p16INK4a基因的功能及其调控

p16INK4a蛋白能抑制CDK4和CDK6的活性,使pRb处于非磷酸化或低磷酸化状态而能与转录因子E2Fs结合,从而抑制DNA 的合成,阻止细胞由G1期进入S期.p16INK4a的表达受Ets1和Ets2的正调控,受Bmi-1的负调控.p16INK4a基因缺失、突变、甲基化、RNA剪接加工错误可导致细胞周期失控和癌变.应用p16INK4a对某些肿瘤进行基因治疗的研究正在进行中.     

The physiology of p16INK4A-mediated G1 proliferative arrest

Phosphorylation of the product of the retinoblastoma susceptibility gene (Rb) physiologically inactivates its growth-suppressive properties. Rb phosphorylation is mediated by cyclin-dependent kinases (CDKs), whose activity is enhanced by cyclins and inhibited by CDK inhibitors. p16^INK4A is a member of a family of inhibitors specific for CDK4 and CDK6. p16^INK4A is deleted and inactivated in a wide variety of human malignancies, including familial melanomas and pancreatic carcinoma syndromes, indicating that it is an authentic human tumor suppressor. Although one mechanism for its tumor suppression may be prevention of Rb phosphorylation, thereby causing G1 arrest, many normal cell types express p16^INK4A, and are still able to traverse the cell cycle. In a search for other mechanisms, we have found that p16^INK4A is required for p53-independent G1 arrest in response to DNA-damaging agents, including topoisomerase I and II inhibitors. Thus, like other tumor suppressors, p16^INK4A plays an essential role in a DNA-damage checkpoint that leads to cell cycle arrest.     

p16INK4a-induced senescence is disabled by melanoma-associated mutations

The p16(INK4a)-Rb tumour suppressor pathway is required for the initiation and maintenance of cellular senescence, a state of permanent growth arrest that acts as a natural barrier against cancer progression. Senescence can be overcome if the pathway is not fully engaged, and this may occur when p16(INK4a) is inactivated. p16(INK4a) is frequently altered in human cancer and germline mutations affecting p16(INK4a) have been linked to melanoma susceptibility. To characterize the functions of melanoma-associated p16(INK4a) mutations, in terms of promoting proliferative arrest and initiating senescence, we utilized an inducible expression system in a melanoma cell model. We show that wild-type p16(INK4a) promotes rapid cell cycle arrest that leads to a senescence programme characterized by the appearance of chromatin foci, activation of acidic beta-galactosidase activity, p53 independence and Rb dependence. Accumulation of wild-type p16(INK4a) also promoted cell enlargement and extensive vacuolization independent of Rb status. In contrast, the highly penetrant p16(INK4a) variants, R24P and A36P failed to arrest cell proliferation and did not initiate senescence. We also show that overexpression of CDK4, or its homologue CDK6, but not the downstream kinase, CDK2, inhibited the ability of wild-type p16(INK4a) to promote cell cycle arrest and senescence. Our data provide the first evidence that p16(INK4a) can initiate a CDK4/6-dependent autonomous senescence programme that is disabled by inherited melanoma-associated mutations.     

小鼠p16~(INK4a)基因位点的结构和功能研究

p1 6INK4a基因的失活与多种肿瘤的发生和发展有联系。通过筛选小鼠基因组文库 ,获得长度为 1 4.5kb的p1 6INK4a基因组DNA片段。对上述 1 4.5kbDNA测序后进行生物信息学分析表明 :该片段包含 3个外显子 ,编码 1个由 1 68个氨基酸残基组成的多肽 ,其相对分子质量的理论计算值为 1 7941 ,有 7个可能的磷酸化位点 ,说明p1 6INK4a蛋白的功能可能受到磷酸化的调控。该DNA片段的非编码区分布着大量短散布元件、长散布元件和简单重复序列 ,这样的结构为转座和同源重组提供了结构基础 ,提示了部分肿瘤细胞中p1 6INK4a基因缺失的可能原因。对第一外显子序列与已发表的相应序列比较发现其DNA序列和所编码的多肽存在多态性     

p16INK4A is a robust in vivo biomarker of cellular aging in human skin

The cell-cycle regulating gene, p16INK4A, encoding an inhibitor of cyclin-dependent kinases 4 and 6, is considered to play an important role in cellular aging and in premature senescence. Although there is an age-dependent increase of p16INK4A expression in human fibroblast senescence in vitro, no data are available regarding the age dependency of p16INK4A in vivo. To determine whether p16INK4A expression in human skin correlates with donor age, p16INK4A expression was analyzed by immunohistochemistry as well as the expression of the p16INK4A repressor BMI1. Samples from the age groups 0-20, 21-70, and 71-95 years were selected from a bank of healthy human skin. We show that the number of p16INK4A positive cells is significantly higher in elderly individuals compared to the younger age groups. The number of p16INK4A positive cells was found to be increased in both epidermis and dermis, compartments with strictly different proliferative activities. BMI1 gene expression was significantly down-regulated with increasing donor age, whereas no striking age differences were observed for Ki67. In immunofluorescence co-expression studies, Ki67-positive cells were negative for p16INK4A and BMI1-expressing cells also stained negatively for Ki67. In conclusion, we provide for the first time evidence that p16INK4A expression directly correlates with chronological aging of human skin in vivo. p16INK4A therefore is a biomarker for human aging in vivo. The data reported here suggest a model for changes in regulatory gene expression that drive aging in human skin.     

Expression and characterization of Syrian golden hamster p16, a homologue of human tumor suppressor p16 INK4A

The p16(INK4A)/CDKN2A tumor suppressor gene is known to be inactivated in up to 98% of human pancreatic cancer specimens and represents a potential target for novel therapeutic intervention. Chemically induced pancreatic tumors in Syrian golden hamsters have been demonstrated to share many morphologic and biological similarities with human pancreatic tumors and this model may be appropriate for studying therapies targeting p16(INK4A)/CDKN2A. The purpose of this study was to investigate the fundamental biochemistry of hamster P16 protein. Using both in vivo and in vitro approaches, the CDK4 binding affinity, kinase inhibitory activity, and thermodynamic stability of hamster and human P16 proteins were evaluated. Furthermore, a structural model of hamster P16 protein was generated. These studies demonstrate that hamster P16 protein is biochemically indistinguishable from human P16 protein. From a biochemical perspective, these data strongly support the study of p16-related pancreatic oncogenesis and cancer therapies in the hamster model.     

p16INK4a deficiency promotes DNA hyper‐replication and genetic instability in melanocytes

Activated oncogenes restrict cell proliferation and transformation by triggering a DNA damage‐dependent senescence checkpoint in response to DNA hyper‐replication. Here, we show that loss of the p16^INK4a cyclin‐dependent kinase inhibitor and melanoma tumour suppressor facilitates a DNA damage response after a hyper‐replicative phase in human melanocytes. Unlike cells expressing activated oncogenes, however, melanocytes depleted for p16^INK4a display enhanced proliferation and an extended replicative lifespan in the presence of replication‐associated DNA damage. Analysis of human benign naevi confirmed that DNA damage and loss of p16^INK4a expression co‐segregate closely. Thus, we propose that loss of p16^INK4a facilitates tumourigenesis by promoting the proliferation of genetically unstable cells.     

Prevalence of aberrant methylation of p14ARF over p16INK4a in some human primary tumors

The INK4a/ARF locus encodes two unrelated tumor suppressor proteins, p16INK4a and p14ARF, which participate in the two main cell-cycle control pathways, p16–Rb and p14–p53. Methylation of CpG promoter islands has been described as a mechanism of gene silencing. Exon 1 of the p16INK4a gene and the p14ARF promoter gene reside within CpG islands. Therefore, both can become methylated de novo and silenced. It has recently been proposed that the methylation changes in certain genes could be used as molecular markers for the detection of almost all forms of human cancer. Here, we analyzed concomitantly in each tumor sample and normal tissue the methylation status of p16INK4a and p14ARF by methylation-specific PCR (MSP) in 100 breast, 95 colon and 27 bladder carcinomas. A series of clinicopathological parameter were obtained from the medical records of the patients, p14ARF showed a higher rate of hypermethylation than p16INK4a in all three tumor types. p16INK4a and p14ARF aberrant methylation was significantly correlated with poor prognosis clinicopathological parameters of the three tumor types. We conclude that both p16INKa and p14ARF hypermethylation may be involved in breast, colon and bladder carcinogenesis, with special emphasis on the role of the lesser studied p14ARF gene, and that tumors with aberrant methylation in the two genes were associated with worse prognosis.     

p16INK4a在早孕小鼠子宫内膜的表达及其对胚泡着床的影响

本研究旨在检测肿瘤抑制基因p16INK4a(inhibitor of cyclin-dependent kinase 4a)在早孕小鼠子宫内膜中的表达规律,探讨p16INK4a在小鼠胚胎着床过程中的作用.采用荧光定量PCR(FQ-PCR)和免疫组织化学方法分别检测未孕小鼠及孕小鼠第2、3、4、5、7天子宫内膜p16INK4a mRNA和蛋白的表达;子宫角注射p16INK4a抗体观察胚泡着床数.FQ-PCR结果显示孕小鼠子宫内膜组织p16INK4amRNA的表达高于未孕小鼠,且随着妊娠天数的增加呈现表达逐渐增强的趋势,到妊娠第5天达到最高,后渐降.免疫组织化学分析显示p16INK4a蛋白在子宫内膜的表达规律与mRNA结果一致.子宫角注射p16INK4a抗体后胚泡着床数明显减少.以上结果提示,P161INK4a在妊娠早期子宫内膜持续表达,可能参与胚泡着床.     

5-Fluorouracil or gemcitabine combined with adenoviral-mediated reintroduction of p16INK4A greatly enhanced cytotoxicity in Panc-1 pancreatic adenocarcinoma cells

BACKGROUND: Pancreatic cancer is one of the most lethal of all the common gastrointestinal malignancies. Although surgery offers the best chance for survival, it is not appropriate for all cases. The only adjuvant treatment to show promise is chemotherapy. Hence new treatments are urgently sought. We previously reported that adenoviral (Ad)-mediated delivery of p53 (Adp53) and p16(INK4A) (Adp16) significantly inhibited the growth of pancreatic cancer cell lines and established subcutaneous pancreatic tumours in nude mice (Ghaneh P, et al. Adenovirus mediated transfer of p53 and p16INK4A results in pancreatic cancer regression in vitro and in vivo. Gene Ther 2001; 8: 199-208). In this study we examine whether combining Ad-mediated delivery of p53 or p16(INK4A) with clinically relevant chemotherapeutic drugs has therapeutic potential for pancreatic cancer. METHODS AND RESULTS: Four pancreatic adenocarcinoma cell lines were evaluated for their sensitivity to 5-fluorouracil (5-FU) and gemcitabine and two of these, Suit-2 and Panc-1, were chosen for combination experiments because they showed moderate and poor sensitivity, respectively, to 5-FU and gemcitabine. We found no evidence for enhanced cytotoxicity when either cell line was transduced with Adp53 before or after incubation with chemotherapeutic drugs. In contrast, incubation of Panc-1 cells with either 5-FU or gemcitabine followed by Ad-mediated overexpression of p16(INK4A) resulted in a substantial reduction in cell viability under conditions where the drugs alone had minimal cytotoxicity. Incubation of Suit-2 cells with 5-FU followed by Ad-mediated overexpression of p16(INK4A) also resulted in a significant reduction in cell viability. This, however, was observed only with higher concentrations of 5-FU and viral vector. Cell cycle analysis of Panc-1 cells showed that the combination of cytotoxic drugs and Adp16 resulted in an increase in the sub-G1 population suggesting an increase in apoptosis. Dual labelling of these cells with annexin V and propidium iodide (PI) confirmed that the combination of 5-FU and Adp16 resulted in a significant increase in early apoptotic cells (annexin V positive and PI negative) compared with controls. Moreover, overexpression of p16(INK4A) was associated with a reduction in pRb levels in these cells-high levels of pRb have been proposed to contribute to chemoresistance in pancreatic cancer cells. CONCLUSIONS: We have shown that the currently used chemotherapeutic drugs for pancreatic adenocarcinoma combined with restoration of p16(INK4A) expression hold promise for the adjuvant treatment of this disease. Importantly, the combination facilitated the use of chemotherapeutic drugs at lower concentrations than would otherwise be effective.     

The number of p16INK4a positive cells in human skin reflects biological age

Cellular senescence is a defense mechanism in response to molecular damage which accumulates with aging. Correspondingly, the number of senescent cells has been reported to be greater in older than in younger subjects and furthermore associates with age-related pathologies. Inter-individual differences exist in the rate at which a person ages (biological age). Here, we studied whether younger biological age is related to fewer senescent cells in middle-aged individuals with the propensity for longevity, using p16INK4a as a marker for cellular senescence. We observed that a younger biological age associates with lower levels of p16INK4a positive cells in human skin.     

三氟拉嗪通过诱导p16INK4a抑制BGC-823细胞增殖

为探索三氟拉嗪（trifluoperazine, TFP）抗肿瘤作用机制,对胃癌BGC-823细胞进 行TFP（5、10 μmol/L）处理后,利用计数法、BrdU脉冲标记法、Western印迹等方法从细胞形态、细胞增殖、S期细胞百分比以及相关因子表达水平等方面进行分析. 结果显示,TFP处理后,细胞形态发生明显改变,细胞增殖受到明显抑制且呈时间计量 效应关系;S期细胞比例下降;p16INK4a表达水平升高.为进一步研究TFP诱导 p16INK4a表达的分子机制,本实验采用插入p16INK4a启动子片段及荧光素酶报告系统 的载体pGL3-Basic-p16INK4a（-967~-165 bp）,研究了TFP在转录水平对p16INK4a启 动子活性的影响.结果表明, TFP能够提高p16INK4a的启动子活性.上述结果提示,TFP 通过诱导p16INK4a表达抑制BGC-823细胞增殖.     

CBX7 and miR‐9 are part of an autoregulatory loop controlling p16INK4a

Polycomb repressive complexes (PRC1 and PRC2) are epigenetic regulators that act in coordination to influence multiple cellular processes including pluripotency, differentiation, cancer and senescence. The role of PRCs in senescence can be mostly explained by their ability to repress the INK4/ARF locus. CBX7 is one of five mammalian orthologues of Drosophila Polycomb that forms part of PRC1. Despite the relevance of CBX7 for regulating senescence and pluripotency, we have a limited understanding of how the expression of CBX7 is regulated. Here we report that the miR‐9 family of microRNAs (miRNAS) downregulates the expression of CBX7. In turn, CBX7 represses miR‐9‐1 and miR‐9‐2 as part of a regulatory negative feedback loop. The miR‐9/CBX7 feedback loop is a regulatory module contributing to induction of the cyclin‐dependent kinase inhibitor (CDKI) p16^INK4a during senescence. The ability of the miR‐9 family to regulate senescence could have implications for understanding the role of miR‐9 in cancer and aging.