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PubMed2004

Proteasome inhibition increases HuR level, restores heat-inducible HSP72 expression and thermotolerance in WI-38 senescent human fibroblasts.

At the end of their replicative potential in vitro, late passage WI-38 human diploid fibroblasts (HDF) have a low basal expression of heat shock protein 72 (HSP72) and an attenuated ability to induce it in response to heat shock. The transient exposure to the specific and reversible proteasome inhibitor MG132 during a mild heat shock induced late passage HDF to synthesize and accumulate high levels of HSP72. This HSP72 expression was long-lasting and appeared to result from both increased cytoplasmic levels and enhanced translation of HSP72 mRNA. The level of HuR, a stabilizing mRNA-binding protein, increased following the MG132 treatment. This result is consistent with the proposed role of HuR in assisting mRNA export to the cytoplasm and in antagonizing its degradation. Furthermore, the previous exposure of late passage HDF to a mild heat shock in the presence of MG132 protected these cells against the otherwise lethal effect of a subsequent severe heat shock. This acquisition of thermotolerance appeared to be correlated with the level of HSP72.

Antigens, Surface↗

PubMedDate not supplied

Diminished in vitro tyrosine kinase activity of the EGF receptor of senescent human fibroblasts.

Fibroblastic cultures derived from normal human tissues undergo a finite number of population doublings when serially subcultivated in vitro (see refs 1, 2 for reviews). Epidermal growth factor (EGF) serves as a mitogen for early doubling level cultures of the human fetal lung-derived cell strain, WI-38, under serum-free conditions. The ability of cells from late doubling level cultures to respond mitogenically to EGF is lost, however, despite undiminished binding of EGF throughout the replicative lifespan. The ultimate effects of EGF, that is DNA synthesis and mitosis (see ref. 4 for review), occur after a sequence of events initiated by binding of ligand to specific cellular receptors. The receptor for EGF has been characterized as a 145,000-165,000 (145 K-165 K) molecular weight doublet, and, like the receptors for platelet-derived growth factor and insulin, and the transforming proteins of certain of the RNA tumour viruses, is a tyrosine-specific protein kinase with autophosphorylating activity. Moreover, several of the cellular target molecules of tyrosine phosphorylation have been found to be substrates for two or more of these kinases. The hypothesis that tyrosine phosphorylation underlies a common mechanism of growth control prompted us to ask whether the loss of responsiveness to EGF by late doubling level WI-38 cells is accompanied by altered expression of the EGF receptor, and specifically whether changes occur in the ability of receptors from populations of cells of various in vitro ages to catalyse tyrosine autophosphorylation. We show here that autophosphorylating activity is absent from the EGF receptor of cells which have lost their mitogenic responsiveness to EGF.

Cell Division↗

PubMedDate not supplied

Insulin-like growth factor binding protein-3 is overexpressed in senescent and quiescent human fibroblasts.

Cellular insulin-like growth factor binding protein-3 (IGFBP-3) mRNA and IGFBP-3 levels in conditioned medium were consistently higher in cultures of late passage normal (old) fibroblasts and prematurely senescent fibroblasts derived from Werner syndrome (WS) during quiescence induced by serum depletion and during the renewed growth ensuing after serum repletion, compared to cultures of early passage normal (young) fibroblasts. Molar ratios of IGFBP-3/IGF-II were always higher in senescent cultures and maintained a hierarchy of old > WS > young human diploid fibroblasts. Transfection into fibroblasts of the normal full-length IGFBP-3 cDNA in an expression vector resulted in a significant reduction in colony formation compared to cells transfected with an empty expression vector (no cDNA) or with IGFBP-3 cDNA altered by a 273 base pair (bp) deletion. Addition to old and young cultures of recombinant human IGFBP-3 and IGF-I at 1:1 or 5:1 molar ratios inhibited IGF-I-mediated DNA synthesis by approximately 70-80%. These data indicate that IGFBP-3 may play an important role in the quiescent and senescent growth arrest of HDF.

Aging↗

PubMed1993

In vitro cellular aging in T-lymphocyte cultures: analysis of DNA content and cell size.

Like other normal diploid mammalian cells, human T-lymphocytes display a limited in vitro lifespan. Long-term cultures of normal adult peripheral blood T cells, activated in vitro and passaged in the presence of interleukin-2 undergo 23 +/- 7 cumulative population doublings. Cell cycle analysis revealed that in senescent T cell cultures, restimulation with the original antigen resulted in 16-22% of the cells entering S phase, compared to 60% entering cycle in young cultures. In addition, within 1 week of restimulation, the senescent cultures do not increase in cell number, but 93% of the cells return to the G1/G0 DNA content, a proportion typically seen in quiescent (nonrestimulated) cultures. Coculture of early- and late-passage cells at two different ratios excluded a putative inhibitory factor produced by the old cells and similarly eliminated a possible stimulatory product in early cultures. Flow cytometry measure of forward angle light scatter revealed no difference in cell size between early- and late-passage cells, in contrast to the findings with senescent fibroblasts. Thus, while increasing cell size may contribute to the senescent phenotype of fibroblast cultures, it is not a factor in the senescence of human T-lymphocytes, and it is therefore doubtful that alterations in cell size are fundamental to in vitro cellular aging.

Cell Cycle↗

PubMed2005

p21WAF/CIP1/SDI1 is upregulated due to increased mRNA stability during hydroxyurea-induced senescence of human fibroblasts.

Hydoxyurea induces senescence-like growth arrest in normal human fibroblasts. p21(WAF/CIP1/SDI1), a cyclin dependent kinase inhibitor, was found to be upregulated during this growth arrest. Levels of p21(WAF/CIP1/SDI1) protein and mRNA were increased nine-fold by hydroxyurea in these cells. In order to determine whether p21(WAF/CIP1/SDI1) mRNA is increased by hydroxyurea at the transcriptional level, human fibroblast cells were transfected with reporter constructs containing a p21(WAF/CIP1/SDI1) promoter fragment and then treated with hydroxyurea. The luciferase activities in the reporter-transfected fibroblast cells were not increased by hydroxyurea, indicating that p21(WAF/CIP1/SDI1) transcription was not elevated by hydroxyurea. The half-life of the p21(WAF/CIP1/SDI1) mRNA was increased by 2.5-fold but that of p21(WAF/CIP1/SDI1) protein was not. Our results suggest that increased mRNA stability is the major mechanism of p21(WAF/CIP1/SDI1) elevation in the hydroxyurea-induced growth arrest of human fibroblasts.

Blotting, Northern↗

PubMedDate not supplied

Replicative senescence of human fibroblasts: the role of Ras-dependent signaling and oxidative stress.

Replicative senescence of human fibroblasts is a widely used cellular model for human aging. While it is clear that telomere erosion contributes to the development of replicative senescence, it is assumed that additional factors contribute to the senescent phenotype. The free radical theory of aging suggests that oxidative damage is a major cause of aging; furthermore, the expression of activated oncogenes, such as oncogenic Ras, can induce premature senescence in primary cells. The functional relation between the various inducers of senescence is not known. The present study was guided by the hypothesis that constitutive activation of normal, unmutated Ras may contribute to senescence-induced growth arrest in senescent human fibroblasts. When various branches of Ras-dependent signaling were investigated, constitutive activation of the Ras/Raf/MEK/ERK pathway was not observed. To evaluate the role of oxidative stress for the senescent phenotype, we also investigated stress-related protein kinases. While we found no evidence for alterations in the activity of p38, we could detect an increased activity of Jun kinase in senescent fibroblasts. We also found higher levels of reactive oxygen species (ROS) in senescent fibroblasts compared to their younger counterparts. The accumulation of ROS in senescent cells may be related to the constitutive activation of Jun kinase.

Blotting, Western↗

PubMed2002

Agonist-specific differential changes of cellular signal transduction pathways in senescent human diploid fibroblasts.

Changes in the signal transduction efficiency of senescent cells led us to compare the signaling events induced by two mitogenic agonists, platelet-derived growth factor (PDGF) and lysophosphatidic acid (LPA) in presenescent and senescent or near-senescent human diploid fibroblasts. When the changes in intracellular [Ca(2+)](i) were analyzed, both PDGF and LPA generated a rhythmic increase in [Ca(2+)](i) in presenescent cells. The frequency of calcium response was reduced and desensitized in PDGF-stimulated senescent cells, while response to a LPA-induced calcium signal was also reduced in frequency, though its magnitude was unaltered. PDGF treatment increased the fibrous actin (F-actin) level in presenescent cells but not in senescent cells in contrast to a reduced but visible increase in F-actin in LPA-treated senescent cells. The effect of PDGF on phospholipase D (PLD) activation was also reduced significantly, as a ca. 60-80% reduction of PLD activity was observed in PDGF-stimulated cells but only a little reduction in LPA-induced cells. Agonist-specific differential changes of cellular signaling events caused a differential effect on DNA synthesis after growth factor stimulation. We observed a dramatic (80-90%) reduction of [3H]thymidine incorporation into DNA in the PDGF-stimulated near-senescent cells. LPA resulted in a 2-3-fold increase in thymidine incorporation even in the near-senescent cells. These differences in the responses of senescent or near-senescent cells to PDGF- and LPA-stimulation raised questions about the differential changes of the respective signaling apparatuses induced by aging. Since PDGF signaling event was affected greatly by aging, we further examined the protein contents involved in PDGF signal transduction pathway. PDGF receptor (PDGFR), protein kinase C-alpha (PKC-alpha), phospholipase C-gamma1 (PLC-gamma1), and PLD1 were examined by Western blot analysis. The protein levels of PKC-alpha and PLC-gamma1 were unchanged, but those of PLD1 and PDGFR were reduced with age. The reduced content of PDGFR protein may be one of the important contributors to the failure of PDGF-stimulated signal transduction in human senescent fibroblasts. Our results strongly suggest that age-dependent agonist-specific changes in signaling events might be in charge of the functional deterioration of senescent cells through imbalance of signal responses.

Actins↗

PubMed2006

Identification of p38MAPK-dependent genes with changed transcript abundance in H2O2-induced premature senescence of IMR-90 hTERT human fibroblasts.

Premature senescence of IMR-90 human diploid fibroblasts expressing telomerase (hTERT) establishes after exposure to an acute sublethal concentration of H2O2. We showed herein that p38(MAPK) was phosphorylated after exposure of IMR-90 hTERT cells to H2O2. Selective inhibition of p38(MAPK) activity attenuated the increase in the proportion of cells positive for senescence associated beta-galactosidase activity. We generated a low density DNA array to study gene expression profiles of 240 senescence-related genes. Using this array, p38(MAPK) inhibitor and p38(MAPK) small interferent RNA, we identified several p38(MAPK)-target genes differentially expressed in H2O2-stressed IMR-90 hTERT fibroblasts.

Cell Line↗

PubMed1988

Absence of three secreted proteins and presence of a 57-kDa protein related to irreversible arrest of cell growth.

Two subsets of proteins from the conditioned medium of normal human diploid fibroblasts were detected by NaDodSO4/polyacrylamide gel electrophoresis analysis; the presence of these subset is related to the nonproliferative state of in vitro aged cells. One subset consists of three proteins (molecular mass from 80 to 87 kDa) secreted by replicating cells in sparse cultures, as well as by quiescent cells in confluent or serum-starved cultures of young fibroblasts. These proteins disappear from the medium when cultures reach the state of senescence. The other subset consists of a single protein of 57 kDa, detected only in conditioned medium of senescent fibroblast cultures. The results suggest that when human fibroblasts reach senescence, they secrete the 57-kDa protein and concomitantly stop secreting the other three proteins normally found in the culture medium of young fibroblasts. Therefore, the alternative secretion of these protein subsets could specifically signify irreversible arrest of cell growth during in vitro cellular aging.

Cell Division↗

PubMedDate not supplied

Drop of connexin 43 in replicative senescence of human fibroblasts HEL-299 as a possible biomarker of senescence.

The expression of connexin 43 (cx43) and cell-cell communication were studied in replicative senescence of cultured HEL-299 fibroblasts. A progressive decrease in fluorescent dye transfer was detected by a scrape-loading technique in aging fibroblasts. This change was accounted for by a marked decrease in the amount of cx43 in aging cells, as detected by western blot analysis (cell extracts) and indirect fluorescence (cells in culture). However, semiquantitative RT-PCR assays of cx43 mRNA did not reveal appreciable changes, which suggests several possible explanations for the mechanism(s) underlying the decrease of cx43 in aging cells. These findings support the idea that the reduced expression of cx43 might be a biomarker of cell senescence.

Biomarkers↗

PubMed1986

Existence of high abundance antiproliferative mRNA's in senescent human diploid fibroblasts.

Polyadenylated RNA isolated from senescent human diploid fibroblasts (HDF) inhibited DNA synthesis in proliferation-competent cells after microinjection, whereas polyadenylated RNA from young HDF had no inhibitory effect. Polyadenylated RNA from young cells made quiescent by removal of serum growth factors had a slight inhibitory effect on DNA synthesis. The abundance level of inhibitor messenger RNA (mRNA) from senescent cells was estimated at 0.8 and that of quiescent cells at 0.005 percent. These results demonstrate the existence of one or more antiproliferative mRNA's in nonproliferating normal human cells; these RNA's code for factors that either work antagonistically to initiators of DNA synthesis or regulate the expression of the initiators in some way. The abundance level of the inhibitory mRNA in senescent cells indicates the feasibility of developing a complementary DNA probe that will be useful in studying cell cycle control mechanisms.

Cell Division↗

PubMed1997

Different kinetics of senescence in human fibroblasts and peritoneal mesothelial cells.

Senescence has been reported for a wide variety of human cell types. In cultures of human fibroblasts the process is due to a percentage of the cells becoming senescent at each passage rather than all the cells entering senescence simultaneously at the end of the life span. By measuring the percentage of fibroblasts which are still cycling at each passage, a rate of decline in the growth fraction, which mirrors the rate of senescence, can be obtained. However, such an analysis has never been undertaken in multiple cell types using the same method to identify cycling cells. It is thus unknown if the rate of senescence is the same or different in cultures of different human cell types. To answer this question the rates of decline in the cycling fractions were simultaneously measured in two cultures of human cells (AGO7086A, peritoneal mesothelial cells; and 2DD, human dermal fibroblasts) which have practically identical in vitro life spans. 2DD fibroblasts showed a rate of decline of 0.89% cycling cells per population doubling when the data obtained were fitted to a simple linear equation. However, AGO7086A gave a decline of approximately 2.2% per population doubling. Thus mesothelial cells enter senescence significantly faster than fibroblasts (P < 0.001). This decline in the growth fraction was accompanied by an increasing fraction of mesothelial cells which retained detectable endogenous beta-galactosidase activity at pH 6. Such activity has previously been shown to be associated with senescent human fibroblasts. These findings suggest that the process of senescence has common features in different cell lineages but that the rate of the process can differ markedly between them.

Biomarkers↗

PubMed2004

Molecular signature of oncogenic ras-induced senescence.

Senescence irreversibly arrests the proliferation of cells that have sustained significant cellular stress. Replicative senescence, due to the shortening and dysfunction of telomeres, appears to provide a barrier to the immortalization of cells and development of cancer. In normal human fibroblasts, senescence induced by oncogenic H-ras displays a nearly identical cellular phenotype to that of replicative senescence, suggesting the activation of a common senescence mechanism. In this study, we investigated the gene expression profile of oncogenic H-ras-induced senescent human diploid fibroblasts. We found altered gene expression of various cell cycle regulators in both oncogenic H-ras-induced senescent cells and replicative senescent cells. Similar to replicative senescent cells, H-ras-induced senescent cells exhibited specific downregulation of genes involved in G2/M checkpoint control and contained tetraploid cells that were arrested in a G1 state. This observation suggests that the inactivation of G2/M checkpoints may be involved in senescence and may play a role in the generation of senescent G1 tetraploid cells. Lastly, we have identified two genes, topoisomerase IIalpha and HDAC9, whose expression was specifically altered under several conditions associated with senescence, suggesting that these two molecules may be novel biomarkers for senescent human fibroblasts.

Antigens, Neoplasm↗

PubMed1993

Toward a molecular understanding of human breast cancer: a hypothesis.

A rate limiting step in most metastatic breast cancers is the development of unlimited proliferative potential by mammary epithelial cells. We describe mechanisms by which these cells can attain this state. The two independent mortality mechanisms controlling fibroblast senescence and immortalization (M1 and M2) are also found in human mammary epithelial cells. However, although both p53 and Rb are involved in the M1 mechanism of fibroblast cellular senescence, in human mammary epithelial cells only p53 is involved. The M1/M2 mechanisms may be induced by the gradual loss of telomere ends that occur as normal cells divide. Loss of telomere ends may result in genomic instability and in altered gene expression due to heterochromatin changes in subtelomeric regions. Events which can abrogate p53 functions are described, as is the current state of knowledge about the function of p53. All these factors are included in a molecular model for the onset of breast cancer.

Breast Neoplasms↗

PubMed2002

Human fibroblasts in vitro senesce with a donor-specific telomere length.

In human fibroblasts, replicative senescence, telomere length and donor age are closely interrelated. We analyzed these relationships for fibroblast strains from 14 healthy human donors in the age range of 28-90 years. In vitro replicative capacity was correlated more closely with donor age than with telomere length ex vivo, especially for healthy donors. Telomere length at senescence was as variable as at cell explantation and increased with donor age. The data suggest a donor-specific, age-dependent regulation of the telomere length threshold that triggers senescence in human fibroblasts.

Adult↗

PubMed1979

On the aging of organisms and their cells.

An explanation of organismic ageing based on a limited division capacity of dividing cells is difficult to reconcile with much of the available data. The physiology of cells in ageing organisms tends, on the contrary, to suggest that organisms age as a result of degeneration in their non-dividing cell populations. Senescence in these non-mitotic cells resmebles the ageing of the non-dividing fraction of cell cultures clonally senescing, or maintained in long-term quiescence in vitro. As cultures of diploid human fibroblasts senesce there is an accumulation of non-dividing cells. Alterations in these post-mitotic cells can explain the senescent properties of late passage cultures. It is proposed that during the in vitro senescence of fibroblast cultures, cell ageing results from, as opposed to causes, the absence of mitosis. Cell ageing may primarily result from changes in the chromatin induced by the non-mitotic state.

Aged↗

PubMed2003

Loss of retinoblastoma but not p16 function allows bypass of replicative senescence in human fibroblasts.

Current models envision replicative senescence to be under dual control by the p53 and retinoblastoma (RB) tumour-suppressor pathways. The role of the p16(INK4a)-RB pathway is controversial, and the function of RB in human cells has not been tested directly. We used targeted homologous recombination to knock out one copy of RB in presenescent human fibroblasts. During entry into senescence, RB+/- cells underwent spontaneous loss of heterozygosity and the resultant RB-/- clones bypassed senescence. The extended lifespan phase was eventually terminated by a crisis-like state. The same phenotype was documented for p21(CIP1/WAF1) and p53 heterozygous cells, indicating that loss of function of all three genes results in failure to establish senescence. By contrast, the abolition of p16 function by the expression of a p16-insensitive cyclin-dependent kinase 4 protein or siRNA-mediated knockdown provided only minimal lifespan extension that was terminated by senescence. We propose that p53, p21 and RB act in a linear genetic pathway to regulate cell entry into replicative senescence.

Cellular Senescence↗

PubMed2002

Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis.

BACKGROUND: To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. METHODS: We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. RESULTS: Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. CONCLUSIONS: The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies.

Cell Division↗