Search

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]

Background. Although alterations in nonspecific (or global) DNA methylation (GDM) in specific cells are known to be involved in the process of lung carcinogenesis, similar associations have not been evaluated in other smoking-related cancers of the head and neck. Methods. We evaluated the status of GDM by using monoclonal antibodies specific for 5-methylcytosine (5-mc) in oral squamous cell carcinoma (SCC) specimens of 48 cigarette smokers who had SCC develop and in 93 age-, race-, and sex-matched smokers who did not. Results. Percentages of cells positive for 5-mc immunostaining of DNA of SCC and dysplastic lesions were significantly higher than those of normal oral epithelial cells from cancer subjects and from noncancer subjects. The degree of DNA methylation was unrelated to DNA content. Conclusions. The pattern of GDM in oral SCCs is different from that of lung SCCs. The differences in nutrient risk factor profiles that are related to GDM and differential activity of DNA methyltranferases between oral and lung SCCs may explain these observations. © 2005 Wiley Periodicals, Inc. Head Neck27: XXX–XXX, 2005 [ABSTRACT FROM AUTHOR]

The implications of global DNA hypomethylation were recently reported in several models of tumorigenesis. Little is known about this epigenetic event in thyroid neoplasia. The study aimed to evaluate the status of global DNA methylation in several types of thyroid tumors using a monoclonal antibody specific for 5-methylcytidine (5-mc) and to define the diagnosis potential of this marker. 5-mc immunostaining scores were calculated in 17 papillary thyroid carcinomas (PTC), 6 follicular thyroid carcinomas (FTC), 16 follicular adenomas (FA), 19 nodular goiters (NG) and ten Hürthle cells adenomas (HCA). The expression of galectin-3 was also evaluated. Computerized image analysis showed a significant lower level of 5-mc immunostaining in thyroid carcinoma when compared with benign tumors or adjacent normal thyroid parenchyma (P<0.0001). Overall, 5-mc accuracy to distinguish malign from benign thyroid tumors was similar to that of galectin-3 (89% versus 87%, P>0.05). The combination of 5-mc with galectin-3 led to an excellent accuracy level of 96%. Among follicular neoplasia 5-mc accuracy to differentiate malign tumors trends to be higher than galectin-3 one (90% versus 66%, P=0.06). These data stress the necessity of epigenetic events evaluation among thyroid nodules and propose global DNA methylation assessment as a potential diagnostic tool to combine with other valuable markers. [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]

Endometrial carcinomas are the most common malignancy of the female genital tract and the third most common cancer in women. Progesterone and oestrogen receptors (PRs, ERs) are the most widely documented prognostic and predictive factors in endometrioid adenocarcinoma. Besides the hormonal pathway involved in the progression of preneoplastic and neoplastic lesions, alterations of the DNA methylation status have been shown to be an early signal of tumorigenesis. In this study, we show that in normal endometrium, during the proliferative phase, DNA methylation and PR expression are high, with a significant decline towards the end of the secretory phase and a gradual increase in non-atypical and atypical endometrial hyperplasia; they reach their highest level in grade I, then decrease significantly in grade-II and grade-III endometrioid adenocarcinomas. During each stage, a significant positive correlation is observed between DNA methylation and PR (P<0.0001). The strong parallelism between DNA methylation and PR expression precludes establishing a precise determination regarding the timing of these events, clearly involved in the genesis of endometrioid adenocarcinoma. [ABSTRACT FROM AUTHOR]

DNA methylation of sex chromosomes was analysed using anti-5-methylcytosine antibodies on metaphase chromosomes of somatic cells from three species: human, lemur and mouse. Germ cells were also studied in male mouse. In female cells (human and mouse), the late replicating X was always the less methylated chromosome. Compared with autosomes, the methylation of both X chromosomes was always lower in fibroblasts than in lymphocytes and the difference was always greater in mouse than in human. In human, mouse and lemur male cells, the labelling of the unique X chromosome was quite similar to that of the early replicating X from female cells. Except for the heterochromatic region of the human Y chromosome, strongly methylated, the overall methylation of the Y chromosome was low. In mouse testicular cells, a variety of DNA methylation patterns was observed according to the cell type and the state of differentiation. Finally, the only structures of sex chromosomes which remain methylated in all conditions correspond to their pseudoautosomal regions. [ABSTRACT FROM AUTHOR]

In situ immunofluorescence detection of antibodies against 5-methylcytosine on metaphase chromosomes prepared by a new procedure allows the display of new 5-methylcytosine-rich sites as compared to previously published methods. In short-term culture lymphocytes, the immunofluorescent signals give a recurrent pattern in which four types of binding sites can be distinguished. Type I sites are the secondary constrictions and a few juxtacentromeric regions, type II sites correspond to T-bands. Both types I and II sites emit a strong fluorescence. Type III sites form an R-band pattern and emit a weaker fluorescence. Type IV sites are the short arms of acrocentrics, they emit strong but polymorphic signals. The results obtained from control experiments suggest that the pattern observed is rather the expression of an uneven distribution of 5-methylcytosine-rich sites than a consequence of the various treatments used. In a lymphoblastoid cell line known to have a reduced 5-methylcytosine content, it was possible to demonstrate a heterogeneous hypomethylation among chromosome structures, principally involving type I sites. The method opens the possibility of studying in situ on chromosomes, regional variations of methylation in pathological conditions. [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]

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]

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.