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.     

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.     

p73 cooperates with Ras in the activation of MAP kinase signaling cascade

The p73 gene is capable of inducing cell cycle arrest, apoptosis, senescence, differentiation and to cooperate with oncogenic Ras in cellular transformation. Ras can be considered as a branch point in signal transduction, where diverse extracellular stimuli converge. The intensity of the mitogen-activated protein kinase (MAPK) cascade activation influences the cellular response to Ras. Despite the fundamental role of p53 in Ras-induced growth arrest and senescence, it remains unclear how the Ras/MEK/ERK pathway induces growth arrest in the absence of p53. We report here that oncogenic Ras stabilizes p73 resulting in p73 accumulation and enhancement of its activity. p73, in turn, induces a sustained activation of the MAP kinase cascade synergizing with oncogenic Ras. We also found that inhibition of p73 function modifies the cellular outcome to Ras activation inhibiting Ras-dependent differentiation. Here, we show for the first time that there is a signaling loop between Ras-dependent MAPK cascade activation and p73 function.     

Escape from premature senescence is not sufficient for oncogenic transformation by Ras

Resistance of primary cells to transformation by oncogenic Ras has been attributed to the induction of replicative growth arrest. This irreversible 'fail-safe mechanism' resembles senescence and requires induction by Ras of p19ARF and p53 (refs 3-5). Mutation of either p19ARF or p53 alleviates Ras-induced senescence and facilitates oncogenic transformation by Ras. Here we report that, whereas Rb and p107 are each dispensable for Ras-induced replicative arrest, simultaneous ablation of both genes disrupts Ras-induced senescence and results in unrestrained proliferation. This occurs despite activation by Ras of the p19ARF /p53 pathway, identifying pRb and p107 as essential mediators of Ras-induced antiproliferative p19ARF/p53 signalling. Unexpectedly, in contrast to p19ARF or p53 deficiency, loss of Rb/p107 function does not result in oncogenic transformation by Ras, as Ras-expressing Rb-/-/p107-/- fibroblasts fail to grow anchorage-independently in vitro and are not tumorigenic in vivo. These results demonstrate that in the absence of both Rb and p107 cells are resistant to p19ARF/p53-dependent protection against Ras-induced proliferation, and uncouple escape from Ras-induced premature senescence from oncogenic transformation.     

PEA-15 is inhibited by adenovirus E1A and plays a role in ERK nuclear export and Ras-induced senescence

Oncogenic ras activates multiple signaling pathways to enforce cell proliferation in tumor cells. The ERK1/2 mitogen-activated protein kinase pathway is required for the transforming effects of ras, and its activation is often sufficient to convey mitogenic stimulation. However, in some settings oncogenic ras triggers a permanent cell cycle arrest with features of cellular senescence. How the Ras/ERK1/2 pathway activates different cellular programs is not well understood. Here we show that ERK1/2 localize predominantly in the cytoplasm during ras-induced senescence. This cytoplasmic localization seems to be dependent on an active nuclear export mechanism and can be rescued by the viral oncoprotein E1A. Consistent with this hypothesis, we showed that E1A dramatically down-regulated the expression of the ERK1/2 nuclear export factor PEA-15. Also, RNA interference against PEA-15 restored the nuclear localization of phospho-ERK1/2 in Ras-expressing primary murine embryo fibroblasts and stimulated their escape from senescence. Because senescence prevents the transforming effect of oncogenic ras, our results suggest a tumor suppressor function for PEA-15 that operates by means of controlling the localization of phospho-ERK1/2.     

The specific role of pRb in p16INK4A-mediated arrest of normal and malignant human breast cells

RB family proteins pRb, p107 and p130 have similar structures and overlapping functions, enabling cell cycle arrest and cellular senescence. pRb, but not p107 or p130, is frequently mutated in human malignancies. In human fibroblasts acutely exposed to oncogenic ras, pRb has a specific role in suppressing DNA replication, and p107 or p130 cannot compensate for the loss of this function; however, a second p53/p21-dependent checkpoint prevents escape from growth arrest. This model of oncogene-induced senescence requires the additional loss of p53/p21 to explain selection for preferential loss of pRb function in human malignancies. We asked whether similar rules apply to the role of pRb in growth arrest of human epithelial cells, the source of most cancers. In two malignant human breast cancer cell lines, we found that individual RB family proteins were sufficient for the establishment of p16-initiated senescence, and that growth arrest in G₁ was not dependent on the presence of functional pRb or p53. However, senescence induction by endogenous p16 was delayed in primary normal human mammary epithelial cells with reduced pRb but not with reduced p107 or p130. Thus, under these circumstances, despite the presence of functional p53, p107 and p130 were unable to completely compensate for pRb in mediating senescence induction. We propose that early inactivation of pRb in pre-malignant breast cells can, by itself, extend proliferative lifespan, allowing acquisition of additional changes necessary for malignant transformation.     

Ing1 mediates p53 accumulation and chromatin modification in response to oncogenic stress

ING proteins are putative tumor suppressor proteins linked to the p53 pathway and to the chromatin modification machinery. Here we have analyzed the role of the products of the murine Ing1 locus in cellular tumor-protective responses, using mouse primary fibroblasts where the Ing1 locus has been inactivated by the integration of a betageo cassette. We show that Ing1-deficient mouse embryonic fibroblasts display a defective senescence-like antiproliferative response against oncogenic Ras, affecting several senescence-specific markers. This phenotype is accompanied by a reduced accumulation of p53, which can be explained by the reduced basal p53 protein stability in the Ing1-deficient background. Ing1 deficiency also results in defects in the appearance of heterochromatic marks upon expression of oncogenic Ras, suggestive of impaired heterochromatin formation during oncogene-induced senescence. Our results support an important role for the Ing1 locus in protection against oncogenic stress in vivo, both as a mediator of p53 activation and as a regulator of chromatin remodeling processes.     

Decreased translation of p21waf1 mRNA causes attenuated p53 signaling in some p53 wild-type tumors

DNA damage induces cell cycle arrest through both Chk1 and the p53 tumor suppressor protein, the latter arresting cells through induction of p21^waf1 protein. Arrest permits cells to repair the damage and recover. The frequent loss of p53 in tumor cells makes them more dependent on Chk1 for arrest and survival. However, some p53 wild type tumor cell lines, such as HCT116 and U2OS, are also sensitive to inhibition of Chk1 due to attenuated p21^waf1 induction upon DNA damage. The purpose of this study is to determine the cause of this attenuated p21^waf1 protein induction. We find that neither the induction of p21^waf1 mRNA nor protein half-life is sufficient to explain the low p21^waf1 protein levels in HCT116 and U2OS cells. The induced mRNA associates with polysomes but little protein is made suggesting these two cell lines have a reduced rate of p21^waf1 mRNA translation. This represents a novel mechanism for disruption of the p53-p21^waf1 pathway as currently known mechanisms involve either mutation of p53 or reduction of p53 protein levels. As a consequence, this attenuated p21^waf1 expression may render some p53 wild type tumors sensitive to a combination of DNA damage plus checkpoint inhibition.     

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.     

Decreased translation of p21waf1 mRNA causes attenuated p53 signaling in some p53 wild-type tumors

DNA damage induces cell cycle arrest through both Chk1 and the p53 tumor suppressor protein, the latter arresting cells through induction of p21^waf1 protein. Arrest permits cells to repair the damage and recover. The frequent loss of p53 in tumor cells makes them more dependent on Chk1 for arrest and survival. However, some p53 wild type tumor cell lines, such as HCT116 and U2OS, are also sensitive to inhibition of Chk1 due to attenuated p21^waf1 induction upon DNA damage. The purpose of this study is to determine the cause of this attenuated p21^waf1 protein induction. We find that neither the induction of p21^waf1 mRNA nor protein half-life is sufficient to explain the low p21^waf1 protein levels in HCT116 and U2OS cells. The induced mRNA associates with polysomes but little protein is made suggesting these two cell lines have a reduced rate of p21^waf1 mRNA translation. This represents a novel mechanism for disruption of the p53-p21^waf1 pathway as currently known mechanisms involve either mutation of p53 or reduction of p53 protein levels. As a consequence, this attenuated p21^waf1 expression may render some p53 wild type tumors sensitive to a combination of DNA damage plus checkpoint inhibition.     

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.     

p53-independent apoptosis is induced by the p19ARF tumor suppressor

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.     

G1阻滞中p21-E2F间的相互作用

前列腺素A2(PGA2)具有强的体内、外抗增殖活性,引起细胞周期阻滞,同时,可诱导cdk抑制物p21蛋白的表达,后者亦可介导多种细胞的G1阻滞.提示p21^waf1/cip1在PGA2诱导的细胞周期阻滞中具有重要作用.主要介绍了近两年来有关p21^waf1/cip1与转录因子E2F间的相互作用的研究,阐述p21^waf1/cip1在PGA2介导的细胞周期阻滞中的作用机制.     

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.     

Klotho RNAi induces premature senescence of human cells via a p53/p21 dependent pathway

Klotho has recently emerged as a regulator of aging. To investigate the role of Klotho in the regulation of cellular senescence, we generated stable MRC-5 human primary fibroblast cells knockdown for Klotho expression by RNAi. Downregulation of Klotho dramatically induces premature senescence with a concomitant upregulation of p21. The upregulation of p21 is associated with cell cycle arrest at G1/S boundary. Knockdown of p53 in the Klotho attenuated MRC-5 cells restores normal growth and replicative potential. These results demonstrate that Klotho normally regulates cellular senescence by repressing the p53/p21 pathway. Our findings implicate Klotho as a regulator of aging in primary human fibroblasts.     

SUMO-modified nuclear cyclin D1 bypasses Ras-induced senescence

Oncogene-induced senescence represents a key tumor suppressive mechanism. Here, we show that Ras oncogene-induced senescence can be mediated by the recently identified haploinsufficient tumor suppressor apoptosis-stimulating protein of p53 (ASPP) 2 through a novel and p53/p19^Arf/p21^waf1/cip1-independent pathway. ASPP2 suppresses Ras-induced small ubiquitin-like modifier (SUMO)-modified nuclear cyclin D1 and inhibits retinoblastoma protein (Rb) phosphorylation. The lysine residue, K33, of cyclin D1 is a key site for this newly identified regulation. In agreement with the fact that its nuclear localization is required for its oncogenic activity, we show that nuclear cyclin D1 is far more potent than wild-type (WT) cyclin D1 in bypassing Ras-induced senescence. Thus, this study identifies SUMO modification as a positive regulator of nuclear cyclin D1, and reveals a new way by which cell cycle entry and senescence are regulated.