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Results for “Senescent fibroblasts”

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PubMed2003

Hypermethylation of the hTERT promoter inhibits the expression of telomerase activity in normal oral fibroblasts and senescent normal oral keratinocytes.

Telomerase activity in human cells closely correlates with the expression of its catalytic subunit, telomerase reverse transcriptase (hTERT). Previously, we reported the lack of telomerase activity in normal human oral fibroblasts (NHOF) and the diminution of telomerase activity during senescence in normal human oral keratinocytes (NHOK). To investigate the underlying mechanisms of telomerase regulation in both cell types, we analysed the expression, promoter activity, and methylation status of the hTERT gene. The expression of hTERT mRNA diminished in senescent NHOK, but was not detected in NHOF at any stage of replication. An exogenous hTERT promoter was active in NHOF and in senescing NHOK, indicating that the lack of hTERT gene expression resulted from alteration of the endogenous hTERT promoter. Since methylation is involved in the silencing of numerous genes, we carried out DNA methylation assays. The assay revealed that the hTERT promoter was hypermethylated in NHOF and was gradually methylated during senescence in NHOK. Treatment of NHOF and senescent NHOK with the demethylating agent 5-aza-2'-deoxycytidine restored the expression of endogenous hTERT mRNA. Our results suggest that hypermethylation of the hTERT promoter plays a critical role in the negative regulation of telomerase activity in normal human oral cells.

Catalytic Domain↗

PubMed2006

Deep senescent human fibroblasts show diminished DNA damage foci but retain checkpoint capacity to oxidative stress.

Critically shortened telomeres trigger a DNA damage response in replicatively senescent cells. Here we report that while DNA damage foci can be detected in newly senescent cells, these foci eventually diminished in deep senescent cells. However, DNA checkpoint signalling and repair machinery in response to oxidative stress remain uncompromised in these deep senescent cells. Activation of p53 by oxidative stress is unaffected despite a marked decrease in expression of platelet-derived growth factor alpha-receptor. These findings suggest that cellular senescence is not a static process hence care must be taken in the selection of biomarkers of senescence in studies of ageing.

Cell Proliferation↗

PubMed2006

Inhibitory effect of peroxiredoxin II (Prx II) on Ras-ERK-NFkappaB pathway in mouse embryonic fibroblast (MEF) senescence.

Intracellular reactive oxygen species (ROS) were attenuated by the expression of peroxiredoxin II (Prx II). Cellular senescence as judged by senescence-associated (SA)-beta-galactosidase (Gal) positive cell formation was increased in Prx II-deficient mouse embryonic fibroblast (MEF). Ras expression was increased following passages. The level of Ras expression was higher in Prx II-/- MEF than wild type MEF. ERK activity was also augmented by the deletion of Prx II. SA-beta-Gal-positive cell formation was reduced by PD98059, ERK inhibitor. Activated nuclear transcription factor, nuclear factor-kappaB (NFkappaB) by the deletion of Prx II was inhibited by the treatment with PD98059. In contrast, no changes in SA-beta-Gal-positive cell formation were detected by NFkappaB inhibitor, N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK). Collectively, results suggest that Prx II deletion activate Ras-ERK-NFkappaB pathways and cellular senescence in Prx II-/- MEF cells was mediated by ERK activation but not by NFkappaB activation.

Animals↗

PubMed1995

Enhanced expression of cyclin D1 in senescent human fibroblasts.

When human fibroblast, TIG-1, was growth-stimulated with fetal bovine serum, the induction level of cell cycle-dependent genes was generally much lower in senescent cells than in young counterparts. Exceptionally, the expression level of cyclin D1 in senescent cells was constitutively higher than in young cells and further increased after serum stimulation, which was confirmed by Northern and Western blots and immunoprecipitation. This was also true in other human diploid fibroblast lines, TIG-3 and MRC-5. However, cyclin D1-dependent kinase activity was not detected in senescent cells. When sense- or antisense-cyclin D1 cDNA driven by beta-actin promoter was transfected into young TIG-1 cells, the number of appeared colonies from sense-strand transfected cultures was lower than that from antisense-strand-transfected ones. However, clones expressing cyclin D1 at low or undetectable level which were isolated after transfection with antisense-cyclin D1 proliferated up to the same division limit as untransfected and sense-strand transfected cells. Four clones of SV40-transformed TIG-1 expressed cyclin D1 at moderate levels during their extended proliferative lifespan. It appears that, if the extremely overexpressed cyclin D1 could cause an inhibition of cell proliferation at senescent stage, cellular senescence occurs regardless of overexpression of cyclin D1.

Animals↗

PubMed1998

Expression of the growth arrest specific genes in rat embryonic fibroblasts undergoing senescence.

Replicative senescence is an in vitro biological phenomenon that results the irreversible growth arrest of mammalian cells. In contrast, cellular quiescence is reversible upon the addition of mitogens or replating. Quiescence is associated with the expression of a particular gene family--the growth arrest specific (GAS) genes. I have determined whether gas genes expression also associates with senescence, as well as whether serum stimulation of senescent cells has the same inhibiting effect as in quiescent cells. Analysis of RNA of cultures of rat embryonic fibroblasts suggests that gas-1 and gas-6 gene expression is associated with the irreversible growth arrest of senescence. However, serum was found to have a different effect upon gas-1 and gas-6 expression in quiescent and senescent cells, because its stimulation of quiescent cells results in a dramatic decrease of their RNA levels, but it had no effect in senescent cells. Finally, analysis of the data is raising the possibility that the gas-1 and gas-6 genes expression may also be associated with conditions of cellular stress.

Animals↗

PubMed1994

Global change of gene expression at late G1/S boundary may occur in human IMR-90 diploid fibroblasts during senescence.

The hallmark of cellular aging is the failure of senescent diploid cells to enter or to complete the S phase of the cell cycle. The cause for such failure may hold the key for our understanding of the molecular basis of cellular aging. We have previously shown that aging of IMR-90 human diploid fibroblasts in culture is accompanied by a five to sevenfold decrease in both thymidine kinase activity and thymidine kinase mRNA level (Chang and Chen, 1988, J. Biol. Chem., 263:11431-11435). To examine whether attenuation of gene expression at G1/S boundary is unique for thymidine kinase or it may involve most, if not all, of other G1/S genes, we compared the expressions of two classes of G1/S genes in young and in old IMR-90 cells following serum stimulation. We found that the expression of all these genes, including thymidylate synthase (TS), dihydrofolate reductase (DHFR), ribonucleotide reductase (PNR), proliferating cell nuclear antigen (PCNA), histone H1, histone H2A + 2B, histone H3, and histone H4, was induced to high levels in young IMR-90 cells but not in old IMR-90 cells. The mRNA levels of all G1/S genes in young cells were more than tenfold higher than that in old cells 12 hr after serum stimulation. The enzymes encoded by TS and DHFR genes and dUTPase also exhibited similar age-dependent attenuation in activities. In contrast, expression of growth-related genes such as eIF-5A, c-Ha-ras, and beta-actin did not show significant differences between young and old cells after serum stimulation. Computer analysis of the promoter region of these G1/S genes revealed an Sp-1 binding site as the most common cis-element. Taken together, our results suggest that the suppression of G1/S gene expressions during senescence may be a global phenomenon and that G1/S genes may be coordinately controlled.

Actins↗

PubMed1998

Increase in telomere sequence-binding activity in normal human fibroblasts in senescence or in cells treated with phorbol ester or N-methyl-N'-nitro-N-nitrosoguanidine.

The telomere is a specialized chromatin structure composed of unique repetitive DNA sequences and specific nuclear proteins. Telomere sequence-binding activity was measured by a mobility shift assay using nuclear extract from normal human fibroblasts. The specific binding activity to the telomere sequence increased in cells that were in a senescence state compared to that in cells at early population doublings. Treatment of cells with tumor promoting phorbol ester TPA induced an increase in the telomere sequence binding activity of nuclear extract in young cells, but the increase was marginal in senescent cells. DNA-damaging N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) also increased the telomere sequence binding activity in young cells, but not in senescent cells. As a reference, we measured the binding activity to NFkB sequence. It was activated by TPA or okadaic acid, but was not affected by MNNG or in senescence. The increase in telomere sequence-binding activity seemed to depend on activation of tyrosine phosphorylation, since an inhibitor of Tyr-kinase abolished the increase in telomere-binding activity. The molecular weight of the major binding factor in the normal human fibroblasts was approximately 32 kDa which is different from that of the telomere-associated protein, TRF-1.

Carcinogens↗

PubMed2000

Impairment of osteocalcin production in senescent periodontal ligament fibroblasts.

Osteocalcin production of senescent periodontal ligament fibroblasts (PDLF) with the expression of senescence-associated beta-galactosidase (SA-beta-Gal) was investigated on clones from 50-80 years old donors (n=20) with teeth extracted due to periodontitis and dental caries, and from 15-19 year old donors (n=20) with normal teeth extracted for orthodontic reasons. Immunohistochemically, the nonsenescent PDLF in all cultures in passage 2 showed strong reactivity with anti-osteocalcin. The reactive intensity of PDLF (passage 2, PD 3.0) was significantly stronger in 50-80 year old donor group than in 15-19 year old donor group, suggesting that osteocalcin production of PDLF cultured in early passage is larger in cells from adult population than in cells from adolescent population. In PDLF cultures in passage 2 from 50-80 year old donor, two types of senescent cells were found: one with strong reactivity to anti-osteocalcin and the other with little detectable reactivity. The culture consisted of senescent PDLF (passage 8, PD 14.8) did not include cells which have a detectable reactivity with anti-osteocalcin immunohistochemically and the reactive intensity was significantly weaker in the senescent culture than in the culture in passage 2 by ELISA. This suggests that the production potential of osteocalcin is impaired in PDLF with aging in culture. Further, the reactive intensity with anti-osteocalcin of PDLF in passage 2 deprived of serum for 48 h was 6% of that of cells cultured with serum and the reaction increased after serum stimulation, suggesting that the osteocalcin production in PDLF in early passage is implicated in mitogenic stimulation.

Adolescent↗

PubMed1995

Origins of G1 arrest in senescent human fibroblasts.

Human diploid fibroblasts have a finite proliferative lifespan in culture, at the end of which they are arrested with G1 phase DNA contents. Upon serum stimulation, senescent cells are deficient in carrying out a subset of early signal transduction events such as activation of protein kinase C and induction of c-fos. Later in G1, they uniformly fail to express late G1 genes whose products are required for DNA synthesis, implying that they are unable to pass the R point. Failure to pass the R point may occur because senescent cells are unable to phosphorylate the retinoblastoma protein, owing to the accumulation of inactive complexes of cyclin E/Cdk2 and possibly cyclin D/Cdk4. Senescent cells contain high amounts of p21, a potent cyclin-dependent kinase inhibitor whose levels are also elevated in cells arrested in G1 following DNA damage, suggesting that both arrests might share a common mechanism. Cell aging is accompanied by a progressive shortening of chromosomal telomeres, which could be perceived by the cells as a form of DNA damage that gives rise to the signals that inactive the cell cycle machinery.

Cell Cycle↗

PubMed2004

Proteasome inhibition induces a senescence-like phenotype in primary human fibroblasts cultures.

Senescent human fibroblasts exhibit several genetic and biochemical differences as compared to their young counterparts including abnormalities of the main proteolytic mechanism, namely the proteasome. Specifically, we and others have shown that there is an impaired function of the proteasome, as senescent cells have reduced proteolytic activities and less proteasome content. In a complementary work we have recently shown that inhibition of the proteasome by a specific inhibitor induces a senescence-like phenotype in young WI38 fibroblasts [Chondrogianni et al. (2003) J Biol Chem 278: 28026-28037]. In this study we tested whether the induction of a senescence-like phenotype following treatment with proteasome inhibitors is a common feature of primary human fibroblasts. A comparative biochemical analysis, after employing three different human fibroblasts cell lines (IMR90, MRC5 and WI38 cells), as well as two proteasome inhibitors (epoxomicin and MG132), has shown that proteasome inhibition results in the appearance of a senescence-like phenotype in all cell lines used. Proteasome inhibitors treated cells were irreversibly stopped dividing, exhibited positive staining to beta-galactosidase as well as reduced CT-L and PGPH activities. In summary, these data reveal the fundamental role of the proteasome in the progression of replicative senescence and open new dimensions towards a better understanding of protein degradation.

Cell Division↗

PubMed2003

An analysis of replicative senescence in dermal fibroblasts derived from chronic leg wounds predicts that telomerase therapy would fail to reverse their disease-specific cellular and proteolytic phenotype.

The accumulation of senescent fibroblasts within tissues has been suggested to play an important role in mediating impaired dermal wound healing, which is a major clinical problem in the aged population. The concept that replicative senescence in wound fibroblasts results in reduced proliferation and the failure of refractory wounds to respond to treatment has therefore been proposed. However, in the chronic wounds of aged patients the precise relationship between the observed alteration in cellular responses with aging and replicative senescence remains to be determined. Using assays to assess cellular proliferation, senescence-associated staining beta-galactosidase, telomere length, and extracellular matrix reorganizational ability, chronic wound fibroblasts demonstrated no evidence of senescence. Furthermore, analysis of in vitro senesced fibroblasts demonstrated cellular responses that were distinct and, in many cases, diametrically opposed from those exhibited by chronic wound fibroblasts. Forced expression of telomerase within senescent fibroblasts reversed the senescent cellular phenotype, inhibiting extracellular matrix reorganizational ability, attachment, and matrix metalloproteinase production and thus produced cells with impaired key wound healing properties. It would appear therefore that the distinct phenotype of chronic wound fibroblasts is not simply due to the aging process, mediated through replicative senescence, but instead reflects disease-specific cellular alterations of the fibroblasts themselves.

Cell Adhesion↗

PubMed2007

Sp1 is essential for p16 expression in human diploid fibroblasts during senescence.

BACKGROUND: p16(INK4a) tumor suppressor protein has been widely proposed to mediate entrance of the cells into the senescent stage. Promoter of p16(INK4a) gene contains at least five putative GC boxes, named GC-I to V, respectively. Our previous data showed that a potential Sp1 binding site, within the promoter region from -466 to -451, acts as a positive transcription regulatory element. These results led us to examine how Sp1 and/or Sp3 act on these GC boxes during aging in cultured human diploid fibroblasts. METHODOLOGY/PRINCIPAL FINDINGS: Mutagenesis studies revealed that GC-I, II and IV, especially GC-II, are essential for p16(INK4a) gene expression in senescent cells. Electrophoretic mobility shift assays (EMSA) and ChIP assays demonstrated that both Sp1 and Sp3 bind to these elements and the binding activity is enhanced in senescent cells. Ectopic overexpression of Sp1, but not Sp3, induced the transcription of p16(INK4a). Both Sp1 RNAi and Mithramycin, a DNA intercalating agent that interferes with Sp1 and Sp3 binding activities, reduced p16(INK4a) gene expression. In addition, the enhanced binding of Sp1 to p16(INK4a) promoter during cellular senescence appeared to be the result of increased Sp1 binding affinity, not an alteration in Sp1 protein level. CONCLUSIONS/SIGNIFICANCE: All these results suggest that GC- II is the key site for Sp1 binding and increase of Sp1 binding activity rather than protein levels contributes to the induction of p16(INK4a) expression during cell aging.

Base Composition↗

PubMed2004

Nuclear accumulation of glycogen synthase kinase-3 during replicative senescence of human fibroblasts.

Activation of the tumor suppressor protein p53 contributes to cellular senescence. As glycogen synthase kinase-3 (GSK3) was recently found to interact with p53 and contribute to the actions of p53, this study examined whether GSK3 accumulated in the nucleus and associated with p53 in senescent cells. Compared with young and middle-aged human WI-38 fibroblasts, senescent cells were found to contain increased nuclear levels of GSK3beta, and also tended to accumulate in the nucleus the other isoform of GSK3, GSK3alpha. Co-immunoprecipitation experiments demonstrated that GSK3beta and p53 formed a complex in the nucleus. Further experiments tested whether inhibition of GSK3 altered the development of senescence using long-term treatment with the selective GSK3 inhibitor lithium. Lithium treatment reduced the senescence-associated accumulation of p53 and caused cells to enter a reversible quiescent state. These results indicate that a portion of the p53 that is activated in senescent cells is modulated by its association with GSK3beta in the nucleus, an association that is known to facilitate the actions of p53 and that may contribute to senescence.

Cell Nucleus↗

PubMed1982

Altered degradation of proteins microinjected into senescent human fibroblasts.

Ribonuclease A was introduced into the cytoplasm of IMR-90 human diploid fibroblasts by red cell-mediated microinjection. Early passage fibroblasts degraded ribonuclease A with a half-life of approximately 90 h in the presence of 10% fetal calf serum and enhanced the degradative rate 1.6-fold upon serum withdrawal. Senescent cells degraded ribonuclease A more slowly with half-lives ranging between 125 and 250 h and had diminished capacities to enhance the catabolism of this protein during serum starvation. Decreased protein degradation in senescent cells was also evident for microinjected RNase S-protein, RNase B, aldolase, lysozyme, and the synthetic copolymer polyglutamate: tyrosine:alanine (1:1:1). These alterations in the mechanisms and regulation of intracellular protein degradation may contribute to several biochemical abnormalities characteristic of aging cells and organisms.

Cell Line↗

PubMed1995

Chromosomal analysis in young vs. senescent human fibroblasts by fluorescence in situ hybridization: a selection hypothesis.

Almost all previous studies on chromosomal analysis related to in vitro aging of human fibroblasts were done using only metaphase chromosomes. However, this procedure may provide only partial information since the aneuploidy presumably hidden in interphase cells would remain undetected. For this reason, we have analyzed aneuploidy both at interphase and at metaphase. Female (IMR-90) and male (IMR-91) cells were grown from the lowest to the highest population doubling levels and aneuploidy analysis was done by FISH with alpha-satellite DNA probes of 15 autosomes and two sex chromosomes. Our data on total aneuploidy in young cells indicate that significantly higher proportions of cells with aneuploidy can be detected at interphase than at metaphase. This presumably indicates that during active division of young cells, more aneuploid than diploid cells are selected against entry to mitosis. In contrast, interphase senescent cells from both strains show significantly fewer aneuploid nuclei than do young cells at interphase. This probably indicates that during senescence, there is greater selective pressure in the culture against long-term survival of aneuploid cells than against survival of diploid cells. Our study shows that cellular dynamics with respect to aneuploidy involving various chromosomes differs significantly at interphase and at mitosis during in vitro aging of human fibroblasts, and we propose a 'Selection Hypothesis' as an explanation to our findings.

Aneuploidy↗

PubMed2000

Re-modelling of nuclear architecture in quiescent and senescent human fibroblasts.

Spatial organisation of the genome within the nucleus can play a role in maintaining the expressed or silent state of some genes [1]. There are distinct addresses for specific chromosomes, which have different functional characteristics, within the nuclei of dividing populations of human cells [2]. Here, we demonstrate that this level of nuclear architecture is altered in cells that have become either quiescent or senescent. Upon cell cycle exit, a gene-poor human chromosome moves from a location at the nuclear periphery to a more internal site in the nucleus, and changes its associations with nuclear substructures. The chromosome moves back toward the edge of the nucleus at a distinctive time after re-entry into the cell cycle. There is a 2-4 hour period at the beginning of G1 when the spatial organisation of these human chromosomes is established. Lastly, these experiments provide evidence that temporal control of DNA replication can be independent of spatial chromosome organisation. We conclude that the sub-nuclear organisation of chromosomes in quiescent or senescent mammalian somatic cells is fundamentally different from that in proliferating cells and that the spatial organisation of the genome is plastic.

Cell Division↗

PubMed2004

Heat shock protein 47 expression in aged normal human fibroblasts: modulation by Salix alba extract.

Heat shock protein (HSP) 47 is a specific chaperone of procollagen. This heat shock protein is responsible for the correct three-dimensional organization of procollagen and its control-quality prior secretion. The aim of the study is to evaluate the level of HSP 47 in aged, photoaged, and senescent fibroblasts and its modulation by a plant extract (Salix alba). The level of HSP 47 and/or procollagen expression in fibroblasts was measured by real-time RT-PCR (mRNA transcripts) and by flow cytometry (immunochemistry technique for measurement of arbitrary fluorescence intensity). Immunochemistry techniques and confocal microscopy were used to visualize the cellular localization of HSP 47 and procollagen. These parameters were compared with different age donors, nonsenescent, and senescent fibroblasts. Fibroblasts were irradiated by a noncytotoxic dose of UVA (6 J/cm(2)), and HSP 47 level was evaluated. S. alba extract was tested for its capacity to modulate HSP 47 expression. Colocalization of HSP 47 and procollagen was shown by confocal microscopy, indicating that HSP 47 could play a role of procollagen molecular chaperone in the cellular model. It was also shown that the HSP 47 level is decreased in old-donor cells, senescent, and irradiated cells. This decrease can be modulated by a S. alba extract (polyphenols rich) in a dose-dependent manner. The evaluation of HSP 47 expression in the experimental conditions can lead to a new approach of aging and photoaging, pointing out the implication of this chaperone in these pathophysiologic phenomena. Modulation of HSP 47 expression by this family of molecules could be of cosmetic and/or dermatologic interest.

Adult↗

PubMed1990

Potential role of anti-oncogenes in aging.

Some observations on cellular senescence are discussed regarding the possibility that the postulated genes, which bring about the DNA synthesis block of senescent fibroblasts, might be similar to those designated as anti-oncogenes or tumour suppressor genes. The latter genes have been defined by tumorogenicity tests of hybrids from fused tumour and normal cells, of which the retinoblastoma (RB) gene is a prototype. Similarities between the two systems are considered, which are consistent with the assumption that senescent growth arrest, like the suppression of tumorogenicity of the hybrids, might be effected by anti-oncogenes via inhibition of cellular oncogenes in their growth signal transduction functions. A link may be provided by the finding of a direct interaction of certain DNA tumour virus oncoproteins with the RB gene product p105-RB, together with observations that the tumour viruses are also able to reinitiate DNA synthesis in growth arrested senescent fibroblasts. The possibility is also discussed that some silent retinoblastoma-like genes might become aberrantly expressed in senescent cells owing to a progressive loss of 5-methyl cytosine residues of DNA.

Aging↗