Inhibitors of DNA synthesis derived from senescent human diploid fibroblasts.
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Aging↗
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Results for “Senescent fibroblasts”
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Gene expression and regulation in H2O2-induced premature senescence of human foreskin fibroblasts expressing or not telomerase.
We compared the DNA-binding activity of transcription factors and gene expression patterns in BJ human diploid fibroblasts (HDFs) expressing or not telomerase (hTERT) in stress-induced premature senescence (SIPS). Senescent BJ cells were also studied. Hydrogen peroxide (H2O2)-induced SIPS modulated gene expression in both BJ and hTERT-BJ1 cells. Increased p21(WAF-1) mRNA level was amongst the common gene expression changes in BJ and hTERT-BJ1 cells induced by SIPS. Telomerase expression markedly changed gene expression in non-stressful conditions. Expression patterns of senescent BJ cells partially overlapped those of BJ and hTERT-BJ1 cells in SIPS. The basal levels of DNA-binding activity of NF-kappaB and phosphorylated ATF-2 were different in BJ and hTERT-BJ1 cells. Both cell lines displayed a higher DNA-binding activity of p53 and HIF-1 72 h after H2O2 exposure. Our results indicate that similar mechanisms involving p21(WAF-1) and probably p53 are at work in BJ and hTERT-BJ1 HDFs under H2O2-induced SIPS, suggesting that generalized DNA damage rather than telomere length/telomerase plays a crucial role in H2O2induced SIPS. We propose that H2O2-induced SIPS involves a rearrangement of proliferative and apoptotic pathways. The marked changes in gene expression induced by telomerase suggest that apart from immortalization of HDFs, telomerase also alters the normal cellular functions but does not protect against SIPS.
Cells, Cultured↗
[Early manifestations of senescence of diabetic fibroblasts in culture. Ultrastructural study].
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Aging↗
Identification of gene sequences overexpressed in senescent and Werner syndrome human fibroblasts.
The phenotype of replicative senescence is a dominant trait in human diploid fibroblasts (HDF). Therefore, we have sought to identify overexpressed and/or newly expressed causal genes by constructing and screening a subtracted cDNA library derived from polyA+RNA of prematurely senescent Werner syndrome (WS) HDF. We have identified 15 cDNA clones that are overexpressed in senescent and WS HDF. Among them are six known sequences coding for: acid sphingomyelinase, fibronectin, SPARC, nm23-metastasis suppressor protein, and two translation factors, eIF-2 beta and EF-1 alpha. Among the 10 unknown clones are: S1-5, which encodes a secreted protein containing EGF-like domains and paradoxically stimulates DNA synthesis of young HDF in an autocrine and paracrine manner, S1-3, which encodes a protein containing "zinc finger" domains, suggesting nucleic acid binding properties; S1-15, which shows sequence similarities to human alpha 2-chimerin; and S2-6, which represents a new member of the LIM family of proteins. The other five clones do not have any significant homology to known sequences. Steady-state mRNA levels of all gene sequences thus far studied are elevated in both WS and senescent normal HDF when compared to young HDF, which suggests that senescent and WS HDF enter a final common pathway where multiple gene overexpression may generate diverse antiproliferative mechanisms and pathogenic sequelae.
Adult↗
A functional screen identifies hDRIL1 as an oncogene that rescues RAS-induced senescence.
Primary fibroblasts respond to activated H-RAS(V12) by undergoing premature arrest, which resembles replicative senescence. This irreversible 'fail-safe mechanism' requires p19(ARF), p53 and the Retinoblastoma (Rb) family: upon their disruption, RAS(V12)-expressing cells fail to undergo senescence and continue to proliferate. Similarly, co-expression of oncogenes such as c-MYC or E1A rescues RAS(V12)-induced senescence. To identify novel genes that allow escape from RAS(V12)-induced senescence, we designed an unbiased, retroviral complementary DNA library screen. We report on the identification of DRIL1, the human orthologue of the mouse Bright and Drosophila dead ringer transcriptional regulators. DRIL1 renders primary murine fibroblasts unresponsive to RAS(V12)-induced anti-proliferative signalling by p19(ARF)/p53/p21(CIP1), as well as by p16(INK4a). In this way, DRIL1 not only rescues RAS(V12)-induced senescence but also causes these fibroblasts to become highly oncogenic. Furthermore, DRIL1 immortalizes mouse fibroblasts, in the presence of high levels of p16(INK4a). Immortalization by DRIL1, whose product binds the pRB-controlled transcription factor E2F1 (ref. 8), is correlated with induction of E2F1 activity. Correspondingly, DRIL1 induces the E2F1 target Cyclin E1, overexpression of which is sufficient to trigger escape from senescence. Thus, DRIL1 disrupts cellular protection against RAS(V12)-induced proliferation downstream of the p19(ARF)/p53 pathway.
Animals↗
Expression of connective tissue growth factor, a biomarker in senescence of human diploid fibroblasts, is up-regulated by a transforming growth factor-beta-mediated signaling pathway.
Molecular changes associated with cellular senescence in human diploid fibroblasts (HDF), IMR-90, were analyzed by two-dimensional differential proteome analysis. A high percentage of replicative senescent cells were positive for senescence-associated beta-galactosidase activity, and displayed elevated levels of p21 and p53 proteins. Comparison of early population doubling level (PDL) versus replicative senescent cells among the 1000 spots resolved on gels revealed that the signal intensities of six spots were increased fivefold, whereas those of four spots were decreased. Proteome analysis data demonstrated that connective tissue growth factor (CTGF) is an age-associated protein. Up-regulation of CTGF expression in senescent cells was further confirmed by Western blotting and RT-PCR. We postulate that CTGF expression is controlled, in part, by transforming growth factor-beta (TGF-beta), in view of the high levels of TGF-beta isoforms as well as type I and II receptors detected only in late PDL of HDF cells. To verify this hypothesis, we stimulated early PDL cells with TGF-beta1 as well as stress inducing agents such as hydrogen peroxide. As expected, CTGF expression and Smad protein phosphorylation were dramatically increased up to observed levels in normal replicative senescent cells. In vivo experiments disclosed that CTGF, pSmad, and p53 were constitutively expressed at basal levels in up to 18-month-old rat liver, and expression was significantly up-regulated in 24-month-old rat tissue. However, expression patterns were not altered at all periods examined in livers of caloric-restricted rats. In view of both in vitro and in vivo data, we propose that the TGF-beta/Smad pathway functions in the induction of CTGF, a novel biomarker protein of cellular senescence in human fibroblasts.
Animals↗
Investigation of the role of G1/S cell cycle mediators in cellular senescence.
Cellular senescence is a state of irreversible cell cycle arrest in which normal cells at the end of their lifespan fail to enter into DNA synthesis upon serum or growth factor stimulation. We examined whether proteins required for G1/S cell cycle progression were irreversibly down-regulated in senescent human fibroblasts. Both the 44- and 42-kDa forms of the MAP-kinase protein were expressed at similar levels in young and senescent cells. In contrast to young cells where both forms were phosphorylated on tyrosine in response to serum, the p42MAP-kinase was not tyrosine phosphorylated upon serum stimulation, whereas p44MAP-kinase was phosphorylated on tyrosine in serum-starved or serum-stimulated senescent cells. Examination of p53 protein in growing, quiescent, and senescent cells revealed no significant differences in levels between the different growth states. In contrast, cdk2 and cyclin A mRNAs were completely down-regulated in stimulated senescent fibroblasts, while the G1 cyclins, C, D1, and E mRNAs, were still expressed in stimulated senescent cells although at reduced levels compared to young cells. The expression of early G1 markers, but not late G1 markers, indicates that senescent cells may be blocked at a point in late G1. We investigated whether transfection of cyclin A, alone or in combination with cdc2, was sufficient for extension of lifespan or escape from senescence. Clones expressing the transfected human cyclin A or cdc2 genes senesced at a population doubling similar to controls, thereby showing that cyclin A or cdc2 expression alone was insufficient for escape from senescence.
Base Sequence↗
Investigation of the signaling pathways involved in the proliferative life span barriers in werner syndrome fibroblasts.
Werner syndrome (WS) fibroblasts enter replicative senescence after a reduced in vitro life span. Although this has been postulated as causal in the accelerated aging seen in this disease, controversy remains as to whether WS is showing the acceleration of a normal cellular aging mechanism or, instead, the occurrence of a novel WS-specific process. To address this, we analyzed the signaling pathways involved in senescence in WS fibroblasts. Cultured WS fibroblasts underwent senescence after approximately 20 population doublings, with the majority of the cells having a 2N DNA content. This was associated with high levels of the CdkIs p16 and p21. Senescent WS cells reentered the cell cycle after microinjection of a p53-neutralizing antibody. Similarly, presenescent WS fibroblasts expressing the E6 and/or E7 oncoproteins bypassed M1 and ultimately reached a second proliferative life span barrier, which strongly resembled the second life span barriers found in normal cells for growth dynamics, cellular morphology, and expression of p16 and p21. The strong similarity between the signaling pathways triggering cell cycle arrest in WS and normal fibroblasts provides support for the defect in WS causing the acceleration of a normal aging mechanism and validates the use of WS as a model for some aspects of human aging.
Aging↗
Cell surface changes in senescent and Werner's syndrome fibroblasts: their role in cell proliferation.
Cell surface is known to participate in the regulation of cell proliferation through interaction with adjacent cell surfaces or the extracellular matrix, or both. A clinical survey of the Werner syndrome suggests some disorders in glycosaminoglycan metabolism. Also, the skin fibroblasts derived from the patients with WS have a reduced proliferation capacity. We here examined, in vitro and in vivo, alterations of the cell-surface properties of WS cells and aging human fibroblasts. Cell-surface negative charges, examined by electrophoretic mobility of dispersed single cells in buffer, were seen to decline steadily as a function of cumulative population doublings. A strict linear relationship was found between electrophoretic mobility (micron/sec/V/cm) and number of cells harvested at each passage in all cell lines examined. The slope of this line in cells from donors of different ages indicated that WS fibroblasts resemble cells from much older normal controls. The same conclusion was drawn from our previous study of Con A-mediated red cell adsorption, which was confirmed as reflecting an alteration of cell-surface coat negative charge. Electrophoretic mobility after treatment of cell surface with degradative enzymes showed that the cell-surface negative charges were attributable to sialic acid, chondroitin sulphates, hyaluronic acid, and heparan sulphate. Two-dimensional electrophoresis of 3H-glucosamine incorporated glycosaminoglycans (GAGs) revealed that heparan sulphate was the main component of GAGs on the fibroblast cell surface and that the relative amount of heparan sulphate among GAGs on the cell surface increased in vitro with the number of passages. Growth kinetics of fibroblasts on sheets of fixed cells treated with a fixative (glutaraldehyde) and degradative enzymes were examined to elucidate the role of cell-surface GAGs in the regulation of cell proliferation. Cell growth was inhibited 40% when the fibroblasts were cultured on the fixed sheets of late passage cells. Treatment of the fixed cell sheets with heparitinase or nitrous acid resulted in complete recovery from the growth inhibition. Cell growth on sheets of fixed cells derived from young, middle, and senescent fibroblasts showed that the surface of the senescent cells had the greatest inhibitory effect. These inhibitory effects of fixed cell sheets correlated well with both the amount of heparan sulphate relative to the total GAGs on the surface and to the saturation density of cell growth at each passage. These findings strongly suggest that heparan sulphate, or its complex, on the cell surface is involved in the regulation of cell proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)
Adolescent↗
Stress-induced premature senescence in hTERT-expressing ataxia telangiectasia fibroblasts.
In addition to replicative senescence, normal diploid fibroblasts undergo stress-induced premature senescence (SIPS) in response to DNA damage caused by oxidative stress or ionizing radiation (IR). SIPS is not prevented by telomere elongation, indicating that, unlike replicative senescence, it is triggered by nonspecific genome-wide DNA damage rather than by telomere shortening. ATM, the product of the gene mutated in individuals with ataxia telangiectasia (AT), plays a central role in cell cycle arrest in response to DNA damage. Whether ATM also mediates signaling that leads to SIPS was investigated with the use of normal and AT fibroblasts stably transfected with an expression vector for the catalytic subunit of human telomerase (hTERT). Expression of hTERT in AT fibroblasts resulted in telomere elongation and prevented premature replicative senescence, but it did not rescue the defect in G(1) checkpoint activation or the hypersensitivity of the cells to IR. Despite these remaining defects in the DNA damage response, hTERT-expressing AT fibroblasts exhibited characteristics of senescence on exposure to IR or H(2)O(2) in such a manner that triggers SIPS in normal fibroblasts. These characteristics included the adoption of an enlarged and flattened morphology, positive staining for senescence-associated beta-galactosidase activity, termination of DNA synthesis, and accumulation of p53, p21(WAF1), and p16(INK4A). The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which mediates signaling that leads to senescence, was also detected in both IR- or H(2)O(2)-treated AT and normal fibroblasts expressing hTERT. These results suggest that the ATM-dependent signaling pathway triggered by DNA damage is dispensable for activation of p38 MAPK and SIPS in response to IR or oxidative stress.
Ataxia Telangiectasia↗
Competition of CUGBP1 and calreticulin for the regulation of p21 translation determines cell fate.
Induction of p21 in senescent human fibroblasts plays a key role in the inactivation of cyclin-dependent kinases and the resulting irreversible growth arrest in the early stages of cell senescence. We found that RNA-binding proteins are critical regulators of p21 during senescence. Two RNA-binding proteins, CUGBP1 and calreticulin (CRT), interact with the same nucleotide sequences within the 5' region of p21 mRNA, but have opposite effects on the translation of p21 mRNA. CUGBP1 increases translation of p21 mRNA, whereas CRT blocks translation of p21 via stabilization of a stem-loop structure within the 5' region of the p21 mRNA. CUGBP1 and CRT compete for binding to p21 mRNA and thereby the regulation of p21 translation. In senescent fibroblasts, CUGBP1 displaces CRT from the p21 mRNA and releases CRT-dependent repression of p21 translation leading to growth arrest and development of a senescent phenotype. These data present evidence that competition between RNA-binding proteins for the regulation of p21 translation determines cell fate.
5' Untranslated Regions↗
Similar gene expression pattern in senescent and hyperoxic-treated fibroblasts.
Different DNA-damaging treatments produce a senescence-like phenotype. Young human fibroblasts are transferred to a senescence-like state after 4 to 6 weeks of culture under 40% ambient oxygen partial pressure. In order to understand the causes of senescence it would be advantageous to know how well this state equals accelerated senescence. Therefore, we measured the expression of genes with known senescence-specific expression pattern in human fibroblasts, which were irreversibly proliferation-inhibited by chronic hyperoxic treatment. A senescence-specific gene expression pattern was confirmed by semiquantitative RT-PCR for eight out of nine examined genes in BJ foreskin fibroblasts and for four out of four genes in MRC-5 lung fibroblasts. For all these cases, gene expression under hyperoxia was similar to that in senescent cells, suggesting that chronic mild hyperoxia is a valid model for accelerated senescence.
Cells, Cultured↗
N-t-butyl hydroxylamine, a hydrolysis product of alpha-phenyl-N-t-butyl nitrone, is more potent in delaying senescence in human lung fibroblasts.
Alpha-phenyl-N-t-butyl nitrone (PBN), a spin trap, scavenges hydroxyl radicals, protects tissues from oxidative injury, and delays senescence of both normal human lung fibroblasts (IMR90) and senescence-accelerated mice. N-t-butyl hydroxylamine and benzaldehyde are the breakdown products of PBN. N-t-Butyl hydroxylamine delays senescence of IMR90 cells at concentrations as low as 10 microM compared with 200 microM PBN to produce a similar effect, suggesting that N-t-butyl hydroxylamine is the active form of PBN. N-Benzyl hydroxylamine and N-methyl hydroxylamine compounds unrelated to PBN were also effective in delaying senescence, suggesting the active functional group is the N-hydroxylamine. All the N-hydroxylamines tested significantly decreased the endogenous production of oxidants, as measured by the oxidation of 2', 7'-dichlorodihydrofluorescin and the increase in the GSH/GSSG ratio. The acceleration of senescence induced by hydrogen peroxide is reversed by the N-hydroxylamines. DNA damage, as determined by the level of apurinic/apyrimidinic sites, also decreased significantly following treatment with N-hydroxylamines. The N-hydroxylamines appear to be effective through mitochondria; they delay age-dependent changes in mitochondria as measured by accumulation of rhodamine-123, they prevent reduction of cytochrome C(FeIII) by superoxide radical, and they reverse an age-dependent decay of mitochondrial aconitase, suggesting they react with the superoxide radical.
Antioxidants↗
Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts.
Human diploid fibroblasts (HDFs) can be grown in culture for a finite number of population doublings before they cease proliferation and enter a growth-arrest state termed replicative senescence. The retinoblastoma gene product, Rb, expressed in these cells is hypophosphorylated. To determine a possible mechanism by which senescent human fibroblasts maintain a hypophosphorylated Rb, we examined the expression levels and interaction of the Rb kinases, CDK4 and CDK6, and the cyclin-dependent kinase inhibitors p21 and p16 in senescent HDFs. Cellular p21 protein expression increased dramatically during the final two to three passages when the majority of cells lost their growth potential and neared senescence but p21 levels declined in senescent HDFs. During this period, p16 mRNA and cellular protein levels gradually rose with the protein levels in senescent HDFs reaching nearly 40-fold higher than early passage cells. In senescent HDFs, p16 was shown to be complexed to both CDK4 and CDK6. Immunodepletion analysis of p21 and p16 from the senescent cell extracts revealed that p16 is the major CDK inhibitor for both CDK4 and CDK6 kinases. Immunoprecipitation of CDK4 and CDK6 and their associated proteins from radiolabeled extracts from senescent HDFs showed no other CDK inhibitors. Based upon these results, we propose that senescence is a multistep process requiring the expression of both p21 and p16. p16 up-regulation is a key event in the terminal stages of growth arrest in senescence, which may explain why p16 but not p21 is commonly mutated in immortal cells and human tumors.
Carrier Proteins↗
Enhanced expression of mitochondrial genes in senescent endothelial cells and fibroblasts.
It has been suggested that some mitochondrial genes are important in cellular senescence. In order to identify the mitochondrial genes that are involved in cellular senescence, we have constructed a cDNA library from senescent human vascular endothelial cells and isolated 86 senescence-specific cDNA clones by differential screening. Among the clones, we identified four distinct mitochondrial genes including NADH dehydrogenase subunit 2 (ND2), ND3, ATPase 6 and 16S ribosomal RNA. We then compared the levels of expression of these genes in young and senescent cells by using two endothelial and two fibroblast cell strains. Northern blot and slot blot hybridization confirmed that the expression levels of ND3, ATPase 6 and 16S rRNA were elevated in senescent cells of all four strains. The expression level of ND2 was also elevated during cellular senescence in three of the four strains. Because mitochondria are actively involved in oxidative phosphorylation and respiratory functions, the altered expression levels of these genes may participate in aging processes.
Adenosine Triphosphatases↗
Insulin-like growth factor binding protein 3 accumulates to high levels in culture medium of senescent and quiescent human fibroblasts.
Insulin-like growth factor binding protein 3 (IGFBP-3) mRNA levels were consistently higher in both senescent normal human diploid fibroblasts (HDFs) at late passage (old cells) and prematurely senescent HDFs from a subject with Werner syndrome (WS) during serum depletion and repletion of growth medium and during proliferation from sparse to high-density inhibited cultures, compared to normal early-passage (young) HDFs. However, IGFBP-3 protein accumulated to higher levels in conditioned medium of old cells than in medium of WS and young cells, in that order, under the same conditions. Insulin-like growth factor I (IGF-I) was not detected in naive medium or in any of the media conditioned by these three cell types, whereas IGF-II was detectable in serum-repleted medium and remained relatively constant. Thus, molar ratios of IGFBP-3/IGF-II were consistently higher in old and WS cells and increased substantially as all three cell types became quiescent, due to either serum depletion or high cell density. These data are consistent with either an adaptive or a causal role for IGFBP-3 protein in the senescent and quiescent growth arrest of HDFs.
Carrier Proteins↗
Defects in signal transduction during replicative senescence of diploid human fibroblasts in vitro.
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Calcium-Calmodulin-Dependent Protein Kinases↗
Bcl-2 promotes premature senescence induced by oncogenic Ras.
The expression of the apoptosis inhibitory protein, Bcl-2, is increased in naturally senescing human fibroblasts and upon induction of their senescence-like growth arrest by oxidative stress, implying its role in maintaining their extended viability. Oncogenic Ras(V12) protein induces signaling cascades that result in the premature senescence of primary fibroblast cells, which are insensitive to oncogene-dependent apoptosis. Here we show that constitutive expression of Bcl-2 accelerates selected features of the Ras-induced senescence program in primary human fibroblasts. Yet, Bcl-2 also inhibits fibroblast apoptosis induced by exogenous H(2)O(2), while both signals induce an increased endogenous Bcl-2 expression in these cells. Together, these data suggest a context-dependent phenotypic function of Bcl-2 in the regulation of overlapping cell fate specification programs, with potential implications for both physiology and multistep tumorigenesis.
Apoptosis↗