脑胶质瘤中p14~(ARF)、MDM2及p53的表达及其意义

目的:探讨p14ARF、MDM2和p53蛋白在脑胶质瘤组织中的表达及p14ARF-MDM2-p53通路在胶质瘤进展中的意义。方法:应用免疫组化SP法检测25例Ⅱ级和23例Ⅳ级胶质瘤组织中-p14ARF、MDM2和-p53蛋白的表达,并分析其与胶质瘤组织学分级之间的关系。结果:Ⅱ级和Ⅳ级胶质瘤中,MDM2蛋白的阳性表达率分别为24.00%(6/25)及56.52%(13/23)(P=0.021),p53蛋白的阳性表达率分别为28.00%(7/25)及60.87%(14/23)(P=0.022),二者阳性表达率均随胶质瘤恶性程度的增加而升高,Spearman等级相关分析显示,MDM2、p53的表达与胶质瘤分级呈正相关;p14ARF的阳性表达率分别为76.00%(19/25)及34.78%(8/23)(P=0.004),其阳性表达率随胶质瘤恶性程度的增加而降低,Spearman等级相关分析显示,p14ARF的表达与胶质瘤分级呈负相关(P<0.05)。结论:脑胶质瘤组织中,MDM2和p53均呈不同程度的过度表达,且随恶性程度的增加表达水平增高;而p14ARF随恶性程度的增加而表达水平降低。MDM2扩增、p53突变及p14ARF蛋白低表达均与胶质瘤的进展有关。     

胶质瘤中p14ARF甲基化分析及其与p53表达的相关性研究

目的:探讨p14ARF甲基化与胶质瘤发生和预后的关系,及其与突变型p53 (mutant type p53,mtp53)蛋白表达的相关性.方法:采用甲基化特异性PCR(methylation specific polymerase chain reaction, MSP)法检测33例胶质瘤和12例正常脑组织中p14ARF的甲基化状况.免疫组织化学法检测58例胶质瘤和12例正常脑组织中p14ARF和mtp53蛋白的表达.结果: 胶质瘤与正常脑组织中p14ARF甲基化率分别为39.4%(13/33)和0(0/12),2组间差异有统计学意义(P<0.01).低级别组胶质瘤甲基化率(6/15)与高级别组(7/18)间差异无统计学意义(P>0.05).p14ARF甲基化情况与患者预后无明显相关性.mtp53蛋白在胶质瘤和正常脑组织中的阳性率分别为56.9%(33/58)和8.3% (1/12),2组间比较,差异有统计学意义(P<0.01),其表达在肿瘤高级别组中明显高于低级别组(P<0.05).胶质瘤中mtp53蛋白表达与p14ARF甲基化呈负相关(P<0.05).结论:p14ARF甲基化与胶质瘤发生密切相关,为胶质瘤发生的早期事件,检测p14ARF甲基化情况可作为胶质瘤早期诊断的分子生物学指标.     

脑胶质瘤组织p14ARF、p53和p21WAF1的表达及其意义

目的:探讨p14ARF、p53和p21WAF1蛋白在不同级别脑胶质瘤组织中的表达及其在胶质瘤发生、发展中的生物学意义.方法:应用免疫组化SP法检测Ⅱ级和Ⅳ级胶质瘤组织中p14ARF、p53和p21WAF1蛋白的表达,并分析其与胶质瘤组织学分级之间的关系.结果:Ⅱ级和Ⅳ级胶质瘤中,p53蛋白的阳性表达率分别为28.00%(7/25)及60.87%(14/23)(P＝0.022),其阳性表达率随胶质瘤恶性程度的增加而升高,Spearman等级相关分析显示,p53的表达与胶质瘤分级呈正相关(P<0.05);p21WAF1的阳性表达率分别为76.00%(19/25)及39.13%(9/23)(P＝0.010) ,p14ARF的阳性表达率分别为76.00%(19/25)及 34.78%(8/23)(P＝0.004),二者阳性表达率均随胶质瘤恶性程度的增加而降低,Spearman等级相关分析显示,p21WAF1、p14ARF的表达与胶质瘤分级呈负相关(P<0.05).结论:胶质瘤组织中,p53呈不同程度的过表达,且随胶质瘤恶性程度的增加而表达水平升高;p21WAF1、p14ARF随胶质瘤恶性程度的增加表达水平降低.突变型p53蛋白的过表达以及p21WAF1、p14ARF蛋白的低表达,可促进胶质瘤的发生、发展.     

p16蛋白和Ki-67抗原在人脑胶质瘤中的表达及其意义

目的　研究 p16蛋白及Ki 6 7抗原在人脑胶质瘤中的表达情况。方法　采用免疫组化方法检测 5 0例胶质瘤中 p16蛋白及Ki 6 7抗原的表达情况。结果　随着胶质瘤病理级别的升高p16蛋白的表达率逐渐降低 ,I IV级阳性率分别为 75 .0 % ,6 6 .7% ,4 1.7% ,2 5 .0 % ,低度恶性组 (I II级 )与高度恶性组 (III IV级 )差异有显著性 (P <0 .0 5 )。p16蛋白与Ki 6 7抗原的表达存在负相关 (r=- 0 .4 73P <0 .0 1)。结论　p16蛋白及Ki 6 7抗原的表达对分析、判断胶质瘤分级的适用性及恶性转化具有一定意义。     

中国人脑瘤中p16、p15基因缺失分析

目的　为了解 p16、p15基因与中国人脑肿瘤遗传易感性的关系。 方法　应用PCR、银染PCR SSCP及免疫组化法对 88例脑肿瘤及相应手术切除正常组织DNA标本 p16、p15基因进行研究。 结果　结果显示在不同病理分级脑胶质瘤中p16、p15基因缺失率分别为Ⅰ -Ⅱ级 4%、0 % ,Ⅲ级 3 2 %、2 8% ,Ⅳ级 3 6 %、2 7% ,p16、p15基因缺失主要见于Ⅲ -Ⅳ级肿瘤中 ;脑转移瘤中也检出缺失该基因 ( 2 /3 ) ,而原发性非胶质细胞瘤 (脑膜瘤、垂体瘤、听神经瘤等 )中未见p16、p15基因缺失 ,各类肿瘤标本PCR SSCP分析均未见 p16、p15基因点突变。 结论　表明国人脑瘤中p16、p15基因异常以缺失为主 ,p16、p15基因缺失在脑胶质瘤中发生频率较高 ,促进肿瘤细胞由良性向恶性演进 ,而对良性非胶质细胞瘤的发病没有作用。     

脑胶质瘤p15基因缺失及5′CPG岛甲基化研究

目的　探讨p15基因变异及其与脑胶质瘤的发生、恶性进展的关系。方法　利用PCR和PCR -based甲基化检测技术检测了5 6例脑胶质瘤中p15基因外显子 1缺失及 5′CPG岛甲基化情况。结果　 43例高级别的脑胶质瘤中 ,14例发生了p15基因缺失( 3 2 6% ) ,而 13例低级别的脑胶质瘤中无一例发生p15基因缺失 ,差异具有显著性 (P <0 0 5 )。 1例低级别的脑胶质瘤、3例高级别的脑胶质瘤发生了p15基因 5′CPG岛甲基化。结论　p15基因异常可能参与脑胶质瘤的发生、恶性进展。基因纯合缺失是脑胶质瘤中p15基因失活的主要机制。     

人脑胶质瘤p53基因突变及MDM2、p16、p53蛋白表达与临床病理特征相关性研究

目的:探讨p53突变在人脑胶质瘤发生发展中的作用及p53蛋白蓄积与突变的符合程度,并研究MDM2、p16、p53蛋白表达与胶质瘤临床病理特征的关系以及三者的相关性。方法:利用聚合酶链反应-单链构象多态性分析法(PCR-SSCP)及LSAB免疫组化法对已明确诊断的48例人脑胶质瘤进行p53基因突变以及MDM2、p16、p53蛋白表达的检测。结果:48例胶质瘤中20例p53蛋白呈阳性表达(41.7%)。PCR-SSCP检测发现17例(35.4%)呈现p53基因的单链构象多态性改变,均位于5～8外显子,突变例数依次为7(41.2%)、1(5.9%)、4(23.5%)、5(29.4%)。两种方法检测的符合率为89.6%(43/48)。MDM2、p53蛋白阳性率分别为22.9%、41.7%,p16表达缺失率为60.4%。在高级别(Ⅲ、Ⅳ级)的肿瘤中p53表达率及p16表达缺失率分别为63.2%、84.2%,均明显高于低级别(Ⅱ级)的肿瘤(分别为27.6%、44.8%)(P<0.05)。在不同分级的胶质瘤中,MDM2表达率及阳性程度没有显著差异。在p53阳性表达的病例中常伴有p16的表达缺失(57.6%),而且大多出现在Ⅲ、Ⅳ级肿瘤中(9/12)。p53突变、三种分子的表达均与患者的年龄、性别、肿瘤的大小及发病部位无相关性。结论:胶质瘤中p53基因的突变与胶质瘤的发生及恶性进展相关,p53蛋白蓄积与突变率较一致。MDM2基因异常参与胶质瘤的发生,是其形成的早期事件;p16、p53在胶质瘤中的异常表达与肿瘤的分化程度相关。     

脑胶质瘤中p16蛋白与PCNA表达及其临床意义

目的　探讨p16蛋白缺失与脑胶质瘤的恶性进展、增殖能力及预后的关系。方法　应用免疫组织化学方法 ,检测了96例不同级别脑胶质瘤存档标本 p16蛋白及增殖细胞核抗原的表达情况。 结果　p16蛋白缺失频率随脑胶质瘤级别的增高而增高 (P <0 .0 1) ;4对复发时级别增高的肿瘤组织均发生了 p16蛋白缺失 ,而另 3对复发时仍保持原来级别者无一发生p16蛋白缺失。同一级别的脑胶质瘤 ,p16蛋白缺失者的PCNA指数明显高于无 p16蛋白缺失者 (P <0 .0 5 )。 结论　p16蛋白缺失与脑胶质瘤的恶性进展及增殖能力有关 ,p16蛋白免疫组织化学检测可作为反映脑胶质瘤预后的 1个指标。     

脑胶质瘤组织中MDM2、p53、p14ARF和p21WAF1蛋白表达及意义

[目的]探讨MDM2、p53、p14ARFF和p21WAF1九蛋白在胶质瘤组织中的表达及意义.[方法]应用免疫组化SP法检测胶质瘤组织中MDM2、p53、p14ARF和p21WAF1蛋白的表达情况,并分析其与胶质瘤组织学分级之间的关系.[结果]Ⅱ级和Ⅳ级胶质瘤中,MDM2阳性表达率分别为24.00%(6/25)及56.52%(13/23)(χ2=5.298,P=0.021);p53阳性表达率分别为28.00%(7/25)及60.87%(14/23)(χ2=5.259,P=0.022).p14ARF的阳性表达率分别为76.00%(19/25)及34.78%(8/23)(χ2=8.270,P=0.004),p21WAF1的阳性表达率分别为76.00%(19/25)及39.13%(9/23)(χ2=6.700,P=0.010).[结论]胶质瘤组织中,MDM2和p53均呈不同程度的过度表达,且随胶质瘤恶性程度的增加而表达水平增高,但p14ARF and p21WAF1蛋白随胶质瘤恶性程度的增加而表达降低.MDM2过表达、p53突变以及p14ARF、p21WAF1蛋白表达降低与胶质瘤的演进有关.     

人脑胶质瘤中PTEN基因突变和蛋白表达的研究

目的 :研究人脑胶质瘤中PTEN蛋白表达和PTEN基因突变情况。方法 :应用免疫组化技术和聚合酶链反应 -单链构象多态性分析 (PCR SSCP)技术 ,检测 10 2例人脑胶质瘤中PTEN蛋白表达及PTEN基因突变。结果 :PTEN阳性染色主要定位于细胞质中。 10 2例中PTEN蛋白表达 5 5 / 10 2(5 3 9% ) ,高分化组 (Ⅰ级和Ⅱ级 ) 32 / 39(82 1% )与低分化组 (Ⅲ级和Ⅳ级 ) 2 3/ 6 3(36 5 % )之间差异有极显著意义 ,P <0 0 1;4 2例胶质母细胞瘤中共有 11例发生PTEN基因突变 ,突变率为 2 6 % (11/ 4 2 ) ;6 0例其他胶质瘤中仅 1例发生突变 ,胶质母细胞瘤突变率显著高于其他胶质瘤 ,χ2 =11 6 2 ,P <0 0 1。结论 :PTEN基因突变或缺失在人脑胶质瘤的发生发展中起重要作用 ,与肿瘤恶性分化程度密切相关     

脑胶质瘤p 15 基因缺失及5′CPG 岛甲基化研究

 目的　探讨 p1 5基因变异及其与脑胶质瘤的发生、恶性进展的关系。方法　利用 PCR和 PCR- based甲基化检测技术检测了 56例脑胶质瘤中 p1 5基因外显子 1缺失及 5′CPG岛甲基化情况。结果　 43例高级别的脑胶质瘤中 ,1 4例发生了 p1 5基因缺失 ( 32 .6% ) ,而 1 3例低级别的脑胶质瘤中无一例发生 p1 5基因缺失 ,差异具有显著性 ( P<0 .0 5)。 1例低级别的脑胶质瘤、3例高级别的脑胶质瘤发生了 p1 5基因 5′CPG岛甲基化。结论　 p1 5基因异常可能参与脑胶质瘤的发生、恶性进展。基因纯合缺失是脑胶质瘤中 p1 5基因失活的主要机制.     

DELETION AND 5'CPG ISLAND METHYLATION OF p15 GENE IN BRAIN GLIOMA

P15geneisananothertumorsuppressorgene,whichislocatedongpZIandadjacenttopl6gene.Itencodesplsproteinthathasbiochemicalfunctionssimilartothoseofp16protein.Therehavebeenreportsaboutabnormalityofplsgeneinbrainglioma.['-']Inthisstudy,wedetecteddeletionand5'CPGislandmethylationofp15genein56casesofbraingliomabythemethodsofPCRandPCR-basedmethylationtoinvestigatethecorrelationbetweenabnormalityofplsgeneandoccurrenceormalignantprogressionofbrainglioma.MATERIALSANDMETHODSSpecimensFifty-sixfre…     

Alterations of p14ARF and p16INK4a genes in salivary gland carcinomas

The p14ARF and p16INK4a genes are localized to 9p21, where genetic alterations have been reported to be frequent in various human neoplasms. To elucidate their status in salivary gland tumorigenesis, we analyzed a series of 36 salivary gland carcinomas (SGCs) using methylation-specific PCR, differential PCR and immunohistochemistry. Homozygous deletion (3 cases) or methylation (7 cases) of p14ARF was detected in 10 (28%) SGCs, one and three showing co-deletion and co-methylation of both p14ARF and p16INK4a genes, respectively. A total of 5 (14%) SGCs demonstrated homozygous deletion (1 case) or methylation (4 cases) of p16INK4a, all but one being adenoid cystic carcinomas. Immunohistochemical study revealed loss of p14ARF and p16INK4a expression in 11 samples (31%), correlating with the gene status. These results indicate that inactivation of p14ARF and p16INK4a genes by either homozygous deletion or promoter hypermethylation may be important for the molecular pathogenesis of salivary malignant tumors, and provide clear evidence that epigenetic changes like methylation are related to salivary gland carcinogenesis.     

p14ARF和p53在子宫内膜癌组织中的表达及其临床意义

目的研究子宫内膜癌组织中p14ARF和p53的表达及意义.方法采用免疫组化LSAB法,检测118例子宫内膜癌及30例正常宫内膜组织中p14ARF及p53的表达.结果p14ARF阴性、弱阳性、中度阳性和强阳性表达率在子宫内膜癌中分别为25.4%(30/118)、38.1%(45/118)、14.4%(17/118)和22.0%(26/118),与正常内膜0(0/30)、56.7%(17/30)、33.3%(10/30)和10.0%(3/30)相比差异有统计学意义,P<0.001,子宫内膜癌中p14ARF表达缺失的比例高于正常宫内膜;p14ARF表达水平与手术病理分期、宫颈间质受累和淋巴结转移相关;与病理分级、组织学类型和肌层侵犯深度无关;不同p14ARF表达水平患者生存率差异无统计学意义,P=0.578 1.p53阴性、弱阳性、中度阳性和强阳性表达率在子宫内膜癌中分别为13.6%(16/118)、40.1%(48/118)、28.0%(33/118)和17.8%(21/118);与正常内膜相比分别为36.7%(11/30)、36.7%(11/30)、16.2%(5/30)和10.0%(3/30)差异有统计学意义,P=0.01,子宫内膜癌中p53阳性表达的比例高于正常宫内膜;p53表达与肿瘤分期、盆腔淋巴结转移和肌层侵犯深度相关,与病理分级、组织学类型和宫颈间质受累无关;不同p53表达水平患者生存率差异无统计学意义,P=0.416 6;在子宫内膜癌组织中p14ARF与p53表达呈负相关,r＝-0.243,P＝0.008.结论p14ARF和p53异常表达与子宫内膜癌发生密切相关,但对其预后的影响有待进一步的研究.     

Ewing sarcomas with p53 mutation or p16/p14ARF homozygous deletion: a highly lethal subset associated with poor chemoresponse.

PURPOSE: EWS-FLI1 fusion type, p53 mutation, and homozygous deletion of p16/p14ARF have each been shown to be prognostically significant in Ewing sarcoma (ES). We provide the first combined prognostic analysis of these three molecular parameters in ES. PATIENTS AND METHODS: We studied 60 patients with ES (stage: localized in 54, metastatic in six). All cases were confirmed to contain the EWS-FLI1 (29 type 1, 12 type 2, 14 other types) or EWS-ERG fusions (five cases). Homozygous deletion of p16/p14ARF, and p53 mutations were determined by fluorescent in situ hybridization and Affymetrix (Santa Clara, CA) p53 GeneChip microarray hybridization, respectively. RESULTS: Eight cases (13.3%) contained point mutations of p53, and eight cases (13.3%) showed p16/p14ARF deletion, including one case with both alterations. Among 32 cases with data on histologic chemoresponse, all 10 with alterations in p53 or p16/p14ARF showed a poor chemoresponse (P = .03). Variables predicting poorer overall survival included p53 mutation alone (P < .001), either p53 or p16/p14ARF alteration (P < .001), and stage (P < .01). In multivariate analysis, alterations of p53 and/or p16/p14ARF as a single variable, was the most adverse prognostic factor (P < .001), followed by stage (P = .04). In a multivariate analysis with alterations of p53 and p16/p14ARF as separate variables, both were significant (P < .001 and P = .03, respectively). Six cases with p16/p14ARF deletion were also studied for co-deletion of the contiguous methylthioadenosine phosphorylase gene, and this was detected in four cases. CONCLUSION: Alterations in p53 or p16/p14ARF are found in a fourth of ES cases and define a subset with highly aggressive behavior and poor chemoresponse.     

Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a genes in human esophageal squamous cell carcinoma.

The 9p21 gene cluster, harboring growth suppressive genes p14ARF, p15INK4b, and p16INK4a, is one of the major aberration hotspots in human cancers. It was shown that p14ARF and p16INK4a play active roles in the p53 and Rb tumor suppressive pathways, respectively, and p15INK4b is a mediator of the extracellular growth inhibition signals. To elucidate specific targets and aberrations affecting this subchromosomal region, we constructed a detailed alteration map of the 9p21 gene cluster by analyzing homozygous deletion, hypermethylation, and mutation of the p14ARF, p15INK4b, and p16INK4a genes individually in 40 esophageal squamous cell carcinomas (ESCCs) and compared the genetic alterations with mRNA expression in 18 of these samples. We detected aberrant promoter methylation of the p16INK4a gene in 16 (40%), of p14ARF in 6 (15%), and of p15INK4b in 5 (12.5%) tumor samples. Most p16INK4a methylations were exclusive, whereas all but one of the p14ARF/p15INK4b methylations were accompanied by concomitant p16INK4a methylation. We detected homozygous deletion of p16INK4a in 7 (17.5%), of p14ARF-E1beta in 13 (33%), and of p15INK4b in 16 (40%) tumor samples. Most deletions occurred exclusively on the E1beta-p15INK4b loci. Two samples contained p14ARF deletion but with p16INK4a and p15INK4b intact. No mutation was detected in the p14ARF and p16INK4a genes. Comparative RT-PCR showed good concordance between suppressed mRNA expression and genetic alteration for p15INK4b and p16INK4a genes in the 18 frozen samples, whereas 5 of the 13 cases with suppressed p14ARF mRNA expression contained no detectable E1beta alteration but aberrations in the p16INK4a locus. Our results show that in human ESCCs, p14ARF is a primary target of homozygous deletion along with p15INK4b, whereas p16INK4a is the hotspot of hypermethylation of the 9p21 gene cluster. The frequent inactivation of the p14ARF and p16INK4a genes may be an important mechanism for the dysfunction of both the Rb and p53 growth regulation pathways during ESCC development.     

外源型P14ARF基因对人脑胶质瘤细胞生长的影响

目的：研究Ｐ１４ＡＲＦ基因对人脑胶质瘤细胞株（Ｕ－２５１）生长的影响。方法：用脂质体介导携带Ｐ１４ＡＰＦ的真核表达载体转染Ｕ－２５１细胞,观察其对该细胞株的生长、增殖周期等生物学行为的影响。结果：导入Ｐ１４ＡＰＦ胶质瘤细胞Ｕ－２５１的生长有明显的抑制作用。     

Alterations of the p16(INK4) locus in human malignant mesothelial tumors

The INK4 locus has two promoters and encodes two unique proteins that share exons in different reading frames, p16(INK4a) and p14(ARF). The p16(INK4a) protein, by inhibiting cyclin-dependent kinase, down regulates Rb-E2F and leads to cell cycle arrest in the G1 phase. The p14(ARF) protein interacts with the MDM2 protein, neutralizing MDM2-mediated degradation of p53. Since p53/Rb genes are not altered in malignant mesothelioma, additional components of these pathways, such as p16(INK4a) and p14(ARF), are candidates for inactivation. In this study, we have examined p16(INK4a) and p14(ARF) alterations (gene deletion, mutation and promoter methylation) in 45 primary malignant mesothelioma specimens. Fourteen patients (31%) had altered p16; four tumors had a methylated promoter region (8.8%), 10 tumors showed p16 to be deleted (22.2%), and one tumor had a point mutation (2%). We did not find any instances of methylation in the p14(ARF) 5'-CpG island. Patients whose tumors had p16 deletion were significantly younger than those with methylation, and, in the patients whose lungs were studied for the prevalence of asbestos fibers, those with any p16 alteration had lower fiber counts than those with no p16 alteration. Hence, p16 gene alteration is relatively common in malignant mesothelioma, while p14(ARF) is rarely, if ever, methylated. Our data suggest that deletion of p16 occurs in a relatively susceptible subset of the population.     

p53 gene mutation and ink4a-arf deletion appear to be two mutually exclusive events in human glioblastoma

P16 and P14ARF are two tumor suppressors encoded by the locus ink4a-arf which is frequently deleted in human tumors. Recent experiments performed with mouse embryonic fibroblasts have shown that P14ARF is an upstream regulator of the P53 pathway. This raises the question as to whether in human tumors the loss of p14arf and mutation of p53 are mutually exclusive events which segregate with genetic alterations at other loci. To examine this question we performed a multigenic analysis on 29 gliomas. We analysed p53 and p14arf in relation with five other genetic loci encoding the most frequently mutated genes in human gliomas: cdkn2a, mdm2, egfr, pten and the chromosomal regions 10q23.3 and 10q25-26. Our study shows for the first time that p53 mutations and p14arf deletions appear mutually exclusive in human glioblastoma, suggesting that they may be functionally redundant in glioma tumorigenesis. The P53 pathway is, therefore, disrupted in 81.8% of malignant gliomas (WHO grades III and IV), either by mutation of the p53 gene (31.8%) or by p14arf deletion (54.5%). These tumors further showed MDM2 overexpression (9.1%), egfr oncogene amplification/egfr overexpression (50%), pten mutations (27.3%) and loss of heterozygosity (LOH) at the chromosomal regions 10q23.3 (86.4%) and 10q25-26 (100%). These alterations did not segregate with p53 mutations or p14arf deletions, while p14arf and cdkn2a were always deleted.