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The purpose of this study was to evaluate whether mandatory fortification of grain products with folic acid in the USA is associated with changes in global DNA methylation in cells involved in cervical carcinogenesis. Archived specimens of cervical intraepithelial neoplasia (CIN) diagnosed before (1990–92) and after mandatory folic acid fortification (2000–02) were used to examine for global DNA methylation in specific lesions involved in cervical carcinogenesis by using a monoclonal antibody specific for 5 methyl cytosine (5-mc). The total number of lesions examined was 152 in the pre-fortification period and 172 in the post-fortification period. Immunohistochemical staining for 5-mc, the assessment of methylation status and data entry were blinded with regard to the fortification status. Age- and race-adjusted mean percentage of cells positive for 5-mc or the 5-mc score was not significantly different (P>0.05) between the pre- and post fortification periods in any of the individual lesions evaluated (i.e., normal cervical epithelium, reactive cervical epithelium, metaplastic cervical epithelium, CIN or carcinoma in situ). The degree of global DNA methylation was significantly higher (P<0.0001) in &ges; CIN 2 lesions compared to &les; CIN 1 lesions, regardless of the fortification group. These results suggest that mandatory fortification with folic acid in the United States has not resulted in a change in the degree or the pattern of global DNA methylation in cells involved in cervical carcinogenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Although there is growing interest in the possibility that alterations in histone methylation may play a role in carcinogenesis, it has not been explored adequately in humans. Similarly, there are no reports of associations between this and a similar epigenetic event, DNA methylation. Using immunohistochemical staining, we compared the methylation of DNA and histones in histopathologically normal oral epithelium, dysplastic oral lesions, and squamous cell cancers (SCCs) from subjects with squamous cell cancer (n=48) with those of normal oral epithelium from subjects without oral cancer (n=93) who were matched on age and race. Monoclonal antibodies specific for 5 methyl cytosine (5-mc), lysine 4 of histone H_3 (H_3-Lys4), and lysine 9 of histone H_3 (H_3-Lys9) were used in this study. The percentages of cells positive and a weighted average of the immunostaining intensity scores were calculated for each of these tissues, and Spearman correlation analyses were employed to study associations between DNA and histone methylation. Correlations between DNA and histone methylation, H_3-Lys4 and H_3-Lys9 were positive and statistically significant in all tissue types; they were strongest in normal oral epithelium from non-cancer subjects (r=0.63, p < 0.001 and r=0.62, p < 0.001 respectively). Similarly, the positive correlations between H_3-Lys4 and H_3-Lys9 were statistically significant in all tissue types and strongest in normal oral epithelium from non-cancer subjects (r=0.77, p< 0.001). Patterns of DNA and histone methylation are similar in tissues across the spectrum of oral carcinogenesis, and there is a significant positive association between these two epigenetic mechanisms. [ABSTRACT FROM AUTHOR]

Antibodies directed against N7-methylguanosine (m7Guo) were prepared and added to a wheat germ cell-free protein-synthesizing system programmed with RNA extracted from monkey cells persistently infected with measles virus. A dose-dependent inhibition of [35S]methionine incorporation was observed when RNA was preincubated with anti-m7Guo immunoglobulins. Antibodies preincubated with m7Guo did not show any inhibiting activity. The inhibitory effect of antibodies was abolished when RNA was preincubated with immunoglobulins in the presence of spermine and spermidine. When polyamines were added to the assay programmed with the IgG-RNA complex, no inhibition was observed. [ABSTRACT FROM AUTHOR]

Antibodies specifically directed to 5-methylcytidine were raised in rabbits and purified by affinity chromatography. The accessibility of 5-methyldeoxycytidine (m5dCyd) to such antibodies was studied with DNAs from various origins. The reaction was followed by measuring the retention of radiolabelled DNA by antibodies on nitrocellulose filters, by immunoprecipitation, by gel filtration and was visualized with the electron microscope. Antibodies did not bind to Escherichia coli B DNA, which is deficient in m5dCyd. Denatured and native DNA from calf thymus, which contains m5dCyd as a minor nucleoside, was weakly retained on the filters whereas DNA extracted from Xanthomonas oryzac XP12 bacteriophage, which is rich in m5dCyd, was well recognized even in the native form. [ABSTRACT FROM AUTHOR]

Reports implications of the demethylation of the zygotic paternal genome. How the paternal genome is demethylated before the onset of DNA replication; Causes of intermediate amounts of methylation, including a combination of under-methylated maternal and methylated paternal DNA; Implications of the findings for genomic imprinting and mammalian cloning.

Evaluates the hypothesis that vertebrate cells possess mechanisms that protect them from epigenomic stress similar to DNA damage checkpoints. Effect of DNA methyltransferase knockdown by an oligonucleotide antisense on the intra-S-phase arrest of DNA replication; Implication on the arrest of cells at different positions throughout the S-phase of the cell cycle; Proposition on intra-S-phase arrest of DNA replication.

: Objective:To determine the relationship between sperm DNA methylation level and sperm characteristics and pregnancy rates.: Design:Prospective study. Quantitation by image analysis of DNA methylation in sperm nucleus.: Setting:Department of Reproduction Biology, Edouard Herriot Hospital, Lyon, France.: Patient(s):Infertile couples undergoing IVF-ET.: Intervention(s):The immunostaining of 5 methyl-cytosine was performed on the spare sperm suspension that was used for an assisted reproduction technology procedure.: Main outcome measure(s):Sperm characteristics according to World Health Organization criteria, sperm motility parameters with computer-assisted semen analysis, sperm DNA methylation level, and heterogeneity index (HI).: Result(s):Sperm DNA methylation level and HI are correlated with sperm DNA characteristics. HI is negatively correlated with fertilization rate; sperm DNA methylation level is correlated with pregnancy rate.: Conclusion(s):The DNA methylation level in human spermatozoa could be a new approach to evaluating the ability of spermatozoa to fertilize and lead to normal embryo development. [Copyright &y& Elsevier]

The levels of genomic DNA methylation in vertebrate species display a wide range of developmental dynamics. Here, we show that in contrast to mice, the paternal genome of the amphibian, Xenopus laevis, is not subjected to active demethylation of 5-methyl cytosine immediately after fertilization. High levels of methylation in the DNA of both oocyte and sperm are maintained in the early embryo but progressively decline during the cleavage stages. As a result, the Xenopus genome has its lowest methylation content at the midblastula transition (MBT) and during subsequent gastrulation. Between blastula and gastrula stages, we detect a loss of methylation at individual Xenopus gene promoters (TFIIIA, Xbra, and c-Myc II) that are activated at MBT. No changes are observed in the methylation patterns of repeated sequences, genes that are inactive at MBT, or in the coding regions of individual genes. In embryos that are depleted of the maintenance methyltransferase enzyme (xDnmt1), these developmentally programmed changes in promoter methylation are disrupted, which may account for the altered patterns of gene expression that occur in these embryos. Our results suggest that DNA methylation has a role in regulating the timing of gene activation at MBT in Xenopus laevis embryos. [Copyright &y& Elsevier]

Whereas accepted models of tumorigenesis exist for genetic lesions, the timing of epigenetic alterations in cancer is not clearly understood. We have analyzed the profile of aberrations in DNA methylation occurring in cells lines and primary tumors of one of the best-characterized mouse carcinogenesis systems, the multistage skin cancer progression model. Initial analysis using high-performance capillary electrophoresis and immunolocalization revealed a loss of genomic 5-methylcytosine associated with the degree of tumor aggressiveness. Paradoxically, this occurs in the context of a growing number of hypermethylated CpG islands of tumor suppressor genes at the most malignant stages of carcinogenesis. We have observed this last phenomenon using two approaches, a candidate gene approach, studying genes with well-known methylation-associated silencing in human tumors, and a mouse cDNA microarray expression analysis after treatment with DNA demethylating drugs. The transition from epithelial to spindle cell morphology is particularly associated with major epigenetic alterations, such as E-cadherin methylation, demethylation of the Snail promoter, and a decrease of the global DNA methylation. Analysis of data obtained from the cDNA microarray strategy led to the identification of new genes that undergo methylation-associated silencing and have growth-inhibitory effects, such as the insulin-like growth factor binding protein-3. Most importantly, all of the above genes were also hypermethylated in human cancer cell lines and primary tumors, underlining the value of the mouse skin carcinogenesis model for the study of aberrant DNA methylation events in cancer cells.

A computer-assisted assay based on the quantitative analysis of DNA methylation in individual interphase nuclei by indirect immunolabelling with anti-5-methylcytosine antibodies was recently developed in our laboratory. In situ analyses were performed on individual nuclei from normal and experimentally hypo- or hypermethylated cultured cells as well as on human peripheral blood B-lymphocytes from normal and chronic lymphoid leukemia (CLL) samples. We present the results obtained on cells from patients affected by different degrees of preneoplastic or neoplastic changes of the uterine cervix as compared to normal controls. The analysis of DNA methylation in individual cells from cytofuge samples was performed as follows: within each nucleus the eu- and heterochromatin methylation levels were quantified in the grey scale range by dedicated software in terms of numbers, areas and optical densities (ODs) of the immunolabeled dense heterochromatic regions ("spots"), and of the optical density of nuclear background, i.e., of nuclear euchromatin. Analogously, in randomly chosen microscope fields of tissue sections from paraffin-embedded samples, progressive tissue demethylation was observed in dysplastic and cancer cells as compared to normal ones. Both methods showed significant and progressive DNA hypomethylation in dysplastic and cancer cells as compared to control specimens.