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PubMed2002

No evidence for hypermethylation of the hSNF5/INI1 promoter in pediatric rhabdoid tumors.

The hSNF5/INI1 gene on chromosome 22 has been implicated as a tumor suppressor gene in pediatric rhabdoid tumor, an aggressive malignancy that generally occurs in the first two years of life. The most common sites for tumor development are the brain and kidney. We and other investigators have identified deletions and mutations of the INI1 gene in the majority of rhabdoid tumors of the central nervous system, kidney, and extrarenal tissues. At least 20% of cases do not have genomic alterations of INI1, although expression at the RNA or protein level may be decreased. The aim of this study was to determine whether hypermethylation or mutation of the 5' promoter region of INI1, or hypermethylation of CpG dinucleotides in a GC-rich repeat region within the first intron, could account for the decreased expression of INI1 observed in these tumors. We employed bisulfite modification, polymerase chain reaction, and sequence analysis to determine the methylation status of the cytosine nucleotides in the predicted promoter region of the INI1 gene, and two GC repeat regions in intron 1. DNA from 24 tumors with or without coding-sequence mutations was analyzed. None of the tumors demonstrated methylation of the promoter or intron 1 regions. This mechanism is unlikely to account for the inactivation of INI1 in rhabdoid tumors without coding-sequence mutations. One tumor demonstrated a potential mutation in the promoter region, but further studies are required for determining its functional significance.

5' Untranslated Regions↗
PubMed2004

Inactivation of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene in squamous cell carcinoma of the larynx.

Defects in the system controlling the cell cycle can lead an increased proliferation of cancer cells. The aim of our study was to analyze the relationship between genetic changes leading to inactivation of the CDKN2A gene and subsequent alteration of protein expression in squamous cell cancer of the larynx (SCCL) in connection with the clinical and histopathological course of the disease. Analysis was carried out on DNA isolated from the blood and primary larynx cancer cells of 62 patients. To investigate loss of heterozygosity (LOH), PCR fragment analysis was applied. The size and quantity of fluorescent PCR products were evaluated in an automated sequencer. Specific chemical methylation with sodium bisulfite in a sequential PCR reaction (MSP) was applied to analyze promoter methylation. Cancer tissue sections served to determine the level of protein expression with immunohistochemical (IHC) staining and commercial antibodies. LOH at the CDKN2A locus was observed in 55.35% of the informative cases. Aberrant methylation was found in 37.5% and a decreased level of protein expression observed in 45% of all informative cases. Whenever P16 expression was decreased, LOH and promoter hypermethylation at CDKN2A were observed with a frequency of 73.33% and 80.95%, respectively (Fisher's test, P<0.005). Sixty-nine percent of G3 tumors had at least one genetic alteration at CDKN2A, compared with 40.9% of G1 cancers. The results indicate that CDKN2A inactivation played a significant role in the development of squamous cell carcinoma of the larynx.

Adult↗
PubMed2002

Loss of genomic imprinting of insulin-like growth factor 2 is strongly associated with cellular proliferation in normal hematopoietic cells.

OBJECTIVE: The human insulin-like growth factor 2 (IGF2) gene was thought to be imprinted and expressed only from the paternal allele in normal tissue. MATERIALS AND METHODS: Initially, we analyzed the imprinting status of IGF2 in bone marrow cells from 49 patients with myelodysplastic syndromes (MDS) utilizing the Apa I polymorphism of IGF2. Thirteen bone marrow and 14 peripheral blood samples from normal individuals served as controls. We utilized normal peripheral blood T lymphocytes to examine the relationship between genomic imprinting and cell proliferation. Expression of IGF2 was quantified by real-time PCR and proliferation of T cells was measured by 3H-thymidine incorporation. Furthermore, methylation status of the imprinting controlling region (ICR) was analyzed by subcloning and sequencing of genomic DNA after sodium bisulfite modification. RESULTS: Among 24 patients who were heterozygous for IGF2, loss of imprinting (LOI) occurred in 22 cases (92%). Surprisingly, LOI of IGF2 occurred in the normal bone marrow cells, but the normal peripheral blood cells showed retention of imprinting (ROI). Unstimulated normal T cells showed ROI. After 24 hours of exposure to PHA, these cells changed their IGF2 imprinting status from ROI to LOI. Concomitantly, their IGF2 RNA levels increased up to sixfold and their proliferation increased 10- to 20-fold. In contrast, normal T cells not stimulated with PHA did not develop LOI of IGF2, had negligible levels of IGF2 RNA, and did not increase their proliferation. In unstimulated T cells, the CpG islands of the ICR were completely methylated on one allele and nearly completely unmethylated on the other allele. After PHA stimulation, the CpG islands at the ICR became completely methylated on both alleles. CONCLUSION: LOI of IGF2 is strongly associated with cell proliferation and is not limited to cancer cells.

Bone Marrow Cells↗
PubMed2003

Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells.

Human CYP1A1, CYP1A2, and CYP1B1 mRNAs were constitutively expressed in MCF-7 (human breast carcinoma) cells and were extensively (6-12-fold) induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, in HeLa (human cervical adenocarcinoma) cells, CYP1A1 and CYP1B1 were induced by TCDD by up to 2-3-fold but CYP1A2 was not detected even when the cells were treated with TCDD. In the present study, the involvement of histone deacetylation and DNA methylation in the cell-specific inducibility of the human CYP1 family was investigated. The treatment of MCF-7 cells with trichostatin A (TSA), an inhibitor of histone deacetylase, and 5-aza-2'-deoxycitidine (AzaC), an inhibitor of DNA methylase, increased the constitutive expression level of CYP1A1, CYP1A2, and CYP1B1 by 2-3-fold. However, these treatments did not affect the levels of induction by TCDD. In HeLa cells, TSA and AzaC treatment increased the constitutive expression levels of CYP1A1 and CYP1B1. The induction of CYP1A2 was enhanced to a detectable level by TSA and AzaC even when the cells were not exposed to TCDD. Interestingly, pretreatment with TSA and AzaC increased the levels of CYP1A1, CYP1A2, and CYP1B1 induced by TCDD in HeLa cells. Furthermore, it was observed that TSA and AzaC treatment increased the constitutive expression level of AhR by 2-fold only in HeLa cells. To compare the methylation status of the 5'-flanking region of the human CYP1A1 gene including five XREs and the promoter region in MCF-7 and HeLa cells, the bisulfite-modified genes were amplified and sequenced. Since there was no remarkable difference in the methylation status within a -1.4 kb region of the human CYP1A1 gene, the methylation status in the CpG sites that exist in other regions of the human CYP1A1 gene might be involved in the cell-specific induction.

5' Flanking Region↗
PubMed2002

Hypermethylation of the TSLC1 gene promoter in primary gastric cancers and gastric cancer cell lines.

The TSLC1 (tumor suppressor in lung cancer-1) gene is a novel tumor suppressor gene on chromosomal region 11q23.2, and is frequently inactivated by concordant promoter hypermethylation and loss of heterozygosity (LOH) in non-small cell lung cancer (NSCLC). Because LOH on 11q has also been observed frequently in other human neoplasms including gastric cancer, we investigated the promoter methylation status of TSLC1 in 10 gastric cancer cell lines and 97 primary gastric cancers, as well as the corresponding non-cancerous gastric tissues, by bisulfite-SSCP analysis followed by direct sequencing. Allelic status of the TSLC1 gene was also investigated in these cell lines and primary gastric cancers. The TSLC1 promoter was methylated in two gastric cancer cell lines, KATO-III and ECC10, and in 15 out of 97 (16%) primary gastric cancers. It was not methylated in non-cancerous gastric tissues, suggesting that this hypermethylation is a cancer-specific alteration. KATO-III and ECC10 cells retained two alleles of TSLC1, both of which showed hypermethylation, associated with complete loss of gene expression. Most of the primary gastric cancers with promoter methylation also retained heterozygosity at the TSLC1 locus on 11q23.2. These data indicate that bi-allelic hypermethylation of the TSLC1 promoter and resulting gene silencing occur in a subset of primary gastric cancers.

Adult↗
PubMed2007

Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is commonly associated with tobacco and alcohol exposures, although dietary factors, particularly folate, and human papillomavirus, are also risk factors. Epigenetic alterations are increasingly implicated in the initiation and progression of cancer. Genome-wide (global) hypomethylation seems to occur in early neoplasia and is a feature of genomic DNA derived from solid tumor tissues, including HNSCC. This study aimed to determine whether global methylation in DNA derived from whole blood, a proxy tissue, is associated with HNSCC and to assess potential modification of this property by environmental or behavioral risk factors. METHODS: Global DNA methylation levels were assessed using a modified version of the combined bisulfite restriction analysis of the LRE1 sequence in a population-based case-control study of HNSCC from the Boston area. RESULTS: Hypomethylation lead to a significant 1.6-fold increased risk for disease (95% confidence interval, 1.1-2.4), in models controlled for other HNSCC risk factors. Smoking showed a significant differential effect (P < 0.03) on blood relative methylation between cases and controls. Furthermore, in cases, variant genotype in the MTHFR gene and low folate intake showed relationships with decreased global methylation, whereas in controls, antibody response to human papillomavirus 16 was associated with an increased global methylation level. DISCUSSION: DNA hypomethylation in nontarget tissue was independently associated with HNSCC and had a complex relationship with the known risk factors associated with the genesis of HNSCC.

Alcohol Drinking↗
PubMed2006

Hypermethylation of CpGs in the promoter of the COL1A1 gene in the aged periodontal ligament.

Although the human periodontal ligament shows age-associated histological alterations, the molecular mechanisms are not yet understood. We previously found that COL1A1 gene expression declines with age. In this study, we asked whether DNA methylation in the regulatory region of the gene alters in the aging process, as a possible cause of the decline. The method used was a bisulfite modification of cytosine and nucleotide sequencing of DNA. While the 1st intron region was kept demethylated at young and old ages, the levels of methylation at most CpG sites in the proximal and distal regions of the promoter showed elevation at older ages. Analysis of the data indicates the possible importance of DNA hypermethylation in the promoter region for the age-associated decrease of COL1A1 gene expression.

Adolescent↗
PubMedDate not supplied

Methylation of a CpG island within the uroplakin Ib promoter: a possible mechanism for loss of uroplakin Ib expression in bladder carcinoma.

Uroplakin Ib is a structural protein on the surface of urothelial cells. Expression of uroplakin Ib mRNA is reduced or absent in many transitional cell carcinomas (TCCs) but molecular mechanisms underlying loss of expression remain to be determined. Analysis of the uroplakin Ib promoter identified a weak CpG island spanning the proximal promoter, exon 1, and the beginning of intron 1. This study examined the hypothesis that methylation of this CpG island regulates uroplakin Ib expression. Uroplakin Ib mRNA levels were determined by reverse transcription polymerase chain reaction and CpG methylation was assessed by bisulfite modification of DNA, PCR, and sequencing. A correlation was demonstrated in 15 TCC lines between uroplakin Ib mRNA expression and lack of CpG methylation. In support of a regulatory role for methylation, incubating uroplakin Ib-negative lines with 5-aza-2'-deoxycytidine reactivated uroplakin Ib mRNA expression. A trend between uroplakin Ib mRNA expression and CpG methylation was also observed in normal urothelium and bladder carcinomas. In particular, loss of uroplakin Ib expression correlated with methylation of a putative Sp1/NFkappaB binding motif. The data are consistent with the hypothesis that methylation of specific sites within the uroplakin Ib promoter may be an important factor in the loss of uroplakin Ib expression in TCCs.

Azacitidine↗
PubMed1998

Novel procedure for the detection of 5-methylcytosine.

Bisulfite converts non-methylated cytosine in DNA to uracil leaving 5-methylcytosine unaltered. In this communication, we present a new approach omitting the conventional PCR amplification step. Bisulfite-converted methylated DNA is directly sequenced. The effectiveness of the new protocol is demonstrated by using it for the detection of 5-methylation of cytosine residues introduced by three different DNA methyltransferases (M.HaeIII, M.HpaII and M.HhaI). A simple experimental system useful to determine the sequence specificity of DNA methyltransferases is also presented.

5-Methylcytosine↗
PubMed2025

DNA Methylation Analysis by Bisulfite Pyrosequencing of Mouse Embryonic Fibroblasts with Reprogramming Enhanced by Thyroid Hormones.

DNA methylation is a widely studied epigenetic mark which in mammals involves the incorporation of a methyl group to the fifth carbon of cytosines, mainly those belonging to CpG dinucleotides. It has been linked to context-dependent regulatory functions ranging from gene and repetitive DNA silencing to gene body transcriptional activity. Because of its important roles during embryonic development and cell differentiation, DNA methylation can be used to track cell reprogramming by measuring the methylation levels of pluripotency-associated factors. In this scenario, bisulfite pyrosequencing is a simple, robust, and widely used technique which allows for the quantification of DNA methylation levels at small, specific regions of the genome. It involves the amplification and biotin tagging of bisulfite-converted DNA. Single amplified strands are then purified using streptavidin and finally pyrosequenced using a sequencing primer. Thus, it is an ideal method for the quantitative profiling of specific genomic regions, with applications ranging from biomarker discovery and epigenetic clock tracking to omic validation studies.

Animals↗
PubMed2005

Isolation and analysis of sequences showing sex-specific cytosine methylation in the mealybug Planococcus lilacinus.

Genomic libraries of Planococcus lilacinus, a mealybug in which paternal chromosomes are facultatively heterochromatic and inactive in sons but not in daughters, were probed with subtraction probes in order to estimate the number of sequences displaying sex-specific cytosine methylation in CpG dinucleotides. Sequences showing male-specific methylation were found to occur approximately 2.5 times more often than those showing female-specific methylation. In order to directly isolate sequences showing sex-specific CpG methylation, we employed methylation-specific arbitrarily primed (MS-AP) polymerase chain reaction (PCR) and identified 72 sex-specific products, of which 51 were from males and 21 from females. Amplification of bisulfite-modified DNA and subsequent Southern hybridization showed that in 33 out of these 72 sex-specific products, there was differential methylation of homologous sequences; i.e., both methylated and unmethylated copies of the same sequence occurred in one sex whereas only unmethylated copies were present in the opposite sex. Sequencing of bisulfite-modified DNA showed an interspersion of CpG and non-CpG methylation among the sex-specifically methylated sequences. Sequences showing male-specific CpG methylation are organized as transcriptionally silent chromatin in males but not in females, whereas those showing female-specific CpG methylation are organized as transcriptionally silent chromatin in females but not in males. The sequences identified in this study that show differential methylation in males, but are unmethylated in females, may prove useful in the study of imprinting in the mealybug system.

Animals↗
PubMed2002

Methylation-specific oligonucleotide microarray: a new potential for high-throughput methylation analysis.

Oligonucleotide microarray-based hybridization is an emerging technology for genome-wide detection of DNA variations. We have extended this principle and developed a novel approach, called methylation-specific oligonucleotide (MSO) microarray, for detecting changes of DNA methylation in cancer. The method uses bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. The amplified product, therefore, may contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. A test sample is hybridized to a set of oligonucleotide (19-23 nucleotides in length) arrays that discriminate methylated and unmethylated cytosine at specific nucleotide positions, and quantitative differences in hybridization are determined by fluorescence analysis. A unique control system is also implemented to test the accuracy and reproducibility of oligonucleotides designed for microarray hybridization. This MSO microarray was applied to map methylated CpG sites within the human estrogen receptor alpha (ERalpha) gene CpG island in breast cancer cell lines, normal fibroblasts, breast tumors, and normal controls. Methylation patterns of the breast cancer cell lines, determined by MSO microarray, were further validated by bisulfite nucleotide sequencing (P <0.001). This proof-of-principle study shows that MSO microarray is a promising technique for mapping methylation changes in multiple CpG island loci and for generating epigenetic profiles in cancer.

Base Sequence↗
PubMed1989

Localized chemical hyperreactivity in supercoiled DNA: evidence for base unpairing in sequences that induce low-salt cruciform extrusion.

Certain A + T-rich DNA sequences (C-type inducing sequences) cause adjacent inverted repeats to undergo cruciform extrusion by a particular pathway (C-type extrusion), which is characterized by large activation energies and extrusion at low salt concentrations and relatively low temperatures. When they are supercoiled, these sequences become reactive toward the normally single-strand-selective reagents bromoacetaldehyde, glyoxal, osmium tetraoxide, and sodium bisulfite. The following evidence is presented: (1) The most reactive sequences are those to the left of the inverted repeat. (2) Chemical reactivity is suppressed by either sodium chloride or micromolar concentrations of distamycin. The suppression of reactivity closely parallels that of C-type cruciform extrusion. (3) Chemical reactivity requires a threshold level of negative supercoiling. The threshold superhelix density depends on the prevailing salt concentration. (4) Analysis of temperature dependences suggests that reaction with osmium tetraoxide involves transient unstacking events, while bromoacetaldehyde requires larger scale helix opening. Thus a variety of opening events may occur in the supercoiled A + T-rich sequences, from small-amplitude breathing to low-frequency, large-amplitude openings. The latter appear to be responsible for C-type cruciform extrusion.

Acetaldehyde↗
PubMed1997

Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE).

We have developed a rapid quantitative method (Ms-SNuPE) for assessing methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by single nucleotide primer extension. Genomic DNA was first reacted with sodium bisulfite to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence was then performed using PCR primers specific for bisulfite-converted DNA and the resulting product isolated and used as a template for methylation analysis at the CpG site(s) of interest. This methylation-sensitive technique has several advantages over existing methods used for detection of methylation changes because small amounts of DNA can be analyzed including microdissected pathology sections and it avoids utilization of restriction enzymes for determining the methylation status at CpG sites.

DNA↗
PubMed1999

Diversity of dissimilatory bisulfite reductase genes of bacteria associated with the deep-sea hydrothermal vent polychaete annelid Alvinella pompejana.

A unique community of bacteria colonizes the dorsal integument of the polychaete annelid Alvinella pompejana, which inhabits the high-temperature environments of active deep-sea hydrothermal vents along the East Pacific Rise. The composition of this bacterial community was characterized in previous studies by using a 16S rRNA gene clone library and in situ hybridization with oligonucleotide probes. In the present study, a pair of PCR primers (P94-F and P93-R) were used to amplify a segment of the dissimilatory bisulfite reductase gene from DNA isolated from the community of bacteria associated with A. pompejana. The goal was to assess the presence and diversity of bacteria with the capacity to use sulfate as a terminal electron acceptor. A clone library of bisulfite reductase gene PCR products was constructed and characterized by restriction fragment and sequence analysis. Eleven clone families were identified. Two of the 11 clone families, SR1 and SR6, contained 82% of the clones. DNA sequence analysis of a clone from each family indicated that they are dissimilatory bisulfite reductase genes most similar to the dissimilatory bisulfite reductase genes of Desulfovibrio vulgaris, Desulfovibrio gigas, Desulfobacterium autotrophicum, and Desulfobacter latus. Similarities to the dissimilatory bisulfite reductases of Thermodesulfovibrio yellowstonii, the sulfide oxidizer Chromatium vinosum, the sulfur reducer Pyrobaculum islandicum, and the archaeal sulfate reducer Archaeoglobus fulgidus were lower. Phylogenetic analysis separated the clone families into groups that probably represent two genera of previously uncharacterized sulfate-reducing bacteria. The presence of dissimilatory bisulfite reductase genes is consistent with recent temperature and chemical measurements that documented a lack of dissolved oxygen in dwelling tubes of the worm. The diversity of dissimilatory bisulfite reductase genes in the bacterial community on the back of the worm suggests a prominent role for anaerobic sulfate-reducing bacteria in the ecology of A. pompejana.

Amino Acid Sequence↗
PubMed1999

Quantitative analysis of aberrant p16 methylation using real-time quantitative methylation-specific polymerase chain reaction.

We have developed a quantitative method for methylation analysis of the p16 gene based on real-time methylation-specific PCR (MSP). Real-time MSP is sensitive enough to detect down to 10 genome equivalents of the methylated p16 sequence. Application of real-time MSP to DNA from tumor-derived cell lines revealed complete concordance with conventional MSP analysis. Quantitative data generated by real-time MSP were expressed as the methylation index, which was defined as the percentage of bisulfite-converted DNA that consisted of methylated target sequences. The methylation index was shown to be inversely correlated with p16 gene transcription during demethylation treatment of cell lines with 5-aza-2'-deoxycytidine. The application of real-time MSP to bone marrow aspirates from patients with multiple myeloma revealed complete concordance with conventional MSP analysis. Real-time quantitative MSP may have applications in elucidating diverse biological processes involving DNA methylation and may become a valuable diagnostic tool for detecting tumor-associated epigenetic changes in cancer patients.

DNA Methylation↗
PubMed1997

Methylation hot spots in the 5' flanking region denote silencing of the O6-methylguanine-DNA methyltransferase gene.

The mechanism whereby the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is silenced in repair-deficient (Mer-) human tumor cells is unknown. The role of methylation of the 5' CpG island in MGMT gene suppression is controversial. Although we previously showed by restriction enzyme analysis that CpG methylation in this region was associated with gene suppression, methylation at such sites was generally incomplete, suggesting heterogeneity. To clarify this issue, we have unequivocally defined the methylation status of every CpG by genomic sequencing of individual cloned copies of bisulfite-modified DNA. The region from -249 to +259 at the transcription start site was virtually methylation free in HT29 cells (Mer+), whereas in BE or HeLa S3 cells (Mer-), this region was substantially methylated in every DNA copy, with "hot spots" from -249 to -103 and from +107 to +196. Up-regulation of MGMT in HeLa S3 cells induced by 5-azacytidine was accompanied by progressive demethylation and the appearance of totally unmethylated copies of DNA. We conclude that, in Mer- cells, the MGMT promoter contains specific CpG methylation hot spots that are tightly linked to and are potential markers of gene silencing.

CpG Islands↗