Weak p53 permits senescence during cell cycle arrest

Cell cycle arrest coupled with hyper-active mTOR leads to cellular senescence. While arresting cell cycle, high levels of p53 can inhibit mTOR (in some cell lines), thus causing reversible quiescence instead of senescence. Nutlin-3a-induced p53 inhibited mTOR and thus caused quiescence in WI-38 cells. In contrast, while arresting cell cycle, the DNA-damaging drug doxorubicin (DOX) did not inhibit mTOR and caused senescence. Super-induction of p53 by either nutlin-3a or high concentrations of DOX (high-DOX) prevented low-DOX-induced senescence, converting it into quiescence. This explains why in order to cause senescence, DNA damaging drugs must be used at low concentrations, which arrest cell cycle but do not induce p53 at levels sufficient to suppress mTOR. Noteworthy, very prolonged treatment with nutlin-3a also caused senescence preventable by rapamycin. In RPE cells, low concentrations of nutlin-3a caused a semi-senescent morphology. Higher concentrations of nutlin-3a inhibited mTOR and caused quiescent morphology. We conclude that low p53 levels during prolonged cell cycle arrest tend to cause senescence, whereas high levels of p53 tend to cause either quiescence or cell death.     

Elimination of proliferating cells unmasks the shift from senescence to quiescence caused by rapamycin

Background

Depending on cellular context, p53-inducing agents (such as nutlin-3a) cause different outcomes including reversible quiescence and irreversible senescence. Inhibition of mTOR shifts the balance from senescence to quiescence. In cell lines with incomplete responses to p53, this shift may be difficult to document because of a high proportion of proliferating cells contaminating arrested (quiescent and senescent) cells. This problem also complicates the study of senescence caused by minimal levels of p21 that are capable to arrest a few cells.

Methodology

During induction of senescence by low levels of endogenous p53 and ectopic p21, cells were co-treated with nocodazole, which eliminated proliferating cells. As a result, only senescent and quiescent cells remained.

Results and Discussion

This approach revealed that rapamycin efficiently converted nutlin-induced-senescence into quiescence. In the presence of rapamycin, nutlin-arrested MCF-7 cells retained the proliferative potential and small/lean morphology. Using this approach, we also unmasked senescence in cells arrested by low levels of ectopic p21, capable to arrest only a small proportion of HT1080-p21-9 cells. When p21 did cause arrest, mTOR caused senescent phenotype. Rapamycin and high concentrations of nutlin-3a, which inhibit the mTOR pathway in these particular cells, suppressed senescence, ensuring quiescence instead. Thus, p21 causes senescence passively, just by causing arrest, while still active mTOR drives senescent phenotype.     

Cooperation between p21 and Akt is required for p53‐dependent cellular senescence

Cellular senescence has been implicated in normal aging, tissue homeostasis, and tumor suppression. Although p53 has been shown to be a central mediator of cellular senescence, the signaling pathway by which it induces senescence remains incompletely understood. In this study, we have shown that both Akt and p21 are required to induce cellular senescence in response to p53 expression. In a p53‐induced senescence model, we found that Akt activation was essential for inducing a cellular senescence phenotype. Surprisingly, Akt inhibition did not abolish p53‐induced cell cycle arrest, but it suppressed the increase in intracellular reactive oxygen species (ROS) levels. The results of the cell cycle and morphological analysis suggest that p53 induced quiescence, not senescence, following Akt inhibition. Conversely, the inhibition of p21 induction abolished cell cycle arrest but did not affect the p53‐induced increase in ROS levels. Additionally, p21 and Akt separately controlled cell cycle arrest and ROS levels, respectively, during H‐Ras‐induced senescence in human normal fibroblasts. The mechanistic analysis revealed that Akt increased ROS levels through NOX4 induction, and increased Akt‐dependent NF‐κB binding to the NOX4 promoter is responsible for NOX4 induction upon p53 expression. We further showed that Akt activation upon p53 expression is mediated by mammalian target of rapamycin complex 2. In addition, p53‐mediated IL6 and IL8 induction was abrogated by Akt inhibition, suggesting that Akt activation is also required for the senescence‐associated secretory phenotype. Collectively, these results suggest that p53 simultaneously controls multiple pathways to induce cellular senescence through p21 and Akt.     

TRPC1: Getting physical in space

We tested a hypothesis that activation of growth-promoting pathways is required for cellular senescence. In the presence of serum, induction of p21 caused senescence, characterized by beta-Galactosidase staining, cell hypertrophy, increased levels of cyclin D1 and active TOR (target of rapamycin, also known as mTOR). Serum starvation and rapamycin inhibited TOR and prevented the expression of some senescent markers, despite high levels of p21 and cell cycle arrest. In the presence of serum, p21-arrested cells irreversibly lost proliferative potential. In contrast, when cells were arrested by p21 in the absence of serum, they retained the capacity to resume proliferation upon termination of p21 induction. In normal human cells such as WI38 fibroblasts and retinal pigment epithelial (RPE) cells, serum starvation caused quiescence, which was associated with low levels of cyclin D1, inactive TOR and slim-cell morphology. In contrast, cellular senescence with high levels of TOR activity was induced by doxorubicin (DOX), a DNA damaging agent, in the presence of serum. Inhibition of TOR partially prevented senescent phenotype caused by DOX. Thus growth stimulation coupled with cell cycle arrest leads to senescence, whereas quiescence (a condition with inactive TOR) prevents senescence.     

A role for p53 in maintaining and establishing the quiescence growth arrest in human cells

The p53 tumor suppressor protein induces transient growth arrest or apoptosis in response to genotoxic stress and mediates the irreversible growth arrest of cellular senescence. We present evidence here that p53 also contributes to the reversible, growth factor-dependent arrest of quiescence (G(0)). Microinjection of expression vectors encoding either MDM2 or a pRb-binding mutant of SV40 T antigen, both of which abrogate p53 function, stimulated quiescent normal human fibroblasts to initiate DNA synthesis and were 40-70% as effective as wild-type T antigen. Electrophoretic mobility shift and p53 transactivation assays showed that p53 activity was higher in quiescent and senescent cells compared with proliferating cells. As proliferating cells entered G(0) after growth factor withdrawal, the p53 mRNA level increased, followed by transient accumulation of the protein. Shortly thereafter, the expression (mRNA and protein) of p21, a p53 target gene and effector of cell cycle arrest, increased. Finally, stable expression of the HPV16 E6 oncogene or dominant negative p53 peptide, GSE-22, both of which inhibit p53 function, delayed entry into quiescence following growth factor withdrawal. Our data indicate that p53 is activated during both quiescence and senescence. They further suggest that p53 activity contributes, albeit not exclusively, to the quiescent growth arrest.     

AMP-activated protein kinase induces a p53-dependent metabolic checkpoint

Replicative cell division is an energetically demanding process that can be executed only if cells have sufficient metabolic resources to support a doubling of cell mass. Here we show that proliferating mammalian cells have a cell-cycle checkpoint that responds to glucose availability. The glucose-dependent checkpoint occurs at the G(1)/S boundary and is regulated by AMP-activated protein kinase (AMPK). This cell-cycle arrest occurs despite continued amino acid availability and active mTOR. AMPK activation induces phosphorylation of p53 on serine 15, and this phosphorylation is required to initiate AMPK-dependent cell-cycle arrest. AMPK-induced p53 activation promotes cellular survival in response to glucose deprivation, and cells that have undergone a p53-dependent metabolic arrest can rapidly reenter the cell cycle upon glucose restoration. However, persistent activation of AMPK leads to accelerated p53-dependent cellular senescence. Thus, AMPK is a cell-intrinsic regulator of the cell cycle that coordinates cellular proliferation with carbon source availability.     

Cellular senescence induced by p53-ras cooperation is independent of p21waf1 in murine embryo fibroblasts

Oncogenic activation in primary murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the p53 tumor suppressor pathway. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras with p53 produced an irreversible cell cycle arrest that displayed features of cellular senescence. Introduction of a conditional murine p53 allele (p53val135) into double p53/p21-null mouse embryonic fibroblasts showed that p21waf1 was not required for this effect, since p53-/-;p21-/- double-null cells undergo terminal growth arrest with features of senescence following coexpression of oncogenic Ras and p53. Our results indicate that oncogenic activation of the Ras pathway in murine fibroblasts converts p53 into a senescence inducer through a p21waf1-independent mechanism.     

lncRNA在细胞衰老中的作用

衰老是身体器官系统功能逐渐衰退的复杂的生物学过程,能诱发多种老年病。 长链非编码RNA（long non-coding RNA, lncRNA）是长度大于200个核苷酸的非编码RNA,在多种生理学和病理学过程中发挥重要作用。 细胞衰老是重要的衰老生物学过程之一,已经发现大量的lncRNA参与了细胞周期、端粒长度和表观遗传学等的调控,并影响关键的细胞周期过程,如细胞的衰老、增殖、分化、静止等;同时,lncRNA还参与了衰老相关重要信号通路的调控,如p53/p21、哺乳动物雷帕霉素靶蛋白（mTOR）、视网膜母细胞瘤蛋白（Rb）/p16和磷脂酰肌醇3激酶/苏氨酸蛋白激酶（PI3K/Akt）信号通路,它们均与许多衰老相关重大疾病密切相关。 本文综述了最近发现的与细胞衰老相关的lncRNA的功能和作用机制,并总结了lncRNA参与调控的细胞衰老信号通路,最后讨论了lncRNA未来的研究方向。     

The DNA Damage Signaling Pathway Connects Oncogenic Stress to Cellular Senescence

The mechanisms of tumor suppression must be linked to the oncogenic threats that may affect a normal cell. An important cancer causing mechanism is the accidental activation of genes that stimulate cell proliferation (oncogenes) by a variety of endogenous or environmental mutagens. This event has been experimentally modelled by enforcing the expression of oncogenes in primary cells. The astonishing outcome of these manipulations is that oncogenes trigger antiproliferative responses preventing progression to malignant transformation. These responses bring to an end proliferation due to cell death or a permanent cell cycle arrest called senescence. Here we review evidence indicating that oncogene induced senescence (OIS) involves activation of p53 via the DNA damage response (DDR). These results imply mechanisms of DNA damage in cells expressing oncogenes, that may be secondary to reactive oxygen species and/or some form of “oncogenic stress” that affect normal DNA replication. Interestingly, DNA damage signals persist in cells that escape from senescence. The implications of these signals for tumorigenesis are also discussed. Given that DNA damage signals have now been observed in cells treated with any stimuli known to induce senescence, the process can be redefined as a metabolically viable but permanent cell cycle arrest with persistent DNA damage signaling.     

Oncogenic ras and p53 cooperate to induce cellular senescence

Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.     

Antroquinonol, a natural ubiquinone derivative, induces a cross talk between apoptosis, autophagy and senescence in human pancreatic carcinoma cells

Pancreatic cancer is a malignant neoplasm of the pancreas. A mutation and constitutive activation of K-ras occurs in more than 90% of pancreatic adenocarcinomas. A successful approach for the treatment of pancreatic cancers is urgent. Antroquinonol, a ubiquinone derivative isolated from a camphor tree mushroom, Antrodia camphorata, induced a concentration-dependent inhibition of cell proliferation in pancreatic cancer PANC-1 and AsPC-1 cells. Flow cytometric analysis of DNA content by propidium iodide staining showed that antroquinonol induced G1 arrest of the cell cycle and a subsequent apoptosis. Antroquinonol inhibited Akt phosphorylation at Ser(473), the phosphorylation site critical for Akt kinase activity, and blocked the mammalian target of rapamycin (mTOR) phosphorylation at Ser(2448), a site dependent on mTOR activity. Several signals responsible for mTOR/p70S6K/4E-BP1 signaling cascades have also been examined to validate the pathway. Moreover, antroquinonol induced the down-regulation of several cell cycle regulators and mitochondrial antiapoptotic proteins. In contrast, the expressions of K-ras and its phosphorylation were significantly increased. The coimmunoprecipitation assay showed that the association of K-ras and Bcl-xL was dramatically augmented, which was indicative of apoptotic cell death. Antroquinonol also induced the cross talk between apoptosis, autophagic cell death and accelerated senescence, which was, at least partly, explained by the up-regulation of p21(Waf1/Cip1) and K-ras. In summary, the data suggest that antroquinonol induces anticancer activity in human pancreatic cancers through an inhibitory effect on PI3-kinase/Akt/mTOR pathways that in turn down-regulates cell cycle regulators. The translational inhibition causes G1 arrest of the cell cycle and an ultimate mitochondria-dependent apoptosis. Moreover, autophagic cell death and accelerated senescence also explain antroquinonol-mediated anticancer effect.     

ATM and MET kinases are synthetic lethal with nongenotoxic activation of p53

The p53 tumor suppressor orchestrates alternative stress responses including cell cycle arrest and apoptosis, but the mechanisms defining cell fate upon p53 activation are poorly understood. Several small-molecule activators of p53 have been developed, including Nutlin-3, but their therapeutic potential is limited by the fact that they induce reversible cell cycle arrest in most cancer cell types. We report here the results of a genome-wide short hairpin RNA screen for genes that are lethal in combination with p53 activation by Nutlin-3, which showed that the ATM and MET kinases govern cell fate choice upon p53 activation. Genetic or pharmacological interference with ATM or MET activity converts the cellular response from cell cycle arrest into apoptosis in diverse cancer cell types without affecting expression of key p53 target genes. ATM and MET inhibitors also enable Nutlin-3 to kill tumor spheroids. These results identify new pathways controlling the cellular response to p53 activation and aid in the design of p53-based therapies.     

p63: A New Link Between Senescence and Aging

Cellular senescence is a distinctive form of cell cycle arrest that has been suggested to modulatethe processes of tumor suppression and aging. Though a detailed understanding of the cellularmachinery regulating this process is emerging, a more thorough understanding of the key playerslinking senescence to organismal aging is needed. The recent discovery that loss of the p53-related protein p63 induces cellular senescence and causes features of accelerated aging providesfurther evidence that cellular senescence is intimately linked with organismal aging, andidentifies p63 as a key regulator of both of these processes.     

p53 can inhibit cell proliferation through caspase-mediated cleavage of ERK2/MAPK

Stimulation of the Ras/MAPK cascade can either activate p53 and promote replicative senescence and apoptosis, or degrade p53 and promote cell survival. Here we show that p53 can directly counteract the Ras/MAPK signaling by inactivating ERK2/MAPK. This inactivation is due to a caspase cleavage of the ERK2 protein and contributes to p53-mediated growth arrest. We found that in Ras-transformed cells, growth arrest induced by p53, but not p21(Waf1), is associated with a strong reduction in ERK2 activity, phosphorylation, and protein half-life, and with the appearance of caspase activity. Likewise, DNA damage-induced cell cycle arrest correlates with p53-dependent ERK2 downregulation and caspase activation. Furthermore, caspase inhibitors or expression of a caspase-resistant ERK2 mutant interfere with ERK2 cleavage and restore proliferation in the presence of p53 activation, indicating that caspase-mediated ERK2 degradation contributes to p53-induced growth arrest. These findings strongly point to ERK2 as a novel p53 target in growth suppression.     

Cell senescence: hypertrophic arrest beyond the restriction point

Withdrawal of mitogens (growth factors) arrests normal cells in G0 (quiescence). All other stresses and factors arrest cell cycle beyond the restriction point in G1 and G2 (non-G0 arrest), in the presence of mitogenic stimulation. Strong mitogenic stimuli by themselves cause non-G0 arrest. Unlike G0, arrest beyond restriction point is characterized by both high levels of cyclins and CDK inhibitors, activated mitogenic pathways with a secondary GF resistance, and continuous mass growth (cell hypertrophy). Prolonged hypertrophic arrest culminates in cell senescence. This review discusses that quiescence and senescence are two opposite, mutually exclusive conditions and that cell senescence can be reversed and prevented.