Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis.

The cyclin-dependent kinase inhibitor p16INK4a can induce senescence of human cells, and its loss by deletion, mutation or epigenetic silencing is among the most frequently observed molecular lesions in human cancer. Overlapping reading frames in the INK4A/ARF gene encode p16INK4a and a distinct tumour-suppressor protein, p19ARF (ref. 3). Here we describe the generation and characterization of a p16Ink4a-specific knockout mouse that retains normal p19Arf function. Mice lacking p16Ink4a were born with the expected mendelian distribution and exhibited normal development except for thymic hyperplasia. T cells deficient in p16Ink4a exhibited enhanced mitogenic responsiveness, consistent with the established role of p16Ink4a in constraining cellular proliferation. In contrast to mouse embryo fibroblasts (MEFs) deficient in p19Arf (ref. 4), p16Ink4a-null MEFs possessed normal growth characteristics and remained susceptible to Ras-induced senescence. Compared with wild-type MEFs, p16Ink4a-null MEFs exhibited an increased rate of immortalization, although this rate was less than that observed previously for cells null for Ink4a/Arf, p19Arf or p53 (refs 4, 5). Furthermore, p16Ink4a deficiency was associated with an increased incidence of spontaneous and carcinogen-induced cancers. These data establish that p16Ink4a, along with p19Arf, functions as a tumour suppressor in mice.     

Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing

Mammalian ageing is associated with reduced regenerative capacity in tissues that contain stem cells. It has been proposed that this is at least partially caused by the senescence of progenitors with age; however, it has not yet been tested whether genes associated with senescence functionally contribute to physiological declines in progenitor activity. Here we show that progenitor proliferation in the subventricular zone and neurogenesis in the olfactory bulb, as well as multipotent progenitor frequency and self-renewal potential, all decline with age in the mouse forebrain. These declines in progenitor frequency and function correlate with increased expression of p16INK4a, which encodes a cyclin-dependent kinase inhibitor linked to senescence. Ageing p16INK4a-deficient mice showed a significantly smaller decline in subventricular zone proliferation, olfactory bulb neurogenesis, and the frequency and self-renewal potential of multipotent progenitors. p16INK4a deficiency did not detectably affect progenitor function in the dentate gyrus or enteric nervous system, indicating regional differences in the response of neural progenitors to increased p16INK4a expression during ageing. Declining subventricular zone progenitor function and olfactory bulb neurogenesis during ageing are thus caused partly by increasing p16INK4a expression.     

BRAFE600-associated senescence-like cell cycle arrest of human naevi

Most normal mammalian cells have a finite lifespan, thought to constitute a protective mechanism against unlimited proliferation. This phenomenon, called senescence, is driven by telomere attrition, which triggers the induction of tumour suppressors including p16(INK4a) (ref. 5). In cultured cells, senescence can be elicited prematurely by oncogenes; however, whether such oncogene-induced senescence represents a physiological process has long been debated. Human naevi (moles) are benign tumours of melanocytes that frequently harbour oncogenic mutations (predominantly V600E, where valine is substituted for glutamic acid) in BRAF, a protein kinase and downstream effector of Ras. Nonetheless, naevi typically remain in a growth-arrested state for decades and only rarely progress into malignancy (melanoma). This raises the question of whether naevi undergo BRAF(V600E)-induced senescence. Here we show that sustained BRAF(V600E) expression in human melanocytes induces cell cycle arrest, which is accompanied by the induction of both p16(INK4a) and senescence-associated acidic beta-galactosidase (SA-beta-Gal) activity, a commonly used senescence marker. Validating these results in vivo, congenital naevi are invariably positive for SA-beta-Gal, demonstrating the presence of this classical senescence-associated marker in a largely growth-arrested, neoplastic human lesion. In growth-arrested melanocytes, both in vitro and in situ, we observed a marked mosaic induction of p16(INK4a), suggesting that factors other than p16(INK4a) contribute to protection against BRAF(V600E)-driven proliferation. Naevi do not appear to suffer from telomere attrition, arguing in favour of an active oncogene-driven senescence process, rather than a loss of replicative potential. Thus, both in vitro and in vivo, BRAF(V600E)-expressing melanocytes display classical hallmarks of senescence, suggesting that oncogene-induced senescence represents a genuine protective physiological process.     

Regulation of cellular response to oncogenic and oxidative stress by Seladin-1

Expression of multiple oncogenes and inactivation of tumour suppressors is required to transform primary mammalian cells into cancer cells. Activated Ha-RasV12 (Ras) is usually associated with cancer, but it also produces paradoxical premature senescence in primary cells by inducing reactive oxygen species followed by accumulation of tumour suppressors p53 and p16(INK4a) (ref. 4). Here we identify, using a direct genetic screen, Seladin-1 (also known as Dhcr24) as a key mediator of Ras-induced senescence. Following oncogenic and oxidative stress, Seladin-1 binds p53 amino terminus and displaces E3 ubiquitin ligase Mdm2 from p53, thus resulting in p53 accumulation. Additionally, Seladin-1 associates with Mdm2 independently of p53, potentially affecting other Mdm2 targets. Ablation of Seladin-1 causes the bypass of Ras-induced senescence in rodent and human fibroblasts, and allows Ras to transform these cells. Wild-type Seladin-1, but not mutants that disrupt its association with either p53 or Mdm2, suppresses the transformed phenotype. The same mutants are also inactive in directing p53-dependent oxidative stress response. These results show an unanticipated role for Seladin-1, previously implicated in Alzheimer's disease and cholesterol metabolism, in integrating cellular response to oncogenic and oxidative stress.     

p15Ink4b is a critical tumour suppressor in the absence of p16Ink4a

The CDKN2b-CDKN2a locus on chromosome 9p21 in human (chromosome 4 in mouse) is frequently lost in cancer. The locus encodes three cell cycle inhibitory proteins: p15INK4b encoded by CDKN2b, p16INK4a encoded by CDKN2a and p14ARF (p19Arf in mice) encoded by an alternative reading frame of CDKN2a (ref. 1). Whereas the tumour suppressor functions for p16INK4a and p14ARF have been firmly established, the role of p15INK4b remains ambiguous. However, many 9p21 deletions also remove CDKN2b, so we hypothesized a synergistic effect of the combined deficiency for p15INK4b, p14ARF and p16INK4a. Here we report that mice deficient for all three open reading frames (Cdkn2ab-/-) are more tumour-prone and develop a wider spectrum of tumours than Cdkn2a mutant mice, with a preponderance of skin tumours and soft tissue sarcomas (for example, mesothelioma) frequently composed of mixed cell types and often showing biphasic differentiation. Cdkn2ab-/- mouse embryonic fibroblasts (MEFs) are substantially more sensitive to oncogenic transformation than Cdkn2a mutant MEFs. Under conditions of stress, p15Ink4b protein levels are significantly elevated in MEFs deficient for p16Ink4a. Our data indicate that p15Ink4b can fulfil a critical backup function for p16Ink4a and provide an explanation for the frequent loss of the complete CDKN2b-CDKN2a locus in human tumours.     

CPEB and two poly(A) polymerases control miR-122 stability and p53 mRNA translation

Cytoplasmic polyadenylation-induced translation controls germ cell development, neuronal synaptic plasticity and cellular senescence, a tumour-suppressor mechanism that limits the replicative lifespan of cells. The cytoplasmic polyadenylation element binding protein (CPEB) promotes polyadenylation by nucleating a group of factors including defective in germline development 2 (Gld2), a non-canonical poly(A) polymerase, on specific messenger RNA (mRNA) 3' untranslated regions (UTRs). Because CPEB regulation of p53 mRNA polyadenylation/translation is necessary for cellular senescence in primary human diploid fibroblasts, we surmised that Gld2 would be the enzyme responsible for poly(A) addition. Here we show that depletion of Gld2 surprisingly promotes rather than inhibits p53 mRNA polyadenylation/translation, induces premature senescence and enhances the stability of CPEB mRNA. The CPEB 3' UTR contains two miR-122 binding sites, which when deleted, elevate mRNA translation, as does an antagomir of miR-122. Although miR-122 is thought to be liver specific, it is present in primary fibroblasts and destabilized by Gld2 depletion. Gld4, a second non-canonical poly(A) polymerase, was found to regulate p53 mRNA polyadenylation/translation in a CPEB-dependent manner. Thus, translational regulation of p53 mRNA and cellular senescence is coordinated by Gld2/miR-122/CPEB/Gld4.     

A Raf-induced allosteric transition of KSR stimulates phosphorylation of MEK

In metazoans, the Ras-Raf-MEK (mitogen-activated protein-kinase kinase)-ERK (extracellular signal-regulated kinase) signalling pathway relays extracellular stimuli to elicit changes in cellular function and gene expression. Aberrant activation of this pathway through oncogenic mutations is responsible for a large proportion of human cancer. Kinase suppressor of Ras (KSR) functions as an essential scaffolding protein to coordinate the assembly of Raf-MEK-ERK complexes. Here we integrate structural and biochemical studies to understand how KSR promotes stimulatory Raf phosphorylation of MEK (refs 6, 7). We show, from the crystal structure of the kinase domain of human KSR2 (KSR2(KD)) in complex with rabbit MEK1, that interactions between KSR2(KD) and MEK1 are mediated by their respective activation segments and C-lobe αG helices. Analogous to BRAF (refs 8, 9), KSR2 self-associates through a side-to-side interface involving Arg?718, a residue identified in a genetic screen as a suppressor of Ras signalling. ATP is bound to the KSR2(KD) catalytic site, and we demonstrate KSR2 kinase activity towards MEK1 by in vitro assays and chemical genetics. In the KSR2(KD)-MEK1 complex, the activation segments of both kinases are mutually constrained, and KSR2 adopts an inactive conformation. BRAF allosterically stimulates the kinase activity of KSR2, which is dependent on formation of a side-to-side KSR2-BRAF heterodimer. Furthermore, KSR2-BRAF heterodimerization results in an increase of BRAF-induced MEK phosphorylation via the KSR2-mediated relay of a signal from BRAF to release the activation segment of MEK for phosphorylation. We propose that KSR interacts with a regulatory Raf molecule in cis to induce a conformational switch of MEK, facilitating MEK's phosphorylation by a separate catalytic Raf molecule in trans.     

人p16~(INK4) cDNA探针在非小细胞肺癌研究中的应用

制备总RNA ,RT PCR克隆p16 INK4 cDNA ,测序验证 ,制备探针 .进行PCR产物Southern杂交检测非小细胞肺癌组织标本中p16 INK4 基因第二外显子阴性杂交率为 12 .9% (4 / 31) .原位杂交显示p16 INK4 基因转录阴性率为2 2 .6 % (7/ 31) .结果说明克隆的p16 INK4 cDNA是正确的 ,可用于临床基因诊断 ,p16 INK4 基因变异及表达在非小细胞肺癌的发生、发展中起作用     

Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice.

CDKN2A (INK4a/ARF) is frequently disrupted in various types of human cancer, and germline mutations of this locus can confer susceptibility to melanoma and other tumours. However, because CDKN2A encodes two distinct cell cycle inhibitory proteins, p16INK4a and p14ARF (p19Arf in mice), the mechanism of tumour suppression by CDKN2A has remained controversial. Genetic disruption of Cdkn2a(p19Arf) (hereafter Arf) alone predisposes mice to tumorigenesis, demonstrating that Arf is a tumour-suppressor gene in mice. We mutated mice specifically in Cdkn2a(p16Ink4a) (hereafter Ink4a). Here we demonstrate that these mice, designated Ink4a*/*, do not show a significant predisposition to spontaneous tumour formation within 17 months. Embryo fibroblasts derived from them proliferate normally, are mortal, and are not transformed by oncogenic HRAS. The very mild phenotype of the Ink4a*/* mice implies that the very strong phenotypes of the original Ink4a/ArfDelta2,3 mice were primarily or solely due to loss of Arf. However, Ink4a*/Delta2,3 mice that are deficient for Ink4a and heterozygous for Arf spontaneously develop a wide spectrum of tumours, including melanoma. Treatment of these mice with the carcinogen 7,12-dimethylbenzanthracene (DMBA) results in an increased incidence of melanoma, with frequent metastases. Our results show that, in the mouse, Ink4a is a tumour-suppressor gene that, when lost, can recapitulate the tumour predisposition seen in humans.     

毛冠鹿p16^INK4基因第二外显子序列分析

采用PCR扩增技术首次克隆毛冠鹿p16^INK4基因第二外显子，DNA杂交和序列分析发现，在307个碱基的第二外显子中仅有5个碱基的差异，同源性达98．4％，p16^INK4基因的第二外显子是高度保守的区域，在其功能中起重要作用。     

Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a

Stem-cell ageing is thought to contribute to altered tissue maintenance and repair. Older humans experience increased bone marrow failure and poorer haematologic tolerance of cytotoxic injury. Haematopoietic stem cells (HSCs) in older mice have decreased per-cell repopulating activity, self-renewal and homing abilities, myeloid skewing of differentiation, and increased apoptosis with stress. Here we report that the cyclin-dependent kinase inhibitor p16INK4a, the level of which was previously noted to increase in other cell types with age, accumulates and modulates specific age-associated HSC functions. Notably, in the absence of p16INK4a, HSC repopulating defects and apoptosis were mitigated, improving the stress tolerance of cells and the survival of animals in successive transplants, a stem-cell-autonomous tissue regeneration model. Inhibition of p16INK4a may ameliorate the physiological impact of ageing on stem cells and thereby improve injury repair in aged tissue.     

紫龙金对BGC-823细胞增殖的抑制作用及对p16~(INK4a)启动子活性的影响

利用记数法和Brdu脉冲标记法研究了紫龙金对细胞增殖的影响,western blot检测结果表明:紫龙金(3,4mg.mL-1)处理可明显抑制人胃癌BGC-823细胞的生长、提高BGC-823细胞中p16的表达水平.进一步利用含有p16INK4a启动子片段(-967~-165区域)及荧光素酶报告系统的载体pGL3-Basic-p16N研究了紫龙金对p16INK4a启动子活性的影响及其作用的分子机制,结果表明,紫龙金可能通过提高p16INK4a启动子活性而促进p16的表达,从而抑制细胞的增殖.     

钙调素拮抗剂TFP对BGC-823细胞增殖的影响及其分子机制的探讨

为了进一步探讨钙调素拮抗剂TFP对人胃癌细胞增殖的影响及其分子机制，利用MTT法及流式细胞光度术检测了TFP对细胞增殖的影响，进一步利用western blot的方法对细胞中p16^INK4a和cyclinD的表达水平进行了检测。结果表明：TFP处理可以明显提高BGC－823细胞中p16^INK4a的表达水平，而cyclinD1的水平则明显下降，提示TFP可能通过影响p16^INK4a的表达水平抑制细胞的增殖。     

PML regulates p53 acetylation and premature senescence induced by oncogenic Ras

The tumour suppressor p53 induces cellular senescence in response to oncogenic signals. p53 activity is modulated by protein stability and post-translational modification, including phosphorylation and acetylation. The mechanism of p53 activation by oncogenes remains largely unknown. Here we report that the tumour suppressor PML regulates the p53 response to oncogenic signals. We found that oncogenic Ras upregulates PML expression, and overexpression of PML induces senescence in a p53-dependent manner. p53 is acetylated at lysine 382 upon Ras expression, an event that is essential for its biological function. Ras induces re-localization of p53 and the CBP acetyltransferase within the PML nuclear bodies and induces the formation of a trimeric p53-PML-CBP complex. Lastly, Ras-induced p53 acetylation, p53-CBP complex stabilization and senescence are lost in PML-/- fibroblasts. Our data establish a link between PML and p53 and indicate that integrity of the PML bodies is required for p53 acetylation and senescence upon oncogene expression.