Hypermethylation of promoter 5＇CpG island of p16 gene in glioma tissue and plasma

ObjectiveTo explore the clinical significance of methylation status of promoter CpG island of p16 gene in glioma tissue and plasma.MethodsMethylation specific polymerase chain reaction (MSP) was used to determine the methylation status of the promoter for p16 gene within glioma tissue and plasma.Immunohistochemicel method (SP) was used to analyze the expressions of p16 and Ki-67 proteins.ResultsHypermethylation was found in 17/40 (42.5%) of brain gliomas,in comparison with 11/40 (27.5%) plasma specimens (x2 = 1.9780,P = 0.1596).Loss of p16 expression was associated (P = 0.0229) with hypermethylation of CpG island of promoter regions.Hypermethylation of p16 gene CpG island was significantly related to the increase of malignant grade of brain glioma (TissueX2 = 11.4288,P = 0.0007;PlasmaX2 = 8.9439,P = 0.0028).The Ki-67 index increased significantly (P＜0.05) in brain gliomas methylated in contrast to those unmethylated.ConclusionP16 hypermethylation may be one of the major mechanisms of tumorigenesis of gliomas.Methylated tumor-specific DNA may be as a plasma biomarker for prognosis in patients with glioma.     

DELETION AND 5

　Objective To investigate the abnormality of p15 gene in brain glioma and the correlation of it with occurrence or malignant progression of brain glioma. Methods Deletion and 5’CPG island methylation of p15 gene were detected by the methods of PCR and PCR-based methylation in 56 cases of brain glioma. Results Out of 43 cases of high grade glioma, 14 cases were found to have homozygous deletion of p15E1, while none of the 13 cases of low grade glioma was found to have deletion of p15E1 (P<0.05). Methylation of 5’CPG Island of p15 gene was found only in four cases of glioma. Conclusion Abnormality of p15 gene may involved in the occurrence and malignant progression of brain glioma. Homozygous deletion of gene is the major mechanism of inactivation for p15 gene in brain glioma.     

Detection of Homozygous Deletions and Mutations in the CDKN2A Gene in Hydatidiform Moles

OBJECTIVE To investigate homozygous deletions and mutations in the CDKN2A gene(p16 INK4a and p14 ARF gene)in hydatidiform moles. METHODS A total of 38 hydatidiform mole samples and 30 villi samples were examined for homozygous deletions in the CDKN2A gene by PCR and for mutations by DHPLC. RESULTS i)Among 38 hydatidiform mole samples, homozygous deletions in the p16 INK4a exon 1 were identified in 5 cases(13.2%),while no homozygous deletions were found in the p16I NK4aexon 1 of 30 early-pregnancy samples.The rates of those deletions in hydatidiform compared to early-pregnancy villi samples was statistically significant(P=0.036).ii)No homozygous deletions in the p14 ARF exon 1 or p16 INK4a exon 2 were found in any of the hydatidiform moles or early-preganancy samples.iii) In all hydatidiform moles and early-pregnancy villi samples,no mutations were detected by DHPLC. CONCLUSION We suggest there may be a close correlation between homozygous deletions in the CDKN2A gene and occurrence of hydatidiform moles variation in the CDKN2A gene is mainly caused by homozygous deletions,while mutations may be not a major cause.     

中国非小细胞肺癌患者血清DAPK基因、p16基因启动子甲基化的分析

Objective： To evaluate the clinical significance of the aberrant methylation of DAPK gene and p16 gene in sera from 65 NSCLC patients from Nanjing General Hospital of Nanjing Command, China. Methods： A methylation-specific PCR （MSP） was performed for the detection of promoter hypermethylation of DAPK gene and p16 gene in blood DNA from 65 cases of NSCLC, and to analyze the relation of the aberrant methylation of DAPK gene and p16 gene and the clinicopathological data. Results： 30.8% （20/65） of the sera from 65 cases of NSCLC showed hypermethylation for DAPK promoter and 43.1% （28/65） the same for p16 promoter, whereas no methylated DAPK gene promoter and p16 gene promoter were found in sera from the patients with lung benign diseases and normal controls. Methylated DAPK gene promoter and p16 gene promoter in sera were not closely correlated with the pathological classification, stage, metastasis and differentiation in NSCLC. Conclusion： Detection of the aberrant methylation of DAPK gene and p16 gene in blood DNA from NSCLC patients might offer an effective means for the earlier auxiliary diagnosis of the malignancy.     

EXPRESSION OF FRAGILE HISTIDINE TRIAD AND P53 IN NON-SMALL CELL LUNG CANCER

Objective： To investigate the expression offragile histidine triad （FHIT） and p53 protein in non-small cell lung cancer （NSCLC） and explore the relationship between their expressions and the clinicopathological features. Methods： FHIT protein and p53 protein were detected by immunohistochemistry in 76 cases of NSCLCs and matched normal lung tissues. Results： Fifty-one cases （67.1%） showed negative expression of FHIT （apparent reduction or loss） and thirty-seven cases （48.7%） showed p53 positive expression （overexpression）. The difference was significant （P=0.04）. However, there was no significant difference in FHIT expression between the p53-positive group and the p53-negative group （64.9% versus 69.2%, P=0.686）. The negative rate of FHIT protein expression was higher in squamous cell carcinoma than in adenocarcinoma, in moderately and poorly differentiated carcinoma than in well-differentiated carcinoma, and in cases with smoking history than in cases without smoking history （P〈0.05）. There was no relationship between FHIT expression and clinical stage or lymph node metastasis. The negative FHIT expression was not an independent predictor of overall survival （P=0.338）. Conclusion： The frequency of negative expression of FHIT protein is higher than that of positive expression of p53 in NSCLCs. The negative expression of FHIT is independent of the expression of p53. The change of expression of FHIT may play a role in the smoking related lung tumorigenesis while it may have no relationship with the progress of NSCLC or prognosis of the patients.     

P27蛋白在人脑胶质瘤组织中的表达及其临床意义

Objective： To study p27/Kipl expression in gliomas and its application value. Methods： Imo munohistochemical technique was used to detect the exprssion of p27/Kipl gene in 48 different malignant grade human brain glioma tissues categorized according to WHO classification and 12 normal human brain tissues,which were analyzed quantitatively by using the image system and compared retrospectively with the patients＇ clinical characteristics. Results： In this series, the immunohistochemical reaction for p27/Kipl was confined to the nuclei. The abnormal positive expression rate of p27/Kipl in gliomas was found to be higher than that in normal tissues （P〈0.05）. The positive nuclei expression of p27/Kipl decreased in number and staining intensity with the increasing degree of histological malignancy （P〈0.05）. Lower expression of p27/Kipl was associated with poor prognosis （P〈0.05）. P27/Kipl expression could be regarded as an independent prognostic factor. Conclusion： The abnormal expression of p27/Kipl may be closely related to the occurrence and development of gliomas, and also can be used to evaluate the prognosis independently.     

Expression of FRAT1 and β-catenin in human brain glioma and their significances

Objective: To investigate the expression of FRAT1 and β-catenin in human brain glioma, analyze the correlation between the expression and clinical pathological grades and the correlation of the two genes. Methods: FRAT1 and β-catenin were detected by immunohistochemistry in 84 human brain glioma tissues and 6 human normal brain tissues. Results: 66.7% (56/84) and 77.4% (65/84) of human brain glioma tissues expressed FRAT1 and β-catenin protein, whereas no FRAT1 and β-catenin protein expression was detected in human normal brain tissues. The expression levels of FRAT1 and β-catenin increased markedly with the ascending of pathologic grade of tumor specimens (r = 0.55, P < 0.01, r = 0.70, P < 0.01), there was a positive correlation between FRAT1 and β-catenin (r = 0.77, P < 0.01). Conclusion: FRAT1 and β-catenin over-expression maybe closely related with occurrence and development of human brain gliomas. The results provide important supplements for the research of Wnt/β-catenin pathway. Meanwhile, FRAT1 may act as a valuable biomarker for molecular diagnosis of glioma and a potential target for gene therapy of glioma.     

THE EXPRESSION OF APOPTOSIS RELATED GENES IN THE PROCESS OF CANCERATION OF ATYPICAL HYPERPLASIA OF MAMMARY DUCT

Objective： To investigate the expression of apoptosis related genes p53 and bcl-2 in atypical hyperplasia of mammary duct and the relationship between the gene expression and oncogenesis of breast. Methods： mRNA of apoptosis related gene p53 and bcl-2 were detected by in situ hybridization in 44 cases of atypical ductal hyperplasia. p53 protein expression was detected by immunohistochemistry. The data were compared with those of 6 cases of benign hyperplasia and 26 cases of breast carcinoma. Results： The expression of p53 mRNA was 66.7% in benign hyperplasia, 40% in atypical ductal hyperplasia （55.6% in mild, 41.7% in medium, 26.1% in severe） and 19.2% in carcinoma （of which 21.4% were intraductal carcinoma and 16.7% were invasive）. The expression of p53 protein was negative in benign hyperplasia, 24% in atypical hyperplasia （mild 11.1%, medium 25%, severe 34.8%）, 38.5% in carcinoma （intraductal carcinoma 35.7%, invasive ductal carcinoma 41.7%）. The expression of bcl-2 was negative in benign hyperplasia, 78.6% in intraductal carcinoma, 83.3% in invasive ductal carcinoma. Conclusion： Loss and mutation of p53 gene and excessive expression bcl-2 mRNA were detected in severe atypical ductal hyperplasia.     

胶质瘤患者血浆与肿瘤组织P16基因甲基化的检测及意义

Objective:To explore the clinical significance of methylation status of promoter CpG island of p16 gene in glioma tissue and plasma.Methods:Methylation specific polymerase chain reaction (MSP) was used to determine the methylation status of the promoter for p16 gene within glioma tissue and plasma.Immunohistochemicel method (SP) was used to analyze the expressions of p16 and Ki-67 proteins.Results:Hypermethylation was found in 17/40 (42.5%) of brain gliomas,in comparison with 11/40 (27.5%) plasma specimens (x2 = 1.9780,P = 0.1596).Loss of p16 expression was associated (P = 0.0229) with hypermethylation of CpG island of promoter regions.Hypermethylation of p16 gene CpG island was significantly related to the increase of malignant grade of brain glioma (Tissue:X2 = 11.4288,P = 0.0007;Plasma:X2 = 8.9439,P = 0.0028).The Ki-67 index increased significantly (P＜0.05) in brain gliomas methylated in contrast to those unmethylated.Conclusion:P16 hypermethylation may be one of the major mechanisms of tumorigenesis of gliomas.Methylated tumor-specific DNA may be as a plasma biomarker for prognosis in patients with glioma.     

p16/MTS1/INK4A gene is frequently inactivated by hypermethylation in childhood acute lymphoblastic leukemia with 11q23 translocation.

The p16 gene encoding a specific inhibitor of cyclin-dependent kinases 4 and 6 has been reported to be inactivated at a variety of rates in malignant tumors. We studied frequency and mechanism of inactivation of the p16 gene in various types of childhood acute lymphoblastic leukemia (ALL) using 36 leukemic cell lines established from children (B precursor-ALL, 28; B-ALL/Burkitt's lymphoma, 3; and T-ALL, 5). On Southern blot, homozygous deletions or hemizygous deletions with rearrangement were detected in 14 cell lines. The expression of p16 protein was not observed on Western blot in 18 of 22 cell lines with intact p16 gene, but induced in 16 cell lines after treatment with the demethylating agent, indicating the silencing of the p16 gene by hypermethylation. Of note, the p16 gene was inactivated by hypermethylation of the 5' CpG island in nine of nine cell lines with 11q23 translocation, but was restored with the treatment of the demethylating agent. Partial methylation of the p16 gene was also demonstrated in three of eight primary leukemia samples with this translocation, suggesting that the p16 gene inactivation by hypermethylation might play a role in the leukemogenesis and disease progression of ALL with 11q23 translocation.     

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.     

P16 deletion and mutation analysis in human brain tumors

We screened human primary and recurrent malignant glioma, juvenile pilocytic astrocytoma, medulloblastoma, and meningioma tissue specimens for alterations in p16 gene structure. Single strand conformation polymorphism (SSCP) analysis was used to screen for point mutations, and a quantitative polymerase chain reaction-based assay was used to screen for homozygous gene deletions. In malignant glioma specimens, homozygous p16 gene deletions were significantly more common in high-grade tumors than in low-grade gliomas. Point mutations causing alteration in predicted protein structure were not detected. Medulloblastomas showed rare homozygous deletions and no point mutations. No mutations were detected in meningiomas.     

The early growth response gene EGR-1 behaves as a suppressor gene that is down-regulated independent of ARF/Mdm2 but not p53 alterations in fresh human gliomas.

PURPOSE: EGR-1 is an immediate early gene with diverse functions that include the suppression of growth. EGR-1 is down-regulated many cancer cell types, suggesting a tumor suppressor role, and may critically involve the p53 pathway. The aim of this work was to measure the expression of EGR-1 and the p16/INK4a/ARF-Mdm2-p53 pathway status in fresh human gliomas. EXPERIMENTAL DESIGN: Thirty-one human gliomas with different grades of malignancy were investigated for Egr-1 mRNA and the protein expression, frequency, and spectrum of p53 gene mutations, mdm2 gene amplification, and p16/INK4a/ARF allele loss. RESULTS: The amplification of Mdm2 and the deletion of the p16/INK4a gene was found in 3 and 5 cases, respectively, whereas mutations of p53, including two novel mutations, were observed in 10 other cases. The three types of changes occurred strictly mutually exclusively, emphasizing that these genes operate in a common pathway critical to glioma progression. EGR-1 mRNA was significantly down-regulated in astrocytomas (14.7 +/- 5.1%) and in glioblastomas (33.6 +/- 10.0%) versus normal brain. Overall, EGR-1 mRNA was strongly suppressed (average, 15.2 +/- 13.9%) in 27 of 31 cases (87%), independent of changes in p16/INK4a/ARF and Mdm2; whereas 4 of 31 cases with residual EGR-1 expression as well as the highest EGR-1 variance segregated with p53 mutations. Immunohistochemical analyses confirmed the suppression of EGR-1 protein. CONCLUSIONS: These results indicate that EGR-1 is commonly suppressed in gliomas independent of p16/INK4a/ARF and Mdm2 and that suppression is less crucial in tumors bearing p53 mutations, and these results implicate an EGR-1 growth regulatory mechanism as a target of inactivation during tumor progression.