Search

Aim: To explore the role of DNA methylation in the innate immunity of the dental pulp, this study investigated the effect of 5‐aza‐2'‐deoxycytidine (AZA) on lipoteichoic acid (LTA)‐induced cytokine production and related intracellular signalling pathways in human odontoblast‐like cells (hOBs). Methodology: hOBs were cultured and differentiated from human dental pulp tissue, and the odontoblastic phenotype of the cells was detected using immunofluorescence, qRT‐PCR and Western blotting. hOBs were pretreated with AZA and then stimulated with 10 μg mL−1 LTA. The levels of 42 cytokines related to immunity and inflammation were examined using a cytokine antibody array and verified using qRT‐PCR and ELISA. The effect of AZA on the LTA‐induced NF‐κB and MAPK signalling pathways was explored using Western blotting. The cells were treated with the specific NF‐κB inhibitor PDTC and MAPK inhibitors (the ERK inhibitor U0126, the p38 inhibitor SB203580, and the JNK inhibitor SP600125) to further confirm the role of the signalling pathways in LTA‐treated hOBs. DNA immunoprecipitation–PCR was used to examine the dynamic methylation status of the gene promoters of myeloid differentiation primary response 88 (MyD88) and tumour necrosis factor receptor‐associated factor 6 (TRAF6) in the LTA‐induced hOBs. Statistical analyses of the differences between two groups were performed using Student's t‐test. One‐way analysis of variance (anova) or repeated‐measures anova with a post hoc Dunnett's test was used to assess the differences between multiple sets of data. P < 0.05 was considered to be statistically significant. Results: The odontoblastic markers were significantly higher in hOBs than those in human dental pulp cells (hDPCs) (P < 0.05). According to the cytokine antibody array results, hOBs pretreated with AZA had significantly increased production of several inflammatory cytokines (P < 0.05), in which the expression levels of IL‐6 and IL‐8 were the most dramatically increased upon LTA stimulation (P < 0.01). Furthermore, AZA resulted in the significant upregulation of p‐IKKα/β, p‐IκBα, p‐p65, p‐p38 and p‐ERK in LTA‐stimulated hOBs (P < 0.01). Treatment with the NF‐κB pathway inhibitor suppressed both IL‐6 and IL‐8 expression (P < 0.05), whereas inhibitors of the MAPK pathway (SB203580 and SP600125) did not. In LTA‐treated hOBs, AZA significantly increased the expression levels of TRAF6 and MyD88 (P < 0.05). AZA induced MyD88 promoter hypomethylation but did not affect TRAF6 methylation. Conclusion: AZA regulated the LTA‐induced inflammatory response through the NF‐κB signal pathway in hOBs. This study highlights the important role of DNA methylation in the immunity defence of odontoblasts during the dental pulp immunity response to caries. [ABSTRACT FROM AUTHOR]

Background: This study was conducted to investigate the effect of P14 promoter aberrant methylation on the biological function of human lung adenocarcinoma cells. Methods: We used nested methylation‐specific PCR (NMSP) to detect the methylation status of the p14ARF promoter region in SPCA1 and BEAS2B cell lines. The experimental groups were treated with 5‐aza‐2′‐deoxycytidine (5‐Aza). Quantitative real‐time PCR, Western blot, flow cytometry, and Cell Counting Kit 8 were used to detect the expression of p14ARF messenger RNA and protein in each group, apoptosis, and cell proliferation inhibition, respectively. Results: NMSP detected that the p14 promoter region of SPCA1 cells has abnormal methylation status. After treatment with 5‐Aza, the expression of p14ARF messenger RNA and protein in SPCA1 cells (P < 0.05) and the inhibition rate of cell proliferation (P < 0.05) were significantly increased, while the apoptosis rate was markedly increased (P < 0.05). However, no differences were observed in BEAS2B cells (P > 0.05). Conclusion: Abnormal methylation of the p14ARF promoter region plays an important role in the development of lung cancer cells. Our results suggest the use of P14 promoter aberrant methylation as a therapeutic target for drug research or to improve the sensitivity of other drugs. [ABSTRACT FROM AUTHOR]

Controlling the functional dynamics of DNA within living cells is essential in biomedical research. Epigenetic modifications such as DNA methylation play a key role in this endeavour. DNA methylation can be controlled by genetic means. Yet there are few chemical tools available for the spatial and temporal modulation of this modification. Herein, we present a small‐molecule approach to modulate DNA methylation with light. The strategy uses a photo‐tuneable version of a clinically used drug (5‐aza‐2′‐deoxycytidine) to alter the catalytic activity of DNA methyltransferases, the enzymes that methylate DNA. After uptake by cells, the photo‐regulated molecule can be light‐controlled to reduce genome‐wide DNA methylation levels in proliferating cells. The chemical tool complements genetic, biochemical, and pharmacological approaches to study the role of DNA methylation in biology and medicine. [ABSTRACT FROM AUTHOR]

Problem The regulatory mechanisms governing differential expression of classical major histocompatibility complex ( MHC) class I ( MHC-Ia) and non-classical MHC class I ( MHC-Ib) genes are poorly understood. Method of study Quantitative reverse transcription- polymerase chain reaction ( PCR) was used to compare the abundance of MHC-I transcripts and related transcription factors in peripheral blood mononuclear cells ( PBMC) and placental trophoblast cells ( PTC). Methylation of MHC-I CpG islands was detected by bisulfite treatment and next-generation sequencing. Demethylation of PBMC and PTC with 5′-aza-deoxycytidine was used to assess the role of methylation in gene regulation. Results MHC-I expression was higher in PBMC than PTC and was correlated with expression of IRF1, class II MHC transactivator ( CIITA), and STAT1. The MHC-Ia genes and Bo LA- NC1 were devoid of CpG methylation in PBMC and PTC. In contrast, CpG sites in the gene body of Bo LA- NC2, - NC3, and - NC4 were highly methylated in PBMC but largely unmethylated in normal PTC and moderately methylated in somatic cell nuclear transfer PTC. In PBMC, demethylation resulted in upregulation of MHC-Ib by 2.8- to 6-fold, whereas MHC-Ia transcripts were elevated less than 2-fold. Conclusion DNA methylation regulates bovine MHC-Ib expression and is likely responsible for the different relative levels of MHC-Ib to MHC-Ia transcripts in PBMC and PTC. [ABSTRACT FROM AUTHOR]

Contents FK506-binding protein 6 ( FKBP6) is essential for meiosis during mammalian spermatogenesis. However, the molecular regulation of FKBP6 during spermatogenesis remains unclear. In the present study, we performed molecular characterization of the meiosis-specific gene FKBP6 in yak testes. Yak FKBP6 encodes a polypeptide of 295 amino acid residues with an FK506-binding domain ( FKBP_C) and three tetratricopeptide repeat domains. The methylation level of the FKBP6 promoter in testes was significantly higher in cattle-yak with male sterility than in yak, and the FKBP6 promoter was methylated in liver tissues in which FKBP6 is not expressed. FKBP6 promoter activity was significantly decreased after treatment with the M.SssI methyltransferase in vitro. Furthermore, the FKBP6 gene was remarkably activated in bovine mammary epithelial cells treated with the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine. Taken together, our results demonstrate for the first time that the FKBP6 promoter is differentially methylated in testes; together with the functional promoter analysis, this suggests that methylation of this promoter may contribute to cattle-yak male infertility. [ABSTRACT FROM AUTHOR]

Background: Epigenetic modifications likely control the fate of hematopoietic stem cells (HSCs). The chromatin-modifying agents (CMAs), 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA), have previously been shown to expand HSCs from cord blood and marrow. Here we assessed whether CMA can also expand HSCs present in growth factor-mobilized human peripheral blood (MPB).Study Design and Methods: 5azaD and TSA were sequentially added to CD34+ MPB cells in the presence of cytokines, and the cells were cultured for 9 days.Results: After culture, a 3.6 ± 0.5-fold expansion of CD34+CD90+ cells, a 10.1 ± 0.5-fold expansion of primitive colony-forming unit (CFU)-mix, and a 2.2 ± 0.5-fold expansion of long-term cobblestone-area-forming cells (CAFCs) was observed in 5azaD/TSA-expanded cells. By contrast, cells cultured in cytokines without 5azaD/TSA displayed no expansion; rather, a reduction in CD34+CD90+ cells (0.7 ± 0.1-fold) and CAFCs (0.3 ± 0.1-fold) from their initial numbers was observed. Global hypomethylation corresponding with increased transcript levels of several genes implicated in HSC self-renewal, including HOXB4, GATA2, and EZH2, was observed in 5azaD/TSA-expanded MPB cells in contrast to controls. 5azaD/TSA-expanded MPB cells retained in vivo hematopoietic engraftment capacity.Conclusion: MPB CD34+ cells from donors can be expanded using 5azaD/TSA, and these expanded cells retain in vivo hematopoietic reconstitution capacity. This strategy may prove to be potentially useful to augment HSC numbers for patients who fail to mobilize. [ABSTRACT FROM AUTHOR]

To test the safety and activity of 5-aza-2′-deoxycytidine (decitabine) in patients with relapsed/refractory acute lymphocytic leukaemia ( ALL), we conducted a phase 1 study with two parts: administering decitabine alone or in combination with Hyper- CVAD (fractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone alternating with high-dose methotrexate and cytarabine). Patients participated in either part of the study or in both parts sequentially. In the initial part, decitabine was administered intravenously at doses of 10-120 mg/m2 per d for 5 d every other week in cycles of 28 d. In the combination part, patients were treated on the first 5 d of Hyper- CVAD with intravenous decitabine at 5-60 mg/m2 per d. A total of 39 patients received treatment in the study: 14 in the first part only, 16 sequentially in both parts and 9 in the second part only. Decitabine was tolerated at all doses administered, and grade 3 or 4 toxic effects included non-life-threatening hepatotoxicity and hyperglycaemia. Induction of DNA hypomethylation was observed at doses of decitabine up to 80 mg/m2. Some patients who had previously progressed on Hyper- CVAD alone achieved a complete response when decitabine was added. Decitabine alone or given with Hyper- CVAD is safe and has clinical activity in patients with advanced ALL. [ABSTRACT FROM AUTHOR]

A 5-formyl-2′-deoxycytidine (fdC) phosphoramidite building block that enables the synthesis of fdC-containing DNA with excellent purity and yield has been developed. In combination with phosphoramidites for 5-methyl-dC, 5-hydroxymethyl-dC, and carboxy-dC, it was possible to prepare a segment of the OCT-4 promoter that contains all four epigenetic bases. Because of the enormous interest in these new epigenetic bases, the ability to insert all four of them into DNA should be of great value for the scientific community. [ABSTRACT FROM AUTHOR]

DNA methylation of cytosine residues constitutes a prominent epigenetic modification of the chromatin fiber which is locked in a transcriptionally inactive conformation leading to gene silencing. Plant developmental processes, as differentiation and proliferation, are accompanied by chromatin remodeling and epigenetic reprogramming. Despite the increasing knowledge gained on the epigenetic mechanisms controlling plant developmental processes, the knowledge of the DNA methylation regulation during relevant developmental programs in flowering plants, such as gametogenesis or embryogenesis, is very limited. The analysis of global DNA methylation levels has been frequently conducted by high performance capillary electrophoresis, and more recently also by ELISA‐based assays, which provided quantitative data of whole organs and tissues. Nevertheless, to investigate the DNA methylation dynamics during plant development in different cell types of the same organ, the analysis of spatial and temporal pattern of nuclear distribution of 5‐methyl‐deoxy‐cytidine (5mdC) constitutes a potent approach. In this work, immunolocalization of 5mdC on sections and subsequent confocal laser microscopy analysis have been applied for in situ cellular analysis of a variety of plant cells, tissues and organs with different characteristics, e.g. hardness, heterogeneity, cell accessibility, tissue compactness, etc.; the results demonstrated the versatility and feasibility of the approach for different plant samples, and revealed defined DNA methylation nuclear patterns associated with differentiation and proliferation events of various plant cell types and developmental programs. Quantification of 5mdC immunofluorescence intensity by image analysis software also permitted to estimate differences in global DNA methylation levels among different cells types of the same organ during development. [ABSTRACT FROM AUTHOR]

Abstract: Medulloblastoma cells exhibit varied responses to therapy by all-trans retinoic acid (RA). The underlying mechanism for such diverse effects however remains largely unclear. In this study, we attempted to elucidate the molecular basis of RA resistance through the study of RA signaling components in both RA-sensitive (Med-3) and RA-resistant (UW228-2 and UW228-3) medulloblastoma cells. The results revealed that RARα/β/γ and RXRα/β/γ were found in the three cell lines. Expression of CRABP-I and CRABP-II was seen in Med-3 cells, up-regulated when treated with RA, but was absent in UW228-2 and UW228-3 cells regardless of RA treatment. Bisulfite sequencing revealed 8 methylated CG sites at the promoter region of CRABP-II in UW228-2 and UW228-3 but not in Med-3 cells. Demethylation by 5-aza-2′-deoxycytidine recovered CRABP-II expression. Upon restoration of CRABP-II expression, both UW228-2 and UW228-3 cells responded to RA treatment by forming neuronal-like differentiation, synaptophysin expression, β-III tubulin upregulation, and apoptosis. Furthermore, CRABP-II specific siRNA reduced RA sensitivity in Med-3 cells. Tissue microarray-based immunohistochemical staining showed variable CRABP-II expression patterns among 104 medulloblastoma cases, ranging from negative (42.3%), partly positive (14.4%) to positive (43.3%). CRABP-II expression was positively correlated with synaptophysin (rs = 0.317; p = 0.001) but not with CRABP-I expression (p > 0.05). In conclusion, aberrant methylation in CRABP-II reduces the expression of CRABP-II that in turn confers RA resistance in medulloblastoma cells. Determination of CRABP-II expression or methylation status may enable a personalized RA therapy in patients with medulloblastomas and other types of cancers. [Copyright &y& Elsevier]