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Results for “Senescent fibroblasts”

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PubMed1996

Protection against impairment of memory and immunoreactivity in senescence-accelerated mice by acidic fibroblast growth factor.

Subcutaneous injection of aFGF once a week into senescence-accelerated mice (SAM)P8 was begun at 3 weeks after birth and continued for 10 months. Saline was injected as a control. Learning and memory and cellular immunological functions in the aFGF group were enhanced significantly, while those of the saline group deteriorated. 1. The number of cholinergic neurons was decreased by less than 20% and choline acetyltransferase activity in individual neurons in the medical septum which send monosynaptic terminals to the hippocampus was significantly decreased in the saline group, but not so much in the aFGF group. 2. The respective densities of muscarinic and NMDA receptors and the aFGF receptor, i.e., FGFR-1 on the hippocampal neurons were also significantly higher in the aFGF group than in the saline group. 3. The long-term potentiation in the hippocampal slice preparations after a brief tetanic stimulation at the Schaffer collateral/commissural afferents was significantly facilitated in the aFGF group, but not in the saline group. 4. These data indicate the normalization caused by aFGF of the medial septohippocampal circuit, which is necessary for learning and memory. 5. The delayed type hypersensitivity reactions (DTH) in the footpad caused by challenge with trinitrophenyl or sheep red blood cells as measured at the end of the 2nd and 7th months, indicated the T cell immune response. Both types of DTHs were reduced in the 7th month as compared with the 2nd month in the saline group, but the aFGF group was protected against this reduction in accordance with age. 6. These results show that aFGF provides protection against impairment of not only learning and memory but also DTH immunoreactivity in SAMP8. They also indicate a close relationship between learning and memory and T cell immune function.

Aging↗

PubMed1995

Effect of cellular senescence and retinoic acid on the expression of cellular retinoic acid binding proteins in skin fibroblasts.

Retinoic acid (RA) has significant effects on a variety of cellular processes, including growth and differentiation. Retinoic acid has also been implicated as a major morphogen during embryogenesis. In the skin, both the epidermis and the dermis are extremely responsive to the effects of retinoids; however, the molecular mechanisms through which retinoids act in this tissue remain poorly understood. Two classes of proteins play roles in mediating the biological effects of retinoic acid. The nuclear receptors for retinoic acid are of two types, the initially described RARs, and the RXRs. Each of these nuclear receptor families has multiple isoforms and can function as a ligand-inducible transcription enhancing factor. A second class of receptor proteins exists for retinoic acid that are found in the cytoplasm, the cellular retinoic acid binding proteins (CRABPs). These proteins play a role in the binding, transport, and metabolism of retinoic acid. In previous studies, we have demonstrated that retinoic acid induces the expression of RAR beta and RAR gamma in human dermal fibroblasts. Moreover, we have also shown the selective transcriptional up-regulation of the RAR beta 2 isoform in senescent dermal fibroblasts and senescent human mammary epithelial cells. In order to further define molecules important in regulating the response of senescent dermal fibroblasts to retinoids, we demonstrate here that retinoic acid induces CRABP-II messenger RNA in human dermal fibroblasts in a dose-dependent manner. Moreover, we show that the induction by RA can be inhibited by actinomycin D, suggesting that the up-regulation may be mediated by a transcriptional mechanism. We further demonstrate that cycloheximide also has an effect on the up-regulation, suggesting a role for protein synthesis in the regulation of CRABP-II gene expression. We show that CRABP-II is a very stable messenger RNA species, in contrast to the mRNAs for RAR alpha, RAR beta, and RAR gamma. Of interest, we demonstrate no significant difference in the expression of CRABP-II between presenescent and senescent fibroblasts. Taken together, these data suggest that retinoic acid plays a central role in the regulation of CRABP-II gene expression in the dermal fibroblast and that this molecule is the major mediator of the cytoplasmic effects of retinoids in dermal fibroblasts. However, in contrast to RAR beta 2, there is no apparent change in the regulation of CRABP-II in senescent dermal fibroblasts.

Cellular Senescence↗

PubMedDate not supplied

Regulation of thymidine kinase expression during cellular senescence.

As human diploid fibroblasts (HDFs) in culture senesce, the expression of thymidine kinase (TK) and the activity of its promoter become attenuated. Herein we analyze the cis-elements involved in transcriptional activation of the hTK promoter, and show that the Sp1 binding site located at -118/-113 and one CCAAT box located at either -71/-67 or -40/-36 are critical for maximal expression of hTK promoter activity in young IMR-90 HDFs. However, the DNA binding activities to TK-CCAAT and Sp1 were not defective in serum-stimulated senescent HDFs. On the other hand, treatment of young HDFs during the late G1 transition with a specific inhibitor of CDK2, roscovitine, blocked the induction of TK RNA expression. Because CDK2 remained inactive during serum stimulation in senescent HDFs, it is likely that the impairment of TK expression in senescent HDFs during serum stimulation is relevant to the inactivation of CDK2, rather than to the controlling mechanism at the level of NF-Y and Sp1 activity.

Blotting, Western↗

PubMed1995

Cataloging altered gene expression in young and senescent cells using enhanced differential display.

Recently, a novel PCR-based technique, differential display (DD), has facilitated the study of differentially expressed genes at the mRNA level. We report here an improved version of DD, which we call Enhanced Differential Display (EDD). We have modified the technique to enhance reproducibility and to facilitate sequencing and cloning. Using EDD, we have generated and verified a catalog of genes that are differentially expressed between young and senescent human diploid fibroblasts (HDF). From 168 genetags that were identified initially, 84 could be sequenced directly from PCR amplified bands. These sequences represent 27 known genes and 37 novel genes. By Northern blot analysis we have confirmed the differential expression of a total of 23 genes (12 known, 11 novel), while 19 (seven known, 12 novel) did not show differential expression. Several of the known genes were previously observed by others to be differentially expressed between young and senescent fibroblasts, thereby validating the technique.

Base Sequence↗

PubMed2000

Protein oxidation and degradation during proliferative senescence of human MRC-5 fibroblasts.

One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The aging process on a cellular level can be treated either as the ongoing proliferation until a certain number of cell divisions is reached (the Hayflick limit) or as the aging of nondividing cells, that is, the age-related changes in cells without proliferation. The present investigation was undertaken to reveal the changes in protein turnover, proteasome activity, and protein oxidation status during proliferative senescence. We were able to demonstrate that the activity of the cytosolic proteasomal system declines dramatically during the proliferative senescence of human MRC-5 fibroblasts. Regardless of the loss in activity, it could be demonstrated that there are no changes in the transcription and translation of proteasomal subunits. This decline in proteasome activity was accompanied by an increased concentration of oxidized proteins. Cells at higher proliferation stages were no longer able to respond with increased degradation of endogenous [(35)S]-Met-radiolabeled proteins after hydrogen peroxide- or quinone-induced oxidative stress. It could be demonstrated that oxidized proteins in senescent human MRC-5 fibroblasts are not as quickly removed as they are in young cells. Therefore, our study demonstrates that the accumulation of oxidized proteins and decline in protein turnover and activity of the proteasomal system are not only a process of postmitotic aging but also occur during proliferative senescence and result in an increased half-life of oxidized proteins.

Cell Division↗

PubMed1993

Effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on proliferation of young and senescent WI-38 human diploid fibroblasts.

The effects of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on the proliferation, protein kinase C activity (PKC), and c-fos gene expression were examined in cultures of young and senescent (90-95% lifespan completed) WI-38 human diploid fibroblasts. We observed that, following stimulation with medium containing 10% fetal bovine serum (FBS), the translocation of PKC from the cytosol to the particulate compartment was less efficient in senescent WI-38 cells than in young cells. However, when PMA was added to the medium, the intracellular distribution of PKC activity in old cells became nearly identical to that observed in young cells. The inducibility of c-fos mRNA by serum addition, which is a protein kinase C-dependent event, was significantly amplified in the presence of PMA. Moreover, the duration of peak c-fos expression, after stimulation by FBS and PMA, increased in senescent cells as compared to young cells. Our results reveal that the normal signal transduction pathway is altered in senescent, slowly proliferating human fibroblasts and that it can be partially restored in the presence of the tumor promoter PMA.

Cell Division↗

PubMed2002

ING1 isoforms differentially affect apoptosis in a cell age-dependent manner.

Recently, several novel human ING1 isoforms have been cloned. However,the biochemical functions and the involvement of these proteins in apoptosis remain uncharacterized. We have examined the apoptotic effects and biochemical functions of the two major human ING1 isoforms p47(ING1a) and p33(ING1b) in young and senescent human diploid fibroblasts induced to enter into apoptosis by diverse treatments. We have found that ING1 displayed isoform-, stimulus- and cell age-dependent apoptotic properties. We present evidence indicating that ING1 proteins bind to chromatin and are regulated in a manner related to their apoptotic properties. In agreement with previous reports, we have found that only young but not senescent fibroblasts were able to enter into apoptosis induced by growth factor deprivation. This effect was accompanied by up-regulation of endogenous p33(ING1b). Ectopic up-regulation of p33(ING1b), but not p47(ING1a), also induced apoptosis and sensitized young but not senescent cells to UV irradiation and hydrogen peroxide-mediated apoptosis. Cotransfection of p33(ING1b) and the tumor suppressor p53 increased the percentage of apoptotic cells yielded by either of these two proteins alone, in agreement with data from tumor cell models. Finally, we found that the chromatin binding affinity of p33(ING1b) was increased in senescent cells, which were resistant to apoptosis. Together, these data support the idea that the apoptotic functions of ING1 may be exerted by chromatin-related functions that are subject to cell age-dependent mechanisms of regulation.

Apoptosis↗

PubMed1996

A role for a p21-E2F interaction during senescence arrest of normal human fibroblasts.

The family of E2F transcription factors forms different multiprotein complexes with cell cycle regulatory proteins to control the expression of genes important in cell proliferation. In this study, we identified four distinct E2F complexes present in aged and senescent normal, human diploid fibroblasts. Two appeared to be identical to the previously described G1-specific p130 and Rb-E2F complexes present in young G0-arrested cells. The other two were novel E2F complexes that contained the cyclin-dependent kinase inhibitor p21 (cip1/WAF1/Sdi1/CAP20/PIC1) complexed with Rb/CDK2/cyclin E or with the Rb-related p107/CDK2/ cyclin D. These p21-E2F complexes, while present in young G1 cells at very low levels, were elevated in senescent cells. The p21 containing E2F complexes were not detected during the S-phase in young cells. The DNA-binding stability of the p21 complexes was approximately 10 times greater than the stability of any other E2F complex or uncomplexed E2F. Addition of purified p21 protein to the S-phase-specific cyclin A/ CDK2-p107-E2F complex from young cells dissociated cyclin A and CDK2 from p107/E2F, suggesting an additional novel function for p21. Finally, expression of p21 specifically inhibited transcription from an E2F-dependent promoter but had no effect on a mutant E2 promoter. In addition to its inhibition of CDK enzymes and proliferating cell nuclear antigen function in DNA replication, these studies reveal a novel mechanism by which p21 mediates growth arrest: direct interaction with E2F complexes and negative regulation of E2F transcription factor activity.

CDC2-CDC28 Kinases↗

PubMed1998

Increased transcription and modified growth state-dependent expression of the plasminogen activator inhibitor type-1 gene characterize the senescent phenotype in human diploid fibroblasts.

The type-1 inhibitor of plasminogen activator (PAI-1) is a major physiologic regulator of pericellular proteolytic activity and, as such, influences matrix integrity, cell-to-substrate adhesion, and cellular proliferation. Excessive accumulation of both PAI-1 mRNA and protein correlates with the progressive acquisition of morphological and growth traits characteristic of the senescent phenotype (Mu and Higgins, 1995, J. Cell. Physiol., 165:647-657). Compared to early-passage IMR-90 human diploid fibroblasts, a late-passage senescence-associated 11-fold elevation in steady-state PAI-1 mRNA content reflected a 15-fold increase in constitutive PAI-1 gene transcription. Differential mRNA stability was not a factor in age-associated PAI-1 overexpression in IMR-90 cells. Upon removal of serum, early-passage human fibroblasts enter into a state of growth arrest with marked down-regulation of PAI-1 synthesis. Rapid induction of both the 3.0- and 2.2-kb PAI-1 mRNA species was evident upon serum-induced "activation" of quiescent early-passage fibroblasts; induced PAI-1 transcripts were maximal at 2 hr post-serum stimulation and declined in late G1 prior to entry into S phase. In contrast, late-passage (p32) fibroblasts maintained a significant level of PAI-1 expression under serum-free culture conditions. Although the PAI-1 gene was further responsive to serum in senescent cells, transcript abundance remained elevated and actually increased over the 12 to 16 hr post-serum addition period (a time when early-passage fibroblasts down-regulate PAI-1 mRNA content). Development of the senescent phenotype in human fibroblasts is associated, therefore, with significant changes in PAI-1 gene regulation. Such reprogramming involves predominantly transcriptional events and results in a marked increase in steady-state PAI-1 transcript abundance involving both the 3.0- and 2.2-kb mRNA species.

Cell Division↗

PubMed1994

Retardation of the senescence of cultured human diploid fibroblasts by carnosine.

We have examined the effects of the naturally occurring dipeptide carnosine (beta-alanyl-L-histidine) on the growth, morphology, and lifespan of cultured human diploid fibroblasts. With human foreskin cells, HFF-1, and fetal lung cells, MRC-5, we have shown that carnosine at high concentrations (20-50 mM) in standard medium retards senescence and rejuvenates senescent cultures. These late-passage cultures preserve a nonsenescent morphology in the presence of carnosine, in comparison to the senescent morphology first described by Hayflick and Moorhead. Transfer of these late-passage cells in medium containing carnosine to unsupplemented normal medium results in the appearance of the senescent phenotype. The serial subculture of cells in the presence of carnosine does not prevent the Hayflick limit to growth, although the lifespan in population doublings as well as chronological age is often increased. This effect is obscured by the normal variability of human fibroblast lifespans, which we have confirmed. Transfer of cells approaching senescence in normal medium to medium supplemented with carnosine rejuvenates the cells but the extension in lifespan is variable. Neither D-carnosine, (beta-alanyl-D-histidine), homocarnosine, anserine, nor beta-alanine had the same effects as carnosine on human fibroblasts. Carnosine is an antioxidant, but it is more likely that it preserves cellular integrity by its effects on protein metabolism.

Carnosine↗

PubMed2005

Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-beta1 signaling pathway.

Premature senescence of human diploid fibroblasts (HDFs) can be induced by exposures to a variety of oxidative stress and DNA damaging agents. In this study we developed a robust model of UVB-induced premature senescence of skin HDFs. After a series of 10 subcytotoxic (non-proapoptotic) exposures to UVB at 250 mJ/cm2, the so-called biomarkers of senescence were markedly expressed: growth arrest, senescence-associated beta-galactosidase activity, senescence-associated gene overexpression, deletion in mitochondrial DNA. A set of 44 stress- and senescence-associated genes were found to be differentially expressed in this model, among which clusterin/apolipoprotein J (apo J) and transforming growth factor-beta1 (TGF-beta1). Transfection of apo J cDNA provided protection against premature senescence-inducing doses of UVB and other stressful agents. Neutralizing antibodies against TGF-beta1 or its receptor II (TbetaRII) sharply attenuated the senescence-associated features, suggesting a role for TGF-beta1 in UVB-induced premature senescence. Both the latent and active forms of TGF-beta1 were increased with time after the last UVB stress. Proteasome inhibition was ruled out as a potential mechanism of UVB-induced stress-induced premature senescence (SIPS). This model represents an alternative in vitro model in photoaging research for screening potential anti-photoaging compounds.

Cell Cycle Proteins↗

PubMed2006

Oxidative stress induces premature senescence by stimulating caveolin-1 gene transcription through p38 mitogen-activated protein kinase/Sp1-mediated activation of two GC-rich promoter elements.

Cellular senescence is believed to represent a natural tumor suppressor mechanism. We have previously shown that up-regulation of caveolin-1 was required for oxidative stress-induced premature senescence in fibroblasts. However, the molecular mechanisms underlying caveolin-1 up-regulation in senescent cells remain unknown. Here, we show that subcytotoxic oxidative stress generated by hydrogen peroxide application promotes premature senescence and stimulates the activity of a (-1,296) caveolin-1 promoter reporter gene construct in fibroblasts. Functional deletion analysis mapped the oxidative stress response elements of the mouse caveolin-1 promoter to the sequences -244/-222 and -124/-101. The hydrogen peroxide-mediated activation of both Cav-1 (-244/-222) and Cav-1 (-124/-101) was prevented by the antioxidant quercetin. Combination of electrophoretic mobility shift studies, chromatin immunoprecipitation analysis, Sp1 overexpression experiments, as well as promoter mutagenesis identifies enhanced Sp1 binding to two GC-boxes at -238/-231 and -118/-106 as the core mechanism of oxidative stress-triggered caveolin-1 transactivation. In addition, signaling studies show p38 mitogen-activated protein kinase (MAPK) as the upstream regulator of Sp1-mediated activation of the caveolin-1 promoter following oxidative stress. Inhibition of p38 MAPK prevents the oxidant-induced Sp1-mediated up-regulation of caveolin-1 protein expression and development of premature senescence. Finally, we show that oxidative stress induces p38-mediated up-regulation of caveolin-1 and premature senescence in normal human mammary epithelial cells but not in MCF-7 breast cancer cells, which do not express caveolin-1 and undergo apoptosis. This study delineates for the first time the molecular mechanisms that modulate caveolin-1 gene transcription upon oxidative stress and brings new insights into the redox control of cellular senescence in both normal and cancer cells.

Animals↗

PubMed1991

A role for both RB and p53 in the regulation of human cellular senescence.

We present evidence for the possible involvement of both the RB and p53 proteins in the regulation of cellular senescence. Human fibroblasts immortalized with an inducible SV40 T-antigen become senescent following the de-induction of T-antigen. Plasmids expressing an alternative source of intact T-antigen restore proliferation but T-antigen deletion mutants lacking either the RB or p53 binding domains are unable to do so. Similarly, combinations of adenovirus E1A + E1B or human papillomavirus E6 + E7 genes are able to replace T-antigen functions and permit cell proliferation, whereas the individual genes do not. These results are discussed in terms of a two-stage model for the escape from in vitro cellular senescence.

Adenoviruses, Human↗

PubMed1995

The growth-inhibitory block of TGF-beta is located close to the G1/S border in the cell cycle.

Transforming growth factor-beta (TGF-beta) inhibits DNA synthesis in dense cultures of young human embryonic fibroblasts and antagonizes the mitogenic action of platelet-derived growth factor (PDGF). The inhibition of the PDGF-BB action by TGF-beta was independent of the induction of mRNAs for the PDGF-A chain and PDGF-beta receptor, the predominant types of PDGF receptor in human fibroblasts. The TGF-beta-mediated inhibition did not influence the expression of various genes that are involved in the transition from the arrested (GO) state to the S phase of the cell cycle. Indeed, TGF-beta upregulated the "early" genes c-myc, c-fos, and junB and downregulated the growth arrest-specific (gas) genes. These results suggest that the inhibition of DNA synthesis by TGF-beta in human fibroblasts is independent of modulation of expression of early and gas genes, placing the TGF-beta block comparatively late in the GO to S transition. In cultures of senescent human fibroblasts TGF-beta stimulated DNA synthesis but, nevertheless, had the same effect as in young cells on the expression of PDGF chains and receptor genes, as well as on early and gas genes, with the exception of a significantly lower induction of c-fos.

Cell Cycle Proteins↗

PubMed1992

Cloning and expression of SAG: a novel marker of cellular senescence.

Unlike immortalized cell lines, normal human fibroblasts in culture undergo replicative senescence in which the number of population doublings is limited. While fibroblasts display a variety of changes as they senesce in vitro, little is known about how gene expression varies as a function of population doubling level. We have used differential hybridization screening to identify human genes that are preferentially expressed in senescent cells. While we found several isolates that were up-regulated in late-passage cells, all appeared to be variants of the same cDNA, which we named senescence-associated gene (SAG). Our data show that SAG expression is threefold higher in senescent fibroblasts and closely parallels the progressive slowdown in growth potential, but is not cell-cycle regulated. Thus, SAG serves as an accurate marker for fibroblast growth potential during replicative senescence. Further studies demonstrated that SAG is a novel gene active in nearly all tissue types tested and that it is conserved through evolution. DNA sequencing data indicate that SAG contains a potential DNA-binding domain, suggesting that SAG may function as a regulatory protein.

Actins↗

PubMedDate not supplied

Alteration in cellular RNAs during the in vitro lifespan of cultured human diploid fibroblasts. II. Synthesis and processing of RNA.

Incorporation of tritiated uridine into cellular RNA is decreased in senescent human fibroblast cultures when measured per cellular RNA content. Since early and late passage cells demonstrate similar kinetics of RNA precursor pool labeling and saturation, this decreased tritiated uridine incorporation reflects decreased RNA synthesis. However, cellular RNA contents are markedly elevated in senescent cell cultures. When data are measured per cell number or DNA content, net RNA synthesis is slightly increased in senescent cultures. Separation of labeled RNAs by polyacrylamide gel electrophoresis revealed the same diminished specific activities (CPM/RNA) and increased net synthesis (CPM/cell number) of major ribosomal and transfer RNAs. However, this slight increase in RNA synthesis is probably not of sufficient magnitude to completely explain the observed increase in cellular RNA content in senescent cells.

Aging↗

PubMed2003

Reversal of human cellular senescence: roles of the p53 and p16 pathways.

Telomere erosion and subsequent dysfunction limits the proliferation of normal human cells by a process termed replicative senescence. Replicative senescence is thought to suppress tumorigenesis by establishing an essentially irreversible growth arrest that requires activities of the p53 and pRB tumor suppressor proteins. We show that, depending on expression of the pRB regulator p16, replicative senescence is not necessarily irreversible. We used lentiviruses to express specific viral and cellular proteins in senescent human fibroblasts and mammary epithelial cells. Expression of telomerase did not reverse the senescence arrest. However, cells with low levels of p16 at senescence resumed robust growth upon p53 inactivation, and limited growth upon expression of oncogenic RAS. In contrast, cells with high levels of p16 at senescence failed to proliferate upon p53 inactivation or RAS expression, although they re-entered the cell cycle without growth after pRB inactivation. Our results indicate that the senescence response to telomere dysfunction is reversible and is maintained primarily by p53. However, p16 provides a dominant second barrier to the unlimited growth of human cells.

Breast↗

PubMed2002

Down-regulation of receptor-mediated endocytosis is responsible for senescence-associated hyporesponsiveness.

Human diploid fibroblasts (HDF) do not divide indefinitely and eventually lead to an arrest of cell division by a process termed cellular or replicative senescence. Irreversible growth arrest of senescent cells is strongly related to the attenuated response to growth factors. Recently, we reported that up-regulation of caveolin in the senescent cells is responsible for the attenuated response to growth factors. Senescent cells did not phosphorylate Erk-1/2 after EGF stimulation, whereas young cells did. In those senescent cells, we found an increased level of caveolin proteins and strong interactions between caveolin-1 and EGFR. When we overexpressed caveolin-1 in young HDF, the activation of Erk-1/2 on EGF stimulation was significantly suppressed. These results suggest that the hyporesponsiveness of senescent fibroblasts to EGF stimulation might be due to the overexpression of caveolin. In addition, the clathrin-dependent endocytosis system plays the more active and dominant role over the caveolae system. Therefore, we monitored the efficiency of clathrin-dependent receptor-mediated endocytosis in the senescent cells in order to elucidate the exact mode of the attenuated response to growth factors in the senescent cells. Using a transferrin-uptake assay and Western blot analysis of endocytosis-related proteins, we found a significant decrease of amphiphysin-1 in human diploid fibroblasts of multipassages. By adjusting the level of amphiphysin, we could modulate the efficiency of receptor-mediated endocytosis either in young or old cells toward growth factors: that is, a dominant negative mutant of amphiphysin-1 blocked the endocytosis in the young cells, while microinjection of the gene resumed its activity in the old cells. Taken together, we conclude that the loss of endocytotic activity of senescent cells is directly related to the down-regulation of amphiphysin-1 and/or up-regulation of caveolins. This opens a new field of functional recovery of the senescent cells simply through adjusting the receptor-mediated endocytosis capacity.

Caveolins↗