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Olivier Toussaint - One of the best experts on this subject based on the ideXlab platform.
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protocols to detect senescence associated beta galactosidase sa βgal activity a biomarker of senescent cells in culture and in vivo
Nature Protocols, 2009Co-Authors: Florence Debacqchainiaux, Jorge D. Erusalimsky, Judith Campisi, Olivier ToussaintAbstract:Normal cells can permanently lose the ability to proliferate when challenged by potentially oncogenic stress, a process termed cellular senescence. Senescence-Associated Beta-Galactosidase (SA-βgal) activity, detectable at pH 6.0, permits the identification of senescent cells in culture and mammalian tissues. Here we describe first a cytochemical protocol suitable for the histochemical detection of individual senescent cells both in culture and tissue biopsies. The second method is based on the alkalinization of lysosomes, followed by the use of 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG), a fluorogenic substrate for βgal activity. The cytochemical method takes about 30 min to execute, and several hours to a day to develop and score. The fluorescence methods take between 4 and 8 h to execute and can be scored in a single day. The cytochemical method is applicable to tissue sections and requires simple reagents and equipment. The fluorescence-based methods have the advantages of being more quantitative and sensitive.
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Protocols to detect Senescence-Associated Beta-Galactosidase (SA-βgal) activity, a biomarker of senescent cells in culture and in vivo
Nature Protocols, 2009Co-Authors: Jorge D. Erusalimsky, Judith Campisi, Olivier ToussaintAbstract:Normal cells can permanently lose the ability to proliferate when challenged by potentially oncogenic stress, a process termed cellular senescence. Senescence-Associated Beta-Galactosidase (SA-betagal) activity, detectable at pH 6.0, permits the identification of senescent cells in culture and mammalian tissues. Here we describe first a cytochemical protocol suitable for the histochemical detection of individual senescent cells both in culture and tissue biopsies. The second method is based on the alkalinization of lysosomes, followed by the use of 5-dodecanoylaminofluorescein di-beta-D-galactopyranoside (C12FDG), a fluorogenic substrate for betagal activity. The cytochemical method takes about 30 min to execute, and several hours to a day to develop and score. The fluorescence methods take between 4 and 8 h to execute and can be scored in a single day. The cytochemical method is applicable to tissue sections and requires simple reagents and equipment. The fluorescence-based methods have the advantages of being more quantitative and sensitive.
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Identification of p53-dependent genes potentially involved in UVB-mediated premature senescence of human skin fibroblasts using siRNA technology.
Mechanisms of ageing and development, 2007Co-Authors: Céline Borlon, Florence Debacq-chainiaux, Sébastien Vankoningsloo, Patrice Godard, Olivier ToussaintAbstract:Premature senescence of skin human diploid fibroblasts is induced after a series of 10 sublethal exposures to UVB at 2.5 kJ/m(2) with appearance of several biomarkers of cellular senescence like Senescence-Associated Beta-Galactosidase activity (SA beta-gal) and cell cycle arrest. Herein it is shown that the induction of UVB-induced premature senescence is associated with a transient increase of protein abundance and DNA-binding activity of p53. Silencing p53 expression with small interfering RNA (siRNA) affected the basal level of SA beta-gal and proliferative potential, but did not prevent UVB-induced increase of SA beta-gal and decrease of DNA synthesis. We used a senescence-specific low-density DNA array and p53 siRNA to study the mRNA abundance of 240 senescence-related genes and identified several potential p53-dependent genes differentially expressed after the repeated exposures to UVB.
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Knocking down p53 with siRNA does not affect the overexpression of p21WAF-1 after exposure of IMR-90 hTERT fibroblasts to a sublethal concentration of H2O2 leading to premature senescence.
Annals of the New York Academy of Sciences, 2007Co-Authors: Stéphanie Zdanov, Florence Debacq-chainiaux, Olivier ToussaintAbstract:Premature senescence of IMR-90 human diploid fibroblasts (HDFs) expressing telomerase was induced by exposure to sublethal concentration of H(2)O(2), with appearance of several biomarkers of cellular senescence like enlarged cell shape, Senescence-Associated Beta-Galactosidase (SA ss-gal) activity, and cell cycle arrest. The induction of stress-induced premature senescence (SIPS) was associated with a transient increase in DNA-binding activity of p53 and an increased expression of p21(WAF-1). p53 small interferent RNA (siRNA) affected the basal level of p21(WAF-1) mRNA but did not affect the overexpression of p21(WAF-1) after stress. This siRNA approach confirms previous results obtained with other methods.
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Identification of potential anti-photoageing algal compounds using an in-vitro model of photoageing
The Journal of pharmacy and pharmacology, 2006Co-Authors: Florence Debacq-chainiaux, José Remacle, Céline Borlon, B. De Hertogh, Pierre Yves Morvan, R. Vallée, Olivier ToussaintAbstract:Stress-induced premature senescence (SIPS) has been proposed as an in-vitro model for testing the long-term effects of stressful events and to find molecules/natural extracts that protect against such stress. Premature senescence of human skin diploid fibroblasts (HDFs) can be induced by repeated subcytotoxic exposure to UVB, with the appearance of so-called biomarkers of senescence such as growth arrest, Senescence-Associated Beta-Galactosidase activity, Senescence-Associated gene over-expression and the common 4977-bp mitochondrial DNA deletion. This model of UVB-induced premature senescence has been acknowledged as a robust in-vitro model in photoageing research. In this study, the potential anti-photoageing effects of a series of algal extracts were tested. The appearance of the biomarkers of UVB-induced premature senescence of HDFs was studied with or without algal extracts. One algal extract was shown to be particularly protective against UVB-induced SIPS. The results obtained here reinforce the notion that UVB-induced premature senescence of HDFs can be used to screen potential anti-photoageing compounds.
Jorge D. Erusalimsky - One of the best experts on this subject based on the ideXlab platform.
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protocols to detect senescence associated beta galactosidase sa βgal activity a biomarker of senescent cells in culture and in vivo
Nature Protocols, 2009Co-Authors: Florence Debacqchainiaux, Jorge D. Erusalimsky, Judith Campisi, Olivier ToussaintAbstract:Normal cells can permanently lose the ability to proliferate when challenged by potentially oncogenic stress, a process termed cellular senescence. Senescence-Associated Beta-Galactosidase (SA-βgal) activity, detectable at pH 6.0, permits the identification of senescent cells in culture and mammalian tissues. Here we describe first a cytochemical protocol suitable for the histochemical detection of individual senescent cells both in culture and tissue biopsies. The second method is based on the alkalinization of lysosomes, followed by the use of 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG), a fluorogenic substrate for βgal activity. The cytochemical method takes about 30 min to execute, and several hours to a day to develop and score. The fluorescence methods take between 4 and 8 h to execute and can be scored in a single day. The cytochemical method is applicable to tissue sections and requires simple reagents and equipment. The fluorescence-based methods have the advantages of being more quantitative and sensitive.
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Protocols to detect Senescence-Associated Beta-Galactosidase (SA-βgal) activity, a biomarker of senescent cells in culture and in vivo
Nature Protocols, 2009Co-Authors: Jorge D. Erusalimsky, Judith Campisi, Olivier ToussaintAbstract:Normal cells can permanently lose the ability to proliferate when challenged by potentially oncogenic stress, a process termed cellular senescence. Senescence-Associated Beta-Galactosidase (SA-betagal) activity, detectable at pH 6.0, permits the identification of senescent cells in culture and mammalian tissues. Here we describe first a cytochemical protocol suitable for the histochemical detection of individual senescent cells both in culture and tissue biopsies. The second method is based on the alkalinization of lysosomes, followed by the use of 5-dodecanoylaminofluorescein di-beta-D-galactopyranoside (C12FDG), a fluorogenic substrate for betagal activity. The cytochemical method takes about 30 min to execute, and several hours to a day to develop and score. The fluorescence methods take between 4 and 8 h to execute and can be scored in a single day. The cytochemical method is applicable to tissue sections and requires simple reagents and equipment. The fluorescence-based methods have the advantages of being more quantitative and sensitive.
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Senescence-Associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells.
Journal of cell science, 2000Co-Authors: David J. Kurz, S Decary, Y. Hong, Jorge D. ErusalimskyAbstract:Senescence-Associated (beta)-galactosidase is widely used as a biomarker of replicative senescence. However, it remains unknown whether this is a distinct enzyme active at pH 6, and differentially expressed in senescence, or a manifestation of an increase in the classic acid lysosomal (beta)-galactosidase. Here we have investigated the origin of Senescence-Associated-(beta)-galactosidase activity by modifying the intracellular and lysosomal pH of young and senescent human umbilical vein endothelial cells and examining the effect of these manipulations on the levels of activity, using a flow cytometric assay. Lysosomal alkalinisation with chloroquine or bafilomycin A(1), as well as equilibration of the intracellular milieu to pH 6 with nigericin, caused a profound (92-99%) inhibition of the total intracellular (beta)-galactosidase activity. However, independent of pH alterations, senescent cells showed levels of (beta)-galactosidase activity three- to sixfold higher than young cells. This increase in activity occurred in parallel to an increase in (beta)-galactosidase protein levels. Acridine Orange staining revealed an increase in lysosomal content with replicative age, which correlated with the increase in (beta)-galactosidase. These findings demonstrate that Senescence-Associated (beta)-galactosidase is a manifestation of residual lysosomal activity at a suboptimal pH, which becomes detectable due to the increased lysosomal content in senescent cells.
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cytochemical detection of a senescence associated beta galactosidase in endothelial and smooth muscle cells from human and rabbit blood vessels
Experimental Cell Research, 1998Co-Authors: Mark Fenton, Jorge D. ErusalimskyAbstract:Abstract 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- d -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 ( 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.
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Cytochemical detection of a Senescence-Associated Beta-Galactosidase in endothelial and smooth muscle cells from human and rabbit blood vessels.
Experimental cell research, 1998Co-Authors: B Van Der Loo, M J Fenton, Jorge D. ErusalimskyAbstract:A Beta-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. Beta-Galactosidase activity was detected by light microscopy using the chromogenic substrate 5-bromo-4-chloro-3-indolyl beta-d-galactopyranoside. In endothelial cell cultures, lysosomal Beta-Galactosidase activity, which is detected at pH 4.0, was present in all cells regardless of their replicative age. In contrast, Senescence-Associated Beta-Galactosidase activity, which is detected at pH 6.0, was absent in the majority of cells in early 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 Beta-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 Beta-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 Beta-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.
Chin-yuan Hsu - One of the best experts on this subject based on the ideXlab platform.
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Ambient temperature influences aging in an annual fish (Nothobranchius rachovii)
Aging cell, 2009Co-Authors: Chin-yuan Hsu, Ya-chi ChiuAbstract:Extending lifespan by lowering ambient temperature in the habitat has been shown in a variety of organisms. Its mechanism, however, remains elusive. In this study, we examined the survivorship and the aging process of the annual fish (Nothobranchius rachovii) reared under high (30 degrees C), moderate (25 degrees C) and low (20 degrees C) ambient temperatures. The results showed that low ambient temperatures prolong survivorship, whereas high ambient temperatures shorten survivorship. At low ambient temperature, expression of Senescence-Associated Beta-Galactosidase, lipofuscin, reactive oxygen species, lipid peroxidation, protein oxidation, mitochondrial density and ADP/ATP ratio were reduced compared with those reared at high and moderate temperatures, whereas catalase activity, Mn-superoxide dismutase activities, mitochondrial membrane potential and the levels of ATP, ADP, Sirt1 and Forkhead box O expression were elevated. The expression levels of Hsp70 and CIRP showed no significant difference under any of the ambient temperatures tested. We concluded that cellular metabolism, energy utilization and gene expression are altered at lower ambient temperature, which is associated with the extension of lifespan of the annual fish.
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Age-Related Markers Assayed at Different Developmental Stages of the Annual Fish Nothobranchius rachovii
The journals of gerontology. Series A Biological sciences and medical sciences, 2008Co-Authors: Chin-yuan Hsu, Ya-chi Chiu, Wei-lun Hsu, Yu-pei ChanAbstract:Although short-lived vertebrates can serve as model animals for understanding the mechanism of aging, whether the annual fish Nothobranchius rachovii is suitable for studying aging remains an open question. In this study, histochemical, biochemical, and genetic techniques were used to determine the age-related markers at three different developmental stages of the annual fish N. rachovii. Histochemical studies revealed that the expression of Senescence-Associated Beta-Galactosidase and accumulation of lipofuscin increased with age. In biochemical assays, lipid peroxidation and protein oxidation increased with age, whereas the activities of catalase, glutathione peroxidase, and superoxide dismutase decreased with age. Genetic analysis established that the activities of telomerase had no apparent relationship with age, but telomere lengths reduced with age from 11.5 +/- 1.98 to 3.58 +/- 0.74 kb. Taken together, these results indicate that the annual fish N. rachovii may be useful as an animal model for the study of aging.
Ya-chi Chiu - One of the best experts on this subject based on the ideXlab platform.
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Ambient temperature influences aging in an annual fish (Nothobranchius rachovii)
Aging cell, 2009Co-Authors: Chin-yuan Hsu, Ya-chi ChiuAbstract:Extending lifespan by lowering ambient temperature in the habitat has been shown in a variety of organisms. Its mechanism, however, remains elusive. In this study, we examined the survivorship and the aging process of the annual fish (Nothobranchius rachovii) reared under high (30 degrees C), moderate (25 degrees C) and low (20 degrees C) ambient temperatures. The results showed that low ambient temperatures prolong survivorship, whereas high ambient temperatures shorten survivorship. At low ambient temperature, expression of Senescence-Associated Beta-Galactosidase, lipofuscin, reactive oxygen species, lipid peroxidation, protein oxidation, mitochondrial density and ADP/ATP ratio were reduced compared with those reared at high and moderate temperatures, whereas catalase activity, Mn-superoxide dismutase activities, mitochondrial membrane potential and the levels of ATP, ADP, Sirt1 and Forkhead box O expression were elevated. The expression levels of Hsp70 and CIRP showed no significant difference under any of the ambient temperatures tested. We concluded that cellular metabolism, energy utilization and gene expression are altered at lower ambient temperature, which is associated with the extension of lifespan of the annual fish.
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Age-Related Markers Assayed at Different Developmental Stages of the Annual Fish Nothobranchius rachovii
The journals of gerontology. Series A Biological sciences and medical sciences, 2008Co-Authors: Chin-yuan Hsu, Ya-chi Chiu, Wei-lun Hsu, Yu-pei ChanAbstract:Although short-lived vertebrates can serve as model animals for understanding the mechanism of aging, whether the annual fish Nothobranchius rachovii is suitable for studying aging remains an open question. In this study, histochemical, biochemical, and genetic techniques were used to determine the age-related markers at three different developmental stages of the annual fish N. rachovii. Histochemical studies revealed that the expression of Senescence-Associated Beta-Galactosidase and accumulation of lipofuscin increased with age. In biochemical assays, lipid peroxidation and protein oxidation increased with age, whereas the activities of catalase, glutathione peroxidase, and superoxide dismutase decreased with age. Genetic analysis established that the activities of telomerase had no apparent relationship with age, but telomere lengths reduced with age from 11.5 +/- 1.98 to 3.58 +/- 0.74 kb. Taken together, these results indicate that the annual fish N. rachovii may be useful as an animal model for the study of aging.
Ke-jie Zhang - One of the best experts on this subject based on the ideXlab platform.
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Synergistic cytotoxic effects of selinexor and chloroquine phosphate in mantle cell lymphoma.
Journal of Clinical Oncology, 2016Co-Authors: Yongyi Zhang, Ke-jie Zhang, Yuhan Chen, Dai-bo Zhang, Qin Yao, Huiqin ZhuoAbstract:e19069Background: Although the cytotoxic effects of novel CRM1 inhibitor, small molecule selective inhibitors of nuclear export (SINE) on Mantle Cell Lymphoma (MCL) cells have now been established, the mechanism of cell death is still not fully understood. The objective of our study was to elucidate the mechanism of Selinexor, a SINE compound, mediated cytotoxic effects and Synergistic cytotoxic effects of Selinexor and Chloroquine phosphate (CQ) in MCL. Methods: CCK-8 assay was used to detect the growth of 4 established MCL cell lines treated with Selinexor and CQ; CalcuSyn 2.0 software was used to detected the Combination index (CI) of Selinexor combined CQ; Apoptosis were detected with Annexin V FITC assay, and Caspase3 activation and PARP cleavage were detected with Western blotting; Senescence associated Beta-Galactosidase (SA-β-gal) were detected with β-gal staining kit; The extent of autophagic flux were determined by evaluation of fluorescent LC3 puncta under a confocal laser scanning microscope b...
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Relation of notch pathway to senescence of murine bone marrow stromal cells
Zhongguo shi yan xue ye xue za zhi, 2010Co-Authors: Ke-jie Zhang, Li-fang Huang, Han-ying Sun, Yan Zhu, Yi Xiao, Mei Huang, Wen-li LiuAbstract:This study was purposed to investigate the relation of the Notch signaling pathway to senescence of murine bone marrow stromal cells in vitro. Intracellular domain of Notch 1 (ICN) was transfected into cultured murine bone marrow stromal cells by lipofectamine transfection. After transfection for three days the proliferation of transfected cells was measured by MTT, cell cycle distribution was analyzed by flow cytometry. The percentage of senescence associated Beta-Galactosidase (SA-beta-Gal) positive cells were measured by cytochemical method, and the expression rates of P53 and p21Cip1/Waf1 at gene and protein levels were analyzed by RT-PCR and Western blot respectively. The results showed that after transfection for 3 days the proliferation of murine bone marrow stromal cells was inhibited with induction of G1 arrest, the percentage of SA-beta-gal positive cells increased and the p53 and p21Cip1/Waf1 mRNA and protein expression levels were upregulated. It is concluded that the activated Notch signaling can induce premature senescence of bone marrow stromal cells through the p53-p21Cip1/Waf1 pathway.
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Role of Notch expression in premature senescence of murine bone marrow stromal cells
Progress in Natural Science, 2009Co-Authors: Ke-jie Zhang, Li-fang Huang, Han-ying Sun, Yan Zhu, Yi Xiao, Mei Huang, Wen-li LiuAbstract:Abstract The aim of the present study was to investigate the role of the Notch signaling pathway in premature senescence of murine bone marrow stromal cells in vitro . The intracellular domain of Notch 1 (ICN) was transfected into cultured murine bone marrow stromal cells by lipofectamine transfection. After three days, the proliferation of transfected cells was measured by MTT assay. Cell cycle distribution was analyzed by flow cytometry. Senescence-Associated Beta-Galactosidase (SA-beta-gal) was measured, and the percentage of positive cells was evaluated by assessing 1000 cells in random fields of view. The expressions of p53 and p21 Cip1/Waf1 were analyzed by both RT-PCR and Western blot analysis. The results showed that activation of Notch signaling inhibited proliferation of murine bone marrow stromal cells with induction of G 1 arrest, increased the percentage of SA-beta-gal positive cells, and upregulated p53 and p21 Cip1/Waf1 mRNA and protein expression levels. Thus, the activated Notch signaling could induce premature senescence of bone marrow stromal cells through the p53-p21 Cip1/Waf1 pathway.
