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Results for “Senescent fibroblasts”

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PubMed1999

The mRNA phenotype of a human RPE cell line at replicative senescence.

PURPOSE: To explore the changes in expression of a set of genes in a single retinal pigment epithelial (RPE) cell line and two fibroblast cell lines as controls under culture conditions previously used for the analysis of senescent gene expression. METHODS: A single human RPE cell line, which had previously been characterized using known markers of senescence, and two fibroblast cell lines were grown to replicative exhaustion. The mRNA phenotype of genes known to be altered by senescence were studied by quantitative Northern analysis. RESULTS: The mRNA phenotype of cells changes at replicative senescence yielding a synthetic phenotype which is similar to cells found in repairing wounds. Of the genes studied, urokinase-type plasminogen activator and plasminogen activator inhibitor-1 were regulated in RPE cells similar to fibroblasts at senescence. The largest changes noted for any single gene were the upregulation of insulin growth factor binding protein 2, and the downregulation of collagen I alpha 2, basic fibroblast growth factor, and fibroblast growth factor-5. CONCLUSIONS: This study demonstrates an altered mRNA phenotype of a human RPE cell line grown to replicative exhaustion. This analysis of a single cell line emphasizes the variability of results based on a single cell line or tissue specimen and indicates the need for additional study.

Blotting, Northern↗

PubMed2001

Energetic stress induces premature aging of diploid human fibroblasts (Wi-38) in vitro.

Oxidative phosphorylation is the main endogenous source for the generation of reactive oxygen species (ROS). In order to investigate the influence of enhanced ROS production on the in vitro senescence of Wi-38 fibroblasts, cells were cultivated in medium with elevated (hypertonic) NaCl concentrations. The number of active Na(+)/K(+)-ATPase molecules per cell was found to be increased. A rise in both respiration and glycolysis as evidenced by the increases in oxygen and glucose consumption and lactate production was revealed. Cells stayed alive in medium with NaCl concentrations of up to 0.30 M and could be adapted to growth under these hypertonic conditions (high-NaCl tolerant cells). These cells exhibited an increased cell size and protein content. A growing number of cells showed stress fibers and granulation. The proliferation rate and the maximum number of cumulative population doublings of these high-NaCl tolerant cultures were reduced and saturation density was decreased. Thus, these cells under energetic stress due to increased energy requirements for active ion transport expressed features typical for aging in vitro. We conclude therefore that energetic stress induces premature aging in human diploid fibroblasts.

Journal Article↗

PubMed1988

Cell surface oligosaccharide modulation during differentiation: IV. Normal and transformed cell growth control.

In previous studies it was shown that cell surface oligosaccharide affinity class distributions and binding capacities were down-regulated as normal cells approach senescence. Using a sensitive, amplified, lectin/specific-ligand competition analysis three other growth regulation states were compared to that of cellular senescence. Non-senescent and senescent low-density and contact-induced growth inhibition was compared with neoplastic cell growth control. Non-senescent human fetal lung fibroblasts (IMR-90) down-regulated their mannosyl and galactosyl specificities in response to both low-density and contact-induced growth inhibition. Senescent IMR-90 down-regulate their mannosyl residues in response to contact conditions while they up-regulate their galactosyl residues under the same conditions. Growth-transformed transplantable canine glioma cells did not show density-dependent regulation of their cell surface oligosaccharide structures. Modulation of the CG cells with a specific alpha-mannosidase II inhibitor, Ricinus communis a galactosyl specific lectin, and pokeweed mitogen a cellular differentiating agent resulted in an altered growth phenotype and up-regulation of the mannosyl and galactosyl surface oligosaccharides. These data indicate a controller function for the cell surface oligosaccharides and a general influence on growth control.

Alkaloids↗

PubMed2002

Overexpression of VDUP1 mRNA sensitizes HeLa cells to paraquat.

5-Bromodeoxyuridine (BrdU) induces or suppresses senescence-associated genes in any types of mammalian cells. From a cDNA library upregulated by BrdU in HeLa cells, we identified the gene encoding VDUP1 as a senescence-associated gene in normal human fibroblasts. To address a role of VDUP1 in senescence, we established HeLa cell clones, V7 and V27, which express its mRNA in a doxycycline-dependent manner. Although their growth in liquid culture was moderately retarded, colony formation on semi-solid medium was strongly inhibited by overexpression of the mRNA. We also examined susceptibility of these clones to various reagents. Consequently, colony formation in liquid culture was strongly inhibited by paraquat in these clones. Their superoxide dismutase activity was normal.

Bromodeoxyuridine↗

PubMed1981

Senescence in vitro and ionising radiations--the human diploid fibroblast model.

The influence of ionising radiations on ageing is still controversial. Since Hayflick established the concept that diploid cells have finite lifespan in vitro, human diploid fibroblast (HDF) cultures have been recognised as a potent experimental model for cyto--gerontological investigations. In this study HDF cultures in phase II were exposed to acute irradiation with either X-rays or fast neutrons. The replicative potentials and labelling indices with [3H]thymidine were measured post irradiation until the cultures ceased growth in phase III. Cell mortality was measured by cloning. The apparent loss in replicative potential of irradiated mass cultures was wholly attributable to the loss of viable clonogenic cells. The current concept of precocious clonal senescence in vitro as a late effect of irradiation in clonogenic survivors is not supported by the present experiments. Instead, our results suggest that exposure to a single dose of ionising radiations either causes total replicative incapacitation (killing) of HDF cells and their progeny early after irradiation or leaves their replicative potentials unperturbed.

Cell Survival↗

PubMed2001

Replicative senescence of human endothelial cells in vitro involves G1 arrest, polyploidization and senescence-associated apoptosis.

Human ageing is characterized by a progressive loss of physiological functions, increased tissue damage and defects in various tissue renewal systems. Age-related decreases of the cellular replicative capacity can be reproduced by in vitro assays of cellular ageing. When diploid human fibroblasts reach their finite lifespan, they enter an irreversible G1 growth arrest status referred to as replicative senescence. While deregulation of programmed cell death (apoptosis) is a key feature of age-related pathology in several tissues, this is not reflected in the standard in vitro senescence model of human fibroblasts, and the role of apoptosis during cellular ageing remains unclear. We have analyzed replicative senescence of human umbilical vein endothelial cells (HUVEC) in vitro and found that senescent HUVEC also arrest in the G1 phase of the cell cycle but, unlike fibroblasts, accumulate with a 4N DNA content, indicative of polyploidization. In contrast to human fibroblasts, senescent endothelial cells display a considerable increase in spontaneous apoptosis. The data imply that age-dependent apoptosis is a regular feature of human endothelial cells and suggest cell type specific differences in human ageing.

Apoptosis↗

PubMed2002

Delaying brain mitochondrial decay and aging with mitochondrial antioxidants and metabolites.

Mitochondria decay with age due to the oxidation of lipids, proteins, RNA, and DNA. Some of this decay can be reversed in aged animals by feeding them the mitochondrial metabolites acetylcarnitine and lipoic acid. In this review, we summarize our recent studies on the effects of these mitochondrial metabolites and mitochondrial antioxidants (alpha-phenyl-N-t-butyl nitrone and N-t-butyl hydroxylamine) on the age-associated mitochondrial decay of the brain of old rats, neuronal cells, and human diploid fibroblast cells. In feeding studies in old rats, these mitochondrial metabolites and antioxidants improve the age-associated decline of ambulatory activity and memory, partially restore mitochondrial structure and function, inhibit the age-associated increase of oxidative damage to lipids, proteins, and nucleic acids, elevate the levels of antioxidants, and restore the activity and substrate binding affinity of a key mitochondrial enzyme, carnitine acetyltransferase. These mitochondrial metabolites and antioxidants protect neuronal cells from neurotoxin- and oxidant-induced toxicity and oxidative damage; delay the normal senescence of human diploid fibroblast cells, and inhibit oxidant-induced acceleration of senescence. These results suggest a plausible mechanism: with age, increased oxidative damage to proteins and lipid membranes, particularly in mitochondria, causes a deformation of structure of enzymes, with a consequent decrease of enzyme activity as well as substrate binding affinity for their substrates; an increased level of substrate restores the velocity of the reaction and restores mitochondrial function, thus delaying mitochondrial decay and aging. This loss of activity due to coenzyme or substrate binding appears to be true for a number of other enzymes as well, including mitochondrial complex III and IV.

Acetylcarnitine↗

PubMed1983

Human diploid fibroblasts (HDF) can induce DNA synthesis in cycling HDF but not in quiescent HDF or senescent HDF.

HeLa cells in S phase induce DNA synthesis in cycling cells, serum-deprived quiescent cells, and non-replicative senescent cells following cell fusion. In contrast normal human diploid fibroblasts (HDF) do not induce DNA synthesis in either quiescent cells or senescent cells. Instead, the replicative HDF nuclei are inhibited from entering S phase in heterokaryons formed with these two types of non-replicative cells. These differences in the inducing capabilities of normal HDF and HeLa cells raise the question whether normal HDF in S phase can induce DNA synthesis in cycling cells. This paper demonstrates that young HDF in S phase can induce DNA synthesis in cycling HDF. Thus, the hypothesis that initiation of DNA synthesis in cycling cells is positively controlled by inducer molecules appears to be valid for normal HDF as well as for transformed cells such as HeLa.

Cell Cycle↗

PubMed2004

Telomere length in leukocytes and cultured gingival fibroblasts from patients with aggressive periodontitis.

BACKGROUND: The association of genetic risk factors with the pathogenesis of aggressive periodontitis (AgP) has been a focus of attention. Telomeres, which are nucleoprotein complexes at the ends of chromosomes, could be a genetic marker for Down's syndrome and Hutchinson-Gilford progeria, in which patients' premature aging is involved in the pathogenesis. It has been reported that these patients tend to experience severe periodontitis. Therefore, we investigated the telomere length of peripheral blood leukocytes (PBL) obtained from patients with AgP and that in the patients' gingival fibroblasts undergoing cellular aging in vitro. METHODS: Twenty-one patients with AgP and 50 age-matched, periodontally healthy subjects (HS) participated in this study. Genomic DNA from PBL and from human gingival fibroblasts (HGF) was analyzed by Southern blotting for telomere length. The percentage of HGF positive for beta-galactosidase (beta-gal), a marker for cellular senescence, was also investigated. RESULTS: There was no significant difference in the telomere length (P = 0.20, Student's t test) between the two groups, and wide interindividual variation was found (5.93 to 11.4 kbp, average 8.35 +/- 1.19 kbp). The telomere length from PBL negatively correlated with donor age, but no significant difference in telomere loss between the two groups was observed. With HGF undergoing aging in culture, the mean telomere length of these cells from six patients with AgP and seven HS decreased an average of -67.5 bp and -81.0 bp, respectively. No association was found in the telomere length between PBL and HGF from the same donors (r = 0.56, P = 0.20). A significant association was found between the telomere length and the percentages of beta-gal-positive HGF during cell passages (r = 0.70, P < 0.001). CONCLUSIONS: These results indicate that patients with AgP do not have excessive telomere loss and thus do not support the notion of the occurrence of a generalized premature cellular aging in patients with AgP. Further studies are required to investigate the association between telomere length and beta-gal in HGF.

Acute Disease↗

PubMed2005

Glycolytic enzymes can modulate cellular life span.

An unbiased screen for genes that can immortalize mouse embryonic fibroblasts identified the glycolytic enzyme phosphoglycerate mutase (PGM). A 2-fold increase in PGM activity enhances glycolytic flux, allows indefinite proliferation, and renders cells resistant to ras-induced arrest. Glucosephosphate isomerase, another glycolytic enzyme, displays similar activity and, conversely, depletion of PGM or glucosephosphate isomerase with short interfering RNA triggers premature senescence. Immortalized mouse embryonic fibroblasts and mouse embryonic stem cells display higher glycolytic flux and more resistance to oxidative damage than senescent cells. Because wild-type p53 down-regulates PGM, mutation of p53 can facilitate immortalization via effects on PGM levels and glycolysis.

Animals↗

PubMed1996

Down-regulation of nuclear binding activities of OXBOX-REBOX transcription factors during cellular senescence.

Functional capacity of mitochondria declines during aging and this impairment may have a major role in aging process. Several observations indicate that transcriptional efficiency is reduced during aging. Our purpose was to find out whether aging and cellular senescence affect the nuclear binding activities of transcription factors which bind to OXBOX-REBOX sequence present in promoter regions of numerous nuclear genes encoding mitochondrial proteins. These factors regulate and coordinate the expression of mitochondrial proteins. We observed a strong down-regulation in the nuclear binding activities of OXBOX-REBOX factors in replicatively senesced human WI-38 and IMR-90 fibroblasts. On the contrary, SV-40 immortalization highly increased the nuclear binding activities. A considerable down-regulation of OXBOX-REBOX factors was also observed in UVB-irradiated WI-38 fibroblasts. Irradiation induced photoaging in fibroblasts which involved cell cycle arrest and senescent morphology. Interestingly, the nuclear binding activities of OXBOX-REBOX factors were also prominently decreased in the liver of Wistar rats at the age of 30 months but not yet at the age of 18 months. Our results suggest that the down-regulation of OXBOX-REBOX factors could affect the expression level of mitochondrial proteins encoded in nucleus and hence induce disturbances in mitochondrial function and promote the cellular aging process.

Animals↗

PubMed2004

Growth retardation and premature aging phenotypes in mice with disruption of the SNF2-like gene, PASG.

Imperfect maintenance of genome integrity has been postulated to be an important cause of aging. Here we provide support for this hypothesis by demonstrating that the disruption of PASG (lsh), a SNF2-like factor that facilitates DNA methylation, causes global hypomethylation, developmental growth retardation and a premature aging phenotype. PASG mutant mice display signs of growth retardation and premature aging, including low birth weight, failure to thrive, graying and loss of hair, reduced skin fat deposition, osteoporosis, kyphosis, cachexia, and premature death. Fibroblasts derived from PASG mutant embryos show a replicative senescence phenotype. Both PASG mutant mice and fibroblasts demonstrate a markedly increased expression of senescence-associated tumor suppressor genes, such as p16(INK4a), that is independent of promoter methylation, but, instead, is associated with down-regulation of bmi-1, a negative regulator of p16(INK4a). These studies show that PASG is essential for properly maintaining DNA methylation and gene expression patterns that are required for normal growth and longevity. PASG mutant mice provide a useful model for the study of aging as well as the mechanisms regulating epigenetic patterning during development and postnatal life.

Aging, Premature↗

PubMed1994

Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells.

Human diploid fibroblast cells lose replicative potential after a certain number of population doublings. We use this experimental system to investigate the role of oxidative damage in cellular aging. Treating cells with H2O2 at < 300 microM did not affect the viability of the majority of cells when judged by morphology, trypan blue exclusion, and protein synthesis. However, the treatment caused a dose-dependent inhibition of DNA synthesis. After a 2-hr treatment with 200 microM H2O2, the cells failed to respond to a stimulus of serum, platelet-derived growth factor, basic fibroblast growth factor, or epidermal growth factor by synthesizing DNA, and the loss of response could not be recovered by 4 days. Subcultivation showed that, as in senescent cells, division of the treated cells was inhibited. The life-time cumulative growth curve showed that the loss of replication due to H2O2 treatment was cumulative and irreversible. The H2O2 treatment decreased the number of the population doublings in the rest of the life span by 35.3 +/- 10.3%. Enzymatic assays indicated that, like the cells in their senescent state, the treated cells were less able to activate ornithine decarboxylase and thymidine kinase. Furthermore, subcultivation after the H2O2 treatment showed that the cells developed the morphology of senescent cells. In conclusion, sublethal treatment of H2O2 "stunned" F65 cells and caused the cells to enter a state resembling senescence.

Amino Acids↗

PubMed2000

Inactivation of p53 and life span extension of human diploid fibroblasts by mot-2.

Normal human lung fibroblasts were transfected with expression plasmids encoding mot-2, an hsp70 family member that is associated with the immortal phenotype. After the empty vector-transfected controls had become senescent and positive for senescence-associated beta-galactosidase (SA-beta-gal), the mot-2-expressing cells continued to proliferate for an additional 12-18 population doublings and showed a young cell morphology and much lower SA-beta-gal activity. The tumor suppressor p53 was found to be transcriptionally inactivated in life span-extended cells. We have thus shown for the first time that overexpression of mot-2 in normal human cells is able to permit their temporary escape from senescence.

Cell Division↗

PubMed2001

Multilineage cells from human adipose tissue: implications for cell-based therapies.

Future cell-based therapies such as tissue engineering will benefit from a source of autologous pluripotent stem cells. For mesodermal tissue engineering, one such source of cells is the bone marrow stroma. The bone marrow compartment contains several cell populations, including mesenchymal stem cells (MSCs) that are capable of differentiating into adipogenic, osteogenic, chondrogenic, and myogenic cells. However, autologous bone marrow procurement has potential limitations. An alternate source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous. In this study, we determined if a population of stem cells could be isolated from human adipose tissue. Human adipose tissue, obtained by suction-assisted lipectomy (i.e., liposuction), was processed to obtain a fibroblast-like population of cells or a processed lipoaspirate (PLA). These PLA cells can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of PLA cells are of mesodermal or mesenchymal origin with low levels of contaminating pericytes, endothelial cells, and smooth muscle cells. Finally, PLA cells differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, the data support the hypothesis that a human lipoaspirate contains multipotent cells and may represent an alternative stem cell source to bone marrow-derived MSCs.

Adipocytes↗

PubMed2004

Disparate effects of telomere attrition on gene expression during replicative senescence of human mammary epithelial cells cultured under different conditions.

Telomere shortening in populations of human mammary epithelial cells (HMECs) that survive early replicative arrest (M0) by the inactivation of p16(INK4A) during cell culture on plastic dishes leads to a state of permanent replicative arrest termed senescence. While culture of HMECs on feeder layers abrogates M0 and p16(INK4A) inactivation, progressive telomere attrition in these cells also eventually results in permanent replicative arrest. Expression of telomerase prevents both senescence on plastic (S-P) and senescence on feeder layers (S-FL) in HMECs, as it does also in cultured primary human fibroblasts. We report here that the gene expression profiles of senescence in HMECs of the same lineage maintained under different culture conditions showed surprisingly little commonality. Moreover, neither of these senescence-associated profiles in HMECs resembles the profile for senescence in human fibroblasts. These results indicate that senescence-associated alterations in gene expression resulting from telomere attrition are affected by culture conditions as well as by cell origins, and argue that replicative senescence at the molecular level is a diverse rather than unique cellular process.

Adult↗

PubMed1993

Suppression of the growth factor-mediated induction of c-fos and down-modulation of AP-1-binding activity are not required for cellular senescence.

It has been suggested that the impaired response of the c-fos gene to serum growth factors and the concomitant loss of AP-1 activity may be a crucial step in the process of cellular senescence. In the present study, we provide evidence arguing against such a conclusion. Data obtained in five independent experiments showed that both c-fos RNA and protein expression were similar in 'young' and in senescent serum-stimulated WI-38 cells, suggesting that the previously reported suppression of c-fos induction is not an obligatory event in the process of cellular senescence. Likewise, expression of fra-1, c-jun and junB continued to be high in serum-stimulated senescent cells, while induction of fosB was reduced approximately fivefold. Among all genes tested fosB thus seems to be the most suitable marker for the detection of senescent cells. Stimulated senescent cells showed only a approximately twofold reduction of AP-1-binding activity, and senescent cells continuously exposed to serum exhibited normal AP-1-binding activity. These observations support the conclusion that a down-modulation of AP-1 is not crucial for human fibroblasts to enter the senescent state.

Cell Line↗

PubMed1995

Cellular aging and transformation suppression: a role for retinoic acid receptor beta 2.

Cellular senescence is characterized by a finite proliferative capacity in vitro. Moreover, the proliferative capacity of dermal fibroblasts harvested from humans is inversely proportional to the age of the donor, suggesting that senescence in culture is a manifestation, at the cellular level, of processes that occur during in vivo human aging. As cellular senescence is a program that ultimately decreases cell proliferation, it has been hypothesized that the genetic mechanisms responsible for the negative growth regulation of senescence may also be involved in the suppression of neoplastic transformation. Retinoic acid (RA) and its derivatives are effective negative growth regulators and are known to inhibit tumor growth, in vitro and in vivo. As a first step in examining a role for retinoic acid in the regulation of cellular aging in human fibroblasts, we examined the expression of the nuclear receptors for RA (RAR alpha, RAR beta, and RAR gamma) in human donors of different ages. These studies demonstrate a selective up-regulation of RAR beta, in response to RA, in fibroblasts that manifest a decreased proliferative capacity. We extend these observations to show that this finding is independent of the age of the donor and correlates with the proliferative capacity of the culture as a whole. Nuclear run-on studies show that the increase in RAR beta mRNA accumulation is mediated by a striking increase in the transcription of the RAR beta 2 isoform. Senescent fibroblasts manifesting the transcriptional increase of the RAR beta 2 isoform also demonstrate transcriptional repression of the protooncogene, c-fos. Functional studies demonstrate that RAR beta 2, like the tumor suppressor gene p53, can inhibit oncogene-induced focus formation. These data provide further support for the contention that genetic events important in cellular senescence may also play a significant role in tumor suppression in humans. Moreover, these observations suggest that RA, through transcriptional regulation of RAR beta 2, may mediate aspects of the negative growth control that characterizes both states.

Actins↗