cytidylyl-3--5--guanosine and Brain-Neoplasms

Topics: Amino Acid Substitution; Arginine; Brain Neoplasms; Citric Acid Cycle; Dinucleoside Phosphates; Fumarate Hydratase; Genes, Tumor Suppressor; Genetic Predisposition to Disease; Glioblastoma; Glutamates; Humans; Hydroxylation; Hypoxia-Inducible Factor 1; Isocitrate Dehydrogenase; Mutation; NADP; Oncogenes; Succinate Dehydrogenase

Tumorigenesis is characterized by alterations of methylation profiles including loss and gain of 5-methylcytosine. Recently, we identified a single CpG, which seemed to be consistently hypomethylated in pilocytic astrocytomas but not in other gliomas. To evaluate its applicability as a biomarker, we examined its methylation status in a large panel of gliomas (n = 97). Methylation-dependent DNA sequence variation may be considered a kind of single nucleotide polymorphism (methylSNP). MethylSNPs can be easily converted into common SNPs of the C/T type by sodium bisulfite treatment of the DNA and afterwards subjected to conventional SNP typing. We adapted SnaPshot trade mark and Pyrosequencing trade mark to determine the methylation of our test CpG in a quantitative manner. The adapted methods, called SNaPmeth and PyroMeth, respectively, gave nearly identical results, however data obtained with PyroMeth showed less scattering. Furthermore, the integrated software for allele frequency determination from Pyrosequencing could be used directly for data analysis while SnaPmeth data had to be exported and processed manually. Although data did not confirm our previous result of a preferential hypomethylation of the tested CpG in pilocytic astrocytomas, we consider quantitative methylSNP analysis by SNaPmeth or PyroMeth a favorable alternative to existing high-throughput methylation assays. It combines single CpG analysis with accurate quantitation and is amenable to high throughput.

Topics: Adolescent; Adult; Astrocytoma; Base Sequence; Brain Neoplasms; Child; Child, Preschool; Dinucleoside Phosphates; DNA Methylation; DNA Primers; DNA, Neoplasm; Electrophoresis, Gel, Two-Dimensional; Female; Genetic Markers; Glioma; Humans; Male; Middle Aged; Oligodendroglioma; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Sequence Analysis, DNA

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

Topics: Adenocarcinoma; Base Sequence; Brain Neoplasms; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Lobular; Colonic Neoplasms; Dinucleoside Phosphates; DNA Methylation; Female; Genome, Human; Humans; Male; Molecular Sequence Data; Neoplasms; Restriction Mapping

The cell-cycle regulator p16 inhibits the complex cdk4-cyclin D1 and controls G1-S transition. In human tumors, p16 inactivation is often accomplished by homozygous deletion (HD) of its encoding gene, CDKN2A. Methylation of the 5' CpG island promoter has been proposed as an alternative mechanism of inactivation. Expression of p16, CDKN2A HD and 5' CDKN2A CpG island methylation was studied in 25 oligodendrogliomas by immunohistochemistry and PCR amplification. Ten oligodendrogliomas were p16-immunonegative, and CDKN2A HD was determined in 8 of these cases. In the 2 immunonegative cases without HD, no CpG island methylation was found. The absence of CpG island methylation in the p16-immunonegative cases without HD suggests either non-genetic regulation of p16 or different genetic changes. CDKN2A HD did not correlate with histological grading (p = n.s.); however, it showed a correlation with survival (p = 0.03), supporting an important role of CDKN2A in the prognosis of oligodendrogliomas.

Topics: Brain Neoplasms; Cell Nucleus; Cyclin-Dependent Kinase Inhibitor p16; Dinucleoside Phosphates; DNA Methylation; Gene Deletion; Genes, p16; Homozygote; Humans; Oligodendroglioma; Prognosis; Survival Analysis

There is considerable evidence that lack of p16 protein expression is a frequent event in human gliomas. Nevertheless, the molecular mechanisms underlying this absence of p16 protein expression are not completely understood. In some gliomas, homozygous deletions are the main cause of p16/CDKN2 gene inactivation. However, other gliomas lacking p16 expression exhibit intact p16/CDKN2 gene, suggesting that p16/CDKN2 is down-regulated at the transcriptional level. In this study we investigated whether aberrant p16/CDKN2 gene methylation correlated with absence of p16 expression in the latter group of gliomas. In a series of 27 gliomas, 12 malignant tumors exhibited loss of p16/CDKN2 expression but not gene deletion. Methylation analysis of the CpG island in the 5' region of the p16/CDKN2 gene showed that exon 1 was extensively methylated in six and partially methylated in the other six of the 12 malignant gliomas. In contrast, no methylation was observed in four other malignant gliomas and two low-grade gliomas that expressed p16 protein. These results indicate that abnormal hypermethylation of the CpG island encompassing the 5' end of the p16/CDKN2 gene may be a mechanism of transcriptional silencing in gliomas without homozygous deletions.

Topics: Brain Neoplasms; Carrier Proteins; Cyclin-Dependent Kinase Inhibitor p16; Dinucleoside Phosphates; Down-Regulation; Glioma; Humans; Immunohistochemistry; Methylation