Glu346Lys polymorphism in the methyl-CpG binding domain 4 gene and the risk of primary lung cancer.
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BACKGROUND: Methyl-CpG binding domain 4 (MBD4) protein functions as a DNA repair enzyme and minimizes mutations at 5-methylcytosine. Polymorphisms in the DNA repair gene MBD4 may be associated with differences in DNA repair capacity and thereby influence an individual's susceptibility to lung cancer. To test this hypothesis, we examined the potential association between the MBD4 Glu346Lys polymorphism and the risk of lung cancer in a Korean population. METHODS: The MBD4 Glu346Lys genotypes were determined in 432 lung cancer patients and 432 healthy age- and gender-matched control subjects. RESULTS: The distribution of the MBD4 Glu346Lys genotypes was not significantly different between the overall lung cancer cases and the controls. However, when the cases were categorized by tumor histology, the Lys346Lys genotype was associated with a significantly decreased risk of adenocarcinoma (AC) as compared with the Glu346Glu genotype [adjusted odds ratio (OR) = 0.50, 95% confidence interval (CI) = 0.26-0.97, P = 0.04]. On the stratification analysis, the protective effect of the Lys346Lys genotype against AC was statistically significant in older individuals and heavier smokers (adjusted OR = 0.08, 95% CI = 0.01-0.64, P = 0.02; and adjusted OR = 0.09, 95% CI = 0.01-0.72, P = 0.02, respectively). CONCLUSIONS: Our findings suggest that the MBD4 Glu346Lys polymorphism could be used as a marker for genetic susceptibility to AC of the lung. (+info)
5-Azacytidine-induced reactivation of the human X chromosome-linked PGK1 gene is associated with a large region of cytosine demethylation in the 5' CpG island.
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Hamster-human cell hybrids containing an inactive human X chromosome were treated with 5-azacytidine and derived clones were examined for phosphoglycerate kinase activity and cytosine methylation in the human PGK1 (X chromosome-linked phosphoglycerate kinase) gene. Comparisons between expressing and nonexpressing clones indicated that demethylation of several methylation-sensitive restriction sites outside of the 5' CpG island were unnecessary for expression. High-resolution polyacrylamide gel analysis of 25 Hpa II, Hha I, and Tha I sites revealed that all clones expressing PGK1 were unmethylated in a large region of the CpG island that includes the transcription start site and 400 base pairs upstream. Many nonexpressing clones had discontinuous patterns of demethylation. Remethylation was often observed in subclones of nonexpressing hybrids. These data suggest that a specific zone of methylation-free DNA within the PGK1 promoter is required for transcription. In addition, the presence of neighboring methylcytosines appears to decrease the heritable stability of unmethylated CpGs in this region. (+info)
Genome-wide hypomethylation in human glioblastomas associated with specific copy number alteration, methylenetetrahydrofolate reductase allele status, and increased proliferation.
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Genome-wide reduction in 5-methylcytosine is an epigenetic hallmark of human tumorigenesis. Experimentally induced hypomethylation in mice promotes genomic instability and is sufficient to initiate tumorigenesis. Here, we report that global hypomethylation is common in primary human glioblastomas [glioblastoma multiforme (GBM)] and can affect up to an estimated 10 million CpG dinucleotides per haploid tumor genome. Demethylation involves satellite 2 (Sat2) pericentromeric DNA at chromosomes 1 and 16, the subtelomeric repeat sequence D4Z4 at chromosomes 4q and 10q, and interspersed Alu elements. Severe hypomethylation of Sat2 sequences is associated with copy number alterations of the adjacent euchromatin, suggesting that hypomethylation may be one factor predisposing to specific genetic alterations commonly occurring in GBMs. An additional apparent consequence of global hypomethylation is reactivation of the cancer-testis antigen MAGEA1 via promoter demethylation, but only in GBMs and GBM cell lines exhibiting a 5-methylcytosine content below a threshold of approximately 50%. Primary GBMs with significant hypomethylation tended to be heterozygous or homozygous for the low-functioning Val allele of the rate-limiting methyl group metabolism gene methylenetetrahydrofolate reductase (MTHFR), or had a deletion encompassing this gene at 1p36. Tumors with severe genomic hypomethylation also had an elevated proliferation index and deletion of the MTHFR gene. These data suggest a model whereby either excessive cell proliferation in the context of inadequate methyl donor production from MTHFR deficiency promotes genomic hypomethylation and further genomic instability, or that MTHFR deficiency-associated demethylation leads to increased proliferative activity in GBM. (+info)
CpG methylation inhibits proenkephalin gene expression and binding of the transcription factor AP-2.
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DNA methylation at HpaII (CmCGG) sites inhibits expression of a human proenkephalin-CAT fusion gene when it is transiently expressed in CV-1 cells or stably expressed in C6-glioma cells. The inhibitory effects of HpaII methylation have been mapped to a site within the human proenkephalin promoter located at position -72 relative to the start site of transcription. This region spans a cAMP and phorbol ester inducible enhancer and methylation at this position inhibits both basal transcription and transcription induced by either cAMP or TPA. The HpaII site is located within an element which binds the transcription factor AP-2. In vitro methylation at this HpaII site inhibits the binding of AP-2. These results suggest that CpG methylation inhibits proenkephalin gene expression by directly interfering with the binding of a positively acting transcription factor previously shown to be essential for maximal basal, cAMP, and TPA inducible transcription. (+info)
Identification of human tRNA:m5C methyltransferase catalysing intron-dependent m5C formation in the first position of the anticodon of the pre-tRNA Leu (CAA).
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We identified a human orthologue of tRNA:m5C methyltransferase from Saccharomyces cerevisiae, which has been previously shown to catalyse the specific modification of C34 in the intron-containing yeast pre-tRNA Leu (CAA). Using transcripts of intron-less and intron-containing human pre-tRNA Leu (CAA) genes as substrates, we have shown that m5C34 is introduced only in the intron-containing tRNA precursors when the substrates were incubated in the HeLa extract. m5C34 formation depends on the nucleotide sequence surrounding the wobble cytidine and on the structure of the prolongated anticodon stem. Expression of the human Trm4 (hTrm4) cDNA in yeast partially complements the lack of the endogenous Trm4p enzyme. The yeast extract prepared from the strain deprived of the endogenous TRM4 gene and transformed with hTrm4 cDNA exhibits the same activity and substrate specificity toward human pre-tRNALeu transcripts as the HeLa extract. The hTrm4 MTase has a much narrower specificity against the yeast substrates than its yeast orthologue: human enzyme is not able to form m5C at positions 48 and 49 of human and yeast tRNA precursors. To our knowledge, this is the first report showing intron-dependent methylation of humanpre-tRNA Leu (CAA) and identification of human gene encoding tRNA methylase responsible for this reaction. (+info)
Interleukin-6 contributes to growth in cholangiocarcinoma cells by aberrant promoter methylation and gene expression.
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The association between chronic inflammation and the development and progression of malignancy is exemplified in the biliary tract where persistent inflammation strongly predisposes to cholangiocarcinoma. The inflammatory cytokine interleukin-6 (IL-6) enhances tumor growth in cholangiocarcinoma by altered gene expression via autocrine mechanisms. IL-6 can regulate the activity of DNA methyltransferases, and moreover, aberrant DNA methylation can contribute to carcinogenesis. We therefore investigated the effect of chronic exposure to IL-6 on methylation-dependent gene expression and transformed cell growth in human cholangiocarcinoma. The relationship between autocrine IL-6 pathways, DNA methylation, and transformed cell growth was assessed using malignant cholangiocytes stably transfected to overexpress IL-6. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine decreased cell proliferation, growth in soft agar, and methylcytosine content of malignant cholangiocytes. However, this effect was not observed in IL-6-overexpressing cells. IL-6 overexpression resulted in the altered expression and promoter methylation of several genes, including the epidermal growth factor receptor (EGFR). EGFR promoter methylation was decreased and gene and protein expression was increased by IL-6. Thus, epigenetic regulation of gene expression by IL-6 can contribute to tumor progression by altering promoter methylation and gene expression of growth-regulatory pathways, such as those involving EGFR. Moreover, enhanced IL-6 expression may decrease the sensitivity of tumor cells to therapeutic treatments using methylation inhibitors. These observations have important implications for cancer treatment and provide a mechanism by which persistent cytokine stimulation can promote tumor growth. (+info)
Lack of 5-methylcytosine in Dictyostelium discoideum DNA.
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We find no evidence for the presence of 5-methylcytosine in the DNA of Dictyostelium discoideum. Methylation was absent from CCGG sites in repetitive DNA and in DNA from the actin multigene family. Nor was 5-methylcytosine detected in total DNA when base composition was determined by means of h.p.l.c. (+info)
Sequence-selective 5-methylcytosine oxidation for epigenotyping.
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Methylation of DNA is an epigenetic modification that can play an important role in the control of gene expression in mammalian cells. The development of a simple and convenient method for site-specific discrimination of cytosine methylation is imperative for genomic studies. Here we report a facile method for distinguishing between cytosine and 5-methylcytosine. Osmium tetroxide caused the dihydroxylation of the C5-C6 double bond of 5-methylcytosine under an appropriate reaction conditions. The oxidation of 5-methylcytosine-containing target DNA was controlled by hybridization with a guide DNA. This technique facilitates the typing of cytosine methylation at a specific site of the target DNA. (+info)