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Real-time in vivo imaging of p16Ink4agene expression: a new approach to study senescence stress signaling in living animals
Cell Division volume 5, Article number: 1 (2010)
Oncogenic proliferative signals are coupled to a variety of growth inhibitory processes. In cultured primary human fibroblasts, for example, ectopic expression of oncogenic Ras or its downstream mediator initiates cellular senescence, the state of irreversible cell cycle arrest, through up-regulation of cyclin-dependent kinase (CDK) inhibitors, such as p16INK4a. To date, much of our current knowledge of how human p16INK4agene expression is induced by oncogenic stimuli derives from studies undertaken in cultured primary cells. However, since human p16INK4agene expression is also induced by tissue culture-imposed stress, it remains unclear whether the induction of human p16INK4agene expression in tissue-cultured cells truly reflects an anti-cancer process or is an artifact of tissue culture-imposed stress. To eliminate any potential problems arising from tissue culture imposed stress, we have recently developed a bioluminescence imaging (BLI) system for non-invasive and real-time analysis of human p16INK4agene expression in the context of a living animal. Here, we discuss the molecular mechanisms that direct p16INK4agene expression in vivo and its potential for tumor suppression.
The INK4a/ARF gene locus encodes two distinct tumor suppressor proteins, p16INK4a and ARF, whose expression enhances the growth-suppressive functions of the retinoblastoma protein (pRb) and the p53 protein, respectively[1–4]. It has been estimated that more than 70% of established human cancer cell lines lack functional p16INK4a due to promoter methylation, mutation, or homozygous deletion[5–10]. In many instances the deletions affect both p16INK4a and ARF, but a substantial proportion of the missense mutations exclusively affect p16INK4a, suggesting that p16INK4a, by itself, plays significant and non-redundant roles in tumor suppression[5–10]. Indeed, accumulating evidence suggest that the p16INK4a gene acts as a sensor of oncogenic stress, its expression being up-regulated upon the detection of various potentially oncogenic stimuli, such as cumulative cell division or oncogenic Ras expression, in cultured human primary cells[11–15]. This unique feature of p16INK4a gene expression, together with its ability to induce the irreversible cell cycle arrest termed cellular senescence, raises the possibility that the p16INK4a gene acts as a safe-guard against neoplasia[3, 4, 16–19]. However since the simple act of placing cells in tissue culture is sufficient to activate p16INK4agene expression and the levels of p16INK4agene expression vary depending on the cell culture conditions[20–23], it remains unclear whether the induction of p16INK4agene expression in cultured human primary cells truly reflects an anti-cancer process or is an artifact of tissue culture-imposed stress.
We believe that p16INK4aknockout mouse is a powerful tool for elucidating the physiological roles of p16INK4agene expression in vivo[24, 25] A limitation of this approach, however, is the developmental or somatic compensation by the remaining p16INK4afamily genes (p15INK4b, p18INK4cand p19INK4d) [26–28]. Moreover, the possibility of cross-species differences between human p16INK4agene expression and mouse p16INK4agene expression also complicates the interpretation of p16INK4aknockout mouse data. Alternative approaches are therefore needed to supplement the knockout mice studies and to assist in understanding the roles and mechanisms regulating human p16INK4agene expression in vivo.
Bioluminescence imaging (BLI) is an emerging approach that is based on the detection of light emission from cells or tissues[29, 30]. Optical imaging by bioluminescence allows a non-invasive and real-time analysis of various biological responses in living animals, such as gene expression, proteolytic processing or protein-protein interactions in living animals [31–36]. Recently, we have generated a new transgenic mouse line (p16-luc) expressing the fusion protein of human p16INK4aand firefly luciferase under the control of human p16INK4a gene regulation. Using this humanized mouse model, we have recently explored the dynamics of human p16INK4agene expression in many different biological processes in living animals. In this commentary, we will introduce the unique utility of BLI in advancing our understanding of the timing and hence, likely roles and mechanisms regulating p16INK4agene expression in vivo.
Real-time imaging of p16INK4agene expression in living animals
In order to monitor human p16INK4agene expression as accurately as possible, we used a large genomic DNA segment of the human chromosome that contains the entire INK4a/ARF gene locus(Figure 1). Furthermore, this human chromosomal segment was engineered to express a fusion protein of human p16INK4a and firefly luciferase without deleting any genomic DNA sequences of the INK4a/ARF gene locus (Figure 1). This is crucial, because BMI-1, which is a negative regulator of p16INK4agene expression, has been shown to bind not only to the promoter region, but also to the intron region of the p16INK4agene locus. Moreover, the expression of the p16-luc fusion protein enables us to specify p16INK4agene expression, but not ARF gene expression, from this overlapping gene locus.
By monitoring and quantifying the bioluminescent signal repeatedly in the same p16-luc mouse throughout its entire lifespan, we were able to unveil the dynamics of human p16INK4agene expression in the aging process of the transgenic mouse (Figure 2). Importantly moreover, the bioluminescence signal levels correlated well with not only exogenous (human) but also endogenous (mouse) p16INK4agene expression, indicating that overall regulation of human p16INK4agene expression is very similar to that of mouse p16INK4agene expression, at least in mouse cells. This is consistent with the previous notion that the levels of p16INK4agene expression were increased during the aging process of both rodents and primates [20, 40–43]. These results illustrate the potential of the p16-luc mice for the analysis of p16INK4agene expression in response to oncogenic stimuli in vivo.
The response of p16INK4agene expression to oncogenic stimuli in vivo
Although ectopic expression of oncogenic Ras initiates cellular senescence through up-regulation of p16INK4a expression in cultured normal human fibroblasts[3, 4, 13, 14, 44], this is not the case in freshly isolated normal human fibroblasts . It remains, therefore, unclear whether the induction of p16INK4agene expression by oncogenic Ras expression in cultured cells truly reflects an anti-cancer process or an artifact of tissue culture-imposed stress. To explore this notion in a more physiological setting rather than using the ectopic expression of oncogenic Ras in cultured cells, the p16-luc mice were subjected to a conventional chemically-induced skin papilloma protocol with a single dose of DMBA, followed by multiple treatments with TPA. Because this protocol induces benign skin papillomas, more than 90% of which harbor an oncogenic-mutation in the H-ras gene[45, 46], it appears to be ideal for studying the physiological response to oncogenic mutation in the endogenous H-ras gene in vivo.
When p16-luc mice were treated with the DMBA/TPA protocol, benign skin papillomas began to appear after 7 weeks of treatment and continued to grow to a larger size for a further 18 weeks (early-stage papilloma). Although bioluminescent signals were hardly detectable during this time, a significant level of bioluminescent signal was induced as the papillomas stopped growing (late-stage papilloma) (Figure 3). The levels of the bioluminescent signals were well correlated with those of endogenous p16INK4aexpression, as well as other senescence markers such as senescence-associated (SA) -galactosidase ( -gal) activity and de-phosphorylation of pRb, indicating that the oncogenic Ras signaling derived from the endogenous H-ras gene indeed provokes p16INK4aexpression, accompanied by senescence cell cycle arrest, in vivo. This also suggests p16INK4amay play important role(s) in late papillomas, presumably preventing the malignant conversion of benign tumors. In agreement with this notion, by 30 weeks after DMBA/TPA treatment, approximately 33% of p16INK4aknock-out mice (C57BL/6 background) had at least one carcinoma, compared with 5% of the wild type mice (unpublished data). These results are also consistent with a previous study showing that the tumor-free survival of DMBA-treated mice was substantially reduced in p16INK4aknockout mice .
Epigenetic regulatory mechanism underlying the p16INK4agene induction
Given that oncogenic mutation in the H-ras gene occurs immediately after DMBA treatment , it was puzzling that p16INK4agene expression was fully induced in the late- but not early- stage papillomas (Figure 3). Interestingly, the levels of DNMT1, which is known to repress p16INK4agene expression, were significantly increased in early-stage papilloma and subsequently reduced in late-stage papillomas. Intriguingly moreover, the status of the histone 3 Lys 9 methylation (H3K9me), but not the CpG methylation around the p16INK4agene promoter, was well correlated with the levels of DNMT1 expression during the course of papilloma development. These results, together with a recent observation that DNMT1 possesses an activity to enhance H3K9 methylation through interacting with G9a, a major H3K9 mono- and di- methyltransferase , suggest that DNMT1 serves to counterbalance the activation of the p16INK4agene promoter mediated by oncogenic Ras during skin papilloma development. Of note, the levels of DNMT1 were initially increased by oncogenic Ras expression and subsequently reduced as cells reached the senescence stage in cultured human primary fibroblasts. Together, these results indicate that a similar mechanism is likely to be involved in the regulation of p16INK4agene expression by oncogenic Ras signaling, both in vitro and in vivo.
DNA damage response regulates p16INK4agene expression through DNMT1
It has previously been shown that oncogenic Ras signaling activates the DNMT1 gene promoter through AP1 . Thus, the induction of DNMT1 expression appears to be caused by a direct effect of oncogenic Ras expression. However, it was unclear how DNMT1 is reduced in the late stage of papilloma development. Our results strongly suggest that the DNA damage response (DDR) triggered by hyper-cell proliferation [50–52] plays critical role(s) in blocking DNMT1 gene expression, at least partly, through the elevation of the reactive oxygen species (ROS) level in late-stage papillomas . Since DNMT1 gene expression is known to be regulated by E2F , and E2F activity is reduced by H2O2 treatment (unpublished data), it is most likely that ROS regulate DNMT1 expression, at least in part, through E2F. These results, together with the observation that depletion of DNMT1 causes up-regulation of p16INK4agene expression in cultured human cells [54, 37], indicate that DDR plays key role(s) in the induction of p16INK4agene expression through blocking DNMT1 expression in the context of Ras-induced senescence in vivo.
Because the p53 tumor suppressor is activated immediately after detection of DNA damage, preventing accumulation of DNA damage[55, 56], it is possible that p53 might block the DDR pathway activating p16INK4agene expression. To explore this idea, we again took advantage of using p16-luc mice, in conjunction with p16-luc mice lacking the p53 gene. Indeed, although bioluminescent signals were only slightly induced after treatment with doxorubicin (DXR), a DNA damaging agent, in p16-luc mice, this effect was dramatically enhanced by p53 deletion, especially in highly proliferating tissues such as the thymus or small intestine. Furthermore, the DDR-pathway activating p16INK4agene expression and consequent cellular senescence was provoked naturally in the thymus of nearly all mice lacking p53 gene at around 10 to 20 weeks after birth. It is therefore possible that p16INK4amay play a back-up tumor suppressor role in case p53 is accidentally inactivated, especially in highly proliferative tissue such as the thymus.
A regulatory circuit between p53 and p16INK4a tumor suppressors
Our results lead to the following model, in which oncogenic Ras signaling has the potential to activate p16INK4agene expression immediately [13–15], but this effect is initially counteracted by elevation of the DNMT1 levels, which thereby causes hyper-cell proliferation. However, since hyper-cell proliferation tends to cause DNA damage and the elevation of ROS, DNMT1 gene expression is eventually reduced by this ROS increase, leading to epigenetic de-repression of p16INK4agene expression and hence senescence cell cycle arrest (see model in Figure 4). Interestingly, moreover, this pathway is potentiated in the setting of p53 deletion, because p53 tends to prevent the proliferation of damaged cells that would cause a further accumulation of DNA damage (Figure 4) [55, 56]. It is therefore most likely that p16INK4a plays a back-up tumor suppressor role if p53 becomes inactivated. In agreement with this notion, it has recently been shown that the levels of p16INK4agene expression are substantially increased in the mice lacking the p53 gene . Moreover, over-expression of Aurora A resulted in a significant induction of p16INK4a expression in the mammary glands of p53 knock-out mice . It is also worth emphasizing that p53 inactivation alone is not sufficient to fully abrogate telomere-directed cellular senescence, but the combined inactivation of p53 and p16Ink4a does do so [59, 60]. These results, together with our recent findings, help to explain why mice doubly deficient for p53 and p16INK4a exhibited an increased rate of tumor formation [61, 62], and why the combination of p53 and p16INK4a loss is frequently observed in human cancer cells .
It is, however, clear that all aspects of p16INK4aregulation cannot be explained by the factors described here, and that the p16INK4agene is subject to multiple levels of control [15, 38, 39, 64–74]. Nonetheless, we have uncovered an unexpected link between p53 and p16INK4agene expression, expanding our understanding of how p16INK4agene expression is induced by oncogenic stimuli in vivo, thus opening up new possibilities for its control. Visualizing the dynamics of p16INK4agene expression in living animals, therefore, provides a powerful tool for not only helping to resolve issues connecting in vitro studies, but also clarifying previously unrecognized functions of this key senescence regulator in various physiological processes in vivo.
DNA damage response
retinoblastoma tumor suppressor protein
DNA methyl transferase 1
histone 3 Lys 9
histone 3 Lys 9 methylation
reactive oxygen species
Serrano M, Hannon GJ, Beach D: A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 1993, 366: 704–707. 10.1038/366704a0
Quelle DE, Zindy F, Ashmun RA, Sherr CJ: Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell 1995, 83: 993–1000. 10.1016/0092-8674(95)90214-7
Gil J, Peters G: Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all. Nat Rev Mol Cell Biol 2006, 7: 667–677. 10.1038/nrm1987
Kim WY, Sharpless NE: The regulation of INK4a/ARF in cancer and aging. Cell 2006, 127: 265–275. 10.1016/j.cell.2006.10.003
Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson DA: Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 1994, 368: 753–756. 10.1038/368753a0
Okamoto A, Demetrick DJ, Spillare EA, Hagiwara K, Hussain SP, Bennett WP, Forrester K, Gerwin B, Serrano M, Beach DH: Mutations and altered expression of p16INK4 in human cancer. Proc Natl Acad Sci USA 1994, 91: 11045–11049. 10.1073/pnas.91.23.11045
Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, Stockert E, Day RS, Johnson BE, Skolnick MH: A cell cycle regulator potentially involved in genesis of any tumor types. Science 1994, 264: 436–40. 10.1126/science.8153634
Ruas M, Peters G: The p16INK4a /CDKN2A tumor suppressor and its relatives. Biochim Biophys Acta 1998, 1378: F115–77.
Rocco JW, Sidransky D: p16MTS-1/CDKN2/INK4a in cancer progression. Exp Cell Res 2001, 264: 42–55. 10.1006/excr.2000.5149
Ortega S, Malumbres M, Barbacid M: Cyclin D-dependent kinases, INK4 inhibitors and cancer. Biochim Biophys Acta 2002, 1602: 73–87.
Hara E, Smith R, Parry D, Tahara H, Stone S, Peters G: Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence. Mol Cell Biol 1996, 16: 859–867.
Palmero I, McConnell B, Parry D, Brookes S, Hara E, Bates S, Jat P, Peters G: Accumulation of p16INK4a in mouse fibroblasts as a function of replicative senescence and not of retinoblastoma gene status. Oncogene 1997, 15: 495–503. 10.1038/sj.onc.1201212
Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW: Oncogenic ras provikes premature cell cenescence associated with accumulation of p53 and p16INK4a . Cell 1997, 88: 593–602. 10.1016/S0092-8674(00)81902-9
Serrano M, Blasco MA: Putting the stress on senescence. Curr Opin Cell 2001, 13: 748–53. 10.1016/S0955-0674(00)00278-7
Ohtani N, Zebedee Z, Huot TJ, Stinson JA, Sugimoto M, Ohashi Y, Sharrocks AD, Peters G, Hara E: Opposing effects of Ets and Id proteins on p16INK4a expression during cellular senescence. Nature 2001, 409: 1067–1070. 10.1038/35059131
Collado M, Blasco MA, Serrano M: Cellular senescence in cancer and aging. Cell 2007, 130: 223–233. 10.1016/j.cell.2007.07.003
McConnell BB, Starborg M, Brookes S, Peters G: Inhibitors of cyclin-dependent kinases induce features of replicative senescence in early passage human diploid fibroblasts. Curr Biol 1998, 8: 351–354. 10.1016/S0960-9822(98)70137-X
Ohtani N, Mann DJ, Hara E: Cellular senescence Its role in tumor suppression and aging. Cancer Science 2009, 100: 792–797. 10.1111/j.1349-7006.2009.01123.x
Takahashi A, Ohtani N, Yamakoshi K, Iida S, Tahara H, Nakayama K, Nakayama KI, Ide T, Saya H, Hara E: Mitogenic signalling and the p16INK4a -Rb pathway cooperate to enforce irreversible cellular senescence. Nat Cell Biol 2006, 8: 1291–1297. 10.1038/ncb1491
Zindy F, Quelle DE, Roussel MF, Sherr CJ: Expression of the p16INK4a tumor suppressor versus other INK4 family members during mouse development and aging. Oncogene 1997, 15: 203–211. 10.1038/sj.onc.1201178
Ince TA, Richardson AL, Bell GW, Saitoh M, Godar S, Karnoub AE, Iglehart JD, Weinberg RA: Transformation of different human breast epithelial cell types leads to distinct tumor phenotypes. Cancer Cell 2007, 12: 160–170. 10.1016/j.ccr.2007.06.013
Ramirez RD, Morales CP, Herbert BS, Rohde JM, Passons C, Shay JW, Wright WE: Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions. Genes Dev 2001, 15: 398–403. 10.1101/gad.859201
Benanti JA, Galloway DA: Normal human fibroblasts are resistant to RAS-induced senescence. Mol Cell Biol 2004, 24: 2842–2852. 10.1128/MCB.24.7.2842-2852.2004
Sharpless NE, Bardeesy N, Lee KH, Carrasco D, Castrillon DH, Aguirre AJ, Wu EA, Horner JW, DePinho RA: Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis. Nature 2001, 413: 86–91. 10.1038/35092592
Krimpenfort P, Quon KC, Mooi WJ, Loonstra A, Berns A: Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice. Nature 2001, 413: 83–86. 10.1038/35092584
Krimpenfort P, Ijpenberg A, Song JY, Valk M, Nawijn M, Zevenhoven J, Berns A: p15Ink4b is a critical tumour suppressor in the absence of p16Ink4a . Nature 2007, 448: 943–946. 10.1038/nature06084
Ramsey MR, Krishnamurthy J, Pei XH, Torrice C, Lin W, Carrasco DR, Ligon KL, Xiong Y, Sharpless NE: Expression of p16Ink4a compensates for p18Ink4c loss in cyclin-dependent kinase 4/6-dependent tumors and tissues. Cancer Res 2007, 67: 4732–4741. 10.1158/0008-5472.CAN-06-3437
Wiedemeyer R, Brennan C, Heffernan TP, Xiao Y, Mahoney J, Protopopov A, Zheng H, Bignell G, Furnari F, Cavenee WK, Hahn WC, Ichimura K, Collins VP, Chu GC, Stratton MR, Ligon KL, Futreal PA, Chin L: Feedback circuit among INK4 tumor suppressors constrains human glioblastoma development. Cancer Cell 2008, 13: 355–364. 10.1016/j.ccr.2008.02.010
Contag PR, Olomu IN, Stevenson DK, Contag CH: Bioluminescent indicators in living mammals. Nat Med 1998, 4: 245–247. 10.1038/nm0298-245
Dothager RS, Flentie K, Moss B, Pan MH, Kesarwala A, Piwnica-Worms D: Advances in bioluminescence imaging of live animal models. Curr Opin Biotechnol 2009, 20: 45–53. 10.1016/j.copbio.2009.01.007
Ohtani N, Imamura Y, Yamakoshi K, Hirota F, Nakayama R, Kubo Y, Ishimaru N, Takahashi A, Hirao A, Shimizu T, Mann DJ, Saya H, Hayashi Y, Arase S, Matsumoto M, Kazuki N, Hara E: Visualizing the dynamics of p21Waf1/Cip1 cyclin-dependent kinase inhibitor expression in living animals. Proc Natl Acad Sci USA 2007, 104: 15034–15039. 10.1073/pnas.0706949104
Uhrbom L, Nerio E, Holland EC: Dissecting tumor maintenance requirements using bioluminescence imaging of cell proliferation in a mouse glioma model. Nat Med 2004, 10: 1257–1260. 10.1038/nm1120
Zhang GJ, Safran M, Wei W, Sorensen E, Lassota P, Zhelev N, Neuberg DS, Shapiro G, Kaelin WG Jr: Bioluminescent imaging of Cdk2 inhibition in vivo. Nat Med 2004, 10: 643–648. 10.1038/nm1047
Li F, Sonveaux P, Rabbani ZN, Liu S, Yan B, Huang Q, Vujaskovic Z, Dewhirst MW, Li CY: Regulation of HIF-1alpha stability through S-nitrosylation. Mol Cell 2007, 26: 63–74. 10.1016/j.molcel.2007.02.024
Vooijs M, Jonkers J, Lyons S, Berns A: Noninvasive imaging of spontaneous retinoblastoma pathway-dependent tumors in mice. Cancer Res 2002, 62: 1862–1867.
Paulmurugan R, Umezawa Y, Gambhir SS: Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies. Proc Natl Acad Sci USA 2002, 99: 15608–15613. 10.1073/pnas.242594299
Yamakoshi K, Takahashi A, Hirota F, Nakayama R, Ishimaru N, Kubo Y, Mann DJ, Ohmura M, Hirao A, Saya H, Arase S, Hayashi Y, Nakao K, Matsumoto M, Ohtani N, Hara E: Real-time in vivo imaging of p16Ink4a reveals cross talk with p53. J Cell Biol 2009, 186: 393–407. 10.1083/jcb.200904105
Jacobs JJ, Kieboom K, Marino S, DePinho RA, van Lohuizen M: The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the Ink4a locus. Nature 1999, 397: 164–168. 10.1038/16476
Kotake Y, Cao R, Viatour P, Sage J, Zhang Y, Xiong Y: pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16INK4a tumor suppressor gene. Genes Dev 2007, 21: 49–54. 10.1101/gad.1499407
Krishnamurthy J, Torrice C, Ramsey MR, Kovalev GI, Al-Regaiey K, Su L, Sharpless NE: Ink4a/Arf expression is a biomarker of aging. J Clin Invest 2004, 114: 1299–1307.
Herbig U, Ferreira M, Condel L, Carey D, Sedivy JM: Cellular senescence in aging primates. Science 2006, 311:-1257. 10.1126/science.1122446
Ressler S, Bartkova J, Niederegger H, Bartek J, Scharffetter-Kochanek K, Jansen-Dürr P, Wlaschek M: p16INK4A is a robust in vivo biomarker of cellular aging in human skin. Aging Cell 2006, 5: 379–389. 10.1111/j.1474-9726.2006.00231.x
Tsygankov D, Liu Y, Sanoff HK, Sharpless NE, Elston TC: A quantitative model for age-dependent expression of the p16INK4a tumor suppressor. Proc Natl Acad Sci USA 2009, 106: 16562–16567. 10.1073/pnas.0904405106
Campisi J: Senescent cells tumor suppression and organismal aging good citizens bad neighbors. Cell 2005, 120: 513–522. 10.1016/j.cell.2005.02.003
Quintanilla M, Brown K, Ramsden M, Balmain A: Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis. Nature 1986, 322: 78–80. 10.1038/322078a0
Kemp CJ: Multistep skin cancer in mice as a model to study the evolution of cancer cells. Semin Cancer Biol 2005, 15: 460–473. 10.1016/j.semcancer.2005.06.003
Sharpless NE, Ramsey MR, Balasubramanian P, Castrillon DH, DePinho RA: The differential impact of p16INK4a or p19ARF deficiency on cell growth and tumorigenesis. Oncogene 2004, 23: 379–385. 10.1038/sj.onc.1207074
Estève PO, Chin HG, Smallwood A, Feehery GR, Gangisetty O, Karpf AR, Carey MF, Pradhan S: Direct interaction between DNMT1 and G9a coordinates DNA and histone methylation during replication. Genes Dev 2006, 20: 3089–103. 10.1101/gad.1463706
MacLeod AR, Rouleau J, Szyf M: Regulation of DNA methylation by the Ras signaling pathway. J Biol Chem 1995, 270: 11327–11337. 10.1074/jbc.270.19.11327
Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N, Vassiliou LV, Kolettas E, Niforou K, Zoumpourlis VC, Takaoka M, Nakagawa H, Tort F, Fugger K, Johansson F, Sehested M, Andersen CL, Dyrskjot L, Ørntoft T, Lukas J, Kittas C, Helleday T, Halazonetis TD, Bartek J, Gorgoulis VG: Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 2006, 444: 633–637. 10.1038/nature05268
Di Micco R, Fumagalli M, Cicalese A, Piccinin S, Gasparini P, Luise C, Schurra C, Garre' M, Nuciforo PG, Bensimon A, Maestro R, Pelicci PG, d'Adda di Fagagna F: Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature 2006, 444: 638–642. 10.1038/nature05327
Mallette FA, Gaumont-Leclerc MF, Ferbeyre G: The DNA damage signaling pathway is a critical mediator of oncogene-induced senescence. Genes Dev 2007, 21: 43–48. 10.1101/gad.1487307
McCabe MT, Davis JN, Day ML: Regulation of DNA methyltransferase 1 by the pRb/E2F1 pathway. Cancer Res 2005,65(9):3624–3632. 10.1158/0008-5472.CAN-04-2158
Robert MF, Morin S, Beaulieu N, Gauthier F, Chute IC, Barsalou A, MacLeod AR: DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells. Nat Genet 2003, 33: 61–65. 10.1038/ng1068
Vousden KH, Lane DP: p53 in health and disease. Nat Rev Mol Cell Biol 2007, 8: 275–283. 10.1038/nrm2147
Riley T, Sontag E, Chen P, Levine A: Transcriptional control of human p53-regulated genes. Nat Rev Mol Cell Biol 2008, 9: 402–412. 10.1038/nrm2395
Leong WF, Chau JF, Li B: p53 Deficiency leads to compensatory up-regulation of p16INK4a . Mol Cancer Res 2009, 7: 354–360. 10.1158/1541-7786.MCR-08-0373
Zhang D, Shimizu T, Araki N, Hirota T, Yoshie M, Ogawa K, Nakagata N, Takeya M, Saya H: Aurora A overexpression induces cellular senescence in mammary gland hyperplastic tumors developed in p53-deficient mice. Oncogene 2008, 27: 4305–4314. 10.1038/onc.2008.76
Jacobs JJ, de Lange T: Significant role for p16INK4a in p53-independent telomere-directed senescence. Curr Biol 2004, 14: 2302–2308. 10.1016/j.cub.2004.12.025
Jacobs JJ, de Lange T: p16INK4a as a second effector of the telomere damage pathway. Cell Cycle 2005, 4: 1364–1368.
Sharpless NE, Alson S, Chan S, Silver DP, Castrillon DH, DePinho RA: p16INK4a and p53 deficiency cooperate in tumorigenesis. Cancer Res 2002, 62: 2761–2765.
Terzian T, Suh YA, Iwakuma T, Post SM, Neumann M, Lang GA, Van Pelt CS, Lozano G: The inherent instability of mutant p53 is alleviated by Mdm2 or p16INK4a loss. Genes Dev 2008, 22: 1337–1344. 10.1101/gad.1662908
Malumbres M, Barbacid M: To cycle or not to cycle a critical decision in cancer. Nat Rev Cancer 2001, 1: 222–231. 10.1038/35106065
Passegué E, Wagner EF: JunB suppresses cell proliferation by transcriptional activation of p16INK4a expression. EMBO J 2000, 19: 2969–2979. 10.1093/emboj/19.12.2969
Ohtani N, Brennan P, Gaubatz S, Sanij E, Hertzog P, Wolvetang E, Ghysdael J, Rowe M, Hara E: Epstein-Barr virus LMP1 blocks p16INK4a-RB pathway by promoting nuclear export of E2F4/5. J Cell Biol 2003, 162: 173–83. Epub 2003 10.1083/jcb.200302085
Gonzalez S, Klatt P, Delgado S, Conde E, Lopez-Rios F, Sanchez-Cespedes M, Mendez J, Antequera F, Serrano M: Oncogenic activity of Cdc6 through repression of the INK4/ARF locus. Nature 2006, 440: 702–706. 10.1038/nature04585
Bracken AP, Kleine-Kohlbrecher D, Dietrich N, Pasini D, Gargiulo G, Beekman C, Theilgaard-Mönch K, Minucci S, Porse BT, Marine JC, Hansen KH, Helin K: The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells. Genes Dev 2007, 21: 525–530. 10.1101/gad.415507
Baker DJ, Perez-Terzic C, Jin F, Pitel K, Niederländer NJ, Jeganathan K, Yamada S, Reyes S, Rowe L, Hiddinga HJ, Eberhardt NL, Terzic A, van Deursen JM: Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency. Nat Cell Biol 2008, 10: 825–836. 10.1038/ncb1744
Tzatsos A, Pfau R, Kampranis SC, Tsichlis PN: Ndy1/KDM2B immortalizes mouse embryonic fibroblasts by repressing the Ink4a/Arf locus. Proc Natl Acad Sci USA 2009, 106: 2641–6. 10.1073/pnas.0813139106
Witcher M, Emerson BM: Epigenetic silencing of the p16INK4a tumor suppressor is associated with loss of CTCF binding and a chromatin boundary. Mol Cell 2009, 34: 271–284. 10.1016/j.molcel.2009.04.001
Agger K, Cloos PA, Rudkjaer L, Williams K, Andersen G, Christensen J, Helin K: The H3K27me3 demethylase JMJD3 contributes to the activation of the INK4A-ARF locus in response to oncogene- and stress-induced senescence. Genes Dev 2009, 23: 1171–6. 10.1101/gad.510809
Barradas M, Anderton E, Acosta JC, Li S, Banito A, Rodriguez-Niedenführ M, Maertens G, Banck M, Zhou MM, Walsh MJ, Peters G, Gil J: Histone demethylase JMJD3 contributes to epigenetic control of INK4a/ARF by oncogenic RAS. Genes Dev 2009, 23: 1177–1182. 10.1101/gad.511109
Kia SK, Gorski MM, Giannakopoulos S, Verrijzer CP: SWI/SNF mediates polycomb eviction and epigenetic reprogramming of the INK4b-ARF-INK4a locus. Mol Cell Biol 2008, 28: 3457–3464. 10.1128/MCB.02019-07
Wong ES, Le Guezennec X, Demidov ON, Marshall NT, Wang ST, Krishnamurthy J, Sharpless NE, Dunn NR, Bulavin DV: p38MAPK controls expression of multiple cell cycle inhibitors and islet proliferation with advancing age. Dev Cell 2009, 17: 142–149. 10.1016/j.devcel.2009.05.009
We thank members of the Hara lab for helpful discussion during the preparation of this manuscript. This work was supported by grants from Ministry of Education, Science, Sports and Technology of Japan, the Mitsubishi Foundation, the Naito Foundation, the Princess Takamatsu Cancer Research Fund, the Takeda Science Foundation, Uehara memorial foundation and the Vehicle Racing Commemorative Foundation.
The authors declare that they have no competing interests.
NO wrote the manuscript. KY collected the information required for this commentary article. AT collected the information required for this commentary article. EH wrote the manuscript.
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Ohtani, N., Yamakoshi, K., Takahashi, A. et al. Real-time in vivo imaging of p16Ink4agene expression: a new approach to study senescence stress signaling in living animals. Cell Div 5, 1 (2010). https://doi.org/10.1186/1747-1028-5-1
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