Methylation of p14(ARF) gene in meningiomas and its correlation to the p53 expression and mutation.

We have previously reported the statistically significant correlation of immunohistochemical expression of MIB-1 and p53 proteins among benign, atypical, and anaplastic meningiomas and p53 protein expression was high in atypical and anaplastic meningiomas. In the present study, we analyzed 22 cases of meningiomas for mutation of p53 gene in its spectrum of exon 5 to 8 using automated genetic analyzer. We did not find any mutation of p53 in any of these cases, thus suggesting the p53 protein expression is wild type. We analyzed 72 cases of meningiomas for determining the methylation status of p14(ARF) gene and the immunohistochemical expression of MDM2 protein to explain p53 protein expression in these meningiomas. We found methylation of p14(ARF) gene in five of 58 cases of benign meningiomas (8.6%), two of 10 cases of atypical meningiomas (20%), and two of four cases of anaplastic meningiomas (50%). In absence of p53 gene mutation, the high percentage of p14(ARF) gene methylation in high-grade meningioma may have been responsible for accumulation of wild-type p53 protein. In addition, we also found the loss of MDM2 protein in high-grade meningiomas. These deregulations of p14-MDM2-p53 pathway may contribute to the malignant progression of meningioma.     

Immunohistochemical inactivation of p14ARF concomitant with MDM2 overexpression inversely correlates with p53 overexpression in oral squamous cell carcinoma

The CDKN2 gene encodes two structurally different proteins: a cyclin-dependent kinase inhibitor called p16, which regulates retinoblastoma protein (pRb)-dependent G1 arrest, and a cell cycle inhibitor designated p14ARF, which arrests cell growth in G1-S and also in G2-M. Whereas inactivation of p16 has been described as a frequent event in various cancers, including oral cancer, the current function of p14ARF is still poorly understood. A physical association between p14ARF and MDM2 blocks MDM2-induced p53 degradation, resulting in increased levels of p53, which in turn leads to cell cycle arrest. The present study immunohistochemically examined the expression of p16 and p14ARF together with pRb, MDM2 and p53 status in a series of oral cancers. The results showed that p14ARF was frequently absent in the oral cancers (15/37, 41%) as was p16 immunostaining. Concomitant immunopositivity for p14ARF and MDM2 overexpression was frequently observed in a subset of the cancers, whereas an inverse correlation between p14ARF and MDM2 expression and the diffuse staining of p53 was clearly detected. Moreover, the results showed that in most cases of oral cancer (35/37, 95%) at least one protein was altered, and lymph node metastasis was more frequent in the tumors with alterations in both the p16/pRb and p14ARF/p53 pathway (8/16, 50%) than in the tumors with one or no alteration of these two major pathways.     

大肠癌组织p14ARF与p53基因变异研究

目的：观察大肠癌组织p14ARF基因遗 传和表观遗传变化及p53基因突变状况，探讨两者改变的关系及p14ARF-p53通路功能 破坏在大肠癌发生中的作用。方法：应用PCR、直接测序、甲基化特异PC R和RT-PCR分别检测56例原发性大肠癌及相应癌旁正常组织p14ARF基因纯合性缺失、 突变、5′CpG岛甲基化、mRNA表达及p53基因突变状况。结果：①大肠 癌组织p14ARF总变异率为27% (15/56)，其中1例纯合性缺失，14例5′CpG岛甲基化,未见突变发生。②15例p14ARF基因变异的大肠癌组织其相应mRNA表达阴性（13例）或 低水平表达（2例），41例未发生基因变异的大肠癌组织和所有癌旁正常组织p14ARF mRNA均显示明显表达。③大肠癌组织p53突变率为48% (27/56)。④56例大肠癌组织中，12例 仅发生p14ARF变异，24例仅显示p53突变，3例同时有p14ARF 5′CpG岛甲基化 和p53突变，17例p14ARF和p53均无变异，p14ARF-p53通路总变异率为70%（39/ 56），p14ARF 5′CpG岛高甲基化与野生型p53状态有关（P＜0.05）。⑤低分化 腺癌组p14ARF变异率（44%，7/16）明显高于高中分化腺癌组（20%，8/40）（P＜ 0.05），但两组间p53突变率无显著差异（P＞0.05）。结论:①大肠癌p14ARF基因5′ CpG岛高甲基化是其表达失活的主要机制；②大肠癌p14 ARF基因高甲基化与p53基因突变可能是相互独立的事件，两者的失活可能各自出现于不同 的大肠癌亚组中；③p14ARF高甲基化或p53突变引起的p14ARF-p53通路功能 破坏在大肠癌的发生中具有重要作用。     

Overexpression of p16 and p14ARF is associated with human papillomavirus infection in cervical squamous cell carcinoma and dysplasia

The CDKN2 gene encodes two structurally different proteins: a cyclin-dependent kinase inhibitor, p16, which regulates retinoblastoma protein (pRb)-dependent G1 arrest, and a cell cycle inhibitor, p14ARF, which blocks MDM2-induced p53 degradation resulting in an increase in p53 levels that leads to cell cycle arrest. Recent studies have revealed that expression of p16 and p14ARF is influenced markedly by the status of pRb and p53, and p16 overexpression has been demonstrated in cervical neoplasia because of functional inactivation of pRb by the human papillomavirus (HPV) E7 protein. To clarify the p14ARF status and the relationship between p16/p14ARF and other cell cycle molecules in cervical carcinogenesis, immunohistochemical analysis of p16, p14ARF, p53 and MDM2 was performed on 65 samples of cervical and genital condylomatous and neoplastic lesions, including nine HPV-negative tumors. In most cervical cancers and preneoplastic lesions with HPV infection of high and intermediate risk, a marked overexpression of p14ARF as well as the p16 protein (i.e. dotted nuclear immunostaining) was observed. All condyloma acuminata except one and low-grade dysplasia with HPV infection of low risk, such as HPV 6, immunohistochemically showed completely negative staining for p14ARF, also seen in non-neoplastic and mesenchymal cells. Our results clearly show that the mode of p14ARF overexpression in cervical neoplastic cells with HPV association differs from that in cancers of other organs without HPV association, and the p14ARF overexpression may be attributable to a negative feedback result in the functional inactivation of the pRb and p53 proteins by HPV oncoproteins.     

Analysis of the p16INK4, p14ARF, p15, TP53, and MDM2 genes and their prognostic implications in osteosarcoma and Ewing sarcoma

We examined alterations of the p16INK4, p14ARF, p15, TP53, and MDM2 genes in 30 osteosarcomas and 24 Ewing sarcomas. Among 21 osteosarcomas and 24 Ewing sarcomas, p16INK4, p14ARF, and p15 abnormalities were found in 4 (19%), 2 (9%), and 3 (14%) osteosarcomas, respectively, and in 4 (17%), 3 (13%), and 4 (17%) Ewing sarcomas, respectively. The alterations of p16INK4, p14ARF, and p15 included homozygous deletions spanning all 3 genes, methylation of p16INK4 or p15, and a nonsense mutation of p16INK4, which simultaneously caused a missense mutation of p14ARF. Alterations of TP53 were found in 15 (50%) of 30 osteosarcomas and 1 (3%) of 24 Ewing sarcomas. None of the sarcomas showed MDM2 amplification. While TP53 abnormalities were far more frequent in osteosarcoma than in Ewing sarcoma, alterations of p16INK4, p14ARF, and p15 were present at similar frequencies in the two types of sarcoma. The event-free survival (EFS) was worse in Ewing sarcoma patients with p16INK4 and p14ARF mutation/deletion than in those without the mutation/deletion (P = 0.019), and EFS was worse in osteosarcoma patients with TP53 alterations than in those without TP53 alterations (P = 0.048). The different incidence of TP53 abnormalities in the 2 types of sarcoma may reflect differences of the molecular processes through which the 2 types of tumor develop.     

HDM2 overexpression and focal loss of p14/ARF expression may deregulate the p53 tumour suppressor pathway in meningeal haemangiopericytomas. Study by double immunofluorescence and laser scanning confocal microscopy

AIMS: To investigate the p53 pathway in meningeal haemangiopericytomas (MHPCs), p14/ARF, p53 protein expression and two wild-type (wt) p53-induced proteins (HDM2 and p21/WAF1) were studied in 18 MHPCs, 11 primary, four of them recurrent on one, one, two and four occasions. METHODS: Immunohistochemical detection of p14/ARF, p53, p21/WAF1, HDM2 and Ki67 proliferative index (PI) protein expression. RESULTS: Ki67 index was > 5% in eight out 18 cases (44.4%). The PI in recurrent cases increased with neoplastic progression. Simultaneous p53 and wt p53 transactivated gene (p21/WAF, HDM2) expression occurred in all cases. This argues against p53 mutation. HDM2 overexpression was observed in 10 cases (55.5%). Double-immunofluorescence staining and laser scanning confocal microscopy (LSCM) displayed HDM2 and p53 colocalization. This strongly suggests that HDM2 binds and inactivates p53 that could be pathogenic for MHPCs, by a different mechanism than point mutation. p14/ARF expression > 5% was observed in 12 cases (66.6%). A normal (diffuse) pattern of expression was seen in 13 cases (72.2%). Focal loss of expression was observed in five patients (27.7%): three primary cases and two recurrences. Therefore, p14/ARF down-regulation may also contribute to the development of MHPC. CONCLUSION: HDM2 overexpression, sometimes combined with focal loss of p14/ARF expression, may play a pathogenic role in MHPCs.     

Aberrant expression of pRb, p16, p14ARF, MDM2, p21 and p53 in stage I adenocarcinomas of the lung

Cancers are always associated with cell cycle abnormalities. To clarify the cell cycle abnormalities present in lung adenocarcinomas, various cell cycle regulatory proteins of both the pRb and p53 pathways were studied immunohistochemically in 50 cases of stage I adenocarcinoma of the lung. In regard to the pRb pathway, most adenocarcinomas showed frequent expression of both p16 and pRb proteins, and aberrant expression in the pRb pathway was observed in about one-quarter of stage I adenocarcinomas. In regard to the p53 pathway, the frequency of immunohistochemical positivity was 8% for p14ARF, 64% for MDM2, 20% for p53 and 24% for p21. In this pathway, the immunohistochemical profile of p14ARF-negative/MDM2-positive/p53-negative is characteristic of stage I adenocarcinoma of the lung. An inverse relationship was found between MDM2 and p53 protein and was associated with the differentiation status of stage I adenocarcinoma of the lung. Our results suggest that the disruption of the pRb and p53 pathways is frequently observed in the early stages of lung adenocarcinoma and might play an important role in the growth and differentiation of adenocarcinoma of the lung.     

Incidence of p14ARF gene deletion in high-grade adult and pediatric astrocytomas

The INK4a-ARF locus encodes 2 separate proteins through differential splicing of alternative first exons to produce p16INK4a (exon 1alpha) and p14ARF (exon 1beta) products in human cells. The p16INK4a protein inhibits the cyclin D-dependent kinases (CDK) that control the phosphorylation of the Rb protein and cell proliferation. The p14ARF gene product can complex with and sequester the MDM2 protein within the nucleus, thus modulating the activity of the p53 protein. Loss of p16INK4a expression would disrupt the retinoblastoma (Rb)/p16INK4a/cyclin D-dependent kinase (CDK4) pathway, whereas loss of p14ARF expression would inactivate both the Rb and p53/ MDM2/p14ARF pathways through MDM2, which can complex with either Rb or p53. Loss of the p16INK4a gene on 9p21 has been documented in a wide range of human tumors, including one third of glioblastomas. However, in tumors showing homozygous loss of exon 2 of the p16INK4a gene, loss of exon 1beta of the p14ARF gene has not been established. In this study, we have assessed deletion of the p14ARF gene in 29 pediatric and 107 adult high-grade astrocytomas and 9 glioma cell lines, using multiplex PCR analysis for exon 1beta. We found homozygous deletions for exon 1alpha and exon 1beta in 3 of 29 (10%) of the pediatric cases (2 grade III, 1 grade IV), 25 of 107 (23%) of the adult cases (6 grade III and 19 grade IV), and 8 of 9 (89%) of the glioma cell lines. Therefore, loss of the INK4a-ARF locus in high-grade astrocytomas may contribute to the highly malignant behavior and treatment resistance of these tumors through elimination of multiple checkpoint cell cycle control proteins.     

Molecular abnormalities of p53, MDM2, and H-ras in synovial sarcoma.

Forty-nine cases of synovial sarcoma were evaluated for mutation of the p53 gene, amplification of the MDM2 gene and mutation of the H-ras gene, and for the relation of these factors to overall survival and clinicopathologic parameters. All investigations were carried out on formalin-fixed paraffin-embedded materials. Furthermore, we evaluated the expression of p53 protein, MDM2, and p21(WAF1/CIP1) immunohistochemically in these cases, together with an assessment of proliferative activities using monoclonal antibody MIB-1. Nine of the 49 cases (18.4%) had p53 gene alteration detected by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct sequencing. Eleven cases (24%) showed nuclear accumulation of p53 protein in more than 10% of the tumor cells. Among them, only three cases contained gene mutations. There was no correlation between p53 nuclear accumulation and p53 gene alteration. MDM2 gene amplification, as shown by differential PCR, was observed in 19 out of 47 cases (40%). Nineteen out of 49 cases (38.8%) showed immunoreactivity for MDM2. MDM2 gene amplification and the expression of MDM2 protein showed a significant positive relationship (P = 0.0004). Moreover, MDM2 immunoreaction was significantly correlated with nuclear accumulation of p53 protein (P = 0.023). Positive immunoreaction for p21(WAF1/CIP1) was observed in 21 out of 48 cases (43.8%). p21(WAF1/CIP1) expression was correlated with p53 protein expression. H-ras gene mutations were seen in only three cases (6.1%). All mutations were in codon 12 (one GGC-to-AGC [Gly-to-Ser] mutation and two GGC-to-GAC [Gly-to-Ap] mutations). The gene alteration of p53, MDM2, and H-ras did not affect the patients' prognosis. Although the cases with positive immunoreaction for p53 tended to have a worse prognosis, the difference was not statistically significant (P = 0.13). No correlation was observed between MIB-1 LI and the immunohistochemical expression of p53, MDM2, and p21(WAF1/CIP1) or the mutation status of p53 and H-ras. On the other hand, high MIB-1 LI (more than 10) significantly correlated with poor prognosis (P < 0.0001). Our results suggest that p53 gene mutation does not appear to be a major prognostic factor and H-ras mutations are infrequent in synovial sarcoma.     

p53 Mutation and MDM2 amplification in inflammatory myofibroblastic tumours

AIMS: The pathogenic mechanism and predictive indicators of biological behaviour of inflammatory myofibroblastic tumour are poorly understood. We investigated molecular abnormalities of p53 and MDM2 in order to assess whether these play an important role in pathogenesis, and whether they also contribute to clinicopathological aggressive phenotype in inflammatory myofibroblastic tumour. METHODS AND RESULTS: We compared the immunohistochemical expression of calponin, h-caldesmon, ALK, and p53 gene mutation and MDM2 gene amplification with clinicopathological findings in 15 cases of inflammatory myofibroblastic tumour. Histologically, cellular atypia was observed in five (33.3%) out of 15 cases. Local recurrences were observed in two (14.3%) of 14 informative cases, but no distant metastasis was observed. The expression of calponin (9/14; 64%) but not h-caldesmon (0/14; 0%) was seen, which suggested myofibroblastic differentiation. ALK expression was seen in eight (53.3%) out of 15 cases, particularly in patients under 40 years old. Nuclear expression of p53 protein was recognized in only one (6.7%) of 15 cases, and polymerase chain reaction single-strand conformation polymorphism followed by direct sequencing revealed p53 gene missense mutations in two (13.3%) of 15 cases. Nuclear expression of MDM2 was seen in four (26.7%) of 15 cases, and the MDM2 gene amplification was observed in two of the four cases. CONCLUSION: Inflammatory myofibroblastic tumour shows a wide spectrum of cellular atypia and biological behaviour with p53 and MDM2 expression. However, the alterations in the p53 pathway seem not to play a major role in the pathogenesis of inflammatory myofibroblastic tumour.     

胰腺癌p53上下游基因mdm2、p21WAF/CIP1以及P14ARF蛋白的表达及相互关系

目的 探讨p14ARF、p53、mdm2及p21WAF/CIP1蛋白在胰腺癌组织中的表达、相互关系及意义。方法 选取167例胰腺癌、101例癌旁与13例良性病变组织构建组织芯片(又称组织微阵列),应用免疫组织化学EnVision二步法检测这4种蛋白在胰腺良恶性病变中的表达。结果 p14ARF、p53、mdm12及p21WAF/CIP1在胰腺癌中表达的阳性率分别为35.3％(59/167)、57.5％(96/167)、64.1％(107/167)和39.5％(66/167)。同癌旁组织相比,p53和mdm2表达明显升高(P<0.01),而p14ARF和p21WAF/CIP1的表达明显降低(P<0.05)。p21WAF/CIP1的阳性表达与年龄、神经受累显著相关(P<0.05);p53的阳性表达与肿瘤的分化、淋巴结转移和神经受累均显著相关(P<0.05);mdm2的阳性表达与肿瘤的分化显著相关(P<0.05);p14ARF与年龄和浸润转移显著相关(P<0.05)。四者阳性表达两两之间统计学上具有关联性(P<0.05)。结论 p53和mdm2的过表达以及p14ARF和p21WAF/CIP1的缺失表达可能会导致胰腺癌的形成和进展;4种蛋白主要以p14ARF-p53-mdm2-p21WAF/CIP1通路的方式作用于细胞的转化和肿瘤的形成;联合检测p53和mdm2的表达可用于评定胰腺癌的恶性程度。     

p53 expression in CMV-infected cells: association with the alternative expression of the p53 transactivated genes p21/WAF1 and MDM2

The p53 tumour suppressor gene is a cell cycle regulator, able to induce cell cycle arrest to allow DNA repair or apoptosis. The molecular mechanisms underlying p53 action imply transactivation of p53 dependent genes such as WAF1 (for wild type p53 associated fragment 1) and the murine double minute (MDM2) gene. In some cases, inactivation of the p53 gene results from p53 gene mutations leading to p53 protein accumulation, but in others it may results from mechanisms other than mutation, such as interaction with viral or cellular proteins. The expression of p53 protein and p53 transactivated gene proteins p21/WAF1 and MDM2, combined with in situ detection of apoptosis, was studied in specimens of CMV-infected patients as an in vivo model of p53 alteration not due to point mutation. p53 positivity was found in CMV + cells in different tissues, in cells with typical inclusion bodies, and in in situ hybridization and immunohistochemistry CMV + cells without inclusions (hidden infection). Although this p53 reactivity was accompanied by the expression of MDM2 and p21/WAF1 proteins, the patterns of MDM2 and p21/WAF1 protein expression were mutually exclusive, and were associated with the presence or absence of inclusion bodies. Nuclei bearing inclusion bodies were usually MDM2 +, p21/WAF1?, while hidden infected cells were usually MDM2?, p21/WAF1 +. Apoptosis was not detected in any tissue section from CMV-infected patients. Two alternative patterns were found in CMV-infected tissues: p53 +, p21/WAF1 +, MDM2?, or p53 +, p21/WAF1?, MDM2 + protein expression. These may represent examples of p53 dependent alternative effects in the course of CMV infection. Early stages are represented by CMV + cells without inclusion bodies, which display p53 and p21/WAF1 expression, suggesting that p53 could be acting as a growth suppressor protein. Late CMV infection is represented by cells harbouring inclusion bodies. These cells showed a p53 +, p21/WAF1?, MDM2 + profile, consistent with MDM2 mediated p53 inactivation. The absence of p21/WAF1 expression and lack of apoptosis suggest that the p53 protein expressed by MDM2 + cells could be functionally inactivated in CMV-infected cells with inclusion bodies. Previous studies have suggested that p53 inactivation by MDM2 over-expression occurs in sarcomas and lymphomas. Our observations seem to indicate that this mechanism of MDM2 mediated p53 inactivation may play a role in the late phase of CMV infection.     

单启动子p53/p14ARF非融合表达载体的构建及其在MG-63中的表达鉴定

目的构建单启动子非融合表达载体pIRES-p53-p14~(ARF)并观察其对骨肉瘤细胞增殖生长的影响及其功能初探。方法将p53与p14~(ARF)全长cDNA编码区依次亚克隆至pIRES载体中,并通过PCR、酶切鉴定重组质粒。用Lipofectamin 2000将野生型pIRES-p53-p14~(ARF)真核表达载体转染MG-63细胞,48h后采用axiovent200型倒置显微镜、荧光免疫细胞化学,观察其在细胞内的表达并初步探究其抑瘤功能。结果成功构建出含p53与p14~(ARF)全长基因片段的单启动子非融合表达载体pIRES-p53-p14~(ARF)。RT-PCR,酶切鉴定质粒大小、位点均符合实验设计。荧光免疫细胞化学检测显示p53阳性细胞同时也表现为p14~(ARF)免疫反应阳性,转染后瘤细胞可见散在凋亡现象。结论pIRES-p53-p14~(ARF)非融合表达载体的构建与真核细胞导入、目的基因表达成功。     

HCV肝炎肝硬化中核心蛋白、p14ARF和p21WAF1的表达

目的检测HCV肝炎、肝硬化组织中的核心蛋白、p14ARF、p21WAF1的表达及其相关性,探讨HCV核心蛋白对p14ARF、p21WAF1的表达作用.方法收集HCV表达阳性的肝炎、肝硬化组织23例,采用免疫组织化学EnVision两步法检测HCV感染的肝炎、肝硬化组织的核心蛋白、p14ARF、p21WAF1的表达,并分析三者间的关系.结果核心蛋白、p14ARF、p21WAF1的阳性表达主要定位于细胞核. 在核心蛋白均阳性的组织中,p14ARF的阳性表达率为69.6%,p21WAF1表达率为13%;三组间的阳性强度比较,差异有显著性(P=0.01),HCV核心蛋白与p14ARF、p21WAF1各组间的P值均<0.01;HCV核心蛋白与p14ARF、p21WAF1阳性强度两者间相关性检验,相关系数rs分别为-0.053、0.45(P值分别为0.72、0.20),表明无相关性.结论 HCV核心蛋白对p14ARF、p21WAF1的表达有影响可能间接促进p14ARF的表达,但抑制p21WAF1的表达.     

High promoter hypermethylation frequency of p14/ARF in supratentorial PNET but not in medulloblastoma

AIMS: Medulloblastoma (MB) is the most common primitive neuroectodermal tumour (PNET) of the central nervous system. Although supratentorial PNET (sPNET) and MB are histologically similar, their clinical behaviour differs, sPNET being more aggressive than MB. The aim of this study was to determine whether sPNET and MB are genetically different entities. METHODS AND RESULTS: We investigated 32 PNET primary tumour samples (23 MB and nine sPNET) and four PNET cell lines, for the presence of CDKN2A homozygous deletions at exon 1-alpha of p16/INK4 and exon 1-beta of p14/ARF, and promoter hypermethylation of both genes. No homozygous deletion of either p16/INK4 or p14/ARF was demonstrated in any of the PNET primary tumour samples. Methylation of p16/INK4 was found in one of six sPNET and in one of 23 MB, while p14/ARF methylation was observed in three of six sPNET and in three of 21 MB. No methylation of p16/INK4 or p14/ARF was found in any of the PNET cell lines analysed. The three MB cell lines did not show p16/INK4 expression, and only the MB Daoy cell line (homozygously deleted at CDKN2A) presented loss of p14/ARF expression. CONCLUSIONS: Our results in this limited series of central PNET show that p14/ARF is frequently involved in PNET carcinogenesis, with a higher frequency, but not statistically significant, for sPNET than for MB.