Werner综合征小鼠模型在早衰与肿瘤研究中的应用

Werner综合征(Werner syndrome, WS)是一种罕见的人类常染色体隐性遗传疾病, 一直以来该病作为研究人类早老综合征的典型病例而受到关注。Werner蛋白(WRN)是Werner综合征中突变的核蛋白, 最近的生化及遗传学研究证明WRN在DNA复制、DNA损伤修复以及端粒的维持方面起着重要的作用。文章综述了Werner综合征的分子遗传学机理及端粒和WRN在Werner综合征发病中的重要作用。通过双敲除Wrn与端粒酶基因建立的小鼠模型忠实地再现了人类Werner综合征, 这种Werner综合征小鼠模型因其同时具有早衰与肿瘤表型而在研究人类肿瘤及衰老的相关性中起到的独特作用。     

Werner protein protects nonproliferating cells from oxidative DNA damage

Werner syndrome, caused by mutations of the WRN gene, mimics many changes of normal aging. Although roles for WRN protein in DNA replication, recombination, and telomere maintenance have been suggested, the pathology of rapidly dividing cells is not a feature of Werner syndrome. To identify cellular events that are specifically vulnerable to WRN deficiency, we used RNA interference (RNAi) to knockdown WRN or BLM (the RecQ helicase mutated in Bloom syndrome) expression in primary human fibroblasts. Withdrawal of WRN or BLM produced accelerated cellular senescence phenotype and DNA damage response in normal fibroblasts, as evidenced by induction of gammaH2AX and 53BP1 nuclear foci. After WRN depletion, the induction of these foci was seen most prominently in nondividing cells. Growth in physiological (3%) oxygen or in the presence of an antioxidant prevented the development of the DNA damage foci in WRN-depleted cells, whereas acute oxidative stress led to inefficient repair of the lesions. Furthermore, WRN RNAi-induced DNA damage was suppressed by overexpression of the telomere-binding protein TRF2. These conditions, however, did not prevent the DNA damage response in BLM-ablated cells, suggesting a distinct role for WRN in DNA homeostasis in vivo. Thus, manifestations of Werner syndrome may reflect an impaired ability of slowly dividing cells to limit oxidative DNA damage.     

Regulation of WRN protein cellular localization and enzymatic activities by SIRT1-mediated deacetylation

Werner syndrome is an autosomal recessive disorder associated with premature aging and cancer predisposition caused by mutations of the WRN gene. WRN is a member of the RecQ DNA helicase family with functions in maintaining genome stability. Sir2, an NAD-dependent histone deacetylase, has been proven to extend life span in yeast and Caenorhabditis elegans. Mammalian Sir2 (SIRT1) has also been found to regulate premature cellular senescence induced by the tumor suppressors PML and p53. SIRT1 plays an important role in cell survival promoted by calorie restriction. Here we show that SIRT1 interacts with WRN both in vitro and in vivo; this interaction is enhanced after DNA damage. WRN can be acetylated by acetyltransferase CBP/p300, and SIRT1 can deacetylate WRN both in vitro and in vivo. WRN acetylation decreases its helicase and exonuclease activities, and SIRT1 can reverse this effect. WRN acetylation alters its nuclear distribution. Down-regulation of SIRT1 reduces WRN translocation from nucleoplasm to nucleoli after DNA damage. These results suggest that SIRT1 regulates WRN-mediated cellular responses to DNA damage through deacetylation of WRN.     

Metabolic and Phenotypic Differences between Mice Producing a Werner Syndrome Helicase Mutant Protein and Wrn Null Mice

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-family DNA helicase, WRN. Mice lacking part of the helicase domain of the WRN orthologue exhibit many phenotypic features of WS, including metabolic abnormalities and a shorter mean life span. In contrast, mice lacking the entire Wrn protein (i.e. Wrn null mice) do not exhibit a premature aging phenotype. In this study, we used a targeted mass spectrometry-based metabolomic approach to identify serum metabolites that are differentially altered in young Wrn helicase mutant and Wrn null mice. An antibody-based quantification of 43 serum cytokines and markers of cardiovascular disease risk complemented this study. We found that Wrn helicase mutants exhibited elevated and decreased levels, respectively, of the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokine IL-18. Wrn helicase mutants also exhibited an increase in serum hydroxyproline and plasminogen activator inhibitor-1, markers of extracellular matrix remodeling of the vascular system and inflammation in aging. We also observed an abnormal increase in the ratio of very long chain to short chain lysophosphatidylcholines in the Wrn helicase mutants underlying a peroxisome perturbation in these mice. Remarkably, the Wrn mutant helicase protein was mislocalized to the endoplasmic reticulum and the peroxisomal fractions in liver tissues. Additional analyses with mouse embryonic fibroblasts indicated a severe defect of the autophagy flux in cells derived from Wrn helicase mutants compared to wild type and Wrn null animals. These results indicate that the deleterious effects of the helicase-deficient Wrn protein are mediated by the dysfunction of several cellular organelles.     

c-Myc Accelerates S-Phase and Requires WRN to Avoid Replication Stress

c-Myc interacts with components of the pre-replication complex and directly regulates DNA replication [1]. However the consequences of this novel c-Myc function on cell cycle dynamics and replication-associated damage are unknown. Here, we show that c-Myc overexpression in primary human fibroblasts markedly accelerates S-phase while c-Myc deficient fibroblasts exhibit a prolonged S-phase. We also show that the Werner DNA helicase protein (WRN) plays a critical role in supporting c-Myc-driven S-phase, as depletion of WRN in c-Myc overexpressing cells increases DNA damage specifically at sites of DNA synthesis. This excess DNA damage activates a “replication stress” pathway involving ATR, CHK1, CHK2, and p53, leading to rapid senescence of WRN deficient c-Myc overexpressing cells. Indeed, depletion of p53 rescues this senescence response. We propose that WRN functions to repair abnormal replication structures caused by the acceleration of DNA replication by c-Myc. This work provides an additional mechanistic explanation for c-Myc-induced DNA damage and senescence, and reveals a vulnerability of c-Myc overexpressing cells that could potentially be exploited in cancer therapy.     

Downregulation of the Werner syndrome protein induces a metabolic shift that compromises redox homeostasis and limits proliferation of cancer cells

The Werner syndrome protein (WRN) is a nuclear protein required for cell growth and proliferation. Loss‐of‐function mutations in the Werner syndrome gene are associated with the premature onset of age‐related diseases. How loss of WRN limits cell proliferation and induces replicative senescence is poorly understood. Here, we show that WRN depletion leads to a striking metabolic shift that coordinately weakens the pathways that generate reducing equivalents for detoxification of reactive oxygen species and increases mitochondrial respiration. In cancer cells, this metabolic shift counteracts the Warburg effect, a defining characteristic of many malignant cells, resulting in altered redox balance and accumulation of oxidative DNA damage that inhibits cell proliferation and induces a senescence‐like phenotype. Consistent with these findings, supplementation with antioxidant rescues at least in part cell proliferation and decreases senescence in WRN‐knockdown cancer cells. These results demonstrate that WRN plays a critical role in cancer cell proliferation by contributing to the Warburg effect and preventing metabolic stress.     

Telomerase expression prevents replicative senescence but does not fully reset mRNA expression patterns in Werner syndrome cell strains.

Reduced replicative capacity is a consistent characteristic of cells derived from patients with Werner syndrome. This premature senescence is phenotypically similar to replicative senescence observed in normal cell strains and includes altered cell morphology and gene expression patterns. Telomeres shorten with in vitro passaging of both WRN and normal cell strains; however, the rate of shortening has been reported to be faster in WRN cell strains, and the length of telomeres in senescent WRN cells appears to be longer than that observed in normal strains, leading to the suggestion that senescence in WRN cell strains may not be exclusively associated with telomere effects. We report here that the telomere restriction fragment length in senescent WRN fibroblasts cultures is within the size range observed for normal fibroblasts strains and that the expression of a telomerase transgene in WRN cell strains results in lengthened telomeres and replicative immortalization, thus indicating that telomere effects are the predominant trigger of premature senescence in WRN cells. Microarray analyses showed that mRNA expression patterns induced in senescent WRN cells appeared similar to those in normal strains and that hTERT expression could prevent the induction of most of these genes. However, substantial differences in expression were seen in comparisons of early-passage and telomerase-immortalized derivative lines, indicating that telomerase expression does not prevent the phenotypic drift, or destabilized genotype, resulting from the WRN defect.     

Myc,Cdk2 and cellular senescence: Old players,new game

The aberrant activation of oncogenic pathways promotes tumor progression, but concomitantly elicits compensatory tumor-suppressive responses, such as apoptosis or senescence. For example, Ras induces senescence, while Myc generally triggers apoptosis. Myc is in fact viewed as an anti-senescence oncogene, as it is a potent inducer of cell proliferation and immortalization, bypasses growth-inhibitory signals, and cooperates with Ras in cellular transformation. Recent reports prompt re-evaluation of Myc-induced senescence, and of its role in tumor progression and therapy. We have shown that the cyclin-dependent kinase Cdk2, although redundant for cell cycle progression, has a unique role in suppressing a Myc-induced senescence program: Myc activation elicited expression of p16^INK4a and p21^Cip1, and caused senescence in cell lacking Cdk2, but not in Cdk2-proficient cells. Additional cellular activities have been identified that suppress Myc-induced senescence, including the Wrn helicase, Telomerase and Miz1. These senescence-suppressing activities were critical for tumor progression, as deficiency in Cdk2, telomerase or Miz1 reduced the onset of Myc-induced lymphoma in transgenic mice. Other gene products like p53, SUV39H1 or TGFß promoted senescence, which together with apoptosis contributed to tumor suppression. Paradoxically, Myc directly counteracted the very same senescence program that it potentially elicits, since it positively regulated Wrn, Telomerase and Cdk2 activity, and Cdk2 inhibition re-activated the latent senescence program in Myc expressing cells. Hence, while these molecules are instrumental to the oncogenic action of Myc, they may simultaneously constitute its Achille's heel for therapeutic development.     

Deficiency in the DNA repair protein ERCC1 triggers a link between senescence and apoptosis in human fibroblasts and mouse skin

ERCC1 (excision repair cross complementing‐group 1) is a mammalian endonuclease that incises the damaged strand of DNA during nucleotide excision repair and interstrand cross‐link repair. Ercc1^?/Δ mice, carrying one null and one hypomorphic Ercc1 allele, have been widely used to study aging due to accelerated aging phenotypes in numerous organs and their shortened lifespan. Ercc1^?/Δ mice display combined features of human progeroid and cancer‐prone syndromes. Although several studies report cellular senescence and apoptosis associated with the premature aging of Ercc1^?/Δ mice, the link between these two processes and their physiological relevance in the phenotypes of Ercc1^?/Δ mice are incompletely understood. Here, we show that ERCC1 depletion, both in cultured human fibroblasts and the skin of Ercc1^?/Δ mice, initially induces cellular senescence and, importantly, increased expression of several SASP (senescence‐associated secretory phenotype) factors. Cellular senescence induced by ERCC1 deficiency was dependent on activity of the p53 tumor‐suppressor protein. In turn, TNFα secreted by senescent cells induced apoptosis, not only in neighboring ERCC1‐deficient nonsenescent cells, but also cell autonomously in the senescent cells themselves. In addition, expression of the stem cell markers p63 and Lgr6 was significantly decreased in Ercc1^?/Δ mouse skin, where the apoptotic cells are localized, compared to age‐matched wild‐type skin, possibly due to the apoptosis of stem cells. These data suggest that ERCC1‐depleted cells become susceptible to apoptosis via TNFα secreted from neighboring senescent cells. We speculate that parts of the premature aging phenotypes and shortened health‐ or lifespan may be due to stem cell depletion through apoptosis promoted by senescent cells.     

Mutations in the WRN gene in mice accelerate mortality in a p53-null background

Werner's syndrome (WS) is a human disease with manifestations resembling premature aging. The gene defective in WS, WRN, encodes a DNA helicase. Here, we describe the generation of mice bearing a mutation that eliminates expression of the C terminus of the helicase domain of the WRN protein. Mutant mice are born at the expected Mendelian frequency and do not show any overt histological signs of accelerated senescence. These mice are capable of living beyond 2 years of age. Cells from these animals do not show elevated susceptibility to the genotoxins camptothecin or 4-NQO. However, mutant fibroblasts senesce approximately one passage earlier than controls. Importantly, WRN(-/-);p53(-/-) mice show an increased mortality rate relative to WRN(+/-);p53(-/-) animals. We consider possible models for the synergy between p53 and WRN mutations for the determination of life span.     

WRN protects against topo I but not topo II inhibitors by preventing DNA break formation

The Werner syndrome helicase/3′-exonuclease (WRN) is a major component of the DNA repair and replication machinery. To analyze whether WRN is involved in the repair of topoisomerase-induced DNA damage we utilized U2-OS cells, in which WRN is stably down-regulated (wrn-kd), and the corresponding wild-type cells (wrn-wt). We show that cells not expressing WRN are hypersensitive to the toxic effect of the topoisomerase I inhibitor topotecan, but not to the topoisomerase II inhibitor etoposide. This was shown by mass survival assays, colony formation and induction of apoptosis. Upon topotecan treatment WRN deficient cells showed enhanced DNA replication inhibition and S-phase arrest, whereas after treatment with etoposide they showed the same cell cycle response as the wild-type. A considerable difference between WRN and wild-type cells was observed for DNA single- and double-strand break formation in response to topotecan. Topotecan induced DNA single-strand breaks 6 h after treatment. In both wrn-wt and wrn-kd cells these breaks were repaired at similar kinetics. However, in wrn-kd but not wrn-wt cells they were converted into DNA double-strand breaks (DSBs) at high frequency, as shown by neutral comet assay and phosphorylation of H2AX. Our data provide evidence that WRN is involved in the repair of topoisomerase I, but not topoisomerase II-induced DNA damage, most likely via preventing the conversion of DNA single-strand breaks into DSBs during the resolution of stalled replication forks at topo I–DNA complexes. We suggest that the WRN status of tumor cells impacts anticancer therapy with topoisomerase I, but not topoisomerase II inhibitors.     

Telomere shortening exposes functions for the mouse Werner and Bloom syndrome genes

The Werner and Bloom syndromes are caused by loss-of-function mutations in WRN and BLM, respectively, which encode the RecQ family DNA helicases WRN and BLM, respectively. Persons with Werner syndrome displays premature aging of the skin, vasculature, reproductive system, and bone, and those with Bloom syndrome display more limited features of aging, including premature menopause; both syndromes involve genome instability and increased cancer. The proteins participate in recombinational repair of stalled replication forks or DNA breaks, but the precise functions of the proteins that prevent rapid aging are unknown. Accumulating evidence points to telomeres as targets of WRN and BLM, but the importance in vivo of the proteins in telomere biology has not been tested. We show that Wrn and Blm mutations each accentuate pathology in later-generation mice lacking the telomerase RNA template Terc, including acceleration of phenotypes characteristic of latest-generation Terc mutants. Furthermore, pathology not observed in Terc mutants but similar to that observed in Werner syndrome and Bloom syndrome, such as bone loss, was observed. The pathology was accompanied by enhanced telomere dysfunction, including end-to-end chromosome fusions and greater loss of telomere repeat DNA compared with Terc mutants. These findings indicate that telomere dysfunction may contribute to the pathogenesis of Werner syndrome and Bloom syndrome.     

Targeting Werner syndrome protein sensitizes U-2 OS osteosarcoma cells to selenium-induced DNA damage response and necrotic death

Mutations in the Werner syndrome protein (WRN), a caretaker of the genome, result in Werner syndrome, which is characterized by premature aging phenotypes and cancer predisposition. Methylseleninic acid (MSeA) can activate DNA damage responses and is a superior compound to suppress tumorigenesis in mouse models of cancer. To test the hypothesis that targeting WRN can potentiate selenium toxicity in cancer cells, isogenic WRN small hairpin RNA (shRNA) and control shRNA U-2 OS osteosarcoma cells were treated with MSeA for 2d, followed by recovery for up to 7d. WRN deficiency sensitized U-2 OS cells to MSeA-induced necrotic death. Co-treatment with the ataxia-telangiectasia mutated (ATM) kinase inhibitor KU55933 desensitized the control shRNA cells, but not WRN shRNA cells, to MSeA treatment. WRN did not affect MSeA-induced ATM phosphorylation on Ser-1981 or H2A.X phosphorylation on Ser-139, but promoted recovery from the MSeA-induced DNA damage. Taken together, WRN protects U-2 OS osteosarcoma cells against MSeA-induced cytotoxicity, suggesting that oxidative DNA repair pathway is a promising target for improving the efficacy of selenium on tumor suppression.     

Telomere instability in a human tumor cell line expressing a dominant-negative WRN protein

Werner Syndrome (WS) is an autosomal recessive disease characterized by premature aging and chromosome instability. The protein involved in WS, WRN, is a RecQ-type helicase that also has exonuclease activity. WRN has been demonstrated to bind to a variety of other proteins, including RPA, DNA-PKcs, and TRF2, suggesting that WRN is involved in DNA replication, repair, recombination, and telomere maintenance. In culture, WS cells show premature senescence, which can be overcome by transfection with an expression vector containing the gene for the catalytic subunit of telomerase. However, telomerase expression does not eliminate chromosome instability in WS cells, which led to the proposal that telomere loss is not the cause of the high rate of chromosome rearrangements in WS cells. In the present study, we have investigated how a WRN protein containing a dominant-negative mutation (K577M-WRN) influences the stability of telomeres in a human tumor cell line expressing telomerase. The results demonstrate an increased rate of telomere loss and chromosome fusion in cells expressing K577M-WRN. Expression of K577M-WRN results in reduced levels of telomerase activity, however, the absence of detectable changes in average telomere length demonstrates that WRN-associated telomere loss results from stochastic events involving complete telomere loss or loss of telomere capping function. Thus, telomere loss can contribute to chromosome instability in cells deficient in WRN regardless of the expression of telomerase activity.     

PLA2R1 promotes DNA damage and inhibits spontaneous tumor formation during aging

Although aging is a major risk factor for most types of cancers, it is barely studied in this context. The transmembrane protein PLA2R1 (phospholipase A2 receptor) promotes cellular senescence, which can inhibit oncogene-induced tumor initiation. Functions and mechanisms of action of PLA2R1 during aging are largely unknown. In this study, we observed that old Pla2r1 knockout mice were more prone to spontaneously develop a wide spectrum of tumors compared to control littermates. Consistently, these knockout mice displayed increased Parp1, a master regulator of DNA damage repair, and decreased DNA damage, correlating with large human dataset analysis. Forced PLA2R1 expression in normal human cells decreased PARP1 expression, induced DNA damage and subsequent senescence, while the constitutive expression of PARP1 rescued cells from these PLA2R1-induced effects. Mechanistically, PARP1 expression is repressed by a ROS (reactive oxygen species)-Rb-dependent mechanism upon PLA2R1 expression. In conclusion, our results suggest that PLA2R1 suppresses aging-induced tumors by repressing PARP1, via a ROS–Rb signaling axis, and inducing DNA damage and its tumor suppressive responses.Subject terms: Cancer, Mechanisms of disease, DNA damage and repair, Ageing     

Asymmetry of DNA replication fork progression in Werner's syndrome

Human aging is associated with accumulation of cells that have undergone replicative senescence. The rare premature aging Werner's syndrome (WS) provides a phenocopy of normal human aging and WS patient cells recapitulate the aging phenotype in culture as they rapidly lose the ability to proliferate or replicate their DNA. WS is associated with loss of functional WRN protein. Although the biochemical properties of WRN protein, which possesses both helicase and exonuclease activities, suggest an involvement in DNA metabolism, its action in cells is not clear. Here, we provide experimental evidence for a role of the WRN protein in DNA replication in normally proliferating cells. Most importantly, we demonstrate that in the absence of functional WRN protein, replication forks from origins of bidirectional replication fail to progress normally, resulting in marked asymmetry of bidirectional forks. We propose that WRN acts in normal DNA replication to prevent collapse of replication forks or to resolve DNA junctions at stalled replication forks, and that loss of this capacity may be a contributory factor in premature aging.     

Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers

Cells respond to genotoxic stress by activating the DNA damage response (DDR). When injury is severe or irreparable, cells induce apoptosis or cellular senescence to prevent transmission of the lesions to the daughter cells upon cell division. Resistance to apoptosis is a hallmark of cancer that challenges the efficacy of cancer therapy. In this work, the effects of ionizing radiation on apoptosis-resistant E1A + E1B transformed cells were investigated to ascertain whether the activation of cellular senescence could provide an alternative tumor suppressor mechanism. We show that irradiated cells arrest cell cycle at G₂/M phase and resume DNA replication in the absence of cell division followed by formation of giant polyploid cells. Permanent activation of DDR signaling due to impaired DNA repair results in the induction of cellular senescence in E1A + E1B cells. However, irradiated cells bypass senescence and restore the population by dividing cells, which have near normal size and ploidy and do not express senescence markers. Reversion of senescence and appearance of proliferating cells were associated with downregulation of mTOR, activation of autophagy, mitigation of DDR signaling, and expression of stem cell markers.