Aging of the cells: Insight into cellular senescence and detection Methods

Cellular theory of aging states that human aging is the result of cellular aging, in which an increasing proportion of cells reach senescence. Senescence, from the Latin word senex, means “growing old,” is an irreversible growth arrest which occurs in response to damaging stimuli, such as DNA damage, telomere shortening, telomere dysfunction and oncogenic stress leading to suppression of potentially dysfunctional, transformed, or aged cells. Cellular senescence is characterized by irreversible cell cycle arrest, flattened and enlarged morphology, resistance to apoptosis, alteration in gene expression and chromatin structure, expression of senescence associated- β-galactosidase (SA-β-gal) and acquisition of senescence associated secretory phenotype (SASP). In this review paper, different types of cellular senescence including replicative senescence (RS) which occurs due to telomere shortening and stress induced premature senescence (SIPS) which occurs in response to different types of stress in cells, are discussed. Biomarkers of cellular senescence and senescent assays including BrdU incorporation assay, senescence associated- β-galactosidase (SA-β-gal) and senescence-associated heterochromatin foci assays to detect senescent cells are also addressed.     

Uncoupling the senescent phenotype from telomere shortening in hydrogen peroxide-treated fibroblasts

Normal human cells have a limited replicative potential and inevitably reach replicative senescence in culture. Replicatively senescent cells show multiple molecular changes, some of which are related to the irreversible growth arrest in culture, whereas others resemble the changes occurring during the process of aging in vivo. Telomeres shorten as a result of cell replication and are thought to serve as a replicometer for senescence. Recent studies show that young cells can be induced to develop features of senescence prematurely by damaging agents, chromatin remodeling, and overexpression of ras or the E2F1 gene. Accelerated telomere shortening is thought to be a mechanism of premature senescence in some models. In this work, we test whether the acquisition of a senescent phenotype after mild-dose hydrogen peroxide (H(2)O(2)) exposure requires telomere shortening. Treating young HDFs with 150 microM H(2)O(2) once or 75 microM H(2)O(2) twice in 2 weeks causes long-term growth arrest, an enlarged morphology, activation of senescence-associated beta-galactosidase, and elevated expression of collagenase and clusterin mRNAs. No significant telomere shortening was observed with H(2)O(2) at doses ranging from 50 to 200 microM. Weekly treatment with 75 microM H(2)O(2) also failed to induce significant telomere shortening. Failure of telomere shortening correlated with an inability to elevate p16 protein or mRNA in H(2)O(2)-treated cells. In contrast, p21 mRNA was elevated over 40-fold and remained at this level for at least 2 weeks after a pulse treatment of H(2)O(2). The role of cell cycle checkpoints centered on p21 in premature senescence induced by H(2)O(2) is discussed here.     

Telomerase expression prevents replicative senescence but does not fully reset mRNA expression patterns in Werner syndrome cell strains.

Reduced replicative capacity is a consistent characteristic of cells derived from patients with Werner syndrome. This premature senescence is phenotypically similar to replicative senescence observed in normal cell strains and includes altered cell morphology and gene expression patterns. Telomeres shorten with in vitro passaging of both WRN and normal cell strains; however, the rate of shortening has been reported to be faster in WRN cell strains, and the length of telomeres in senescent WRN cells appears to be longer than that observed in normal strains, leading to the suggestion that senescence in WRN cell strains may not be exclusively associated with telomere effects. We report here that the telomere restriction fragment length in senescent WRN fibroblasts cultures is within the size range observed for normal fibroblasts strains and that the expression of a telomerase transgene in WRN cell strains results in lengthened telomeres and replicative immortalization, thus indicating that telomere effects are the predominant trigger of premature senescence in WRN cells. Microarray analyses showed that mRNA expression patterns induced in senescent WRN cells appeared similar to those in normal strains and that hTERT expression could prevent the induction of most of these genes. However, substantial differences in expression were seen in comparisons of early-passage and telomerase-immortalized derivative lines, indicating that telomerase expression does not prevent the phenotypic drift, or destabilized genotype, resulting from the WRN defect.     

胸苷激酶基因在人二倍体成纤维细胞衰老过程中的表达调控

为研究人胸苷激酶 (humanthymidinekinase ,hTK)基因在复制衰老细胞及早衰细胞中表达下调的分子机制 ,构建了含hTK启动子的荧光素酶报告基因载体 .转染结果显示 ,复制衰老细胞与早衰细胞中hTK启动子的转录活性比年轻细胞中下降了近 3倍 ,表明转录水平的调控是hTK在衰老细胞中表达下降的主要调控机制 .定点突变的结果显示 ,转录因子Sp1、NF Y结合位点的突变可使hTK启动子活性降低近 5 0 % ,而E2F结合位点的突变可使其活性升高 2倍多 ,提示Sp1和NF Y是hTK基因的转录活化因子 ,而E2F为转录抑制因子 .电泳迁移率变更实验发现 ,与年轻细胞相比 ,Sp1、NF Y与hTK启动子的DNA结合活性在复制衰老细胞和早衰细胞中无明显改变 ,提示转录活化因子Sp1、NF Y并非hTK在衰老细胞中下调的主要因素 .染色质免疫共沉淀结果显示 ,在细胞内Rb结合在hTK启动子上 ,且同年轻细胞相比 ,复制衰老细胞及早衰细胞中的hTK启动子结合着更多的Rb ,这提示细胞衰老过程中Rb的去磷酸化可能与hTK基因在衰老过程中的下调有关 .     

Stochastic variation in telomere shortening rate causes heterogeneity of human fibroblast replicative life span

The replicative life span of human fibroblasts is heterogeneous, with a fraction of cells senescing at every population doubling. To find out whether this heterogeneity is due to premature senescence, i.e. driven by a nontelomeric mechanism, fibroblasts with a senescent phenotype were isolated from growing cultures and clones by flow cytometry. These senescent cells had shorter telomeres than their cycling counterparts at all population doubling levels and both in mass cultures and in individual subclones, indicating heterogeneity in the rate of telomere shortening. Ectopic expression of telomerase stabilized telomere length in the majority of cells and rescued them from early senescence, suggesting a causal role of telomere shortening. Under standard cell culture conditions, there was a minor fraction of cells that showed a senescent phenotype and short telomeres despite active telomerase. This fraction increased under chronic mild oxidative stress, which is known to accelerate telomere shortening. It is possible that even high telomerase activity cannot fully compensate for telomere shortening in all cells. The data show that heterogeneity of the human fibroblast replicative life span can be caused by significant stochastic cell-to-cell variation in telomere shortening.     

Decreased tumorigenicity in vivo when transforming growth factor beta treatment causes cancer cell senescence

We have previously reported that transforming growth factor beta (TGF-beta) triggers two independent senescence programs, 1) replicative senescence dependent upon telomere shortening and 2) premature senescence independent of telomere shortening, in the cell line of A549 human lung adenocarcinoma. In this study, we examined the possibility that cancer cell tumor phenotypes could be suppressed by forced senescence. We used A549 cells treated with TGF-beta for a long time (over 50 days), where senescence was induced in a telomere-shortening-dependent or an independent way. Fully senescent A549 cells were elongated, acquired contact inhibition capabilities when reaching confluence, and secreted the senescence-associated cytokine IL-6. Furthermore, senescent A549 cells had no tumorigenicity in nude mice. These results indicate that the forced induction of senescence in cancer cells may be a novel and potentially powerful method for advancing anti-cancer therapy.     

Senescence: a telomeric limit to immortality or a cellular response to physiologic stresses?

Cells entering a state of senescence undergo a irreversible cell cycle arrest, associated by a set of functional and morphological changes. Senescence occurs following telomeres shortening (replicative senescence) or exposure to other acute or chronic physiologic stress signals (a phenomenon termed stasis: stress or aberrant signaling-induced senescence). In this review, I discuss the pathways of cellular senescence, the mechanisms involved and the role that these pathways have in regulating the initiation and progression of cancer. Telomere-initiated senescence or loss of telomere function trigger focal recruitement of protein sensors of the DNA double-strand breaks leading to the activation of the DNA damage checkpoint responses and the tumour suppressor gene product, p53, which in turn induces the cell-cycle inhibitor, p21(WAF1). Loss of p53 and pRb function allows continued cell division despite increasing telomere dysfunction and eventually entry into telomere crisis. Immortalisation is an essential prerequisite for the formation of a tumour cell. Therefore, a developing tumour cell must circumvent at least two proliferative barriers--cellular senescence and crisis--to achieve neoplastic transformation. These barriers are regulated by telomere shortening and by the p16(INK4a)/Rb and p53 tumour suppressor pathways. Elucidation of the genes and emerging knowledge about the regulatory mechanisms that lead to senescence and determine the pattern of gene expression in senescent cells may lead to more effective treatments for cancer.     

Mitogen stimulation cooperates with telomere shortening to activate DNA damage responses and senescence signaling

Replicative senescence is induced by critical telomere shortening and limits the proliferation of primary cells to a finite number of divisions. To characterize the activity status of the replicative senescence program in the context of cell cycle activity, we analyzed the senescence phenotypes and signaling pathways in quiescent and growth-stimulated primary human fibroblasts in vitro and liver cells in vivo. This study shows that replicative senescence signaling operates at a low level in cells with shortened telomeres but becomes fully activated when cells are stimulated to enter the cell cycle. This study also shows that the dysfunctional telomeres and nontelomeric DNA lesions in senescent cells do not elicit a DNA damage signal unless the cells are induced to enter the cell cycle by mitogen stimulation. The amplification of senescence signaling and DNA damage responses by mitogen stimulation in cells with shortened telomeres is mediated in part through the MEK/mitogen-activated protein kinase pathway. These findings have implications for the further understanding of replicative senescence and analysis of its role in vivo.     

Senescent mouse cells fail to overtly regulate the HIRA histone chaperone and do not form robust Senescence Associated Heterochromatin Foci

Background

Cellular senescence is a permanent growth arrest that occurs in response to cellular stressors, such as telomere shortening or activation of oncogenes. Although the process of senescence growth arrest is somewhat conserved between mouse and human cells, there are some critical differences in the molecular pathways of senescence between these two species. Recent studies in human fibroblasts have defined a cell signaling pathway that is initiated by repression of a specific Wnt ligand, Wnt2. This, in turn, activates a histone chaperone HIRA, and culminates in formation of specialized punctate domains of facultative heterochromatin, called Senescence-Associated Heterochromatin Foci (SAHF), that are enriched in the histone variant, macroH2A. SAHF are thought to repress expression of proliferation-promoting genes, thereby contributing to senescence-associated proliferation arrest. We asked whether this Wnt2-HIRA-SAHF pathway is conserved in mouse fibroblasts.

Results

We show that mouse embryo fibroblasts (MEFs) and mouse skin fibroblasts, do not form robust punctate SAHF in response to an activated Ras oncogene or shortened telomeres. However, senescent MEFs do exhibit elevated levels of macroH2A staining throughout the nucleus as a whole. Consistent with their failure to fully activate the SAHF assembly pathway, the Wnt2-HIRA signaling axis is not overtly regulated between proliferating and senescent mouse cells.

Conclusions

In addition to the previously defined differences between mouse and human cells in the mechanisms and phenotypes associated with senescence, we conclude that senescent mouse and human fibroblasts also differ at the level of chromatin and the signaling pathways used to regulate chromatin. These differences between human and mouse senescence may contribute to the increased propensity of mouse fibroblasts (and perhaps other mouse cell types) to become immortalized and transformed, compared to human cells.     

Senescence delay of human diploid fibroblast induced by anti-sense p16INK4a expression

p16(INK4a), a tumor suppressor gene that inhibits cyclin-dependent kinase 4 and cyclin-dependent kinase 6, is also implicated in the mechanisms underlying replicative senescence, because its RNA and protein accumulate as cells approach their finite number of population doublings in tissue culture. To further explore the involvement of p16(INK4a) in replicative senescence, we constructed a retroviral vector containing antisense p16(INK4a), pDOR-ASp16, and introduced it into early passages of human diploid fibroblasts. The introduction of this construct significantly suppressed the expression of wild-type p16(INK4a). It also imposed a finite increase in proliferative life span and significant delay of several other cell senescent features, such as cell flattening, cell cycle arrest, and senescence-associated beta-galactosidase positivity. Moreover, telomere shortening and decline in DNA repair capacity, which normally accompany cell senescence, are also postponed by the ASp16 transfection. The life span of fibroblasts was significantly extended, but the onset of replicative senescence could not be totally prevented. Telomerase could not be activated even though telomere shortening was slowed. These observations suggest that the telomere pathway of senescence cannot be bypassed by ASp16 expression. These data not only strongly support a role for p16(INK4a) in replicative senescence but also raise the possibility of using the antisense p16(INK4a) therapeutically.     

BTG2 antagonizes Pin1 in response to mitogens and telomere disruption during replicative senescence

Cellular senescence limits the replicative capacity of normal cells and acts as an intrinsic barrier that protects against the development of cancer. Telomere shortening–induced replicative senescence is dependent on the ATM‐p53‐p21 pathway but additional genes likely contribute to senescence. Here, we show that the p53‐responsive gene BTG2 plays an essential role in replicative senescence. Similar to p53 and p21 depletion, BTG2 depletion in human fibroblasts leads to an extension of cellular lifespan, and ectopic BTG2 induces senescence independently of p53. The anti‐proliferative function of BTG2 during senescence involves its stabilization in response to telomere dysfunction followed by serum‐dependent binding and relocalization of the cell cycle regulator prolyl isomerase Pin1. Pin1 inhibition leads to senescence in late‐passage cells, and ectopic Pin1 expression rescues cells from BTG2‐induced senescence. The neutralization of Pin1 by BTG2 provides a critical mechanism to maintain senescent arrest in the presence of mitogenic signals in normal primary fibroblasts.     

Microenvironment Determinants of Brain Metastasis

The Inhibitor of Growth (ING) proteins represent a type II tumor suppressor family comprising five conserved genes, ING1 to ING5. While ING1, ING2 and ING3 proteins are stable components of the mSIN3a-HDAC complexes, the association of ING1, ING4 and ING5 with HAT protein complexes was also reported. Among these the ING1 and ING2 have been analyzed more deeply. Similar to other tumor suppressor factors the ING proteins are also involved in many cellular pathways linked to cancer and cell proliferation such as cell cycle regulation, cellular senescence, DNA repair, apoptosis, inhibition of angiogenesis and modulation of chromatin. A common structural feature of ING factors is the conserved plant homeodomain (PHD), which can bind directly to the histone mark trimethylated lysine of histone H3 (H3K4me3). PHD mutants lose the ability to undergo cellular senescence linking chromatin mark recognition with cellular senescence. ING1 and ING2 are localized in the cell nucleus and associated with chromatin modifying enzymes, linking tumor suppression directly to chromatin regulation. In line with this, the expression of ING1 in tumors is aberrant or identified point mutations are mostly localized in the PHD finger and affect histone binding. Interestingly, ING1 protein levels increase in replicative senescent cells, latter representing an efficient pathway to inhibit cancer proliferation. In association with this, suppression of p33ING1 expression prolongs replicative life span and is also sufficient to bypass oncogene-induced senescence. Recent analyses of ING1- and ING2-deficient mice confirm a tumor suppressive role of ING1 and ING2 and also indicate an essential role of ING2 in meiosis. Here we summarize the activity of ING1 and ING2 as tumor suppressors, chromatin factors and in development.     

Recent advances in cellular senescence, cancer and aging

How much do we know about the biology of aging from cell culture studies? Most normal somatic cells have a finite potential to divide due to a process termed cellular or replicative senescence. A growing body of evidence suggests that senescence evolved to protect higher eukaryotes, particularly mammals, from developing cancer. We now know that telomere shortening, due to the biochemistry of DNA replication, induces replicative senescence in human cells. However, in rodent cells, replicative senescence occurs despite very long telomeres. Recent findings suggest that replicative senescence is just the tip of the iceberg of a more general process termed cellular senescence. It appears that cellular senescence is a response to potentially oncogenic insults, including oxidative damage. In young organisms, growth arrest by cell senescence suppresses tumor development, but later in life, due to the accumulation of senescent cells which secret factors that can disrupt tissues during aging, cellular senescence promotes tumorigenesis. Therefore, antagonistic pleiotropy may explain in part, if not in whole, the apparently paradoxical effects of cellular senescence, though this still remains an open question.