Real-time in vivo imaging of p16 Ink4a gene expression: a new approach to study senescence stress signaling in living animals
© Ohtani et al. 2010
Received: 14 December 2009
Accepted: 14 January 2010
Published: 14 January 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 p16 INK4a gene expression is induced by oncogenic stimuli derives from studies undertaken in cultured primary cells. However, since human p16 INK4a gene expression is also induced by tissue culture-imposed stress, it remains unclear whether the induction of human p16 INK4a gene 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 p16 INK4a gene expression in the context of a living animal. Here, we discuss the molecular mechanisms that direct p16 INK4a gene expression in vivo and its potential for tumor suppression.
Abbreviations used in this paper
DNA damage response
retinoblastoma tumor suppressor protein
DNA methyl transferase 1
histone 3 Lys 9
histone 3 Lys 9 methylation
reactive oxygen species
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 p16 INK4a gene expression and the levels of p16 INK4a gene expression vary depending on the cell culture conditions[20–23], it remains unclear whether the induction of p16 INK4a gene expression in cultured human primary cells truly reflects an anti-cancer process or is an artifact of tissue culture-imposed stress.
We believe that p16 INK4a knockout mouse is a powerful tool for elucidating the physiological roles of p16 INK4a gene expression in vivo[24, 25] A limitation of this approach, however, is the developmental or somatic compensation by the remaining p16 INK4a family genes (p15 INK4b , p18 INK4c and p19 INK4d ) [26–28]. Moreover, the possibility of cross-species differences between human p16 INK4a gene expression and mouse p16 INK4a gene expression also complicates the interpretation of p16 INK4a knockout mouse data. Alternative approaches are therefore needed to supplement the knockout mice studies and to assist in understanding the roles and mechanisms regulating human p16 INK4a gene 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 p16 INK4a and firefly luciferase under the control of human p16 INK4a gene regulation. Using this humanized mouse model, we have recently explored the dynamics of human p16 INK4a gene 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 p16 INK4a gene expression in vivo.
Real-time imaging of p16 INK4a gene expression in living animals
The response of p16 INK4a gene 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 p16 INK4a gene 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.
Epigenetic regulatory mechanism underlying the p16 INK4a gene induction
Given that oncogenic mutation in the H-ras gene occurs immediately after DMBA treatment , it was puzzling that p16 INK4a gene expression was fully induced in the late- but not early- stage papillomas (Figure 3). Interestingly, the levels of DNMT1, which is known to repress p16 INK4a gene 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 p16 INK4a gene 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 p16 INK4a gene 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 p16 INK4a gene expression by oncogenic Ras signaling, both in vitro and in vivo.
DNA damage response regulates p16 INK4a gene 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 p16 INK4a gene expression in cultured human cells [54, 37], indicate that DDR plays key role(s) in the induction of p16 INK4a gene 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 p16 INK4a gene 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 p16 INK4a gene 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 p16 INK4a may 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
It is, however, clear that all aspects of p16 INK4a regulation cannot be explained by the factors described here, and that the p16 INK4a gene is subject to multiple levels of control [15, 38, 39, 64–74]. Nonetheless, we have uncovered an unexpected link between p53 and p16 INK4a gene expression, expanding our understanding of how p16 INK4a gene expression is induced by oncogenic stimuli in vivo, thus opening up new possibilities for its control. Visualizing the dynamics of p16 INK4a gene 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.
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.
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