Polycomb CBX7 has a unifying role in cellular lifespan

In contrast to cancer cells and embryonic stem cells, the lifespan of primary human cells is finite. After a defined number of population doublings, cells enter in an irreversible growth-arrested state termed replicative senescence. Mutations of genes involved in immortalization can contribute to cancer. In a genetic screen for cDNAs bypassing replicative senescence of normal human prostate epithelial cells (HPrEC), we identified CBX7, a gene that encodes a Polycomb protein, as shown by sequence homology, its interaction with Ring1 and its localization to nuclear Polycomb bodies. CBX7 extends the lifespan of a wide range of normal human cells and immortalizes mouse fibroblasts by downregulating expression of the Ink4a/Arf locus. CBX7 does not inter-function or colocalize with Bmi1, and both can exert their actions independently of each other as shown by reverse genetics. CBX7 expression is downregulated during replicative senescence and its ablation by short-hairpin RNA (shRNA) treatment inhibited growth of normal cells though induction of the Ink4a/Arf locus. Taken together, these data show that CBX7 controls cellular lifespan through regulation of both the p16(Ink4a)/Rb and the Arf/p53 pathways.     

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.     

Losses of both products of the Cdkn2a/Arf locus contribute to asbestos-induced mesothelioma development and cooperate to accelerate tumorigenesis

The CDKN2A/ARF locus encompasses overlapping tumor suppressor genes p16(INK4A) and p14(ARF), which are frequently co-deleted in human malignant mesothelioma (MM). The importance of p16(INK4A) loss in human cancer is well established, but the relative significance of p14(ARF) loss has been debated. The tumor predisposition of mice singly deficient for either Ink4a or Arf, due to targeting of exons 1α or 1β, respectively, supports the idea that both play significant and nonredundant roles in suppressing spontaneous tumors. To further test this notion, we exposed Ink4a(+/-) and Arf(+/-) mice to asbestos, the major cause of MM. Asbestos-treated Ink4a(+/-) and Arf(+/-) mice showed increased incidence and shorter latency of MM relative to wild-type littermates. MMs from Ink4a(+/-) mice exhibited biallelic inactivation of Ink4a, loss of Arf or p53 expression and frequent loss of p15(Ink4b). In contrast, MMs from Arf(+/-) mice exhibited loss of Arf expression, but did not require loss of Ink4a or Ink4b. Mice doubly deficient for Ink4a and Arf, due to deletion of Cdkn2a/Arf exon 2, showed accelerated asbestos-induced MM formation relative to mice deficient for Ink4a or Arf alone, and MMs exhibited biallelic loss of both tumor suppressor genes. The tumor suppressor function of Arf in MM was p53-independent, since MMs with loss of Arf retained functional p53. Collectively, these in vivo data indicate that both CDKN2A/ARF gene products suppress asbestos carcinogenicity. Furthermore, while inactivation of Arf appears to be crucial for MM pathogenesis, the inactivation of both p16(Ink4a) and p19(Arf) cooperate to accelerate asbestos-induced tumorigenesis.     

The yin and yang of the Cdkn2a locus in senescence and aging

Senescence of cultured cells involves activation of the p19Arf-p53 and the p16Ink4a-Rb tumor suppressor pathways. This, together with the observation that p19Arf and p16Ink4a expression increases with age in many tissues of humans and rodents, led to the speculation that these pathways drive in vivo senescence and natural aging. However, it has been difficult to test this hypothesis using a mammalian model system because inactivation of either of these pathways results in early death from tumors. One approach to bypass this problem would be to inactivate these pathways in a murine segmental progeria model such as mice that express low amounts of the mitotic checkpoint protein BubR1 (BubR1 hypomorphic mice). These mice have a five-fold reduced lifespan and develop a variety of early-aging associated phenotypes including cachetic dwarfism, skeletal muscle degeneration, cataracts, arterial stiffening, (subcutaneous) fat loss, reduced stress tolerance and impaired wound healing. Importantly, BubR1 hypomorphism elevates both p16Ink4a and p19Arf expression in skeletal muscle and fat. Inactivation of p16Ink4a in BubR1 mutant mice delays both cellular senescence and aging specifically in these tissues. Surprisingly, however, inactivation of p19Arf has the opposite effect; it exacerbates in vivo senescence and aging in skeletal muscle and fat. These mouse studies suggest that p16Ink4a is indeed an effector of aging and in vivo senescence, but p19Arf an attenuator. Thus, the role of the p19Arf-p53 pathway in aging and in vivo senescence seems far more complex than previously anticipated.     

Nucleolar Arf sequesters Mdm2 and activates p53

The Ink4/Arf locus encodes two tumour-suppressor proteins, p16Ink4a and p19Arf, that govern the antiproliferative functions of the retinoblastoma and p53 proteins, respectively. Here we show that Arf binds to the product of the Mdm2 gene and sequesters it into the nucleolus, thereby preventing negative-feedback regulation of p53 by Mdm2 and leading to the activation of p53 in the nucleoplasm. Arf and Mdm2 co-localize in the nucleolus in response to activation of the oncoprotein Myc and as mouse fibroblasts undergo replicative senescence. These topological interactions of Arf and Mdm2 point towards a new mechanism for p53 activation.     

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.     

The histone demethylases Jhdm1a/1b enhance somatic cell reprogramming in a vitamin-C-dependent manner

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenome to an embryonic-like state. Vitamin C enhances the reprogramming process, but the underlying mechanisms are unclear. Here we show that the histone demethylases Jhdm1a/1b are key effectors of somatic cell reprogramming downstream of vitamin C. We first observed that vitamin C induces H3K36me2/3 demethylation in mouse embryonic fibroblasts in culture and during reprogramming. We then identified Jhdm1a/1b, two known vitamin-C-dependent H3K36 demethylases, as potent regulators of reprogramming through gain- and loss-of-function approaches. Furthermore, we found that Jhdm1b accelerates cell cycle progression and suppresses cell senescence during reprogramming by repressing the Ink4/Arf locus. Jhdm1b also cooperates with Oct4 to activate the microRNA cluster 302/367, an integral component of the pluripotency machinery. Our results therefore reveal a role for H3K36me2/3 in cell fate determination and establish a link between histone demethylases and vitamin-C-induced reprogramming.     

Upregulation of p16(INK4A) promotes cellular senescence of bone marrow-derived mesenchymal stem cells from systemic lupus erythematosus patients

Previous studies have indicated that bone marrow-derived mesenchymal stem cells (MSCs) from patients with systemic lupus erythematosus (SLE) exhibited impaired proliferation, differentiation, and immune modulation capacities. Thus, MSCs may be associated with the pathogenesis of SLE. The aim of this study was to determine whether MSCs from SLE patients were senescent and to determine the mechanism underlying this phenomenon. MSCs from both untreated and treated SLE patients showed characteristics of senescence. The expression of p16(INK4A) was significantly increased, whereas levels of CDK4, CDK6 and p-Rb expression were decreased in the MSCs from both untreated and treated SLE patients. Knockdown of p16(INK4A) expression reversed the senescent features of MSCs and upregulated TGF-β expression. In vitro, when purified CD4+ T cells were incubated with p16(INK4A)-silenced SLE MSCs, the percentage of regulatory T cells was significantly increased. Further, we have found that p16(INK4A) promotes MSC senescence via the suppression of the extracellular signal regulated kinase (ERK) pathway. p16(INK4A) knockdown up-regulated ERK1/2 activation. Our results demonstrated that MSCs from SLE patients were senescent and that p16 (INK4A) plays an essential role in the process by inhibiting ERK1/2 activation.     

Loss of heterozygosity at the Ink4a/Arf locus facilitates Abelson virus transformation of pre-B cells

In many tumor systems, analysis of cells for loss of heterozygosity (LOH) has helped to clarify the role of tumor suppressor genes in oncogenesis. Two important tumor suppressor genes, p53 and the Ink4a/Arf locus, play central roles in the multistep process of Abelson murine leukemia virus (Ab-MLV) transformation. p53 and the p53 regulatory protein, p19Arf, are required for the apoptotic crisis that characterizes the progression of primary transformed pre-B cells to fully malignant cell lines. To search for other tumor suppressor genes which may be involved in the Ab-MLV transformation process, we used endogenous proviral markers and simple-sequence length polymorphism analysis to screen Abelson virus-transformed pre-B cells for evidence of LOH. Our survey reinforces the role of the p53-p19 regulatory pathway in transformation; 6 of 58 cell lines tested had lost sequences on mouse chromosome 4, including the Ink4a/Arf locus. Consistent with this pattern, a high frequency of primary pre-B-cell transformants derived from Ink4a/Arf +/- mice became established cell lines. In addition, half of them retained the single copy of the locus when the transformation process was complete. These data demonstrate that a single copy of the Ink4a/Arf locus is not sufficient to fully mediate the effects of these genes on transformation.     

Increased gene dosage of Ink4/Arf and p53 delays age‐associated central nervous system functional decline

The impairment of the activity of the brain is a major feature of aging, which coincides with a decrease in the function of neural stem cells. We have previously shown that an extra copy of regulated Ink4/Arf and p53 activity, in s‐Ink4/Arf/p53 mice, elongates lifespan and delays aging. In this work, we examined the physiology of the s‐Ink4/Arf/p53 brain with aging, focusing on the neural stem cell (NSC) population. We show that cells derived from old s‐Ink4/Arf/p53 mice display enhanced neurosphere formation and self‐renewal activity compared with wt controls. This correlates with augmented expression of Sox2, Sox9, Glast, Ascl1, and Ars2 NSC markers in the subventricular zone (SVZ) and in the subgranular zone of the dentate gyrus (DG) niches. Furthermore, aged s‐Ink4/Arf/p53 mice express higher levels of Doublecortin and PSA‐NCAM (neuroblasts) and NeuN (neurons) in the olfactory bulbs (OB) and DG, indicating increased neurogenesis in vivo. Finally, aged s‐Ink4/Arf/p53 mice present enhanced behavioral and neuromuscular coordination activity. Together, these findings demonstrate that increased but regulated Ink4/Arf and p53 activity ameliorates age‐related deterioration of the central nervous system activity required to maintain the stem cell pool, providing a mechanism not only for the extended lifespan but also for the health span of these mice.     

Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells

Dicer, an enzyme involved in microRNA (miRNA) maturation, is required for proper cell differentiation and embryogenesis in mammals. Recent evidence indicates that Dicer and miRNA may also regulate tumorigenesis. To better characterize the role of miRNA in primary cell growth, we generated Dicer-conditional mice. Ablation of Dicer and loss of mature miRNAs in embryonic fibroblasts up-regulated p19(Arf) and p53 levels, inhibited cell proliferation, and induced a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and in adult skin. Furthermore, deletion of the Ink4a/Arf or p53 locus could rescue fibroblasts from premature senescence induced by Dicer ablation. Although levels of Ras and Myc oncoproteins appeared unaltered, loss of Dicer resulted in increased DNA damage and p53 activity in these cells. These results reveal that loss of miRNA biogenesis activates a DNA damage checkpoint, up-regulates p19(Arf)-p53 signaling, and induces senescence in primary cells.     

Premeiotic germ cell defect in seminiferous tubules of Atm-null testis

Lifelong spermatogenesis is maintained by coordinated sequential processes including self-renewal of stem cells, proliferation of spermatogonial cells, meiotic division, and spermiogenesis. It has been shown that ataxia telangiectasia-mutated (ATM) is required for meiotic division of the seminiferous tubules. Here, we show that, in addition to its role in meiosis, ATM has a pivotal role in premeiotic germ cell maintenance. ATM is activated in premeiotic spermatogonial cells and the Atm-null testis shows progressive degeneration. In Atm-null testicular cells, differing from bone marrow cells of Atm-null mice, reactive oxygen species-mediated p16(Ink4a) activation does not occur in Atm-null premeiotic germ cells, which suggests the involvement of different signaling pathways from bone marrow defects. Although Atm-null bone marrow undergoes p16(Ink4a)-mediated cellular senescence program, Atm-null premeiotic germ cells exhibited cell cycle arrest and apoptotic elimination of premeiotic germ cells, which is different from p16(Ink4a)-mediated senescence.     

DNA methyltransferase controls stem cell aging by regulating BMI1 and EZH2 through microRNAs

Epigenetic regulation of gene expression is well known mechanism that regulates cellular senescence of cancer cells. Here we show that inhibition of DNA methyltransferases (DNMTs) with 5-azacytidine (5-AzaC) or with specific small interfering RNA (siRNA) against DNMT1 and 3b induced the cellular senescence of human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs) and increased p16(INK4A) and p21(CIP1/WAF1) expression. DNMT inhibition changed histone marks into the active forms and decreased the methylation of CpG islands in the p16(INK4A) and p21(CIP1/WAF1) promoter regions. Enrichment of EZH2, the key factor that methylates histone H3 lysine 9 and 27 residues, was decreased on the p16(INK4A) and p21(CIP1/WAF1) promoter regions. We found that DNMT inhibition decreased expression levels of Polycomb-group (PcG) proteins and increased expression of microRNAs (miRNAs), which target PcG proteins. Decreased CpG island methylation and increased levels of active histone marks at genomic regions encoding miRNAs were observed after 5-AzaC treatment. Taken together, DNMTs have a critical role in regulating the cellular senescence of hUCB-MSCs through controlling not only the DNA methylation status but also active/inactive histone marks at genomic regions of PcG-targeting miRNAs and p16(INK4A) and p21(CIP1/WAF1) promoter regions.