[Clinical significance of the expression of DNA methyltransferase genes (DNMT) in acute leukemia patients].
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Abstract:
To investigate the relationship between the expression of DNMT and clinical prognosis in adult patients with acute leukemia (AL), the mRNA expressions of DNMT, p15(INK4B), mdr1 were measured in 72 AL patients and 20 normal controls by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR); the ratio of p15 CpG land methylation was measured in 56 AL patients and 14 normal controls by methylation-specific PCR (MSP-PCR). The results showed that all three DNMT mRNA expressions in AL patients were significantly higher than that in normal controls (P < 0.01). When the internal control was changed into PCNA, a kind of cell proliferation marker gene, the difference still showed a statistic significance. All three DNMT genes were significantly expressed and positively correlated with AL patients, showing high synergistic expression, and there was a negative correlation between the levels of p15, mdr1 gene expression and DNMT. The complete remission (CR) rate in AL patients with the positive expression of all DNMT genes was significantly higher than that of AL patients with partially positive or negative expression (P < 0.01) of DNMT genes. In 56 AL patients, the P15I(NK4B) was completely methylated in 55.4% (31 of 56), partly methylated in 21.4% (12 of 56) and all 14 cases of normal controls were not methylated. It is concluded that DNMT genes are abnormally high expressed in adult AL patients, which lead to methylation-silence of tumor suppressor genes by CpG land hypermethylation, the AL patients with high expression of DNMT are more sensitive to chemotherapy, which may be a good prognostic factor for AL patients.Keywords:
DNA methyltransferase
CpG site
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Overexpression of DNA methyltransferases(DNMTs)is observed in many tumors. Overexpressed DNMTs silence tumor suppressor genes by catalyzing the methylation of CpG islands within the promoter regions, and therefore promote the neoplastic transformation of normal cells. Highly elevated DNMT activities have been observed in cancers including gastrointestinal cancers, breast cancer, and lung cancer and have been associated with cancer development and prognosis. Drugs and targeted therapies that inhibit DNMT activities can reactivate methylated tumor suppressor genes, promote apoptosis, and thus inhibit tumor growth.
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DNA(Cytosine-5-)-Methyltransferase; DNA methylation; Neoplasms
DNA methyltransferase
Neoplastic transformation
CpG site
DNMT1
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CpG site
DNA methyltransferase
Bisulfite sequencing
Arsenic Trioxide
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This study examines the efficacy of using plasmid expression vectors containing sense and antisense DNA MTase cDNA to both up- and downregulate intracellular DNA MTase levels in human glioma cells. The effects of the changes in MTase levels on global genomic DNA methylation and on the methylation status of CpG dinucleotides in the GSTP1 gene were determined in a glioma cell line that overexpresses the GSTP1 gene. In cells transfected with sense DNA MTase cDNA, MTase gene transcripts increased to a maximum of 2. 5-fold at 24 h, while MTase activity increased to a maximum of 3. 6-fold at 48 h. The effects of antisense MTase cDNA transfections were less pronounced, and levels of MTase gene transcripts and enzyme activity in transfectants were decreased to only, approximately, one-half the levels of controls. The alterations in DNA MTase expression were associated with corresponding changes in the level of global DNA methylation and in the methylation of the GSTP1 gene in the cells, however, with no detectable morphological or cytotoxic effects on the cells. No significant changes in GSTP1 gene expression were detected after the transfections, presumably because of the high levels of basal GSTP1 expression in the cells. Consequently, the p16 gene, known to be repressed transcriptionally by DNA methylation, was examined for the functional effects of the altered MTase levels. The results showed a 2-fold decrease in p16 gene transcripts with the sense MTase transfectants, while in the MTase antisense-transfected cells p16 transcript levels increased by 30%. Together, these results demonstrate the feasibility of using both sense and antisense DNA MTase expression vectors to regulate DNA MTase levels in glioma cells and that, over relatively short periods of time, the alterations in MTase activities are not deleterious to the cells. The system provides a model with which the role of DNA methylation in critical genes and DNA sequences can be investigated in glioma cells.
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Strategies to detect the methylation of site specific DNA and assay of M.SssI methyltransferase (M.SssI MTase) activity are important in determining human cancers due to aberrant methylation linked to cancer initiation and progression. Herein, we report a label-free fluorescence detection method for DNA methylation and MTase activity based on restriction endonuclease HpaII and exonuclease III (Exo III). A label-free probe DNA was designed, which hybridized with target DNA (one 32-mer DNA from the exon 8 promoter region of the Homo sapiens p53 gene) to form double stranded DNA (dsDNA). Upon the cleavage action of HpaII and degradation reaction of Exo III, dsDNA changed to single stranded DNA (ssDNA) and the fluorescence intensity of thiazole orange (TO) is weak. After the resulting dsDNA was methylated by M.SssI MTase, the action of HpaII and Exo III was prevented, then TO intercalates into the dsDNA and emits strong fluorescence. This method can determine DNA methylation at the site of CpG and distinguish a one-base mismatched target sequence. The fluorescence intensity has a linear relationship with M.SssI MTase activities in the range of 1–10 U mL−1 with a detection limit of 0.16 U mL−1 in terms of 3 times deviation of the blank sample. The methylation of DNA by a hydroxyl radical triggered by DMSO and CH3CHO was also measured. These results show that the proposed method can specifically and selectively detect DNA methylation and M.SssI MTase activity. Human serum has no obvious effects on the assay performance, indicating that the method has great potential for further application in complex samples.
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Previous studies have documented extensive methylation of CpG islands and abundant methyltransferase gene (DNMT) in Fugacium kawagutii (formerly Symbiodinium kawagutii) genome. However, whether DNA methylation plays a role in regulating gene expression in this and other dinoflagellates remains unclear. Here, we characterized gene body methylation levels using methylation-specific PCR (MS-PCR) and bisulfite sequencing PCR (BSP) and measured transcriptional levels for three photosystem genes in F. kawagutii under different light conditions (20, 100, and 600 µE · m-2 · s-1 ). To explore the association of methylation with DNA methylase, the expression of DNA methyltransferase (Symbio-DIRS-Dnmt3) was also measured. Our results showed that peridinin-chlorophyll a-binding protein (PCP), light-harvesting complex (LHC), and chlorophyll a-c-binding protein complex (acpPC) gene expression was all significantly up-regulated under low light in which their methylation level was down-regulated, constant, and elevated, respectively. Symbio-DIRS-Dnmt3 exhibited elevated transcriptional level under increased light intensity. The results led us to hypothesize that DNA methylation level can be modulated by environmental conditions such as irradiance, probably through the regulation of Symbio-DIRS-Dnmt3 transcription level, and in turn may regulate the expression of genes in F. kawagutii. Further study is needed to determine whether the same gene methylation and expression characteristics reported here occur in other dinoflagellates and to explore their ecological implications.
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3238 Aberrant methylation is implicated in the development of a variety of solid tumors, including hepatocellular carcinoma (HCC). We show here that genome wide hypomethylation and CpG hypermethylation correlate with biological features and clinical outcome of human HCC. Both parameters reached the highest levels in HCCs from patients with shorter survival length, in parallel with increasing genomic instability and upregulation of de novo DNA Methyltransferases (DNMTs) 1, 3A, and 3b. A progressive rise in Interleukin 6 and Fli-1 levels was responsible for upregulation of DNMT1, but not DNMT3s, in surrounding non-neoplastic livers and HCCs. Methylation-specific PCR and combined bisulphite restriction analyses of 100 putative tumor suppressor gene promoters identified the genes both universally and specifically inactivated in distinct subgroups of HCCs based on the length of patient’s survival. In particular, we identified activation of the Ras, Jak/Stat, and canonical Wnt/β-catenin pathways via epigenetic silencing on of Ras (ARHI, LOX, NORE1A, RASSF1A, and RIG), Jak/Stat (CIS, PIAS-1, PIAS-γ, SOCS1, 2, 3, and SHP1), and Wnt/β-catenin (APC, E-cadherin, SFRP1, 2, 4, 5, and WIF-1) inhibitors in the majority of HCCs, regardless of clinicopathological features of HCC patients. Furthermore, selective inactivation of genes controlling EGFR signaling and angiogenesis (e.g. SPRY1, 2, SOCS4, 5, EGLN2, BNIP3) was associated with an unfavorable outcome. Taken together, our results assign a therapeutic significance to methylation patterns in human HCC. Treatment approaches aimed at modifying the methylation status and utilizing novel molecular targets identified in this study may inhibit HCC development and progression.
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Our previous studies have shown that short-term treatment with phenobarbital (PB) resulted in cytosine methylation of CpG sites on the p53 gene promoter in male Wistar rats' liver. Furthermore, PB induced DNA-methyltransferases (DNMTs) activity was also demonstrated; being the enzymes that catalyze methyl group transfer to cytosine in CpG dinucleotides.Since DNA methylation is involved in regulating gene transcription and that DNMT1 is implicated in regulating DNA methylation, this study assessed whether PB-induced hypermethylation of the p53 promoter region was associated with an altered expression of p53 and Dnmt1 genes.Male Wistar rats received PB in three daily oral doses (at 24-h intervals) of 92,8 mg/kg b.w. x day-1. Levels of mRNA for p53 and Dnmt1 and levels of relevant proteins were respectively examined by Real-Time PCR and Western blot analysis.Gene expression analysis revealed that exposure of Wistar rats to PB caused statistically significant alternations in the expression of tested genes. We found that both mRNA and protein expression of p53 was down-regulated, whereas expression of Dnmt1 (both mRNA and protein) was up-regulated after PB treatment.Suppression of p53 mRNA and protein expression, which is probably a result of epigenetic changes, (in particular aberrant p53 promoter hypermethylation), can be associated with tumour promoting activity of phenobarbital.
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To assess the role of DNA cytosine methylation in the expression of the O-6-methylguanine DNA methyltransferase (MGMT) gene, the methylation status of selected CpG-containing dinucleotides in and surrounding the coding regions of the gene were examined and correlated with steady state expression of MGMT mRNA in 13 human cell lines. Additionally, tumor cells which exhibited very high levels of MGMT expression were chronically exposed to 5-azacytidine to assess the effects of changes in gene methylation on MGMT expression. Results of these studies demonstrate that the degree of methylation of multiple MGMT gene regions correlates with gene expression, but in a direct rather than an inverse fashion, and that 5-azacytidine-induced demethylation of the MGMT gene correlates with a significant reduction, rather than induction, of MGMT steady-state mRNA expression. These results suggest a unique, potentially alterable methylation-related regulatory mechanism for the MGMT gene.
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RNA-Directed DNA Methylation
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