Effect of cell cycle inhibitor p19ARF on senescence of human diploid cell

To investigate the effect of cell cycle inhibitor p19ARF on replicative senescence of human diploid cell,recombinant p19ARF eukaryotic expression vector was constructed and p19ARF gene was transfected into human diploid fibroblasts (WI-38 cells) by liposome-mediated transfection for overexpression.Then, the effects of p19ARF on replicative senescence of WI-38 cells were observed. The results revealed that, compared with control cells, the WI-38 cells in which p19ARFgene was introduced showed significant up-regulation of p53 and p21 expression level, decrease of cell generation by 10-12 generations, decline of cell growth rate with cell cycle being arrested at G1 phase, increase of positive rate of senescent marker SA-β-gal staining, and decrease of mitochondrial membrane potential. The morphology of the transfected fibroblasts presented the characteristics changes similar to senescent cells.These results indicated that high expression of p19ARF may promote the senescent process of human diploid cells.     

Effect of cell cycle inhibitor p19ARF on senescence of human diploid cell

To investigate the effect of cell cycle inhibitor p19ARF on replicative senescence of human diploid cell, recombinant p19ARF eukaryotic expression vector was constructed and p19ARF gene was transfected into human diploid fibroblasts (WI-38 cells) by liposome-mediated transfection for overexpression. Then, the effects of p19ARF on replicative senescence of WI-38 cells were observed. The results re- vealed that, compared with control cells, the WI-38 cells in which p19ARF gene was introduced showed significant up-regulation of p53 and p21 expression level, decrease of cell generation by 10 12 generations, decline of cell growth rate with cell cycle being arrested at G1 phase, increase of positive rate of senescent marker SA-β-gal staining, and decrease of mitochondrial membrane potential. The morphology of the transfected fibroblasts presented the characteristics changes similar to senescent cells. These results indicated that high expression of p19ARF may promote the senescent process of human diploid cells.     

BTG2 antagonizes Pin1 in response to mitogens and telomere disruption during replicative senescence

Cellular senescence limits the replicative capacity of normal cells and acts as an intrinsic barrier that protects against the development of cancer. Telomere shortening–induced replicative senescence is dependent on the ATM‐p53‐p21 pathway but additional genes likely contribute to senescence. Here, we show that the p53‐responsive gene BTG2 plays an essential role in replicative senescence. Similar to p53 and p21 depletion, BTG2 depletion in human fibroblasts leads to an extension of cellular lifespan, and ectopic BTG2 induces senescence independently of p53. The anti‐proliferative function of BTG2 during senescence involves its stabilization in response to telomere dysfunction followed by serum‐dependent binding and relocalization of the cell cycle regulator prolyl isomerase Pin1. Pin1 inhibition leads to senescence in late‐passage cells, and ectopic Pin1 expression rescues cells from BTG2‐induced senescence. The neutralization of Pin1 by BTG2 provides a critical mechanism to maintain senescent arrest in the presence of mitogenic signals in normal primary fibroblasts.     

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.     

An altered repertoire of fos/jun (AP-1) at the onset of replicative senescence.

With multiple divisions in culture, normal diploid cells suffer a loss of growth potential that leads to replicative senescence and a finite replicative capacity. Using quantitative RT-PCR, we have monitored mRNA expression levels of c-fos, c-jun, JunB, c-myc, p53, H-ras, and histone H4 during the replicative senescence of human fibroblasts. The earliest and the largest changes in gene expression occurred in c-fos and junB at mid-senescence prior to the first slowing in cell growth rates. The basal level of c-fos mRNA decreased to one-ninth that of the early-passage levels, while junB declined to one-third and c-jun expression remained constant. The decline in the basal c-fos mRNA level in mid-senescence should lead to an increase in Jun/Jun AP-1 homodimers at the expense of Fos/Jun heterodimers and may trigger a cascade of further changes in c-myc, p53, and H-ras expression in late-passage senescent fibroblasts.     

Partial proteasome inhibition in human fibroblasts triggers accelerated M1 senescence or M2 crisis depending on p53 and Rb status

Proteasome-dependent degradation has been extensively investigated and has been shown to play a vital role in the maintenance of cellular homeostasis. Proteasome activity and expression are reduced during aging and replicative senescence. Its activation has been shown to confer lifespan extension in human diploid fibroblasts (HDFs), whereas partial proteasome inhibition triggers an irreversible premature senescent state in young HDFs. As p53 and Rb tumor suppressors regulate both replicative and premature senescence (RS and PS, respectively), in this study we investigated their implication in proteasome inhibition-mediated PS. By taking advantage of a variety of HDFs with defective p53 or/and Rb pathways, we reveal that proteasome activity inhibition to levels normally found in senescent human cells results in immediate growth arrest and/or moderate increase of apoptotic death. These effects are independent of the cellular genetic context. However, in the long term, proteasome inhibition-mediated PS can only be initiated and maintained in the presence of functional p53. More specifically, we demonstrate that following partial proteasome inhibition, senescence is dominant in HDFs with functional p53 and Rb molecules, crisis/death is induced in cells with high p53 levels and defective Rb pathway, whereas stress recovery and restoration of normal cycling occurs in cells that lack functional p53. These data reveal the continuous interplay between the integrity of proteasome function, senescence and cell survival.     

Nek6 overexpression antagonizes p53-induced senescence in human cancer cells

Nek6 is an NIMA-related kinase that plays a critical role in mitotic cell cycle progression. Recent studies have shown that Nek6 is upregulated in various human cancers, but the function of Nek6 in tumorigenesis is largely unknown. Here, we examined the role of Nek6 in cellular senescence. Our data revealed that Nek6 expression is decreased both in both the replicative senescence of human normal fibroblasts and premature senescence induced by p53 expression in EJ human bladder cancer cells and H1299 human lung cancer cells. Interestingly, the enforced expression of Nek6 in EJ and H1299 cells completely suppresses p53-induced senescence, whereas the expression of kinase-dead Nek6 did not affect p53-induced senescence. Mechanistic studies revealed that cell cycle arrest in the G1 and G2/M phases, as well as the reduction of cyclin B and cdc2 protein level upon p53 expression were significantly reduced by Nek6 overexpression. In addition, p53-induced increases in intracellular levels of ROS were also inhibited in cells overexpressing Nek6. These results suggest that the downregulation of Nek6 expression is required for the onset of p53-induced cellular senescence and imply a possible role of Nek6 in tumorigenesis.     

Inhibition of phosphatidylinostol 3-kinase uncouples H2O2-induced senescent phenotype and cell cycle arrest in normal human diploid fibroblasts

Exposure of WI38 human diploid fibroblasts (HDFs) to hydrogen peroxide (H2O2) induced premature senescence. The senescent HDFs were permanently arrested and exhibited a senescent phenotype including enlarged and flattened cell morphology and increased senescence-associated beta-galactosidase (SA-beta-gal) activity. The induction of HDF senescence was associated with an activation of p53, increased expression of p21Cip1/WAF1, and hypophosphorylation of retinoblastoma protein (Rb), while no changes in the expression of p16Ink4a, p27Kip1, and p14Arf were observed. Exposure of WI38 cells to H2O2 also selectively activated phosphatidylinostol 3-kinase (PI3 kinase) and mitogen-activated protein kinase (MAPK) kinase (MEK), while no changes in p38 MAPK and Jun kinase (JNK) activities were observed. Selective inhibition of PI3 kinase activity with LY294002 abrogated H2O2-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-beta-gal activity, but did not affect H2O2-induced cell cycle arrest. In contrast, selective inhibition of MEK and p38 MAPK with PD98059 and SB203580, respectively, produced no significant effect on H2O2-induced senescent phenotype and cell cycle arrest. These findings demonstrate that expression of the senescent phenotype can be uncoupled from cell cycle arrest in prematurely senescent cells induced by H2O2 and does not contribute to the maintenance of permanent cell cycle arrest.     

Klotho RNAi induces premature senescence of human cells via a p53/p21 dependent pathway

Klotho has recently emerged as a regulator of aging. To investigate the role of Klotho in the regulation of cellular senescence, we generated stable MRC-5 human primary fibroblast cells knockdown for Klotho expression by RNAi. Downregulation of Klotho dramatically induces premature senescence with a concomitant upregulation of p21. The upregulation of p21 is associated with cell cycle arrest at G1/S boundary. Knockdown of p53 in the Klotho attenuated MRC-5 cells restores normal growth and replicative potential. These results demonstrate that Klotho normally regulates cellular senescence by repressing the p53/p21 pathway. Our findings implicate Klotho as a regulator of aging in primary human fibroblasts.     

Regulation of c-fos expression in senescing Werner syndrome fibroblasts differs from that observed in senescing fibroblasts from normal donors

The Werner syndrome (WS) is a segmental progeroid syndrome caused by a recessive mutation (WRN) mapped to 8p12. The replicative life spans of somatic cells cultured from WS patients are substantially reduced compared to age-matched controls. Certain molecular concomitants of the replicative decline of normal fibroblast cultures have recently been defined, and it appears that multiple changes in gene expression accompany normal cell senescence. If the mechanisms by which WS cells exit the cell cycle were entirely comparable, the molecular markers of senescence should be identical in normal and WS cells. We find that this is not the case. The constitutive expression of statin, a nuclear protein associated with the nonproliferating state, was comparably expressed in normal and WS senescent cells. Likewise, the steady state levels of p53, a protein known to be involved in the G1 checkpoint of the cell cycle, were similar in early-passage fibroblasts from normal and WS subjects. The levels of p53 were not increased in senescent fibroblasts, whether derived from normal or WS subjects. By contrast, the inducibility of mRNA and protein expression of the c-fos protooncogene is preserved in late-passage WS cells. This is in contrast to what is observed in late-passage fibroblasts from normal subjects. Additional genotypes will have to be examined, however, to determine the specificity of this new aspect of the WS phenotype. © 1995 Wiley-Liss, Inc.     

Elevated miR-34c-5p Mediates Dermal Fibroblast Senescence by Ultraviolet Irradiation

Previous studies showed that several miRNAs can regulate pathways involved in UVB-induced premature senescence and response to ultraviolet irradiation. It has also been reported that miR-34c-5p may be involved in senescence-related mechanisms. We propose that miR-34c-5p may play a crucial role in senescence of normal human primary dermal fibroblasts. Here, we explored the roles of miR-34c-5p in UVB-induced premature senescence on dermal fibroblasts. MiR-34c-5p expression was increased in dermal fibroblasts after repeated subcytotoxic UVB treatments. Underexpression of miR-34c-5p in dermal fibroblasts led to a marked delay of many senescent phenotypes induced by repeated UVB treatments. Furthermore, underexpression of miR-34c-5p in dermal fibroblasts can antagonize the alteration of G1-arrested fibroblasts. Moreover, E2F3, which can inactivate p53 pathway and play a role in cell cycle progression, is a down-stream target of miR-34c-5p. Forced down-expression of miR-34c-5p decreased the expression of UVB-SIPS induced P21 and P53 at both mRNA and protein levels. Our data demonstrated that down-regulation of miR-34c-5p can protect human primary dermal fibroblasts from UVB-induced premature senescence via regulations of some senescence-related molecules.     

Suppression of replicative senescence by rapamycin in rodent embryonic cells

The TOR (target of rapamycin) pathway is involved in aging in diverse organisms from yeast to mammals. We have previously demonstrated in human and rodent cells that mTOR converts stress-induced cell cycle arrest to irreversible senescence (geroconversion), whereas rapamycin decelerates or suppresses geroconversion during cell cycle arrest. Here, we investigated whether rapamycin can suppress replicative senescence of rodent cells. Mouse embryonic fibroblasts (MEFs) gradually acquired senescent morphology and ceased proliferation. Rapamycin decreased cellular hypertrophy, and SA-beta-Gal staining otherwise developed by 4-6 passages, but it blocked cell proliferation, masking its effects on replicative lifespan. We determined that rapamycin inhibited pS6 at 100-300 pM and inhibited proliferation with IC50 around 30 pM. At 30 pM, rapamycin partially suppressed senescence. However, the gerosuppressive effect was balanced by the cytostatic effect, making it difficult to suppress senescence without causing quiescence. We also investigated rat embryonic fibroblasts (REFs), which exhibited markers of senescence at passage 7, yet were able to slowly proliferate until 12–14 passages. REFs grew in size, acquired a large, flat cell morphology, SA-beta-Gal staining and components of DNA damage response (DDR), in particular, γH2AX/53BP1 foci. Incubation of REFs with rapamycin (from passage 7 to passage 10) allowed REFs to overcome the replicative senescence crisis. Following rapamycin treatment and removal, a fraction of proliferating REFs gradually increased and senescent phenotype disappeared completely by passage 24.     

Stress-induced premature senescence in hTERT-expressing ataxia telangiectasia fibroblasts

In addition to replicative senescence, normal diploid fibroblasts undergo stress-induced premature senescence (SIPS) in response to DNA damage caused by oxidative stress or ionizing radiation (IR). SIPS is not prevented by telomere elongation, indicating that, unlike replicative senescence, it is triggered by nonspecific genome-wide DNA damage rather than by telomere shortening. ATM, the product of the gene mutated in individuals with ataxia telangiectasia (AT), plays a central role in cell cycle arrest in response to DNA damage. Whether ATM also mediates signaling that leads to SIPS was investigated with the use of normal and AT fibroblasts stably transfected with an expression vector for the catalytic subunit of human telomerase (hTERT). Expression of hTERT in AT fibroblasts resulted in telomere elongation and prevented premature replicative senescence, but it did not rescue the defect in G(1) checkpoint activation or the hypersensitivity of the cells to IR. Despite these remaining defects in the DNA damage response, hTERT-expressing AT fibroblasts exhibited characteristics of senescence on exposure to IR or H(2)O(2) in such a manner that triggers SIPS in normal fibroblasts. These characteristics included the adoption of an enlarged and flattened morphology, positive staining for senescence-associated beta-galactosidase activity, termination of DNA synthesis, and accumulation of p53, p21(WAF1), and p16(INK4A). The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which mediates signaling that leads to senescence, was also detected in both IR- or H(2)O(2)-treated AT and normal fibroblasts expressing hTERT. These results suggest that the ATM-dependent signaling pathway triggered by DNA damage is dispensable for activation of p38 MAPK and SIPS in response to IR or oxidative stress.     

Contribution of p16INK4a and p21CIP1 pathways to induction of premature senescence of human endothelial cells: permissive role of p53

We have previously found that nonenzymatically glycated collagen I (GC), mimicking diabetic microenvironment, can induce senescent phenotype in early passage human umbilical vein endothelial cells (HUVECs). In the present study, we explored the functional involvement of cell cycle checkpoint pathways in initiating GC-induced premature endothelial cell senescence. When compared with native collagen, early passage HUVECs showed increased p53, p21(CIP1) (p21), and p16(INK4a) (p16) mRNA expression after exposure to GC. Twenty-four hours after transfection of p16, p21, and p53-enhanced green fluorescent protein (EGFP) recombinant plasmids, HUVECs entered G(1)-phase cell cycle arrest. By days 3 and 5, HUVECs transfected with p16-EGFP showed an increased proportion of senescent cells, and this increase was more prominent in the GFP-positive cell population, which exhibited 68% of senescent cells. Transfection of p21 also induced senescence but only by day 5. Cotransfection of p16 and p21 showed no additive effect. Transfection of p21 or p53 induced apoptosis in HUVECs. Next, we suppressed endogenous p53, p21, p16, or retinoblastoma (Rb) gene expression through small interference RNA strategy and investigated their influence in p16- and p21-initiated endothelial cell senescence. Analysis indicated that suppression of p53 expression can abolish senescence induced by p16 overexpression. Paradoxically, this effect was not observed when p21 was suppressed. On the other hand, suppression of Rb eliminated senescence initiated by either p16 or p21 overexpression. In summary, the p53/p21 pathway is mainly responsible for GC-induced apoptosis, but the coordinated activation of the p53/p21 and p16 pathway is responsible for GC-induced endothelial cell senescence through a Rb-dependent mechanism.     

Expression of connective tissue growth factor, a biomarker in senescence of human diploid fibroblasts, is up-regulated by a transforming growth factor-beta-mediated signaling pathway

Molecular changes associated with cellular senescence in human diploid fibroblasts (HDF), IMR-90, were analyzed by two-dimensional differential proteome analysis. A high percentage of replicative senescent cells were positive for senescence-associated beta-galactosidase activity, and displayed elevated levels of p21 and p53 proteins. Comparison of early population doubling level (PDL) versus replicative senescent cells among the 1000 spots resolved on gels revealed that the signal intensities of six spots were increased fivefold, whereas those of four spots were decreased. Proteome analysis data demonstrated that connective tissue growth factor (CTGF) is an age-associated protein. Up-regulation of CTGF expression in senescent cells was further confirmed by Western blotting and RT-PCR. We postulate that CTGF expression is controlled, in part, by transforming growth factor-beta (TGF-beta), in view of the high levels of TGF-beta isoforms as well as type I and II receptors detected only in late PDL of HDF cells. To verify this hypothesis, we stimulated early PDL cells with TGF-beta1 as well as stress inducing agents such as hydrogen peroxide. As expected, CTGF expression and Smad protein phosphorylation were dramatically increased up to observed levels in normal replicative senescent cells. In vivo experiments disclosed that CTGF, pSmad, and p53 were constitutively expressed at basal levels in up to 18-month-old rat liver, and expression was significantly up-regulated in 24-month-old rat tissue. However, expression patterns were not altered at all periods examined in livers of caloric-restricted rats. In view of both in vitro and in vivo data, we propose that the TGF-beta/Smad pathway functions in the induction of CTGF, a novel biomarker protein of cellular senescence in human fibroblasts.     

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.     

Role of p14(ARF) in replicative and induced senescence of human fibroblasts

Following a proliferative phase of variable duration, most normal somatic cells enter a growth arrest state known as replicative senescence. In addition to telomere shortening, a variety of environmental insults and signaling imbalances can elicit phenotypes closely resembling senescence. We used p53(-/-) and p21(-/-) human fibroblast cell strains constructed by gene targeting to investigate the involvement of the Arf-Mdm2-p53-p21 pathway in natural as well as premature senescence states. We propose that in cell types that upregulate p21 during replicative exhaustion, such as normal human fibroblasts, p53, p21, and Rb act sequentially and constitute the major pathway for establishing growth arrest and that the telomere-initiated signal enters this pathway at the level of p53. Our results also revealed a number of significant differences between human and rodent fibroblasts in the regulation of senescence pathways.