Kimi Yamakoshi

Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence.

The p16INK4a cyclin-dependent kinase inhibitor has a key role in establishing stable G1 cell-cycle arrest through activating the retinoblastoma (Rb) tumour suppressor protein pRb in cellular senescence. Here, we show that the p16INK4a /Rb-pathway also cooperates with mitogenic signals to induce elevated intracellular levels of reactive oxygen species (ROS), thereby activating protein kinase Cδ (PKCδ) in human senescent cells. Importantly, once activated by ROS, PKCδ promotes further generation of ROS, thus establishing a positive feedback loop to sustain ROS–PKCδ signalling. Sustained activation of ROS–PKCδ signalling irreversibly blocks cytokinesis, at least partly through reducing the level of WARTS (also known as LATS1), a mitotic exit network (MEN) kinase required for cytokinesis, in human senescent cells. This irreversible cytokinetic block is likely to act as a second barrier to cellular immortalization ensuring stable cell-cycle arrest in human senescent cells. These results uncover an unexpected role for the p16INK4a–Rb pathway and provide a new insight into how senescent cell-cycle arrest is enforced in human cells.

DNA damage signaling triggers degradation of histone methyltransferases through APC/C Cdh1 in senescent cells

Both the DNA damage response (DDR) and epigenetic mechanisms play key roles in the implementation of senescent phenotypes, but very little is known about how these two mechanisms are integrated to establish senescence-associated gene expression. Here we show that, in senescent cells, the DDR induces proteasomal degradation of G9a and GLP, major histone H3K9 mono- and dimethyltransferases, through Cdc14B- and p21(Waf1/Cip1)-dependent activation of APC/C(Cdh1) ubiquitin ligase, thereby causing a global decrease in H3K9 dimethylation, an epigenetic mark for euchromatic gene silencing. Interestingly, induction of IL-6 and IL-8, major players of the senescence-associated secretory phenotype (SASP), correlated with a decline of H3K9 dimethylation around the respective gene promoters and knockdown of Cdh1 abolished IL-6/IL-8 expression in senescent cells, suggesting that the APC/C(Cdh1)-G9a/GLP axis plays crucial roles in aspects of senescent phenotype. These findings establish a role for APC/C(Cdh1) and reveal how the DDR integrates with epigenetic processes to induce senescence-associated gene expression.

Intrinsic Cooperation between p16INK4a and p21Waf1/Cip1 in the Onset of Cellular Senescence and Tumor Suppression In vivo

Although the p16(INK4a) and p21Waf1/Cip1 cyclin-dependent kinase (CDK) inhibitors are known to play key roles in cellular senescence in vitro, their roles in senescence remain rather poorly understood in vivo. This situation is partly due to the possibility of compensatory effect(s) between p16INK4a and p21Waf1/Cip1 or to the upregulation of functionally related CDK inhibitors. To directly address the cooperative roles of p16INK4a and p21Waf1/Cip1 in senescence in vivo, we generated a mouse line simply lacking both p16INK4a and p21Waf1/Cip1 genes [double-knockout (DKO)]. Mouse embryonic fibroblasts (MEF) derived from DKO mice displayed no evidence of cellular senescence when cultured serially in vitro. Moreover, DKO MEFs readily escaped Ras-induced senescence and overrode contact inhibition in culture. This was not the case in MEFs lacking either p16INK4a or p21Waf1/Cip1, indicating that p16(INK4a) and p21Waf1/Cip1 play cooperative roles in cellular senescence and contact inhibition in vitro. Notably, we found the DKO mice to be extremely susceptible to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-induced skin carcinogenesis that involves oncogenic mutation of the H-ras gene. Mechanistic investigations suggested that the high incidence of cancer in DKO mice likely reflected a cooperative effect of increased benign skin tumor formation caused by p21Waf1/Cip1 loss, with increased malignant conversion of benign skin tumors caused by p16(INK4a) loss. Our findings establish an intrinsic cooperation between p16INK4a and p21Waf1/Cip1 in the onset of cellular senescence and tumor suppression in vivo.

Reduction of total E2F/DP activity induces senescence-like cell cycle arrest in cancer cells lacking functional pRB and p53.

E2F/DP complexes were originally identified as potent transcriptional activators required for cell proliferation. However, recent studies revised this notion by showing that inactivation of total E2F/DP activity by dominant-negative forms of E2F or DP does not prevent cellular proliferation, but rather abolishes tumor suppression pathways, such as cellular senescence. These observations suggest that blockage of total E2F/DP activity may increase the risk of cancer. Here, we provide evidence that depletion of DP by RNA interference, but not overexpression of dominant-negative form of E2F, efficiently reduces endogenous E2F/DP activity in human primary cells. Reduction of total E2F/DP activity results in a dramatic decrease in expression of many E2F target genes and causes a senescence-like cell cycle arrest. Importantly, similar results were observed in human cancer cells lacking functional p53 and pRB family proteins. These findings reveal that E2F/DP activity is indeed essential for cell proliferation and its reduction immediately provokes a senescence-like cell cycle arrest.

Real-time in vivo imaging of p16Ink4agene expression: a new approach to study senescence stress signaling in living animals

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.

Visualizing the dynamics of p21Waf1/Cip1 cyclin-dependent kinase inhibitor expression in living animals

Although the role of p21Waf1/Cip1 gene expression is well documented in various cell culture studies, its in vivo roles are poorly understood. To gain further insight into the role of p21Waf1/Cip1 gene expression in vivo, we attempted to visualize the dynamics of p21Waf1/Cip1 gene expression in living animals. In this study, we established a transgenic mice line (p21-p-luc) expressing the firefly luciferase under the control of the p21Waf1/Cip1 gene promoter. In conjunction with a noninvasive bioluminescent imaging technique, p21-p-luc mice enabled us to monitor the endogenous p21Waf1/Cip1 gene expression in vivo. By monitoring and quantifying the p21Waf1/Cip1 gene expression repeatedly in the same mouse throughout its entire lifespan, we were able to unveil the dynamics of p21Waf1/Cip1 gene expression in the aging process. We also applied this system to chemically induced skin carcinogenesis and found that the levels of p21Waf1/Cip1 gene expression rise dramatically in benign skin papillomas, suggesting that p21Waf1/Cip1 plays a preventative role(s) in skin tumor formation. Surprisingly, moreover, we found that the level of p21Waf1/Cip1 expression strikingly increased in the hair bulb and oscillated with a 3-week period correlating with hair follicle cycle progression. Notably, this was accompanied by the expression of p63 but not p53. This approach, together with the analysis of p21Waf1/Cip1 knockout mice, has uncovered a novel role for the p21Waf1/Cip1 gene in hair development. These data illustrate the unique utility of bioluminescence imaging in advancing our understanding of the timing and, hence, likely roles of specific gene expression in higher eukaryotes.

Identification of a gene required for de novo DNA methylation of the zebrafish no tail gene

The zebrafish no tail gene (ntl) is indispensable for tail and notochord development. We have shown previously that ntl is de novo methylated during early embryogenesis. To find the gene that de novo methylates ntl and understand the meaning of this methylation, we cloned seven genes that encode the conserved catalytic domain of methyltransferases. We found that injection of antisense morpholino oligonucleotides against one of them, termed dnmt7, into eggs significantly reduced the level of ntl methylation, although no apparent phenotype was induced by the injection. Inhibition of Dnmt7 activity did not change the level of genome‐wide methylation nor did it affect de novo methylation of injected plasmid DNA, indicating that Dnmt7 specifically methylates ntl in the genome. Developmental Dynamics 233:1509–1516, 2005.

Ablation of the p16 INK4a tumour suppressor reverses ageing phenotypes of klotho mice

The p16INK4a tumour suppressor has an established role in the implementation of cellular senescence in stem/progenitor cells, which is thought to contribute to organismal ageing. However, since p16INK4a knockout mice die prematurely from cancer, whether p16INK4a reduces longevity remains unclear. Here we show that, in mutant mice homozygous for a hypomorphic allele of the α-klotho ageing-suppressor gene (klkl/kl), accelerated ageing phenotypes are rescued by p16INK4a ablation. Surprisingly, this is due to the restoration of α-klotho expression in klkl/kl mice and does not occur when p16INK4a is ablated in α-klotho knockout mice (kl−/−), suggesting that p16INK4a is an upstream regulator of α-klotho expression. Indeed, p16INK4a represses α-klotho promoter activity by blocking the functions of E2Fs. These results, together with the observation that the expression levels of p16INK4a are inversely correlated with those of α-klotho throughout ageing, indicate that p16INK4a plays a previously unrecognized role in downregulating α-klotho expression during ageing.

Dysregulation of the Bmi-1/p16Ink4a pathway provokes an aging-associated decline of submandibular gland function

Bmi‐1 prevents stem cell aging, at least partly, by blocking expression of the cyclin‐dependent kinase inhibitor p16Ink4a. Therefore, dysregulation of the Bmi‐1/p16Ink4a pathway is considered key to the loss of tissue homeostasis and development of associated degenerative diseases during aging. However, because Bmi‐1 knockout (KO) mice die within 20 weeks after birth, it is difficult to determine exactly where and when dysregulation of the Bmi‐1/p16Ink4a pathway occurs during aging in vivo. Using real‐time in vivo imaging of p16Ink4a expression in Bmi‐1‐KO mice, we uncovered a novel function of the Bmi‐1/p16Ink4a pathway in controlling homeostasis of the submandibular glands (SMGs), which secrete saliva into the oral cavity. This pathway is dysregulated during aging in vivo, leading to induction of p16Ink4a expression and subsequent declined SMG function. These findings will advance our understanding of the molecular mechanisms underlying the aging‐related decline of SMG function and associated salivary gland hypofunction, which is particularly problematic among the elderly.

PCR-based cloning of an intronless zebrafish no tail gene.

The zebrafish no tail gene (ntl) is indispensable for the formation of the notochord and tail structure. Here, we show the presence of an intronless ntl gene in zebrafish, which we designated cryptail (ctl). ctl could not be found in any zebrafish genomic resources examined and was only just cloned by a PCR-based approach that relied on its lack of introns and homology to ntl. The amplifiable region of ctl was confined to the transcribed region of ntl. ctl thus appeared to have been generated by reverse transcription of ntl mRNA, like a processed pseudogene. ctl was very polymorphic even in individual fish, but had no missense mutations. This may suggest that ctl has a physiological function in zebrafish.