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PubMed1998

Independent induction of senescence by p16INK4a and p21CIP1 in spontaneously immortalized human fibroblasts.

In this work, we address the question of whether replicative senescence can be induced in immortal nontumorigenic human fibroblasts. The immortal fibroblasts used in this study were derived from two Li-Fraumeni (LF) patients who carry in their germ line one wild-type and one mutant p53 allele. Both immortal lines have lost the wtp53 allele and express no detectable p16INK4a protein, although they carry the p16INK4a gene. In contrast to immortal human fibroblasts, senescent human fibroblasts have a low content of 5-methyl-cytosine in their DNA. This observation suggested the possibility that a demethylating agent could revert the immortal phenotype and induce replicative senescence in the immortal cell lines. Cells of the two LF lines were exposed to the demethylating agent 5-aza-2'-deoxycytidine. Within 6 days, all cells were growth arrested and showed the enlarged and flat morphology characteristic of senescent cells, an accumulation of lipofuscin granules and senescence-associated beta-galactosidase activity at pH6, both biomarkers for senescence. Immunoblots of 5-aza-2'-deoxycytidine-treated cells showed a greatly increased expression of p16INK4a protein but no detectable change in the expression of p21CIP1, a gene known to be strongly expressed in senescent normal human fibroblasts. In two other experimental series, cells of the two LF lines were infected with retroviral constructs encoding either p16INK4a or p21CIP1. Each of the transduced genes induced senescence without affecting the expression of the other endogenous gene. The results show that induction of senescence in immortal LF fibroblasts can occur by different pathways: (a) by demethylation-dependent pathways that induce the expression of p16INK4a; and (b) by demethylation-independent pathways involving the expression of p21CIP1. The induction of senescence by p16INK4a and p21CIP1 occurred equally in the two human immortal fibroblast lines, which differed in the length of their telomeres and the activity of their telomerase.

5-Methylcytosine↗

PubMed1993

Modifications of proteoglycans produced by human skin fibroblast cultures during replicative senescence.

The properties of proteoglycans (PGs) produced by normal human skin fibroblasts were investigated with increasing passage. The increase of subculture number was associated with a constant increase in PG molecular size, which was particularly evident in cell layer extracts. In the cell layer, the ratio of DS-PGs/HS-PGs was markedly higher in early passage cultures. Moreover, the cell layer from young cells contained lower amounts of radioactivity incorporated into the most hydrophobic PG populations, suggesting that the PG core protein might also undergo significant modification with increasing subcultures. There was no significant difference in energy charge value between early and late passage cultures, whereas the NAD/NADH ratio was found to decrease markedly in senescent cells.

Cell Division↗

PubMed2006

p21Waf1/Cip1 plays a critical role in modulating senescence through changes of DNA methylation.

It has been reported that genomic DNA methylation decreases gradually during cell culture and an organism's aging. However, less is known about the methylation changes of age-related specific genes in aging. p21(Waf1/Cip1) and p16(INK4a) are cyclin-dependent kinase (Cdk) inhibitors that are critical for the replicative senescence of normal cells. In this study, we show that p21(Waf1/Cip1) and p16(INK4a) have different methylation patterns during the aging process of normal human 2BS and WI-38 fibroblasts. p21(Waf1/Cip1) promoter is gradually methylated up into middle-aged fibroblasts but not with senescent fibroblasts, whereas p16(INK4a) is always unmethylated in the aging process. Correspondently, the protein levels of DNA methyltransferase 1 (DNMT1) and DNMT3a increase from young to middle-aged fibroblasts but decrease in the senescent fibroblasts, while DNMT3b decreases stably from young to senescent fibroblasts. p21(Waf1/Cip1) promoter methylation directly represses its expression and blocks the radiation-induced DNA damage-signaling pathway by p53 in middle-aged fibroblasts. More importantly, demethylation by 5-aza-CdR or DNMT1 RNA interference (RNAi) resulted in an increased p21(Waf1/Cip1) level and premature senescence of middle-aged fibroblasts demonstrated by cell growth arrest and high beta-Galactosidase expression. Our results suggest that p21(Waf1/Cip1) but not p16(INK4a) is involved in the DNA methylation mediated aging process. p21(Waf1/Cip1) promoter methylation may be a critical biological barrier to postpone the aging process.

Azacitidine↗

PubMed2004

Cloning and characterization of cellular senescence-associated genes in human fibroblasts by suppression subtractive hybridization.

Cellular senescence marks the end of the proliferative life span of normal cells in tissue culture and occurs after cells have undergone a certain number of population doublings (PDLs). It is accompanied by alterations in the pattern of gene expression. A specific human embryonic lung diploid fibroblast cell line, 2BS, has been studied as a model of senescence in our laboratory. Here, we report a set of cellular senescence-associated genes identified from suppression subtractive cDNA libraries from senescent and young 2BS cells. They include three novel genes and six previously identified genes of unknown function. The genes whose functions are known belong to various functional pathways that have been reported to change with the onset of senescence. These include three pre-mRNA splicing factors with reduced expression in senescent cells, indicating that the regulation of mRNA splicing is altered during cell senescence. In addition, the expression of the gene TOM1 (target of Myb 1), which has not previously been associated with cellular senescence, is shown to increase in senescent cells, and we demonstrate that the expression of antisense TOM1 gene in 2BS cells can delay the progress of senescence.

Cell Line↗

PubMed2001

p53 and its homologues, p63 and p73, induce a replicative senescence through inactivation of NF-Y transcription factor.

Recent studies have identified two p53 homologues, p63 and p73. They activate p53-responsive promoters and induce apoptosis when overexpressed in certain human tumors. Here, we report that p63, like p53 and p73, induces replicative senescence when expressed in a tetracycline-regulated manner in EJ cells lacking a functional p53. In addition to transcription activation of p53-responsive genes, we found that p63 and p73 repress transcription of the cdk1 and cyclin B genes, both of which are irreversibly repressed in senescent human fibroblast. In transient transfection assay, p63 and p73 repress the cdk1 promoter regardless of the presence of a dominant negative mutant form of p53. Furthermore, we found that DNA binding activity of NF-Y transcription factor, which is essential for transcription of the cdk1 and cyclin B genes and inactivated in senescent fibroblast, is significantly decreased by expression of either of p53, p63, or p73. Since NF-Y binds to many promoters besides the cdk1 and cyclin B promoters, inactivation of NF-Y by p53 family genes may be a general mechanism for transcription repression in replicative senescence.

CCAAT-Binding Factor↗

PubMed1985

Histone H1 in G1 arrested, senescent, and Werner syndrome fibroblasts.

Histone H1 content and synthesis were examined in normal, Werner-syndrome, and transformed fibroblasts. Analysis of 3H-lysine incorporation indicated that senescent cells, but not G1-arrested young cells, had a lower ratio of molar synthesis of H1 histone to nucleosome histones than did growing young cells or gamma-ray-transformed cells. Furthermore, a biochemical study of histone H1 content plotted as a function of DNA synthesis activity and an immunocytological study using antiserum against histone H1 revealed that senescent cells had a lower histone H1 content than did young cultures at all stages of cell proliferation. Werner syndrome skin fibroblasts at early passage, however, had amounts of histone H1 comparable to those of age-matched normal control fibroblasts. We conclude that a decline, with increasing passage number, in content and synthesis of H1 histone relative to nucleosomal histones (Mitsui et al., 1980) was not simply due to passage-related accumulation of G1-arrested cells, but actually reflected age specific changes of cultured human fibroblasts. The depletion of histone H1 in the chromatin of senescent cells is a possible cause of DNA strand breakage or relaxation of gene repression.

Cell Cycle↗

PubMed2000

Cell cycle regulation in H(2)O(2)-induced premature senescence of human diploid fibroblasts and regulatory control exerted by the papilloma virus E6 and E7 proteins.

Many biomarkers of replicative senescence appear in stress-induced premature senescence (SIPS) of human diploid fibroblasts (HDFs). The mRNA level of key cell cycle regulators was studied in H(2)O(2)-induced premature senescence of HDFs expressing or not the papillomavirus E6 and E7 proteins, which enhanced, respectively, the proteolysis of p53 and Rb. The CdKI's p21(waf-1) and p16(Ink-4a) were found overexpressed in H(2)O(2)-induced premature senescence, while p19(Ink-4d)and p27(Kip-1) were repressed. The results obtained in E6 HDFs suggest that p21(waf-1) and p16(Ink-4a) overexpressions are p53-independent, while p27(Kip-1) and p19(Ink-4d) down-regulations are p53-dependent.E6 regulated Rb, p130, p53 and p16(Ink-4a) mRNA level in non-stressing conditions, and regulated p130, p107, p53, p19(Ink-4d), p27(Kip-1) mRNA level in SIPS. SIPS modified the E6-mediated regulatory control on p107, p16(Ink-4a), p19(Ink-4d) and p27(Kip-1) mRNA level, when compared to normal conditions.E7 regulated the mRNA level of all the genes studied, in all conditions, suggesting that the Rb family or other E7-interacting proteins might modify the expression of these genes. SIPS modified strongly the E7-mediated regulatory control on p107, p16(Ink-4a), p19(Ink-4d), p27(Kip-1), p21(Waf-1) and Rb mRNA level, when compared to normal conditions. Further work is ongoing to test whether this E7-mediated regulatory control takes place through interactions with Rb or other E7-interacting proteins.

Cell Cycle↗

PubMed2006

Busulfan selectively induces cellular senescence but not apoptosis in WI38 fibroblasts via a p53-independent but extracellular signal-regulated kinase-p38 mitogen-activated protein kinase-dependent mechanism.

Busulfan (BU) is a unique alkylating agent that primarily targets slowly proliferating or nonproliferating cells in the body, leading to various normal tissue damage while killing leukemia cells. However, the mechanism(s) of action whereby BU injures normal cells has not been well defined and, therefore, was investigated in the present study by using the normal human diploid WI38 fibroblasts as a model system. We found that WI38 fibroblasts incubated with BU (from 7.5-120 microM) for 24 h underwent senescence but not apoptosis in a dose-independent manner, whereas cells incubated with 80 and 20 microM etoposide (Etop) were committed to apoptosis and senescence, respectively. The induction of WI38 cell senescence by Etop was associated with p53 activation and could be attenuated by down-regulation of p53 using alpha-pifithrin (alpha-PFT) or p53 small interference RNA (siRNA). In contrast, WI38 cell senescence induced by BU was associated with prolonged activation of extracellular signal-regulated kinase (Erk), p38 mitogen-activated protein kinase (p38), and c-Jun NH(2)-terminal kinase (JNK) and could be suppressed by the inhibition of Erk and/or p38 with PD98059 (2'-amino-3'-methoxyflavone) and/or SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], respectively. However, inhibition of p53 with alpha-PFT or p53 siRNA or JNK with SP600125 (1,9-pyrazoloanthrone) failed to protect WI38 cells from BU-induced senescence. These findings suggest that BU is a distinctive chemotherapeutic agent that can selectively induce normal human fibroblast senescence through the Erk and p38 pathways.

Antineoplastic Agents, Alkylating↗

PubMed2002

Biallelic mutations in p16(INK4a) confer resistance to Ras- and Ets-induced senescence in human diploid fibroblasts.

The INK4a/ARF tumor suppressor locus is implicated in the senescence-like growth arrest provoked by oncogenic Ras in primary cells. INK4a and ARF are distinct proteins encoded by transcripts in which a shared exon is decoded in alternative reading frames. Here we analyze dermal fibroblasts (designated Q34) from an individual carrying independent missense mutations in each copy of the common exon. Both mutations alter the amino acid sequence of INK4a and functionally impair the protein, although they do so to different degrees. Only one of the mutations affects the sequence of ARF, causing an apparently innocuous change near its carboxy terminus. Unlike normal human fibroblasts, Q34 cells are not permanently arrested by Ras or its downstream effectors Ets1 and Ets2. Moreover, ectopic Ras enables the cells to grow as anchorage-independent colonies, and in relatively young Q34 cells anchorage independence can be achieved without addition of telomerase or perturbation of the p53 pathway. Whereas ARF plays the principal role in Ras-induced arrest of mouse fibroblasts, our data imply that INK4a assumes this role in human fibroblasts.

Adult↗

PubMed1997

[Studies on cell senescence induced by D-galactose in cultured neurons and fibroblasts].

The cell senescence induced by D-galactose (D-gal) in cultured rat fetal brain neurons and human fetal lung fibroblast was investigated in vivo and in vitro. The results showed pronounced decreases in nerve cell diameters, number of process and percentage of multiprocess neurons including "pyramidal" and "stellate" shaped and a rather high level of mortality rate observed in D-gal-treated (8 g/L) neurons. Lessening of the proliferating population of lung fibroblast taken from rat treated with D-gal (50 mg/kg.d, subcutanous injection for 6 weeks) was observed. We have also determined the changes of cell cycle in human fetal lung fibroblast cells treated with D-gal (8 g/L): the proportion of the G0-G1 cells increased but the G2-M and DNA level and the results showed that decreased and the cell growth rate declined in comparison with those of the control. Lipid peroxidation was observed in both kinds of cells because D-gal was proved to have action of decreasing SOD activity and increasing MDA content, the results found in D-gal-treated rats were similar to those observed in hypoxanthine-xanthine oxidation reaction system (O2-), indicating that reactive oxygen was generated in the course of D-gal metabolism. It is concluded that D-gal has the hastening effect on cell senescence and oxidative stress might be responsible for this aging effect.

Aging↗

PubMed2003

A P53-dependent, telomere-independent proliferative life span barrier in human astrocytes consistent with the molecular genetics of glioma development.

An in vitro model, based on normal (primary) human astrocytes (NHAs), was used to investigate the nature of the selection pressures for events that occur during the progression of astrocyte-derived tumors and, in particular, the potential role of proliferative life span barriers (PLBs). As with fibroblasts, NHAs senesced with elevated p21(WAF1) and senescence-associated beta-galactosidase activities. Unlike fibroblasts, replicative senescence (M1) occurred much earlier, after approximately 20 pd and was not bypassed by hTERT expression. Abrogation of p53 function, by expression of human papillomavirus type 16 E6, led to an extension of life span, implying that replicative senescence in NHAs was p53-dependent but telomere-independent. human papillomavirus type16 E6 expression promoted additional growth of up to 12 pd, until a second telomere-independent PLB (termed M(INT)) was imposed associated with elevated p16(INK4A) levels. A proportion of cells escaped from M(INT) lost p16(INK4A) expression and achieved approximately an additional 25 pd until a crisis-like third PLB (M2) was reached. Expression of hTERT in post-M(INT) cells allowed these cells to become immortal and bypass this third PLB. The in vitro PLBs appear, in order of occurrence, dependent upon p53, p16(INK4A), and telomere erosion, a situation that mirrors an equivalent order of mutational events during tumor progression in vivo. This study describes a model that provides a plausible explanation for the selective pressures driving mutational events in this tumor type and provides direct evidence of a p53-dependent, telomere-independent PLB.

Astrocytes↗

PubMed2002

Telomerase positive human diploid fibroblasts are resistant to replicative senescence but not premature senescence induced by chemical reagents.

Human diploid fibroblasts in tissue culture undergo replicative senescence after a finite number of divisions that is characterized by a permanent loss of their dividing potential. However, senescence-like phenotypes, including growth cessation, morphological changes, and appearance of senescence-associated beta-galactosidae (SA-gal) activity, can be induced by treating early passage cells with C(6)-ceramide, H(2)O(2), LY294002, or trichostatin A. While there is convincing evidence that telomere shortening is causally related to replicative senescence, the role of telomere shortening in the chemical-induced premature senescence is unclear. Here we employed a normal human BJ cell strain and its telomerase-transfected counterpart, termed BJ-T cells, to examine whether active telomerase in BJ-T can block or delay the premature senescence induced by various chemicals and, if not, whether telomere shortening still occurs. We found that, although all four chemicals tested could induce growth arrest, and in some cases SA-gal activity, in both BJ and BJ-T cells, only H(2)O(2) clearly caused an irreversible loss of dividing potential. H(2)O(2) treatment did not inhibit the cellular telomerase activity, nor did it cause any appreciable telomere shortening in BJ-T cells. These results suggest that oxidative stress and other chemical reagents can target at sites unrelated to the telomere-associated clocking mechanism. Alternatively these chemicals may bypass the telomere length maintenance machinery and target at its downstream sites.

Base Sequence↗

PubMed2001

Attenuation of EGF signaling in senescent cells by caveolin.

One of the characteristics of senescent cells is unresponsiveness to external stimuli like EGF. Although they have a normal level of receptors and downstream signaling molecules, EGF cannot induce the activation of Erk kinases and DNA synthesis in senescent cells as much as in young cells. Caveolin proteins directly interact with signaling molecules including EGF receptor and suppress the activation of EGFR upon EGF stimulation. We found that Erk activation after EGF stimulation in senescent human diploid fibroblasts was down-regulated. Those senescent cells showed an increased level of three isoforms of caveolin proteins. This change seems to lie in transcriptional control in senescent cells. We also demonstrated up-regulated caveolin proteins were co-localized with EGFR proteins in detergent-insoluble fractions. From these results, we suggest that the up-regulated expression of caveolin might explain the unresponsiveness of senescent fibroblasts to EGF stimulation.

Animals↗

PubMed1995

An overexpressed gene transcript in senescent and quiescent human fibroblasts encoding a novel protein in the epidermal growth factor-like repeat family stimulates DNA synthesis.

We carried out subtractive enrichment of a cDNA library derived from mRNA of senescent human diploid fibroblasts (HDF) established from a subject with Werner syndrome of premature aging. By differential screening, we isolated an overexpressed cDNA sequence (S1-5) that codes for a novel protein containing epidermal growth factor (EGF)-like domains which match the EGF-like consensus sequences within several known extracellular proteins that play a role in cell growth, development, and cell signalling. S1-5 mRNA is overexpressed in Werner syndrome and senescent normal HDF, is induced by growth arrest of young normal cells, but is significantly decreased by high serum, conditions which promote cellular proliferation. Paradoxically, microinjection into young HDF of two different lengths of S1-5 mRNA, containing different putative AUG translational start sites, consistently stimulated rather than inhibited DNA synthesis by an apparent autocrine/paracrine mechanism. Thus, the S1-5 gene product may represent a negative and/or positive factor whose ultimate activity is modulated by the cell environment as occurs with other members of EGF-like family.

Amino Acid Sequence↗

PubMed1978

Protein turnover in senescent cultured chick embryo fibroblasts.

The over-all rates of protein synthesis, degradation and net accumulation were estimated in rapidly growing young and slowly doubling old cultures of chick fibroblasts. We find that not only the rate of protein synthesis is reduced in senescent cultures, but the average rate of protein degradation is also slowed down considerably. This decrease in the rate of protein breakdown in aging cells stands in contrast with the previously observed acceleration of this process by other conditions (such as serum deprivation or overcrowding) that lead to the cessation of cellular growth. Though the retarded protein degradation may contribute to the acculation of abnormal proteins in senescent cells we find that the breakdown of grossly abnormal puromycin peptides proceeds equally rapidly in young and old cultures. The protein content of senescent cells increases by 1.8-fold as compared to young cells, while the average cell volume is increased even more (almost 5-fold). By contrast, consideration of the over-all balance of protein metabolism in these cells indicates that the average concentration of metabolically turning-over proteins is somewhat higher in senescent than in young fibroblasts.

Cell Count↗

PubMed1999

Murine fibroblasts lacking p21 undergo senescence and are resistant to transformation by oncogenic Ras.

The cell-cycle inhibitor p21 is upregulated during senescence and upon induction of senescence-like arrest by oncogenic Ras. We have used primary fibroblasts derived from p21-null mice to evaluate the role of p21 in these processes. We find that primary p21-/- cells enter senescence and have a lifespan similar to wild-type cells. Upon immortalization, most wild-type and p21-/- cultures acquire alterations in either p53 or p16INK4a, further indicating that p21-deficiency is not sufficient by itself to allow immortalization. Primary p21-/- cells, like wild-type cells, respond to oncogenic Ras by accumulating p53 and p16INK4a, and by decreasing their proliferation rate. In agreement with this, p21-/- cells are refractory to neoplasic transformation by oncogenic Ras when compared to p53-/- cells. We conclude that, in murine fibroblasts, p21 is not essential neither for senescence nor for preventing neoplasic transformation by oncogenic Ras.

3T3 Cells↗

PubMed1995

Oxidative DNA damage and senescence of human diploid fibroblast cells.

Human diploid fibroblast cells cease growth in culture after a finite number of population doublings. To address the cause of growth cessation in senescent IMR-90 human fibroblast cells, we determined the level of oxidative DNA damage by using 8-oxoguanine excised from DNA and 8-oxo-2'-deoxyguanosine in DNA as markers. Senescent cells excise from DNA four times more 8-oxoguanine per day than do early-passage young cells. The steady-state level of 8-oxo-2'-deoxyguanosine in DNA is approximately 35% higher in senescent cells than in young cells. Measurement of protein carbonyls shows that senescent cells did not appear to have elevated protein oxidation. To reduce the level of oxidative damage, we cultured cells under a more physiological O2 concentration (3%) and compared the replicative life span to the cells cultured at the O2 concentration of air (20%). We found that cells grown under 3% O2 achieved 50% more population doublings during their lifetime. Such an extension of life span resulted from the delayed onset of senescence and elevation of growth rate and saturation density of cells at all passages. The spin-trapping agent alpha-phenyl-t-butyl nitrone (PBN), which can act as an antioxidant, also effectively delayed senescence and rejuvenated near senescent cells. The effect is dose-dependent and is most pronounced for cells at the stage just before entry into senescence. Our data support the hypothesis that oxidative DNA damage contributes to replicative cessation in human diploid fibroblast cells.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2003

Phosphatidylinositol 3-kinase inhibitor, LY294002, induced senescence-like changes in human diploid fibroblasts.

OBJECTIVE: To reveal the role of Phosphatidylinositol 3-kinases (PI3Ks) in regulating human diploid fibroblast (2BS cell) senescence as well as the possible mechanisms involved. METHODS: Using a PI3Ks specific inhibitor, LY294002, cell cycle, apoptosis, proliferation, senescence association beta-galactosidase staining as well as senescence association CKIs, p16(INK4) and p21(Cip1) protein expressions were all measured in the low passages of 2BS cells. RESULTS: Both 25 micro mol/L and 50 micro mol/L concentrations of LY294002 could cause a significant decrease in cells entering into S phase, and this cell cycle of G(1) phase arrest was dose-dependent. Meanwhile, LY294002 contributed to apoptosis, caused 2BS cell growth arrest, and activated senescence association beta-galactosidase (P < 0.05). In addition, LY294002 could induce time-course expressions of p16(INK4) and p21(Cip1) in 2BS cell lines. CONCLUSIONS: PI3Ks inhibitor LY294002 could induce senescence-like changes in 2BS cell lines. Two senescence associated CKIs, p16(INK4) and p21(Cip1), might be involved in this senescence phenotype proceeding in 2BS cell lines.

Cells, Cultured↗