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Results for “Senescent fibroblasts”

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At least 865 records · Page 48Linked to original sources
PubMed1997

The effects of age on rabbit MCL fibroblast matrix synthesis in response to TGF-beta 1 or EGF.

In this study, we examined the effects of age on collagen and total protein synthesis by ligament fibroblasts in response to growth factors. Three different doses of transforming growth factor-beta 1 (TGF-beta 1) or epidermal growth factor (EGF) were individually added to in vitro fibroblast cultures from the medial collateral ligament (MCL) of skeletally immature (age 3 months), mature (age 12 months) and senescent (age 48-51 months) rabbits. Analysis of the effects of age revealed that fibroblasts from senescent rabbits produced significantly less collagen in response to TGF-beta 1 or EGF stimulation when compared to fibroblasts from immature rabbits. Furthermore, increased age was found to result in significant reductions in the baseline levels of collagen synthesis but not total protein synthesis. Additionally, collagen and total protein synthesis by MCL fibroblasts were significantly affected by the TFG-beta 1 dose, but not by the EGF dose. When fibroblasts were normalized to their own controls, the increase in collagen and total protein synthesis due to TGF-beta 1 and EGF for the senescent group were found to be greater than those for the skeletally immature rabbits at all doses. This demonstrates that MCL fibroblasts from senescent rabbits are responsive to growth factors.

Aging↗
PubMed2006

Downregulation of protein kinase CKII is associated with cellular senescence.

Protein kinase CKII (CKII) plays a critical role in cell growth and proliferation. In this study, we examine how CKII activity is regulated during cellular senescence. Our results demonstrate that CKII activity apparently decreases during both replicative and H2O2-induced senescence in human diploid fibroblast IMR-90 cells. The mRNA and protein levels of CKIIalpha decreases significantly during replicative and H2O2-induced senescence, while only slight reduction in those of CKIIbeta is observed during replicative senescence. Treatment of IMR-90 cells with CKII inhibitors 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole and apigenin led cells to acquire a senescent phenotype as judged by the senescence-associated beta-galactosidase marker and overexpression of p53 and p21(Waf-1). Knockdown of CKIIalpha in IMR-90 cells by RNA interference also dramatically induced the senescent phenotype. In parallel, CKII activity was transcriptional downregulated in rat liver and testis with advancing age. Taken together, these results suggest that downregulation of CKII activity is tightly associated not only with cellular senescence but also with organism aging.

Aging↗
PubMed2006

Death-associated protein 3 regulates cellular senescence through oxidative stress response.

Death-associated protein 3 (DAP3) has been originally identified as a positive mediator of apoptosis. It has been revealed recently that the predominant localization of DAP3 to mitochondria implies its functional involvement in mitochondrial metabolism in addition to apoptosis. However, little is known about the molecular basis of these physiological functions of DAP3. Here, we demonstrate that DAP3 is reduced in both replicative and premature senescence induced by oxidative stress, and the DAP3 reduction induced by oxidative stress is observed mostly in a mitochondrial fraction. Using DAP3-specific short hairpin RNA (shRNA) in a clonogenic survival assay, we reveal that reduction of DAP3 induces resistance to oxidative stress and decreases intracellular reactive oxygen species (ROS) production. Furthermore, this strategy allows us to show that loss of DAP3 is involved in the avoidance of replicative senescence in mouse embryonic fibroblasts (MEFs). Thus, our study offers an insight into the potential regulatory function of mitochondrial DAP3 involved in cellular senescence.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Regulation of a novel gene encoding a lysyl oxidase-related protein in cellular adhesion and senescence.

We report here a novel cDNA clone with a predicted protein sequence similar to lysyl oxidase. This full-length cDNA clone of 3432 base pairs (WS9-14) was isolated from human fibroblasts on the basis of its overexpression in senescent cells. It encodes an 87-kDa polypeptide, whose protein is a member of the scavenger receptor cysteine-rich family, because it contains four scavenger receptor cysteine-rich domains that are found in several secreted or cell surface proteins. The WS9-14 protein has a 48% identity with both lysyl oxidase and lysyl oxidase-like protein at a region corresponding to exons 2-6, implying the existence of a lysyl oxidase gene family. The pattern of WS9-14 gene expression by fibroblasts parallels pro-collagen I-alpha1 expression. Its mRNA level is induced by transforming growth factor beta-1 and indomethacin and inhibited by phorbol ester and retinoic acid. WS9-14 is abundantly expressed in all tumor cell lines examined that attach to culture dishes but not in cell lines that grow in suspension and is also up-regulated in senescent fibroblasts. These results suggest that WS9-14 gene encodes an extracellular protein that may be specifically involved in cell adhesion and senescence.

3T3 Cells↗
PubMed1994

Altered regulation of fibronectin gene expression in Werner syndrome fibroblasts.

Fibronectin (FN) production was quantified in the extracellular medium and in extracts of human diploid fibroblasts (HDF) by enzyme-linked immunosorbent assay and by immunoprecipitation of [35S]methionine biosynthetically labeled proteins. FN output was increased in normal late passage (old) HDF and in prematurely senescent Werner syndrome (WS) fibroblasts, compared to normal early passage (young) HDF. Output was maximal when cells were preconfluent, exceeding the level found at confluence and postconfluence. For all cell types the highest proportion of newly synthesized FN was found in the extracellular compartment. Both young and old HDF exposed to regular growth medium containing 15% fetal bovine serum (FBS) secreted twice as much FN as when exposed to serum-free medium, and cognate mRNA levels were commensurate with protein output. WS fibroblasts secreted 1.6-fold more FN than old HDF when both cell types were exposed to medium containing 15% FBS. Immunofluorescent analysis revealed greater association of FN with WS and old HDF than with young HDF. Furthermore, WS cells exposed to 15% FBS contained 3.4-fold more FN mRNA and produced seven times more FN protein compared to WS cells exposed to serum-free medium. Thus, WS fibroblasts secrete more FN than old normal and young normal HDF in the presence of 15% FBS due to augmentation of FN mRNA levels and enhanced efficiency of FN mRNA translation and/or post-translational processing.

Animals↗
PubMed2000

Expression of transcriptional repressor proteins mSin3A and 3B during aging and replicative senescence.

Sin3 proteins have a key role in transcriptional repression mediated by histone deacetylation. Mammalian Sin3 proteins, mSin3A and 3B, act as adapter molecules which bind both to repressive transcription factors and to the methyl-CpG-binding proteins (MeCPs) and recruit histone deacetylases to assemble a multiprotein repressor complex. We have recently observed (Biochem. Biophys. Res. Commun. 252, 274-277, 1998) that the expression of mSin3A but not mSin3B protein is induced during neuronal apoptosis. The purpose of this study was to find out whether aging and replicative senescence affect the expression levels of mSin3A and 3B repressor proteins. We studied the expression levels of mSin3A and 3B mRNAs and proteins both in replicative senescence model of WI-38 fibroblasts and in liver and brain tissues of young (4-6 months) and old (26-30 months) male Wistar rats. Replicative senescence of human WI-38 fibroblasts did not affect the expression levels of mSin3A and 3B mRNAs. However, the late passage WI-38 fibroblasts showed a significant decline in the expression level of mSin3A protein. Immortalization of WI-38 fibroblasts with SV-40 transformation increased the expression level of 6.0 kb mSin3A mRNA. Aging of Wistar rats did not affect the expression levels of either mSin3A or mSin3B mRNAs in the liver and frontal cortex. Similarly, the protein levels of mSin3A and 3B were unaffected in the hippocampus, cerebellum and liver tissues during aging. These results show that aging in vivo, in contrast to replicative senescence, does not affect the expression levels of mSin3A and 3B repressor proteins. However, this does not exclude the possible age-related functional changes mediated by mSin3-histone deacetylase complexes.

Animals↗
PubMed1997

Sodium butyrate induces NIH3T3 cells to senescence-like state and enhances promoter activity of p21WAF/CIP1 in p53-independent manner.

Sodium butyrate, a histone deacetylase inhibitor, has been shown to induce differentiation of many cancer cells and senescence-like state of human fibroblasts. Here we show that sodium butyrate also induces senescence-like state of NIH3T3 cells. The treated cells were blocked at G1 phase and featured morphologically like senescent cells with enlarged cytoplasm and multiple nuclei. The expression of p21(WAF/CIP1) (p21) increased after sodium butyrate treatment at transcriptional level. To analyze the induction of promoter activity, we isolated 4.6 kb of murine p21 promoter and inserted it upstream of a luciferase reporter gene. When this construct was transiently transfected into NIH3T3 cells, sodium butyrate enhanced the luciferase activity. p53 independency of sodium butyrate-inducible p21 promoter activity was confirmed by using the deletion mutants lacking p53 binding sites and p53 deficient cells in transfection experiments.

3T3 Cells↗
PubMed1998

Telomerase expression in chickens: constitutive activity in somatic tissues and down-regulation in culture.

Although human and rodent telomeres have been studied extensively, very little is known about telomere dynamics in other vertebrates. Moreover, our current dependence on mice as a model for human tumorigenesis and aging poses a problem because human and mouse telomere biology is very different. To explore whether chickens might provide a more useful model, we have examined telomerase activity and telomere length in chicken tissues as well as in primary cell cultures. Although chicken telomeres resemble human telomeres in that they are 8-20 kb in length, the distribution of telomerase activity in chickens resembles what is found in mice. Active enzyme is present in germline tissue as well as in a wide range of somatic tissues. Because chicken cells exhibit extremely low rates of spontaneous immortalization, this finding indicates that constitutive telomerase expression does not necessarily lead to an increased immortalization frequency. Finally, we found that telomerase activity is greatly down-regulated when primary cultures are established from chicken embryos. Although this down-regulation explains the telomere loss and replicative senescence that we observed in fibroblast cultures, it raises questions concerning how relevant studies of senescence in primary cell cultures are to aging in whole animals.

Animals↗
PubMed2003

Ras induces vascular smooth muscle cell senescence and inflammation in human atherosclerosis.

BACKGROUND: Vascular cells have a finite cell lifespan and eventually enter an irreversible growth arrest, cellular senescence. The functional changes associated with cellular senescence are thought to contribute to human aging and age-related vascular disorders. Ras, an important signaling molecule involved in atherogenic stimuli, is known to promote aging in yeast and cellular senescence in primary human fibroblasts. The aim of this study was to investigate the role of Ras-induced vascular smooth muscle cell (VSMC) senescence in atherogenesis. METHODS AND RESULTS: We introduced an activated ras allele (H-rasV12) into human VSMCs using retroviral infection. Introduction of H-rasV12 induced a growth arrest with phenotypic characteristics of cellular senescence, such as enlarged cell shapes and increases in expression of cyclin-dependent kinase inhibitors and senescence-associated beta-galactosidase (SA-beta-gal) activity. Activation of Ras drastically increased expression of proinflammatory cytokines, in part through extracellular signal-regulated kinase activation. To determine whether Ras activation induces cellular senescence in vivo, we transduced the adenoviral vector encoding H-rasV12 into rat carotid arteries injured by a balloon catheter. Introduction of Ras into the arteries enhanced vascular inflammation and senescence compared with mock-infected injured arteries. Moreover, SA-beta-gal-positive VSMCs were detected in the intima of advanced human atherosclerotic lesions and exhibited increased levels of extracellular signal-regulated kinase activity and proinflammatory cytokine expression. CONCLUSIONS: Our results suggest that atherogenic stimuli mediated by Ras induce VSMC senescence and vascular inflammation, thereby contributing to atherogenesis. This novel mechanism of atherogenesis may provide insights into a new antisenescence treatment for atherosclerosis.

Adult↗
PubMed1998

Premature senescence involving p53 and p16 is activated in response to constitutive MEK/MAPK mitogenic signaling.

Oncogenic Ras transforms immortal rodent cells to a tumorigenic state, in part, by constitutively transmitting mitogenic signals through the mitogen-activated protein kinase (MAPK) cascade. In primary cells, Ras is initially mitogenic but eventually induces premature senescence involving the p53 and p16(INK4a) tumor suppressors. Constitutive activation of MEK (a component of the MAPK cascade) induces both p53 and p16, and is required for Ras-induced senescence of normal human fibroblasts. Furthermore, activated MEK permanently arrests primary murine fibroblasts but forces uncontrolled mitogenesis and transformation in cells lacking either p53 or INK4a. The precisely opposite response of normal and immortalized cells to constitutive activation of the MAPK cascade implies that premature senescence acts as a fail-safe mechanism to limit the transforming potential of excessive Ras mitogenic signaling. Consequently, constitutive MAPK signaling activates p53 and p16 as tumor suppressors.

Animals↗
PubMed2000

LMP1 of Epstein-Barr virus suppresses cellular senescence associated with the inhibition of p16INK4a expression.

Epstein-Barr virus is associated with a number of human proliferative and malignant diseases. It is capable of immortalizing human primary B-lymphocytes in vitro. Studies indicate that latent membrane protein LMP1 is one of the viral proteins essential for this process. In this report, LMP1 was shown to prevent primary mouse embryonic fibroblasts from entering into replicative senescence in vitro. It further suppresses the senescence-associated induction of p16INK4a, commonly believed to be a key regulator of replicative senescence. In addition, LMP1 was shown to prevent premature senescence provoked by oncogenic ras in mouse embryonic fibroblasts, and to inhibit the oncogene ras-mediated induction of p16INK4a and p21WAF1. In parallel, LMP1 also prevents ras-induced premature senescence in rat embryonic fibroblasts REF52 and human diploid fibroblasts IMR90. Moreover, LMP1 is capable of suppressing the p16INK4a promoter in REF52 and Saos-2 cells in a promoter reporter assay. Our findings suggest that with the expression of p16INK4a and replicative senescence being suppressed, LMP1 may play a key role in Epstein-Barr virus-associated proliferative diseases, and it may further contribute to cancer development by preventing premature senescence induced by mitogenic oncogenes.

Animals↗
PubMedDate not supplied

Nuclear matrix composition and in vitro cellular senescence.

The synthesis of nuclear matrix components from human diploid fibroblasts of different in vitro ages was analyzed. Radiolabeled nuclear matrices were prepared from human diploid fibroblasts at various stages of the cell cycle, and their components were separated by two dimensional electrophoresis. The same general electrophoretic pattern was observed at all cell cycle points analyzed, regardless of in vitro age. However, several of the more than 150 peptides that were observed exhibited some cell cycle or age-related variation in radiolabeling. Ten of these were chosen for further analysis. One peptide, with an approximate molecular weight of 47 kDa and pI of 6.8 exhibited the most significant cell cycle and age-related alterations. In matrices from younger cells, incorporation into this peptide was very low in GO but increased as these cells moved through the cell cycle, with maximum incorporation occurring in S phase. As cells neared the end of their in vitro lifespan, labeling of this peptide was elevated at all stages of the cell cycle. Since many of the functional alterations observed in senescent human diploid fibroblasts are nuclear-matrix-associated activities, these results suggest that the inappropriate expression of nuclear matrix components contribute to the functional changes which characterize in vitro senescence.

Antigens, Nuclear↗
PubMed2004

Regulation of cellular senescence and p16(INK4a) expression by Id1 and E47 proteins in human diploid fibroblast.

Id1, a member of Id family of helix-loop-helix transcriptional regulatory proteins, is implicated in cellular senescence by repressing p16(INK4a) expression, but the mechanisms and cellular effects in human diploid fibroblasts remain unknown. Here we analyzed the patterns of p16(INK4a) and Id1 expression during the lifespan of 2BS cells and presented the inverse correlation between these two proteins. Immunoprecipitation assays demonstrated the presence of endogenous interaction of Id1 and E47 proteins that was strong in young 2BS cells and weakened during replicative senescence and, thereby, influenced the transcription activation of p16(INK4a) by E47. Furthermore, we found that E47 protein could bind to the E-box-containing region in p16(INK4a) promoter in senescent cells by chromatin immunoprecipitation analyses, suggesting that E47 is indeed ultimately involved in the regulation of p16(INK4a) transcription in vivo. Silencing Id1 expression in young cells by RNA interference induced an increased p16(INK4a) level and premature cellular senescence, whereas silencing E47 expression inhibited the expression of p16(INK4a) and delayed the onset of senescent phenotype. The present study demonstrated not only the capacity of Id1 to regulate p16(INK4a) gene expression by E47, but also the phenotypic consequence of the regulation on cellular senescence, moreover, raised the possibility of Id1-specific gene silencing for human cancer therapy.

Base Sequence↗
PubMed2006

HIF1alpha delays premature senescence through the activation of MIF.

Premature senescence in vitro has been attributed to oxidative stress leading to a DNA damage response. In the absence of oxidative damage that occurs at atmospheric oxygen levels, proliferation of untransformed cells continues for extended periods of time. We have investigated the role of the hypoxia-inducible factor 1alpha (HIF1alpha) transcription factor in preventing senescence in aerobic and hypoxic conditions. Using embryonic fibroblasts from a conditional HIF1alpha knockout mouse, we found that loss of HIF1alpha under aerobic conditions significantly accelerated the onset of cellular senescence, and decreased proliferation under hypoxia. Furthermore, we identify the macrophage migration inhibitory factor (MIF) as a crucial effector of HIF1alpha that delays senescence. Inhibition of MIF phenocopies loss of HIF1alpha. Our findings highlight a novel role for HIF1alpha under aerobic conditions, and identify MIF as a target responsible for this function.

Aerobiosis↗
PubMed1994

Cloning of cDNAs with possible association with senescence and immortalization of human cells.

Normal human diploid fibroblasts (HDF) have a finite life span in vitro and have been used as a model system for the study of in vivo aging. Little is known about how changes in gene expression may affect the immortalization of human fibroblasts. We looked for cDNA clones whose mRNAs were differentially expressed between mortal senescent SV40-transformed human fibroblasts (B-32) and the immortal counterparts (B-32F) derived from B-32 cells. We identified three cDNA isolates by subtractive differential hybridization with 32P-labeled cDNA probes from B-32 cells and B-32F cells. Nucleotide sequence analysis of these cDNA clones revealed that they were homologous to the human vimentin, a human mitochondrial gene and a human gene of unknown nature. Slot blot and Northern blot analyses demonstrated that the former two were preferentially expressed in senescent B-32 cells and the last one was less expressed in B-32F immortal cells.

Brain Chemistry↗
PubMed1992

Characterization of nuclear morphology and nuclear matrices in ageing human fibroblasts.

It is believed that the mechanisms for cellular senescence may reside within the genome, however, the changes which occur in the DNA and the surrounding nuclear environment have not been well documented. As the dynamic skeletal framework of the nucleus, the nuclear matrix is poised to play a critical role in the ageing process. The nuclear matrix plays a central role in DNA organization and nuclear structural morphology. The important roles of the nuclear matrix in cell structure and function are demonstrated by its properties of tissue specificity and that it is altered by viral infection, differentiation and carcinogenesis. We therefore undertook a study to investigate the morphologic alterations which occur in ageing nuclei and to determine whether compositional changes in the nuclear matrix occur with age in human skin fibroblasts. We found that as the nucleus increases in size and becomes more round with age, the qualitative pattern of the prominent nuclear matrix proteins does not appear to undergo major changes with age. There do, however, appear to be quantitative alterations in these proteins.

Cell Line↗