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PubMed1998

Senescent WI-38 fibroblasts overexpress LPC-1, a putative transmembrane shock protein.

We have recently reported the isolation of cDNAs for a number of genes that are differentially expressed between nonproliferating early (young) and late (senescent) population doubling level (PDL) WI-38 human, fetal lung-derived, fibroblast-like cells. We now demonstrate that one of these isolates, LPC-1 (Late PDL cDNA-1), derives from an approximately 2.9-kb mRNA species that is expressed at a two- to fivefold higher level in serum-starved, confluent, senescent versus similarly treated young WI-38 cells. Nucleotide sequence analysis of this cDNA confirms its identity with that of a cDNA encoding a marker (p63) for the rough endoplasmic recticulum and a related swine hepatic cardiogenic shock protein. We show that LPC-1 expression in early PDL WI-38 cells is strictly cell cycle-regulated and its expression peaks 9-12 h after serum stimulation of G0 cultures. The steady state levels of LPC-1 transcript in early PDL cells preceeding and following its peak expression are low, reflecting basal levels seen in G0 upon removal of serum. Late PDL cells, however, seem to have lost this tight cell cycle regulation seen in early PDL cells and inappropriately express high levels of the transcript after serum stimulation. Specific antiserum detects a protein of approximately 63 kDa by Western analysis and elicits intense cytoplasmic staining of senescent fibroblasts by immunohistochemistry. Related genomic sequences are found in all mammalian species examined as well as in the chicken. These findings are consistent with the hypothesis that senescent WI-38 cells exhibit a state of growth arrest fundamentally distinct from that of quiescent (G0) young cells.

Animals↗

PubMed1999

Proliferation and mitogenic response to PDGF-BB of fibroblasts isolated from chronic venous leg ulcers is ulcer-age dependent.

Several pathophysiologic mechanisms have been proposed to explain slow-healing leg ulcers, but little is known about the growth behavior of cells in these wounds. Platelet-derived growth factor-BB applied topically to chronic wounds has shown beneficial effects, although the effects have been less pronounced than would have been expected based on studies on acute wounds. The objective of this study was to compare fibroblasts in culture obtained from chronic wounds (non-healing chronic venous leg ulcers), acute wounds and normal dermis regarding growth, mitogenic response to platelet-derived growth factor-BB and levels ofplatelet-derived growth factor alpha-receptor and beta-receptor. Fibroblasts were obtained by an explant technique and expanded in vitro using fibroblast growth medium supplemented with 10% fetal bovine serum and used for the assays at their third passage. Growth of chronic wound fibroblasts (n = 8) was significantly (p < 0.05) decreased compared with those from acute wounds (n = 10) and normal dermis (n = 5). Fibroblasts from ulcers older than 3 y grew significantly (p < 0.01) slower than those from ulcers that had been present for less than 3 y. Morphology and size of fibroblasts from the oldest chronic wounds deviated substantially from those of acute wounds and normal dermis, and resembled in vitro aged or senescent fibroblasts. Mitogenic response of chronic wound fibroblasts to human recombinant platelet-derived growth factor-BB was also reduced with ulcer age. No significant differences were found in the amount of either platelet-derived growth factor alpha-receptor or beta-receptor among the three groups. The features decreased growth related to ulcer age, altered morphology, and reduced response to platelet-derived growth factor, indicating that fibroblasts in some chronic wounds have approached or even reached the end of their lifespan (phase III). This might provide one explanation for the non-healing state and therapy resistance to topical platelet-derived growth factor-BB of some venous leg ulcers.

Aged↗

PubMed1986

Failure in S6 protein phosphorylation by serum stimulation of senescent human diploid fibroblasts, TIG-1.

When quiescent young or senescent human diploid cells, TIG-1, were metabolically labeled with 32Pi and stimulated with 10% fetal bovine serum, the phosphorylation of ribosomal S6 protein was enhanced in young cells but not in senescent cells while that of some other proteins were increased in both cells. Inability to stimulate the phosphorylation of S6 protein in senescent cells after serum addition may be the primary cause of the failure of enhancement in protein synthesis followed by the block of prereplicative events dependent on protein synthesis and thus of the failure of cells to enter S phase. However, when the cell-free preparation from serum-stimulated senescent cells was incubated with [gamma-32P]ATP, S6-kinase activity was stimulated and S6 in ribosomal fraction was susceptible to phosphorylation as observed in young cells. Differences in S6 phosphorylation of senescent cells between in vivo and in vitro was discussed.

Blood↗

PubMed2000

Mutations in the WRN gene in mice accelerate mortality in a p53-null background.

Werner's syndrome (WS) is a human disease with manifestations resembling premature aging. The gene defective in WS, WRN, encodes a DNA helicase. Here, we describe the generation of mice bearing a mutation that eliminates expression of the C terminus of the helicase domain of the WRN protein. Mutant mice are born at the expected Mendelian frequency and do not show any overt histological signs of accelerated senescence. These mice are capable of living beyond 2 years of age. Cells from these animals do not show elevated susceptibility to the genotoxins camptothecin or 4-NQO. However, mutant fibroblasts senesce approximately one passage earlier than controls. Importantly, WRN(-/-);p53(-/-) mice show an increased mortality rate relative to WRN(+/-);p53(-/-) animals. We consider possible models for the synergy between p53 and WRN mutations for the determination of life span.

4-Nitroquinoline-1-oxide↗

PubMed2003

Superoxide dismutase 1 knock-down induces senescence in human fibroblasts.

Reactive oxygen species (ROS) such as superoxide radicals are responsible for the pathogenesis of various human diseases. ROS are generated during normal metabolic process in all of the oxygen-utilizing organisms. The copper-zinc-containing SOD (SOD1) acts as a major defense against ROS by detoxifying the superoxide anion. In model organisms, SOD1 has been shown to play a role in the aging process. However, the exact role of the SOD1 protein in the human aging process remains to be resolved. We show that SOD1 RNA interference (RNAi) induces senescence in normal human fibroblasts. This premature senescence depends on p53 induction. In contrast, in human fibroblastic cells with inactivated p53, the SOD1 RNAi is without effect. Surprisingly, in cancer cells (HeLa), the SOD1 RNAi induces cell death rather then senescence. Together, these findings support the notion that in normal human cells the SOD1 protein may play a role in the regulation of cellular lifespan by p53 and may also regulate the death signals in cancer cells.

Aging↗

PubMed2001

Two-dimensional gel electrophoretic studies on the cellular aging: accumulation of alpha-2-macroglobulin in human fibroblasts with aging.

To understand the mechanisms that control life span and age-related phenotypes, we used two-dimensional (2D) gel electrophoresis to study the intracellular proteins whose amounts change during the process of cellular aging. We found that the amount of an alpha-2-macroglobulin (A2M) fragment derived from culture medium increased in the cells with aging. A2M is linked to Alzheimer's disease both genetically and functionally. This is the first report of accumulation of an A2M fragment in senescent fibroblasts. We also studied 2D gel profiles of human fibroblasts immortalized by treatment with either 60Co gamma rays or 4-nitroquinoline 1-oxide. As immortalized cells overcome cellular senescence to gain an unlimited life span, the proteins whose amounts change after immortalization may be relevant to the age-related phenotypes. 2D gel analysis revealed that the A2M fragment was down-regulated in the immortalized cells, compared with their normal counterparts, regardless of their passage. We also found that the other four proteins increased in amount with aging and decreased in amount after immortalization. Our results suggest: (1) the A2M incorporation into the cells is increased in the process of cellular aging; and (2) A2M may be linked to the age-related phenotypes that were lost during the process of immortalization of human cells.

Cell Line↗

PubMed1995

Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence?

Mild oxidative stress as exerted by culture of human WI-38 fibroblasts under 40% oxygen partial pressure blocks proliferation irreversibly after one to three population doublings. Hyperoxically blocked cells are similar to senescent ones in terms of general morphology and lipofuscin accumulation. Moreover, they, like senescent fibroblasts, are blocked preferentially in G1 as evident from DNA content measurements by flow cytometry. Southern blotting of AluI- and HinfI-restricted genomic DNA shows an increase of the rate of telomere shortening from 90 bp per population doubling under normoxia to more than 500 bp per population doubling under hyperoxia. In every case, proliferation is blocked if a telomere cutoff length of about 4 kb is arrived at. The fact that telomere length correlates with the final inhibition of proliferation under conditions of varied oxidative stress, while the population doubling level does not, suggests that telomere shortening provides the signal for cell cycle exit in senescence. In postmitotic cells, no further telomere shortening occurs. However, the sensitivity of terminal restriction fragments to S1 nuclease increases, indicating the accumulation of single-strand breaks in telomeres of nondividing fibroblasts. This effect is found both under normoxic and hyperoxic culture, although it is more pronounced under conditions of higher oxidative stress. It might be speculated that accumulation of single-strand breaks and the resultant loss of distal single-stranded fragments during replication could be a major cause of telomere shortening, possibly more important than incomplete replication per se.

Aerobiosis↗

PubMed1979

Patterns of peptide synthesis in senescent and presenescent human fibroblasts.

Peptide production in senescent and presenescent human foreskin fibroblasts was measured using 2-dimensional polyacrylamide gel electrophoresis. This procedure permits the visualization of a cohort of the major peptides being produced. Among this cohort of over 500 peptides only two were found to differ in relative amount in that more was being produced in senescent cells. This difference was confirmed by measurements of the relative intensity of the peptide spot. This difference was senescent cell-specific and not due to the differences in rate of growth of senescent and non-senescent cells.

Cell Differentiation↗

PubMed2000

p16INK4A mediates cyclin dependent kinase 4 and 6 inhibition in senescent prostatic epithelial cells.

The senescence checkpoint constrains the proliferative potential of normal cells in culture to a finite number of cell doublings. In this study, we investigated the mechanism of cyclin dependent kinase (cdk) inhibition in senescent human prostatic epithelial cells (HPECs). Progression of HPECs from early passage to senescence was accompanied by a gradual loss of cells in S phase and an accumulation of cells containing 2N DNA. Furthermore, G1-S phase-associated kinase activities progressively diminished with increasing cell passage. In senescent HPECs, cdk4 and cyclin E1- and A-associated kinases were catalytically inactive. In contrast to observations in senescent fibroblasts, levels of the kinase inhibitor protein (KIP) inhibitor p21CIP1 diminished over the proliferative life span of HPECs. p27KIP1 levels fell as cells approached senescence, and the association of both p21CIP1 and p27KIP1 with cdk4/6 complexes was decreased. However, the level of cyclin E1-associated KIP molecules was unaltered as cells progressed into senescence. Progression to senescence was accompanied by a progressive increase in both the level of p16(INK4A) and in its association with cdk4 and cdk6. As HPECs approached senescence, cdk4- and cdk6-bound p16(INK4A) showed a shift to a slower mobility due to a change in its phosphorylation profile. As p16(INK4A) increased in cdk4 and cdk6 complexes, there was a loss of cyclin D1 binding. The altered phosphorylation of p16(INK4A) in senescent prostatic epithelial cells may facilitate its association with cdk4 and cdk6 and play a role in the inactivation of these kinases.

CDC2-CDC28 Kinases↗

PubMed2006

Cellular senescence in aging primates.

The aging of organisms is characterized by a gradual functional decline of all organ systems. Mammalian somatic cells in culture display a limited proliferative life span, at the end of which they undergo an irreversible cell cycle arrest known as replicative senescence. Whether cellular senescence contributes to organismal aging has been controversial. We investigated telomere dysfunction, a recently discovered biomarker of cellular senescence, and found that the number of senescent fibroblasts increases exponentially in the skin of aging baboons, reaching >15% of all cells in very old individuals. In addition, the same cells contain activated ataxia-telangiectasia mutated kinase and heterochromatinized nuclei, confirming their senescent status.

Aging↗

PubMed1986

Membrane-associated inhibitor of DNA synthesis in senescent human diploid fibroblasts: characterization and comparison to quiescent cell inhibitor.

Cell membranes prepared from senescent human diploid fibroblasts (HDF) inhibited entry into S phase by 35% when added to the medium of replicating young HDF. This membrane-associated inhibitory activity was (i) sensitive to trypsin, heat, and periodate, which suggests that the inhibitor is a glycoprotein, and (ii) not able to inhibit DNA synthesis in simian virus 40-transformed HDF, which indicates that not all types of cells are sensitive to this inhibitor. Quiescent young HDF also have a surface membrane-associated inhibitor of DNA synthesis. A comparison of the senescent HDF and quiescent HDF inhibitor activities indicates that they may have the same chemical and physical nature and the same specific activity, but their regulation is different. The inhibitory activity of quiescent young HDF is abolished within 20 hr after refeeding with fresh serum-containing medium, whereas that of senescent HDF remains unchanged. Quiescent old HDF (two or three population doublings remaining) exhibit an intermediate response to serum with approximately two-thirds of the inhibitory activity abolished. The fraction of cells in S phase at 20-24 hr post-stimulation (37% in young HDF, 24% in old HDF, and 0% in senescent HDF) is inversely proportional to inhibitor levels. This suggests that inability to neutralize the inhibitory activity in response to serum stimulation could be involved in the inability of senescent HDF to enter S phase. Disappearance of the inhibitory activity from quiescent young HDF occurs late in G1 phase. Thus, the inhibitor may play a role in determining the length of the G0 to S phase transition in these cells.

Cell Cycle↗

PubMed2006

Modulation of replicative senescence of diploid human cells by nuclear ERK signaling.

Normal somatic cells have a limited replicative lifespan, and serial subcultivation ultimately results in senescence. Senescent cells are irreversibly growth-arrested and show impaired responses to mitogens. Activation of the ERK signaling pathway, an absolute requirement for cell proliferation, results in nuclear relocalization of active ERKs, an event impaired in senescent fibroblasts. This impairment coincides with increased activity of the nuclear ERK phosphatase MKP2. Here we show that replicative lifespan can be altered by changes in nuclear ERK activity. Ectopic expression of MKP2 results in premature senescence. In contrast, knock-down of MKP2 expression, through transduction of MKP2 sequence-specific short hairpin RNA, or expression of the phosphatase resistant ERK2(D319N) mutant, abrogates the effects of increased endogenous MKP2 levels and senescence is postponed. Nuclear targeting of ERK2(D319N) significantly augments its effects and the transduced cultures show higher than 60% increase in replicative lifespan compared with cultures transduced with wt ERK2. Long-lived cultures senesce with altered molecular characteristics and retain the ability to express c-fos, and Rb is maintained in its inactive form. Our results support that MKP2-mediated inactivation of nuclear ERK2 represents a key event in the establishment of replicative senescence. Although it is evident that senescence can be imposed through multiple mechanisms, restoration of nuclear ERK activity can bypass a critical senescence checkpoint and, thus, extend replicative lifespan.

Amino Acid Substitution↗

PubMed2006

A novel role for high-mobility group a proteins in cellular senescence and heterochromatin formation.

Cellular senescence is a stable state of proliferative arrest that provides a barrier to malignant transformation and contributes to the antitumor activity of certain chemotherapies. Senescent cells can accumulate senescence-associated heterochromatic foci (SAHFs), which may provide a chromatin buffer that prevents activation of proliferation-associated genes by mitogenic transcription factors. Surprisingly, we show that the High-Mobility Group A (HMGA) proteins, which can promote tumorigenesis, accumulate on the chromatin of senescent fibroblasts and are essential structural components of SAHFs. HMGA proteins cooperate with the p16(INK4a) tumor suppressor to promote SAHF formation and proliferative arrest and stabilize senescence by contributing to the repression of proliferation-associated genes. These antiproliferative activities are canceled by coexpression of the HDM2 and CDK4 oncogenes, which are often coamplified with HMGA2 in human cancers. Our results identify a component of the senescence machinery that contributes to heterochromatin formation and imply that HMGA proteins also act in tumor suppressor networks.

Animals↗

PubMed1998

Cloned transgenic calves produced from nonquiescent fetal fibroblasts.

An efficient system for genetic modification and large-scale cloning of cattle is of importance for agriculture, biotechnology, and human medicine. Here, actively dividing fetal fibroblasts were genetically modified with a marker gene, a clonal line was selected, and the cells were fused to enucleated mature oocytes. Out of 28 embryos transferred to 11 recipient cows, three healthy, identical, transgenic calves were generated. Furthermore, the life-span of near senescent fibroblasts could be extended by nuclear transfer, as indicated by population doublings in fibroblast lines derived from a 40-day-old fetal clone. With the ability to extend the life-span of these primary cultured cells, this system would be useful for inducing complex genetic modifications in cattle.

Animals↗

PubMed1986

Expression of cell cycle-dependent genes in young and senescent WI-38 fibroblasts.

We studied the expression of 11 cell cycle-dependent genes in senescent WI-38 fibroblasts and compared the results to those obtained in WI-38 cells from early passages (young cells). Every gene we examined is expressed in the senescent cells at levels similar to those in the young cells, including two genes maximally expressed at the G1/S phase boundary--genes for thymidine kinase and histone H3. The results clearly show that senescent, noncycling WI-38 cells are not similar to quiescent cells. Rather, such senescent WI-38 cells may be blocked just prior to the onset of DNA synthesis.

Cell Cycle↗

PubMed1994

Age related induction of platelet-derived growth factor A-chain mRNA in normal human fibroblasts.

We have previously found that stimulation of normal neonatal fibroblasts with PDGF or EGF leads to a transient induction of PDGF A-chain mRNA and the synthesis of PDGF-AA proteins. This finding may imply the existence of an autocrine feedback mechanism to amplify the mitogenic signal under certain conditions. We have now studied the PDGF-BB mediated PDGF A-chain induction in a set of fibroblasts from young and old donors to clarify if the levels of induction are correlated to the donor age and the replicative capacity of the cells. The PDGF A-chain induction was found to be reduced in cells from old donors compared with cells from embryonic and neonatal donors. The different cell strains were also characterized further with respect to PDGF receptor expression and PDGF binding properties. PDGF beta-receptors were found to be enhanced in old donor cell strains, whereas the PDGF alpha-receptors showed more variability in expression between the strains. The PDGF A-chain mRNA induction was also decreased or absent in late passage human fibroblasts (senescent cells) when compared with early passage cells. These data suggest that the PDGF A-chain mRNA induction is regulated by an age related mechanism in human fibroblasts.

Aged↗

PubMed1983

Inhibition of DNA synthesis in proliferating human diploid fibroblasts by fusion with senescent cytoplasts.

Cytoplasts were prepared from senescent human diploid fibroblasts. The cytoplasts were fused to young human diploid fibroblasts and DNA synthesis was analyzed in the fusion products. DNA synthesis was inhibited (greater than or equal to 40%) in the senescent cytoplast fusion products when compared to unfused young cells or young cytoplasts fused with young cells. These results are consistent with previous experiments that have shown the blockage of DNA synthesis in both nuclei of heterokaryons from fusions of senescent and young human diploid fibroblast cells. Furthermore, these results support the postulate that senescent cells synthesize a specific substance(s), which is present in the cytoplasm of the senescent cell that inhibits DNA synthesis.

Cell Division↗

PubMedDate not supplied

Telomere loss: mitotic clock or genetic time bomb?

The Holy Grail of gerontologists investigating cellular senescence is the mechanism responsible for the finite proliferative capacity of somatic cells. In 1973, Olovnikov proposed that cells lose a small amount of DNA following each round of replication due to the inability of DNA polymerase to fully replicate chromosome ends (telomeres) and that eventually a critical deletion causes cell death. Recent observations showing that telomeres of human somatic cells act as a mitotic clock, shortening with age both in vitro and in vivo in a replication dependent manner, support this theory's premise. In addition, since telomeres stabilize chromosome ends against recombination, their loss could explain the increased frequency of dicentric chromosomes observed in late passage (senescent) fibroblasts and provide a checkpoint for regulated cell cycle exit. Sperm telomeres are longer than somatic telomeres and are maintained with age, suggesting that germ line cells may express telomerase, the ribonucleoprotein enzyme known to maintain telomere length in immortal unicellular eukaryotes. As predicted, telomerase activity has been found in immortal, transformed human cells and tumour cell lines, but not in normal somatic cells. Telomerase activation may be a late, obligate event in immortalization since many transformed cells and tumour tissues have critically short telomeres. Thus, telomere length and telomerase activity appear to be markers of the replicative history and proliferative potential of cells; the intriguing possibility remains that telomere loss is a genetic time bomb and hence causally involved in cell senescence and immortalization.

Animals↗