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Results for “DNA Methylation Detection”

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PubMed2006

Epigenetic profiling of synchronous colorectal neoplasias by quantitative DNA methylation analysis.

Transcriptional inactivation of tumor-suppressor genes by promoter CpG island methylation is thought to be an important mechanism in human carcinogenesis. The CpG island methylator phenotype (CIMP) with extensive promoter methylation appears to be a distinct epigenetic subtype of colorectal carcinoma. Most previous studies on CpG island methylation in colorectal carcinoma used methylation-specific PCR, which may detect low levels of DNA methylation with little or no biological significance. In contrast, quantitative DNA methylation assays have been shown to provide useful information beyond that which can be achieved with methylation-specific PCR. Synchronous neoplasias provide a unique model for investigators to examine molecular alterations in multistep tumorigenesis within one individual. However, no study to date has quantified DNA methylation of CIMP-specific promoters in synchronous colorectal neoplasias. Utilizing real-time PCR (MethyLight), we quantified DNA methylation in five CIMP-specific gene promoters [CACNA1G (calcium channel, voltage-dependent, T type alpha-1G subunit), CDKN2A (p16/INK4A), CRABP1 (cellular retinoic acid binding protein-1), MLH1 and NEUROG1 (neurogenin 1)] and MGMT in six synchronous carcinoma pairs (12 carcinomas) and eight synchronous carcinoma and adenoma pairs (16 tumors). We found that while some synchronous tumor pairs showed discordant promoter methylation patterns, other tumor pairs showed similar, but not exactly identical, patterns of promoter methylation. All but two pairs showed concordant patterns of CIMP status (CIMP positive vs CIMP negative) (P = 0.05 in cancer pairs). BRAF mutations were present in only CIMP-positive tumors. A high degree of microsatellite instability (MSI-H) was observed in both CIMP-positive and CIMP-negative tumors. KRAS mutations were not concordant in any synchronous neoplasia pair. In conclusion, epigenetic alterations at CIMP-specific promoter CpG islands in synchronous colorectal neoplasias likely have both random and nonrandom components.

Adenocarcinoma↗

PubMed2004

Prognostic DNA methylation marker in serum of cancer patients.

Changes in the status of DNA methylation are among the most common molecular alterations in human neoplasia. Recent demonstrations of tumor-derived methylated DNA in the blood stream of cancer patients allow the use of these epigenetic markers for risk assessment in cancer patients. We were interested in evaluating the prognostic value of several methylated genes in the serum of cancer patients. Using MethyLight, a high-throughput DNA methylation assay, we analyzed 215 serum samples from patients with cervical (n = 93) or breast cancer (n = 122) for DNA methylation changes. In cervical cancer, hypermethylation of three genes (MYOD1, CDH1, and CDH13) in pretreatment sera was statistically significantly associated with a poorer disease outcome. Additionally, for the first time we used a so-called gene evaluation set to identify the most important DNA methylation changes in the serum of breast cancer patients from a long list of candidate genes. In the gene evaluation set, we detected five genes (ESR1, APC, HSD17B4, HIC1, and RASSF1A) using our criteria for further analysis. Finally, two of the evaluated genes (APC and RASSF1A) proved to be independent prognostic parameters in breast cancer patients. In summary, we detected several prognostic DNA methylation markers in the serum of cervical and breast cancer patients. This finding indicates great potential for the use of these epigenetic markers in clinical, routine risk assessment in patients with various malignancies.

Biomarkers, Tumor↗

PubMed1997

Association of arsenic-induced malignant transformation with DNA hypomethylation and aberrant gene expression.

Inorganic arsenic, a human carcinogen, is enzymatically methylated for detoxication, consuming S-adenosyl-methionine (SAM) in the process. The fact that DNA methyltransferases (MeTases) require this same methyl donor suggests a role for methylation in arsenic carcinogenesis. Here we test the hypothesis that arsenic-induced initiation results from DNA hypomethylation caused by continuous methyl depletion. The hypothesis was tested by first inducing transformation in a rat liver epithelial cell line by chronic exposure to low levels of arsenic, as confirmed by the development of highly aggressive, malignant tumors after inoculation of cells into Nude mice. Global DNA hypomethylation occurred concurrently with malignant transformation and in the presence of depressed levels of S-adenosyl-methionine. Arsenic-induced DNA hypomethylation was a function of dose and exposure duration, and remained constant even after withdrawal of arsenic. Hyperexpressibility of the MT gene, a gene for which expression is clearly controlled by DNA methylation, was also detected in transformed cells. Acute arsenic or arsenic at nontransforming levels did not induce global hypomethylation of DNA. Whereas transcription of DNA MeTase was elevated, the MeTase enzymatic activity was reduced with arsenic transformation. Taken together, these results indicate arsenic can act as a carcinogen by inducing DNA hypomethylation, which in turn facilitates aberrant gene expression, and they constitute a tenable theory of mechanism in arsenic carcinogenesis.

Animals↗

PubMed1984

Maternal inheritance of the mouse mitochondrial genome is not mediated by a loss or gross alteration of the paternal mitochondrial DNA or by methylation of the oocyte mitochondrial DNA.

To evaluate whether the absence or modification of paternal mitochondrial DNA or methylation of the oocyte mitochondrial DNA could be the molecular basis for maternal inheritance of mitochondria in mammals, the mitochondrial genome has been analyzed in four meiotic and postmeiotic testicular cell types, and in oocytes from the mouse. All four testicular cell types including spermatozoa contain mitochondrial DNA. Between meiosis and the end of spermatogenesis the number of mitochondrial genomes per haploid genome decreases 8- to 10-fold with spermatozoa containing approximately one copy of the mitochondrial genome per mitochondrion. Restriction enzyme digestions with six different enzymes indicate no gross differences in DNA sequence in the testicular mitochondrial DNA from meiotic cells, early haploid cells, late haploid cells, and spermatozoa. By the criterion of differential digestion with the isoschizomers, MspI and HpaII, the mitochondrial DNA is not differentially methylated during spermatogenesis. No methylation differences were detected in mitochondrial DNA from sperm and oocytes following digestion with seven methylation-sensitive restriction enzymes.

Animals↗

PubMed2006

Epigenetic deregulation of imprinting in congenital diseases of aberrant growth.

Human chromosome 11p15 comprises two imprinted domains important in the control of fetal and postnatal growth. Novel studies establish that imprinting at one of these, the IGF2-H19 domain, is epigenetically deregulated (with loss of DNA methylation) in Silver-Russell Syndrome (SRS), a congenital disease of growth retardation and asymmetry. Previously, the exact opposite epigenetic alteration (gain of DNA methylation) had been detected at the domain's 'imprinting control region' (ICR) in patients with Beckwith-Wiedemann Syndrome (BWS), a complex disorder of fetal overgrowth. However, more frequently, BWS is caused by loss of DNA methylation at the ICR that regulates the second imprinted domain at 11p15. Interestingly, a similar epigenetic alteration (with loss of methylation) at a putative ICR on human chromosome 6q24, is involved in transient neonatal diabetes mellitus (TNDM), a congenital disease with intrauterine growth retardation and a transient lack of insulin. Thus, fetal and postnatal growth is epigenetically controlled by different ICRs, at 11p15 and other chromosomal regions.

Beckwith-Wiedemann Syndrome↗

PubMed1980

Identification of 5-methylcytosine in DNA fragments immobilized on nitrocellulose paper.

A method to identify 5-methylcytosine (m5Cyt) in DNA immobilized on nitrocellulose paper by using antibody against m5Cyt raised in rabbits is described. Immobilized restriction fragments of DNA are incubated first with purified antibody against m5Cyt and then with goat anti-rabbit IgG labeled with 125I. Restriction fragments containing m5Cyt are visualized by autoradiography. By using this method, a heavily methylated fragment of about 1700 base pairs was identified in nuclear DNA fom Chinese hamster cells, the methylation pattern of calf thymus satellite I DNA was examined, and chloroplast DNAs that were extracted from various stage of the Chlamydomonas life cycle were compared. Little if any methylation was detected in chloroplast DNA from vegetative cells or from male gametes, whereas homologous DNAs from female gametes and from zygotes were heavily methylated. The sensitivity of the method was examined with calf thymus satellite I DNA (which contains approximately 40 m5Cyt residues per repeat unit of 1400 base pairs) and with phi X174 virion DNA (which contains a single m5Cyt per molecule). The presence of m5Cyt was detected with as little as 40 ng of phi X174 DNA containing 0.02 pmol of m5Cyt and with 100 ng of satellite DNA containing about 0.5 pmol of m5Cyt. Thus, the method makes possible the identification of individual methylated sites in purified DNAs in the size range of single genes.

5-Methylcytosine↗

PubMed2006

Evidence for an instructive mechanism of de novo methylation in cancer cells.

DNA methylation has a role in the regulation of gene expression during normal mammalian development but can also mediate epigenetic silencing of CpG island genes in cancer and other diseases. Many individual genes (including tumor suppressors) have been shown to undergo de novo methylation in specific tumor types, but the biological logic inherent in this process is not understood. To decipher this mechanism, we have adopted a new approach for detecting CpG island DNA methylation that can be used together with microarray technology. Genome-wide analysis by this technique demonstrated that tumor-specific methylated genes belong to distinct functional categories, have common sequence motifs in their promoters and are found in clusters on chromosomes. In addition, many are already repressed in normal cells. These results are consistent with the hypothesis that cancer-related de novo methylation may come about through an instructive mechanism.

Animals↗

PubMed2005

High frequency of promoter hypermethylation of RASSF1A in tumorous and non-tumourous tissue of breast cancer.

AIM: To determine the presence of RASSF1A promoter methylation in tumorous and non-tumorous tissues of breast cancer. METHODS: Methylation-specific PCR was used to detect RASSF1A methylation in DNA extracted from tumorous and paired non-tumorous tissues of 40 breast cancer patients. The associations of RASSF1A hypermethylation with clinicopathological characteristics in tumorous and non-tumorous breast tissues were analysed. RESULTS: RASSF1A promoter hypermethylation was detected in 38 of the 40 breast cancer tissues (95%) and 37 of the paired non-tumorous tissues (92.5%). When compared with the non-tumorous tissues, aberrant methylation was detected to be higher in 24 of the tumorous tissues (60%). The latter was found to be associated with lower histological grade tumours (p=0.048). CONCLUSION: RASSF1A promoter hypermethylation occurred at a high frequency in breast cancer tumorous and non-tumorous tissues; the majority of tumours have a higher level of methylation status when compared with non-tumorous tissues. This supports the notion that RASSF1A methylation is an early and premalignant alteration.

Adenocarcinoma↗

PubMed2002

Detection of aberrant p16 methylation in the serum of colorectal cancer patients.

PURPOSE: This study was designed to detect aberrant p16 promoter methylation in the serum of patients with colorectal cancer (CRC) and to explore the possibility of using this assay in early detection or as a prognostic marker of CRC patients. EXPERIMENTAL DESIGN: Methylation-specific PCR was used to detect p16 methylation in DNA extracted from 52 CRCs and matching serum samples and control serum samples from 34 patients with adenomatous polyps and 10 healthy individuals. The association of p16 hypermethylation in serum DNA of CRC patients with clinicopathological characteristics was then analyzed. RESULTS: P16 hypermethylation was found in 20 of 52 (38%) CRCs. Among the 20 cases with aberrant methylation in the tumor tissues, similar changes were also detected in the serum of 14 (70%) cases. No methylated p16 sequences were detected in the peripheral serum of the other 32 CRC cases without these changes in the tumor, in 34 patients with adenomatous polyps, or in 10 healthy control subjects. Clinicopathological analysis revealed that p16 methylation in serum was significantly associated with later Dukes' stage (P = 0.03). CONCLUSIONS: This assay offers a potential means for the serum-based detection and/or monitoring of CRC patients.

Adenomatous Polyps↗

PubMed2005

Detection of p16 promoter methylation in the serum of oral cancer patients.

P16 promoter methylation occurs frequently in oral squamous cell carcinoma (OSCC). For the early detection of tumour-related aberrant DNA, we examined p16 methylation using the methylation-specific polymerase chain reaction (MSP) in tumour and serum samples of 17 OSCC patients. Aberrant p16 methylation was detected in 11 (64.7%) cases of primary OSCC. Of these 11 patients, 6 (54.5%) showed the same alteration in their serum. No methylation was found in control groups. Interestingly, DNA was detected in the serum of 3 out of 4 patients with recurrence. These results suggest that the MSP may be a sensitive and useful method for detecting recurrent OSCC.

Adult↗

PubMed2002

High-performance capillary electrophoretic method for the quantification of 5-methyl 2'-deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues.

A new approach to the evaluation of the relative degree of genomic DNA methylation through the quantification of 2'-deoxynucleosides is proposed. Detection and quantification of 5-methyl 2'-deoxycytidine in genomic DNA has been performed using micellar high-performance capillary electrophoresis (HPCE) with UV-Vis detection. This approach has been demonstrated to be more sensitive and specific than other HPCE methods for the quantification of DNA methylation degree and also to be faster than other HPLC-based methods. The detection and quantification of nucleosides through enzymatic hydrolyses notably increases the specificity of the technique and allows its exploitation in the analysis of poorly purified and/or concentrated DNA samples such as those obtained from meristematic plant regions and paraffin-embedded tissues.

Animals↗

PubMed2004

DNA methylation in bovine adult and fetal fibroblast cells.

The aim of this project was to develop a simple screening tool to measure the DNA methylation of fibroblast cells, and to determine if differences in DNA methylation could be detected in adult and fetal fibroblast cells after serum starvation (SS). Four adult and four fetal tissue explants were collected to produce presumptive fibroblast cell cultures for this experiment. All cell lines underwent three repetitions of serum starvation for 0 (control), 2, 5, or 7 days. The DNA was extracted from the cells and analyzed for DNA methylation content using methylation sensitive restriction enzyme digestion, gel electrophoresis and image analysis. There was no difference (p = 0.11) between the DNA methylation of the adult and fetal nonclonal cell lines. A cubic trend (p = 0.09) of increased DNA methylation at 2 days of serum starvation followed by periods of decreasing DNA methylation at 5 and 7 days were observed for the adult nonclonal cell lines. A significant interaction (p = 0.03) was observed between fetal cell line and day. This simple, rapid DNA methylation assay may be beneficial when evaluating cells' DNA methylation content.

Adult↗

PubMed1989

The methylation state of the T cell antigen receptor beta chain gene in subpopulations of mouse thymocytes.

Previous analyses of T cell receptor beta chain (TcR beta) genomic DNA from subsets of human peripheral blood leukocytes suggested that the TcR beta methylation pattern might reflect distinct differentiation pathways. The studies presented here, using murine thymocyte subsets, have specifically addressed the question of whether methylation of TcR beta DNA is related to the cellular maturity and type of TcR beta mRNA expressed in the different subsets. We have observed that the DNA isolated from either CD4+ or CD8+ thymocytes, the more mature thymic subsets, is less methylated in the TcR beta region than DNA isolated from the CD4-CD8-, double-negative population containing the more immature thymocytes. In addition, this pattern of DNA methylation is directly related to the ratio of 1.3-kb to 1.0-kb TcR beta mRNA seen in these different cell types. Although a quantitative difference in the level of TcR beta mRNA was noted for the two mature subsets, no qualitative difference in the ratio of 1.3-kb to 1.0-kb mRNA was detected. Furthermore, these DNA methylation patterns appear to be lineage related, because the TcR beta region of genomic DNA isolated from mouse macrophages is heavily methylated.

Animals↗

PubMed1980

Uptake and binding of 1-methyl-1-nitrosourea (MNU) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) by the isolated guinea pig pancreas.

1-Methyl-1-nitrosourea (MNU) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) are carcinogens which methylate nucleic acids and proteins and covalently modify proteins by carbamoylation (MNU) or guanidination (MNNG). Using MNU and MNNG labeled with carbon-14 in the individual carbon positions, the above reactions were quantitated in the isolated guinea pig pancreas, an organ susceptible to tumorigenesis by MNU. Freshly prepared pancreatic lobules were incubated with the labeled drugs (0.03, 0.3 and 1.0 mM) for one hour at 37 degrees C. Alkylated purines from hydrolyzed DNA were separated on Sephadex G10 and acid-soluble nuclear proteins were extracted and separated on polyacrylamide gels. Total uptake of all four labels into lobules was linear with concentration. Acid insoluble radioactivity also increased linearly except for MNNG methylation which plateaued between 0.3 and 1.0 mM. 7-Methylguanine formation by both compounds was approximately ten fold greater than 06-methylation. However, DNA modification by MNU exceeded that by MNNG, especially at the higher drug concentrations. No carbamoylation or guanidination of DNA was detected. Total binding (methylation plus carbamoylation/guanidination) to acid-extractable chromatin proteins was equivalent to DNA modification on a molar basis (approximately 0.35 and 0.08 pmol/microg for exposure to 1.0 mM MNU and MNNG, respectively). All histones were labeled by all drug preparations, with H2A being the principal site of methylation and H2B, H3 and H1 being the major targets of carbamoylation and guanidination. H4 was the least modified histone. Drug binding to cytoplasmic organelles also occurred. These results show a broad spectrum of nuclear and cytoplasmic modification of pancreatic cells by MNU and, to a smaller extent, MNNG.

Animals↗

PubMed2004

Methylated DNA collected by tampons--a new tool to detect endometrial cancer.

This proof of principle study aimed to define a new and simple strategy for detection of endometrial cancer using epigenetic markers. We investigated DNA isolated from vaginal secretion collected from tampon for aberrant methylation of five genes (CDH13, HSPA2, MLH1, RASSF1A, and SOCS2) using MethyLight in 15 patients with endometrial cancer and 109 patients without endometrial cancer. All endometrial cancer patients revealed three or more methylated genes, whereas 91% (99 of 109) of the patients without endometrial cancer had no or fewer than three genes methylated in their vaginal secretion. The methods developed in this study provide the basis for a prospective clinical trial to screen asymptomatic women who are at high risk for endometrial cancer.

Base Sequence↗

PubMed1992

A colorimetric microassay for the detection of agents that interact with DNA.

A simple microtiter assay for the detection of compounds that bind DNA is described. Agents that displace methyl green from DNA are detected spectrophotometrically by a decrease in absorbance at 630 nm. The feasibility of using the assay for detecting DNA-active compounds in fermentation extracts was assessed, and the activities of reference compounds in the methyl green assay and an ethidium bromide displacement method were compared.

Aminoglycosides↗

PubMedDate not supplied

Role of the cytosine DNA-methyltransferase and p16INK4a genes in the development of mouse lung tumors.

CpG island methylation is an epigenetic modification of DNA associated with the silencing of gene transcription. The p16INK4a (p16) tumor suppressor gene is inactivated in human non-small cell lung cancers (NSCLCs) by either homozygous deletion or aberrant methylation. Inactivation of tumor suppressor genes by methylation has been linked in part to altered activity of the cytosine DNA-methyltransferase (DNA-MTase), the enzyme that catalyzes DNA methylation at CpG sites. The purpose of these studies was to define the role of DNA-MTase and p16 in the development of murine lung cancer. DNA-MTase activity was determined in alveolar type II and Clara cells from A/J and C3H mice that exhibit high and low susceptibility, respectively, for lung tumor formation. Increased DNA-MTase activity leading to an increase in overall DNA methylation was found only in alveolar type II cells, the target for murine adenocarcinomas. Both DNA-MTase and DNA methylation changes were detected 7 days after carcinogen exposure and, thus, were early events in neoplastic evolution. In addition, enzyme activity increased incrementally during lung cancer progression. Expression of p16 was detected in all primary lung tumors from A/J mice; however, levels of expression differed by up to 15-fold between tumors. The apparent low levels of expression seen in approximately half of the tumors was not attributed to methylation of the p16 gene. In contrast to the detection of p16 expression in primary tumors, this gene was deleted in four tumor-derived cell lines induced in the A/J mouse by NNK. The results from these studies indicate that the modulation of DNA-MTase activity was cell specific, segregated with susceptibility, and occurred early in neoplastic evolution. Thus, the marked increase in enzyme activity detected in alveolar type II cells after carcinogen treatment could be a major factor contributing to the high susceptibility for chemical-induced neoplasia associated with the A/J mouse strain. The inactivation of the p16 gene in murine cancers induced by NNK most likely arises as a late event via homozygous deletion.

Adenocarcinoma↗

PubMed1980

Loss of reiterated DNA sequences during serial passage of human diploid fibroblasts.

A specific family of tandemly repeated DNA sequences was found to diminish in the human genome after serial passage of three strains of diploid fibroblasts. Eco RI restriction fragments of 340 and 680 bp were significantly reduced in quantity at late passage as determined by autoradiography of 14C-DNA and also by ethidium bromide fluorescence. The reduction in these closely related DNA sequences was confirmed by saturation hybridization to excess 14H-RNA transcribed from a homogeneous restriction fragment recleaved from the 340 bp DNA. The maximal fraction of DNA hybridizing to the 3H-RNA probe declined by 33-50% over 21-41 population doublings. Divergence and/or methylation of such sequences could not account for these results since the thermal stability of cRNA:DNA duplexes actually increased by 0.3 degrees C at late passage. Total highly repetitive sequences assayed by reassociation kinetics were also substantially reduced at late passage, implying that depletion may be common to many repeat families in DNA. The denaturation temperature for such rapidly reassociated duplexes again increased slightly at late passage, possibly reflecting the minor decreases in DNA methylation which were detected in two of the cell strains. Karyotype analyses demonstrated that over 95% euploidy was maintained, with no specific chromosome loss and no visible deletions at late passage. The depletion of reiterated sequences during repeated cell division is thus attributed to numerous small DNA deletions, which may arise from unequal recombination coupled with selection or from a nonreciprocal mechanism such as excision.

Cell Division↗