In order to monitor human p16^INK4a gene 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 p16^INK4a 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 p16^INK4a gene expression38, has been shown to bind not only to the promoter region, but also to the intron region of the p16^INK4a gene locus39. Moreover, the expression of the p16-luc fusion protein enables us to specify p16^INK4a gene 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 p16^INK4a gene 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) p16^INK4a gene expression, indicating that overall regulation of human p16^INK4a gene expression is very similar to that of mouse p16^INK4a gene expression, at least in mouse cells37. This is consistent with the previous notion that the levels of p16^INK4a gene expression were increased during the aging process of both rodents and primates 2040414243. These results illustrate the potential of the p16-luc mice for the analysis of p16^INK4a gene expression in response to oncogenic stimuli in vivo.

Although ectopic expression of oncogenic Ras initiates cellular senescence through up-regulation of p16^INK4a expression in cultured normal human fibroblasts34131444, this is not the case in freshly isolated normal human fibroblasts 23. 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 gene4546, 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 p16^INK4a expression, as well as other senescence markers such as senescence-associated (SA) -galactosidase ( -gal) activity and de-phosphorylation of pRb37, indicating that the oncogenic Ras signaling derived from the endogenous H-ras gene indeed provokes p16^INK4a expression, accompanied by senescence cell cycle arrest, in vivo. This also suggests p16^INK4a may 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 p16^INK4a knock-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 p16^INK4a knockout mice 47.

Given that oncogenic mutation in the H-ras gene occurs immediately after DMBA treatment 45, 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 papillomas37. 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 development37. 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 48, 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 fibroblasts37. 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.

It has previously been shown that oncogenic Ras signaling activates the DNMT1 gene promoter through AP1 49. 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 505152 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 37. Since DNMT1 gene expression is known to be regulated by E2F 53, 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 5437, 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 damage5556, 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 gene37. 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 intestine37. 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 birth37. 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.

Our results lead to the following model, in which oncogenic Ras signaling has the potential to activate p16^INK4a gene expression immediately 131415, 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 p16^INK4a gene 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) 5556. It is therefore most likely that p16^INK4a 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 p16^INK4a gene expression are substantially increased in the mice lacking the p53 gene 57. Moreover, over-expression of Aurora A resulted in a significant induction of p16^INK4a expression in the mammary glands of p53 knock-out mice 58. 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 p16^Ink4a does do so 5960. These results, together with our recent findings37, help to explain why mice doubly deficient for p53 and p16^INK4a exhibited an increased rate of tumor formation 6162, and why the combination of p53 and p16^INK4a loss is frequently observed in human cancer cells 63.

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 1538396465666768697071727374. Nonetheless, we have uncovered an unexpected link between p53 and p16^INK4a gene expression37, 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.