抑制性消减杂交筛选细胞衰老相关基因

目的 研究人胚肺二倍体成纤维细胞2BS在衰老过程中的基因表达变化。方法 对年轻2BS细胞[低于30PD(population doubling)]和衰老2BS细胞(高于55PD)样品进行双向抑制性消减杂交，并筛选分析。结果 构建了2个抑制性消减文库，分别代表年轻细胞和衰老细胞高表达基因，点杂交筛选获得30个差异表达片断(差异比例大于2)包含多种细胞功能的变化。部分差异基因表达在新生儿脐带血和老年人外周血样中差异也有显著性。结论首次报道了RBM4、FBXO7、TOM1等基因在2BS细胞衰老时的表达变化，并表明体外培养细胞与个体的衰老变化过程有一定相似性。     

NKG2D ligands mediate immunosurveillance of senescent cells

Cellular senescence is a stress response mechanism that limits tumorigenesis and tissue damage. Induction of cellular senescence commonly coincides with an immunogenic phenotype that promotes self-elimination by components of the immune system, thereby facilitating tumor suppression and limiting excess fibrosis during wound repair. The mechanisms by which senescent cells regulate their immune surveillance are not completely understood. Here we show that ligands of an activating Natural Killer (NK) cell receptor (NKG2D), MICA and ULBP2 are consistently up-regulated following induction of replicative senescence, oncogene-induced senescence and DNA damage - induced senescence. MICA and ULBP2 proteins are necessary for efficient NK-mediated cytotoxicity towards senescent fibroblasts. The mechanisms regulating the initial expression of NKG2D ligands in senescent cells are dependent on a DNA damage response, whilst continuous expression of these ligands is regulated by the ERK signaling pathway. In liver fibrosis, the accumulation of senescent activated stellate cells is increased in mice lacking NKG2D receptor leading to increased fibrosis. Overall, our results provide new insights into the mechanisms regulating the expression of immune ligands in senescent cells and reveal the importance of NKG2D receptor-ligand interaction in protecting against liver fibrosis.     

Endothelial cellular senescence is inhibited by nitric oxide: implications in atherosclerosis associated with menopause and diabetes

Senescence may contribute to the pathogenesis of atherosclerosis. Although the bioavailability of nitric oxide (NO) is limited in senescence, the effect of NO on senescence and its relationship to cardiovascular risk factors have not been investigated fully. We studied these factors by investigating senescence-associated beta-galactosidase (SA-beta-gal) and human telomerase activity in human umbilical venous endothelial cells (HUVECs). Treatment with NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-aminoethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) and transfection with endothelial NO synthase (eNOS) into HUVECs each decreased the number of SA-beta-gal positive cells and increased telomerase activity. The NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) abolished the effect of eNOS transfection. The physiological concentration of 17beta-estradiol activated hTERT, decreased SA-beta-gal-positive cells, and caused cell proliferation. However, ICI 182780, an estrogen receptor-specific antagonist, and L-NAME each inhibited these effects. Finally, we investigated the effect of NO bioavailability on high glucose-promoted cellular senescence of HUVECs. Inhibition by eNOS transfection of this cellular senescence under high glucose conditions was less pronounced. Treatment with L-arginine or L-citrulline of eNOS-transfected cells partially inhibited, and combination of L-arginine and L-citrulline with antioxidants strongly prevented, high glucose-induced cellular senescence. These data demonstrate that NO can prevent endothelial senescence, thereby contributing to the anti-senile action of estrogen. The ingestion of NO-boosting substances, including L-arginine, L-citrulline, and antioxidants, can delay endothelial senescence under high glucose. We suggest that the delay in endothelial senescence through NO and/or eNOS activation may have clinical utility in the treatment of atherosclerosis in the elderly.     

Hypoxia-reoxygenation induces premature senescence in FA bone marrow hematopoietic cells

Hematopoietic cells are often exposed to transient hypoxia and reoxygenation as they develop and migrate. Given that bone marrow (BM) failure occurred in patients with Fanconi anemia (FA), we reason that hypoxia-then-reoxygenation represents a physiologically relevant stress for FA hematopoietic progenitor/stem cells. Here we show that expansion of Fancc-/- BM cells enriched for progenitor and stem cells was significantly decreased after 2 continuous cycles of hyperoxic-hypoxic-hyperoxic treatments compared with wild-type (WT) BM cells. This inhibition was attributable to a marked decrease of lineage-depleted (Lin-) ScaI- c-kit+ cells and more primitive Lin- ScaI+ c-kit+ cells in Fancc-/- BM cells following reoxygenation. Evaluation of the cell-cycle profile of long-term BM culture (LTBMC) revealed that a vast majority (70.6%) of reoxygenated Fancc-/- LTBMC cells was residing in the G0 and G1 phases compared with 55.8% in WT LTBMC cells. Fancc-/- LTBMC cells stained intensely for SA-beta-galactosidase activity, a biomarker for senescence; this was associated with increased expression of senescence-associated proteins p53 and p21(WAF1/CIP1). Taken together, these results suggest that reoxygenation induces premature senescence in Fancc-/- BM hematopoietic cells by signaling through p53, up-regulating p21, and causing senescent cell-cycle arrest. Thus, reoxygenation-induced premature senescence may be a novel mechanism underlying hematopoietic cell depletion and BM failure in FA.     

Stress chaperones, mortalin, and pex19p mediate 5-aza-2' deoxycytidine-induced senescence of cancer cells by DNA methylation-independent pathway

DNA demethylating agents are used to reverse epigenetic silencing of tumor suppressors in cancer therapeutics. Understanding of the molecular and cellular factors involved in DNA demethylation-induced gene desilencing and senescence is still limited. We have tested the involvement of two stress chaperones, Pex19p and mortalin, in 5-Aza-2' deoxycytidine (5AZA-dC; DNA demethylating agent)-induced senescence. We found that the cells overexpressing these chaperones were highly sensitive to 5AZA-dC, and their partial silencing eliminated 5AZA-dC-induced senescence in human osteosarcoma cells. We demonstrate that these chaperones modulate the demethylation and chromatin remodeling-dependent (as accessed by p16(INK4A) expression) and remodeling-independent (such as activation of tumor suppressor p53 pathway) senescence response of cells. Furthermore, we found the direct interactions of 5AZA-dC with these chaperones that may alter their functions. We conclude that both mortalin and Pex19p are important mediators, prognostic indicators, and tailoring tools for 5AZA-dC-induced senescence in cancer cells.     

Replicatively senescent cells are arrested in G1 and G2 phases

Most human somatic cells do not divide indefinitely but enter a terminal growth arrest termed replicative senescence. Replicatively senescent cells are generally believed to arrest in G1 or G0 stage of the cell cycle. While doing cell cycle analysis on three different lines of normal human fibroblasts we observed that 36-60% of the replicatively senescent cells had 4N DNA content. Only up to 5% of senescent cells had more than one nucleus ruling out the possibility that the 4N cell population were G1-arrested bi-nucleated cells. Furthermore, it is unlikely that the 4N cells are tetraploids, because actively dividing pre-senescent cultures lacked the 8N tetraploid G2 population. Collectively these results suggest that the 4N population consists of G2 arrested cells. The notion that a large fraction of senescent cell population is arrested in G2 is important for understanding the biology of replicative senescence.     

Pathological aging of the vascular endothelium: are endothelial progenitor cells the sentinels of the cardiovascular system?

Aging is associated with vascular endothelial dysfunction, which ultimately leads to atherosclerosis. On the other hand, it is clear that in young patients with risk factors for cardiovascular diseases (CVD), endothelial dysfunction is an early marker of the ongoing atherogenic process. It is therefore tempting to speculate that risk factors for CVD accelerate the aging process. The aging of an endothelial cell (EC) is not chronological but rather dependent on its replication rate. ECs have a finite number of divisions and enter replicative senescence after exhaustion of this potential. Telomere attrition is believed to be responsible for this phenomenon. Upon reaching a critical minimal telomere length, ECs enter a nondividing state of replicative senescence. Recently, endothelial progenitor cells originating from the bone marrow have been isolated from the circulation. They integrate into the endothelial layer of the vessel and contribute to healing, ischemic repair and angiogenesis. A completely new field of investigation is now open. Are endothelial progenitor cells sensitive to the aging process? Do they prevent endothelial dysfunction? Are they the ultimate shield against the damages induced by risk factors for CVD? There are no definite answers to these questions, but the potential of these cells is tremendous and understanding their physiology is essential.     

Molecular mechanisms of replicative senescence in endothelial cells

As human somatic cells age, they stop replicating and enter an irreversible state of growth arrest known as replicative senescence. Senescent cells are viable, metabolically active, and display altered gene and protein expression compared to proliferating cells. Endothelial cells, both in vitro and in vivo, are known to undergo senescence. As endothelial cells are a critical component of the vasculature, senescence of these cells can have a significant impact of vascular integrity, function, and overall homeostasis. This review will summarize recent work to understand the molecular mechanisms of endothelial cell senescence and the resulting alterations in gene/protein expression in these cells. Endothelial cell senescence will then be discussed in the context of disease development with a focus on atherosclerosis, an important age-associated disease of the vasculature.     

Does hereditary metabolic disease modulate senescence and ageing?

Hereditary metabolic diseases in the context of evolutionary biology elicit interesting questions about ageing and senescence: Will persons successfully treated for inborn errors of metabolism, age and die prematurely because of compromised longevity? Because some unhealthy longevity has its origins in germline and somatic mutational processes, and in an inability to withstand metabolic stress, are there lessons to be learned about senescence from hereditary metabolic disease? Why are ageing, senescence and death necessary for Homo sapiens and how do they happen? These questions form the theme upon which several variations are played during the course of this essay. The theory of the disposable soma recognizes genomic and environmental events, well-seasoned by Chance, as determinants of ageing and senescence. Together, they cause the somatic damage that results in death. Genomics will reveal genes involved in longevity, both healthy and unhealthy. There will be schedules of gene expression behind our life-history traits. As in the field of hereditary metabolic disease, analogous genetic enquiries about ageing can be formulated. For example, how will heterozygotes age? Will association studies in centenarians reveal 'longevity genes'? Will disparate longevity in sib pairs reveal genetic factors? If there are 'ageing' mutations, of what types and with what effects? Will these initiatives lead to healthier longevity? A deeper question yet remains: why has human biology invested so greatly in grandparenthood?     

Estimation of replicative senescence via a population dynamics model of cells in culture

A simple mathematical model is developed for determining the time-varying fraction of senescent cells in culture in terms of the underlying probability distribution of the number of population doublings until senescence. This functional relationship is inverted, which allows for the estimation of the probability distribution of the number of population doublings until senescence given experimental data on the time-varying fraction of senescent cells. The relationship - in particular, the lag - between these two quantities is analyzed under the assumption that the number of population doublings until senescence follows the Weibull distribution. If the number of population doublings until senescence is geometrically distributed (i.e. the Weibull with shape parameter equal to one) then the cell culture appears immortal.     

Down-regulation of Dicer1 promotes cellular senescence and decreases the differentiation and stem cell-supporting capacities of mesenchymal stromal cells in patients with myelodysplastic syndrome

Although it has been reported that mesenchymal stromal cells are unable to provide sufficient hematopoietic support in myelodysplastic syndrome, the underlying mechanisms remain elusive. In this study, we found that mesenchymal stromal cells from patients with myelodysplastic syndrome displayed a significant increase in senescence, as evidenced by their decreased proliferative capacity, flattened morphology and increased expression of SA-β-gal and p21. Senescent mesenchymal stromal cells from patients had decreased differentiation potential and decreased stem cell support capacity. Gene knockdown of Dicer1, which was down-regulated in mesenchymal stromal cells from patients, induced senescence. The differentiation and stem cell-supporting capacities were significantly inhibited by Dicer1 knockdown. Overexpression of Dicer1 in mesenchymal stromal cells from patients reversed cellular senescence and enhanced stem cell properties. Furthermore, we identified reduced expression in the microRNA-17 family (miR-17-5p, miR-20a/b, miR-106a/b and miR-93) as a potential factor responsible for increased p21 expression, a key senescence mediator, in Dicer1 knockdown cells. Moreover, we found that miR-93 and miR-20a expression levels were significantly reduced in mesenchymal stromal cells from patients and miR-93/miR-20a gain of function resulted in a decrease of cellular senescence. Collectively, the results of our study show that mesenchymal stromal cells from patients with myelodysplastic syndrome are prone to senescence and that Dicer1 down-regulation promotes cellular senescence and decreases the differentiation and stem cell-supporting capacities of mesenchymal stromal cells. Dicer1 down-regulation seems to contribute to the insufficient hematopoietic support capacities of mesenchymal stromal cells from patients with myelodysplastic syndrome.     

p63-microRNA feedback in keratinocyte senescence

We investigated the expression of microRNAs (miRNAs) associated with replicative senescence in human primary keratinocytes. A cohort of miRNAs up-regulated in senescence was identified by genome-wide miRNA profiling, and their change in expression was validated in proliferative versus senescent cells. Among these, miRNA (miR)-138, -181a, -181b, and -130b expression increased with serial passages. miR-138, -181a, and -181b, but not miR-130b, overexpression in proliferating cells was sufficient per se to induce senescence, as evaluated by inhibition of BrdU incorporation and quantification of senescence-activated β-galactosidase staining. We identified Sirt1 as a direct target of miR-138, -181a, and -181b, whereas ΔNp63 expression was inhibited by miR-130b. We also found that ΔNp63α inhibits miR-138, -181a, -181b, and -130b expression by binding directly to p63-responsive elements located in close proximity to the genomic loci of these miRNAs in primary keratinocytes. These findings suggest that changes in miRNA expression, by modulating the levels of regulatory proteins such as p63 and Sirt1, strongly contribute to induction of senescence in primary human keratinocytes, thus linking these two proteins. Our data also indicate that suppression of miR-138, -181a, -181b, and -130b expression is part of a growth-promoting strategy of ΔNp63α in epidermal proliferating cells.     

Premature aging/senescence in cancer cells facing therapy: good or bad?

Normal and cancer cells facing their demise following exposure to radio-chemotherapy can actively participate in choosing their subsequent fate. These programmed cell fate decisions include true cell death (apoptosis-necroptosis) and therapy-induced cellular senescence (TIS), a permanent “proliferative arrest” commonly portrayed as premature cellular aging. Despite a permanent loss of proliferative potential, senescent cells remain viable and are highly bioactive at the microenvironment level, resulting in a prolonged impact on tissue architecture and functions. Cellular senescence is primarily documented as a tumor suppression mechanism that prevents cellular transformation. In the context of normal tissues, cellular senescence also plays important roles in tissue repair, but contributes to age-associated tissue dysfunction when senescent cells accumulate. Theoretically, in multi-step cancer progression models, cancer cells have already bypassed cellular senescence during their immortalization step (see hallmarks of cancer). It is then perhaps surprising to find that cancer cells often retain the ability to undergo TIS, or premature aging. This occurs because cellular senescence results from multiple signalling pathways, some retained in cancer cells, aiming to prevent cell cycle progression in damaged cells. Since senescent cancer cells persist after therapy and secrete an array of cytokines and growth factors that can modulate the tumor microenvironment, these cells may have beneficial and detrimental effects regarding immune modulation and survival of remaining proliferation-competent cancer cells. Similarly, while normal cells undergoing senescence are believed to remain indefinitely growth arrested, whether this is true for senescent cancer cells remains unclear, raising the possibility that these cells may represent a reservoir for cancer recurrence after treatment. This review discusses our current knowledge on cancer cell senescence and highlight questions that must be addressed to fully understand the beneficial and detrimental impacts of cellular senescence during cancer therapy.     

The Role of Cellular Senescence in the Gastrointestinal Mucosa

Cellular senescence is a biologically irreversible state of cell-growth arrest that occurs following either a replicative or an oncogenic stimulus. This phenomenon occurs as a response to the presence of premalignant cells and appears to be an important anticancer mechanism that keeps these transformed cells at bay. Many exogenous and endogenous triggers for senescence have been recognized to act via genomic or epigenomic pathways. The most common stimulus for senescence is progressive loss of telomeric DNA, which results in the loss of chromosomal stability and eventual unregulated growth and malignancy. Senescence is activated through an interaction between the p16 and p53 tumor-suppressor genes. Senescent cells can be identified in vitro because they express senescence-associated β-galactosidase, a marker of increased lysosomal activity. Cellular senescence plays an integral role in the prevention and development of both benign and malignant gastrointestinal diseases. The senescence cascade and the cell-cycle checkpoints that dictate the progression and maintenance of senescence are important in all types of gastrointestinal cancers, including pancreatic, liver, gastric, colon, and esophageal cancers. Understanding the pathogenic mechanisms involved in cellular senescence is important for the development of agents targeted toward the treatment of gastrointestinal tumors.     

Resistance to discodermolide, a microtubule-stabilizing agent and senescence inducer, is 4E-BP1-dependent

Discodermolide is a microtubule-stabilizing agent that induces accelerated cell senescence. A discodermolide-resistant cell line, AD32, was generated from the human lung cancer cell line A549. We hypothesize that the major resistance mechanism in these cells is escape from accelerated senescence. AD32 cells have decreased levels of 4E-BP1 mRNA and protein, relative to the parental discodermolide-sensitive A549 cells. Lentiviral-mediated re-expression of wild-type 4E-BP1 in AD32 cells increased the proliferation rate and reverted resistance to discodermolide via restoration of discodermolide-induced accelerated senescence. Consistent with this, cell growth and response to discodermolide was confirmed in vivo using tumor xenograft models. Furthermore, reintroduction of a nonphosphorylatable mutant (Thr-37/46 Ala) of 4E-BP1 was able to partially restore sensitivity and enhance proliferation in AD32 cells, suggesting that these effects are independent of phosphorylation by mTORC1. Microarray profiling of AD32-resistant cells versus sensitive A549 cells, and subsequent unbiased gene ontology analysis, identified molecular pathways and functional groupings of differentially expressed mRNAs implicated in overcoming discodermolide-induced senescence. The most statistically significant classes of differentially expressed genes included p53 signaling, G2/M checkpoint regulation, and genes involved in the role of BRCA1 in the DNA damage response. Consistent with this, p53 protein expression was up-regulated and had increased nuclear localization in AD32 cells relative to parental A549 cells. Furthermore, the stability of p53 was enhanced in AD32 cells. Our studies propose a role for 4E-BP1 as a regulator of discodermolide-induced accelerated senescence.