Background: Somatic mutations in the hotspot region of the DNA-methyltransferase 3A (DNMT3A) gene were recurrently identified in acute myeloid leukemia (AML). It is believed that DNMT3A mutations confer an adverse prognosis for AML patients. These lines of evidence support the need for a rapid and cost-efficient method for the detection of these mutations. The present study aimed to establish high resolution melting (HRM) curve analysis as a rapid and sensitive test to identify DNMT3A gene mutations in AML patients.
Materials and Methods: In this retrospective cohort study, a total of 220 AML patients who referred to hematology-oncology and stem cell transplantation centers (referral center) at Shariati hospital in Tehran, Iran, were included. AML-M3 and therapy-related AML patients were excluded. The HRM assay was used to identify R882 mutations in DNMT3A gene, and the results were compared with those of Sanger sequencing as the gold standard test for detection of such mutations.
Results: Among 220 samples from AML patients, Sanger sequencing detected 25 (11.4%) patients as having DNMT3A R882 mutations. HRM assay detected mutations in 23 (92%) samples and reported two false-negative results that were related to poor-quality DNA samples. There was an overall good agreement between direct sequencing and HRM assay (kappa value of 0.95) (p<0.001). Sensitivity assay showed that the analytical detection limits for HRM were 10% for the detection of R882H mutation compared with Sanger sequencing at 25%. Both Sanger sequencing and HRM assay reported no false-positive results.
Conclusion: HRM curve analysis can be considered as a sensitive, fast, and high-throughput method for the detection of DNMT3A R882 mutation in AML patients. These results validate HRM analysis as an alternative method to Sanger sequencing because of its simplicity along with the lower cost and less required time.

Background: DNA molecule of the eukaryotic cells is found in the form of a nucleoprotein complex named chromatin. Two epigenetic modifications are critical for transcriptional control of genes, including acetylation and DNA methylation. Hypermethylation of tumor suppressor genes is catalyzed by various DNA methyltransferase enzymes (DNMTs), including DNMT1, DNMT2, and DNMT3. The most well characterized DNA demetilating and histone deacetylase inhibitor drugs are 5-aza-2ˈ-deoxycytidine (5-Aza-CdR) and valproic acid (VPA), respectively. The purpose of the current study was to analyze the effects of 5-Aza-CdR and VPA on cell growth, apoptosis, and DNMT1 gene expression in the WCH-17 hepatocellular carcinoma (HCC) cell line.
Materials and Methods: In this descriptive analytical study, MTT assay, flow cytometry assay, and Quantitative Real-Time RT-PCRwere done to evaluate proliferative and apoptotic effects and also gene expression.
Results: Both compounds inhibited the cell growth and induced apoptosis significantly in a dose and time depended fashion. Additionally, 5-Aza-CdR down-regulated DNMT1 gene expression. The relative expression of DNMT1 was 0.40 and 0.20 (P < 0.001) at different times, respectively. The percentage of VPA- treated apoptotic cells were reduced by about 28 and 34 % (P˂0.001) and that of 5-Aza-CdR-treated were reduced by about 34 and 44 % (P˂0.001) after treatment time periods.
Conclusion: In the current study, it was observed that 5-Aza-CdR and VPA could significantly inhibit the growth of WCH-17 cell and played a significant role in apoptosis. It was also found that 5-Aza-CdR could decrease DNMT1 gene expression.

Background: 6-thioguanine (6-TG) is one of the thiopurine drugs with successful use in oncology, especially for acute lymphoblastic leukemia (ALL). 6-TG is proposed to act as an epigenetic drug affecting DNA methylation. The aim of this study was to clarify the effect of 6-TG on the proliferation, viability and expression of genes coding for the enzymes DNA methyltransferase 3A and DNA methyltransferase 3B (DNMTs) as well as histone deacetylase 3 (HDAC3) in the human B cell-ALL cell line Nalm6.
Materials and Methods: In this experimental study, Nalm6 cells and also normal peripheral blood mononuclear cells (PBMCs) were grown in RPMI 1640 medium containing 10% fetal bovine serum. They were then treated with 6-TG at their exponential growth phase. Cell viability was monitored using the Cell Counting Kit-8 assay with an enzyme-linked immunosorbent assay (ELISA) reader. The expressions of the above-mentioned 3 genes were quantified using real-time PCR.
Results: 6-TG could inhibit the proliferation of Nalm6 cells and decrease their viability. In Nalm6 cells, as compared to normal PBMCs, 6-TG significantly decreased HDAC3 (p = 0.008) as well as DNMT3B (p = 0.003) gene expressions, but increased the expression of DNMT3A gene (p = 0.02) after normalization to GAPDH, as the housekeeping gene.
Conclusion: These findings suggested that the altered expression of DNMT3A, DNMT3B and HDAC3 genes was responsible for at least part of the antitumoral properties of 6-TG, providing an insight into mechanism of its action as an epigenetic drug.
 

Background: Decitabine (5-aza-2'-deoxycytidine, DAC) is a deoxycytidine analog currently used as an effective drug against myelodysplastic syndromes and acute myeloid leukemia. Although various studies have pointed out the epigenetic effects of this drug, its epigenetic mechanisms in different leukemic cell lines are not specified. In this lab trial study, possible epigenetic effects of decitabine on leukemia cell lines Hl-60 (acute promyelocytic leukemia) and Nalm-6 (acute pre-B cell lymphoblastic leukemia) vs. normal peripheral blood mononuclear cells (PBMCs) are compared.
Materias and Methods: At the logarithmic phase of growth, the cultured cells Hl-60 and Nalm-6 obtained from Tehran Pasteur Institute, Iran, were treated for 24 hr with 1 μM of decitabine, a dose selected from literature and the MTT viability assay. Normal PBMCs were obtained from a pool of 3 healthy adult volunteer males, and cultured simultaneously in the same manner. The gene expressions of epigenetic enzymes DNA methyltransferases (DNMTs) were assessed with the real-time PCR technique before and after treatment. The GAPDH gene expression served as the calibrator, while normal PBMCs were used for comparison.
Results: The expressions of DNMT1 and DNMT3B in lymphoblasts were significantly (p=0.0017 and p=0.0489, respectively) decreased after treatment with decitabine, while the expression of DNMT3A was significantly (p=0.0022) increased. In leukemic promyelocytes the expressions of DNMT1 and DNMT3B in lymphoblasts were significantly (p=0.0222 and p=0.0452, respectively) decreased after treatment with decitabine, while the expression of DNMT3A was significantly (p=0.0013) increased.
Conclusion: One of the mechanisms by decitabine to inhibit the proliferation of both myeloid and lymphoid acute leukemia cells maybe by altering the gene expression of DNMTs.

Background: DNA methylation is catalyzed by DNA methyltransferases (DNMTs) which are encoded by DNMT1, DNMT3A, and DNMT3B. DNMTs play a major role in the abnormal methylation of tumor suppressors and cancer-related genes. Herein, this study explored the expression profile of DNMTs in pediatric patients with B-cell acute lymphoblastic leukemia (ALL), before and after methotrexate (MTX)/mercaptopurine (6-MP) treatment.
Materials and Methods: This before-after prospective study included 30 matched children in sex and age (20 children with B-cell ALL and 10 healthy children used as a control or calibrator group). The expression profile of DNMTs was assessed at two-time points; at the diagnosis time and after MTX/6-MP treatment in the consolidation-maintenance phase of therapy. Notable, all pediatric patients included in this study continued the therapy without adverse events, except two children who were excluded from the study.
Results: The average age of the patient group was 7.1 ± 1.3 years (in the range of 4-9 years), and the average age of the control group was 8.3 ± 1.7 years (6-10 years). The expression profile of DNMTs in B-cell ALL children was obtained completely different from that in the healthy group. After MTX/6-MP treatment of B-cell ALL children, the expression levels of DNMT1 and 3A were increased (p <0.01 & 0.04, respectively), and the expression level of DNMT3B was decreased (p <0.01), significantly.
Conclusions: In ALL, the expression profile of DNMTs would be changed whereby contribute to abnormal growth and maturation capacity of leukemic stem cells and MTX/6-MP treatment could reverse this profile from a cancerous phenotype to the normal one.DNA Methyltransferases (DNMTs), Methotrexate (MTX), Mercaptopurine (6-MP)