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PubMed1990

Replicative senescence: the human fibroblast comes of age.

Human diploid fibroblasts undergo replicative senescence predominantly because of arrest at the G1/S boundary of the cell cycle. Senescent arrest resembles a process of terminal differentiation that appears to involve repression of proliferation-promoting genes with reciprocal new expression of antiproliferative genes, although post-transcriptional factors may also be involved. Identification of participating genes and clarification of their mechanisms of action will help to elucidate the universal cellular decline of biological aging and an important obverse manifestation, the rare escape of cells from senescence leading to immortalization and oncogenesis.

Cell Cycle↗

PubMedDate not supplied

Molecular markers of senescence in fibroblast-like cultures.

The loss of replicative capacity in vitro of normal human diploid fibroblasts is a model for studying molecular changes that accompany both regulated growth control and cellular senescence. We describe the molecular phenotype of senescent fibroblasts in terms of markers that are altered with proliferative decline. We describe these markers by analyzing pathways and associated mechanisms related to the responsiveness of proliferatively competent and senescent cells to growth signals including changes in the extracellular environment, growth factors, growth factor receptors, secondary messengers, cell-cycle progression, transcription factors, and the fidelity of DNA synthesis. There is an abundance of molecular markers for senescence in culture at every level of information transfer. Although it seems clear that some alterations in gene expression with senescence are the result of specific changes in upstream events, more global dysregulation of coordinated growth control point to as yet undefined mechanisms.

Cell Cycle↗

PubMed2003

Reversal of senescence in mouse fibroblasts through lentiviral suppression of p53.

Senescence is generally defined as an irreversible state of G(1) cell cycle arrest in which cells are refractory to growth factor stimulation. In mouse embryo fibroblasts (MEFs), induction of senescence requires the presence of p19(ARF) and p53, as genetic ablation of either of these genes allows escape from senescence and leads to immortalization. We have developed a lentiviral vector that directs the synthesis of a p53-specific short hairpin transcript, which mediates stable suppression of p53 expression through RNA interference. We show that suppression of p53 expression in senescent MEFs leads to rapid cell cycle re-entry, is associated with loss of expression of senescence-associated genes, and leads to immortalization. These data indicate that senescence in MEFs is reversible and demonstrate that both initiation and maintenance of senescence is p53-dependent.

Animals↗

PubMed1991

Ultrastructure of dermal fibroblasts during development and aging: relationship to in vitro senescence of dermal fibroblasts.

One approach to understanding the relationship between in vitro cellular senescence and in vivo aging is to define the development and aging of cells in vivo and then to compare these in vivo properties with the in vitro behavior of the same cells. The Syrian hamster is being used as an experimental aging model to investigate the intrinsic developmental program of dermal fibroblasts in vivo (proliferation, extracellular matrix (ECM) production, quiescence, and reactivation during wound repair) in order to determine whether the in vivo differentiation program and mature function of these cells is related to their in vitro proliferation and senescence pattern. The ultrastructure of dermal fibroblasts from midfetal development through old age is described, and a working hypothesis of the development and aging of dermal fibroblasts is proposed as a framework for further evaluation of the relationship between in vitro proliferative capacity of dermal fibroblasts and in vivo developmental and age-related changes in the dermis.

Aging↗

PubMed2000

Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part I--effects of proliferative senescence.

Oxidized and cross-linked proteins tend to accumulate in aging cells. Declining activity of proteolytic enzymes, particularly the proteasome, has been proposed as a possible explanation for this phenomenon, and direct inhibition of the proteasome by oxidized and cross-linked proteins has been demonstrated in vitro. We have further examined this hypothesis during both proliferative senescence (this paper) and postmitotic senescence (see the accompanying paper, ref 1 ) of human BJ fibroblasts. During proliferative senescence, we found a marked decline in all proteasome activities (trypsin-like activity, chymotrypsin-like activity, and peptidyl-glutamyl-hydrolyzing activity) and in lysosomal cathepsin activity. Despite the loss of proteasome activity, there was no concomitant change in cellular levels of actual proteasome protein (immunoassays) or in the steady-state levels of mRNAs for essential proteasome subunits. The decline in proteasome activities and lysosomal cathepsin activities was accompanied by dramatic increases in the accumulation of oxidized and cross-linked proteins. Furthermore, as proliferation stage increased, cells exhibited a decreasing ability to degrade the oxidatively damaged proteins generated by an acute, experimentally applied oxidative stress. Thus, oxidized and cross-linked proteins accumulated rapidly in cells of higher proliferation stages. Our data are consistent with the hypothesis that proteasome is progressively inhibited by small accumulations of oxidized and cross-linked proteins during proliferative senescence until late proliferation stages, when so much proteasome activity has been lost that oxidized proteins accumulate at ever-increasing rates. Lysosomes attempt to deal with the accumulating oxidized and cross-linked proteins, but declining lysosomal cathepsin activity apparently limits their effectiveness. This hypothesis, which may explain the progressive intracellular accumulation of oxidized and cross-linked proteins in aging, is further explored during postmitotic senescence in the accompanying paper (1).

Cell Division↗

PubMed1986

Fibronectin synthesis and degradation in human fibroblasts with aging.

Fibronectin was measured in early and late passaged human skin fibroblasts utilizing immunoprecipitation and polyacrylamide gel electrophoresis. A progressive increase in the rate of fibronectin and total cellular protein synthesis per cell was observed by late passaged human diploid fibroblasts. The absolute protein concentration increased in the late passaged fibroblasts. There was no significant difference in the [3H]leucine incorporation into fibronectin or total cellular protein/mg protein. The turnover of fibronectin and total cellular protein did not differ between early and late passaged fibroblasts. The transport of fibronectin to the cell membrane was similar in late passaged fibroblasts. The increased fibronectin synthesis in senescent fibroblasts appeared to correlate with the general increase in rate of protein synthesis/cell.

Cell Survival↗

PubMedDate not supplied

Effects of aging on ribosomal protein L7 messenger RNA levels in cultured rat preadipocytes.

Ribosomal protein L7 mRNA is a cell cycle-independent message whose levels are lower in late passage "senescent" fibroblasts than early passage cultures. To determine whether decreases in L7 mRNA levels also occur during aging in tissues in vivo and whether reduced L7 mRNA is caused by terminal differentiation, we measured L7 and adipsin (a differentiation-dependent serine protease) mRNA levels in undifferentiated and differentiated preadipocytes and glyceraldehyde-3-phosphate dehydrogenase mRNA in differentiated preadipocytes cultured from perirenal fat depots of 3-, 17-, and 24-month-old male rats. L7 mRNA levels decreased with increasing age and were not affected by differentiation. In the same cultures, adipsin mRNA levels did not increase with age but did increase with differentiation, confirming that the preadipocytes exposed to enriched medium had, in fact, differentiated. Glyceraldehyde-3-phosphate dehydrogenase mRNA levels did not change with age indicating that the decrease in L7 mRNA was not a result of a general decrease in mRNA with age. These observations are consistent with the hypotheses that decreasing L7 mRNA levels are associated with aging and that late passage fibroblasts have features in common with senescence. The observations are not consistent with the hypothesis that senescent changes in cellular function are caused by terminal differentiation.

Adipocytes↗

PubMed2000

Fas-mediated apoptosis of proliferating, transiently growth-arrested, and senescent normal human fibroblasts.

Previous studies suggest that apoptotic signaling may require proteins that are critical to cellular proliferation and cell cycle regulation. To further examine this question, proliferating, transiently growth-arrested, and senescent normal human fibroblasts were induced to undergo apoptosis in response to two distinct mediators of apoptosis-Fas (APO-1/CD95) death receptor and staurosporine. Ligation of the Fas receptor in the presence of cycloheximide or actinomycin D resulted in apoptosis of proliferating cells, cells transiently growth arrested by gamma-irradiation or serum starvation (i.e., G(0) arrest), and permanently growth-arrested senescent fibroblasts. Proliferating and G(0)-arrested cells were also susceptible to staurosporine-mediated apoptosis. Surprisingly, gamma-irradiated cells did not undergo staurosporine-mediated apoptosis, and remained viable for a prolonged time. Fas-mediated apoptosis of senescent fibroblasts was evidenced by chromosome condensation and by activation of caspase-8 and -3, proteases crucial for the execution of the Fas apoptosis pathway. In addition, ligation of the Fas receptor in G(0)-arrested cells did not result in the activation of p34(cdc2) kinase, arguing that activation of this kinase is not essential in this apoptotic process. From these studies we conclude that proliferating, transiently growth-arrested, and senescent normal human fibroblasts are susceptible to apoptotic signals and that apoptosis is not necessarily dependent upon cell cycle or proliferative state of the cell.

Apoptosis↗

PubMedDate not supplied

Characterization of the absence of an unique DNA-binding protein in senescent but not in their young growing and nongrowing counterparts provides the means to mark the final stage of the cellular aging process.

In senescent fibroblasts, the incapability of cell replication is permanent, and may involve the irreversible loss of gene expressions potentiating the engagement in DNA replication. The initial attempt is to identify gene products that are permanently lost in irreversibly growth-arrested cells. In this article, we report the success in identifying a DNA-associated S6 antigen found in the nuclei of growing and growth-arrested young cells, but not in the nuclei of their senescent counterparts. The presence of the S6 antigen is uniform throughout the nucleoplasm, except in the regions of the nucleoli, and found to be associated with condensed chromosomes in mitotic cells. Treatment with RNase does not abolish the antibody staining activity in the nuclei, while treatment with DNase does remove the activity. Equal intensity of S6 antibody staining is observed in transformed, growing, and contact-inhibited young fibroblasts. Significant reduced level of S6 antibody staining activity is found in the nuclei of senescent fibroblasts, indicating the loss of the expression of this protein during cellular aging process. Immunoblotting assay shows that the S6 antigen is of 50 kDa and with a possibility of a 100 kDa as a dimeric precursor. Our results suggests that a permanent turning off of unique gene expression is associated with the onset of senescence or terminal differentiation, and this hypothesis is supported by the characterization of S6 antigen's absence in in vitro-aged fibroblasts.

Cell Division↗

PubMed2006

Establishment of H2O2-induced premature senescence in human fibroblasts concomitant with increased cellular production of H2O2.

Premature senescence of human fibroblasts is established after exposure to an acute sublethal concentration of H(2)O(2). Overexpression of transforming growth factor-beta1 (TGF-beta1) was shown to be responsible for the appearance of the biomarkers of senescence in these conditions. Other studies have shown that incubation of human fibroblasts with TGF-beta1 leads to overexpression of H(2)O(2). In this work, we show an increased production of H(2)O(2) by human fibroblasts as premature senescence is established after an initial exposure to H(2)O(2).

Cells, Cultured↗

PubMed2003

Altered cAMP signaling induced by lysophosphatidic acid in senescent human diploid fibroblasts.

Lysophosphatidic acid (LPA) is a lipid mitogen that acts through G-protein-coupled receptors. LPA responsiveness has been reported to be dependent on the senescent state of the cells. To solve the mechanism underlying, we observed LPA-dependent cAMP status and found its age-dependent contrasting profile such as high level of cAMP in the senescent cells vs its low level in the young cells. In order to clarify the molecular mechanism of the ageing effect, we examined various molecular species involved in the cAMP signaling pathway by semi-quantitative RT-PCR. EDG-1 and EDG-4 were unchanged, but EDG-2 and EDG-7 were reduced with age. Senescent cells showed a partial reduction of Gi1, Gi2, and Gi3, but no change in the level of Gq. Decreased Gis and Gi-coupled LPA receptors may reduce the inhibitory effect of Gi alpha on adenylyl cyclases (ACs), resulting in cAMP accumulation via activation of adenylyl cyclase in senescent fibroblasts. We also observed an age-dependent increase in some of AC isoforms: II, IV, and VI. In conclusion, multiple changes in the cAMP signaling pathway of the senescent cells might explain the altered responsiveness to the mitogenic stimuli.

Adenylyl Cyclases↗

PubMed1996

Regulation of dihydrofolate reductase gene expression and E2F components in human diploid fibroblasts during growth and senescence.

The induction of dihydrofolate reductase (DHFR), a key enzyme in DNA biosynthesis that is induced just before the onset of S phase, is markedly attenuated in senescent human fibroblasts (Pang and Chen, 1994, J. Cell. Physiol., 160:531-538). Footprinting analysis of the 365 bp promoter region of the human DHFR gene (-381 to -17) indicated that nuclear proteins bind to a cluster of cis-elements, including two overlapping E2F binding sequences, two Sp1 sites, and one Yi sequence. Gel mobility shift assays were performed to assess the role of each cis-element in the regulation of DHFR gene expression. We found that 1) Sp1 binding activity was constitutively expressed throughout the cell cycle in early passage and senescent cells; 2) Yi binding activity was undetectable in both early passage and senescent cells; and 3) E2F binding activity was serum-inducible, senescence-dependent, and prominent in presenescent cells but strikingly diminished in senescent cells. Northern blot analysis of the expression of E2F and DP family members showed that the E2F-1, E2F-4, and E2F-5 mRNA was growth- and senescence-dependent, whereas E2F-3, DP-1, and DP-2 expression was constitutive and senescence-independent. In contrast, E2F-2 mRNA was not detectable in IMR-90 or WI-38 human fibroblasts. Western blot analysis showed that among the E2F-associated proteins, the expression of E2F-1, cyclin A, and cyclin B but not p107 was cell cycle- and senescence-dependent. A nuclear extract mixing experiment suggested that an inhibitory factor may further reduce E2F binding activity in senescent cells.

Base Sequence↗

PubMedDate not supplied

Cultured pressure ulcer fibroblasts show replicative senescence with elevated production of plasmin, plasminogen activator inhibitor-1, and transforming growth factor-beta1.

In a 16-patient study, cultured fibroblast populations from normal skin were able to replicate an average of 14.8 +/- 2.2 times before becoming senescent, while fibroblast populations from the ulcer bed reached the end of their replicative life span after 7.2 +/- 1.9 population doublings (p= 0.001). Fibroblast populations from 10 of 16 pressure ulcers became senescent after fewer than five population doublings, whereas when populations of fibroblasts from adjacent normal skin were studied, only 2 of 16 became senescent within this same time period. In addition, only an occasional fibroblast from normal skin stained positively for senescence-associated beta-galactosidase compared to approximately 50% of equally aged ulcer bed fibroblasts (p = 0.0060). Senescent ulcer bed fibroblasts secreted significantly more plasmin than early passage ulcer bed fibroblasts (p= 0.0237), nearly six times as much plasmin as early passage normal skin fibroblasts (p < 0.0001), three and a half times the level of normal skin fibroblasts of the same age (11.52 +/- 4.58 microg/mg protein; p= 0.0003), and more than one and a half times the level of senescent normal skin fibroblasts (p= 0.0525). Senescent pressure ulcer fibroblasts generated significantly more plasminogen activator inhibitor-1 (1179.27 +/- 25.37 ng/mg protein) than normal skin fibroblasts of the same age (132.16 +/- 16.20 ng/mg protein; p = 0.0357). Also, senescent ulcer bed fibroblasts produced higher levels of transforming growth factor-beta1, but these were not significantly different from senescent normal skin fibroblasts. Although senescent ulcer fibroblasts produce elevated levels of plasminogen activator inhibitor-1 and transforming growth factor-beta1, the ratio of these factors to plasmin levels suggests that this may have little influence on extracellular matrix synthesis or maintenance in the chronic wound. These data show that cultured fibroblasts from most patient pressure ulcers profile a wound environment that is associated with an increasing population of senescent fibroblasts; however, factors within the chronic wound environment that promote cellular senescence remain unclear. We have proposed that a prolonged inflammatory response may be a contributing factor to the chronic wound condition.

Cells, Cultured↗

PubMed2006

Senescence of human fibroblasts after psoralen photoactivation is mediated by ATR kinase and persistent DNA damage foci at telomeres.

Cellular senescence is a phenotype that is likely linked with aging. Recent concepts view different forms of senescence as permanently maintained DNA damage responses partially characterized by the presence of senescence-associated DNA damage foci at dysfunctional telomeres. Irradiation of primary human dermal fibroblasts with the photosensitizer 8-methoxypsoralen and ultraviolet A radiation (PUVA) induces senescence. In the present study, we demonstrate that senescence after PUVA depends on DNA interstrand cross-link (ICL) formation that activates ATR kinase. ATR is necessary for the manifestation and maintenance of the senescent phenotype, because depletion of ATR expression before PUVA prevents induction of senescence, and reduction of ATR expression in PUVA-senesced fibroblasts releases cells from growth arrest. We find an ATR-dependent phosphorylation of the histone H2AX (gamma-H2AX). After PUVA, ATR and gamma-H2AX colocalize in multiple nuclear foci. After several days, only few predominantly telomere-localized foci persist and telomeric DNA can be coimmunoprecipitated with ATR from PUVA-senesced fibroblasts. We thus identify ATR as a novel mediator of telomere-dependent senescence in response to ICL induced by photoactivated psoralens.

Ataxia Telangiectasia Mutated Proteins↗

PubMed1981

Heat-labile glucose-6-phosphate dehydrogenase in cultured fibroblasts from patients with De Sanctis-Cacchione Syndrome.

The normal senescent fibroblasts in culture accumulate a significantly high proportion of altered enzymes, and the alterations are considered to be the manifestation of ageing in molecular terms. To detect the possible molecular alterations in patients with De Sanctis-Cacchione syndrome, the severest form of xeroderma pigmentosum, in which repair processes to UV light-damaged DNA are defective and the neurologic abnormalities are considered to reflect accelerated ageing, we studied the heat stability of glucose-6-phosphate dehydrogenase (G6PD) in crude extracts of cultured skin fibroblasts. Three patients with the syndrome were the center of our investigation. Even at early passage in culture the heat-labile portion of G6PD was increased in the cells from patients in comparison to normal controls. The life span of the cells in culture from patients was not reduced below normal age-matched controls, and no appreciable senescent appearance was observed. The increase in the heat-labile portion of G6PD from cells of DeSanctis-Cacchione syndrome patients to reflect that defective repair of DNA damage occurs, rather than being a direct result of ageing of cultured cells.

Adult↗

PubMed1995

Diminished responsiveness of senescent normal human fibroblasts to TNF-dependent proliferation and interleukin production is not due to its effect on the receptors or on the activation of a nuclear factor NF-kappa B.

The limited life span in culture of normal human diploid fibroblasts (HDF) has provided a model of cellular senescence. The short-term growth of these cells in culture is regulated by a number of different cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1), and fibroblast growth factor (FGF). However, the effect of senescence on the responsiveness of HDF to these cytokines is not known. In the present report, we examined the effects of TNF on foreskin-derived HDF at different passage levels. We compared the response of HDF cells at population doubling (PD) 23 (young) with that of cells at PD 70 (senescent). Young cells proliferated in response to TNF in a dose-dependent manner. Under these conditions TNF had no effect on senescent HDF. The decrease in TNF responsiveness was found to be dependent on PD. The lack of response of senescent HDF was not unique to TNF, since FGF and IL-1 were also ineffective. In contrast to senescent HDF, TNF-dependent proliferation of young HDF could be further potentiated by IL-1 and FGF, suggesting an independent signaling mechanism. On exposure to TNF, senescent HDF produced IL-6 and IL-8, but to a much lower degree than that produced by young HDF. The diminished responsiveness of senescent HDF to TNF does not appear to be due to the difference in either receptor number or affinity, since senescent cells had two- to threefold higher number of TNF receptors than young HDF but the same affinity. TNF induced the activation of a nuclear transcriptional factor, NF-kappa B, equally in both young and senescent cells, which indicates the lack of a defect in the early events of TNF signal transduction in senescent fibroblasts. Overall, our results indicate that there is an age-dependent decline in TNF-induced proliferation and in the production of interleukins by fibroblasts; this unresponsiveness appears not to be due to TNF receptors or NF-kappa B activation. These results may have importance in understanding the diminished immune response, inflammation, and wound healing associated with aging.

Base Sequence↗

PubMed1989

Replicative senescence of human skin fibroblasts correlates with a loss of regulation and overexpression of collagenase activity.

The atrophy of extracellular matrix is a common event during the aging of connective tissues. In this study, we tested the hypothesis that the altered ability of senescent cells to be modulated by serum growth factors correlated with a loss of regulation of collagenase synthesis. We examined the levels of immunoreactive procollagenase and collagenase inhibitor (the tissue inhibitor of metalloproteinases, TIMP) associated with young and senescent fibroblasts cultured in vitro. Young fibroblasts cultured in low (0.5%) concentrations of fetal bovine serum respond to increased (10%) serum by increasing levels of procollagenase and TIMP beginning 4.0 h after serum stimulation. In contrast, senescent fibroblasts constitutively produce relatively high levels of procollagenase even when cultured in low levels of serum and do not respond to serum stimulation by increasing procollagenase synthesis. In addition, senescent fibroblasts constitutively express a relatively small amount of TIMP which is not induced upon serum stimulation. This altered expression of collagenase and TIMP appears unique to the senescent phenotype and not merely a result of growth inhibition, since young cells growth arrested by density-dependent growth inhibition displayed a temporal pattern of procollagenase and TIMP expression upon serum stimulation similar to that of subconfluent young cultures. An assay of net collagenase activity revealed a greater than 20-fold elevation of activity in trypsin-activated extracts from senescent versus young fibroblasts when cultured in a low concentration of fetal bovine serum. These results demonstrate for the first time a direct correlation between alterations in the molecular pathways regulating connective tissue homeostasis and those of replicative senescence. The increased collagenolytic activity of senescent compared to young fibroblasts cultured in the presence of a low serum concentration suggests that aging fibroblasts may become increasingly fibroclastic causing many of the age-associated alterations in dermal collagen observed during aging in vivo.

Blotting, Western↗

PubMed2005

Heterogeneity of dimer excision in young and senescent human dermal fibroblasts.

We have examined the relationship between nucleotide excision of the main UV-induced photoproduct, the cyclobutane pyrimidine dimer and in vitro cellular senescence. An in situ semiquantitative immunocytochemical assay has demonstrated that, following a UV-C dose of 15 J m-2, young human dermal fibroblasts maintained in a high level of serum are more efficient than senescent fibroblasts in the removal of dimers. However, in G0-arrested cultures (serum-starved), young fibroblasts are compromised in their ability to remove dimers and are significantly less efficient than senescent cells in this process. Supplementation of the culture medium with 0.1 mm deoxyribonucleosides enhances the removal of dimers in both young and senescent fibroblasts in proliferating or serum-starved cells. These data indicate that overall there is a modest but significant reduction in nucleotide excision of dimer photoproducts in cells as they age in vitro. In addition, G0-arrested young cells exhibit reduced removal of dimers, although this can be complemented by deoxyribonucleoside addition. In addition, this in situ assay has revealed heterogeneity in both susceptibility to UV-C-induced damage and excision. Overall, we provide evidence of reduced UV-induced damage excision in senescent compared with young fibroblasts, and demonstrate modulation of these processes in young and senescent cells under specific growth conditions.

Biomarkers↗