuning of p21 levels by Wig1 is essential for the prevention of cellular senescence.     

Wild-type p53-induced phosphatase 1 (Wip1) forestalls cellular premature senescence at physiological oxygen levels by regulating DNA damage response signaling during DNA replication

Wip1 (protein phosphatase Mg²⁺/Mn²⁺-dependent 1D, Ppm1d) is a nuclear serine/threonine protein phosphatase that is induced by p53 following the activation of DNA damage response (DDR) signaling. Ppm1d^−/− mouse embryonic fibroblasts (MEFs) exhibit premature senescence under conventional culture conditions; however, little is known regarding the role of Wip1 in regulating cellular senescence. In this study, we found that even at a representative physiological concentration of 3% O₂, Ppm1d^−/− MEFs underwent premature cellular senescence that depended on the functional activation of p53. Interestingly, Ppm1d^−/− MEFs showed increased H2AX phosphorylation levels without increased levels of reactive oxygen species (ROS) or DNA base damage compared with wild-type (Wt) MEFs, suggesting a decreased threshold for DDR activation or sustained DDR activation during recovery. Notably, the increased H2AX phosphorylation levels observed in Ppm1d^−/− MEFs were primarily associated with S-phase cells and predominantly dependent on the activation of ATM. Moreover, these same phenotypes were observed when Wt and Ppm1d^−/− MEFs were either transiently or chronically exposed to low levels of agents that induce replication-mediated double-stranded breaks. These findings suggest that Wip1 prevents the induction of cellular senescence at physiological oxygen levels by attenuating DDR signaling in response to endogenous double-stranded breaks that form during DNA replication.     

Cellular senescence induced by p53-ras cooperation is independent of p21waf1 in murine embryo fibroblasts

Oncogenic activation in primary murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the p53 tumor suppressor pathway. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras with p53 produced an irreversible cell cycle arrest that displayed features of cellular senescence. Introduction of a conditional murine p53 allele (p53val135) into double p53/p21-null mouse embryonic fibroblasts showed that p21waf1 was not required for this effect, since p53-/-;p21-/- double-null cells undergo terminal growth arrest with features of senescence following coexpression of oncogenic Ras and p53. Our results indicate that oncogenic activation of the Ras pathway in murine fibroblasts converts p53 into a senescence inducer through a p21waf1-independent mechanism.     

The ING1b tumor suppressor facilitates nucleotide excision repair by promoting chromatin accessibility to XPA

ING1b is the most studied ING family protein and perhaps the most ubiquitously and abundantly expressed. This protein is involved in the regulation of various biological functions ranging from senescence, cell cycle arrest, apoptosis, to DNA repair. ING1b is upregulated by UV irradiation and enhances the removal of bulky nucleic acid photoproducts. In this study, we provide evidence that ING1b mediates nucleotide excision repair by facilitating the access to damaged nucleosomal DNA. We demonstrate that ING1b is not recruited to UV-induced DNA lesions but enhances nucleotide excision repair only in XPC-proficient cells, implying an essential role in early steps of the 'access, repair, restore' model. We also find that ING1b alters histone acetylation dynamics upon exposure to UV radiation and induces chromatin relaxation in microccocal nuclease digestion assay, revealing that ING1b may allow better access to nucleotide excision repair machinery. More importantly, ING1b associates with chromatin in a UV-inducible manner and facilitates DNA access to nucleotide excision repair factor XPA. Furthermore, depletion of the endogenous ING1b results to the sensitization of cells at S-phase to UV irradiation. Taken together, these observations establish a role of ING1b acting as a chromatin accessibility factor for DNA damage recognition proteins upon genotoxic injury.     

The PPARγ‐SETD8 axis constitutes an epigenetic,p53‐independent checkpoint on p21‐mediated cellular senescence

Cellular senescence is a permanent proliferative arrest triggered by genome instability or aberrant growth stresses, acting as a protective or even tumor‐suppressive mechanism. While several key aspects of gene regulation have been known to program this cessation of cell growth, the involvement of the epigenetic regulation has just emerged but remains largely unresolved. Using a systems approach that is based on targeted gene profiling, we uncovered known and novel chromatin modifiers with putative link to the senescent state of the cells. Among these, we identified SETD8 as a new target as well as a key regulator of the cellular senescence signaling. Knockdown of SETD8 triggered senescence induction in proliferative culture, irrespectively of the p53 status of the cells; ectopic expression of this epigenetic writer alleviated the extent doxorubicin‐induced cellular senescence. This repressive effect of SETD8 in senescence was mediated by directly maintaining the silencing mark H4K20me1 at the locus of the senescence switch gene p21. Further in support of this regulatory link, depletion of p21 reversed this SETD8‐mediated cellular senescence. Additionally, we found that PPARγ acts upstream and regulates SETD8 expression in proliferating cells. Downregulation of PPARγ coincided with the senescence induction, while its activation inhibited the progression of this process. Viewed together, our findings delineated a new epigenetic pathway through which the PPARγ‐SETD8 axis directly silences p21 expression and consequently impinges on its senescence‐inducing function. This implies that SETD8 may be part of a cell proliferation checkpoint mechanism and has important implications in antitumor therapeutics.     

过表达人p33ING1b促进UV诱导的衰老细胞凋亡

p33 ^ING1b是肿瘤抑制基因ING1的主要表达形式,已有的研究表明,p33^ING1b参与了细胞的生长抑制、凋亡、染色质重塑、DNA损伤修复、肿瘤抑制等.但是,它在细胞衰老过程中的作用目前还不清楚.本研究分析了p33 ^ING1b基因在细胞衰老过程中的表达情况.结果发现,无论在mRNA水平还是在蛋白水平,p33 ^ING1b在衰老细胞中的表达均降低.通过构建和包装含p33^ING1b基因的重组腺病毒,将p33 ^ING1b导入衰老细胞中使其过表达,结果显示,p33^ING1b的过表达明显促进UV诱导的衰老细胞凋亡,提示p33I^NG1b在衰老细胞中的表达下调与衰老细胞抗凋亡有关.     

Insulin‐like growth factor‐1 regulates the SIRT1‐p53 pathway in cellular senescence

Cellular senescence, which is known to halt proliferation of aged and stressed cells, plays a key role against cancer development and is also closely associated with organismal aging. While increased insulin‐like growth factor (IGF) signaling induces cell proliferation, survival and cancer progression, disrupted IGF signaling is known to enhance longevity concomitantly with delay in aging processes. The molecular mechanisms involved in the regulation of aging by IGF signaling and whether IGF regulates cellular senescence are still poorly understood. In this study, we demonstrate that IGF‐1 exerts a dual function in promoting cell proliferation as well as cellular senescence. While acute IGF‐1 exposure promotes cell proliferation and is opposed by p53, prolonged IGF‐1 treatment induces premature cellular senescence in a p53‐dependent manner. We show that prolonged IGF‐1 treatment inhibits SIRT1 deacetylase activity, resulting in increased p53 acetylation as well as p53 stabilization and activation, thus leading to premature cellular senescence. In addition, either expression of SIRT1 or inhibition of p53 prevented IGF‐1‐induced premature cellular senescence. Together, these findings suggest that p53 acts as a molecular switch in monitoring IGF‐1‐induced proliferation and premature senescence, and suggest a possible molecular connection involving IGF‐1‐SIRT1‐p53 signaling in cellular senescence and aging.     

The p53/miRNAs/Ccna2 pathway serves as a novel regulator of cellular senescence: Complement of the canonical p53/p21 pathway

Aging is a multifactorial process characterized by the progressive deterioration of physiological functions. Among the multiple molecular mechanisms, microRNAs (miRNAs) have increasingly been implicated in the regulation of Aging process. However, the contribution of miRNAs to physiological Aging and the underlying mechanisms remain elusive. We herein performed high‐throughput analysis using miRNA and mRNA microarray in the physiological Aging mouse, attempted to deepen into the understanding of the effects of miRNAs on Aging process at the “network” level. The data showed that various p53 responsive miRNAs, including miR‐124, miR‐34a and miR‐29a/b/c, were up‐regulated in Aging mouse compared with that in Young mouse. Further investigation unraveled that similar as miR‐34a and miR‐29, miR‐124 significantly promoted cellular senescence. As expected, mRNA microarray and gene co‐expression network analysis unveiled that the most down‐regulated mRNAs were enriched in the regulatory pathways of cell proliferation. Fascinatingly, among these down‐regulated mRNAs, Ccna2 stood out as a common target of several p53 responsive miRNAs (miR‐124 and miR‐29), which functioned as the antagonist of p21 in cell cycle regulation. Silencing of Ccna2 remarkably triggered the cellular senescence, while Ccna2 overexpression delayed cellular senescence and significantly reversed the senescence‐induction effect of miR‐124 and miR‐29. Moreover, these p53 responsive miRNAs were significantly up‐regulated during the senescence process of p21‐deficient cells; overexpression of p53 responsive miRNAs or knockdown of Ccna2 evidently accelerated the cellular senescence in the absence of p21. Taken together, our data suggested that the p53/miRNAs/Ccna2 pathway might serve as a novel senescence modulator independent of p53/p21 pathway.     

The PCNA-associated factor KIAA0101/p15(PAF) binds the potential tumor suppressor product p33ING1b

The KIAA0101/p15(PAF)/OEATC-1 protein was initially isolated in a yeast two-hybrid screen for proliferating cell nuclear antigen (PCNA) binding partners, and was shown to bind PCNA competitively with the cell cycle regulator p21(WAF). PCNA is involved in DNA replication and damage repair. Using polyclonal antisera raised against a p15(PAF) fusion protein, we have shown that in a range of mammalian tumor and non-tumor cell lines the endogenous p15(PAF) protein localises to the nucleus and the mitochondria. Under normal conditions no co-localisation with PCNA could be detected, however following exposure to UV it was possible to co-immunoprecipitate p15(PAF) and PCNA from a number of cell lines, suggesting a UV-enhanced association of the two proteins. Overexpression of p15(PAF) in mammalian cells was also found to protect cells from UV-induced cell death. Based on similarities between the behaviour of p15(PAF) and the potential tumor suppressor product p33ING1b, we have further shown that these two proteins interact in the same complex in cell cultures. This suggests that p15(PAF) forms part of a larger protein complex potentially involved in the regulation of DNA repair, apoptosis and cell cycle progression.     

Bmal1-deficient mouse fibroblast cells do not provide premature cellular senescence in vitro

Bmal1 is a core circadian clock gene. Bmal1^?/? mice show disruption of the clock and premature aging phenotypes with a short lifespan. However, little is known whether disruption of Bmal1 leads to premature aging at cellular level. Here, we established primary mouse embryonic fibroblast (MEF) cells derived from Bmal1^?/? mice and investigated its effects on cellular senescence. Unexpectedly, Bmal1^?/? primary MEFs that showed disrupted circadian oscillation underwent neither premature replicative nor stress-induced cellular senescence. Our results therefore uncover that Bmal1 is not required for in vitro cellular senescence, suggesting that circadian clock does not control in vitro cellular senescence.     

腺病毒介导的p33ING1b过表达显著促进衰老细胞的DNA损伤修复

p33^ING1b是一个较晚发现的肿瘤抑制基因ING1的主要表达形式,自从被成功克隆以后得到了广泛的研究,已有的研究表明,p33^ING1b参与了细胞的生长抑制、凋亡、染色质重塑、DNA损伤修复、肿瘤抑制和细胞衰老等。但是它在细胞衰老过程中的作用特别是对衰老细胞DNA损伤修复的影响还没有被地阐明,在本研究中,我们首先用2BS细胞构建了细胞衰老模型,通过RT-PCR和Western blot技术证实p33^ING1b在衰老细胞中的表达水平是下调的,然后通过构建和包装包含p33^ING1b基因的腺病毒,将p33^ING1b导入年轻和衰老细胞中并使其过表达,用HCR（host cell reactivation）方法检测年轻细胞和衰老细胞DNA损伤修复能力。我们的实验首次表明,相对于年轻细胞,p33^ING1b的过表达使衰老细胞的DNA的损伤修复能力显著增加,这说明p33^ING1b在衰老细胞中的表达下调与衰老细胞DNA损伤修复能力的下降有关,也进一步证实了p33^ING1b在细胞衰老过程中起着十分重要的作用。     

Cell senescence contributes to tissue regeneration in zebrafish

Cellular senescence is a stress response that limits the proliferation of damaged cells by establishing a permanent cell cycle arrest. Different stimuli can trigger senescence but excessive production or impaired clearance of these cells can lead to their accumulation during aging with deleterious effects. Despite this potential negative side of cell senescence, its physiological role as a pro‐regenerative and morphogenetic force has emerged recently after the identification of programmed cell senescence during embryogenesis and during wound healing and limb regeneration. Here, we explored the conservation of tissue injury‐induced senescence in a model of complex regeneration, the zebrafish. Fin amputation in adult fish led to the appearance of senescent cells at the site of damage, and their removal impaired tissue regeneration. Despite many conceptual similarities, this tissue repair response is different from developmental senescence. Our results lend support to the notion that cell senescence is a positive response promoting tissue repair and homeostasis.     

The replicometer is broken: telomeres activate cellular senescence in response to genotoxic stresses

Telomeres, the ends of our linear chromosomes, can function as ‘replicometers’, capable of counting cell division cycles as they progressively erode with every round of DNA replication. Once they are critically short, telomeres become dysfunctional and consequently activate a proliferative arrest called replicative senescence. For many years, telomeres were thought to be autonomous structures, largely isolated from cell intrinsic and extrinsic signals, whose function is to prevent limitless cellular proliferation, a characteristic of most cancer cells. It is becoming increasingly evident, however, that telomeres not only count cell divisions, but also function as sensors of genotoxic stresses to stop cell cycle progression prematurely and long before cells would have entered replicative senescence. This stable growth arrest, triggered by dysfunctional telomeres that are not necessarily critically short, likely evolved as a tumor‐suppressing mechanism as it prevents proliferation of cells that are at risk for acquiring potentially hazardous and transforming mutations both in vitro and in vivo. Here, we review studies supporting the concept that telomeres are important cellular structures whose function not only is to count cell divisions, but also to act as molecular switches that can rapidly stop cell cycle progression permanently in response to a variety of stresses, including oncogenic signals.