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.     

Chemokine receptor CXCR2 is transactivated by p53 and induces p38‐mediated cellular senescence in response to DNA damage

Mammalian cells may undergo permanent growth arrest/senescence when they incur excessive DNA damage. As a key player during DNA damage response (DDR), p53 transactivates an array of target genes that are involved in various cellular processes including the induction of cellular senescence. Chemokine receptor CXCR2 was previously reported to mediate replicative and oncogene‐induced senescence in a DDR and p53‐dependent manner. Here, we report that CXCR2 is upregulated in various types of cells in response to genotoxic or oxidative stress. Unexpectedly, we found that the upregulation of CXCR2 depends on the function of p53. Like other p53 target genes such as p21, CXCR2 is transactivated by p53. We identified a p53‐binding site in the CXCR2 promoter that responds to changes in p53 functional status. Thus, CXCR2 may act downstream of p53. While the senescence‐associated secretory phenotype (SASP) exhibits a kinetics that is distinct from that of CXCR2 expression and does not require p53, it reinforces senescence. We further showed that the cellular senescence caused by CXCR2 upregulation is mediated by p38 activation. Our results thus demonstrate CXCR2 as a critical mediator of cellular senescence downstream of p53 in response to DNA damage.     

Accumulation of apoptosis‐insensitive human bone marrow‐mesenchymal stromal cells after long‐term expansion

Cells undergo replicative senescence during in vitro expansion, which is induced by the accumulation of cellular damage caused by excessive reactive oxygen species. In this study, we investigated whether long‐term‐cultured human bone marrow mesenchymal stromal cells (MSCs) are insensitive to apoptotic stimulation. To examine this, we established replicative senescent cells from long‐term cultures of human bone marrow MSCs. Senescent cells were identified based on declining population doublings, increased expression of senescence markers p16 and p53 and increased senescence‐associated β‐gal activity. In cell viability assays, replicative senescent MSCs in late passages (i.e. 15–19 passages) resisted damage induced by oxidative stress more than those in early passages did (i.e. 7–10 passages). This resistance occurred via caspase‐9 and caspase‐3 rather than via caspase‐8. The senescent cells are gradually accumulated during long‐term expansion. The oxidative stress‐sensitive proteins ataxia‐telangiectasia mutated and p53 were phosphorylated, and the expression of apoptosis molecules Bax increased, and Bcl‐2 decreased in early passage MSCs; however, the expression of the apoptotic molecules did less change in response to apoptotic stimulation in late‐passage MSCs, suggesting that the intrinsic apoptotic signalling pathway was not induced by oxidative stress in long‐term‐cultured MSCs. Based on these results, we propose that some replicative senescent cells may avoid apoptosis signalling via impairment of signalling molecules and accumulation during long‐term expansion. Copyright © 2016 John Wiley & Sons, Ltd.     

Quantitative proteomic analysis reveals induction of premature senescence in human umbilical vein endothelial cells exposed to chronic low‐dose rate gamma radiation

Chronic low‐dose ionizing radiation induces cardiovascular disease in human populations but the mechanism is largely unknown. We suggested that chronic radiation exposure may induce endothelial cell senescence that is associated with vascular damage in vivo. We investigated whether chronic radiation exposure is causing a change in the onset of senescence in endothelial cells in vitro. Indeed, when exposed to continuous low‐dose rate gamma radiation (4.1 mGy/h), primary human umbilical vein endothelial cells (HUVECs) initiated senescence much earlier than the nonirradiated control cells. We investigated the changes in the protein expression of HUVECs before and during the onset of radiation‐induced senescence. Cellular proteins were quantified using isotope‐coded protein label technology after 1, 3, and 6 weeks of radiation exposure. Several senescence‐related biological pathways were influenced by radiation, including cytoskeletal organization, cell–cell communication and adhesion, and inflammation. Immunoblot analysis showed an activation of the p53/p21 pathway corresponding to the progressing senescence. Our data suggest that chronic radiation‐induced DNA damage and oxidative stress result in induction of p53/p21 pathway that inhibits the replicative potential of HUVECs and leads to premature senescence. This study contributes to the understanding of the increased risk of cardiovascular diseases seen in populations exposed to chronic low‐dose irradiation.     

Single‐cell analysis of p16INK4a and p21WAF1 expression suggests distinct mechanisms of senescence in normal human and Li‐Fraumeni Syndrome fibroblasts

Herein we used single‐cell observation methods to gain insight into the roles of p16^INK4A and p21^WAF1 (hereafter p16 and p21) in replicative senescence and ionizing radiation‐induced accelerated senescence in human [normal, ataxia telangiectasia (AT) and Li‐Fraumeni syndrome (LFS)] fibroblast strains. Cultures of all strains entered a state of replicative senescence at late passages, as evident from inhibition of growth, acquisition of flattened and enlarged cell morphology, and positive staining for senescence‐associated β‐galactosidase. In addition, proliferating early‐passage cultures of these strains exhibited accelerated senescence in response to ionizing radiation. Immunofluorescence microscopy revealed the heterogeneous expression of p16 in normal and AT fibroblast strains, with the majority of the cells exhibiting undetectable levels of p16 irrespective of in vitro culture age. Importantly, replicative senescence as well as accelerated senescence triggered by ionizing radiation were accompanied by sustained nuclear accumulation of p21, but did not correlate with p16 expression in p53‐proficient (normal and AT) fibroblasts. In p53‐deficient (LFS) fibroblasts, on the other hand, replicative senescence and ionizing radiation‐triggered accelerated senescence strongly correlated with expression of p16 but not of p21. Furthermore, senescence in LFS fibroblasts was associated with genomic instability encompassing polyploidy. Our findings are compatible with a model in which p16 serves as a backup regulator of senescence, triggering this response preferentially in the absence of wild‐type p53 activity. The possibility that one of the tumor‐suppressor functions of p16 may be associated with genomic instability, preventing the emergence of malignant progeny from polyploid giant cells, is also supported by these results. J. Cell. Physiol. 223: 57–67, 2010. © 2009 Wiley‐Liss, Inc.     

ING2 regulates the onset of replicative senescence by induction of p300-dependent p53 acetylation

ING2 is a candidate tumor suppressor gene that can activate p53 by enhancing its acetylation. Here, we demonstrate that ING2 is also involved in p53-mediated replicative senescence. ING2 protein expression increased in late-passage human primary cells, and it colocalizes with serine 15-phosphorylated p53. ING2 and p53 also complexed with the histone acetyltransferase p300. ING2 enhanced the interaction between p53 and p300 and acted as a cofactor for p300-mediated p53 acetylation. The level of ING2 expression directly modulated the onset of replicative senescence. While overexpression of ING2 induced senescence in young fibroblasts in a p53-dependent manner, expression of ING2 small interfering RNA delayed the onset of senescence. Hence, ING2 can act as a cofactor of p300 for p53 acetylation and thereby plays a positive regulatory role during p53-mediated replicative senescence.     

氧化应激诱导的细胞衰老过程中细胞生长相关基因的表达

应激诱导的细胞早衰与复制性细胞衰老有相似的细胞表型,但其机制不尽相同.分析二者的衰老相关基因表达特点对了解应激因素诱导细胞衰老的机制有重要意义. 本文对过氧化氢诱导的HeLa细胞早衰过程中的关键衰老相关基因及其转录后调控因子的表达做了分析.结果发现,在复制性衰老过程中明显降低的cyclin A、cyclin B1、c-fos及HuR,在温和过氧化氢诱导的细胞早衰过程中并无明显改变;在氧化应激诱导的细胞早衰过程中,p21与p16表达升高,AUF1则降低,与复制性衰老过程一致;p21 mRNA半衰期在复制性衰老过程中无明显变化,但在氧化应激诱导的细胞早衰过程中则显著延长.上述结果提示,尽管氧化应激诱导的细胞早衰与复制性衰老存在相似基因表达变化,调控机制则不尽相同.     

Increased expression of SIRT2 is a novel marker of cellular senescence and is dependent on wild type p53 status

Sirtuins (SIRT) belonging to the NAD+ dependent histone deacetylase III class of enzymes have emerged as master regulators of metabolism and longevity. However, their role in prevention of organismal aging and cellular senescence still remains controversial. In the present study, we now report upregulation of SIRT2 as a specific feature associated with stress induced premature senescence but not with either quiescence or cell death. Additionally, increase in SIRT2 expression was noted in different types of senescent conditions such as replicative and oncogene induced senescence using multiple cell lines. Induction of SIRT2 expression during senescence was dependent on p53 status as depletion of p53 by shRNA prevented its accumulation. Chromatin immunoprecipitation revealed the presence of p53 binding sites on the SIRT2 promoter suggesting its regulation by p53, which was also corroborated by the SEAP reporter assay. Overexpression or knockdown of SIRT2 had no effect on stress induced premature senescence, thereby indicating that SIRT2 increase is not a cause of senescence; rather it is an effect linked to senescence-associated changes. Overall, our results suggest SIRT2 as a promising marker of cellular senescence at least in cells with wild type p53 status.     

CUL4B impedes stress-induced cellular senescence by dampening a p53-reactive oxygen species positive feedback loop

Tumor suppressor p53 is known to regulate the level of intracellular reactive oxygen species (ROS). It can either alleviate oxidative stress under physiological and mildly stressed conditions or exacerbate oxidative stress under highly stressed conditions. We here report that a p53–ROS positive feedback loop drives a senescence program in normal human fibroblasts (NHFs) and this senescence-driving loop is negatively regulated by CUL4B. CUL4B, which can assemble various ubiquitin E3 ligases, was found to be downregulated in stress-induced senescent cells, but not in replicative senescent cells. We observed that p53-dependent ROS production was significantly augmented and stress-induced senescence was greatly enhanced when CUL4B was absent or depleted. Ectopic expression of CUL4B, on the other hand, blunted p53 activation, reduced ROS production, and attenuated cellular senescence in cells treated with H₂O₂. CUL4B was shown to promote p53 ubiquitination and proteosomal degradation in NHFs exposed to oxidative stress, thus dampening the p53-dependent cellular senescence. Together, our results established a critical role of CUL4B in negatively regulating the p53–ROS positive feedback loop that drives cellular senescence.     

Telomeric plasmid induces human cancer cell dysfunction depending on ATM activity

Telomeres are essential for chromosome stability and the regulation of the replicative life‐span of somatic cells. Many studies showed that exogenous telomeric repeats could activate p53 protein. It is not known how cell dysfunction is induced by telomeric plasmids. A covalent closed circular (ccc) double‐stranded plasmid containing (TTAGGG)₉₆ repeats (pRST5) was transiently transfected into the human gastric cancer MGC‐803 cells. We first confirmed that the cell viabilities decreased by 27%, cell senescence increased by 62% and G2/M cycle arrested in pRST5 plasmid transfected cells. Compared to control groups, cells transfected with telomeric plasmids showed an ATM‐dependent increasing of p53, TRF1, and TRF2 expression. Furthermore, telomere dysfunction‐induced foci (TIF) were observed. In conclusion, telomeric plasmids can elicit endogenous telomere dysfunction and induce cell senescence by activating ATM‐p53 pathway. Copyright © 2010 John Wiley & Sons, Ltd.     

Senescence: a telomeric limit to immortality or a cellular response to physiologic stresses?

Cells entering a state of senescence undergo a irreversible cell cycle arrest, associated by a set of functional and morphological changes. Senescence occurs following telomeres shortening (replicative senescence) or exposure to other acute or chronic physiologic stress signals (a phenomenon termed stasis: stress or aberrant signaling-induced senescence). In this review, I discuss the pathways of cellular senescence, the mechanisms involved and the role that these pathways have in regulating the initiation and progression of cancer. Telomere-initiated senescence or loss of telomere function trigger focal recruitement of protein sensors of the DNA double-strand breaks leading to the activation of the DNA damage checkpoint responses and the tumour suppressor gene product, p53, which in turn induces the cell-cycle inhibitor, p21(WAF1). Loss of p53 and pRb function allows continued cell division despite increasing telomere dysfunction and eventually entry into telomere crisis. Immortalisation is an essential prerequisite for the formation of a tumour cell. Therefore, a developing tumour cell must circumvent at least two proliferative barriers--cellular senescence and crisis--to achieve neoplastic transformation. These barriers are regulated by telomere shortening and by the p16(INK4a)/Rb and p53 tumour suppressor pathways. Elucidation of the genes and emerging knowledge about the regulatory mechanisms that lead to senescence and determine the pattern of gene expression in senescent cells may lead to more effective treatments for cancer.     

Mammalian SIRT1 limits replicative life span in response to chronic genotoxic stress

The Saccharomyces cerevisiae chromatin silencing factor Sir2 suppresses genomic instability and extends replicative life span. In contrast, we find that mouse embryonic fibroblasts (MEFs) deficient for SIRT1, a mammalian Sir2 homolog, have dramatically increased resistance to replicative senescence. Extended replicative life span of SIRT1-deficient MEFs correlates with enhanced proliferative capacity under conditions of chronic, sublethal oxidative stress. In this context, SIRT1-deficient cells fail to normally upregulate either the p19(ARF) senescence regulator or its downstream target p53. However, upon acute DNA damage or oncogene expression, SIRT1-deficient cells show normal p19(ARF) induction and cell cycle arrest. Together, our findings demonstrate an unexpected SIRT1 function in promoting replicative senescence in response to chronic cellular stress and implicate p19(ARF) as a downstream effector in this pathway.     

Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence

p53 limits the proliferation of primary diploid fibroblasts by inducing a state of growth arrest named replicative senescence - a process which protects against oncogenic transformation and requires integrity of the p53 tumour suppressor pathway. However, little is known about the downstream target genes of p53 in this growth-limiting response. Here, we report that suppression of the p53 target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference (RNAi) leads to escape from replicative senescence both in primary mouse embryo fibroblasts and primary human BJ fibroblasts. PAI-1 knockdown results in sustained activation of the PI(3)K-PKB-GSK3beta pathway and nuclear retention of cyclin D1, consistent with a role for PAI-1 in regulating growth factor signalling. In agreement with this, we find that the PI(3)K-PKB-GSK3beta-cyclin D1 pathway is also causally involved in cellular senescence. Conversely, ectopic expression of PAI-1 in proliferating p53-deficient murine or human fibroblasts induces a phenotype displaying all the hallmarks of replicative senescence. Our data indicate that PAI-1 is not merely a marker of senescence, but is both necessary and sufficient for the induction of replicative senescence downstream of p53.     

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.     

Signaling pathway requirements for induction of senescence by telomere homolog oligonucleotides

Cellular senescence is a major defense against cancer. In human fibroblasts, suppressing both the p53 and pRb pathways is necessary to bypass replicative senescence as well as senescence induced by ectopic expression of a dominant negative form of the telomere repeat binding factor 2, TRF2(DN). We recently reported that exposure to oligonucleotides homologous to the telomere 3' overhang (T-oligos) activates both the p53 and pRb pathways and leads to senescence in primary human fibroblasts. To further characterize T-oligo-induced senescence, we compared established isogenic fibroblast cell lines lacking functional p53 and/or pRb pathways to the normal parental line. Here, we report that, as in physiologic senescence, inactivation of both the p53 and pRb pathways is necessary to suppress T-oligo-induced senescence. Moreover, T-oligo rapidly induces senescence in a malignant fibroblast-derived cell line, demonstrating the potential of using T-oligo as a novel anticancer therapeutic. Our data support the hypothesis that exposure of the TTAGGG tandem repeat telomere 3' overhang sequence is the event that initiates signaling through DNA damage response pathways after experimental telomere disruption, serial passage, or acute genomic damage of normal cells.     

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.     

Telomere dysfunction suppresses spontaneous tumorigenesis in vivo by initiating p53-dependent cellular senescence

Dysfunctional telomeres induce p53-dependent cellular senescence and apoptosis, but it is not known which function is more important for tumour suppression in vivo. We used the p53 ( R172P ) knock-in mouse, which is unable to induce apoptosis but retains intact cell-cycle arrest and cellular senescence pathways, to show that spontaneous tumorigenesis is potently repressed in Terc -/- p53 ( R172P ) mice. Tumour suppression is accompanied by global induction of p53, p21 and the senescence marker senescence-associated-beta-galactosidase. By contrast, cellular senescence was unable to suppress chemically induced skin carcinomas. These results indicate that suppression of spontaneous tumorigenesis by dysfunctional telomeres requires the activation of the p53-dependent cellular senescence pathway.