Increased ezrin expression and activation by CDK5 coincident with acquisition of the senescent phenotype

Passage of normal cells in culture leads to senescence, an irreversible cell cycle exit characterized by biochemical changes and a distinctive morphology. Cellular stresses, including oncogene activation, can also lead to senescence. Consistent with an anti-oncogenic role for this process, the tumor suppressor pRb plays a critical role in senescence. Reexpression of pRb in human tumor cells results in senescence-like changes including cell cycle exit and shape changes. Here we show that senescence is accompanied by increased expression and altered localization of ezrin, an actin binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of CDK5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells.     

TGF-beta1 Does Not Induce Senescence of Multipotent Mesenchymal Stromal Cells and Has Similar Effects in Early and Late Passages

Transforming growth factor-beta 1 (TGF-β1) stimulates a broad range of effects which are cell type dependent, and it has been suggested to induce cellular senescence. On the other hand, long-term culture of multipotent mesenchymal stromal cells (MSCs) has a major impact on their cellular physiology and therefore it is well conceivable that the molecular events triggered by TGF-β1 differ considerably in cells of early and late passages. In this study, we analyzed the effect of TGF-β1 on and during replicative senescence of MSCs. Stimulation with TGF-β1 enhanced proliferation, induced a network like growth pattern and impaired adipogenic and osteogenic differentiation. TGF-β1 did not induce premature senescence. However, due to increased proliferation rates the cells reached replicative senescence earlier than untreated controls. This was also evident, when we analyzed senescence-associated DNA-methylation changes. Gene expression profiles of MSCs differed considerably at relatively early (P 3 - 5) and later passages (P 10). Nonetheless, relative gene expression differences provoked by TGF-β1 at individual time points or in a time course dependent manner (stimulation for 0, 1, 4 and 12 h) were very similar in MSCs of early and late passage. These results support the notion that TGF-β1 has major impact on MSC function, but it does not induce senescence and has similar molecular effects during culture expansion.     

Stable Cellular Senescence Is Associated with Persistent DDR Activation

The DNA damage response (DDR) is activated upon DNA damage generation to promote DNA repair and inhibit cell cycle progression in the presence of a lesion. Cellular senescence is a permanent cell cycle arrest characterized by persistent DDR activation. However, some reports suggest that DDR activation is a feature only of early cellular senescence that is then lost with time. This challenges the hypothesis that cellular senescence is caused by persistent DDR activation. To address this issue, we studied DDR activation dynamics in senescent cells. Here we show that normal human fibroblasts retain DDR markers months after replicative senescence establishment. Consistently, human fibroblasts from healthy aged donors display markers of DDR activation even three years in culture after entry into replicative cellular senescence. However, by extending our analyses to different human cell strains, we also observed an apparent DDR loss with time following entry into cellular senescence. This though correlates with the inability of these cell strains to survive in culture upon replicative or irradiation-induced cellular senescence. We propose a model to reconcile these results. Cell strains not suffering the prolonged in vitro culture stress retain robust DDR activation that persists for years, indicating that under physiological conditions persistent DDR is causally involved in senescence establishment and maintenance. However, cell strains unable to maintain cell viability in vitro, due to their inability to cope with prolonged cell culture-associated stress, show an only-apparent reduction in DDR foci which is in fact due to selective loss of the most damaged cells.     

Uncoupling the senescent phenotype from telomere shortening in hydrogen peroxide-treated fibroblasts

Normal human cells have a limited replicative potential and inevitably reach replicative senescence in culture. Replicatively senescent cells show multiple molecular changes, some of which are related to the irreversible growth arrest in culture, whereas others resemble the changes occurring during the process of aging in vivo. Telomeres shorten as a result of cell replication and are thought to serve as a replicometer for senescence. Recent studies show that young cells can be induced to develop features of senescence prematurely by damaging agents, chromatin remodeling, and overexpression of ras or the E2F1 gene. Accelerated telomere shortening is thought to be a mechanism of premature senescence in some models. In this work, we test whether the acquisition of a senescent phenotype after mild-dose hydrogen peroxide (H(2)O(2)) exposure requires telomere shortening. Treating young HDFs with 150 microM H(2)O(2) once or 75 microM H(2)O(2) twice in 2 weeks causes long-term growth arrest, an enlarged morphology, activation of senescence-associated beta-galactosidase, and elevated expression of collagenase and clusterin mRNAs. No significant telomere shortening was observed with H(2)O(2) at doses ranging from 50 to 200 microM. Weekly treatment with 75 microM H(2)O(2) also failed to induce significant telomere shortening. Failure of telomere shortening correlated with an inability to elevate p16 protein or mRNA in H(2)O(2)-treated cells. In contrast, p21 mRNA was elevated over 40-fold and remained at this level for at least 2 weeks after a pulse treatment of H(2)O(2). The role of cell cycle checkpoints centered on p21 in premature senescence induced by H(2)O(2) is discussed here.     

Analysis of the impact of indole-3-acetic acid (IAA) on gene expression during leaf senescence in Arabidopsis thaliana

Leaf senescence is an important developmental process for the plant life cycle. It is controlled by endogenous and environmental factors and can be positively or negatively affected by plant growth regulators. It is characterised by major and significant changes in the patterns of gene expression. Auxin, especially indole-3-acetic acid (IAA), is a plant growth hormone that affects plant growth and development. The effect of IAA on leaf senescence is still unclear. In this study, we performed microarray analysis to investigate the role of IAA on gene expression during senescence in Arabidopsis thaliana. We sprayed IAA on plants at 3 different time points (27, 31 or 35 days after sowing). Following spraying, PSII activity of the eighth leaf was evaluated daily by measurement of chlorophyll fluorescence parameters. Our results show that PSII activity decreased following IAA application and the IAA treatment triggered different gene expression responses in leaves of different ages.

     

Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics

Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16(INK4a) cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16(INK4a)-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT(+) keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16(INK4a) expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16(INK4a)-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems.     

Cell senescence and human aging: where's the link?

Normal human cells will not divide forever in culture. After a defined number of passages, every culture enters a viable non-dividing state termed senescence. This led to the proposal that the progressive accumulation of senescent cells contributes to (but does not exclusively cause) the aging process. Data now suggest that cell senescence, like apoptosis, occurs as an anti-cancer mechanism. In vivo, extremely low rates of division occur over very long periods of time. Thus, by the end of its lifespan, an organism has performed a surprisingly large amount of cell turnover. In a normal culture the fraction of senescent cells increases smoothly with proliferation in vitro. This process can be accurately modelled by the progressive loss of telomeric sequence (proposed as a counting mechanism for senescence). Telomere length is usually maintained by the enzyme telomerase (repressed in many human tissues). Re-introduction of telomerase prevents the onset of senescence. However, data suggest that a separate telomere-independent pathway exists. Evidence that this pathway is present in humans is now emerging.     

Changes in peroxidases in the suspension culture of Rubus fruticosus during growth

The growth parameters of a cell suspension culture of Rubus fruticosus L. were determined over a culture period including exponential growth, stationary phase and a glucose starvation period at the end of the normal culture cycle. Peroxidase activities were measured in the cytoplasm, in the cell wall, and in the culture medium by the guaiacol assay. There is a relationship between the activity found in the spent medium and the dry matter mass of the cells during the exponential growth. In the three compartments a bimodal repartition of peroxidase activities was observed, with the two peaks at day 4 and day 26, respectively. This suggests that the first peak corresponds to actively dividing cells whereas the second is associated with senescence, or stress due to starvation. Fractionation of the peroxidases from the culture mediuim revealed the presence of two sets of cationic isoenzymes, with minor amount of anionic peroxidases. Interestingly, the second peak of cationic enzymes which was of weak intensity at day 10 of the culture, becameprevalent at day 26. This indicates that not only the total amount of peroxidases varies as a function of culture time, but also that the nature of the peroxidases secreted into the medium changes during growth.Abbreviations DW dry weight - FW fresh weight - MV medium volume - SV suspension volume - BSA bovine serum albumin     

Inhibition of p21-mediated ROS accumulation can rescue p21-induced senescence

The cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1/Sdi1) was identified initially as a gene induced in senescent cells and itself has been shown to cause permanent growth arrest/senescence. Reactive oxygen species (ROS), a byproduct of oxidative processes, can also induce an irreversible growth arrest similar to senescence. Here we show that p21 increased intracellular levels of ROS both in normal fibroblasts and in p53-negative cancer cells. N-acetyl-L-cysteine, an ROS inhibitor, rescued p21-induced senescence, showing that ROS elevation is necessary for induction of the permanent growth arrest phenotype. p16(Ink4a), a CDK4- and CDK6-specific inhibitor, failed to increase ROS levels, and cell cycle arrest induced by p16 was reversible following its down-regulation, demonstrating the specificity of this p21 effect. A p21 mutant that lacked the ability to bind proliferating cell nuclear antigen (PCNA) retained the ability to induce both ROS and permanent growth arrest. All of these findings establish that p21 mediates senescence by a mechanism involving ROS accumulation which does not require either its PCNA binding or the CDK inhibitory functions shared with p16.     

Genetic manipulation of telomerase in HIV-specific CD8+ T cells: enhanced antiviral functions accompany the increased proliferative potential and telomere length stabilization

A large proportion of the CD8(+) T cell pool in persons chronically infected with HIV consists of cells that show features of replicative senescence, an end stage characterized by irreversible cell cycle arrest, multiple genetic and functional changes, and shortened telomeres. The objective of our research was to determine whether constitutive expression of the gene for the human telomerase (hTERT) can prevent senescence-induced impairments in human virus-specific CD8(+) T cells, particularly in the context of HIV-1 disease. Our results indicate that hTERT-expressing HIV-specific CD8(+) lymphocytes show both an enhanced and sustained capacity to inhibit HIV-1 replication in in vitro coculture experiments, as well as prolonged ability to produce IFN-gamma and TNF-alpha in response to stimulation with HIV-1-derived peptides, as compared with vector-transduced controls. Loss of CD28 expression, the signature change of replicative senescence in cell culture, was retarded in those CD8(+) T cell cultures that had high levels of CD28 at the time of hTERT transduction. These findings suggest that telomere shortening may be the primary driving force behind several aspects of CD8(+) T cell dysfunction associated with replicative senescence. We also demonstrate reduced accumulation of the p16(INK4a) and p21(WAF1) cell cycle inhibitors in hTERT-transduced lymphocytes, providing a possible mechanism by which stable hTERT expression is able to circumvent the senescence barrier in CD8(+) T cells. Given the key role of CD8(+) T cell function in controlling a variety of acute and latent viral infections, approaches to retard the functional decrements associated with replicative senescence may lead to novel types of immunotherapy.     

How might replicative senescence contribute to human ageing?

Cell senescence is the limited ability of primary human cells to divide when cultured in vitro. This eventual cessation of division is accompanied by a specific set of changes in cell physiology, morphology, and gene expression. Such changes in phenotype have the potential to contribute to human ageing and age-related diseases. Until now, senescence has largely been studied as an in vitro phenomenon, but recent data have for the first time directly demonstrated the presence of senescent cells in aged human tissues. Although a direct causal link between the ageing of whole organisms and the senescence of cells in culture remains elusive, a large body of data is consistent with cell senescence contributing to a variety of pathological changes seen in the aged. This review considers the in vitro phenotype of cellular senescence and speculates on the various possible routes whereby the presence of senescent cells in old bodies may affect different tissue systems.     

Overexpression of the p21 sdi1 gene induces senescence-like state in human cancer cells: implication for senescence-directed molecular therapy for cancer.

Normal cells in a culture enter a nondividing state after a finite number of population doubling, which is termed replicative senescence, whereas cancer cells have unlimited proliferative potential and are thought to exhibit an immmortal phenotype by escaping from senescence. The p21 gene (also known as sdi1), which encodes the cyclin-dependent kinase inhibitor, is expressed at high levels in senescent cells and contributes to the growth arrest. To examine if the p21sdi1 gene transfer could induce senescence in human cancer cells, we utilized an adenoviral vector-based expression system and four human cancer cell lines differing in their p53 status. Transient overexpression of p21sdi1 on cancer cells induced quiescence by arresting the cell cycle at the G1 phase and exhibited morphological changes, such as enlarged nuclei as well as a flattened cellular shape, specific to the senescence phenotype. We also showed that p21sdi1-transduced cancer cells expressed beta-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Moreover, the polymerase chain reaction-based assay demonstrated that levels of telomerase activity were significantly lower in p21sdi1-expressing cells compared to parental cancer cells. These observations provide the evidence that p21sdi1 overexpression could induce a senescence-like state and reduce telomerase activity in human cancer cells, suggesting that these novel p21sdi1 functions may have important implications for anticancer therapy.     

When Celibacy Matters: Incorporating Non-Breeders Improves Demographic Parameter Estimates

In long-lived species only a fraction of a population breeds at a given time. Non-breeders can represent more than half of adult individuals, calling in doubt the relevance of estimating demographic parameters from the sole breeders. Here we demonstrate the importance of considering observable non-breeders to estimate reliable demographic traits: survival, return, breeding, hatching and fledging probabilities. We study the long-lived quasi-biennial breeding wandering albatross (Diomedea exulans). In this species, the breeding cycle lasts almost a year and birds that succeed a given year tend to skip the next breeding occasion while birds that fail tend to breed again the following year. Most non-breeders remain unobservable at sea, but still a substantial number of observable non-breeders (ONB) was identified on breeding sites. Using multi-state capture-mark-recapture analyses, we used several measures to compare the performance of demographic estimates between models incorporating or ignoring ONB: bias (difference in mean), precision (difference is standard deviation) and accuracy (both differences in mean and standard deviation). Our results highlight that ignoring ONB leads to bias and loss of accuracy on breeding probability and survival estimates. These effects are even stronger when studied in an age-dependent framework. Biases on breeding probabilities and survival increased with age leading to overestimation of survival at old age and thus actuarial senescence and underestimation of reproductive senescence. We believe our study sheds new light on the difficulties of estimating demographic parameters in species/taxa where a significant part of the population does not breed every year. Taking into account ONB appeared important to improve demographic parameter estimates, models of population dynamics and evolutionary conclusions regarding senescence within and across taxa.     

MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation

Keratinocyte replicative senescence has an important role in time-dependent changes of the epidermis, a tissue with high turnover. Senescence encompasses growth arrest during which cells remain metabolically active but acquire a typical enlarged, vacuolar and flattened morphology. It is also accompanied by the expression of endogenous senescence-associated-β-galactosidase and specific gene expression profiles. MicroRNAs levels have been shown to be modulated during keratinocytes senescence, playing key roles in inhibiting proliferation and in the acquisition of senescent markers. Here, we identify miR-191 as an anti-proliferative and replicative senescence-associated miRNA in primary human keratinocytes. Its overexpression is sufficient per se to induce senescence, as evaluated by induction of several senescence-associated markers. We show that SATB1 and CDK6 3'UTRs are two miR-191 direct targets involved in this pathway. Cdk6 and Satb1 protein levels decrease during keratinocytes replicative senescence and their silencing by siRNA is able to induce a G1 block in cell cycle, accompanied by an increase in senescence-associated markers.     

ULTRASTRUCTURE OF SPONGIOCHLORIS TYPICA DURING SENESCENCE12

The ultrastructural changes which occurred during senescence in the stationary phase of growth of the unicellular green alga Spongiochloris typica were observed. The cell wall consists of a membrane like primary wall and an inner secondary wall which becomes progressively thickened with age of the culture. During senescence the lamellae become more compact within the chloroplast. The major feature of aging is the appearance of lipid bodies which eventually come to occupy a major portion of the cell lumen. The ultrastructural changes observed to occur during senescence are discussed in relation to physiological data.     

Cisplatin-induced premature senescence with concomitant reduction of gap junctions in human fibroblasts

To examine the role of gap junctions in cell senescence, the changes of gap junctions in cisplatin-induced premature senescence of primary cultured fibroblasts were studied and compared with the replicative senescent human fibroblasts.Dye transfer assay for gap junction function and immunofluorescent staining for connexin 43 protein distribution were done respectively. Furthermore, cytofluorimetry and DAPI fluorescence staining were performed for cell cycle and apoptosis analysis, p53 gene expression level was detected with indirect immunofluorescence. We found that cisplatin(10mM) treatment could block cell growth cycle at G1 and induced premature senescence. The premature senescence changes included high frequency of apoptosis, elevation of p53 expression, loss of membranous gap junctions and reduction of dye-transfer capacity. These changes were comparable to the changes of replicative senescence of human fibroblasts. It was also concluded that cisplatin could induce premature senescence concomitant with inhibition of gap junctions in the fibroblasts. Loss of functional gap junctions from the cell membrane may account for the reduced intercellular communication in the premature senescent fibroblasts. The cell system we used may provide a model useful for the study of the gap junction thus promoting agents against premature senescence.     

Immortalization of primary cells by DNA tumor viruses

Cellular senescence is characterized by a decline in sensitivity to growth factors resulting in cessation of cellular growth. The expression of cellular or viral oncogenes may result in the establishment of cell lines with unlimited proliferative potential ("immortalization"). A variety of viral and cellular oncogenes have been reported to immortalize cells, suggesting that multiple mechanisms may lead to an escape from senescence. Immortalization has been reported to occur as a result of an interaction of viral proteins with cellular suppressor gene products or may result from the elevated expression of "transforming" oncoproteins (such as the polyomavirus middle-t antigen). Here we speculate that a selection for cells with a further decreased probability of cell cycle withdrawal can occur during the growth of cells expressing viral early genes, resulting in a process of tumor progression. Explaining immortalization in terms of mitogenic stimulation due to the expression of viral oncogenes followed by genetic/epigenetic changes may help to explain why lytic DNA viruses have a biological activity which may not be necessary for their life cycle.