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.     

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.     

Ataxia Telangiectasia Mutated (ATM)-mediated DNA Damage Response in Oxidative Stress-induced Vascular Endothelial Cell Senescence

Oxidative stress regulates dysfunction and senescence of vascular endothelial cells. The DNA damage response and its main signaling pathway involving ataxia telangiectasia mutated (ATM) have been implicated in playing a central role in mediating the actions of oxidative stress; however, the role of the ATM signaling pathway in vascular pathogenesis has largely remained unclear. Here, we identify ATM to regulate oxidative stress-induced endothelial cell dysfunction and premature senescence. Oxidative stress induced senescence in endothelial cells through activation/phosphorylation of ATM by way of an Akt/p53/p21-mediated pathway. These actions were abrogated in cells in which ATM was knocked down by RNA interference or inhibited by specific inhibitory compounds. Furthermore, the in vivo significance of this regulatory pathway was confirmed using ATM knock-out mice in which induction of senescent endothelial cells in the aorta in a diabetic mouse model of endothelial dysfunction and senescence was attenuated in contrast to pathological changes seen in wild-type mice. Collectively, our results show that ATM through an ATM/Akt/p53/p21-dependent signaling pathway mediates an instructive role in oxidative stress-induced endothelial dysfunction and premature senescence.     

Akt-Induced Cellular Senescence: Implication for Human Disease

Reduction-of-function mutations in components of the insulin/insulin-like growth factor-1/Akt pathway have been shown to extend the lifespan in organisms ranging from yeast to mice. It has also been reported that activation of Akt induces proliferation and survival of mammalian cells, thereby promoting tumorigenesis. We have recently shown that Akt activity increases with cellular senescence and that inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Constitutive activation of Akt promotes senescence-like arrest of cell growth via a p53/p21-dependent pathway, leading to endothelial dysfunction. This novel role of Akt in regulating the cellular lifespan may contribute to various human diseases including atherosclerosis and diabetes mellitus.     

Akt-induced cellular senescence: implication for human disease

Reduction-of-function mutations in components of the insulin/insulin-like growth factor-1/Akt pathway have been shown to extend the lifespan in organisms ranging from yeast to mice. It has also been reported that activation of Akt induces proliferation and survival of mammalian cells, thereby promoting tumorigenesis. We have recently shown that Akt activity increases with cellular senescence and that inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Constitutive activation of Akt promotes senescence-like arrest of cell growth via a p53/p21-dependent pathway, leading to endothelial dysfunction. This novel role of Akt in regulating the cellular lifespan may contribute to various human diseases including atherosclerosis and diabetes mellitus.     

Senescence evasion in melanoma progression: uncoupling of DNA‐damage signaling from p53 activation and p21 expression

The best‐established function of the melanoma‐suppressor p16 is mediation of cell senescence, a permanent arrest following cell proliferation or certain stresses. The importance of p16 in melanoma suggests indolence of the other major senescence pathway through p53. Little or no p53 is expressed in senescent normal human melanocytes, but p16‐deficient melanocytes can undergo p53‐mediated senescence. As p16 expression occurs in nevi but falls with progression toward melanoma, we here investigated whether p53‐dependent senescence occurs at some stage and, if not, what defects were detectable in this pathway, using immunohistochemistry. Phosphorylated checkpoint kinase 2 (CHEK2) can mediate DNA‐damage signaling, and under some conditions senescence, by phosphorylating and activating p53. Remarkably, we detected no prevalent p53‐mediated senescence in any of six classes of lesions. Two separate defects in p53 signaling appeared common: in nevi, lack of p53 phosphorylation by activated CHEK2, and in melanomas, defective p21 upregulation by p53 even when phosphorylated.     

miRNAs与衰老相关信号通路的研究进展

miRNAs是一类负调控基因表达的内源性非编码小分子RNA,在细胞衰老过程中发挥重要作用. 细胞衰老是指可增殖细胞在各种应激下出现细胞周期阻滞,并且丧失增殖能力,进入一种不可逆的、相对稳定的状态. p53、p21、p16、SIRT1、胰岛素/IGF-1及mTOR等蛋白是衰老相关信号通路中的重要分子,参与细胞衰老过程. 研究表明,miRNAs可以通过调控这些衰老相关蛋白所在的信号通路,促进或延缓细胞衰老. 本文综述细胞衰老相关的miRNAs,以及它们对衰老相关信号通路的影响,为深化认识衰老和衰老相关疾病的分子机制奠定基础.     

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.     

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.     

Reduction of SIRT1 blunts the protective effects of ischemic post-conditioning in diabetic mice by impairing the Akt signaling pathway

Ischemic post-conditioning (IPO) activates Akt signaling to confer cardioprotection. The responsiveness of diabetic hearts to IPO is impaired. We hypothesized that decreased cardiac SIRT1, a positive regulator of Akt, may be responsible for the impaired responsiveness of diabetic hearts to IPO-mediated cardioprotection. High-fat diet and streptozotocin-induced diabetic mice were subjected to myocardial ischemia/reperfusion (MI/R, 30 min ischemia and 180 min reperfusion) or IPO (three cycles of 10 s of reperfusion and ischemia at the onset of reperfusion). Adenoviral vectors encoding GFP or SIRT1 (Ad-SIRT1) were administered by direct injection into the left ventricular. Our results showed that IPO activated the Akt signaling pathway and reduced MI/R injury in non-diabetic hearts but not in diabetic hearts, in which reduced expression of SIRT1 and increased Akt acetylation were observed. Delivery of Ad-SIRT1 into the diabetic hearts reduced Akt acetylation and restored the cardioprotective effects of IPO by modulating Akt signaling pathway. In contrast, cardiac-specific SIRT1 knockout increased Akt acetylation and blunted the cardioprotective effects of IPO. In in vitro study, transfection with wild-type SIRT1 but not inactive mutant SIRT1 reduced the expression of Akt acetylation and restored the protective effects of hypoxic post-conditioning in high glucose-incubated cardiomyocytes. Moreover, the cardiomyocytes transfected with constitutive Akt acetylation showed repressed Akt phosphorylation and blunted protective effects against hypoxia/reoxygenation injury. These findings demonstrate that the reduction of SIRT1 blunts the protective effects of IPO by impairing Akt signaling pathway and that SIRT1 up-regulation restores IPO-mediated cardioprotection in diabetic mice via deacetylation-dependent activation of Akt signaling pathway.     

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.     

Differential regulation of signal transduction pathways in wild type and mutated p53 breast cancer epithelial cells by copper and zinc

Previous studies have suggested that cells may differ in their response to metal stress. This study was undertaken to investigate the role of PI3K/Akt signaling pathway in metal resistance in human breast cancer epithelial cells with different p53 and estrogen receptor status. Exposure to copper and zinc increased Akt phosphorylation with its nuclear localization only in MDA-MB-231 cells with no estrogen receptor and mutated p53. Cyclin D1 expression and cell-cycle progression followed the metal-induced Akt phosphorylation. Treatment with LY294002 abrogated these effects, suggesting the essential role of PI3-kinase. In contrast, in MCF-7 cells with wild type p53 and estrogen receptor, there was no change in Akt activation, while suppression of p53 activity by pifithrin-alpha increased phosphorylation of Akt after the treatment with copper. In MCF-7 cells, the metal treatment increased the phosphorylation of p53 at serine 15, up-regulated p21 expression, and resulted in cell-cycle arrest in G1 phase with apoptosis. These results demonstrate that copper-induced apoptosis in MCF-7 cells is p53 dependent, whereas the metal resistance in MDA-MB-231 cells may be due to activation of Akt in the absence of a functional p53.     

Abrogation of p53 by its antisense in MCF-7 breast carcinoma cells increases cyclin D1 via activation of Akt and promotion of cell proliferation

The p53 protein has been a subject of intense research interest since its discovery as about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancers suggesting a critical role of p53 in breast cancer development, growth and chemosensitivity. This report describes the derivation and characterization of MCF-7As53, an isogenic cell line derived from MCF-7 breast carcinoma cells in which p53 was abrogated by antisense p53 cDNA. Similar to MCF-7 and simultaneously selected hygromycin resistant MCF-7H cells, MCF-7As53 cells have consistent basal epithelial phenotype, morphology, and estrogen receptor expression levels at normal growth conditions. Present work documents investigation of molecular variations, growth kinetics, and cell cycle related studies in relation to absence of wild-type p53 protein and its transactivation potential as well. Even though wild-type tumor suppressor p53 is an activator of cell growth arrest and apoptosis-mediator genes such as p21, Bax, and GADD45 in MCF-7As53 cells, no alterations in expression levels of these genes were detected. The doubling time of these cells decreased due to depletion of G0/G1 cell phase because of constitutive activation of Akt and increase in cyclin D1 protein levels. This proliferative property was abrogated by wortmannin, an inhibitor of PI3-K/Akt signaling pathway. Therefore this p53 null cell line indicates that p53 is an indispensable component of cellular signaling system which is regulated by caveolin-1 expression, involving Akt activation and increase in cyclin D1, thereby promoting proliferation of breast cancer cells.     

细胞衰老与肿瘤发生

细胞衰老（cell senescence）是指细胞在信号转导作用下不可逆地脱离细胞周期并丧失增殖能力后进入的一种相对稳定的状态。细胞衰老有增殖衰老与早熟衰老两种形式：增殖衰老由端粒缩短激发的信号转导激发,与TP53/CDKN1a（p21^WAF-1/Cip1）/pRB/E2F信号通路密切相关;早熟衰老由细胞内在或外在急慢性应激信号引发,与TP53/CDKN1a（p21^WAF-1/Cip1）/pRB/E2F或CDKN2a（p16^ink4A）/pRB/E2F信号通路相关。目前研究已经证实早熟衰老是细胞在癌变过程中的天然屏障,是继DNA修复、细胞凋亡后的第三大细胞内在抗癌机制,在机体防止肿瘤形成中起重要作用。     

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.     

AMPK induces vascular smooth muscle cell senescence via LKB1 dependent pathway

Vascular cells have a limited lifespan with limited cell proliferation and undergo cellular senescence. The functional changes associated with cellular senescence are thought to contribute to age-related vascular disorders. AMP-activated protein kinase (AMPK) has been discussed in terms of beneficial or harmful effects for aging-related diseases. However, the detailed functional mechanisms of AMPK are largely unclear. An aging model was established by stimulating vascular smooth muscle cell (VSMC) with adriamycin. Adriamycin progressively increased the mRNA and protein expressions of AMPK. The phosphorylation levels of LKB1 and acetyl-CoA carboxylase (ACC), the upstream and downstream of AMPK, were dramatically increased by adriamycin stimulation. The expressions of p53 and p21, which contribute to vascular senescence, were also increased. Inhibition of AMPK diminished senescence-associated β-galactosidase (SA-β-gal) staining, and restored VSMC proliferation. Cytosolic translocation of LKB1 by adriamycin could be a mechanism for AMPK activation in senescence. Furthermore, p53 siRNA and p21 siRNA transfection attenuated adriamycin-induced SA-β-gal staining. These results suggest that LKB1 dependent AMPK activation elicits VSMC senescence and p53–p21 pathway is a mediator of LKB1/AMPK-induced senescence.