A progressive reduction in autophagic capacity contributes to induction of replicative senescence in Hs68 cells

Autophagy has been implicated in delayed aging and extended longevity. Here, we aimed to study the possible effects of autophagy during the progression of replicative senescence, which is one of the major features of aging. Human foreskin fibroblasts, Hs68 cells, at an initial passage of 15 were serially cultured for several months until they reached cellular senescence. A decrease in cell proliferation was observed during the progression of senescence. Induction of replicative senescence in aged cells (at passage 40) was confirmed by senescence-associated β-galactosidase (SA-β-gal) activity that represents a sensitive and reliable marker for quantifying senescent cells. We detected a significantly increased percentage (%) of SA-β-gal-positive cells at passage 40 (63%) when compared with the younger SA-β-gal-positive cells at passage 15 (0.5%). Notably, the gradual decrease in basal autophagy coincided with replicative senescence induction. However, despite decreased basal autophagic activity in senescent cells, autophagy inducers could induce autophagy in senescent cells. RT-PCR analysis of 11 autophagy-related genes revealed that the decreased basal autophagy in senescent cells might be due to the downregulation of autophagy-regulatory proteins, but not autophagy machinery components. Moreover, the senescence phenotype was not induced in the cells in which rapamycin was added to the culture to continuously induce autophagy from passage 29 until passage 40. Together, our findings suggest that reduced basal autophagy levels due to downregulation of autophagy-regulatory proteins may be the mechanism underlying replicative senescence in Hs68 cells.     

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.     

α-Fucosidase as a novel convenient biomarker for cellular senescence

Due to its role in aging and antitumor defense, cellular senescence has recently attracted increasing interest. However, there is currently no single specific marker that can unequivocally detect senescent cells. Here, we identified α-L-fucosidase (α-Fuc) as a novel sensitive biomarker for cellular senescence. Regardless of the stress stimulus and cell type, α-Fuc activity was induced in all canonical types of cellular senescence, including replicative, DNA damage- and oncogene-induced senescence. Strikingly, in most models the degree of α-Fuc upregulation was higher than the induction of senescence-associated β-galactosidase (SA-β-Gal), the current gold standard for senescence detection. As α-Fuc is convenient and easy to measure, we suggest its utility as a valuable marker, in particular in cells with low SA-β-Gal activity.     

Murine CD8+ T cells but not macrophages express the vitamin D 1α-hydroxylase

The active form of vitamin D, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] is synthesized by the 1α-hydroxylase, which is encoded by the Cyp27B1 gene. Using transgenic mice that have replaced the Cyp27B1 gene with the bacterial lacZ reporter gene (β-galactosidase), the inflammatory conditions that induce Cyp27B1 in the immune system were probed. A variety of stimuli including lipopolysaccharide, anti-CD3 or PMA/ionomycin were used to stimulate splenocytes and bone marrow derived macrophage in vitro. Only anti-CD3 stimulation resulted in a low induction of β-galactosidase activity in the spleen, indicating that T cells might be a source of Cyp27B1. In vivo, challenge with lipopolysaccharide, α-galactosylceramide, and Listeria monocytogenes failed to induce β-galactosidase activity outside of the kidneys. During more prolonged and severe inflammation there was staining in both the lungs and the gastrointestinal tract for β-galactosidase. Furthermore, wild-type reconstitution of the hematopoietic cell population in Cyp27B1 KO mice protected the mice from experimental colitis. T cell production of Cyp27B1 activity was shown to be from the CD8+ but not the CD4+ T cell population. CD8+ T cells expressed the reporter gene only after 48 h of stimulation. The data is consistent with a model where CD8+ T cells are activated to produce Cyp27B1 and 1,25(OH)₂D₃ that serves to turn off the local immune response.     

Simvastatin rises reactive oxygen species levels and induces senescence in human melanoma cells by activation of p53/p21 pathway

Recent studies demonstrated that simvastatin has antitumor properties in several types of cancer cells, mainly by inducing apoptosis and inhibiting growth. The arrest of proliferation is a feature of cellular senescence; however, the occurrence of senescence in melanoma cells upon simvastatin treatment has not been investigated until now. Our results demonstrated that exposure of human metastatic melanoma cells (WM9) to simvastatin induces a senescent phenotype, characterized by G1 arrest, positive staining for senescence-associated β-galactosidase assay, and morphological changes. Also, the main pathways leading to cell senescence were examined in simvastatin-treated human melanoma cells, and the expression levels of phospho-p53 and p21 were upregulated by simvastatin, suggesting that cell cycle regulators and DNA damage pathways are involved in the onset of senescence. Since simvastatin can act as a pro-oxidant agent, and oxidative stress may be related to senescence, we measured the intracellular ROS levels in WM9 cells upon simvastatin treatment. Interestingly, we found an increased amount of intracellular ROS in these cells, which was accompanied by elevated expression of catalase and peroxiredoxin-1. Collectively, our results demonstrated that simvastatin can induce senescence in human melanoma cells by activation of p53/p21 pathway, and that oxidative stress may be related to this process.     

PFG acted as an inducer of premature senescence in TIG-1 normal diploid fibroblast and an inhibitor of mitosis in the HeLa cells

Our previous work has reported an anti-proliferative compound from moutan cortex, paeoniflorigenone which can induce cancer-selective apoptosis. However, its anti-proliferative mechanism is still unknown. According to morphology changes (hypertrophy and flattening), we hypothesized that PFG can induce senescence or inhibit cell mitosis. Here we show that PFG can induce cellular senescence, evidenced by the expression of senescence-associated β-galactosidase, G0/G1 cell cycle arrest and permanent loss of proliferative ability, in normal TIG-1 diploid fibroblast but not cancerous HeLa cells. In cancerous HeLa cells, PFG inhibited proliferation by inducing S and G2/M cell cycle arrest and mitosis inhibition. DNA damage response was activated by PFG, interestingly the reactive oxygen species level was suppressed instead of escalated. To sum up, we report 3 new roles of PFG as, 1. inducer of premature senescence in normal TIG-1 cells, 2. inhibitor of mitosis in cancerous HeLa cells, 3. ROS scavenger.

Abbreviations: PFG: Paeoniflorigenone; ROS: reactive oxygen species; ATM: ataxia telangiectasia mutated; t-BHP: tert-butyl hydroperoxide; SA-β-gal: senescence-associatedβ-galactosidase; DNA-PK_cs: DNA-dependent protein kinase; γ-H2AX: H2AX phosphoryla-tion at Ser-139     

MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation

Keratinocyte replicative senescence has an important role in time-dependent changes of the epidermis, a tissue with high turnover. Senescence encompasses growth arrest during which cells remain metabolically active but acquire a typical enlarged, vacuolar and flattened morphology. It is also accompanied by the expression of endogenous senescence-associated-β-galactosidase and specific gene expression profiles. MicroRNAs levels have been shown to be modulated during keratinocytes senescence, playing key roles in inhibiting proliferation and in the acquisition of senescent markers. Here, we identify miR-191 as an anti-proliferative and replicative senescence-associated miRNA in primary human keratinocytes. Its overexpression is sufficient per se to induce senescence, as evaluated by induction of several senescence-associated markers. We show that SATB1 and CDK6 3'UTRs are two miR-191 direct targets involved in this pathway. Cdk6 and Satb1 protein levels decrease during keratinocytes replicative senescence and their silencing by siRNA is able to induce a G1 block in cell cycle, accompanied by an increase in senescence-associated markers.     

Upregulation of the gene encoding a cytoplasmic dynein intermediate chain in senescent human cells

Normal human somatic cells, unlike cancer cells, stop dividing after a limited number of cell divisions through the process termed cellular senescence or replicative senescence, which functions as a tumor-suppressive mechanism and may be related to organismal aging. By means of the cDNA subtractive hybridization, we identified eight genes upregulated during normal chromosome 3-induced cellular senescence in a human renal cell carcinoma cell line. Among them is the DNCI1 gene encoding an intermediate chain 1 of the cytoplasmic dynein, a microtubule motor that plays a role in chromosome movement and organelle transport. The DNCI1 mRNA was also upregulated during in vitro aging of primary human fibroblasts. In contrast, other components of cytoplasmic dynein showed no significant change in mRNA expression during cellular aging. Cell growth arrest by serum starvation, contact inhibition, or gamma-irradiation did not induce the DNCI1 mRNA, suggesting its specific role in cellular senescence. The DNCI1 gene is on the long arm of chromosome 7 where tumor suppressor genes and a senescence-inducing gene for a group of immortal cell lines (complementation group D) are mapped. This is the first report that links a component of molecular motor complex to cellular senescence, providing a new insight into molecular mechanisms of cellular senescence.     

Cytochemical Detection of a Senescence-Associated β-Galactosidase in Endothelial and Smooth Muscle Cells from Human and Rabbit Blood Vessels

A β-galactosidase activity has recently been used as a histochemical marker of replicative senescence in human fibroblasts and keratinocytes. To establish whether this marker could be used to detect senescence of vascular cells, we have investigated its presence in cultures of serially passaged human umbilical vein endothelial cells and rabbit aortic smooth muscle cells. β-Galactosidase activity was detected by light microscopy using the chromogenic substrate 5-bromo-4-chloro-3-indolyl β- -galactopyranoside. In endothelial cell cultures, lysosomal β-galactosidase activity, which is detected at pH 4.0, was present in all cells regardless of their replicative age. In contrast, senescence-associated β-galactosidase activity, which is detected at pH 6.0, was absent in the majority of cells in early passage cultures (<15 cumulative population doublings), but was present in a large proportion of cells (up to 62%) in late passage cultures (>30 cumulative population doublings); in intermediate passage cultures (15–30 cumulative population doublings) it was found in fewer than 15% of the cells. The increase in the percentage of senescence-associated β-galactosidase-positive cells correlated with a decrease in the cell density at confluence and with a marked increase in cell size. Counterstaining with an antibody directed against the endothelial cell marker CD31 showed that senescent cells retained the expression of this antigen. Senescence-associated β-galactosidase was also detected in serially passaged, but not in primary explant cultures of rabbit aortic vascular smooth muscle cells. The presence of senescence-associated β-galactosidase in cultured vascular smooth muscle cells and endothelial cells suggests that this marker could be used to study the role of cellular senescence in vascular disease.     

Effects of p21 deletion in mouse models of premature aging

An approach to investigate the role of cellular senescence in organismal aging has been to abrogate signaling pathways known to induce cellular senescence and to assess the effects in mouse models of premature aging. Recently, we reported the effect of loss of function of p21, a gene implicated in p53-induced cellular senescence, in the background of the Ku80-/- premature aging mouse (Zhao et al., EMBO reports, 2009). Here, we provide an overview of the effects of p21 deletion in different models of premature aging.     

Cell surface sphingolipid glycohydrolases in neuronal differentiation and aging in culture

Qualitative and quantitative changes in glycosphingolipids, together with changes in the expression of the corresponding glycosyltransferases, have been reported along neuronal differentiation and aging. Plasma membrane (PM) glycosphingolipid pattern and content are the result of a complex network of metabolic pathways, including those potentially involving the activity of PM glycohydrolases. We analyzed the total cell activities of sialyltransferase I, II and IV, sialidase, β-galactosidase and β-glucosidase, and the PM-associated activities of sialidase Neu3, β-galactosidase, Conduritol B Epoxide-sensitive β-glucosidase and β-glucosidase GBA2 in rat cerebellar granule cells along differentiation and aging in culture. Sialyltransferase activities increased during cell differentiation, in agreement with the known increase of the total ganglioside content during neuronal maturation. The remodeling of ganglioside pattern could be because of the augmented activities of total sialidase and, within PM, to the action of the cell surface associated sialidase Neu3. Sialidase activities remained high during aging, in agreement with the known progressive ganglioside reduction in brain senescence. As PM β-galactosidase and β-glucosidase activities and parallely ceramide levels markedly increased along in vitro aging, PM ceramide production in neurons might be because of local catabolism of glycosphingolipids and not only to that of sphingomyelin, as already reported in human fibroblasts.     

Induction of apoptosis in human replicative senescent fibroblasts

Cellular senescence is an irreversible growth phase characteristic of normal cells. We have found that human senescent fibroblasts can be induced to undergo programmed cell death (apoptosis) by ceramide, TNF-alpha, or okadaic acid. The most profound effects were induced by TNF-alpha and okadaic acid treatment. In the present study, we also evaluated the contribution of lysosomal activation as a possible mechanism underlying the induction of apoptosis. Four lysosomal enzyme activities were measured: beta-galactosidase, alpha-galactosidase A, beta-glucoronidase, and acid phosphatase. Using an in situ assay, we have found that the activity of beta-galactosidase, which is also a biochemical marker of senescence, is induced in young proliferating fibroblasts following exposure to all three apoptotic inducing agents. The other enzymes were not significantly induced in young fibroblasts following exposure to agents that induce apoptosis. During replicative senescence, three of the four lysosomal enzymes tested (beta-galactosidase, alpha-galactosidase A, and beta-glucoronidase) are constitutively expressed at high levels. TNF-alpha was the only agent that induced lysosomal activity in senescent fibroblasts, of which only alpha-galactosidase A activity was induced. Our studies show that senescent fibroblasts can be induced to undergo apoptosis in a signal-dependent manner. However, the lysosomal enzymes examined do not appear to be correlated with apoptotic induction.     

Exploiting tumor cell senescence in anticancer therapy

Cellular senescence is a physiological process of irreversible cell-cycle arrest that contributes to various physiological and pathological processes of aging. Whereas replicative senescence is associated with telomere attrition after repeated cell division, stress-induced premature senescence occurs in response to aberrant oncogenic signaling, oxidative stress, and DNA damage which is independent of telomere dysfunction. Recent evidence indicates that cellular senescence provides a barrier to tumorigenesis and is a determinant of the outcome of cancer treatment. However, the senescence-associated secretory phenotype, which contributes to multiple facets of senescent cancer cells, may influence both cancer-inhibitory and cancer-promoting mechanisms of neighboring cells. Conventional treatments, such as chemo- and radiotherapies, preferentially induce premature senescence instead of apoptosis in the appropriate cellular context. In addition, treatment-induced premature senescence could compensate for resistance to apoptosis via alternative signaling pathways. Therefore, we believe that an intensive effort to understand cancer cell senescence could facilitate the development of novel therapeutic strategies for improving the efficacy of anticancer therapies. This review summarizes the current understanding of molecular mechanisms, functions, and clinical applications of cellular senescence for anticancer therapy. [BMB Reports 2014; 47(2): 51-59]     

Geroconversion: irreversible step to cellular senescence

Cellular senescence happens in 2 steps: cell cycle arrest followed, or sometimes preceded, by gerogenic conversion (geroconversion). Geroconvesrion is a form of growth, a futile growth during cell cycle arrest. It converts reversible arrest to irreversible senescence. Geroconversion is driven by growth-promoting, mitogen-/nutrient-sensing pathways such as mTOR. Geroconversion leads to hyper-secretory, hypertrophic and pro-inflammatory cellular phenotypes, hyperfunctions and malfunctions. On organismal level, geroconversion leads to age-related diseases and death. Rapamycin, a gerosuppressant, extends life span in diverse species from yeast to mammals. Stress–and oncogene-induced accelerated senescence, replicative senescence in vitro and life-long cellular aging in vivo all can be described by 2-step model.     

58-kDa microspherule protein (MSP58) is novel Brahma-related gene 1 (BRG1)-associated protein that modulates p53/p21 senescence pathway

The nucleolar 58-kDa microspherule protein (MSP58) protein is a candidate oncogene implicated in modulating cellular proliferation and malignant transformation. In this study, we show that knocking down MSP58 expression caused aneuploidy and led to apoptosis, whereas ectopic expression of MSP58 regulated cell proliferation in a context-dependent manner. Specifically, ectopic expression of MSP58 in normal human IMR90 and Hs68 diploid fibroblasts, the H184B5F5/M10 mammary epithelial cell line, HT1080 fibrosarcoma cells, primary mouse embryonic fibroblasts, and immortalized NIH3T3 fibroblasts resulted in induction of premature senescence, an enlarged and flattened cellular morphology, and increased senescence-associated β-galactosidase activity. MSP58-driven senescence was strictly dependent on the presence of functional p53 as revealed by the fact that normal cells with p53 knockdown by specific shRNA or cells with a mutated or functionally impaired p53 pathway were effective in bypassing MSP58-induced senescence. At least two senescence mechanisms are induced by MSP58. First, MSP58 activates the DNA damage response and p53/p21 signaling pathways. Second, MSP58, p53, and the SWI/SNF chromatin-remodeling subunit Brahma-related gene 1 (BRG1) form a ternary complex on the p21 promoter and collaborate to activate p21. Additionally, MSP58 protein levels increased in cells undergoing replicative senescence and stress-induced senescence. Notably, the results of analyzing expression levels of MSP58 between tumors and matched normal tissues showed significant changes (both up- and down-regulation) in its expression in various types of tumors. Our findings highlight new aspects of MSP58 in modulating cellular senescence and suggest that MSP58 has both oncogenic and tumor-suppressive properties.     

尖顶羊肚菌胞外多糖提取物对皮肤成纤维细胞增殖和衰老的影响

不同浓度尖顶羊肚菌胞外多糖提取物作用人皮肤成纤维细胞（human skin fibroblasts,HSF）,检测对HSF细胞形态、细胞增殖、衰老相关β-半乳糖苷酶活性、羟脯氨酸含量的影响,探究尖顶羊肚菌胞外多糖提取物对HSF增殖和衰老的影响。结果显示,125μg/mL尖顶羊肚菌胞外多糖提取物使HSF细胞活力增加了25.2%,羟脯氨酸含量增加了12.1%,β-半乳糖苷酶活性降低了48.1%。说明适宜浓度尖顶羊肚菌胞外多糖提取物具有促进HSF细胞增殖、胶原蛋白合成,延缓细胞衰老的作用。     

Cyclin-Dependent Kinase 5 Activity Enhances Monocytic Phenotype and Cell Cycle Traverse in 1,25-Dihydroxyvitamin D3-Treated HL60 Cells

The function of most cyclin-dependent kinases (Cdks) is to facilitate progression through the checkpoints of the cell cycle, but Cdk5 is known to be involved in differentiation of CNS, muscle, and lens cells, though not in the cell cycle traverse. Here we show an additional role for Cdk5, an enhancement of monocytic differentiation with abrogation of the G1 checkpoint. Human leukemia HL60 cells exposed to 1alpha,25-dihydroxyvitamin D3 (1,25D3) displayed monocytic phenotype and increased Cdk5 kinase activity. An analog of 1,25D3 which does not induce differentiation failed to upregulate Cdk5, and 1,25D3-resistant cells had reduced Cdk5 activity. Active or inactive Cdk5 was associated with cyclin D1, but only active Cdk5 exhibited threonine phosphorylation. Inhibition of Cdk5 expression by an antisense construct reduced the intensity of 1, 25D3-induced expression of CD14, a marker of monocytes, and increased the 1,25D3-induced G1 block. These findings demonstrate a novel aspect of Cdk5 activity-facilitation of the G1- to S-phase transition in cells which are approaching replicative quiescence and a concomitant enhancement of monocytic differentiation.