Expression of caveolin-1 induces premature cellular senescence in primary cultures of murine fibroblasts

Caveolae are vesicular invaginations of the plasma membrane. Caveolin-1 is the principal structural component of caveolae in vivo. Several lines of evidence are consistent with the idea that caveolin-1 functions as a "transformation suppressor" protein. In fact, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). We have previously demonstrated that overexpression of caveolin-1 arrests mouse embryonic fibroblasts in the G(0)/G(1) phase of the cell cycle through activation of a p53/p21-dependent pathway, indicating a role of caveolin-1 in mediating growth arrest. However, it remains unknown whether overexpression of caveolin-1 promotes cellular senescence in vivo. Here, we demonstrate that mouse embryonic fibroblasts transgenically overexpressing caveolin-1 show: 1) a reduced proliferative lifespan; 2) senescence-like cell morphology; and 3) a senescence-associated increase in beta-galactosidase activity. These results indicate for the first time that the expression of caveolin-1 in vivo is sufficient to promote and maintain the senescent phenotype. Subcytotoxic oxidative stress is known to induce premature senescence in diploid fibroblasts. Interestingly, we show that subcytotoxic level of hydrogen peroxide induces premature senescence in NIH 3T3 cells and increases endogenous caveolin-1 expression. Importantly, quercetin and vitamin E, two antioxidant agents, successfully prevent the premature senescent phenotype and the up-regulation of caveolin-1 induced by hydrogen peroxide. Also, we demonstrate that hydrogen peroxide alone, but not in combination with quercetin, stimulates the caveolin-1 promoter activity. Interestingly, premature senescence induced by hydrogen peroxide is greatly reduced in NIH 3T3 cells harboring antisense caveolin-1. Importantly, induction of premature senescence is recovered when caveolin-1 levels are restored. Taken together, these results clearly indicate a central role for caveolin-1 in promoting cellular senescence and they suggest the hypothesis that premature senescence may represent a tumor suppressor function mediated by caveolin-1 in vivo.     

Bleomycin treatment of A549 human lung cancer cells results in association of MGr1-Ag and caveolin-1 in lipid rafts

Bleomycin treatment of A549 cells induces senescence rather than apoptosis, a more usual response of cancer cells to cytotoxic drugs. We have previously shown that upregulation of caveolin-1, the main structural component of caveolae, plays a key role in this process. In order to gain a better understanding of the molecular basis of this phenomenon, caveolin-1-enriched microdomains of untreated and bleomycin-treated growth-arrested A549 cells were analysed for differential protein expression using 2-D DIGE followed by LC-MS/MS. One of these differentially expressed proteins was found to be the multidrug resistance-associated protein (MGr1-Ag). We show that MGr1-Ag becomes partly localised in lipid rafts following bleomycin treatment, and that MGr1-Ag and caveolin-1 occur in a common protein complex in vivo using co-immunoprecipitation studies. GST pull-down assays demonstrated an increased interaction between MGr1-Ag and caveolin-1 following bleomycin treatment in vitro. Our results reveal MGr1-Ag as a novel lipid raft protein; its increased association with caveolin-1 in bleomycin-induced cell cycle arrest and subsequent cellular senescence might contribute to the success of chemotherapy.     

DNA damage caused by ionizing radiation in embryonic diploid fibroblasts WI-38 induces both apoptosis and senescence

Cellular response to ionizing radiation-induced damage depends on the cell type and the ability to repair DNA damage. Some types of cells undergo apoptosis, whereas others induce a permanent cell cycle arrest and do not proliferate. Our study demonstrates two types of response of embryonic diploid fibroblasts WI-38 to ionizing radiation. In the WI-38 cells p53 is activated, protein p21 increases, but the cells are arrested in G2 phase of cell cycle. Some of the cells die by apoptosis, but in remaining viable cells p16 increases, senescence associated DNA-damage foci occur, and senescence-associated beta-galactosidase activity increases, which indicate stress-induced premature senescence.     

Simvastatin rises reactive oxygen species levels and induces senescence in human melanoma cells by activation of p53/p21 pathway

Recent studies demonstrated that simvastatin has antitumor properties in several types of cancer cells, mainly by inducing apoptosis and inhibiting growth. The arrest of proliferation is a feature of cellular senescence; however, the occurrence of senescence in melanoma cells upon simvastatin treatment has not been investigated until now. Our results demonstrated that exposure of human metastatic melanoma cells (WM9) to simvastatin induces a senescent phenotype, characterized by G1 arrest, positive staining for senescence-associated β-galactosidase assay, and morphological changes. Also, the main pathways leading to cell senescence were examined in simvastatin-treated human melanoma cells, and the expression levels of phospho-p53 and p21 were upregulated by simvastatin, suggesting that cell cycle regulators and DNA damage pathways are involved in the onset of senescence. Since simvastatin can act as a pro-oxidant agent, and oxidative stress may be related to senescence, we measured the intracellular ROS levels in WM9 cells upon simvastatin treatment. Interestingly, we found an increased amount of intracellular ROS in these cells, which was accompanied by elevated expression of catalase and peroxiredoxin-1. Collectively, our results demonstrated that simvastatin can induce senescence in human melanoma cells by activation of p53/p21 pathway, and that oxidative stress may be related to this process.     

The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21(Cip1/Waf1) axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H(2)O(2). The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H(2)O(2)-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21(Cip1/Waf1) axis; but inhibiting the canonical NF-κB pathway exacerbated H(2)O(2)-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H(2)O(2)-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.     

H2O2 accelerates cellular senescence by accumulation of acetylated p53 via decrease in the function of SIRT1 by NAD+ depletion.

It has been reported that p53 acetylation, which promotes cellular senescence, can be regulated by the NAD(+)-dependent deacetylase SIRT1, the human homolog of yeast Sir2, a protein that modulates lifespan. To clarify the role of SIRT1 in cellular senescence induced by oxidative stress, we treated normal human diploid fibroblast TIG-3 cells with H(2)O(2) and examined DNA cleavage, depletion of intracellular NAD(+), expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated beta-galactosidase (SA-beta-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H(2)O(2), though no cell death was observed. NAD(+) levels in TIG-3 cells treated with H(2)O(2) were also decreased significantly. Pre-incubation with the poly (ADP-ribose) polymerase (PARP) inhibitor resulted in preservation of intracellular NAD(+) levels. The amount of acetylated p53 was increased in TIG-3 cells at 4h after H(2)O(2) treatment, while there was little to no decrease in SIRT1 protein expression. The expression level of p21 was increased at 12h and continued to increase for up to 24h. Additionally, exposure of TIG-3 cells to H(2)O(2) induced cell cycle arrest at 24h and increased SA-beta-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.     

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.     

The ATM/p53/p21 pathway influences cell fate decision between apoptosis and senescence in reoxygenated hematopoietic progenitor cells

Hematopoietic cells are often exposed to transient hypoxia as they develop and migrate between blood and tissues. We tested the hypothesis that hypoxia-then-reoxygenation represent a stress for hematopoietic progenitor cells. Here we report that reoxygenation-generated oxidative stress induced senescence, tested as staining for SA-beta-galactosidase (SA-beta-gal), of bone marrow progenitor cells. Reoxygenation induced significant DNA damage and inhibited colony formation in lineage-depleted bone marrow cells enriched for progenitor cells. These reoxygenated cells exhibited a prolonged G(0)/G(1) accumulation without significant apoptosis after 24 h of treatments. Reoxygenated bone marrow progenitor cells expressed SA-beta-gal and senescence-associated proteins p53 and p21(WAF1). Reoxygenated Fancc-/- progenitor cells, which underwent significant apoptosis and senescence, tested as staining for SA-beta-gal, also expressed p16(INK4A). Suppression of apoptosis by the pan-caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone dramatically increased senescent Fancc-/- progenitor cells. Senescence induction, tested as staining for SA-beta-gal, in reoxygenated progenitor cells was closely correlated with extent of DNA damage and phosphorylation of ATM at Ser-1981 and p53 at Ser-15. Moreover, inhibition of ATM signaling reduced SA-beta-gal positivity but increased apoptosis of reoxygenated progenitor cells. Thus, these results suggest that the ATM/p53/p21 pathway influences cell fate decision between apoptosis and senescence in reoxygenated hematopoietic progenitor cells.     

Homologous recombination-mediated irreversible genome damage underlies telomere-induced senescence

Loss of telomeric DNA leads to telomere uncapping, which triggers a persistent, p53-centric DNA damage response that sustains a stable senescence-associated proliferation arrest. Here, we show that in normal cells telomere uncapping triggers a focal telomeric DNA damage response accompanied by a transient cell cycle arrest. Subsequent cell division with dysfunctional telomeres resulted in sporadic telomeric sister chromatid fusions that gave rise to next-mitosis genome instability, including non-telomeric DNA lesions responsible for a stable, p53-mediated, senescence-associated proliferation arrest. Unexpectedly, the blocking of Rad51/RPA-mediated homologous recombination, but not non-homologous end joining (NHEJ), prevented senescence despite multiple dysfunctional telomeres. When cells approached natural replicative senescence, interphase senescent cells displayed genome instability, whereas near-senescent cells that underwent mitosis despite the presence of uncapped telomeres did not. This suggests that these near-senescent cells had not yet acquired irreversible telomeric fusions. We propose a new model for telomere-initiated senescence where tolerance of telomere uncapping eventually results in irreversible non-telomeric DNA lesions leading to stable senescence. Paradoxically, our work reveals that senescence-associated tumor suppression from telomere shortening requires irreversible genome instability at the single-cell level, which suggests that interventions to repair telomeres in the pre-senescent state could prevent senescence and genome instability.     

Inhibition of phosphatidylinostol 3-kinase uncouples H2O2-induced senescent phenotype and cell cycle arrest in normal human diploid fibroblasts

Exposure of WI38 human diploid fibroblasts (HDFs) to hydrogen peroxide (H2O2) induced premature senescence. The senescent HDFs were permanently arrested and exhibited a senescent phenotype including enlarged and flattened cell morphology and increased senescence-associated beta-galactosidase (SA-beta-gal) activity. The induction of HDF senescence was associated with an activation of p53, increased expression of p21Cip1/WAF1, and hypophosphorylation of retinoblastoma protein (Rb), while no changes in the expression of p16Ink4a, p27Kip1, and p14Arf were observed. Exposure of WI38 cells to H2O2 also selectively activated phosphatidylinostol 3-kinase (PI3 kinase) and mitogen-activated protein kinase (MAPK) kinase (MEK), while no changes in p38 MAPK and Jun kinase (JNK) activities were observed. Selective inhibition of PI3 kinase activity with LY294002 abrogated H2O2-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-beta-gal activity, but did not affect H2O2-induced cell cycle arrest. In contrast, selective inhibition of MEK and p38 MAPK with PD98059 and SB203580, respectively, produced no significant effect on H2O2-induced senescent phenotype and cell cycle arrest. These findings demonstrate that expression of the senescent phenotype can be uncoupled from cell cycle arrest in prematurely senescent cells induced by H2O2 and does not contribute to the maintenance of permanent cell cycle arrest.     

DNA double strand break responses and chromatin alterations within the aging cell

Cellular senescence is a state of permanent replicative arrest that allows cells to stay viable and metabolically active but resistant to apoptotic and mitogenic stimuli. Specific, validated markers can identify senescent cells, including senescence-associated β galactosidase activity, chromatin alterations, cell morphology changes, activated p16- and p53-dependent signaling and permanent cell cycle arrest. Senescence is a natural consequence of DNA replication-associated telomere erosion, but can also be induced prematurely by telomere-independent events such as failure to repair DNA double strand breaks. Here, we review the molecular pathways of senescence onset, focussing on the changes in chromatin organization that are associated with cellular senescence, particularly senescence-associated heterochromatin foci formation. We also discuss the altered dynamics of the DNA double strand break response within the context of aging cells. Appreciating how, mechanistically, cellular senescence is induced, and how changes to chromatin organization and DNA repair contributes to this, is fundamental to our understanding of the normal and premature human aging processes associated with loss of organ and tissue function in humans.     

Induction of senescence by adenosine suppressing the growth of lung cancer cells

Extracellular adenosine is well reported to suppress tumor growth by induction of apoptosis. However, in this study we found that adenosine treatment results in cellular senescence in A549 lung cancer cells both in vitro and in vivo; adenosine induces cell cycle arrest and senescence in a p53/p21 dependent manner; adenosine elevates the level of phosphor-γH2AX, pCHK2 and pBRCA1, the markers for prolonged DNA damage response which are likely responsible for initiating the cellular senescence. Our study first demonstrates that adenosine suppresses growth of cancer cells by inducing senescence and provides additional evidence that adenosine could act as an effective anticancer agent for targeted cancer therapy.     

综述: 从分子水平看c-Myc在细胞衰老中的作用

细胞衰老是指细胞生长永久阻滞于细胞周期的G1期,出现形态、生化及表观遗传的变化特性.细胞衰老由端粒缩短、DNA损伤、缺氧或癌基因失调等因素引起,它是抵抗肿瘤发生的主要壁垒.原癌基因c-myc编码转录因子,可调控很多基因,进而影响细胞周期演进、衰老、凋亡、代谢等生物学过程.c-Myc蛋白与细胞衰老密切相关,它可影响hTERT、p16、p53、Bmi-1和p27等衰老相关基因转录.c-Myc不仅可抑制复制性衰老,也能抑制癌基因诱发的衰老.c-Myc抑制ras诱导的细胞衰老取决于CDK2.c-Myc失活不仅能够诱导非恶性细胞(如人成纤维细胞)衰老,而且在许多肿瘤细胞中也可诱导衰老.然而,与ras基因类似,在特定条件下,c-Myc也可诱导细胞衰老,并可促进维氏综合症(Werner syndrome,WRN)缺失细胞的衰老.     

Respiratory syncytial virus matrix protein induces lung epithelial cell cycle arrest through a p53 dependent pathway

Respiratory syncytial virus (RSV) is the major cause of viral respiratory infections in children. Our previous study showed that the RSV infection induced lung epithelial cell cycle arrest, which enhanced virus replication. To address the mechanism of RSV-induced cell cycle arrest, we examined the contribution of RSV-matrix (RSV-M) protein. In this report, we show that in both the A549 cell line and primary human bronchial epithelial (PHBE) cells, transfection with RSV-M protein caused the cells to proliferate at a slower rate than in control cells. The cell cycle analysis showed that RSV-M protein induced G1 phase arrest in A549 cells, and G1 and G2/M phase arrest in PHBE cells. Interestingly, RSV-M expression induced p53 and p21 accumulation and decreased phosphorylation of retinoblastoma protein (Rb). Further, induction of cell cycle arrest by RSV-M was not observed in a p53-deficient epithelial cell line (H1299). However, cell cycle arrest was restored after transfection of p53 cDNA into H1299 cells. Taken together, these results indicate that RSV-M protein regulates lung epithelial cell cycle through a p53-dependent pathway, which enhances RSV replication.