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Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis.

ABSTRACT: BACKGROUND:Hepatocellular carcinoma (HCC) presents one of the major health threats in China today. A better understanding of the molecular genetics underlying malignant transformation of hepatocytes is critical to success in the battle against this disease. The methylation state of C5 of the cytosine in the CpG di-nucleotide that is enriched within or near the promoter region of over 50 % of the polymerase II genes has a drastic effect on transcription of these genes. Changes in the methylation profile of the promoters represent an alternative to genetic lesions as causative factors for the tumor-specific aberrant expression of the genes. METHODS:We have used the methylation specific PCR method in conjunction with DNA sequencing to assess the methylation state of the promoter CpG islands of twenty genes. Aberrant expression of these genes have been attributed to the abnormal methylation profile of the corresponding promoter CpG islands in human tumors. RESULTS:While the following sixteen genes remained the unmethylated in all tumor and normal tissues: CDH1, APAF1, hMLH1, BRCA1, hTERC, VHL, RARbeta, TIMP3, DAPK1, SURVIVIN, p14ARF, RB1, p15INK4b, APC, RASSF1c and PTEN, varying degrees of tumor specific hypermethylation were associated with the p16INK4a, RASSF1a, CASP8 and CDH13 genes. For instance, the p16INK4a was highly methylated in HCC (17/29, 58.6%) and less significantly methylated in non-cancerous tissue (4/29. 13.79%). The RASSF1a was fully methylated in all tumor tissues (29/29, 100%), and less frequently methylated in corresponding non-cancerous tissue (24/29, 82.75%). CONCLUSIONS:Furthermore, co-existence of methylated with unmethylated DNA in some cases suggested that both genetic and epigenetic (CpG methylation) mechanisms may act in concert to inactivate the p16INK4a and RASSF1a in HCC. Finally, we found a significant association of cirrhosis with hypermethylation of the p16INK4a and hypomethylation of the CDH13 genes. For the first time, the survey was carried out on such an extent that it would not only provide new insights into the molecular mechanisms underscoring the aberrant expression of the genes in this study in HCC, but also offer essential information required for a good methylation-based diagnosis of HCC.

SUBMITTER: Yu J

PROVIDER: S-EPMC139988 | biostudies-literature | 2002 Nov

REPOSITORIES: biostudies-literature

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Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis.

Yu Jian J Ni Min M Xu Jian J Zhang Hongyu H Gao Baomei B Gu Jianren J Chen Jianguo J Zhang Lisheng L Wu Mengchao M Zhen Sushen S Zhu Jingde J

BMC cancer 20021115

<h4>Background</h4>Hepatocellular carcinoma (HCC) presents one of the major health threats in China today. A better understanding of the molecular genetics underlying malignant transformation of hepatocytes is critical to success in the battle against this disease. The methylation state of C5 of the cytosine in the CpG di-nucleotide that is enriched within or near the promoter region of over 50 % of the polymerase II genes has a drastic effect on transcription of these genes. Changes in the meth ...[more]

PMID: 12433278

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Project description:BackgroundAstrocytoma is a common aggressive intracranial tumor and presents a formidable challenge in the clinic. Association of altered DNA methylation patterns of the promoter CpG islands with the expression profile of cancer-related genes, has been found in many human tumors. Therefore, DNA methylation status as such may serve as an epigenetic biomarker for both diagnosis and prognosis of human tumors, including astrocytoma.MethodsWe used the methylation specific PCR in conjunction with sequencing verification to establish the methylation profile of the promoter CpG island of thirty four genes in astrocytoma tissues from fifty three patients (The WHO grading: I: 14, II: 15, III: 12 and IV: 12 cases, respectively). In addition, compatible tissues (normal tissues distant from lesion) from three non-astrocytoma patients were included as the control.ResultsSeventeen genes (ABL, APC, APAF1, BRCA1, CSPG2, DAPK1, hMLH1, LKB1, PTEN, p14ARF, p15INK4b, p27KIP1, p57KIP2, RASSF1C, RB1, SURVIVIN, and VHL) displayed a uniformly unmethylated pattern in all the astrocytoma and non-astrocytoma tissues examined. However, the MAGEA1 gene that was inactivated and hypermethylated in non-astrocytoma tissues, was partially demethylated in 24.5% of the astrocytoma tissues (co-existence of the hypermethylated and demethylated alleles). Of the astrocytoma associated hypermethylated genes, the methylation pattern of the CDH13, cyclin a1, DBCCR1, EPO, MYOD1, and p16INK4a genes changed in no more than 5.66% (3/53) of astrocytoma tissues compared to non-astrocytoma controls, while the RASSF1A, p73, AR, MGMT, CDH1, OCT6, MT1A, WT1, and IRF7 genes were more frequently hypermethylated in 69.8%, 47.2%, 41.5%, 35.8%, 32%, 30.2%, 30.2%, 30.2% and 26.4% of astrocytoma tissues, respectively. Demethylation mediated inducible expression of the CDH13, MAGEA1, MGMT, p73 and RASSF1A genes was established in an astrocytoma cell line (U251), demonstrating that expression of these genes is likely regulated by DNA methylation. AR gene hypermethylation was found exclusively in female patients (22/27, 81%, 0/26, 0%, P < 0.001), while the IRF7 gene hypermethylation preferentially occurred in the male counterparts (11/26, 42.3% to 3/27, 11%, P < 0.05). Applying the mathematic method "the Discovery of Association Rules", we have identified groups consisting of up to three genes that more likely display the altered methylation patterns in concert in astrocytoma.ConclusionsOf the thirty four genes examined, sixteen genes exhibited astrocytoma associated changes in the methylation profile. In addition to the possible pathological significance, the established concordant methylation profiles of the subsets consisting of two to three target genes may provide useful clues to the development of the useful prognostic as well as diagnostic assays for astrocytoma.

Project description:AIM:To establish the DNA methylation patterns of the promoter CpG islands of 14 "drug-resistance" genes in hepatocellular carcinoma (HCC). METHODS:The methylation specific polymerase chain reaction in conjunction with sequencing verification was used to establish the methylation patterns of the 14 genes in the liver tissues of four healthy liver donors, as well as tumor and the paired non-cancerous tissues of 30 HCC patients. RESULTS:While 11 genes (ATP-binding cassette, sub-family G (WHITE), member 2(ABCG2), activating transcription factor (ATF2), beta-2-microglobulin (B2M), deoxycytidine kinase (DCK), occludin (OCLN), v-raf-1 murine leukemia viral oncogene homolog (RAF1), ralA binding protein 1 (RALBP1), splicing factor (45 kD) (SPF45), S-phase kinase-associated protein 2 (p45) (SKP2), tumor protein p53 (Li-Fraumeni syndrome) (TP53) and topoisomerase (DNA) II beta (TOP2B)) maintained the unmethylated patterns, three genes displayed to various extents the hypermethylation state in tumor tissues in comparison with the normal counterparts. The catalase (CAT) was hypermethylated in tumor and the neighboring non-cancerous tissue of one case (3.3%). Both glutathione S-transferase pi (GSTpi) (80%, 24/30 in tumor and 56.7%, 17/30 in the paired non-cancerous tissues) and cystic fibrosis transmembrane conductance regulator, ATP-binding cassette (sub-family C, member 7) (CFTR) (77%, 23/30 in tumor and 50%, 15/30 in the paired non-cancerous tissues) genes were prevalently hypermethylated in HCC as well as their neighboring non-cancerous tissues. No significant difference in the hypermethylation occurrence was observed between the HCC and its neighboring non-cancerous tissues. CONCLUSION:Hypermethylation of promoter CpG islands of both CFTR and GSTpi genes occurs prevalently in HCC, which may correlate with the low expression of these two genes at the mRNA level and has the profound etiological and clinical implications. It is likely to be specific to the early phase of HCC carcinogenesis.

Project description:Background?-Glutamyl hydrolase (GGH) regulates intracellular folates and antifolates such as methotrexate (MTX) for proper nucleotide biosynthesis and antifolate-induced cytotoxicity, respectively. In addition to genetic polymorphism and karyotypic abnormalities, methylation of CpG island 1 (CpG1) in the promoter region is found to modulate GGH activity by reducing GGH mRNA expression in acute lymphoblastic leukemia (ALL) cells. We aim to investigate methylation status of two CpG islands (CpG1 and CpG2) in the GGH promoter region in pediatric patients with ALL and acute myelogenous leukemia (AML).Methods70B-ALL, 29 AML, 10 ITP (idiopathic thrombocytopenic purpura) and 40 healthy children are recruited in the present study. MS-HRM (methylation-sensitive high-resolution melting) and bisulfite sequencing PCR (BSP) are used to detect methylation change and its level in CpG1 and CpG2 in the GGH promoter region. GGH mRNA expression is quantified by real-time PCR. Correlation between CpG island methylation and GGH mRNA expression is assessed by statistical software.ResultsMethylations of CpG1 are detected in leukemia cells samples obtained from 30.9% (21/68) of patients with ALL and 20.7% (6/29) of patients with AML. These methylations are not detected in the controls. Methylations of CpG2 are detected in leukemia cell samples obtained from 44.1% (30/68) of the ALL patients and 37.9% (11/29) of the AML patients. These percentages are significantly higher than that observed in the control cell samples: 6.0% (3/50) (Fisher's exact test, P = 0.000). The abundance of CpG1 methylation in all leukemia cell samples is classified as Grade I (methylation level is less than 10%) and the abundance of CpG2 methylation in leukemia cell samples is classified in separate grades. Our results indicate that methylation of CpG1 or hypermethylation (the methylation level is greater than 10%) of CpG2 could significantly reduce GGH mRNA expression in leukemia cells from the ALL and AML patients (ALL-CpG1: t = 4.632, P = 0.000; ALL-CpG2: t = 3.250, P = 0.006; AML-CpG1: t = -2.254, P = 0.037; AML-CpG2: t = 1.328, P = 0.202).ConclusionEither methylation of CpG1 or hypermethylation of CpG2 in GGH promoter region can significantly reduce GGH mRNA expression in pediatric patients with acute leukemia, which can improve the response to treatment.

Dataset Information

Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis.

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Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis.

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