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Results for “Senescent fibroblasts”

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PubMed2000

Activation of heat shock factor 1 by hyperosmotic or hypo-osmotic stress is drastically attenuated in normal human fibroblasts during senescence.

We have previously reported that osmotic stress prominently induces the DNA binding activity of the heat shock transcription factor 1 (HSF1). In the present study, we examined the effects of medium osmolarity on both the activation of HSF1 and the programmed cell death in normal human fibroblasts during cellular senescence. The activation of HSF1 occurred rapidly in presenescent (early passage) IMR-90 cells when exposed to either hypo-osmotic or hyperosmotic stress. In contrast, the activation of HSF1 was significantly attenuated in senescent cells. Western blot analysis indicated that equal amounts of HSF1 were present as monomers in the cytoplasm of both presenescent and senescent cells in normal growth medium. Under either hypo-osmotic or hyperosmotic stress, trimerization and nuclear localization of HSF1 occurred in presenescent cells but not in senescent cells. More than 80% of HSF1 in senescent cells remained as monomers in the cytoplasm under osmotic stress, suggesting a defect in the signal transduction pathways that lead to HSF1 trimerization or a dysfunction in the HSF1 protein itself. Possible involvement of mitogen-activated protein kinase (MAPK) signal transduction pathways in the activation HSF1 was investigated by monitoring the activation of the three MAPKs, ERK1/2, JNK1/2, and p38, in cells exposed to hypo-osmotic or hyperosmotic stress. All three MAPKs were activated by hyperosmotic stress but not hypo-osmotic stress, suggesting that the MAPK signal transduction pathways may not be directly linked to the osmotic stress-induced activation of HSF1. In contrast to the rapid heat shock transcription factor (HSF) activation, apoptosis occurred only after long-term exposure to hypo-osmotic or hyperosmotic stress. Despite the prominent induction of HSF1 activation, the presenescent cells were more sensitive than the senescent cells to the osmotic stress-induced apoptosis.

Apoptosis↗

PubMed2005

The comet assay approach to senescent human diploid fibroblasts identifies different phenotypes and clarifies relationships among nuclear size, DNA content, and DNA damage.

The comet assay methodology was used to monitor nuclear changes occurring in MRC5 human fibroblasts during transition from young to senescent cultures and to study heterogeneity of senescent populations. Nuclear morphology and size, DNA content per nucleus, and DNA damage (basal strand break, total damage, and oxidized base levels) were evaluated; moreover, visually identified large and small nuclei were analyzed separately and arranged in classes of increasing DNA damage. Oxidized base levels were definitely lower in young versus senescent fibroblasts of which, however, a significant proportion showed negligible DNA damage. Nuclear size enlargement accompanying senescence was almost equally influenced by cell ploidy increase and also by a chromatin decondensation process involving diploid cells. It is noteworthy that DNA damage in senescent fibroblasts correlated significantly to nuclear size, but not to DNA content. The comet assay allowed us to identify different senescent phenotypes and to investigate changes in nuclear features and/or DNA damage irrespective of time elapsed in culture.

Cell Nucleus↗

PubMedDate not supplied

Demethylation of classical satellite 2 and 3 DNA with chromosomal instability in senescent human fibroblasts.

Demethylation of genomic 5-methylcytosine is reported in aged human tissues and senesced human cells, although it is not understood to what extent this phenomenon contributes to replicative senescence. We examined methylation status of satellite 2 and 3 sequences during passages of normal human fibroblasts. These sequences are abundant in the juxtacentromeric heterochromatin of human chromosomes 1, 9 and 16, and heavily methylated in tissues of normal individuals. The decrease in DNA methylation level was two times faster in satellite 3 DNA than in satellite 2 and total DNA. Then we monitored appearance of micronuclei during the passages since they are indicative of heterochromatin decondensation or chromosome breakage. Concomitant with the DNA demethylation, micronuclei containing the heterochromatin of chromosomes 1, 9 or 16, appeared specifically. These results suggest that demethylation of heterochromatin has a role in replicative senescence through chromosome instability.

Cell Line↗

PubMed2003

Comparative levels of DNA breaks and sensitivity to oxidative stress in aged and senescent human fibroblasts: a distinctive pattern for centenarians.

Basal and H(2)O(2)-induced DNA breaks as well as DNA repair activity and efficacy of the antioxygenic system were determined in human dermal fibroblasts explanted from either (i) young donors and passaged serially to reach replicative senescence or (ii) young, old and centenarian donors and shortly propagated in culture. These fibroblasts have been employed as an in vitro and ex vivo model, respectively, to evaluate comparatively DNA integrity during senescence (increasing population doubling levels) and aging (increasing donor age). Constitutive levels of DNA total strand breaks, as determined by the alkaline extraction procedure, changed moderately among the different cell lines, which exhibited, however, significant differences in the amount of either single or double strand breaks. The former decreased along with both aging and senescence; the latter augmented during senescence while being virtually steady during aging. Moreover, fibroblasts from centenarians showed to be less sensitive to H(2)O(2)-induced DNA damage than other ex vivo fibroblasts. This feature could not account for either increased DNA repair activity or higher efficacy of the antioxygenic system and pointed, instead, to an intrinsic nuclear stability which might be typical of centenarian fibroblasts and potentially functional to longevity.

Adolescent↗

PubMed2005

Budded karyoplasts from multinucleated fibroblast cells contain centrosomes and change their morphology to mitotic cells.

Cell changes to accelerated rates of growth are determined by random, new gene mutations that have their origin in vitro in a morphologically visible process of cell alterations. It is a continuous process that, step by step, transforms the normal cell into extended life (EL) cells. These latter cells with limited life spans can further transform to immortalized cells (i.e., cell lines) by the same sequence of morphological cell changes. In contrast to human epithelial cells, fibroblasts in culture have not given rise spontaneously to EL cells. Therefore, it was assumed that some of the cell changes (i.e., cell indicators of the process of transformation) might not be present in near senescent fibroblast cell cultures. As a positive control to normal fibroblast expansion to senescence, the same cells were stressed by inadequate nutrition and confluence. Another positive control was cell indicators induced by SV40 infections. The consecutive sequence of the cell indicators in the transformation process reported previously were: (1) large polyploid cells with nuclei that contained more than two genomes, (2) fragmentation/amitosis of the polyploid nuclei to bi- and multinucleated cells (MNCs), and (3) nuclear budding (i.e., karyoplasts) from MNCs that gave rise to EL cell colonies with various longevities beyond the senescent phase. The present study shows that MNCs and karyoplasts were present in the near-senescent fibroblast cell cultures. Furthermore, new data on the following aspect of the cell transformation process are presented: (1) association of the nuclear fragmentation process with death of cells, (2) cytological markers that distinguish between fragmentation-MNCs versus MNCs from cell fusion, (3) cytological changes of karyoplasts that go through mitotic division to produce daughter cells, and (4) presence of two centrosomes (spindle polar bodies) in the budded karyoplasts. These new findings are discussed in regard to the nuclear fragmentation process in polyploid cells which gives rise to smaller, viable nuclei in MNCs with reduced numbers of whole genomes.

Biomarkers↗

PubMed1994

Regulation of two E2F-related genes in presenescent and senescent human fibroblasts.

Several mammalian genes expressed in late G1 are positively regulated by E2F, a heterodimeric transcription factor. Genes encoding two E2F proteins, E2F-1 and DP-1, were regulated differently during the cell cycle and replicative senescence of normal human fibroblasts. In presenescent cells, E2F-1 mRNA was cell-cycle regulated, appearing a few hours before S phase. By contrast, DP-1 mRNA was constitutively expressed, independent of position in the cell cycle. After a finite number of divisions, normal cells enter a state of irreversible growth arrest termed senescence. Many genes remain mitogen-inducible in senescent cells; there are, however, exceptions, including several late G1 genes potentially regulated by E2F. Senescent cells expressed DP-1 at the presenescent level, but did not express E2F-1 mRNA. Senescent cells were also markedly deficient in E2F binding activity associated with the dihydrofolate reductase promoter. E2F-1 and DP-1 expression vectors only weakly induced DNA synthesis in quiescent or senescent human cells and immortal murine NIH3T3 cells, although the E2F-1 vector stimulated DNA synthesis in immortal murine A31 cells, and transactivated E2F-responsive promoters in NIH3T3 cells. The results suggest that senescent cells may fail to express late G1 genes due to repression of E2F-1, leading to a deficiency of E2F activity. Furthermore, although E2F-1 stimulates DNA synthesis in some cells, other cells, including normal human fibroblasts, require additional factors.

Animals↗

PubMed2004

Loss of proliferative capacity and induction of senescence in oxidatively stressed human fibroblasts.

Cellular senescence can result from short, dysfunctional telomeres, oxidative stress, or oncogene expression, and may contribute to aging. To investigate the role of cellular senescence in aging it is necessary to define the time-dependent molecular events by which it is characterized. Here we investigated changes in levels of key proteins involved in cell cycle regulation, DNA replication, and stress resistance in senescing human fibroblasts following oxidative stress. An immediate response in stressed cells was dephosphorylation of retinoblastoma (Rb) and cessation of DNA synthesis. This was followed by sequential induction of p53, p21, and p16. Increase in hypophosphorylated Rb and induction of p53 and p21 by a single stress treatment was transient, whereas sustained induction or dephosphorylation were achieved by a second stress. Down-regulation of the critical DNA replication initiation factor Cdc6 occurred early after stress concurring with p53 induction, and was followed by a decrease in Mcm2 levels. A late event in the stress-induced molecular sequence was the induction of SOD1, catalase, and HSP27 coinciding with development of the fully senescent phenotype. Our data suggest that loss of proliferative capacity in oxidatively stressed cells is a multistep process regulated by time-dependent molecular events that may play differential roles in induction and maintenance of cellular senescence.

Blotting, Western↗

PubMed2000

Possible role of subunit A of nuclear factor Y (NF-YA) in normal human diploid fibroblasts during senescence.

NF-Y, a heterotrimeric CCAAT binding protein, may have a role in regulating some G1/S genes whose expressions are attenuated during replicative senescence [Matuoka and Chen (1999) Exp Cell Res 253: 365-371]. The hallmark of replicative senescence is the loss of dividing potential. Hence, attenuation of G1/S gene expressions may be causally related to aging. To understand how NF-Y is involved in regulating G1/S genes during replicative senescence, we have examined the expressions of three NF-Y subunit genes in human IMR-90 cells over the entire course of their life-span. The mRNA levels of NF-YA, B, and C did not show any age-dependent change. In contrast, the protein level of NF-YA exhibited a significant and progressive decrease during cell senescence. Cross-linking experiments indicated that NF-Y may interact with proteins such as GCN5 and P/CAF. Co-transfection of cells with plasmid encoding NF-YA protein enhanced the expression of reporter gene fused with G1/S gene promoter that contains NF-Y sites. In contrast, co-transfection with plasmid encoding the dominant negative NF-YA mutant suppressed the expression of the reporter genes. Transient transfection of human cells with dominant negative NF-YA mutant could lead to an increase in neutral beta-galactosidase activity, a marker of cell senescence. These results support the view that NF-Y may have a role in cell senescence.

Acetyltransferases↗

PubMed2003

Mutant p53 can delay growth arrest and loss of CDK2 activity in senescing human fibroblasts without reducing p21(WAF1) expression.

Functional wild-type p53 is required for human diploid fibroblasts (HDF) to enter an irreversible growth arrest known as replicative senescence. Experimentally, abrogation of p53 function by expression of human papillomavirus type 16 E6 or disruption of a key downstream effector p21 by homologous recombination both extended HDF life span. However, although sufficient to extend life span, p21 down-regulation is not necessary, because expression of a dominant-negative mutant p53 (143(ala)) extends life span without apparently decreasing p21 expression. Given the importance of p53 in cellular senescence and the general assumption that p21 may be the sole mediator of its action in this process, we have investigated how abrogation of p53 function can overcome senescence without lowering expression of p21. We have found up-regulated levels of the cyclin-dependent kinase 2 (cdk2) protein in HDF expressing 143(ala) mutant p53 as compared to senescent controls, together with an increase in p21-free cdk2 which, in conjunction with cyclin E, is able to form an active kinase which can phosphorylate the retinoblastoma protein. However, forced overexpression of cdk2 in near-senescent HDF failed to restore cdk2-associated kinase activity. Our data suggest that p53-mediated senescence depends on factor(s) other than p21 which modulate formation of cyclin E-cdk2 complexes.

CDC2-CDC28 Kinases↗

PubMed1995

Evidence for a critical telomere length in senescent human fibroblasts.

Telomeres, the G/C-rich DNA sequences capping the ends of all eukaryotic chromosomes, have been shown to shorten during replicative aging of normal cells in vitro and in vivo. Moreover, variation in the initial length of terminal restriction fragments (TRF) accounts for much of the variation in replicative capacity of fibroblast cultures from different donors. Since replicative capacity also varies significantly between clones in a mass culture of fibroblasts from a single donor, we wished to further test the hypothesis that the shortening of telomeres to a critical or threshold length acts as a signal for cell senescence. Thus, we measured TRF length and total telomeric signal intensity for 35 clonal fibroblast populations at early passage and at senescence. Replicative capacity was found to be directly proportional to mean TRF length (m = 7.2 population doublings/kbp, r = 0.65, P = 0.0004) and total signal intensity (m = 25.0 population doublings/unit, r = 0.63, P < 0.003) at early passage. More importantly, the variability in both mean TRF length and signal intensity (F = 2.0 and 2.9; P = 0.02 and 0.03, respectively) at senescence was markedly less than that at early passage. Although initial telomere length cannot account for all of the interclonal variability in replicative capacity, our observations support the existence of a critical telomere length in senescing cells and a causal role of telomere shortening in cell senescence.

Adult↗

PubMed2006

Role of protein kinase C-dependent A-kinase anchoring proteins in lysophosphatidic acid-induced cAMP signaling in human diploid fibroblasts.

Previously, we reported that lysophosphatidic acid (LPA)-induced adenosine 3',5'-cyclic monophosphate (cAMP) production by human diploid fibroblasts depends on the age of the fibroblasts. In this study, we examined the role of A-kinase anchoring proteins (AKAP) in the regulation of LPA-stimulated cAMP production in senescent fibroblasts. We found that levels of protein kinase C (PKC)-dependent AKAPs, such as Gravin and AKAP79, were elevated in senescent cells. Co-immunoprecipitation experiments revealed that Gravin and AKAP79 do not associate with adenylyl cyclase type 2 (AC2) but bind to AC4/6, which interacts with calcium-dependent PKCs alpha/beta both in young and senescent fibroblasts. When the expression of Gravin and AKAP79 was blocked by small interference RNA transfection, the basal level of cAMP was greatly reduced and the cAMP status after LPA treatment was also reversed. Protein kinase A showed a similar pattern in terms of its basal activity and LPA-dependent modulation. These data suggest that Gravin and to a lesser extent, AKAP79, may play important roles in maintaining the basal AC activity and in coupling the AC systems to inhibitory signals such as Gialpha in young cells, and to stimulatory signals such as PKCs in senescent cells. This study also demonstrates that Gravin is especially important for the long-term activation of PKC by LPA in senescent cells. We conclude that LPA-dependent increased level of cAMP in senescent human diploid fibroblasts is associated with increases in Gravin levels resulting in its increased binding with and activation of calcium-dependent PKC alpha/beta and AC4/6.

A Kinase Anchor Proteins↗

PubMed1985

A 57,000-mol-wt protein uniquely present in nonproliferating cells and senescent human fibroblasts.

Mouse monoclonal antibody, S-30, was produced from hybridoma preparation from mice injected with the cytoskeleton extract of an in vitro aged culture of human fibroblasts derived from a 66-yr-old donor. The antibody stains positively the nuclei of the nonproliferating cells present predominantly in the senescent cultures of five selected fibroblast strains derived from donors of different age groups, whereas a negative reaction is observed in the cultures of their young counterparts. In the intermediate stage of the in vitro life span of these cell strains, a heterogeneous positive reaction for staining with S-30 antibody is observed in different subfractions of cell cultures. However, the expression of S-30 can be induced in the young fibroblasts at the early stage of their life by prolonged culturing to confluence. This induced expression of S-30 nuclear staining can be depleted upon subculturing at low cell density. Immunoelectron microscopy with colloidal gold-protein A complex demonstrates that the S-30 proteins are present in the nuclear plasma and at the region of nuclear envelope in a clustered arrangement. Immunoprecipitation of [3H]leucine labeled cell specimens shows that the antibody S-30 reacts with a protein with a molecular weight of approximately 57,000.

Animals↗

PubMed1991

Senescent human fibroblasts have a post-transcriptional block in the expression of the proliferating cell nuclear antigen gene.

The product of the proliferating cell nuclear antigen (PCNA) gene is the co-factor of DNA polymerase delta, which is required for cellular and viral DNA replication. Its steady-state mRNA levels are growth-regulated in young human diploid fibroblasts (HDF) as well as in many other cell types. In senescent HDF, PCNA mRNA is not detectable. However, the PCNA gene is transcribed in senescent HDF as efficiently as in young cells. Furthermore, PCNA hnRNA is easily detectable by reverse transcriptase-polymerase chain reaction in both senescent and young HDF, and the levels are essentially similar. These results indicate that in senescent HDF which are incapable of synthesizing cellular DNA, one of the genes coding for a protein of the DNA-synthesizing apparatus is still transcribed, but the product fails to be processed into mature mRNA.

Cell Cycle↗

PubMed1999

Senescent human fibroblasts have elevated Ku86 proteolytic cleavage activity.

A proteolytic activity capable of cleaving the Ku86 subunit of Ku protein to two polypeptides, with molecular masses of 69 and 17 kDa in vitro, is present in a human diploid fibroblast (HDF) cell line. The activity is elevated in late-passaged and senescent cells, and the cleaved 69-kDa product seems able to form complex with Ku70 to bind DNA ends. However, further studies indicate that cleavage of Ku86 could be inhibited by including leupeptin in the extraction buffer, and no 69 kDa variant was evident in the cell. In fact, the levels of Ku86, Ku70 and DNA-end binding activity of Ku remained unchanged during replicative senescence. Thus, this study reveals an intriguing protease in HDFs, and also indicates that inconsistent results of Ku86 expression will be obtained if the protease activity is not completely inhibited.

Antigens, Nuclear↗

PubMed2004

Down-regulation of p21WAF1 promotes apoptosis in senescent human fibroblasts: involvement of retinoblastoma protein phosphorylation and delay of cellular aging.

It has been suggested that genes which exercise checkpoint control during cell cycle traverse are equally important to the process of apoptotic cell death. In this study, we show that the key cell cycle regulatory gene p21(WAF1) is also involved in the execution of apoptosis. p21(WAF1) expression was down-regulated during NaBu-induced apoptosis of senescent normal diploid human 2BS fibroblasts. Conversely, when p21(WAF1) expression was actively suppressed in 2BS cells by a stably transfected antisense p21(WAF1) construct, apoptosis was accelerated and senescence was delayed, as shown by several markers of cell aging. Down-regulation of p21(WAF1) by antisense caused an increase in the phosphorylation and inactivation of pRb. Phosphorylation of pRb was further enhanced upon induction of apoptosis by NaBu. Our results suggest that p21(WAF1), acting through the phosphorylation of pRb, regulates whether 2BS cells cease to proliferate and become senescent but resistant to apoptosis, or whether they accelerate proliferation while becoming more susceptible to apoptotic stimuli.

Apoptosis↗

PubMed1992

Senescent human fibroblasts are more sensitive to the effects of a phorbol ester on macromolecular synthesis and growth characteristics.

4-beta-phorbol-12-beta-myristate-13-alpha-acetate (PMA) alters cellular growth properties by modulating gene expression in a wide variety of cell types. Human diploid fibroblasts MRC-5 were treated with PMA at different phases of their lifespan in vitro and the alterations of their short-term growth characteristics and macromolecular synthesis in response to PMA were analysed. PMA stimulates DNA and RNA synthesis in both Phase II (young) and Phase III (senescent) MRC-5 cells. Treatment of senescent cells with various PMA concentrations results in a greater stimulation of DNA and RNA synthesis than that in young cells. Senescent cells are also more sensitive to the PMA-induced alterations of growth characteristics and higher concentrations of PMA become toxic for them. The age-related alterations of cellular responsiveness are also apparent in the gradual loss of responsiveness to serum, observed in parallel with the increased sensitivity to PMA. Furthermore, serum-induced stimulation of macromolecular synthesis is inhibited by PMA. Since it is known that serum and PMA elicit their effects via different signal transduction pathways, our results point to suggest the differential regulation of the signalling mechanisms during cellular ageing.

Cell Division↗

PubMed2002

Microarray analysis of E-box binding-related gene expression in young and replicatively senescent human fibroblasts.

An E-box (CACGTG) designer microarray was developed to monitor a group of genes whose expressions share a particular regulatory mode. Sensitivity and specificity of microarray hybridization, as well as variability of microarray data, were evaluated. This designer microarray was used to generate expression profiles of E-box binding-related genes in WI-38 fibroblast cultures at three different growth states: low-passage replicating, low-passage contact-inhibited quiescent, and replicatively senescent. Microarray gene screening reveals that quiescent and senescent cells, in comparison with replicating ones, are characterized by downregulation of Pam, a protein associated with c-Myc, and upregulation of Mad family genes, Max dimerization proteins. Moreover, quiescence and senescence can be distinguished by increased expression of Irlb, c-Myc transcription factor, and Miz-1, c-Myc-interacting Zn finger protein 1, only in the former state. Senescence is characterized by downregulation of Id4, inhibitor of DNA binding 4, and Mitf, microphthalmia-associated transcription factor, in comparison with young replicating and quiescent states. Differential expression of genes detected by microarray hybridization was independently confirmed by reverse transcription polymerase chain reaction technique. Alterations in the expression of E-box-binding transcription factors and c-Myc-binding proteins demonstrate the importance of these genes in establishing the contact-inhibited quiescent or senescent phenotypes.

Cell Division↗

PubMed1996

The helix-loop-helix protein Id-1 and a retinoblastoma protein binding mutant of SV40 T antigen synergize to reactivate DNA synthesis in senescent human fibroblasts.

Normal somatic cells of higher organisms do not divide indefinitely. After a finite number of divisions, normal cells irreversibly cease proliferation by a process termed replicative or cellular senescence. Replicative senescence is controlled by multiple, dominant-acting genes about which very little is known. The only genes known to reactivate DNA synthesis in senescent cells are viral oncogenes encoding proteins that bind and inactivate the p53 and retinoblastoma (pRb) tumor suppressor proteins. SV40 T antigen is the best studied of these viral oncoproteins. T[K1] is a T antigen point mutant that selectively is defective in binding pRb and the pRb-related proteins p107 and p130. We show that T[K1] stimulated quiescent human fibroblasts to synthesize DNA nearly as well as wild-type T but was incapable of stimulating senescent cells. We tested several growth regulatory genes that are repressed in senescent cells for ability to restore activity to T[K1]. These included c-fos, c-jun, Id-1, Id-2, E2F-1, and cdc2. Only the helix-loop-helix (HLH) protein, Id-1, restored the ability of T[K1] to reactivate DNA synthesis in senescent cells. This activity of Id-1 was not shared by Id-2, a related protein, and depended on an intact HLH domain. It did not appear that Id-1 interacted directly with pRb or p107. Constitutive Id-1 expression failed to rescue proliferating cells from growth inhibition by pRb, p107, or p130, and failed to interact with pRb in the yeast two hybrid system. Because Id proteins negatively regulate basic-HLH (bHLH) transcription factors, we suggest that senescent cells express one or more bHLH factor that cooperates with pRb, or pRb-related proteins, to suppress proliferation.

Antigens, Polyomavirus Transforming↗