The role of p38 kinase in the activation of the premature senescence program in transformed mouse fibroblasts

The role of p38α stress-kinase in the regulation of the premature senescence program induced by the histone deacetylase inhibitor sodium butyrate (NaB) was studied in rodent transformed cell lines. The study was carried out on E1A+cHa-ras transformants obtained from mouse embryonic fibroblasts null for the Mapk14 gene encoding p38α stress-kinase (the mERasp38^?/? cell line), or for the PPM1D gene encoding the Wip1 phosphatase (the mERas Wip1^?/? cell line), whose absence led to constitutive activation of p38α kinase. It was found that after NaB treatment both cell lines completely stopped proliferation due to irreversible G₁/S cell cycle arrest. In both lines a marker of senescence appeared—the activity of β-galactosidase (SA-β-Gal). As well, treatment of the cells with NaB for several days led to morphological cell changes, such as partial readjustment of the actin cytoskeleton, spreading on the substrate, and heterochromatin focus formation (SAHF) in the senescent cell nuclei. These data allow us to suggest that, in the absence of functionally active p38α kinase, the NaB-induced irreversible process of cellular senescence may occur via alternative pathways for downregulation of the cell cycle.     

8-Hydroxydeoxyguanosine induces senescence-like changes in KG-1, human acute myelocytic leukemia cell line

8-Hydroxydeoxyguanosine (oh⁸dG) treatment induced senescence-like changes in KG-1 cells, a human acute myelocytic leukemia cell line. The oh⁸dG-treated cells stained positive for senescence associated β-galactosidase (SA-β-galactosidase) and had enlarged cell shape, both of which are senescence indexes. The oh⁸dG-treated cells were also cell growth inhibited and arrested at G₁ in the cell cycle. The accumulation of cdk (cyclin dependent kinase) inhibitors, such as p16, p21, and p27, also implies that cellular senescence was induced in oh⁸dG-treated cells. However, these changes were not accompanied by cell differentiation or telomerase activity. Taken together, we conclude that oh⁸dG treatment of KG-1 cells induces cellular senescence.     

Escherichia coli Producing Colibactin Triggers Premature and Transmissible Senescence in Mammalian Cells

Cellular senescence is an irreversible state of proliferation arrest evoked by a myriad of stresses including oncogene activation, telomere shortening/dysfunction and genotoxic insults. It has been associated with tumor activation, immune suppression and aging, owing to the secretion of proinflammatory mediators. The bacterial genotoxin colibactin, encoded by the pks genomic island is frequently harboured by Escherichia coli strains of the B2 phylogenetic group. Mammalian cells exposed to live pks+ bacteria exhibit DNA-double strand breaks (DSB) and undergo cell-cycle arrest and death. Here we show that cells that survive the acute bacterial infection with pks+ E. coli display hallmarks of cellular senescence: chronic DSB, prolonged cell-cycle arrest, enhanced senescence-associated β-galactosidase (SA-β-Gal) activity, expansion of promyelocytic leukemia nuclear foci and senescence-associated heterochromatin foci. This was accompanied by reactive oxygen species production and pro-inflammatory cytokines, chemokines and proteases secretion. These mediators were able to trigger DSB and enhanced SA-β-Gal activity in bystander recipient cells treated with conditioned medium from senescent cells. Furthermore, these senescent cells promoted the growth of human tumor cells. In conclusion, the present data demonstrated that the E. coli genotoxin colibactin induces cellular senescence and subsequently propel bystander genotoxic and oncogenic effects.     

Program of premature senescence is not induced by sodium butyrate in transformants with JNK1,2 knockout

The role of JNK1,2 stress kinases in the regulation of premature senescence stimulated by sodium butyrate (NaB), a histone deacetylase inhibitor, has been studied. It was found that NaB did not block the cell cycle in E1A+cHa-ras transformants selected from embryonic mouse fibroblasts with jnk1,2 stress-kinase gene knockout (mERasJNK^?/? cells). Even long-term (five days) NaB treatment did not block cell cycle distribution or cell proliferation, nor did it induce cellular hypertrophy or activate SA-β-galactosidase activity, a senescence marker. The data show that JNK stress kinases are involved in senescence induced in E1A+cHa-ras mouse transformants by NaB. It is possible to suggest that JNK1,2 have tumor suppressor properties because the process of senescence, which prevents tumor cell proliferation, does not occur if they are absent.     

Role of p38alpha kinase in activation of premature senescence program in transformed mouse fibroblasts

We investigated the role of p38alpha stress-kinase in regulation of premature senescence program, stimulated by histone deacetylase inhibitor--sodium butyrate (NaB)--after application to rodent transformed cell lines. Investigation was performed on the E1A + cHa-ras transformants selected from mice embryonic fibroblasts null at the p38alpha kinase gene or null fibroblasts at the PPM1D gene, which encoded phosphatase Wip1. Absence of Wip1 led to constitutive activation of p38alpha kinase. It was revealed that after NaB treatment both cell lines completely stopped proliferation due to irreversible cell cycle arrest in G1/S phase. In both cell lines sodium butyrate induced sustained block of prolifaration due to irreversible cell cycle arrest in G1/S phase. Following sodium butyrate treatment cells expressed marker of senescence--beta-galactosidase activity (SA-beta-Gal). Long-term (during several days) NaB treatment of cells led to partial restoration of actin cytoskeleton, focal adhesion contacts and heterochromatin focus formation (SAHF) in the nucleus of senescent cells. Obtained data allow us to suppose that irreversible process of cellular senescence activated by sodium butyrate can occur in the absence of functionally active p38 kinase by means of other ways of cell cycle suppression.     

Cellular Senescence in Livers from Children with End Stage Liver Disease

Background

Senescent cells occur in adults with cirrhotic livers independent of the etiology. Aim: Investigate the presence rate of cellular senescence and expression of cell cycle check points in livers from children with end stage disease.

Methodology/Principal Findings

Livers of five children aged three years or less undergoing liver transplantation due to tyrosinemia (n = 1), biliary atresia (n = 2), or fulminant hepatitis (n = 2) were analyzed for senescence associated β-galactosidase (SA-βgal) activity and p16INK4a, p21cip1 and p53. All livers displayed positive cellular staining for SA-βgal in the canals of Hering and interlobular biliary ducts. In the presence of cirrhosis (3/5 cases) SA-βgal was found at the cholangioles and hepatocytes surrounding the regenerative nodules. Children with fulminant hepatic failure without cirrhosis had significant ductular transformation with intense SA-βgal activity. No SA-βgal activity was evident in the fibrous septa. Staining for p53 had a similar distribution to that observed for SA-βgal. Staining for p16^INK4a and p21^cip1 was positive in the explanted liver of the patient with tyrosinemia, in the hepatocytes, the canals of Hering, cholangioles and interlobular bile ducts. In the livers with fulminant hepatitis, p21^cip1 staining occurred in the areas of ductular transformation and in the interlobular bile ducts.

Conclusions/Significance

Cellular senescence in livers of children with end stage disease is associated with damage rather than corresponding to an age dependent phenomenon. Further studies are needed to support the hypothesis that these senescence markers correlate with disease progression.     

Silencing p53 inhibits interleukin 10-induced activated hepatic stellate cell senescence and fibrotic degradation in vivo

Activated hepatic stellate cells are reported to play a significant role in liver fibrogenesis. Beside the phenotype reversion and apoptosis of activated hepatic stellate cells, the senescence of activated hepatic stellate cells limits liver fibrosis. Our previous researches have demonstrated that interleukin-10 could promote hepatic stellate cells senescence via p53 signaling pathway in vitro. However, the relationship between expression of p53 and senescence of activated hepatic stellate cells induced by interleukin-10 in fibrotic liver is unclear. The purpose of present study was to explore whether p53 plays a crucial role in the senescence of activated hepatic stellate cells and degradation of collagen mediated by interleukin-10. Hepatic fibrosis animal model was induced by carbon tetrachloride through intraperitoneal injection and transfection of interleukin-10 gene to liver was performed by hydrodynamic-based transfer system. Depletions of p53 in vivo and in vitro were carried out by adenovirus-based short hairpin RNA against p53. Regression of fibrosis was assessed by liver biopsy and collagen staining. Cellular senescence in the liver was observed by senescence-associated beta-galactosidase (SA-β-Gal) staining. Immunohistochemistry, immunofluorescence double staining, and Western blot analysis were used to evaluate the senescent cell and senescence-related protein expression. Our data showed that interleukin-10 gene treatment could lighten hepatic fibrosis induced by carbon tetrachloride and induce the aging of activated hepatic stellate cells accompanied by up-regulating the expression of aging-related proteins. We further demonstrated that depletion of p53 could abrogate up-regulation of interleukin-10 on the expression of senescence-related protein in vivo and vitro. Moreover, p53 knockout in fibrotic mice could block not only the senescence of activated hepatic stellate cells, but also the degradation of fibrosis induced by interleukin-10 gene intervention. Taken together, our results suggested that interleukin-10 gene treatment could attenuate carbon tetrachloride-induced hepatic fibrosis by inducing senescence of activated hepatic stellate cells in vivo, and this induction was closely related to p53 signaling pathway.     

Brief report: a new profile of terminal <Emphasis Type="Italic">N</Emphasis>-acetyllactosamines glycans on pig red blood cells and different expression of α-galactose on Sika deer red blood cells and nucleated cells

It has been reported that: (1) large variations were found in the number of sialic acid (SA) capped with N-acetyllactosamines (SA-Galβ1-4GlcNAc-R) and α-Gal epitopes (Galα1-3Galβ1-4GlcNAc-R) or uncapped N-acetyllactosamines (Galβ1-4GlcNAc-R) on different mammalian red blood cells, and on nucleated cells originating from a given tissue in various species; (2) goat, sheep, horse and mouse red blood cells lack α-Gal epitopes, despite the expression of this epitope on a variety of nucleated cells in these species, including lymphocytes differentiated from the same hematopoietic origin. In this study, flow cytometry and Western blot analyses of pig red blood cells showed that α-Gal epitopes on pig red cells developed concomitantly after treatment with neuraminidase, suggesting that the terminal N-acetyllactosaminide glycans were capped with SA-α-Gal epitopes. Whereas, the expression of the α-Gal epitopes on red blood cells from Sika deer (Cevus nippon hortulorum) were found to be absent even though the epitopes were present on their white blood cells. Thus, these results add new data not only for the terminal carbohydrate structures on cell surface glycans of various mammalian cells, but also for wide variety of epitope expression on the cells from different tissues, which might be useful for understanding their unique states resulting from differentiation and evolution.     

Effect of cell cycle inhibitor p19ARF on senescence of human diploid cell

To investigate the effect of cell cycle inhibitor p19ARF on replicative senescence of human diploid cell, recombinant p19ARF eukaryotic expression vector was constructed and p19ARF gene was transfected into human diploid fibroblasts (WI-38 cells) by liposome-mediated transfection for overexpression. Then, the effects of p19ARF on replicative senescence of WI-38 cells were observed. The results re- vealed that, compared with control cells, the WI-38 cells in which p19ARF gene was introduced showed significant up-regulation of p53 and p21 expression level, decrease of cell generation by 10 12 generations, decline of cell growth rate with cell cycle being arrested at G1 phase, increase of positive rate of senescent marker SA-β-gal staining, and decrease of mitochondrial membrane potential. The morphology of the transfected fibroblasts presented the characteristics changes similar to senescent cells. These results indicated that high expression of p19ARF may promote the senescent process of human diploid cells.     

p21Waf1 is required for cellular senescence but not for cell cycle arrest induced by the HDAC inhibitor sodium butyrate

Cell senescence is characterized by senescent morphology and permanent loss of proliferative potential. HDAC inhibitors (HDACI) induce senescence and/or apoptosis in many types of tumor cells. Here, we studied the role of cyclin-kinase inhibitor p21^waf1(Cdkn1n gene) in cell cycle arrest, senescence markers (cell hypertrophy, SA-bGal staining and accumulation of gH2AX foci) in p21^Waf1+/+ versus p21^Waf1-/- mouse embryonic fibroblast cells transformed with E1A and cHa-Ras oncogenes (mERas). While short treatment with the HDACI sodium butyrate (NaB) induced a reversible G1 cell cycle arrest in both parental and p21^Waf1-/- cells, long-term treatment led to dramatic changes in p21^Waf1+/+ cells only: cell cycle arrest became irreversible and cells become hypertrophic, SA-bGal-positive and accumulated gH2AX foci associated with mTORC1 activation. The p21^Waf1+/+ cells lost their ability to migrate into the wound and through a porous membrane. Suppression of migration was accompanied by accumulation of vinculin-staining focal adhesions and Ser3-phosphorylation of cofilin, incapable for F-actin depolymerization. In contrast, the knockout of the p21^Waf1 abolished most of the features of NaB-induced senescence, including irreversibility of cell cycle arrest, hypertrophy, additional focal adhesions and block of migration, gH2AX foci accumulation and SA-bGal staining. Rapamycin, a specific inhibitor of mTORC1 kinase, decreased cellular hypertrophy, canceled coffilin phosphorylation and partially restored cell migration in p21^Waf1+/+ cells. Taken together, our data indicate a new role of p21^Waf1 in cell senescence, which may be connected not with execution of cell cycle arrest, but also with the development of mTOR-dependent markers of cellular senescence.     

Sodium butyrate induces cell senescence in transformed rodent cells resistant to apoptosis

The capacity of HDAC inhibitor sodium butyrate to induce senescence in cells derived from rat embryonic fibroblasts and transformed by E1A + E1B19 kDa oncogene has been studied. These transformants are resistant to apoptosis in response to γ-irradiation and the deprivation of growth factors. The process of cell senescence was investigated by analyzing cell growth curves, G1/S and G2/M cell cycle arrest and senescent associated β-galactosidase expression. The irreversibility of the antiproliferative activity of sodium butyrate was analyzed by clonogenic assay.     

Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation

The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene) has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol’s anti-aging effects both in vitro and in vivo attributed to activation of a (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. In mammals seven members (SIRT1-7) of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ) and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated β-galactosidase (SA-β-gal) activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21^CIP1 and p16^INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-β-gal activity, γ-H2AX phosphorylation and p53, p21^CIP1 and p16^INK4A levels. Interestingly DNA damaging agent doxorubicin also induced senescence in BJ fibroblasts associated with decreased SIRT1/2 levels. In conclusion our data reveal that resveratrol induced premature senescence is associated with SIRT1 and SIRT2 down regulation in human dermal fibroblasts. Here we suggest that the concomitant decline in SIRT1/2 expression in response to resveratrol treatment may be a cause for induction of senescence, which is most likely mediated by a regulatory mechanism activated by DNA damage response.     

Cell senescence induced by histone deacetylase inhibitor sodium butyrate in rodent transformed cells resistant to apoptosis

The capacity of HDAC inhibitor sodium butyrate to induce senescence in cells derived from rat embryonic fibroblasts transformed by E1A+E1B19 kDa oncogenes has been studied. These transformants are resistant to apoptosis in response to gamma-irradiation and growth factor deprivation. The process of cell senescence was investigated by the analysis of cell growth curves, G1/S and G2/M cell cycle arrest, and senescent associated beta-galactosidase expression. The irreversibility of sodium butyrate antiproliferative activity was analyzed by clonogenic assay. We show that sodium butyrate suppresses proliferation and induces senescence in the E1A+E1B19 kDa transformed cells. Interestingly, NaB induces growth arrest due to accumulation of cells in G2/M phase, these cells are not tetraploid but mainly binuclear. Thus, in case of NaB induced senescence in E1A+E1B19 kDa transformed fibroblasts, the observed suppression of cell proliferation may be the result of cytokinesis failure leading to formation of binuclear and multinuclear cells incapable to proliferate.