p16INK4a基因的功能及其调控

p16^INK4a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.p16^INK4a的表达受Ets1和Ets2的正调控,受Bmi-1的负调控.p16^INK4a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p16^INK4a对某些肿瘤进行基因治疗的研究正在进行中.     

P16、P27蛋白在食管鳞状细胞癌中的表达及意义

目的为探讨P16、P27蛋白在食管鳞状上皮、增生上皮和癌变上皮中表达状况及其与鳞状细胞癌发生、进展和转移的相关性.方法采用SP免疫组织化学方法,检测72例(其中活检标本13例)食管癌组织中P16、P27蛋白的表达情况.结果 P16、P27在食管癌和增生性上皮均有阳性表达,但两者相比均无统计学意义(P>0.05),在正常上皮组均无阳性表达;P16、P27在高分化鳞癌组阳性表达率均显著高于低分化鳞癌组(P<0.05);P16、P27在伴有淋巴结转移的食管癌组与不伴有淋巴结转移组比较差异显著(P<0.05).P16在原发食管癌组阳性表达率和淋巴结转移癌中比较具有显著性差异(P<0.05).结论 P16、P27基因蛋白在食管癌组织中的表达与病理分化程度有关;P16的表达与转移癌的形成有关;P16、P27表达与患者性别、年龄、肿瘤发生部位、浸润深度无明显相关性.     

宫颈HPV16持续感染阶段宫颈P16和Ki67的表达及其与宫颈癌变的关系

目的:探讨宫颈人乳头状瘤病毒(HPV)16持续感染阶段宫颈P16和Ki67的表达及其与宫颈癌变的相关性。方法:采用P16/Ki67免疫组化双染法检测102例HPV16持续感染者、136例非持续感染者宫颈组织P16、Ki67蛋白的表达,并根据免疫组化结果分组为双染阳性组、双染阴性组。所有患者随访观察2年,比较两组患者的结局及宫颈癌前病变的发生率。结果:P16、Ki67及P16/Ki67双染的阳性率分别为40.3%、44.5%及34.0%,HPV16持续感染患者P16、Ki67及P16/Ki67双染的阳性率均显著高于非持续感染患者(P0.05)。HPV16持续感染患者的P16、Ki67蛋白表达呈显著正相关(P0.05)。HPV16持续感染患者中,双染阳性组的病情持续和进展比例明显高于双染阴性组,也明显高于HPV16非持续感染(双染阴性组、双染阳性组)患者(P0.05)。HPV16持续感染患者中,双染阳性组进展为HSIL及以上病变发生率为32.5%(13/40),显著高于双染阴性组6.5%(P0.05)。结论:P16,Ki67双染阳性在HPV16持续感染阶段与宫颈上皮内病变疾病进展成正相关,对HPV16持续感染进展为宫颈高度病变有预警价值,可作为HPV16阳性早期治疗的敏感指标。     

人脑胶质瘤CDK4、CyclinD1和P16基因蛋白表达的免疫组织化学研究

采用检测人脑胶质瘤中CDK4、CyclinD1和P16基因蛋白的表达情况,探讨G1→S期细胞周期调节蛋白在胶质瘤发生,发展过程中的作用。对39例近期手术的胶质瘤标本和8例瘤旁正常脑组织标本进行免疫组织化学检测。结果显示：CDK4蛋白在良性、交界性胶质瘤和瘤旁正常脑组织中的表达差异没有显性意义（P＞0.05）,在恶性胶质瘤中的表达却有明显地增高,与前两比较差异均有显性意义（P＜0.05）。P16蛋白在恶性胶质瘤中的表达显低于在瘤旁正常脑组织和良性、交界性胶质瘤中的表达（P＜0.01）,但在后两中的表达差异没有显性意义（P＞0.05）。CyclinD1蛋白在三中的表达水平均较低且差异无显性意义（P＞0.05）。结果表明：CDK4蛋白的表达增高和P16蛋白的表达下调发生在胶质瘤的较晚期阶段,与胶质瘤的恶性变和恶性胶质瘤的发生有关,而CyclinD1蛋白的表达可能与胶质瘤的发生和发展无关。     

外源基因转染导致小鼠体细胞过快衰老 及p16INK4a表达水平变化

对小鼠胎儿成纤维细胞进行外源基因转染时发现, 外源基因转染后的小鼠体细胞染色体端粒的长度以每代47 bp碱基缩短; 在转染后的衰老细胞中, 或细胞随着增龄, p16^INK4a 5′-调控区DNA甲基化程度逐渐降低; 利用RT-PCR与Northern blot证明, 衰老细胞与年轻细胞中的p16^INK4a基因的表达水平存在显著差异, 传代45代的细胞和外源基因转染后的衰老细胞p16^INK4a基因的表达水平大约是原代细胞的12~16倍, 而原代细胞与20代细胞间的差异很小。外源基因转染后的衰老细胞核移植后能支持克隆胚胎的体外早期发育。     

高危型人乳头瘤病毒感染后TopoⅡα和p16INK4A蛋白的表达及诊断价值

目的 探讨高危型人乳头瘤病毒(HR-HPV)感染后TopoⅡα和p16INK4A在宫颈组织中的表达及其对癌前病变的诊断价值.方法 选取在温州医学院附属二院行宫颈病变诊治的患者135例,包括各级宫颈上皮内瘤变(CIN)和宫颈鳞癌(SCC)的患者.随机选取同期就诊的慢性宫颈炎患者30例作为对照.运用第二代杂交捕获法(HC2)检测高危型人乳头瘤病毒(HR-HPV)的表达.活体提取组织,运用免疫组织化学法(SP法)检测宫颈组织中Topo Ⅱα和p16INK4A蛋白表达.结果 在HR-HPV阳性患者的宫颈组织中Topo Ⅱα和p16INK4A表达率高于HR-HPV阴性患者,差异有统计学意义.Topo Ⅱα和p16INK4A在正常宫颈上皮组织、各级CIN和SCC中的表达率逐渐增加,差异有统计学意义.p16INK4A用于筛查宫颈癌前病变(包括SCC)时阳性预测值99.3％.Topo Ⅱα用于筛查宫颈严重病变(CINⅢ和SCC)时阳性预测值67.9％.结论 HR-HPV感染后Topo Ⅱα和p16INK4A蛋白表达明显增高,并且Topo Ⅱα和p16INK4A蛋白表达率随着宫颈癌前病变的严重程度而上调,参与宫颈上皮的异常增殖和恶性转变.p16INK4A免疫组化检测提高了宫颈癌前病变筛查的特异性,Topo Ⅱα免疫组化检测提高了宫颈严重病变筛查的特异性,当Topo Ⅱα蛋白表达阳性,宫颈组织可能已发生不可逆性病变.     

宫颈癌组织中MCM5和P16~(INK4A) mRNA的表达及其临床意义

目的:检测宫颈癌组织中微小染色体维持蛋白-5(minichromosome maintenance protein 5,MCM5)与P16INK4AmRNA的表达,并探讨其在宫颈癌中的临床意义。方法:采用实时荧光定量PCR(real-time PCR)检测40例宫颈鳞状细胞癌、15例低度宫颈上皮内瘤变(CINⅠ)、20例高度宫颈上皮内瘤变(CINⅡ-Ⅲ)中MCM5和P16INK4AmRNA的相对表达量,并以20例正常宫颈组织作为对照,分析其与宫颈癌临床病理特征的关系。结果:(1)随着宫颈病变程度的加重,MCM5和P16INK4AmRNA的表达量逐渐增高。宫颈癌组织中MCM5和P16INK4AmRNA的表达量分别是正常宫颈组织的(3.026±1.210)倍和(2.540±0.718)倍,差异具有统计学意义(P0.05)。宫颈癌组织中MCM5 mRNA的表达量明显高于CINⅠ、CINⅡ-Ⅲ(P0.05),CINⅠ、CINⅡ-Ⅲ中MCM5 mRNA的表达量均显著高于正常宫颈组织,差异具有统计学意义(P0.05),而CINⅠ与CINⅡ-Ⅲ比较差异无统计学意义(P0.05);宫颈癌组织中P16INK4AmRNA的表达量为正常宫颈组织的(2.54±0.86)倍,差异有统计学意义(P0.05),亦显著高于CINⅠ,差异具有统计学意义(P0.05),但与CINⅡ-Ⅲ比较差异无统计学意义(P0.05)。(2)在宫颈癌组织中,MCM5 mRNA的表达量与肿瘤的临床期别、分化程度显著相关(P0.01),但与患者的年龄无关(P0.05);P16INK4AmRNA的表达量与肿瘤的临床期别、年龄均无关(P0.05),但与肿瘤的分化程度相关(P0.01)。结论:MCM5、P16INK4A的高表达可能在宫颈癌的发展中起重要作用。MCM5基因检测有助于区分癌前病变和宫颈癌,有望成为宫颈癌肿瘤增生的新标志物。P16INK4A的检测在宫颈病变筛查中具有重要意义,有助于CIN的分级并预测转归,从而提高宫颈癌筛查率。     

P16^（INK4α）及Ki67在正常宫颈、慢性宫颈炎及宫颈上皮内瘤变中的表达及意义

目的探讨联合检测P16^INK4α、Ki67的表达在子宫颈上皮内瘤变（CIN）中的诊断价值。方法用免疫组化S-P法对10例正常宫颈、20例慢性宫颈炎、CINⅠ、CINⅡ、CINⅢ各30例中P16^INK4α和Ki67的表达进行检测。结果P16^INK4α在CIN的阳性表达明显高于与正常宫颈和宫颈慢性炎症组织中的表达,差异有显著性意义（P〈0.05）。ki67在CINⅠ、CINⅡ、CINⅢ的表达三者比较差异有显著性意义（P〈0.05）。结论P16^INK4α在判断是否有宫颈上皮内瘤变（CIN）时具有很高的特异性和敏感性,结合Ki67标记可以准确判断CIN级别。联合检测P16^INK4α和Ki67可以作为判断慢性宫颈炎与CIN的客观指标。     

HPV16+治疗性无佐剂蛋白疫苗—HPV16Z- Hsp65-E6/E7的构建、表达及纯化工艺研究

目的：研制针对我国宫颈癌高危相关的HPV16型的治疗性无佐剂蛋白疫苗。方法：应用PCR技术自我国山西宫颈癌高发现场分离到的毒株-HPV16z中获得E6/E7转化基因片段,自卡介苗菌株中克隆获得Hsp65基因片段,对E6/E7基因片段定点突变修饰,构建pET28a-Hsp65-E6/E7表达载体,在大肠杆菌BL21(DE3)中表达融合蛋白,并研究重组蛋白的纯化方案和工艺。结果：成功构建pET28a-Hsp65-E6/E7重组表达载体,E6/E7突变位点正确,融合蛋白在亲和层析柱上正确复性和初步纯化,经阴离子交换色谱纯化后蛋白纯度达到95%。结论：该研究为无佐剂治疗性重组蛋白疫苗Hsp65-E6/E7的进一步功能研究奠定了基础。     

Inactivation of p16INK4a in Primary Tumors and Cell Lines of Head and Neck Squamous Cell Carcinoma

Inactivation of the p16^INK4a gene by mutation and deletion is common in head and neck squamous cell carcinoma (HNSCC). The present study demonstrates that hypermethylation of the 5 CpG islands can serve as an alternative mechanism for the inactivation of the p16^INK4a gene in this tumor. We studied 11 HNSCC cell lines and 17 oral squamous cell carcinoma (OSCC) primary tumors for p16^INK4a gene status by protein/mRNA and DNA genetic/epigenetic analyses to determine the incidence of its inactivation. Our study indicates that: (1) inactivation of p16 protein is frequent in HNSCC cell lines (6/11, 54.5%) and OSCC primary tumors (15/17, 88.2%), (2) inactivation of p16^INK4a protein is commonly associated with the presence of gene alteration such as mutation, homozygous deletion and especially aberrant methylation, and (3) genomic sequencing of bisulfite-modified DNA shows that the carcinoma develops a heterogeneous pattern of hypermethylation.     

Identification and characterization of P15RS, a novel P15(INK4b) related gene on G1/S progression

To screen genes involved in P15(INK4b) regulation during cell cycle, differential display method was applied to compare mRNAs from G(1) synchronized cells of MLIK6, which overexpressed P15(INK4b) gene, and its control MLC2. By using this approach, 15 cDNA fragments that were preferentially expressed in MLIK6 cells, but not in MLC2 cells, were screened out. A novel gene named P15RS was identified with further analysis. Combining the sequence from DD-PCR, homology analysis against EST database and RACE, a 4,404 bp complete cDNA sequence of P15RS was generated. Sequence analysis revealed that P15RS cDNA encoded a 312-amino-acid peptide containing a RAR domain that is involved in regulation of nuclear pre-mRNA, which suggests that P15RS may be a nuclear regulation protein. Genomic sequence analysis demonstrated that human P15RS gene was localized on chromosome 18q12 with seven exons and six introns. Expressing antisense P15RS in MLIK6 cells can up-regulate the expression of cyclinD1 and cyclinE. These data indicate that P15RS may act as a negative regulator in G(1) phase.     

High Expression of SOX2 and OCT4 Indicates Radiation Resistance and an Independent Negative Prognosis in Cervical Squamous Cell Carcinoma

Radiotherapy (RT) as a preoperative or postoperative adjuvant or primary treatment is the most common management modality for locally advanced cervical cancer. Radioresistance of tumor cells remains a major therapeutic problem. Consequently, we aimed to explore if the stem cell biomarkers SOX2 and OCT4 protein could be used to predict radioresistance in patients with locally advanced cervical squamous cell carcinoma (LACSCC). These 132 patients were divided into two groups (radiation-resistant and radiation-sensitive groups) according to progress-free survival (PFS). Using pretreatment paraffin-embedded tissues, we evaluated SOX2 and OCT4 expression using immunohistochemical staining. The percentage of overexpression of SOX2 and OCT4 in the radiation-resistant group was much higher than that in the radiation-sensitive group (p<0.001 and p <0.001, respectively). The patients with high expression of SOX2 and OCT4 showed a shorter PFS than those with low expression. Our study suggests that the expression of SOX2 and OCT4 in tumor cells indicates resistance to radiotherapy and that these two factors were important predictors of poor survival in patients with LACSCC (hazard ratio [95% CI], 2.294 [1.013, 5.195] and 2.300 [1.050, 5.037], respectively; p=0.046 and p=0.037, respectively).     

Design and characterization of a hyperstable p16INK4a that restores Cdk4 binding activity when combined with oncogenic mutations

Cyclin-dependent kinase inhibitor p16(INK4a) is the founding member of the INK4 family of tumor suppressors capable of arresting mammalian cell division. Missense mutations in the p16(INK4a) gene (INK4a/CDKN2A/MTS1) are strongly linked to several types of human cancer. These mutations are evenly distributed throughout this small, ankyrin repeat protein and the majority of them disrupt the native secondary and/or tertiary structure, leading to protein unfolding, aggregation and loss of function. We report here the use of multiple stabilizing substitutions to increase the stability of p16(INK4a) and furthermore, to restore Cdk4 binding activity of several defective, cancer-related mutant proteins. Stabilizing substitutions were predicted using four different techniques. The three most effective substitutions were combined to create a hyperstable p16(INK4a) variant that is 1.4 kcal/mol more stable than wild-type. This engineered construct is monomeric in solution with wild-type-like secondary and tertiary structure and cyclin-dependent kinase 4 binding activity. Interestingly, these hyperstable substitutions, when combined with oncogenic mutations R24P, P81L or V126D, can significantly restore Cdk4 binding activity, despite the divergent features of each destabilizing mutation. Extensive biophysical studies indicate that the hyperstable substitutions enhance the binding activity of mutant p16 through several different mechanisms, including an increased amount of secondary structure and thermostability, reduction in exposed hydrophobic surface(s) and/or a reduced tendency to aggregate. This apparent global suppressor effect suggests that increasing the thermodynamic stability of p16 can be used as a general strategy to restore the biological activity to defective mutants of this important tumor suppressor protein.     

喉癌中p15、p16基因纯合缺失与EGFR基因扩增相关性研究

研究p15、p16基因缺失和EGFR基因扩增的相关性及其与喉癌发生、发展的关系。提取喉癌新鲜组织中基因组DNA,采用聚合酶链反应技术,分别对30例喉癌进行p15基因第2外显子(p15E2)和p16基因第2外显子(p16E2)进行纯合缺失研究;应用FISH方法进行喉癌实体瘤EGFR基因扩增研究。p15E2纯合缺失率为13．3％(4／30),p16E2纯合缺失率为16．7％(5／30),p15E2、p16E2共同缺失率为6．7％(2／30)。在30例喉癌实体瘤EGFR基因扩增频率为30％(9／30),扩增2～8倍。p15E2和p16E2纯合缺失以及二者共同缺失与EGFR基因扩增相关,可能引起细胞周期失控而导致细胞增殖紊乱,在喉癌的发生及恶性进展中发挥一定作用。     

Identification and characterization of a novel protein ISOC2 that interacts with p16INK4a

p16(INK4a) is a multiple tumor suppressor, playing an important role in proliferation and tumorigenesis. To screen the p16(INK4a)-associated proteins, we performed a yeast two-hybrid assay and identified a novel protein isochorismatase domain containing 2 (ISOC2). ISOC2 conserves in different species, and encodes 205 and 210 amino acids in human and mouse, respectively. The expression of ISOC2 in mouse is universal but predominantly in uterus, stomach, and urinary tract system. Interaction between ISOC2 and p16(INK4a) was verified using in vitro pull-down assays and in vivo co-immunoprecipitation. Confocal microscopy studies using green and cyan fluorescent fusion proteins determined that ISOC2 co-localizes with p16(INK4a). Over-expressed ISOC2 is able to inhibit p16(INK4a) in dose-dependent manner. Our data indicated that ISOC2 is a novel functional protein, which is able to bind and co-localize with a tumor suppressor gene p16(INK4a). Over-expressed ISOC2 inhibits the expression of p16(INK4a), suggesting that this novel gene may play a role during the tumor development by interacting with p16(INK4a).