sodium-bisulfite and Neoplasms

DNA methylation, the addition of a methyl group to cytosines and adenosines, regulates gene expression on a level that is usually referred to as epigenetic, that is, stably maintained during cell divisions. In humans, aberrant DNA methylation is associated with several malignancies, including cancer and so-called imprinting disorders, making it an attractive target for diagnostic purposes. Here we give a brief introduction to the biology of DNA methylation and present the use of methylation biomarkers in laboratory medicine. DNA methylation assays have become the standard procedure in the diagnosis of imprinting disorders, and they are about to shift cancer diagnostics and prognostics to the next level of molecular medicine. However, there is evidence of problems associated with the introduction of such cancer assays in routine diagnostics. We review several assays that have been proposed for DNA methylation analysis. The assays presented analyse the methylation status of single loci and are based either on a bisulphite-treatment or on methylation-sensitive restriction of the DNA under investigation.

Topics: DNA; DNA Methylation; Epigenesis, Genetic; Genomic Imprinting; Humans; Neoplasms; Polymerase Chain Reaction; Sulfites

Topics: Cell Transformation, Neoplastic; Chromosome Aberrations; Chromosome Disorders; Chromosome Mapping; Gene Amplification; Gene Expression Regulation; Humans; Karyotyping; Mutation; Neoplasms; Nucleic Acid Hybridization; Proto-Oncogenes; Sulfites; Translocation, Genetic

The methylation of the promoter region of DNA is an important regulatory mechanism for the downstream gene expression, and the extent of methylation has been linked to cancer formation. In this study, we report a simple method to screen for the degree of DNA methylation by combined bisulfite restriction analysis (COBRA) and capillary electrophoresis with laser-induced fluorescence (CE-LIF). After treating genomic DNA with sodium bisulfite, nested-PCR amplification and endonuclease (Taq I) digestion were performed. The digested DNA fragments were then separated by capillary electrophoresis using 1.5% poly(ethylene) oxide (M(ave), 8,000,000 g/mol) in the presence of electroosmotic flow. The improvement for DNA amplification using the nested PCR described here corresponded to approximately ten cells. In addition, the level of DNA methylation shown in the electropherograms obtained corresponded to the original percentage of DNA methylation from commercial available standard sample (0-100%). The electrophoretic patterns demonstrated that the six cancer cell lines tested displayed different degrees of DNA methylation and could be differentiated by hierarchical cluster analysis. Furthermore, the DNA methylation level was eliminated after treating the cells with an anti-cancer drug (5'-aza-2'-deoxycytidine). Together, these results suggest that CE-LIF is a potentially useful and cost-effective tool for cancer diagnosis or prognosis based on the heterogeneity in a patient's DNA.

Topics: Base Sequence; Cell Line, Tumor; DNA; DNA Methylation; Electrophoresis, Capillary; Fluorescence; Humans; Neoplasms; Sulfites

DNA methylation is one of the most important heritable epigenetic modifications of the genome and is involved in the regulation of many cellular processes. Aberrant DNA methylation has been frequently reported to influence gene expression and subsequently cause various human diseases, including cancer. Recent rapid advances in next-generation sequencing technologies have enabled investigators to profile genome methylation patterns at single-base resolution. Remarkably, more than 20 eukaryotic methylomes have been generated thus far, with a majority published since November 2009. Analysis of this vast amount of data has dramatically enriched our knowledge of biological function, conservation and divergence of DNA methylation in eukaryotes. Even so, many specific functions of DNA methylation and their underlying regulatory systems still remain unknown to us. Here, we briefly introduce current approaches for DNA methylation profiling and then systematically review the features of whole genome DNA methylation patterns in eight animals, six plants and five fungi. Our systematic comparison provides new insights into the conservation and divergence of DNA methylation in eukaryotes and their regulation of gene expression. This work aims to summarize the current state of available methylome data and features informatively.

Topics: Animals; CpG Islands; Cytosine; DNA Methylation; Embryonic Stem Cells; Epigenomics; Eukaryota; Evolution, Molecular; Fungi; Genome; Humans; Neoplasms; Plants; Sequence Analysis, DNA; Sulfites

Cytosine methylation provides an epigenetic level of cellular plasticity that is important for development, differentiation and cancerogenesis. We adopted microdroplet PCR to bisulfite treated target DNA in combination with second generation sequencing to simultaneously assess DNA sequence and methylation. We show measurement of methylation status in a wide range of target sequences (total 34 kb) with an average coverage of 95% (median 100%) and good correlation to the opposite strand (rho = 0.96) and to pyrosequencing (rho = 0.87). Data from lymphoma and colorectal cancer samples for SNRPN (imprinted gene), FGF6 (demethylated in the cancer samples) and HS3ST2 (methylated in the cancer samples) serve as a proof of principle showing the integration of SNP data and phased DNA-methylation information into "hepitypes" and thus the analysis of DNA methylation phylogeny in the somatic evolution of cancer.

Topics: Aged; Colonic Neoplasms; DNA Methylation; DNA, Neoplasm; Female; Fibroblast Growth Factor 6; Genetic Predisposition to Disease; Genome-Wide Association Study; Genome, Human; Haplotypes; Humans; Lymphoma, Follicular; Male; Middle Aged; Neoplasms; Phylogeny; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Sequence Analysis, DNA; snRNP Core Proteins; Sulfites; Sulfotransferases

Circulating extracellular nucleic acids derived from body fluids such as blood are commonly analyzed to assess malignant diseases. Efficient isolation, extraction, quantification, modification, and analysis methods remain important for utilizing circulating nucleic acids as potential molecular biomarkers. Our refined techniques of DNA isolation from serum, sodium bisulfite modification of extracted DNA, and methylation analysis provide a robust approach for quantitative analysis of circulating tumor-related DNA. The approach allows direct comparison of methylated and nonmethylated genomic sequences in a specimen.

Topics: DNA Methylation; DNA, Neoplasm; Electrophoresis, Capillary; Humans; Neoplasms; Polymerase Chain Reaction; Sulfites

Topics: CpG Islands; DNA Methylation; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Humans; Neoplasms; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA; Sulfites

Topics: Chromatin Immunoprecipitation; CpG Islands; DNA Methylation; Epigenesis, Genetic; Genomics; Health; Humans; Mass Spectrometry; Microarray Analysis; Neoplasms; Sulfites

Loss of p16(INK4a) protein expression has frequently been related to DNA methylation in association with gene silencing. Although the methylation status of exon1alpha for p16(INK4a) involvement in various cancers has been extensively analyzed, it has been pointed out that some inconsistencies existed in its relationship to gene silencing of p16(INK4a). In this study, we focused on the expression and methylation status in the regions of nt -478 to -201, containing a putative TATA box (nt -401 to -396), and nt -233 to 26, both in a recently cloned 5' upstream region of rat p16(INK4a). We showed that rat lung adenocarcinoma RLCNR did not express the p16(INK4a) gene, whereas rat osteosarcoma COS1NR and malignant fibrous histiocytoma MFH1NR both expressed it at levels similar to normal fibroblasts, even though the region of nt -233 to 26 was hypermethylated in COS1NR rather than RLCNR. In contrast, the CpG islands near the putative TATA box region were consistently methylated in RLCNR, but not in COS1NR and MFH1NR, as well as in normal fibroblasts. Treatment with 5-aza 2'-deoxycytidine induced expression of p16(INK4a) gene in RLCNR after 48 h, but no changes were observed in COS1NR and MFH1NR. The results indicated that methylation of CpG islands near a TATA box region played a critical role for gene silencing of the rat p16(INK4a) gene, rather than that of other regions.

Topics: Adenocarcinoma; Animals; Azacitidine; CpG Islands; Cyclin-Dependent Kinase Inhibitor p16; Decitabine; DNA Methylation; Fibroblasts; Gene Silencing; Histiocytoma, Benign Fibrous; Lung Neoplasms; Neoplasms; Osteosarcoma; Promoter Regions, Genetic; Rats; RNA, Messenger; Sulfites; TATA Box; Tumor Cells, Cultured

ID4 gene is a member of the inhibitor of DNA binding (ID) family proteins that inhibit DNA binding of basic helix-loop-helix transcription factors. The epigenetic inactivation of ID4 gene on colorectal cancer (CRC) development and its clinical significance was assessed.. In CRC cell lines, ID4 methylation status of the promoter region was assessed by methylation-specific PCR and bisulfite sequencing. The mRNA expression level was assessed by quantitative real-time reverse transcription-PCR. The methylation status of 9 normal epithelia, 13 adenomas, 92 primary CRCs, and 26 liver metastases was assessed by methylation-specific PCR. ID4 protein expression was assessed by immunohistochemistry analysis of tissue specimen.. CRC cell lines were shown to be hypermethylated, and mRNA expression was suppressed and could be restored by 5-aza-cytidine treatment. In clinical specimens from normal epithelia, adenomas, primary CRCs, and liver metastases, the frequency of ID4 hypermethylation was 0 of 9 (0%), 0 of 13 (0%), 49 of 92 (53%), and 19 of 26 (73%), respectively, with a significant elevation according to CRC pathological progression. Methylation status of primary CRCs significantly correlated with histopathological tumor grade (P = 0.028). Immunohistochemistry analysis showed ID4 expression of normal colon epithelia, adenomas, and unmethylated primary CRCs but not hypermethylated CRC specimens. Among 76 American Joint Committee on Cancer stage I to IV patients who had undergone curative surgical resection, overall survival was significantly poorer in patients with hypermethylated ID4 bearing tumors (P = 0.0066).. ID4 gene is a potential tumor suppressor gene for which methylation status may play an important role in the CRC progression.

Topics: Adult; Aged; Aged, 80 and over; Azacitidine; Carcinoma; Cell Differentiation; Cell Line, Tumor; Colon; Colorectal Neoplasms; Disease Progression; DNA Methylation; DNA Primers; DNA-Binding Proteins; Female; Humans; Immunohistochemistry; Inhibitor of Differentiation Protein 2; Male; Middle Aged; Models, Genetic; Neoplasms; Polymerase Chain Reaction; Prognosis; Repressor Proteins; Risk; RNA, Messenger; Sequence Analysis, DNA; Sulfites; Time Factors; Transcription Factors

The gene hypermethylated in cancer-1 (HIC1) encodes a zinc-finger transcription factor that belongs to a group of proteins known as the POZ family. HIC1 is hypermethylated and transcriptionally silent in several types of human cancer. Homozygous disruption of Hic1 impairs development and results in embryonic and perinatal lethality in mice. Here we show that mice disrupted in the germ line for only one allele of Hic1 develop many different spontaneous malignant tumors, including a predominance of epithelial cancers in males and lymphomas and sarcomas in females. The complete loss of Hic1 function in the heterozygous mice seems to involve dense methylation of the promoter of the remaining wild-type allele. We conclude that HIC1 is a candidate tumor-suppressor gene for which loss of function in both mouse and human cancers is associated only with epigenetic modifications.

Topics: Animals; Blotting, Southern; DNA Methylation; Female; Gene Silencing; Gene Transfer Techniques; Genes, Tumor Suppressor; Heterozygote; Homozygote; Humans; Immunoenzyme Techniques; Immunoglobulin G; Kruppel-Like Transcription Factors; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Neoplasms; Peptide Fragments; Polymerase Chain Reaction; Promoter Regions, Genetic; Rabbits; Ribonuclease, Pancreatic; Sex Factors; Sulfites; Syndrome; Transcription Factors