58-kDa microspherule protein (MSP58) is novel Brahma-related gene 1 (BRG1)-associated protein that modulates p53/p21 senescence pathway

The nucleolar 58-kDa microspherule protein (MSP58) protein is a candidate oncogene implicated in modulating cellular proliferation and malignant transformation. In this study, we show that knocking down MSP58 expression caused aneuploidy and led to apoptosis, whereas ectopic expression of MSP58 regulated cell proliferation in a context-dependent manner. Specifically, ectopic expression of MSP58 in normal human IMR90 and Hs68 diploid fibroblasts, the H184B5F5/M10 mammary epithelial cell line, HT1080 fibrosarcoma cells, primary mouse embryonic fibroblasts, and immortalized NIH3T3 fibroblasts resulted in induction of premature senescence, an enlarged and flattened cellular morphology, and increased senescence-associated β-galactosidase activity. MSP58-driven senescence was strictly dependent on the presence of functional p53 as revealed by the fact that normal cells with p53 knockdown by specific shRNA or cells with a mutated or functionally impaired p53 pathway were effective in bypassing MSP58-induced senescence. At least two senescence mechanisms are induced by MSP58. First, MSP58 activates the DNA damage response and p53/p21 signaling pathways. Second, MSP58, p53, and the SWI/SNF chromatin-remodeling subunit Brahma-related gene 1 (BRG1) form a ternary complex on the p21 promoter and collaborate to activate p21. Additionally, MSP58 protein levels increased in cells undergoing replicative senescence and stress-induced senescence. Notably, the results of analyzing expression levels of MSP58 between tumors and matched normal tissues showed significant changes (both up- and down-regulation) in its expression in various types of tumors. Our findings highlight new aspects of MSP58 in modulating cellular senescence and suggest that MSP58 has both oncogenic and tumor-suppressive properties.     

The M‐type receptor PLA2R regulates senescence through the p53 pathway

Senescence is a stable proliferative arrest induced by various stresses such as telomere erosion, oncogenic or oxidative stress. Compelling evidence suggests that it acts as a barrier against tumour development. Describing new mechanisms that favour an escape from senescence can thus reveal new insights into tumorigenesis. To identify new genes controlling the senescence programme, we performed a loss‐of‐function genetic screen in primary human fibroblasts. We report that knockdown of the M‐type receptor PLA2R (phospholipase A2 receptor) prevents the onset of replicative senescence and diminishes stress‐induced senescence. Interestingly, expression of PLA2R increases during replicative senescence, and its ectopic expression results in premature senescence. We show that PLA2R regulates senescence in a reactive oxygen species–DNA damage–p53‐dependent manner. Taken together, our study identifies PLA2R as a potential new tumour suppressor gene crucial in the induction of cellular senescence through the activation of the p53 pathway.     

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.     

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.     

Stress-induced premature senescence in hTERT-expressing ataxia telangiectasia fibroblasts

In addition to replicative senescence, normal diploid fibroblasts undergo stress-induced premature senescence (SIPS) in response to DNA damage caused by oxidative stress or ionizing radiation (IR). SIPS is not prevented by telomere elongation, indicating that, unlike replicative senescence, it is triggered by nonspecific genome-wide DNA damage rather than by telomere shortening. ATM, the product of the gene mutated in individuals with ataxia telangiectasia (AT), plays a central role in cell cycle arrest in response to DNA damage. Whether ATM also mediates signaling that leads to SIPS was investigated with the use of normal and AT fibroblasts stably transfected with an expression vector for the catalytic subunit of human telomerase (hTERT). Expression of hTERT in AT fibroblasts resulted in telomere elongation and prevented premature replicative senescence, but it did not rescue the defect in G(1) checkpoint activation or the hypersensitivity of the cells to IR. Despite these remaining defects in the DNA damage response, hTERT-expressing AT fibroblasts exhibited characteristics of senescence on exposure to IR or H(2)O(2) in such a manner that triggers SIPS in normal fibroblasts. These characteristics included the adoption of an enlarged and flattened morphology, positive staining for senescence-associated beta-galactosidase activity, termination of DNA synthesis, and accumulation of p53, p21(WAF1), and p16(INK4A). The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which mediates signaling that leads to senescence, was also detected in both IR- or H(2)O(2)-treated AT and normal fibroblasts expressing hTERT. These results suggest that the ATM-dependent signaling pathway triggered by DNA damage is dispensable for activation of p38 MAPK and SIPS in response to IR or oxidative stress.     

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.     

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.     

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.     

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.     

An altered repertoire of fos/jun (AP-1) at the onset of replicative senescence.

With multiple divisions in culture, normal diploid cells suffer a loss of growth potential that leads to replicative senescence and a finite replicative capacity. Using quantitative RT-PCR, we have monitored mRNA expression levels of c-fos, c-jun, JunB, c-myc, p53, H-ras, and histone H4 during the replicative senescence of human fibroblasts. The earliest and the largest changes in gene expression occurred in c-fos and junB at mid-senescence prior to the first slowing in cell growth rates. The basal level of c-fos mRNA decreased to one-ninth that of the early-passage levels, while junB declined to one-third and c-jun expression remained constant. The decline in the basal c-fos mRNA level in mid-senescence should lead to an increase in Jun/Jun AP-1 homodimers at the expense of Fos/Jun heterodimers and may trigger a cascade of further changes in c-myc, p53, and H-ras expression in late-passage senescent fibroblasts.     

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.