UBTD1 induces cellular senescence through an UBTD1–Mdm2/p53 positive feedback loop

The tumour suppressor p53 plays an important role in tumourigenesis. Besides inducing apoptosis, it regulates cellular senescence, which constitutes an important barrier to tumourigenesis. The mechanism of regulation of cellular senescence by p53 and its downstream pathway are poorly understood. Here, we report that the ubiquitin domain‐containing 1 (UBTD1) gene, a new downstream target of p53, induces cellular senescence and acts as a novel tumour suppressor by a mechanism that depends on p53. Expression of UBTD1 increased upon cellular senescence induced by serial passageing of cultures, as well as by exposure to DNA‐damageing drugs that induce premature senescence. Over‐expression of UBTD1 induces senescence in human fibroblasts and cancer cells and attenuation of the transformed phenotype in cancer cells. UBTD1 is down‐regulated in gastric and colorectal cancer tissues, and its lower expression correlates with a more aggressive phenotype and worse prognosis. Multivariate analysis revealed that UBTD1 expression was an independent prognostic factor for gastric cancer patients. Furthermore, UBTD1 increased the stability of p53 protein, by promoting the degradation of Mdm2 protein. Importantly, UBTD1 and p53 function mutually depend on each other in regulating cellular senescence and proliferation. Thus, our data suggest that, upon DNA damage, p53 induction by UBTD1 creates a positive feedback mechanism to further increase p53 expression. Our results establish UBTD1 as a regulator of cellular senescence that mediates p53 function, and provide insights into the mechanism of Mdm2 inhibition that impacts p53 dynamics during cellular senescence and tumourigenesis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

p53依赖的X-连锁凋亡抑制蛋白调节与卵巢癌顺铂耐药的关系

目的本研究旨在探讨X-连锁凋亡抑制蛋白（XIAP）在卵巢癌顺铂耐药中的作用。方法应用RT—PCR、Western Blot、流式细胞仪等检测卵巢癌顺铂敏感细胞株0V2008、A2780s和耐药株C13＊、A2780cp中XIAP表达和卵巢癌细胞的凋亡率,并将反义XIAP寡核苷酸（XIAPAs—ODN）、正义XIAP寡核苷酸（XIAPs—ODN）和随机对照链导入顺铂耐药细胞C13＊（p53野生型）和A2780cp（p53突变型）中,比较转染前、后耐药细胞Caspase-3活性和顺铂耐药性的改变。结果卵巢癌顺铂敏感细胞和耐药细胞中XIAP在mRNA水平的表达无明显差异（P〉0．05）。顺铂可以引起OV2008和A2780s中XIAP蛋白表达明显下降（P〈0．05）,而对C13＊和A2780cp的XIAP蛋白含量无明显影响（P〉0．05）。转染XIAPAs—ODN可降调p53野生型耐药细胞C13＊中XIAP的表达,并显著增加Caspase-3活性和对顺铂敏感性（P〈0．05）,而XIAP As—ODN对p53突变型耐药细胞A2780cp无此作用。结论卵巢癌细胞对顺铂产生耐药可能与XIAP蛋白相对高表达有关,反义XIAP可在一定程度上逆转卵巢癌顺铂耐药,该作用与卵巢癌细胞的p53表型有关。     

A chromatin-independent role of Polycomb-like 1 to stabilize p53 and promote cellular quiescence

Polycomb-like proteins 1–3 (PCL1–3) are substoichiometric components of the Polycomb-repressive complex 2 (PRC2) that are essential for association of the complex with chromatin. However, it remains unclear why three proteins with such apparent functional redundancy exist in mammals. Here we characterize their divergent roles in both positively and negatively regulating cellular proliferation. We show that while PCL2 and PCL3 are E2F-regulated genes expressed in proliferating cells, PCL1 is a p53 target gene predominantly expressed in quiescent cells. Ectopic expression of any PCL protein recruits PRC2 to repress the INK4A gene; however, only PCL2 and PCL3 confer an INK4A-dependent proliferative advantage. Remarkably, PCL1 has evolved a PRC2- and chromatin-independent function to negatively regulate proliferation. We show that PCL1 binds to and stabilizes p53 to induce cellular quiescence. Moreover, depletion of PCL1 phenocopies the defects in maintaining cellular quiescence associated with p53 loss. This newly evolved function is achieved by the binding of the PCL1 N-terminal PHD domain to the C-terminal domain of p53 through two unique serine residues, which were acquired during recent vertebrate evolution. This study illustrates the functional bifurcation of PCL proteins, which act in both a chromatin-dependent and a chromatin-independent manner to regulate the INK4A and p53 pathways.     

乳腺癌中p53基因丢失与p53 mRNA高表达相关性研究

为探讨乳腺癌组织中ｐ５３基因丢失与ｐ５３ ｍＲＮＡ表达的相关性，应用Ｓｏｕｔｈｅｒｎ杂交及反转录－定量ＰＣＲ技术对４７例乳腺癌组织进行了检测。结果发现：ｐ５３基因杂合性缺失率为３４．０％；在乳腺正常腺体中有中度ｐ５３ＲＮＡ表达，面 肿瘤组织中有４０．０％，患者伴ｐ５３ ｍＲＮＡ高表达。ｐ５３基因与ｐ５３ ｍＲＮＡ高表达之间呈显著性相关。     

Glycogen synthase kinase 3 promotes p53 mRNA translation via phosphorylation of RNPC1

The RNPC1 RNA-binding protein, also called Rbm38, is a target of p53 and a repressor of p53 mRNA translation. Thus, the p53–RNPC1 loop is critical for modulating p53 tumor suppression, but it is not clear how the loop is regulated. Here, we showed that RNPC1 is phosphorylated at Ser195 by glycogen synthase kinase 3 (GSK3). We also showed that GSK3 promotes p53 mRNA translation through phosphorylation of RNPC1. Interestingly, we found that the phosphor-mimetic mutant S195D and the deletion mutant Δ189–204, which lacks the GSK3 phosphorylation site, are unable to repress p53 mRNA translation due to loss of interaction with eukaryotic translation factor eIF4E on p53 mRNA. Additionally, we found that phosphorylated RNPC1, RNPC1-S195D, and RNPC1(Δ189–204) promote p53 mRNA translation through interaction with eukaryotic translation factor eIF4G, which then facilitates the assembly of the eIF4F complex on p53 mRNA. Furthermore, we showed that upon inhibition of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway, GSK3 is activated, leading to increased RNPC1 phosphorylation and increased p53 expression in a RNPC1-dependent manner. Together, we postulate that the p53–RNPC1 loop can be explored to increase or decrease p53 activity for cancer therapy.     

Co-expression of p53 and MDM2 in human atherosclerosis: implications for the regulation of cellularity of atherosclerotic lesions

Atherosclerosis is a fibroproliferative disease of the arterial intima. It was recently found that wild-type p53 (wt p53) accumulates in human atherosclerotic tissue. Wt p53 is a cell cycle regulator involved in DNA repair, DNA synthesis, cell differentiation, and apoptosis and might therefore make an important contribution to the cellularity of atherosclerotic plaques. The product of the MDM2 gene is a nuclear protein which forms a complex with p53, thereby inhibiting the negative regulatory effects of wt p53 on cell cycle progression. In order to address a potential role of the interaction of p53 with MDM2 for the regulation of cellularity in atherosclerotic tissue, 22 carotid atheromatous plaques from patients undergoing endarterectomy were studied to determine the presence of p53 immunoreactivity (IR), MDM2 IR, cell proliferation as evidenced by MIB1/Ki-67 IR and DNA fragmentation by in situterminal transferase-mediated dUTP 3′ end labelling (TUNEL), as a marker for apoptosis. p53 IR localized to areas with evidence of chronic inflammation (22/22) and was observed in virtually all cell types in 68·79±7·51 per cent of the nuclei. p53 staining in the control tissue from human internal mammary arteries was present in 0·2±0·29 per cent of the cells (P≤0·002). MDM2 IR was present in all cases (22/22) in macrophages and smooth muscle cells (SMCs) in 60·53±8·32 per cent of the nuclei (controls: 0·8±0·65 per cent, P≤0·002) and co-localized with p53 IR as shown by examination of adjacent sections and by double immunofluorescence labelling. Importantly, co-immunoprecipitation and western blot analysis revealed that p53 and MDM2 were physically associated, indicating that MDM2–p53 complex formation takes place in vivoin human atherosclerotic tissue. Positive TUNEL staining and MIB1/Ki-67 IR present in 3·01±1·27 per cent of the nuclei (controls: 0 per cent, P≤0·002) localized to the same plaque compartments as p53 IR and MDM2 IR. Thus, the fate of cells with p53 accumulation may depend on the interaction and the stoichiometry of the p53 and MDM2 proteins. Cells were indeed found with strong p53 accumulation and nuclear morphology typical for apoptosis and there were a few MIB1/Ki-67-positive cells with co-expression of MDM2, indicating a possible role for MDM2 in reversing the negative regulatory effects of p53 for cell cycle progression. The nuclear co-localization of p53 IR with MDM2 IR and the co-immunoprecipitation assay indicate the presence of p53–MDM2 complex formation in vivo in human atherosclerotic tissue. The destiny of individual p53 and MDM2-co-expressing cells either to undergo p53-dependent apoptosis or to re-enter the cycle of cell proliferation may depend on the relative ratios of the two proteins. p53 and MDM2 may therefore play an important role in regulating cellularity and inflammatory activity in human atherosclerotic plaques. © 1998 John Wiley & Sons, Ltd.     

胃癌组织中p21、p27、p53和Rb的表达及临床意义

目的 探讨p21、p27、p53和Rb蛋白表达在胃癌发生、发展中的作用.方法 应用免疫组化EnVision法检测111例胃癌,38例不典型增生、47例肠上皮化生、25例慢性萎缩性胃炎和53例正常胃黏膜组织中p21、p27、p53和Rb蛋白的表达.结果 p21和p27蛋白表达水平在不典型增生组织最高,其次是胃癌组织,与正常胃黏膜组织相比差异均有显著性(P<0.001).在胃癌组织中p21、p27、p53和Rb蛋白表达均高于正常胃黏膜组织(P均＜0.01).p21、p27蛋白表达与胃癌的类型和分化相关(P均＜0.01),p53表达水平与患者的年龄、性别、胃癌的类型和患者的生存率相关.女性患者Rb蛋白表达明显高于男性,差异有显著性(P<0.05).p21、p27、p53和Rb蛋白表达水平之间均呈正相关.结论 p21、p27、p53和Rb蛋白表达可以作为胃癌的辅助诊断指标,四种蛋白在判断胃癌的生物学行为上具有协同作用.     

p66shc, but not p53, is involved in early arrest of in vitro-produced bovine embryos

High embryo loss occurs in the first week of bovine embryo development, with a high percentage of embryonic arrest. We hypothesized that arrested embryos enter a 'senescence-like state' and that both the cell cycle regulatory protein p53 and the stress-related protein p66(shc), which are involved in the onset of senescence in somatic cells, are responsible for this early embryonic arrest. In our in vitro production system, 13.5 +/- 0.5% of embryos arrest at the 2-4-cell stage. First cleavage occurs between 26 and 48 h post insemination (hpi), with early cleaving embryos showing only 0.6 +/- 0.3% arrest, with later cleaving embryos exhibiting up to 14.2 +/- 0.9% arrest. We compared 2-4-cell embryos collected at 28 hpi with those arrested at the 2-4-cell stage collected at day 8 post insemination. Quantification by real-time PCR and by semi-quantitative immunofluorescence showed significantly higher p66(shc) mRNA and protein levels in both arrested and late cleaving embryos versus 28 hpi embryos. By comparison, no significant changes in p53 mRNA, protein and phosphorylation levels were detected. Taken together, these results demonstrate that embryonic developmental potential is related to the time of first cleavage and that p66(shc), but not p53, is up-regulated in early arrested in vitro-produced bovine embryos.     

Altered senescence, apoptosis, and DNA damage response in a mutant p53 model of accelerated aging

The tumor suppressors p16^INK4a and p53 have been implicated as contributors to age-associated stem cell decline. Key functions of p53 are the induction of cell cycle arrest, senescence, or apoptosis in response to DNA damage. Here, we examine senescence, apoptosis, and DNA damage responses in a mouse accelerated aging model that exhibits increased p53 activity, the p53^+/m mouse. Aged tissues of p53^+/m mice display higher percentages of senescent cells (as determined by senescence-associated β-galactosidase staining and p16^INK4a and p21 accumulation) compared to aged tissues from p53^+/+ mice. Surprisingly, despite having enhanced p53 activity, p53^+/m lymphoid tissues exhibit reduced apoptotic activity in response to ionizing radiation compared to p53^+/+ tissues. Ionizing radiation treatment of p53^+/m tissues also induces higher and prolonged levels of senescence markers p16^INK4a and p21, suggesting that in p53^+/m tissues the p53 stress response is enhanced and is shifted away from apoptosis toward senescence. One potential mechanism for accelerated aging in the p53^+/m mouse is a failure to remove damaged or dysfunctional cells (including stem and progenitor cells) through apoptosis. The increased accumulation of dysfunctional and senescent cells may contribute to reduced tissue regeneration, tissue atrophy, and some of the accelerated aging phenotypes in p53^+/m mice.     

粘液表皮样癌p16、p53、nm23蛋白及其mRNA表达的临床病理意义

目的：了解粘液表皮样癌中p16、p53、nm23蛋白及其mRNA表达的情况,探讨它们与粘液表皮样癌的病理形态学特征的关系。方法：用HE和组织化学方法对41例粘液表皮样癌诊断,并按WHO标准分为高分化、低分化两组,用免疫组织化学SABC法和原位杂交方法,分别检测41例粘液表皮样癌p16、p53、nm23蛋白及mRNA,用统计学方法分析它们之间的关系。结果：粘液表皮样癌中低分化组,高分化组的p16蛋白失表达率分别是62．5％（15／24）和29．4（5／17）,P＝0．037;p53蛋白的阳性率分别为70．8％（17／24）和23．5％（4／17）,P＝0．003;nm23蛋白的阳性率分别为37．5％（9／24）和64．7（11／17）P＝0．086。p16、p53、nm23mRNA的表达与病理分级无明显相关。结论：p16、p53、nm23mRNA表达与病理分级无明显相关。     

Cell fusion induced by ERVWE1 or measles virus causes cellular senescence

Cellular senescence limits proliferation of potentially detrimental cells, preventing tumorigenesis and restricting tissue damage. However, the function of senescence in nonpathological conditions is unknown. We found that the human placental syncytiotrophoblast exhibited the phenotype and expressed molecular markers of cellular senescence. During embryonic development, ERVWE1-mediated cell fusion results in formation of the syncytiotrophoblast, which serves as the maternal/fetal interface at the placenta. Expression of ERVWE1 caused cell fusion in normal and cancer cells, leading to formation of hyperploid syncytia exhibiting features of cellular senescence. Infection by the measles virus, which leads to cell fusion, also induced cellular senescence in normal and cancer cells. The fused cells activated the main molecular pathways of senescence, the p53- and p16–pRb-dependent pathways; the senescence-associated secretory phenotype; and immune surveillance-related proteins. Thus, fusion-induced senescence might be needed for proper syncytiotrophoblast function during embryonic development, and reuse of this senescence program later in life protects against pathological expression of endogenous fusogens and fusogenic viral infections.     

p53、p63及p73在皮肤血管瘤组织中的表达

目的　为探讨p5 3p6 3及p73蛋白在皮肤血管瘤组织中的表达及意义。方法　采用免疫组织化学S P法和图像分析技术检测 4 0例血管瘤组织和 2 0例正常皮肤组织中p5 3、p6 3及p73基因的表达。结果　在增生期血管瘤、退化期血管瘤和正常皮肤组之间 ,p73蛋白免疫组化阳性反应颗粒的平均光密度分别为 6 4 0 8± 2 15 1、1 0 73± 0 5 16和 0 95 3± 0 12 0。p6 3蛋白免疫组化阳性反应颗粒的平均光密度分别为 8 2 71± 1 95 3、0 92 3± 0 191和 0 92 0± 0 187。p5 3蛋白免疫组化阳性反应颗粒的平均光密度分别为 7 2 4 0± 1 874、0 934± 0 187和 0 92 3± 0 16 5。增生期血管瘤与退化期血管瘤、正常皮肤组分别组比 ,p73、p6 3及p5 3阳性表达的差异均有高度显著性差异 (P <0 0 0 1) ,消退期血管瘤与正常皮肤组之间 ,p73、p6 3及p5 3阳性表达差异无显著性 (P >0 0 5 )。结论　在增生期血管瘤组织中p73、p6 3及p5 3呈高表达 ,促进了内皮细胞的增殖 ,是导致血管瘤发生、发展的主要因素