Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency

Expression of p16(Ink4a) and p19(Arf) increases with age in both rodent and human tissues. However, whether these tumour suppressors are effectors of ageing remains unclear, mainly because knockout mice lacking p16(Ink4a) or p19(Arf) die early of tumours. Here, we show that skeletal muscle and fat, two tissues that develop early ageing-associated phenotypes in response to BubR1 insufficiency, have high levels of p16(Ink4a) and p19(Arf). Inactivation of p16(Ink4a) in BubR1-insufficient mice attenuates both cellular senescence and premature ageing in these tissues. Conversely, p19(Arf) inactivation exacerbates senescence and ageing in BubR1 mutant mice. Thus, we identify BubR1 insufficiency as a trigger for activation of the Cdkn2a locus in certain mouse tissues, and demonstrate that p16(Ink4a) is an effector and p19(Arf) an attenuator of senescence and ageing in these tissues.     

p16(Ink4a) interferes with Abelson virus transformation by enhancing apoptosis

Pre-B-cell transformation by Abelson virus (Ab-MLV) is a multistep process in which primary transformants are stimulated to proliferate but subsequently undergo crisis, a period of erratic growth marked by high levels of apoptosis. Inactivation of the p53 tumor suppressor pathway is an important step in this process and can be accomplished by mutation of p53 or down-modulation of p19(Arf), a p53 regulatory protein. Consistent with these data, pre-B cells from either p53 or Ink4a/Arf null mice bypass crisis. However, the Ink4a/Arf locus encodes both p19(Arf) and a second tumor suppressor, p16(Ink4a), that blocks cell cycle progression by inhibiting Cdk4/6. To determine if p16(Ink4a) plays a role in Ab-MLV transformation, primary transformants derived from Arf(-/-) and p16(Ink4a(-/-)) mice were compared. A fraction of those derived from Arf(-/-) animals underwent crisis, and even though all p16(Ink4a(-/-)) primary transformants experienced crisis, these cells became established more readily than cells derived from +/+ mice. Analyses of Ink4a/Arf(-/-) cells infected with a virus that expresses both v-Abl and p16(Ink4a) revealed that p16(Ink4a) expression does not alter cell cycle profiles but does increase the level of apoptosis in primary transformants. These results indicate that both products of the Ink4a/Arf locus influence Ab-MLV transformation and reveal that in addition to its well-recognized effects on the cell cycle, p16(Ink4a) can suppress transformation by inducing apoptosis.     

Reactive oxygen-induced carcinogenesis causes hypermethylation of p16(Ink4a) and activation of MAP kinase

BACKGROUND: Implantation of foreign materials into mice and humans has been noted to result in the appearance of soft tissue sarcomas at the site of implantation. These materials include metal replacement joints and Dacron vascular grafts. In addition, occupational exposure to nickel has been shown to result in an increased risk of carcinogenesis. The molecular mechanisms of foreign body-induced carcinogenesis are not fully understood. MATERIALS AND METHODS: In order to gain insight into these mechanisms, we implanted nickel sulfide into wild type C57BL/6 mice as well as a mouse heterozygous for the tumor suppressor gene, p53. Malignant fibrous histiocytomas arose in all mice, and we have characterized the profile of tumor suppressor genes and signal transduction pathways altered in these cells. RESULTS: All tumors demonstrated hypermethylation of the tumor suppressor gene p16, as well as activation of the mitogen activated protein kinase (MAP kinase) signaling pathway. This knowledge may be beneficial in the prevention and treatment of tumors caused by foreign body implantation. CONCLUSIONS: Oxidative stress induced by nickel sulfide appears to cause loss of p16 and activation of MAP kinase signaling. These findings support the hypothesis of synergistic interactions between MAP kinase activation and p16 loss in carcinogenesis.     

Anti-aging activity of the Ink4/Arf locus

The proteins encoded by the Ink4/Arf locus, p16^Ink4a, p19^Arf and p15^Ink4b are major tumour suppressors that oppose aberrant mitogenic signals. The expression levels of the locus are progressively increased during aging and genome-wide association studies have linked the locus to a number of aging-associated diseases and frailty in humans. However, direct measurement of the global impact of the Ink4/Arf locus on organismal aging and longevity was lacking. In this work, we have examined the fertility, cancer susceptibility, aging and longevity of mice genetically modified to carry one ( Ink4/Arf -tg) or two ( Ink4/Arf -tg/tg) intact additional copies of the locus. First, increased gene dosage of Ink4/Arf impairs the production of male germ cells, and in the case of Ink4/Arf -tg/tg mice results in a Sertoli cell-only-like syndrome and a complete absence of sperm. Regarding cancer, there is a lower incidence of aging-associated cancer proportional to the Ink4/Arf gene dosage. Interestingly, increased Ink4/Arf gene dosage resulted in lower scores in aging markers and in extended median longevity. The increased survival was also observed in cancer-free mice indicating that cancer protection and delayed aging are separable activities of the Ink4/Arf locus. In contrast to these results, mice carrying one or two additional copies of the p53 gene ( p53 -tg and p53 -tg/tg) had a normal longevity despite their increased cancer protection. We conclude that the Ink4/Arf locus has a global anti-aging effect, probably by favouring quiescence and preventing unnecessary proliferation.     

Real-time in vivo imaging of p16Ink4a reveals cross talk with p53

Expression of the p16^Ink4a tumor suppressor gene, a sensor of oncogenic stress, is up-regulated by a variety of potentially oncogenic stimuli in cultured primary cells. However, because p16^Ink4a expression is also induced by tissue culture stress, physiological mechanisms regulating p16^Ink4a expression remain unclear. To eliminate any potential problems arising from tissue culture–imposed stress, we used bioluminescence imaging for noninvasive and real-time analysis of p16^Ink4a expression under various physiological conditions in living mice. In this study, we show that oncogenic insults such as ras activation provoke epigenetic derepression of p16^Ink4a expression through reduction of DNMT1 (DNA methyl transferase 1) levels as a DNA damage response in vivo. This pathway is accelerated in the absence of p53, indicating that p53 normally holds the p16^Ink4a response in check. These results unveil a backup tumor suppressor role for p16^Ink4a in the event of p53 inactivation, expanding our understanding of how p16^Ink4a expression is regulated in vivo.     

p19Ink4d Is a Tumor Suppressor and Controls Pituitary Anterior Lobe Cell Proliferation

Pituitary tumors develop in about one-quarter of the population, and most arise from the anterior lobe (AL). The pituitary gland is particularly sensitive to genetic alteration of genes involved in the cyclin-dependent kinase (CDK) inhibitor (CKI)–CDK-retinoblastoma protein (Rb) pathway. Mice heterozygous for the Rb mutation develop pituitary tumors, with about 20% arising from the AL. Perplexingly, none of the CKI-deficient mice reported thus far develop pituitary AL tumors. In this study, we show that deletion of p19^Ink4d (p19), a CKI gene, in mice results in spontaneous development of tumors in multiple organs and tissues. Specifically, more than one-half of the mutant mice developed pituitary hyperplasia or tumors predominantly in the AL. Tumor development is associated with increased cell proliferation and enhanced activity of Cdk4 and Cdk6 and phosphorylation of Rb protein. Though Cdk4 is indispensable for postnatal pituitary cell proliferation, it is not required for the hyperproliferative pituitary phenotype caused by p19 loss. Loss of p19 phosphorylates Rb in Cdk4^−/− pituitary AL cells and mouse embryonic fibroblasts (MEFs) and rescues their proliferation defects, at least partially, through the activation of Cdk6. These results provide the first genetic evidence that p19 is a tumor suppressor and the major CKI gene that controls pituitary AL cell proliferation.     

Rescue of key features of the p63‐null epithelial phenotype by inactivation of Ink4a and Arf

Mice lacking p63 cannot form skin, exhibit craniofacial and skeletal defects, and die soon after birth. The p63 gene regulates a complex network of target genes, and disruption of p63 has been shown to affect the maintenance of epithelial stem cells, the differentiation of keratinocytes, and the preservation of the adhesive properties of stratified epithelium. Here, we show that inactivation of p63 in mice is accompanied by aberrantly increased expression of the Ink4a and Arf tumour suppressor genes. In turn, anomalies of the p63‐null mouse affecting the skin and skeleton are partially ameliorated in mice lacking either Ink4a or Arf. Rescue of epithelialization is accompanied by restoration of keratinocyte proliferative capacity both in vivo and in vitro and by expression of markers of squamous differentiation. Thus, in the absence of p63, abnormal upregulation of Ink4a and Arf is incompatible with skin development.     

Detection of methylation of human p16(Ink4a) gene 5'-CpG islands by electrochemical method coupled with linker-PCR

Aberrant DNA methylation of the CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or the detection of recurrence. p16, an inhibitor of the cyclin D-dependent protein kinases, is a classical tumor suppressor gene, and its inactivation is closely associated with carcinogenesis. p16 hypermethylation could be detected in each stage, which is consistent with the finding that aberrant methylation of p16 is a very early event in carcinogenesis. We have developed an electrochemical procedure for detecting DNA methylation of the human p16(Ink4a) gene. The procedure is based on the coupling of DNA electrochemical sensors with linker-PCR- amplified DNA from human gastric tumor tissue and whole blood cells of healthy human. The synthesized oligonucleotide was immobilized on the modified gold electrode to fabricate a DNA biosensor. The hybridization reaction on the electrode surface was monitored by cyclic voltammogram (CV) and square wave voltammogram (SWV), using [Co(phen)(3)](ClO(4))(3) as a redox indicator. Methylation status of human p16(Ink4a) gene was detected and the results were validated by bisulfite DNA sequencing. A good reproducibility was observed in several parallel experiments. The coupling of DNA electrochemical sensors with PCR allowed quick detection and have the potential of the quantitative evaluation of the methylation status of the human p16(Ink4a) gene.     

p16(INK4a) translation suppressed by miR-24

Background

Expression of the tumor suppressor p16^INK4a increases during aging and replicative senescence.

Methodology/Principal Findings

Here, we report that the microRNA miR-24 suppresses p16 expression in human diploid fibroblasts and cervical carcinoma cells. Increased p16 expression with replicative senescence was associated with decreased levels of miR-24, a microRNA that was predicted to associate with the p16 mRNA coding and 3′-untranslated regions. Ectopic miR-24 overexpression reduced p16 protein but not p16 mRNA levels. Conversely, introduction of antisense (AS)-miR-24 blocked miR-24 expression and markedly enhanced p16 protein levels, p16 translation, and the production of EGFP-p16 reporter bearing the miR-24 target recognition sites.

Conclusions/Significance

Together, our results suggest that miR-24 represses the initiation and elongation phases of p16 translation.     

Nucleophosmin is required for DNA integrity and p19Arf protein stability

Nucleophosmin (NPM) is a nucleolar phosphoprotein that binds the tumor suppressors p53 and p19(Arf) and is thought to be indispensable for ribogenesis, cell proliferation, and survival after DNA damage. The NPM gene is the most frequent target of genetic alterations in leukemias and lymphomas, though its role in tumorigenesis is unknown. We report here the first characterization of a mouse NPM knockout strain. Lack of NPM expression results in accumulation of DNA damage, activation of p53, widespread apoptosis, and mid-stage embryonic lethality. Fibroblasts explanted from null embryos fail to grow and rapidly acquire a senescent phenotype. Transfer of the NPM mutation into a p53-null background rescued apoptosis in vivo and fibroblast proliferation in vitro. Cells null for both p53 and NPM grow faster than control cells and are more susceptible to transformation by activated oncogenes, such as mutated Ras or overexpressed Myc. In the absence of NPM, Arf protein is excluded from nucleoli and is markedly less stable. Our data demonstrate that NPM regulates DNA integrity and, through Arf, inhibits cell proliferation and are consistent with a putative tumor-suppressive function of NPM.     

The induction of H3K9 methylation by PIWIL4 at the p16Ink4a locus

The field of epigenetics has made progress by the identification of the small RNA-mediated epigenetic modification. However, little is known about the key proteins. Here, we report that the human PIWI-like family is a candidate protein that is involved in the pathway responsible for chromatin remodeling. The PIWI-like family proteins, expressed as the Flag-fusion proteins, formed a bulky body and localized to the nuclear periphery. Transient transfection of PIWI-like 4 (PIWIL4), only member of the PIWI-like family that was ubiquitously expressed in human tissues, induced histone H3 lysine 9 methylation at the p16(Ink4a) (CDKN2A) locus. The elevated level of histone methylation resulted in the downregulation of the p16(Ink4a) gene. These results suggest PIWIL4 plays important roles in the chromatin-modifying pathway in human somatic cells.     

小鼠p16~(INK4a)基因位点的结构和功能研究

p1 6INK4a基因的失活与多种肿瘤的发生和发展有联系。通过筛选小鼠基因组文库 ,获得长度为 1 4.5kb的p1 6INK4a基因组DNA片段。对上述 1 4.5kbDNA测序后进行生物信息学分析表明 :该片段包含 3个外显子 ,编码 1个由 1 68个氨基酸残基组成的多肽 ,其相对分子质量的理论计算值为 1 7941 ,有 7个可能的磷酸化位点 ,说明p1 6INK4a蛋白的功能可能受到磷酸化的调控。该DNA片段的非编码区分布着大量短散布元件、长散布元件和简单重复序列 ,这样的结构为转座和同源重组提供了结构基础 ,提示了部分肿瘤细胞中p1 6INK4a基因缺失的可能原因。对第一外显子序列与已发表的相应序列比较发现其DNA序列和所编码的多肽存在多态性     

Murine mesenchymal cells that express elevated levels of the CDK inhibitor p16(Ink4a) in vivo are not necessarily senescent

Age-related health decline has been attributed to the accumulation of senescent cells recognized in vivo by p16(Ink4a) expression. The pharmacological elimination of p16(Ink4a)-positive cells from the tissues of mice was shown to extend a healthy lifespan. Here, we describe a population of mesenchymal cells isolated from mice that are highly p16(INK4a)-positive are proficient in proliferation but lack other properties of cellular senescence. These data, along with earlier reports on p16(Ink4a)-positive macrophages, indicate that p16(Ink4a)-positive and senescent cell populations only partially intersect, therefore, extending the list of potential cellular targets for anti- aging therapies.     

Ink4a/Arf regulation by let-7b and Hmga2: a genetic pathway governing stem cell aging

Stem cell self-renewal capacity declines with age. In a recent issue of Cell, Nishino and colleagues (2008) show that Hmga2 maintains neural stem cell (NSC) function in young mice through repression of the Ink4a/Arf locus; in contrast, during aging, elevated let-7b blocks Hmga2 and contributes to declining NSC function.     

C/EBPbeta represses p53 to promote cell survival downstream of DNA damage independent of oncogenic Ras and p19(Arf)

CCAAT/enhancer-binding protein-beta (C/EBPbeta) is a mediator of cell survival and tumorigenesis. When C/EBPbeta(-/-) mice are treated with carcinogens that produce oncogenic Ras mutations in keratinocytes, they respond with abnormally elevated keratinocyte apoptosis and a block in skin tumorigenesis. Although this aberrant carcinogen-induced apoptosis results from abnormal upregulation of p53, it is not known whether upregulated p53 results from oncogenic Ras and its ability to induce p19(Arf) and/or activate DNA-damage response pathways or from direct carcinogen-induced DNA damage. We report that p19(Arf) is dramatically elevated in C/EBPbeta(-/-) epidermis and that C/EBPbeta represses a p19(Arf) promoter reporter. To determine whether p19(Arf) is responsible for the proapoptotic phenotype in C/EBPbeta(-/-) mice, C/EBPbeta(-/-);p19(Arf-/-) mice were generated. C/EBPbeta(-/-);p19(Arf-/-) mice responded to carcinogen treatment with increased p53 and apoptosis, indicating p19(Arf) is not essential. To ascertain whether oncogenic Ras activation induces aberrant p53 and apoptosis in C/EBPbeta(-/-) epidermis, we generated K14-ER:Ras;C/EBPbeta(-/-) mice. Oncogenic Ras activation induced by 4-hydroxytamoxifen did not produce increased p53 or apoptosis. Finally, when C/EBPbeta(-/-) mice were treated with differing types of DNA-damaging agents, including alkylating chemotherapeutic agents, they displayed aberrant levels of p53 and apoptosis. These results indicate that C/EBPbeta represses p53 to promote cell survival downstream of DNA damage and suggest that inhibition of C/EBPbeta may be a target for cancer cotherapy to increase the efficacy of alkylating chemotherapeutic agents.     

TRADD contributes to tumour suppression by regulating ULF-dependent p19Arf ubiquitylation

Tumour necrosis factor receptor (TNFR)-associated death domain (TRADD) protein is a central adaptor in the TNFR1 signalling complex that mediates both cell death and inflammatory signals. Here, we report that Tradd deficiency in mice accelerated tumour formation in a chemical-induced carcinogenesis model independently of TNFR1 signalling. In vitro, primary cells lacking TRADD were less susceptible to HRas-induced senescence and showed a reduced level of accumulation of the p19(Arf) tumour suppressor protein. Our data indicate that TRADD shuttles dynamically from the cytoplasm into the nucleus to modulate the interaction between p19(Arf) and its E3 ubiquitin ligase ULF, thereby promoting p19(Arf) protein stability and tumour suppression. These results reveal a previously unknown tumour-suppressive role for nuclear TRADD, augmenting its long-established cytoplasmic functions in inflammatory and immune signalling cascades. Our findings also make an important contribution to the rapidly expanding field of p19(Arf) post-translational regulation.     

Heterogeneous activation of p19Arf in pulmonary artery smooth muscle cells

p19(ARF) is a tumor suppressor that leads to cell cycle arrest or apoptosis by stabilizing p53. p19(ARF) is not critical for cell cycle regulation under normal conditions, but loss of p19(ARF) is seen in many human cancers, and a murine p19(Arf) knockout model leads to malignant proliferation and tumor formation; its role in controlling nonmalignant proliferation is less defined. To examine this question, pulmonary artery smooth muscle cells (PASMC) were expanded in culture from a transgenic mouse in which the coding sequence of the p19(Arf) gene was replaced with a cDNA encoding green fluorescent protein (GFP), leaving the promoter intact. During the first 10 days in culture, wild-type, heterozygous, and knockout PASMC grew similarly, but, by day 14, p19(Arf)-deficient PASMC proliferated faster than p19(Arf) heterozygous or wild-type cells; reexpression of p19(Arf) prevented the increased proliferation. This time course correlated with activation of the p19(Arf) promoter, as indicated by the appearance of GFP positivity in p19(Arf)-deficient PASMC. By day 42, ～80% of p19(Arf)-deficient cells were GFP-positive. When GFP-positive, p19(Arf)-deficient cells were sorted and subcultured separately, they remained GFP-positive, indicating that once cells had activated the p19(Arf) promoter, the promoter remained active in those and all subsequent daughter cells. In contrast, GFP-negative p19(Arf)-deficient cells gave rise to a combination of GFP-positive and -negative daughter cells over time. These results suggest that a subpopulation of PASMC are resistant to the signals that activate the p19(Arf) promoter, an event that would normally target these cells for arrest or cell death.