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Results for “Senescent fibroblasts”

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PubMed1997

Down-regulation of Ku autoantigen, DNA-dependent protein kinase, and poly(ADP-ribose) polymerase during cellular senescence.

During aging and cellular senescence mutations accumulate in genomic and mitochondrial DNA. Ku autoantigens, DNA-dependent protein kinase, and poly (ADP-ribose) polymerase have an essential role in DNA damage recognition. Our purpose was to find out whether cellular senescence of fibroblasts affects the protein components that recognize DNA damage and induce the repair process. We compared presenescent and replicatively senescent human WI-38 fibroblasts with each other and with SV-40 immortalized and serum-deficient quiescent WI-38 cells. Our results showed that replicative senescence significantly decreased the nuclear level of both p70 and p86 components of Ku autoantigen. SV-40 immortalization and cellular quiescence did not affect the level of the p86 component but slightly increased that of p70. Both replicative senescence and cellular quiescence decreased the activity of DNA-dependent protein kinase in WI-38 fibroblasts. On the other hand, SV-40 immortalization increased the activity of DNA-dependent protein kinase. The protein level of poly(ADP-ribose) polymerase (PARP) was strongly decreased in replicatively senescent fibroblasts. Quiescence of early-passage fibroblasts also slightly reduced the protein level of PARP. Apoptosis was not observed in replicatively senescent fibroblasts. Our results show that replicative senescence and to some extent cellular quiescence down-regulate the recognition system of DNA damage involving Ku autoantigens, DNA-dependent protein kinase, and PARP and hence could enhance the accumulation of DNA damage during aging.

Amino Acid Sequence↗

PubMed1985

Variable transglutaminase activity in human diploid fibroblasts during in vitro senescence.

The specific activity of transglutaminase (TGase) was followed in human diploid fibroblasts (HDF) as a function of in vitro age. It was determined that at least 90% of the TGase activity was found in a soluble fraction at all in vitro ages; but the activity was variable with age. It was high in cells that had completed less than 50% of their lifespan (%LSC), declined to a minimum between 60 and 85% LSC, and again became elevated at more than 90% LSC. These age related variations in TGase activity could not be attributed to cellular growth characteristics, enzyme amount, or clonal selection processes. It is postulated that the variable TGase activity observed during in vitro senescence of HDF may reflect a change in affinity of the enzyme for a particular molecule; possibly fibronectin.

Cell Division↗

PubMed2004

Senescence occurs with hTERT repression and limited telomere shortening in human oral keratinocytes cultured with feeder cells.

We investigated the phenotypic and molecular alterations in normal human oral keratinocytes (NHOK) during in vitro replication in two different culture conditions. The cells were cultured either in chemically defined Keratinocyte Growth Medium (KGM) without feeder layers or in serum-containing flavin-adenine dinucleotide (FAD) medium with feeder layers. Primary NHOK underwent 22 +/- 3 population doublings (PDs) in KGM and 42 +/- 4 PDs in FAD medium, reflecting 52% increase in replication capacity with feeder layers. In both culture conditions, exponentially replicating NHOK demonstrated telomerase activity and expression of human telomerase reverse transcriptase (hTERT) gene. Telomerase activity and hTERT expression were rapidly diminished in senescing NHOK, which exhibited small decrease of telomere length for the remaining limited cellular replications until the complete arrest of cell division. However, telomere length in senescent NHOK was 6.7 +/- 0.5 kilobase pairs (kbps), significantly longer than that (5.12 kbps) of senescent human fibroblasts. The onset of senescence was accompanied with marked induction of p16(INK4A), and this occurred in both culture systems using either KGM or FAD medium. These results indicate that replicative senescence of NHOK is associated with loss of telomerase activity followed by limited telomere shortening.

Blotting, Southern↗

PubMed2002

Cancer and aging: a model for the cancer promoting effects of the aging stroma.

The incidence of cancer rises exponentially with age in humans and many other mammalian species. Malignant tumors are caused by an accumulation of oncogenic mutations. In addition, malignant tumorigenesis requires a permissive tissue environment in which mutant cells can survive, proliferate, and express their neoplastic phenotype. We propose that the age-related increase in cancer results from a synergy between the accumulation of mutations and age-related, pro-oncogenic changes in the tissue milieu. Most age-related cancers derive from the epithelial cells of renewable tissues. An important element of epithelial tissues is the stroma, the sub-epithelial layer composed of extracellular matrix and several cell types. The stroma is maintained, remodeled and repaired by resident fibroblasts, supports and instructs the epithelium, and is essential for epithelial function. One change that occurs in tissues during aging is the accumulation of epithelial cells and fibroblasts that have undergone cellular senescence. Cellular senescence irreversibly arrests proliferation in response to damage or stimuli that put cells at risk for neoplastic transformation. Senescent cells secrete factors that can disrupt tissue architecture and/or stimulate nearby cells to proliferate. We therefore speculate that their presence may create a pro-oncogenic tissue environment that synergizes with oncogenic mutations to drive the rise in cancer incidence with age. Recent evidence lends support to this idea, and suggests that senescent stromal fibroblasts may be particularly adept at creating a tissue environment that can promote the development of age-related epithelial cancers.

Age Factors↗

PubMed2002

Differential proteome analysis of replicative senescence in rat embryo fibroblasts.

Normal somatic cells undergo a finite number of divisions and then cease dividing whereas cancer cells are able to proliferate indefinitely. To identify the underlying mechanisms that limit the mitotic potential, a two-dimensional differential proteome analysis of replicative senescence in serially passaged rat embryo fibroblasts was undertaken. Triplicate independent two-dimensional gels containing over 1200 spots each were run, curated, and analyzed. This revealed 49 spots whose expression was altered more than 2-fold. Of these, 42 spots yielded positive protein identification by mass spectrometry comprising a variety of cytoskeletal, heat shock, and metabolic proteins, as well as proteins involved in trafficking, differentiation, and protein synthesis, turnover, and modification. These included gelsolin, a candidate tumor suppressor for breast cancer, and alpha-glucosidase II, a member of the family of glucosidases that includes klotho; a defect in klotho expression in mice results in a syndrome that resembles human aging. Changes in expression of TUC-1, -2, -4, and -4 beta, members of the TUC family critical for neuronal differentiation, were also identified. Some of the identified changes were also shown to occur in two other models of senescence, premature senescence of REF52 cells and replicative senescence of mouse embryo fibroblasts. The majority of these candidate proteins were unrecognized previously in replicative senescence. They are now implicated in a new role.

Animals↗

PubMed1998

Psoralen photoactivation promotes morphological and functional changes in fibroblasts in vitro reminiscent of cellular senescence.

Premature aging of the skin is a prominent side effect of psoralen photoactivation, a treatment used widely for various skin disorders. The molecular mechanisms underlying premature aging upon psoralen photoactivation are as yet unknown. Here we show that treatment of fibroblasts with 8-methoxypsoralen (8-MOP) and subsequent ultraviolet A (UVA) irradiation resulted in a permanent switch of mitotic to stably postmitotic fibroblasts which acquired a high level of de novo expression of SA-beta-galactosidase, a marker for fibroblast senescence in vitro and in vivo. A single exposure of fibroblasts to 8-MOP/UVA resulted in a 5.8-fold up-regulation of two matrix-degrading enzymes, interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), over a period of >120 days, while TIMP-1, the major inhibitor of MMP-1 and MMP-3, was only slightly induced. This imbalance between matrix-degrading metalloproteases and their inhibitor may lead to connective tissue damage, a hallmark of premature aging. Superoxide anion and hydrogen peroxide, but not singlet oxygen, were identified as important intermediates in the downstream signaling pathway leading to these complex fibroblast responses upon psoralen photoactivation. Collectively, the end phenotype induced upon psoralen photoactivation shares several criteria of senescent cells. In the absence of detailed molecular data on what constitutes normal aging, it is difficult to decide whether the changes reported here reflect mechanisms underlying normal cellular aging/senescence or rather produce a mimic of cellular aging/senescence by quite different pathways.

Cell Division↗

PubMed1994

Involvement of protein kinase C and arachidonate signaling pathways in the alteration of proliferative response of senescent IMR-90 human fibroblasts.

The proliferative response of IMR-90 fibroblasts at low and high population doubling level (PDL) to protein kinase C activation has been investigated to clarify whether the reduced mitogenic responsiveness of senescent cells can be ascribed to an alteration in protein kinase C signal transduction pathway. The results show that the signaling pathway leading to DNA synthesis through protein kinase C activation, appears to be modified in senescent IMR-90 human fibroblasts. High PDL fibroblasts exhibit a different sensitivity to phorbol 12-myristate 13-acetate (PMA) and dioctanoylglycerol (diC8); high glucose reduced responsiveness to PMA only in these cells. In addition, high PDL fibroblasts are characterized by an increase in diacylglycerol (DAG) cellular mass that could contribute to the different regulatory properties of the signaling pathway. On the other hand, the ability of the cyclooxygenase inhibitor indomethacin to strikingly improve the proliferative response of high PDL cells to PMA indicates that an altered overall metabolism of arachidonate may represent a crucial step in the reduced mitogenic response involving protein kinase C activation.

Arachidonic Acid↗

PubMed1994

Cleavage of the epidermal growth factor receptor by a membrane-bound leupeptin-sensitive protease active in nonionic detergent lysates of senescent but not young human diploid fibroblasts.

Numerous studies suggest that epidermal growth factor (EGF) signaling is impaired in nonproliferating senescent human diploid fibroblasts downstream of receptor binding. One possible explanation for these results is that senescent cells possess unique enzymatic activities capable of regulating functional levels of the EGF receptor. To test that hypothesis, nonionic detergent lysates of young and senescent cells were compared for proteolytic activity directed towards the EGF receptor, and a protease that cleaves the 170 kDa EGF receptor was identified in lysates from senescent but not young cells. Although studies presented here were carried out with WI-38 cells, our data indicate that other senescent fibroblasts possess a similar activity. The degradation product immunoprecipitated by a monoclonal antibody specific for an EGF receptor exocytosolic epitope had an approximate molecular weight of 100,000. This product was also detected following cell surface labeling with 125I, and by cross-linking 125I-EGF to intact cells with disuccinimidyl suberate. The proteolytic activity in senescent cell lysates was specifically inhibited by leupeptin and did not require divalent cations; it was also inactivated by aprotic solvents such as dimethylsulfoxide (DMSO) or ethylene carbonate. Interestingly, this protease was not active during ligand-induced intracellular processing of the EGF receptor, suggesting that it does not normally function in endocytic or lysosomal compartments. The susceptibility of the protease to inactivation by cell surface trypsinization is consistent with a plasma membrane localization. Since EGF receptor cleavage is not observed unless senescent cells are solubilized with nonionic detergents, it seems likely that the protease is confined to specialized regions of the plasma membrane. Whether or not the EGF receptor is a physiologic target for this protease is unclear. Its expression at the cell surface is nevertheless significant, since it suggests there are mechanisms for regulating membrane-bound proteins, or biologically active peptides in the extracellular space, in senescent cells that are either absent or inactive in young cells.

Cell Line↗

PubMed1999

Dephosphorylation of specific proteins during induction of senescence in immortal human fibroblasts expressing thermolabile SV40 T antigen.

Particular protein kinase inhibitors block a senescence-like phenomenon in SVts8 cells induced by a shift up in temperature. We characterized cellular proteins with affinity chromatography using one such inhibitor as a ligand. Two proteins of 56 and 100 kDa were found to be dephosphorylated specifically, probably due to induction of a protein phosphatase activity(s).

Aging↗

PubMed1984

Synthesis of the nuclear protein cyclin in growing, senescent and morphologically transformed human skin fibroblasts.

Quantitative two-dimensional gel electrophoretic analysis (IEF) of the nuclear polypeptide cyclin in normal human skin biopsies, growing and senescent fibroblasts and morphologically transformed skin fibroblasts (limited life span) has revealed a direct correlation between the levels of this protein and proliferative state of the cells. These results strengthen the notion that cyclin may be a key component of the pathway(s) that control cell proliferation.

Cell Division↗

PubMed2005

Down-regulation of a forkhead transcription factor, FOXO3a, accelerates cellular senescence in human dermal fibroblasts.

The signaling pathway of insulin/insulin-like growth factor/phosphatidylinositol-3 kinase/Akt/forkhead transcription factors is known to control life span and senescence in organisms ranging from yeast to mice. The FOXO family of forkhead transcription factors, FOXO1, FOXO3a, and FOXO4, play a critical role in this signal transduction pathway. However, the impact of FOXO3a activation on life span of primary cultured human dermal fibroblasts (HDFs) is unknown. To investigate the role of FOXO3a in the regulation of cellular senescence, we prepared FOXO3a-siRNA stable HDFs. We found that the down-regulation of FOXO3a RNA and protein in HDFs induced many senescent phenotypes, including changes in cell morphology, increases in population doubling times, senescence-associated beta-galactosidase staining and the cellular reactive oxygen species, and up-regulation of p53/p21 protein expression. Our data provide evidence of the key role of FOXO3a transcription factor as a mediator of cellular senescence in HDFs, and suggest that the mechanism of senescence is conserved in HDFs.

Cells, Cultured↗

PubMed1985

Ribonucleotide reductase activity during the senescence of normal human diploid fibroblasts in culture.

Changes in ribonucleotide reductase occur during the senescence of normal human diploid fibroblasts in culture. Enzyme activity is significantly lower in cells and extracts at high mean population doublings (MPD) as compared to fibroblasts at low MPD. Although many of the kinetic properties of the enzyme remain unaltered in cells at high MPD, changes in the extent and kinetic mechanism of inhibition of CDP and ADP reductase activity by dATP, the overall negative effector of ribonucleotide reductase, were observed. These results and a previous observation that the four deoxyribonucleotide pools are markedly altered during in vitro senescence of human diploid fibroblasts, provide evidence for a link between ribonucleotide reduction, deoxyribonucleotide pools, and the establishment of the non-proliferative or senescent state.

Cell Division↗

PubMed2004

Selective COX-2 inhibitor, NS-398, inhibits the replicative senescence of cultured dermal fibroblasts.

Cyclooxygenase 2 (COX-2) is known to be increased in aged cells. Recent studies suggest that the increased expression of COX-2 may be involved in the pathogenesis of age-associated diseases such as rheumatoid arthritis and cancer. We investigated the role of COX-2 in cell cycle arrest and collagen deficiency during the aging process. Using the replicative senescence model of dermal fibroblasts, we demonstrated the increased expression of COX-2 and increased PGE(2) levels associated with replicative senescence. Replicative senescent cells showed a decreased ability to induce cell proliferation, probably due to the increased expression of the p53 protein and the decreased expression of the PCNA protein, and also showed increased expression of MMP-1, and decreased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and procollagen. The selective COX-2 inhibitor, NS-398, can inhibit the senescence-associated increases of COX-2, PGE(2), p53 and MMP-1 expression, and the senescence-associated decreases of PCNA, TIMP-1 and procollagen expression. These results suggest that the increased level of COX-2 and higher level of PGE(2) in aged cells may play an important role in cellular senescence, and that selective COX-2 inhibitors may be useful for the intervention of skin aging.

Arthritis, Rheumatoid↗

PubMed1997

Downregulation of DNA topoisomerase I in old versus young human diploid fibroblasts.

DNA topoisomerase I (Topo I) is an enzyme that alters the superhelicity of DNA. It has been implicated in such critical cellular functions as transcription, DNA replication, and recombination. Roles for Topo I in DNA repair following DNA damage have also been studied extensively. In the present investigation, we examined the regulation of Topo I expression and activity during cellular replicative senescence. We found that the capacity of Topo I to relax supercoiled DNA molecules is significantly decreased in senescent diploid fibroblasts when compared to young (early passage) fibroblasts. We also found that the steady-state expression level of Topo I mRNA is correlated with enzyme activity, i.e., decreased in early vs. late passage cells. We also treated early and late passage cells with agents that may modulate the process of cellular senescence: UV light, retinoic acid, and interleukin-1 beta. We found that all three agents decreased the activity of Topo I in young fibroblasts and increased the activity of Topo I in senescent fibroblasts. This effect was most striking following exposure of the cells to retinoic acid, so to analyze this effect, we postulated an age-dependent kinetics of Topo I mRNA induction in response to retinoic acid. Consistent with this postulate, we found that whereas exposure of early passage cells to retinoic acid results, in a matter of hours, in a decrease in the expression of Topo I mRNA, exposure of the senescent cells to retinoic acid results in an increased expression. These observations suggest that processes that are altered in senescent fibroblasts, such as DNA replication and repair, may be due, in part, to alteration in the expression and activity of DNA Topo I.

Cells, Cultured↗

PubMed1994

Senescence of immortal human fibroblasts by the introduction of normal human chromosome 6.

In these studies we show that introduction of a normal human chromosome 6 or 6q can suppress the immortal phenotype of simian virus 40-transformed human fibroblasts (SV/HF). Normal human fibroblasts have a limited life span in culture. Immortal clones of SV/HF displayed nonrandom rearrangements in chromosome 6. Single human chromosomes present in mouse/human monochromosomal hybrids were introduced into SV/HF via microcell fusion and maintained by selection for a dominant selectable marker gpt, previously integrated into the human chromosome. Clones of SV/HF cells bearing chromosome 6 displayed limited potential for cell division and morphological characteristics of senescent cells. The loss of chromosome 6 from the suppressed clones correlated with the reappearance of immortal clones. Introduced chromosome 6 in the senescing cells was distinguished from those of parental cells by the analysis for DNA sequences specific for the donor chromosome. Our results further show that suppression of immortal phenotype in SV/HF is specific to chromosome 6. Introduction of individual human chromosomes 2, 8, or 19 did not impart cellular senescence in SV/HF. In addition, introduction of chromosome 6 into human glioblastoma cells did not lead to senescence. Based upon these results we propose that at least one of the genes (SEN6) for cellular senescence in human fibroblasts is present on the long arm of chromosome 6.

Animals↗

PubMed1979

Effects of glutaraldehyde and other drugs on concanavalin A-mediated red blood cell adsorption to nonsenescent, senescent and transformed human fibroblasts.

Concanavalin A (Con A)-mediated red blood cell (RBC) adsorption with the RBC coating method (in which Con A-coated RBC's are adsorbed to fibroblasts) was greatly increased by glutaraldehyde fixation of RBCs before Con A-coating and decreased by the fixation of fibroblasts. On the other hand, RBC adsorption with the fibroblast coating method (in which RBCs are adsorbed to Con A-coated fibroblasts) was decreased by the fixation of RBCs and increased by the fixation of fibroblasts before Con A coating. The fixation of RBCs or fibroblasts after Con A coating did not have these effects. In addition, the fixation of both RBCs and fibroblasts nearly completely abolished RBC adsorption with either method. However, the amount of [3H] Con A binding was not affected by the fixation. RBC adsorption with the fibroblast coating method was also affected by cytochalasin B, colchicine, NaN3 and dibucane treatments of fibroblasts. These drug treatments of fibroblasts, however, did not affect RBC adsorption with the RBC coating method, except cytochalasin B. In addition, the effects of drug treatments of fibroblasts examined occurred nearly to the same extent for nonsenescent, senescent, and transformed cells. Our results suggest that secondary processes after Con A binding, receptor mobility and receptor association with cytoskeletals, play important roles in RBC adsorption, but that the roles do not change with aging or transformation.

Aldehydes↗

PubMed1994

Accelerated aging of dermal fibroblast-like cells from senescence-accelerated mouse (SAM). 1. Acceleration of population aging in vitro.

Fibroblast-like cells were isolated from the senescence accelerated mouse (SAM) and cultured, after which evidence of accelerated senescence was sought. Fibroblast-like cell lines were established from the dorsal dermis of neonate mice of both the accelerated senescence-prone strain, SAMP11 and the accelerated senescence-resistant strain, SAMR1. All cell lines from both strains showed a crisis in growth and were immortalized. At crisis, all cultures were composed of morphologically characteristic senescent cells. However, in cell lines from SAMP11, this change was more rapid and at earlier population doublings (PDs) than seen in cell lines from SAMR1. Crises (SAMP11; SAMR1) were also operationally taken to be the point of the least change in PDs (11.2 +/- 1.1; 15.4 +/- 0.5 PDs), the least saturation density (11.3 +/- 0.8; 19.1 +/- 2.6 PDs), and the longest population doubling time (10.1 +/- 0.8; 14.2 +/- 0.6 PDs). Crisis occurred significantly earlier (P < 0.05) and the aging process was accelerated in cell lines from SAMP11, compared with lines from SAMR1. This evidence tends to support various observations made in the accelerated senescence-prone strains of SAMP, in vivo.

Aging↗

PubMed2000

Cytoplasmic retention of p-Erk1/2 and nuclear accumulation of actin proteins during cellular senescence in human diploid fibroblasts.

In order to investigate the role of signal transduction and the related changes of actin cytoskeleton organization in the process of cellular senescence, H-ras double mutants--V12S35, V12G37 and V12C40--proteins were expressed constitutively in human diploid fibroblast (HDF) cells by retrovirus infection at PD26. Constitutive expression of V12S35, V12G37 and V12C40 proteins induced premature senescence at PD38, PD47 and PD50, respectively, in contrast to the control cells at PD59. Premature senescence was evidenced by the slow cellular growth rate and SA-beta-galactosidase expression accompanied by morphological changes such as flat and large cell shape. Senescent HDF cells as well as the H-ras mutant expressers accumulated p-Erk1/2 in the cytoplasm with increased MEK activity and failed to translocate it to nuclei on EGF stimulation. Senescent HDF cells as well as V12S35 and V12G37 expressers were unable to export actin fibers from nucleus to cytoplasm, form stress fibers through the MAPK and Ral.GDS pathways. Perinuclear expression of Racl was prominent in the HDF cells and V12C40 expresser, while translocation of Racl from perinucleus to nucleus and strong expression of RhoA were observed in the V12S35 expresser. In summary, the induced premature senescence by H-ras double mutants were accompanied by nuclear accumulation of actin and Racl proteins, cytoplasmic retention of p-Erk1/2 and marked induction of RhoA expression mainly through dysregulation of the MEK pathway.

Actins↗