• Asian Pacific Journal of Cancer Prevention
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• v.15 no.3
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• pp.1247-1253
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• 2014

Mutations in the DNMT3A and IDH genes represent the most common genetic alteration after FLT3/NPM1 in acute myeloid leukemia (AML). We here analyzed the frequency and distribution pattern of DNMT3A and IDH mutations and their associations with other molecular markers in normal karyotype AML patients. Fortyfive patients were screened for mutations in DNMT3A (R882), IDH1 (R132) and IDH2 (R140 and R172) genes by direct sequencing. Of the 45 patients screened, DNMT3A and IDH mutations were observed in 6 (13.3%) and 7 (15.4%), respectively. Patients with isolated DNMT3A mutations were seen in 4 cases (9%), isolated IDH mutations in 5 (11.1%), while interestingly, two cases showed both DNMT3A and IDH mutations (4.3%). Nucleotide sequencing of DNMT3A revealed missense mutations (R882H and R882C), while that of IDH revealed R172K, R140Q, R132H and R132S. Both DNMT3A and IDH mutations were observed only in adults, with a higher frequency in males. DNMT3A and IDH mutations were significantly associated with NPM1, while trends towards higher coexistence with FLT3 mutations were observed. This is the first study to evaluate DNMT3A/IDH mutations in Indian patients. Significant associations among the various molecular markers was observed, that highlights cooperation between them and possible roles in improved risk stratification.

• Journal of Animal Science and Technology
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• v.53 no.3
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• pp.269-274
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• 2011

DNA methyltransferases (DNMTs) are closely associated with the epigenetic change and the gene silencing through the regulation of methylation status in animal genome. But, the role of DNMTs in transgene silencing has remained unclear. So, we examined whether the knockdown of DNMT influences the reactivation of transgene expression in the transgenic quails. In this study, we investigated the expression of DNMT3a, and DNMT3b in blastoderm, quail embryonic fibroblasts (QEFs) and limited embryonic tissues such as gonad, kidney, heart and liver of E6 transgenic quails (TQ2) by RT-PCR. We further analyzed the expression of DNMT3a at different stages of whole embryos during early embryonic development by qRT-PCR. DNMT3a expression was detected in all test samples; however, it showed the highest expression in E6 whole embryo. Embryonic fibroblasts collected from TQ2 quails were treated with two DNMT3a-targeted siRNAs (siDNMT3a-51 and siDNMT3a-88) for RNA interference assay, and changes in expression were then analyzed by qRT-PCR. The siDNMT3a-51 and siDNMT3a-88 reduced 53.34% and 64.64% of DNMT3a expression in TQ2 QEFs, respectively. Subsequently the treatment of each siRNA reactivated enhanced green fluorescent protein (EGFP) expression in TQ2 (224% and 114%). Our results might provide a clue for understanding the DNA methylation mechanism responsible for transgenic animal production and stable transgene expression.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.1
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• pp.17-24
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• 2018

Objective: To investigate sperm chromatin/DNA integrity, global DNA methylation, and DNMT mRNA transcription in men with oligoasthenoteratozoospermia (OAT) compared with normozoospermic men. Methods: Semen samples from 32 OAT patients who comprised the case group and 32 normozoospermic men who comprised the control group were isolated and purified using a standard gradient isolation procedure according to World Health Organization criteria. DNMT1, DNMT3A, and DNMT3B transcripts were then compared between groups using real-time quantitative reverse-transcription polymerase chain reaction. Global DNA methylation in sperm was determined by an enzyme-linked immunosorbent assay. Protamine deficiency and the proportion of apoptotic spermatozoa were evaluated using chromomycin A3 (CMA3), aniline blue (AB), and toluidine blue (TB) staining, as well as the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The p-values < 0.05 were considered to indicate statistical significance. Results: Significantly higher proportions of AB+, TB+, CMA3+, and TUNEL+ spermatozoa, as well as DNMT3A and DNMT3B transcription, were found in the OAT group. Positive correlations were detected between sperm parameters, DNA/chromatin damage, and DNMT3A and DNMT3B transcripts. Global DNA methylation was significantly higher in the OAT patients and had a significant correlation with abnormal results of all sperm chromatin integrity tests, but was not associated with DNMT1, DNMT3A, or DNMT3B expression. Conclusion: Oligoasthenoteratozoospermic men showed abnormal sperm parameters, abnormal chromatin/DNA integrity, and a higher global DNA methylation rate, as well as overexpression of DNMT mRNA.

• Reproductive and Developmental Biology
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• v.30 no.4
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• pp.255-261
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• 2006

There are replete numbers of reports which have apparently shown that established patterns of methylation are critical for normal mammalian development. Here, we report expression of the DNA methyltransferases (Dnmts) family during mouse early development. Transcription of Dnmt1o occurs in one-cell and morula stage embryos, whereas Dnmtls transcripts were detectable in all cells and tissues examined during the study. Dnmt3a1 transcript was detected in all cells and Dnmt3a2 transcript was particularly detected in the oocyte and 1-cell stages. Low level Dnmt3b1 transcripts were expressed ubiquitously in oocyte, 1-cell, and preimplantation embryos except $2{\sim}4cell$ stages. Dnmt3b3 transcripts were only detected in E7.5 embryo and ovary. Furthermore, Dnmt31 transcripts were detectable in all cells and tissues examined. Unlike Dnmtl, both Dnmt3a and Dnmt3b proteins existed in the nucleus of preimplantation embryos till the morula stage. These Results suggest that differences Dnmts expression level exist and genomic DNA methylation patterns may be determined partly through differential expression of Dnmts during early development.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.10
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• pp.5713-5718
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• 2013

Background: DNA methyltransferase-3a (DNMT3a) plays significant roles in embryogenesis and the generation of aberrant methylation in carcinogenesis. This study aimed to investigate associations between single nucleotide polymorphisms (SNPs) of the DNMT3a gene and risk of Helicobacter pylori infection, gastric atrophy and gastric cancer. Methods: The subjects comprised 447 patients with gastric cancer; 111 individuals with gastric atrophy and 961 healthy controls. Two SNPs (rs1550117 and rs13420827) of the DNMT3a gene were genotyped by Taqman assay. DNMT3a expression was analyzed in cancer tissues from 89 patients by tissue microarray technique. Odds ratio (ORs) and 95% confidence intervals were calculated by multivariate logistic regression. Results: Among healthy controls, risk of H.pylori infection was significantly higher in subjects with the rs1550117 AA genotype, compared to those with GG/AG genotypes of DNMT3a [OR=2.08, (95%CI: 1.02-4.32)]. However, no significant correlation was found between the two SNPs and risk of developing gastric atrophy or gastric cancer. In addition, no increase in DNMT3a expression was observed in the gastric cancer with H.pylori infection. Conclusions: This study revealed that DNMT3a rs1550117 polymorphism is significantly associated with an increased risk of H. pylori infection, but did not support any evidence for contributions of DNMT3a rs1550117 and rs13420827 to either gastric atrophy or gastric cancer. The biological roles of DNMT3a polymorphisms require further investigation.

• Molecules and Cells
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• v.46 no.10
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• pp.611-626
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• 2023

Acute myeloid leukemia (AML) is a heterogeneous disease caused by distinctive mutations in individual patients; therefore, each patient may display different cell-type compositions. Although most patients with AML achieve complete remission (CR) through intensive chemotherapy, the likelihood of relapse remains high. Several studies have attempted to characterize the genetic and cellular heterogeneity of AML; however, our understanding of the cellular heterogeneity of AML remains limited. In this study, we performed single-cell RNA sequencing (scRNAseq) of bone marrow-derived mononuclear cells obtained from same patients at different AML stages (diagnosis, CR, and relapse). We found that hematopoietic stem cells (HSCs) at diagnosis were abnormal compared to normal HSCs. By improving the detection of the DNMT3A R882 mutation with targeted scRNAseq, we identified that DNMT3A-mutant cells that mainly remained were granulocyte-monocyte progenitors (GMPs) or lymphoid-primed multipotential progenitors (LMPPs) from CR to relapse and that DNMT3A-mutant cells have gene signatures related to AML and leukemic cells. Copy number variation analysis at the single-cell level indicated that the cell type that possesses DNMT3A mutations is an important factor in AML relapse and that GMP and LMPP cells can affect relapse in patients with AML. This study advances our understanding of the role of DNMT3A in AML relapse and our approach can be applied to predict treatment outcomes.

• Korean Journal of Clinical Laboratory Science
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• v.52 no.1
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• pp.69-77
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• 2020

The tumor suppressor gene, p53, is inactivated in the human hepatocellular carcinoma cells line, Hep3B. Berberine has been reported to inhibit the proliferation of cancer cells. This study examined whether apoptosis was induced in berberine-treated Hep3B cells and observed the association between apoptosis and the expression of p53 and DNA methyltransferase (DNMT). The cell viability was measured using an MTT assay. Apoptosis of Hep3B was measured using annexin V flow cytometry. Berberine-treated cells were examined for their DNMT enzymatic activity, mRNA expression, and protein synthesis. The p53 levels were examined by Western blot analysis. The berberine treatment resulted in increased Hep3B cell death and apoptosis in a time- and dose-dependent manner. The DNMT3b activity, mRNA expression, and protein levels all decreased after the berberine treatment. In contrast, the p53 protein levels increased with a concomitant decrease in DNMT3b. No change in the expression of ERK was observed, but the P-ERK levels decreased in a dose dependent manner. These results indicate that a treatment of Hep3B cells with berberine can reduce the expression of DNMT3b, leading to an increase in the tumor suppressant gene p53 and an increase in cell apoptosis. This shows that berberine can effectively suppress the proliferation of liver cancer cells.

• Biomolecules & Therapeutics
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• v.31 no.3
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• pp.319-329
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• 2023

Resistance to hypomethylating agents (HMAs) in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is a concerning problem. Polo-like kinase 1 (PLK1) is a key cell cycle modulator and is known to be associated with an activation of the PI3K pathway, which is related to the stabilization of DNA methyltransferase 1 (DNMT1), a target of HMAs. We investigated the effects of volasertib on HMA-resistant cell lines (MOLM/AZA-1 and MOLM/DEC-5) derived from MOLM-13, and bone marrow (BM) samples obtained from patients with MDS (BM blasts >5%) or AML evolved from MDS (MDS/AML). Volasertib effectively inhibited the proliferation of HMA-resistant cells with suppression of DNMTs and PI3K/AKT/mTOR and ERK pathways. Volasertib also showed significant inhibitory effects against primary BM cells from patients with MDS or MDS/AML, and the effects of volasertib inversely correlated with DNMT3B expression. The DNMT3B-overexpressed AML cells showed primary resistance to volasertib treatment. Our data suggest that volasertib has a potential role in overcoming HMA resistance in patients with MDS and MDS/AML by suppressing the expression of DNMT3 enzymes and PI3K/AKT/mTOR and ERK pathways. We also found that DNMT3B overexpression might be associated with resistance to volasertib.

• Journal of Gastric Cancer
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• v.7 no.1
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• pp.9-15
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• 2007

Purpose: DNA methylation is an important epigenetic factor in tumorigenesis. We hypothesized that polymorphism of the promoter of the DNA methyltransferase 3b (DNMT3b) genes, which are responsible for regulating the methylation status of tumor suppressor genes, are associated with increased risk of gastric cancer. Materials and Methods: In this hospital-based case-control study, to determine the role of this polymorphism of the promoter of DNA methyltransferase 3b (DNMT3b) genes in gastric cancer, we genotyped 176 cases and 70 control subjects. To determine the genotype, we used a polymerase chain reaction restriction fragment length polymorphism assay. We compared alleles and genotypes between the two groups and revealed an association of DNMT3b promoter polymorphism with increased risk of gastric cancer in the Korean population. Results: Genotype frequencies were 14.8% (Cytosine-Cytosine), 71.6% (Cytosine-Thymine), and 13.6% (Thymine- Thymine) in the case patients and 40.0% (Cytosine-Cytosine), 42.9% (Cytosine-Thymine), and 17.1% (Thymine-Thymine) in the control subjects, respectively. Compared with CC homozygotes, CT heterozygotes had a 4.523-fold increased risk (OR, 2.13; 95% CI, 2.324~8.803), and the TT homozygotes had a 2.154-fold elevated risk (OR, 1.42; 95% CI, 0.899~85.165). For the T variant genotype (CT+TT), there was a 3.846-fold increased risk (OR, 1.88; 95% CI, 2.040~7.251). However, no significance was observed in the genotype distributions of both polymorphisms according to histopathology, stage of stomach cancer. The Ssame results were observed with Helicobacter infection. Conclusion: DNMT3b promoter polymorphism, especially the T variant genotype, is associated significantly with thean increased risk of gastric cancer.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.6
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• pp.3015-3020
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• 2016

Background: Numerous studies have investigated associations of DNA methyltransferase (DNMT) -149 C>T and -579 G>T polymorphisms with gastric cancer (GC) and colorectal cancer (CRC) susceptibility; however, the findings are inconsistent prompting the present meta-analysis. Materials and Methods: Related studies were identified from PubMed, Google scholar, and SID until 10 October 2015. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the associations. Results: Eleven studies were included based on the search criteria for CRC and GC related to the DNMT3B 149 C>T (3,353 cases and 4,936 controls) and DNMT3B 579 G>T (1,387 cases and 2,064 controls) polymorphisms. There was no significant association overall between DNMT3B -149 and 579 polymorphisms and the risk of cancer. In the stratified analysis by cancer type, DNMT3B 579G>T polymorphism was associated with the risk of CRC and GC. While the DNMT3B -149C/T polymorphism was related with a significantly increased risk of CRC in two tested models, dominant (GG+GT vs. TT: OR 0.51, 95 % CI 0.38-0.69; P = 0.00, Pheterogeneity=0.69, $I^2=0%$) and heterozygote (GT vs. TT: OR 0.50, 95 % CI 0.37-0.69; P=0.00, Pheterogeneity=0.41, $I^2=0%$), no evidence of any association with GC risk was observed as in the pooled analyses. Conclusions: More studies are needed to assess associations of DNMT3B -149C/T and DNMT3B 579G>T polymorphisms with cancer in different ethnicities with large population sizes to generate comprehensive conclusions.