Uncoupling between phenotypic senescence and cell cycle arrest in aging p21-deficient fibroblasts

Irreversible G(1) arrest in senescent human fibroblasts is mediated by two inhibitors of cyclin-dependent kinases (Cdks), p21(Cip1/SDI1/WAF1) and p16(Ink4A). To determine the physiological and molecular events that specifically require p21, we studied senescence in human diploid fibroblasts expressing the human papillomavirus type 16 E6 oncogene, which confers low p21 levels via enhanced p53 degradation. We show that in late-passage E6 cells, high Cdk activity drives the cell cycle, but population expansion is slowed down by crisis-like events, probably owing to defective cell cycle checkpoints. At the end of lifespan, terminal-passage E6 cells exhibited several aspects of the senescent phenotype and accumulated unphosphorylated pRb and p16. However, both replication and cyclin-Cdk2 kinase activity were still not blocked, demonstrating that phenotypic and replicative senescence are uncoupled in the absence of normal p21 levels. At this stage, E6 cells also failed to upregulate p27 and inactivate cyclin-Cdk complexes in response to serum deprivation. Eventually, irreversible G(1) arrest occurred coincident with inactivation of cyclin E-Cdk2 owing to association with p21. Similarly, when p21(-/-) mouse embryo fibroblasts reached the end of their lifespan, they had the appearance of senescent cells yet, in contrast to their wild-type counterparts, they were deficient in downregulating bromodeoxyuridine incorporation, cyclin E- and cyclin A-Cdk2 activity, and inhibiting pRb hyperphosphorylation. These data support the model that the critical event ensuring G(1) arrest in senescence is p21-dependent Cdk inactivation, while other aspects of senescent phenotype appear to occur independently of p21.     

Cellular aging beyond cellular senescence: Markers of senescence prior to cell cycle arrest in vitro and in vivo

The field of research on cellular senescence experienced a rapid expansion from being primarily focused on in vitro aspects of aging to the vast territories of animal and clinical research. Cellular senescence is defined by a set of markers, many of which are present and accumulate in a gradual manner prior to senescence induction or are found outside of the context of cellular senescence. These markers are now used to measure the impact of cellular senescence on aging and disease as well as outcomes of anti‐senescence interventions, many of which are at the stage of clinical trials. It is thus of primary importance to discuss their specificity as well as their role in the establishment of senescence. Here, the presence and role of senescence markers are described in cells prior to cell cycle arrest, especially in the context of replicative aging and in vivo conditions. Specifically, this review article seeks to describe the process of “cellular aging”: the progression of internal changes occurring in primary cells leading to the induction of cellular senescence and culminating in cell death. Phenotypic changes associated with aging prior to senescence induction will be characterized, as well as their effect on the induction of cell senescence and the final fate of cells reviewed. Using published datasets on assessments of senescence markers in vivo, it will be described how disparities between quantifications can be explained by the concept of cellular aging. Finally, throughout the article the applicational value of broadening cellular senescence paradigm will be discussed.     

Peroxisomes, cell senescence, and rates of aging

The peroxisome is functionally integrated into an exquisitely complex network of communicating endomembranes which is only beginning to be appreciated. Despite great advances in identifying essential components and characterizing molecular mechanisms associated with the organelle's biogenesis and function, there is a large gap in our understanding of how peroxisomes are incorporated into metabolic pathways and subcellular communication networks, how they contribute to cellular aging, and where their influence is manifested on the initiation and progression of degenerative disease. In this review, we summarize recent evidence pointing to the organelle as an important regulator of cellular redox balance with potentially far-reaching effects on cell aging and the genesis of human disease. The roles of the organelle in lipid homeostasis, anaplerotic reactions, and other critical metabolic and biochemical processes are addressed elsewhere in this volume. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.     

Cellular aging and senescence

Differentiated eukaryotic cells have only a finite capacity for cell division. This limitation is thought to be a cellular manifestation of organismal aging, and a restraint to tumor progression. The molecular basis for cellular senescence is not known, but a molecular framework for understanding this phenomenon has recently been established.     

Cellular senescence in cancer and aging

Cellular senescence, a state of irreversible growth arrest, can be triggered by multiple mechanisms including telomere shortening, the epigenetic derepression of the INK4a/ARF locus, and DNA damage. Together these mechanisms limit excessive or aberrant cellular proliferation, and so the state of senescence protects against the development of cancer. Recent evidence suggests that cellular senescence also may be involved in aging.     

Cellular senescence in the aging and diseased kidney

The program of cellular senescence is involved in both the G1 and G2 phase of the cell cycle, limiting G1/S and G2/M progression respectively, and resulting in prolonged cell cycle arrest. Cellular senescence is involved in normal wound healing. However, multiple organs display increased senescent cell numbers both during natural aging and after injury, suggesting that senescent cells can have beneficial as well as detrimental effects in organismal aging and disease. Also in the kidney, senescent cells accumulate in various compartments with advancing age and renal disease. In experimental studies, forced apoptosis induction through the clearance of senescent cells leads to better preservation of kidney function during aging. Recent groundbreaking studies demonstrate that senescent cell depletion through INK-ATTAC transgene-mediated or cell-penetrating FOXO4-DRI peptide induced forced apoptosis, reduced age-associated damage and dysfunction in multiple organs, in particular the kidney, and increased performance and lifespan. Senescence is also involved in oncology and therapeutic depletion of senescent cells by senolytic drugs has been studied in experimental and human cancers. Although studies with senolytic drugs in models of kidney injury are lacking, their dose limiting side effects on other organs suggest that targeted delivery might be needed for successful application of senolytic drugs for treatment of kidney disease. In this review, we discuss (i) current understanding of the mechanisms and associated pathways of senescence, (ii) evidence of senescence occurrence and causality with organ injury, and (iii) therapeutic strategies for senescence depletion (senotherapy) including targeting, all in the context of renal aging and disease.     

Autophagy controls mesenchymal stem cell properties and senescence during bone aging

Bone marrow‐derived mesenchymal stem cells (BMMSCs) exhibit degenerative changes, including imbalanced differentiation and reduced proliferation during aging, that contribute to age‐related bone loss. We demonstrate here that autophagy is significantly reduced in aged BMMSCs compared with young BMMSCs. The autophagy inhibitor 3‐methyladenine (3‐MA) could turn young BMMSCs into a relatively aged state by reducing their osteogenic differentiation and proliferation capacity and enhancing their adipogenic differentiation capacity. Accordingly, the autophagy activator rapamycin could restore the biological properties of aged BMMSCs by increasing osteogenic differentiation and proliferation capacity and decreasing adipogenic differentiation capacity. Possible underlying mechanisms were explored, and the analysis revealed that autophagy could affect reactive oxygen species and p53 levels, thus regulating biological properties of BMMSCs. In an in vivo study, we found that activation of autophagy restored bone loss in aged mice. In conclusion, our results suggest that autophagy plays a pivotal role in the aging of BMMSCs, and activation of autophagy could partially reverse this aging and may represent a potential therapeutic avenue to clinically treat age‐related bone loss.     

Collagenase‐resistant collagen promotes mouse aging and vascular cell senescence

Collagen fibrils become resistant to cleavage over time. We hypothesized that resistance to type I collagen proteolysis not only marks biological aging but also drives it. To test this, we followed mice with a targeted mutation (Col1a1^r/r) that yields collagenase‐resistant type I collagen. Compared with wild‐type littermates, Col1a1^r/r mice had a shortened lifespan and developed features of premature aging including kyphosis, weight loss, decreased bone mineral density, and hypertension. We also found that vascular smooth muscle cells (SMCs) in the aortic wall of Col1a1^r/r mice were susceptible to stress‐induced senescence, displaying senescence‐associated ß‐galactosidase (SA‐ßGal) activity and upregulated p16^INK4A in response to angiotensin II infusion. To elucidate the basis of this pro‐aging effect, vascular SMCs from twelve patients undergoing coronary artery bypass surgery were cultured on collagen derived from Col1a1^r/r or wild‐type mice. This revealed that mutant collagen directly reduced replicative lifespan and increased stress‐induced SA‐ßGal activity, p16^INK4A expression, and p21^CIP1 expression. The pro‐senescence effect of mutant collagen was blocked by vitronectin, a ligand for αvß3 integrin that is presented by denatured but not native collagen. Moreover, inhibition of αvß3 with echistatin or with αvß3‐blocking antibody increased senescence of SMCs on wild‐type collagen. These findings reveal a novel aging cascade whereby resistance to collagen cleavage accelerates cellular aging. This interplay between extracellular and cellular compartments could hasten mammalian aging and the progression of aging‐related diseases.     

Catastrophic senescence and semelparity in the Penna aging model

The catastrophic senescence of the Pacific salmon is among the initial tests used to validate the Penna aging model. Based on the mutation accumulation theory, the sudden decrease in fitness following reproduction may be solely attributed to the semelparity of the species. In this work, we report other consequences of mutation accumulation. Contrary to earlier findings, such dramatic manifestation of aging depends not only on the choice of breeding strategy but also on the value of the reproduction age, R, and the mutation threshold, T. Senescence is catastrophic when T ￡ R.T le R. As the organism’s tolerance for harmful genetic mutations increases, the aging process becomes more gradual. We observe senescence that is threshold dependent whenever T > R. That is, the sudden drop in survival rate occurs at age equal to the mutation threshold value.     

Quantitative model of cell cycle arrest and cellular senescence in primary human fibroblasts

Primary human fibroblasts in tissue culture undergo a limited number of cell divisions before entering a non-replicative "senescent" state. At early population doublings (PD), fibroblasts are proliferation-competent displaying exponential growth. During further cell passaging, an increasing number of cells become cell cycle arrested and finally senescent. This transition from proliferating to senescent cells is driven by a number of endogenous and exogenous stress factors. Here, we have developed a new quantitative model for the stepwise transition from proliferating human fibroblasts (P) via reversibly cell cycle arrested (C) to irreversibly arrested senescent cells (S). In this model, the transition from P to C and to S is driven by a stress function γ and a cellular stress response function F which describes the time-delayed cellular response to experimentally induced irradiation stress. The application of this model based on senescence marker quantification at the single-cell level allowed to discriminate between the cellular states P, C, and S and delivers the transition rates between the P, C and S states for different human fibroblast cell types. Model-derived quantification unexpectedly revealed significant differences in the stress response of different fibroblast cell lines. Evaluating marker specificity, we found that SA-β-Gal is a good quantitative marker for cellular senescence in WI-38 and BJ cells, however much less so in MRC-5 cells. Furthermore we found that WI-38 cells are more sensitive to stress than BJ and MRC-5 cells. Thus, the explicit separation of stress induction from the cellular stress response, and the differentiation between three cellular states P, C and S allows for the first time to quantitatively assess the response of primary human fibroblasts towards endogenous and exogenous stress during cellular ageing.     

Restoration of CPEB4 prevents muscle stem cell senescence during aging

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Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence

The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21^/Cip and p27^/Kip1. Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.     

Change and aging senescence as an adaptation

Understanding why we age is a long-lived open problem in evolutionary biology. Aging is prejudicial to the individual, and evolutionary forces should prevent it, but many species show signs of senescence as individuals age. Here, I will propose a model for aging based on assumptions that are compatible with evolutionary theory: i) competition is between individuals; ii) there is some degree of locality, so quite often competition will be between parents and their progeny; iii) optimal conditions are not stationary, and mutation helps each species to keep competitive. When conditions change, a senescent species can drive immortal competitors to extinction. This counter-intuitive result arises from the pruning caused by the death of elder individuals. When there is change and mutation, each generation is slightly better adapted to the new conditions, but some older individuals survive by chance. Senescence can eliminate those from the genetic pool. Even though individual selection forces can sometimes win over group selection ones, it is not exactly the individual that is selected but its lineage. While senescence damages the individuals and has an evolutionary cost, it has a benefit of its own. It allows each lineage to adapt faster to changing conditions. We age because the world changes.     

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.     

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.     

Adult Sox2+ stem cell exhaustion in mice results in cellular senescence and premature aging

Aging is characterized by a gradual functional decline of tissues with age. Adult stem and progenitor cells are responsible for tissue maintenance, repair, and regeneration, but during aging, this population of cells is decreased or its activity is reduced, compromising tissue integrity and causing pathologies that increase vulnerability, and ultimately lead to death. The causes of stem cell exhaustion during aging are not clear, and whether a reduction in stem cell function is a cause or a consequence of aging remains unresolved. Here, we took advantage of a mouse model of induced adult Sox2+ stem cell depletion to address whether accelerated stem cell depletion can promote premature aging. After a short period of partial repetitive depletion of this adult stem cell population in mice, we observed increased kyphosis and hair graying, and reduced fat mass, all of them signs of premature aging. It is interesting that cellular senescence was identified in kidney after this partial repetitive Sox2+ cell depletion. To confirm these observations, we performed a prolonged protocol of partial repetitive depletion of Sox2+ cells, forcing regeneration from the remaining Sox2+ cells, thereby causing their exhaustion. Senescence specific staining and the analysis of the expression of genetic markers clearly corroborated that adult stem cell exhaustion can lead to cellular senescence induction and premature aging.     

Stress kinase MKK7: savior of cell cycle arrest and cellular senescence

The c-Jun N-terminal kinase (JNK/SAPK) signaling cascade controls a spectrum of cellular processes, including cell growth, differentiation, transformation, and apoptosis. We recently demonstrated that stress kinase MKK7, a direct activator of JNKs, couples stress signaling to G2/M cell cycle progression, CDC2 expression, and cellular senescence. We further explored other molecules involved in JNK pathway and found that both MKK4, another direct activator of JNK, and c-Jun, a direct substrate of JNK, have similar roles to MKK7. Here we discuss the importance of the MKK4/MKK7-JNK-c-Jun pathway linking stress and developmental signals to cell proliferation, cell cycle progression, cellular senescence, and apoptosis including recent unpublished data from our lab.