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Results for “DNA Methylation Detection”

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PubMed2006

Transcriptional silencing of multiple genes in trophozoites of Entamoeba histolytica.

In a previous work we described the transcriptional silencing of the amoebapore A (AP-A) gene (Ehap-a) of Entamoeba histolytica strain HM-1:IMSS. The silencing occurred following transfection with a plasmid containing a 5' upstream region (473 bp) of Ehap-a that included a truncated segment (140 bp) of a short interspersed nuclear element (SINE1). Silencing remained in effect even after removal of the plasmid (clone G3). Neither short interfering RNA nor methylated DNA were detected, but the chromatin domain of Ehap-a in the gene-silenced trophozoites was modified. Two other similar genes (Ehap-b and one encoding a Saposin-like protein, SAPLIP 1) also became silenced. In the present work we demonstrate the silencing of a second gene of choice, one that encodes the light subunit of the Gal/GalNAc inhibitable lectin (Ehlgl1) and the other, the cysteine proteinase 5 (EhCP-5). This silencing occurred in G3 trophozoites transfected with a plasmid in which the 473 bp 5' upstream Ehap-a fragment was directly ligated to the second gene. Transcriptional silencing occurred in both the transgene and the chromosomal gene. SINE1 sequences were essential, as was a direct connection between the Ehap-a upstream region and the beginning of the open reading frame of the second gene. Gene silencing did not occur in strain HM-1:IMSS with any of these plasmid constructs. The trophozoites with two silenced genes were virulence-attenuated as were those of clone G3. In addition, trophozoites not expressing Lgl1 and AP-A proteins had a significantly reduced ability to cap the Gal/GalNAc-lectin to the uroid region when incubated with antibodies against the heavy (170 kDa) subunit of the lectin. Lysates of trophozoites lacking cysteine proteinase 5 and AP-A proteins had 30% less cysteine proteinase activity than those of HM-1:IMSS strain or the G3 clone. Silencing of other genes in G3 amoebae could provide a model to study their various functions. In addition, double gene-silenced, virulence-attenuated trophozoites may be an important tool in vaccine development.

Animals↗

PubMed2004

Methylation assay by nucleotide incorporation: a quantitative assay for regional CpG methylation density.

Although the aberrant methylation in CpG islands is of great interest as a causative role in human malignancies, it has been very difficult to accurately determine methylation density. Here we report a novel microplate-based quantitative methylation assay, designated MANIC, for a region containing a number of CpG sites based on incorporation of hapten-labeled dCTP at cytosine sites where the methylated cytosines have not been converted to uracil by the bisulfite treatment. Validation using control DNAs revealed that the method was sensitive enough to detect < 1.25% methylated DNA and that calibration curve was linear. With this approach, we determined relative methylation density of O6-methylguanine-DNA methyltransferase gene promoter containing 12 CpG sites among the 12 colorectal cancers and corresponding normal mucosal tissues. Consequently, MANIC showed a high concordance with results by a quantitative method, bisulfite PCR single-stranded conformational polymorphism (BiPS). MANIC is a technique that avoids cumbersome procedures such as electrophoresis or the use of radiolabeling and is applicable to any sequence regardless of the total number of CpG sites or heterogeneity in methylation status.

Chromosome Mapping↗

PubMed1992

Flow cytometric detection of drugs altering the DNA methylation pattern.

We have developed a model system for assessing the demethylating potential of external agents. Disruption in the DNA methylation pattern was evaluated at the translational level of the Escherichia coli beta-galactosidase coding gene (lacZ). We have constructed a clonal cell line (A4/4 cells) derived from the adenovirus-transformed human embryonic kidney 293 strain. The A4/4 cells contain the E. coli lacZ gene under the control of the mouse metallothionein 1 promoter which is down-regulated by a natural DNA methylation pattern. Furthermore, the lacZ transcription is also regulated by the E. coli lac operator/repressor system and by mouse metallothionein 1 metal responsiveness offering a wide range in lacZ expression. In this system, the beta-galactosidase activity was only recovered in the presence of a demethylating agent such as 5-azacytidine. The demethylating potential of 5-azacytidine, 5-aza 2'-deoxycytidine and sodium butyrate was rapidly assessed by a flow cytometric method using fluorescein di-beta-D galactopyranoside as a fluorescent probe. A tremendous induction of lacZ expression was triggered by these drugs. Analysis of cell cycles showed little disruptions with 5-azacytidine and sodium butyrate, but an important blockage in the S-phase following 5-aza 2'-deoxycytidine treatment was observed. This approach allows a rapid identification and study of environmental demethylating agents.

Azacitidine↗

PubMed2006

Molecular diagnosis of Beckwith-Wiedemann syndrome using quantitative methylation-sensitive polymerase chain reaction.

PURPOSE: Beckwith-Wiedemann Syndrome is caused by defects in imprinted gene expression at 11p15. Currently, quantitative Southern analysis using DNA methylation-sensitive restriction enzymes is used in molecular diagnosis of this syndrome. METHODS: We describe a rapid and highly quantitative test for assessing DNA methylation at 11p15 using sodium bisulfite treatment of genomic DNA coupled with quantitative TaqMan methylation-sensitive polymerase chain reaction. RESULTS: TaqMan MSP can assess DNA methylation at both differentially methylated region (DMR)1 and DMR2 at 11p15. In addition, by using TaqMan MSP we were able to determine the parent of origin of a duplication of 11p15 by quantification of both DMR1 and DMR2 DNA methylation. CONCLUSION: TaqMan MSP method is a robust and rapid method for detecting changes in DNA methylation that compares favorably to the current standard of Southern blot for DNA methylation analysis. Assessment of DMR1 and DMR2 provides the most comprehensive assay for methylation defects in Beckwith Wiedemann Syndrome, accounting for more than 70% of the cases. The advantages of TaqMan MSP are that it requires less DNA and that it is rapid, less labor-intensive, and amenable to high-throughput analysis. Moreover, this approach can be modified to assess DNA methylation changes anywhere in the genome.

Beckwith-Wiedemann Syndrome↗

PubMed1983

Exposure of JB-6 mouse epidermal cells to 12-O-tetradecanoyl-phorbol-13-acetate is not accompanied by a significant change in total DNA-cytosine methylation.

The extent of methylation of the cytosine bases in DNA is believed to be a major factor influencing gene expression in eukaryotic cells. We have asked whether the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alters the amount of 5-methylcytosine in DNA. The amount and relative distribution of 5-methylcytosine in the DNA of two subclones of the JB-6 mouse epidermal cell line were determined respectively by high performance liquid chromatography and digestion with the restriction enzymes MspI and HpaII. Exposure to TPA for up to several cell generations had no detectable effect on the degree of DNA methylation (3.9% of the total cytosine) in the two JB-6 lines or Friend erythroleukemia cells. Reduced methylation was readily detected in DNA extracted from cells exposed to 5-azacytidine. The data suggest that tumor promotion (at least that induced by TPA) is likely not the consequence of a generalized elevation or reduction in the amount of 5-methyl-cytosine in the DNA.

5-Methylcytosine↗

PubMed1992

Increase in susceptibility to EcoRII restriction of bacteriophage lambda produced by propagation on host cells growing in 5-azacytidine: a new in-vivo method for demonstration of DNA-methylation inhibition.

The efficiency of plating on EcoRII-restricting cells of bacteriophage lambda vir propagated on an Escherichia coli K-12 dcm+ host decreased with increase in concentration of 5-azacytidine (5-azaC) in the propagating medium. This illustrates, in-vivo, the inhibition of DNA-cytosine methylation induced by 5-azaC and provides a simple system for the detection of DNA-methylation inhibitors.

Azacitidine↗

PubMed2006

A comparative study of using comet assay and gammaH2AX foci formation in the detection of N-methyl-N'-nitro-N-nitrosoguanidine-induced DNA damage.

Comet assay is a useful technique in the detection of DNA damages, particularly DNA strand breaks; and it has been utilized to show that a potent carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), can induce such damages. Recently, gammaH2AX foci formation has been suggested as another sensitive way to detect DNA double strand breaks (DSBs). However, there is no systematic comparison being conducted to evaluate the consistency of these two methods. Using MNNG as a model chemical, the sensitivity of neutral comet assay and gammaH2AX foci formation in detecting MNNG-induced damage was studied. It was found that at concentrations of 0.1 and 1 microg/ml, both methods can detect MNNG-induced damage in human amnion FL cells. However, at 0.1 microg/ml, comet assay revealed more percentage of cells with DNA damage than gammaH2AX fluorescence revealed. On the other hand, while gammaH2AX foci were readily formed at very early times by 10 microg/ml MNNG treatment, neutral comet assay did not detect any significant DNA damage at the same time points. In addition, 10 microg/ml MNNG induced a distinct whole nuclei staining pattern of gammaH2AX, a type of DNA damage which was not detected by neutral comet assay but could be detected by alkaline comet assay. Therefore, gammaH2AX may be used as a sensitive indicator for DNA damage.

Cells, Cultured↗

PubMed2005

Gene promoter methylation in plasma and sputum increases with lung cancer risk.

PURPOSE: Lung cancer is the leading cause of cancer mortality in the United States, due in part to the lack of a validated and effective screening approach for early detection. The prevalence for methylation of seven and three genes was examined in DNA from sputum and plasma, respectively, from women at different risk for lung cancer. EXPERIMENTAL DESIGN: Lung cancer survivors (n = 56), clinically cancer-free smokers (n = 121), and never smokers (n = 74) comprised the study population. Plasma was collected from all three groups, whereas sputum was collected from lung cancer survivors and smokers. RESULTS: Methylation was detected in plasma DNA from 10 of 74 women who never smoked. Prevalence for methylation of the p16 gene in plasma was highest in lung cancer survivors. Lung cancer survivors showed a significant increase in the odds of having at least one or more genes methylated in plasma (odds ratio, 3.6; 95% confidence interval, 1.9-9.1) than never smokers. The prevalence for methylation of the O(6)-methylguanine-DNA methyltransferase, ras effector homologue 1, death associated protein kinase, and PAX5alpha genes in sputum was significantly higher in lung cancer survivors compared with smokers. Lung cancer survivors had 6.2-fold greater odds (95% confidence interval, 2.1-18.5) for methylation of three or more genes in sputum compared with smokers. Methylation was more commonly detected in sputum than plasma for O(6)-methylguanine-DNA methyltransferase and ras effector homologue 1, but not p16, in lung cancer survivors. CONCLUSION: Concomitant methylation of multiple gene promoters in sputum is strongly associated with lung cancer risk.

Adult↗

PubMed2006

Methylation enrichment pyrosequencing: combining the specificity of MSP with validation by pyrosequencing.

It has been suggested that detection of aberrant DNA methylation in clinical specimens such as sputum or saliva may be a valuable tumour biomarker. Any clinically applicable detection technique must combine high sensitivity with high specificity. In this study we describe methylation enrichment pyrosequencing (MEP), which benefits from the high sensitivity and specificity of methylation-specific PCR (MSP) but has a second, confirmatory, pyrosequencing step. The pyrosequencing reaction is rapid, relatively inexpensive and offers significant logistical advantages over previously described validation methods. As proof of principle, we illustrate MEP using assays of p16 and cyclin A1 promoters in a methylated DNA dilution matrix and also in a clinical setting using paired saliva and oral tumour specimens. Our results confirm that mis-priming of MSP, with subsequent false positive results, can occur frequently (perhaps 10%) in assays combining high numbers of PCR cycles and low concentrations of starting DNA. In our clinical example, MEP of saliva-derived DNA was more sensitive than standard non-methylation-specific pyrosequencing as illustrated using p16 and cyclin A1 promoter methylation assays.

Base Sequence↗

PubMed2003

DNA methylation analysis techniques.

DNA methylation contributes to the control of gene expression and plays an essential role in cellular physiology. Well-defined patterns of DNA methylation are established and fixed during embryonic development, and changes in these patterns may be a contributing factor in developmental disorders, cancer and aging. Not least the possibility of using DNA methylation as a marker for disease has created a strong need for techniques to detect and measure DNA methylation. Different techniques provide information on DNA methylation at different levels, spanning from genome-wide methylation content to methylation of single residues in specific genes. The limitations of individual techniques strongly affect interpretation of data. In this review, we discuss some general themes in DNA methylation analysis and outline the basic principles of current key techniques. We discuss the advantages and disadvantages of these techniques, including potential artifacts and pitfalls, and suggest some overall guidelines that may be instructive for a rational choice of methodology.

Blotting, Southern↗

PubMed2005

High frequency of promoter hypermethylation of RASSF1A in tumor and plasma of patients with hepatocellular carcinoma.

PURPOSE: To determine the presence of ras association domain family 1A (RASSF1A) promoter methylation in the tumor tissues and plasma of patients with hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: Methylation-specific polymerase chain reaction was used to detect RASSF1A methylation in DNA extracted from HCC tumors and paired plasma samples of 40 patients. The association of RASSF1A hypermethylation in tumor and plasma DNA of HCC patients with clinicopathological characteristics was also analyzed. RESULTS: RASSF1A promoter hypermethylation was detected in 37 of the 40 HCC tissues (92.5%). Of the paired plasma from the 40 HCC patients, aberrant methylation was detected in 17 (42.5%). No RASSF1A methylation was detected in the plasma in the absence of methylation in the corresponding tumor. The presence of RASSF1A promoter hypermethylation in plasma DNA was found to associate with HCC size of > or =4 cm (P = 0.035). CONCLUSION: RASSF1A promoter hypermethylation occurred at a high frequency in HCC. The aberrant methylation was also detectable in over 40% of matched plasma. The latter should be evaluated as a screening tool and/or prognosticator of HCC patients.

Base Sequence↗

PubMed2003

Microarrays assembled in microfluidic chips fabricated from poly(methyl methacrylate) for the detection of low-abundant DNA mutations.

Low-density arrays were assembled into microfluidic channels hot-embossed in poly(methyl methacrylate) (PMMA) to allow the detection of low-abundant mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. Following spotting, the chip was assembled with a cover plate and the array accessed using microfluidics in order to enhance the kinetics associated with hybridization. The array was configured with zip code sequences (24-mers) that were complementary to sequences present on the target. The hybridization targets were generated using an allele-specific ligase detection reaction (LDR), in which two primers (discriminating primer that carriers the complement base to the mutation being interrogated and a common primer) that flank the point mutation and were ligated joined together) only when the particular mutation was present in the genomic DNA. The discriminating primer contained on its 5'-end the zip code complement (directs the LDR product to the appropriate site of the array), and the common primer carried on its 3' end a fluorescent dye (near-IR dye IRD-800). The coupling chemistry (5'-amine-containing oligonucleotide tethered to PMMA surface) was optimized to maximize the loading level of the zip code oligonucleotide, improve hybridization sensitivity (detection of low-abundant mutant DNAs in high copy numbers of normal sequences), and increase the stability of the linkage chemistry to permit re-interrogation of the array. It was found that microfluidic addressing of the array reduced the hybridization time from 3 h for a conventional array to less than 1 min. In addition, the coupling chemistry allowed reuse of the array > 12 times before noticing significant loss of hybridization signal. The array was used to detect a point mutation in a K-ras oncogene at a level of 1 mutant DNA in 10,000 wild-type sequences.

DNA↗

PubMed1988

Detection of heavy methylation in human repetitive DNA subsets by a monoclonal antibody against 5-methylcytosine.

A monoclonal antibody (IgM) against 5-methylcytosine (mC) was isolated and characterized. It showed a high specificity for mC with a cross-reactivity of less than 1% with cytosine and 0.1% with thymidine. An improved immunohybridization method, originally developed with polyclonal antibodies (Sano et al. (1980) Proc. Natl. Acad. Sci. USA 77, 3581), was applied to detect mC in immobilized DNA using the new monoclonal preparation. Human genomic DNA was cleaved with the restriction enzyme EcoRI and successively fractionated by malachite-green affinity chromatography and agarose gel electrophoresis. The fractionated DNA was transferred to nitrocellulose paper and treated with the anti-mC monoclonal antibody. Heavy methylation was observed in EcoRI-ladders of repetitive sequences of 1360, 1750, 2200 and 3400 bp, while 340, 660 and 2700 bp fragments were less methylated. The results show that methylation occurs in limited subsets of satellite II and III repetitive DNAs that contain high amounts of methylatable CpG dinucleotides, or CpG clusters.

5-Methylcytosine↗

PubMed2006

Effects of Helicobacter pylori eradication on methylation status of E-cadherin gene in noncancerous stomach.

PURPOSE: Promoter hypermethylation of E-cadherin plays an important role on gastric cancer development. Whereas E-cadherin methylation was frequently detected in the stomach of Helicobacter pylori-infected individuals, we tested whether eradication of H. pylori alters the methylation status of the noncancerous gastric epithelium. EXPERIMENTAL DESIGN: Endoscopic biopsies were taken from the antrum and corpus of H. pylori-infected subjects without gastric cancer. Presence of methylated E-cadherin sequences in the gastric specimens was detected by methylation-specific PCR. Bisulfite DNA sequencing was done to determine the topographical distribution and changes in methylation profiles with H. pylori eradication. RESULTS: Among the 28 H. pylori-infected subjects (median age, 44.5 years), 15 (53.6%) had E-cadherin methylation detected in stomach at baseline. Discordant methylation patterns between the antrum and corpus were noted in six patients. One year after successful H. pylori eradication, there was a significant reduction in the methylation density of the promoter region and exon 1 of the E-cadherin gene as detected by bisulfite DNA sequencing (P < 0.001). CONCLUSION: Promoter methylation in E-cadherin was frequently detected in the stomach of H. pylori-infected individuals. Eradication of H. pylori might possibly reduce the methylation density in E-cadherin gene and the chance of subsequent neoplastic transformation.

Adult↗

PubMed2006

6,6'-Disubstituted benzothiazole trimethine cyanines--new fluorescent dyes for DNA detection.

The influence of methyl-, 2-hydroxyethyl-, dimethyl-, diethyl- and benzoyl-amino substituents in the 6,6'-positions of benzothiazole heterocycle of trimethine cyanines on their spectral-luminescent properties and behavior in presence of DNA, RNA and BSA was studied. It was shown that incorporation of 6,6'-substituents generally leads to the increase in dyes tendency to aggregation, resulting in the considerable decrease in the emission intensity of the disubstituted dyes as compared to the unsubstituted ones. Emission of the studied 6,6'-disubstited dyes in DNA presence is considerably more intensive than in presence of RNA, that points on the existing of DNA binding preference for the mentioned dyes. Insertion of benzoyl-amino groups into the 6,6'-positions permitted us to design the DNA-sensitive dyes on the basis of symmetric trimethine cyanines with unsubstituted polymethine chain, while typically such dyes slightly respond on the presence of biopolymers. 6,6'-Benzoyl-amino-disubstituted trimethine cyanines are proposed as efficient dyes for DNA detection.

Animals↗

PubMedDate not supplied

Improvement of the methylation specific PCR technical conditions for the detection of p16 promoter hypermethylation in small amounts of tumor DNA.

Methylation specific PCR (MSP) is a technique that enables the detection of hypermethylation at a CpG island. The objective of this study is the introduction of small modifications to the MSP technique to make it more suitable for the study of promoter hypermethylation at tumor suppressor genes whenever there is a shortage of material available for study. This commonly happens in the case of using archival material from the Pathology departments. Tumor DNA was extracted from a collection of 40 fresh-frozen soft tissue sarcomas and 19 paraffin-embedded PNETs (primitive neuroectodermal tumors). The MSP technique was performed to detect hypermethylation at the p16 promoter. Also, blood genomic DNA was mixed with herry sperm genomic DNA as a carrier, in nine different combinations, in order to test for the best conditions that could produce MSP bands even when low amounts of genomic tumor DNA is available for study. We demonstrate the benefit of using herry sperm carrier DNA up to 10 microg together with small quantities of tumor DNA. This result will facilitate the incorporation of paraffin-embedded samples for study of promoter hypermethylation at tumor suppressor genes. Other technical conditions for the MSP technique are also studied.

Brain Neoplasms↗

PubMed1991

The combination of DNA methylation and H1 histone binding inhibits the action of a restriction nuclease on plasmid DNA.

To investigate the potentials of DNA methylation and H1 histone in regulating the action of DNA binding proteins, well ordered complexes were formed by slow salt gradient dialysis of mixtures of H1 histone with either methylated or nonmethylated DNA. The sites methylated in the plasmids were CCGG. Methylation of cytosine in this site protects the DNA against HpaII endonuclease but not against MspI. However, when the methylated DNA was complexed to H1, it was protected against MspI. The protection was only effective for a subset of the MspI restriction sites. The protection of DNA afforded by the combination of H1 binding and DNA methylation did not apply to EcoRI, PstI, or BamHI sites and so did not seem to be due to aggregation of the DNA by H1 histone. Gel retardation assays indicated that the affinity of H1 for methylated DNA was not detectably different from its affinity for nonmethylated DNA. Probably methylated DNA when bound to H1 is in a conformation that is resistant to MspI endonuclease. Such conformational changes induced by DNA methylation and H1 binding might affect the action of other DNA binding proteins, perhaps in chromatin as well as in H1.DNA complexes.

Animals↗

PubMed1990

Combined high-performance liquid chromatography/32P-postlabeling assay of N7-methyldeoxyguanosine.

A highly sensitive and specific assay for the detection of N7-methyl-2'-deoxyguanosine (N7methyldG) has been developed by combining high-performance liquid chromatography, 32P-postlabeling, and nucleotide chromatography. Separation of normal nucleotides and adducts by high-performance liquid chromatography and then combining a portion of 2'-deoxyguanosine to the N7methyldG allows for quantitation using an internal standard. The directly determined molar ratio is not subject to errors in digestion, variable ATP-specific activity, or assumptions in relative adduct-labeling efficiency. The detection limit was one N7methyldG adduct in 10(7) unmodified 2'-deoxyguanosine bases. N7methyldG adducts have been detected in 5 human lung samples in which O6-methyl-2'-deoxyguanosine adducts had been previously determined. The mean ratio of N7methyldG to O6-methyl-2'-deoxyguanosine was determined to be approximately 10. The current assay complements the high-performance liquid chromatography/32P-postlabeling assay for O6-methyl-2'-deoxyguanosine and increases the detection sensitivity of DNA methylated by exogenous alkylating agents.

Adult↗