Induced DNA Hypomethylation Research Articles

Oxytocin prevents cue-induced reinstatement of oxycodone seeking: Involvement of DNA methylation in the hippocampus.

Oxycodone is one of the most commonly used analgesics in the clinic. However, long-term use can contribute to drug dependence. Accumulating evidence of changes in DNA methylation after opioid relapse has provided insight into mechanisms underlying drug-associated memory. The neuropeptide oxytocin is reported to be a potential treatment for addiction. The present study sought to identify changes in global and synaptic gene methylation after cue-induced reinstatement of oxycodone conditioned place preference (CPP) and the effect of oxytocin. We analyzed hippocampal mRNA of synaptic genes and also synaptic density in response to oxycodone CPP. We determined the mRNA levels of DNA methyltransferases (Dnmts) and ten-eleven translocations (Tets), observed global 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) levels, and measured DNA methylation status of four synaptic genes implicated in learning and memory (Arc, Dlg1, Dlg4, and Syn1). Both synaptic density and the transcription of 15 hippocampal synaptic genes significantly increased following cue-induced reinstatement of oxycodone CPP. Oxycodone relapse was also related to markedly decreased 5-mC levels and decreased transcription of Dnmt1, Dnmt3a, and Dnmt3b; in contrast, 5-hmC levels and the transcription of Tet1 and Tet3 were increased. Oxycodone exposure induced DNA hypomethylation at the exons of the Arc, Dlg1, Dlg4, and Syn1 genes. Intracerebroventricular (ICV) administration of oxytocin (2.5μg/μl) specifically blocked oxycodone relapse, possibly by inhibition of Arc, Dlg1, Dlg4, and Syn1 hypomethylation in oxycodone-treated rats. Together, these data indicate the occurrence of epigenetic changes in the hippocampus following oxycodone relapse and the potential role of oxytocin in oxycodone addiction.

English

β-expansin 2 (EXPB2) gene induces drought tolerance in different plant species including maize. Different epigenetic mechanisms like DNA methylation, histone modification and RNA interference affect the gene activities under stress conditions. DNA methylation, an important epigenetic mechanism, could be involved in the regulation of ZmEXPB2 gene under drought stress in maize. Plants of drought sensitive variety „Jalal‟ were grown till 4th leaf stage under well-watered conditions. At 5th leaf stage, plants were divided in two groups i.e., well-watered (100% water holding capacity) or drought stress (0% water holding capacity for 15 days). Plants subjected to drought stress showed clear signs of stress by significant decrease in fresh weight of whole plant, 6th leaf length, stunted secondary root growth and increased primary root length. DNA methylation profile of three regions (denoted as -1.7 k, -1.3 k and -0.8 k) in the promoter of ZmEXPB2 gene, of root DNA, were evaluated. Under well-watered conditions, heterogeneity in DNA methylation profile along the promoter sequence was observed. Regions -1.7 k and -1.3 k were methylated whereas the region -0.8 k was nonmethylated. After the comparison of DNA methylation profile of well-watered and drought stress plants, no change in -1.7 k and -0.8 k regions was observed. However, the -1.3k region had significant decrease in the DNA methylation at symmetric cytosine sites i.e., cytosine-guanine (CG) dinucleotides and cytosine-adenine/cytosine/thymine-guanine (CHG where H = A, C or T) trinucleotide and significant increase at asymmetric cytosine sites (CHH) under the stress condition. In addition, significant increase in the gene expression of ZmEXPB2 under drought was also observed. In conclusion, drought stress conditions induce DNA hypomethylation at CG, and CHG sites and DNA hypermethylation at CHH sites in the middle region of the promoter of ZmEXPB2 gene. This shift can be associated with the up regulation of ZmEXPB2 gene which in turn increased primary root length as a plant stress response mechanism. © 2021 Friends Science Publishers

The Regulatory Effect of ERK1 Pathway in the DNA Hypomethylation of MRL/lpr Mice

Background: Defective ERK1 activity may regulate the DNA hypomethylation in T cells of lupus mice. Aims: To investigate the function of ERK1 in regulating DNA hypomethylation and explore the potential molecular mechanisms involved in lupus mice. Methods: CD4+T cells were isolated from MRL/lpr and BALB/c mice, activated in vitro in the presence or absence of 5-azacytidine (5-Aza) (a DNA methyltransferase inhibitor)/U0126 (a selective inhibitor of the ERK signaling pathway) respectively. The positive rate of CD4+T cell was measured by flow cytometry, and expression levels of CD70, ERK1, DNA transferase 1 (DNMT1) were estimated by RT-PCR and Western blot. The methylated DNA is detected using the Capture and Detection antibodies, then quantified colorimetrically. Results: The expression level of p-ERK1 in MRL/lpr mice was significantly lower than healthy control (P<0.05); Meanwhile, down regulation of DNMT1 and DNA hypomethylation were found in MRL/lpr mice T cells. In contrary, the expression levels of CD70 increased when compared with healthy control (P<0.05). The same changes were seen in healthy CD4+T cells treated with U0126. Conclusions: DNA hypomethylation regulating mechanisms in lupus T cells were associated with ERK1 signal pathway. U0126 could inhibit ERK1 signal transduction pathway in normal T cells, and induce DNA hypomethylation by regulating the expression of methylation sensitive gene CD70.

Silver Nanoparticles Induce DNA Hypomethylation through Proteasome-Mediated Degradation of DNA Methyltransferase 1.

Nanoparticles are used in many fields and in everyday products. Silver nanoparticles are the most frequently used nanoparticles; for example, in food-related products, owing to their antibacterial activity. However, it has been pointed out that they might have unexpected biological effects, and evaluation of their effects is underway. Although there is a growing body of evidence that nanoparticles can also induce epigenetic changes, there is still little information on the underlying mechanisms. Here, we evaluated changes in DNA methylation induced by silver nanoparticles and attempted to elucidate the induction mechanism. Immunofluorescence staining analysis revealed that silver nanoparticles with a diameter of 10, 50, or 100 nm (nAg10, nAg50, and nAg100, respectively) decreased the content of methylated DNA in A549 alveolar epithelial cells. The level of DNA methyltransferase 1 (Dnmt1) protein, which is involved in maintaining methylation during DNA replication, was significantly decreased, whereas that of Dnmt3b, which is responsible for de novo DNA methylation, was significantly increased by nAg10 treatment. Co-treatment with nAg10 and cycloheximide, which inhibits translation by inhibiting the translocation step of protein synthesis, decreased the level of Dnmt1 in comparison with nAg10-treated A549 cells, indicating a post-translational effect of nAg10. Furthermore, pretreatment with the proteasome inhibitor lactacystin restored the levels of Dnmt1 protein and DNA methylation in nAg10-treated cells. Collectively, these results suggest that nAg10 induced DNA hypomethylation through a proteasome-mediated degradation of Dnmt1.

Induced DNA hypomethylation by Folic Acid Deprivation in Bovine Fibroblast Donor Cells Improves Reprogramming of Somatic Cell Nuclear Transfer Embryos

Aberrant patterns of DNA methylation are consistent events in SCNT derived embryos and mechanistically are believed to be related to abnormal development. While some epigenetic drugs have been used in attempts to improve SCNT efficiency but some concerns remained toward the safety of these drugs on the health of future offspring. Folate is an essential cofactor in one‐carbon cycle for conversion of homocysteine to methionine, thereby ensuring supply of SAM, the universal methyl donor for many biological methylation reactions including DNA methylation. Therefore, in vitro DNA hypo-methylation can be induced by folate deprivation and this study aims at deciphering the role of folic acid deprivation in culture medium of BFFs for 6 days on SCNT efficiency. Our data revealed that culture of fibroblast cells in folate− medium containing 0.5% FBS did not alter the cell cycle compared to other groups. Flowcytometric analysis revealed that DNA methylation (5-mC level) in folate deprived cells cultured in 0.5% serum was decreased compared to folate+ group. The result of bisulfite sequencing was in accordance with flowcytometric analysis, which indicated a decrease in DNA methylation of POU5F1 promoter. Gene expression analysis revealed an increase in expression of POU5F1 gene in folate− group. The nuclear area of the cells in folate− group was significantly larger than folate+ group. Induced DNA hypomethylation by folate deprivation in the folate− group significantly improved blastocyst rate compared to the folate+ group. DNA methylation level in POU5F1 promoter and ICR of H19 and IGF2 of SCNT derived embryos in the folate− group was similar to the IVF derived blastocysts. In conclusion, our results proposes a promising “non-chemical” instead of “chemical” approach using inhibitors of epigenetic modifier enzymes for improving mammalian SCNT efficiency for agricultural and biomedical purposes.

Decitabine Augments Chemotherapy-Induced PD-L1 Upregulation for PD-L1 Blockade in Colorectal Cancer.

Programmed cell death-1 (PD-1) has demonstrated impressive clinical outcomes in several malignancies, but its therapeutic efficacy in the majority of colorectal cancers is still low. Therefore, methods to improve its therapeutic efficacy in colorectal cancer (CRC) patients need further investigation. Here, we demonstrate that immunogenic chemotherapeutic agents trigger the induction of tumor PD-L1 expression in vitro and in vivo, a fact which was validated in metastatic CRC patients who received preoperatively neoadjuvant chemotherapy (neoCT) treatment, suggesting that tumor PD-L1 upregulation by chemotherapeutic regimen is more feasible via PD-1/PD-L1 immunotherapy. However, we found that the epigenetic control of tumor PD-L1 via DNA methyltransferase 1 (DNMT1) significantly influenced the response to chemotherapy. We demonstrate that decitabine (DAC) induces DNA hypomethylation, which not only directly enhances tumor PD-L1 expression but also increases the expression of immune-related genes and intratumoral T cell infiltration in vitro and in vivo. DAC was found to profoundly enhance the therapeutic efficacy of PD-L1 immunotherapy to inhibit tumor growth and prolong survival in vivo. Therefore, it can be seen that DAC remodels the tumor microenvironment to improve the effect of PD-L1 immunotherapy by directly triggering tumor PD-L1 expression and eliciting stronger anti-cancer immune responses, providing potential clinical benefits to CRC patients in the future.

Application of 5-azacytidine induces DNA hypomethylation and accelerates dormancy release in buds of tree peony

Release of bud dormancy is a prerequisite for the growth resumption and production in perennial plants such as tree peony. DNA methylation plays a pivotal role in regulating gene expression. In this study, combination of morphologic observation and DNA methylation analysis indicated that 5-azacytidine (5-azaC) application for 7 d declined 5 mC quantities and promoted dormancy release. After 5-azaC treatment, total 174,341 unigenes and 1818 differentially expression genes (DEGs) were obtained by RNA-seq, of which there were 1194 DEGs after 1 d 5-azaC treatment (AD1 vs CD1), and 624 DEGs after 7 d (AD7 vs CD7), respectively. The KEGG pathway analysis identified that totally 10 DEGs annotated in DNA replication pathway were enriched when AD7 compared with CD7. Furthermore, the expression patterns of several DEGs by real-time quantitative RT-PCR were consistent with that of RNA-seq data. 5-azaC application significantly decreased the expression levels of DNA methyltransferase genes, PsCMT3, PsMET1 and PsDRM2, and increased the transcript of demethylase gene PsROS1. Simultaneously, total methyltransferases activity decreased, and demethylase activity was induced by 5-azaC. In summary, application of 5-azaC inhibited the expression of the genes related to growth and development in short-term, indicating a possible toxic effect to plant, and its long-term effect was to induce hypomethylation by increasing demethylase genes transcripts and decreasing the expressions of methyltransferase genes, and then activate cell cycle, DNA replication and glycol-metabolism processes, which subsequently accelerated dormancy release. All these would provide a new strategy to further understand the molecular mechanism of dormancy release in tree peony.

Histone acetylation together with DNA demethylation empowers higher plasticity in adipocytes to differentiate into osteoblasts

Bone regeneration has been a challenge for both researchers and clinicians. In the field of tissue engineering, much effort has been made to identify cell sources including stem cells. The present study aimed to induce trans-differentiation from adipocytes to osteoblasts using epigenetic modifiers; 5-aza-dC and/or trichostatin-A (TSA). 3 T3-L1 preadipocytes were treated with TSA (100 nM) and then with Wnt3a (50 ng/ml). Microscopic observation showed trans-differentiated cell morphology. Methylation-specific PCR and immunoblotting were performed to analyze the DNA methylation and histone acetylation patterns. The gene expression was determined by real-time PCR. Based on these in vitro experiments, in vivo mouse experiments supplemented the possibility of trans-differentiation by epigenetic modification. TSA induced the acetylation of lysine9 on histone H3, and a sequential Wnt3a treatment stimulated the expression of bone marker genes in adipocytes, suppressing adipogenesis and stimulating osteogenesis. Furthermore, TSA induced DNA hypomethylation, and a combined treatment with TSA and 5-aza-dC showed a synergistic effect in epigenetic modifications. The number of adipocytes and DNA methylation patterns of old (15 months) and young (6 weeks) mice were significantly different, and TSA and sequential Wnt3a treatments increased bone formation in the old mice. Collectively, our results confirmed cell trans-differentiation via epigenetic modifications and osteogenic signaling from adipocytes to osteoblasts for the bone regeneration in vitro and in vivo, and indicated that histone acetylation could induce DNA hypomethylation, enhancing the chance of trans-differentiation.

MiR-29b interacts with IFN-γ and induces DNA hypomethylation in CD4+ T cells of oral lichen planus

Oral lichen planus (OLP) is an autoimmune inflammatory disease mediated by T cells, in whose pathogenesis CD4+ T helper cells are supposed to play vital roles. MiR-29b has recently been recognized as a crucial regulator in immune response and inflammation. The current research focuses on exploring how miR-29b functions in the immunopathogenesis of OLP. Our findings showed that miR-29b expression in CD4+ T cells was upregulated in OLP, especially in its erosive form. MiR-29b in CD4+ T cells repressed IFN-γ mRNA and IFN-γ secretion, but not T-bet and EOMES; in turn, IFN-γ increased the expression of STAT1 and miR-29b in CD4+ T cells. Moreover, miR-29b in CD4+ T cells suppressed DNMT1 expression and induced global DNA hypomethylation. In conclusion, elevated miR-29b interacts with IFN-γ via a regulatory feedback loop and induces global DNA hypomethylation in CD4+ T cells, which consequently modulates Type 1 T helper immune response, thus contributing to the immune dysregulation of OLP.

Modulation of Mitochondrial and Epigenetic Targets by Polyphenols-rich Extract from Araucaria angustifolia in Larynx Carcinoma.

Araucaria angustifolia extract (AAE) is a polyphenol-rich extract that has gained interest as a natural anticancer agent. Recent work suggests that AAE induces oxidative damage and apoptosis through its action on decreasing complex I activity of the mitochondrial Electron Transport Chain (ETC). In the present study, we aimed to further examine the specific targets by which AAE exerts proapoptotic effects in HEp-2 cancer cells. Specifically, the effect of AAE on the: 1) levels of pyruvate dehydrogenase was assessed by ELISA assay; 2) levels of mitochondrial ETC complexes, focusing on complex I at the gene transcript and protein level relevant to ROS generation was evaluated by multiplex ELISA followed by qRT-PCR and immunoblotting; 3) mitochondrial network distribution analysis was assessed by MitoTracker Red CMXRos; and 4) chemical variations on DNA was evaluated by dot-blotting in HEp-2 cells. Results demonstrated that AAE increased protein levels of PDH, switching energy metabolism to oxidative metabolism. Protein expression levels of complex I and III were found decreased in AAE-treated HEp-2 cells. Analyzing the subunits of complex I, changes in protein and gene transcript levels of NDUFS7 and NDUFV2 were found. Mitochondria staining after AAE incubation revealed changes in the mitochondrial network distribution. AAE was able to induce DNA hypomethylation and decreased DNA (cytosine-5)-methyltransferase 1 activity. Our data demonstrate for the first time that AAE alters expression of NDUFS7 and NDUFV2 mitochondrial subunits and induce epigenetic changes in HEp-2 cancer cells leading to a possible suppression of oncogenes.

Effect of 5-azacytidine induced DNA demethylation on abiotic stress tolerance in Arabidopsis thaliana

The effect of 5-azacytidine (5A)-induced DNA hypomethylation on the growth and abiotic stress tolerance of Arabidopsis thaliana were analysed. Growth analysis revealed that aqueous solutions of 5A added to the soil did not affect the fresh and dry biomass accumulation but led to a higher percentage of flowering A. thaliana plants after four weeks of cultivation. The 5A treatment considerably lowered survival rates of Arabidopsis plants under high soil salinity, heat stress, and drought, while it did not affect the survival rates after freezing stress. 5A eliminated the stimulatory effect of the heat and drought stresses on the transcriptional levels of a number of stress-inducible genes, such as DREB1, LEA, SOS1, or RD29A. A less clear but similar trend has been detected for the effect of 5A on expression of the stress-inducible genes under salt and cold stresses. The data indicate that DNA methylation is an important mechanism regulating plant abiotic stress resistance.

Oxytocin inhibits methamphetamine-associated learning and memory alterations by regulating DNA methylation at the Synaptophysin promoter.

Methamphetamine (METH) causes memory changes, but the underlying mechanisms are poorly understood. Epigenetic mechanisms, including DNA methylation, can potentially cause synaptic changes in the brain. Oxytocin (OT) plays a central role in learning and memory, but little is known of the impact of OT on METH-associated memory changes. Here, we explored the role of OT in METH-induced epigenetic alterations that underlie spatial and cognitive memory changes. METH (2.0mg/kg, i.p.) was administered to male C57BL/6 mice once every other day for 8days. OT (2.5μg, i.c.v.) or aCSF was given prior to METH. Spatial and cognitive memory were assessed. In Hip and PFC, synaptic structures and proteins were examined, levels of DNA methyltransferases (DNMTs) and methyl CpG binding protein 2 (MECP2) were determined, and the DNA methylation status at the Synaptophysin (Syn) promoter was assessed. METH enhanced spatial memory, decreased synapse length, downregulated DNMT1, DNMT3A, DNMT3B, and MECP2, and induced DNA hypomethylation at the Syn promoter in Hip. In contrast, METH reduced cognitive memory, increased synapse thickness, upregulated DNMT1, DNMT3A, and MECP2, and induced DNA hypermethylation at the Syn promoter in PFC. OT pretreatment specifically ameliorated METH-induced learning and memory alterations, normalized synapse structures, and regulated DNMTs and MECP2 to reverse the DNA methylation status changes at the Syn promoter in Hip and PFC. DNA methylation is an important gene regulatory mechanism underlying METH-induced learning and memory alterations. OT can potentially be used to specifically manipulate METH-related memory changes.

A novel FLI1 exonic circular RNA promotes metastasis in breast cancer by coordinately regulating TET1 and DNMT1

BackgroundFriend leukemia virus integration 1 (FLI1), an ETS transcription factor family member, acts as an oncogenic driver in hematological malignancies and promotes tumor growth in solid tumors. However, little is known about the mechanisms underlying the activation of this proto-oncogene in tumors.ResultsImmunohistochemical staining showed that FLI1 is aberrantly overexpressed in advanced stage and metastatic breast cancers. Using a CRISPR Cas9-guided immunoprecipitation assay, we identify a circular RNA in the FLI1 promoter chromatin complex, consisting of FLI1 exons 4-2-3, referred to as FECR1.Overexpression of FECR1 enhances invasiveness of MDA-MB231 breast cancer cells. Notably, FECR1 utilizes a positive feedback mechanism to activate FLI1 by inducing DNA hypomethylation in CpG islands of the promoter. FECR1 binds to the FLI1 promoter in cis and recruits TET1, a demethylase that is actively involved in DNA demethylation. FECR1 also binds to and downregulates in trans DNMT1, a methyltransferase that is essential for the maintenance of DNA methylation.ConclusionsThese data suggest that FECR1 circular RNA acts as an upstream regulator to control breast cancer tumor growth by coordinating the regulation of DNA methylating and demethylating enzymes. Thus, FLI1 drives tumor metastasis not only through the canonical oncoprotein pathway, but also by using epigenetic mechanisms mediated by its exonic circular RNA.

All-Trans Retinoic Acid and Decitabine Synergistically Induce Anti-Leukemia Effect on U937 Cell Line and Newly Diagnosed Elder AML Patients

To investigate the effect of all transretinoicacid(ATRA) combined with decitabine (5-Aza-2'-deoxycytidine;DAC) on DNA methylation and gene expression of p16INK4a (p16) and retinoic acid receptor β (RARβ), and to explore their combined anti neoplastic effect on U937 cells and newly diagnose delder acute myeloid leukemia(AML) patients. The expression levels of p16 and RARβ were determined by qRT-PCR and Western blot. Methylation-specific PCR was used to analyze their methylation status. WST-1 and flow cytometry were performed to detect growth inhibition, differentiation, apoptosis and cell cycle of U937 cells respectively. The expression p16 and RARβ was down-regulated by promoter hypermethylation in newly diagnose delder AML patients and U937 cells. Combination treatment of ATRA and DAC induced DNA hypomethylation as well as gene expression of p16 and RARβ, which contributed to the growth inhibition, differentiation, apoptosis and cell cycle arrest of U937 cells. In addition for elder AML patients intolerable to standard chemotherapy, the combination regimen of ATRA and DAC showed antineoplastic activity accompamied by up-regulation of p16 and RARβ expression and decrease of bone marrow blast, moreover the parients showed good tolerence to the reginen. The regimen of ATRA combined with DAC as the combination therapeutic strategy for inducing differentiation and demethylation possesses the anti-AML potency, and contributes to optimizing the therapeutic strategy for elder AML patients and promoting the clinical prognosis.

Abstract 2867: Glycomimetic antagonist of E-selectin, GMI-1271, enhances therapeutic activity of the hypomethylating agent 5-azacitidine in the KG1 model of AML

Abstract Binding of AML blasts to E-selectin activates cellular survival pathways leading to chemoresistance. GMI-1271 is a novel E-selectin antagonist that when used in combination with chemotherapy results in improved survival in mouse syngeneic and xenogeneic AML tumor models. GMI-1271 in combination with two chemotherapy regimens is in early clinical trials for the treatment of AML. Azacitidine (5-AC) is a DNA-methyltransferase inhibiting cytosine nucleoside analog that at low doses induces DNA hypomethylation and transcriptional activation, while at higher doses is directly cytotoxic to neoplastic cells including AML blasts. 5-AC is approved in Europe for the treatment of limited populations with AML. We evaluated GMI-1271 in combination with 5-AC in the KG1 AML tumor model to assess the potential for therapeutic benefit of the combination. NSG mice (10/group) received i.v. injections of KG1 cells, and were treated with saline, GMI-1271 alone, 5-AC alone, or the combination of GMI-1271 and 5-AC. The median survival time (MST) of mice treated with 5-AC was 88 days and statistically different (P&amp;lt;0.002) to groups treated with saline (MST=69.5 days) or GMI-1271 alone (MST=69 days). All mice treated with saline or GMI-1271 alone succumbed to progressive tumor growth. At study conclusion (Day 104 post tumor injection) 20% of mice treated with 5-AC remained alive. Importantly, the therapeutic activity of 5-AC was significantly enhanced when combined with GMI-1271 (MST&amp;gt;104 days, P=0.0140 compared to 5-AC alone) with 70% of mice surviving to study conclusion. These results indicate that E-selectin/AML blast interaction in the KG1 model protects from the anti-tumor activity of 5-AC and that GMI-1271 attenuates this protection. To investigate the nature of the observed in vivo activity of GMI-1271 and 5-AC, KG1 cells were cultured for 96 h in a noncytotoxic concentration (100 nM) of 5-AC and the reactivity of the cells to HECA-452 (an antibody that recognizes an E-selectin carbohydrate ligand) and binding to E-selectin were determined by flow cytometry. Treatment with 5-AC resulted in a 28% increase in reactivity of cells to HECA-452 and a 32% increase in binding to E-selectin. Further in vitro assays of static adhesion revealed an increase in the adhesion of 5-AC-treated KG1 cells to E-selectin. Notably, the enhanced adhesion of KG-1 cells to E-selectin was reversed using GMI-1271. Collectively, these results demonstrate that 5-AC can lead to increased expression of E-selectin ligands on AML cells and that the therapeutic potential of 5-AC could be improved by combination with GMI-1271. Citation Format: Theodore A. Smith, William E. Fogler, Ji-Won Lee, John L. Magnani. Glycomimetic antagonist of E-selectin, GMI-1271, enhances therapeutic activity of the hypomethylating agent 5-azacitidine in the KG1 model of AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2867.

Incorporating DNA Methyltransferase Inhibitors (DNMTis) in the Treatment of Genitourinary Malignancies: A Systematic Review.

Inhibition of DNA methyltransferases (DNMTs) has emerged as a novel treatment strategy in solid tumors. Aberrant hypermethylation in promoters of critical tumor suppressor genes is the basis for the idea that treatment with hypomethylating agents may lead to the restoration of a "normal" epigenome and produce clinically meaningful therapeutic outcomes. The aim of this review article is to summarize the current state of knowledge of DNMT inhibitors in the treatment of genitourinary malignancies. The efficacy of these agents in genitourinary malignancies was reported in a number of studies and suggests a role of induced DNA hypomethylation in overcoming resistance to conventional cytotoxic treatments. The clinical significance of these findings should be further investigated.

Homocysteine inhibits endothelial progenitor cells proliferation via DNMT1-mediated hypomethylation of Cyclin A

Endothelial progenitor cells (EPCs) contribute to neovasculogenesis and reendothelialization of damaged blood vessels to maintain the endothelium. Dysfunction of EPCs is implicated in the pathogenesis of vascular injury induced by homocysteine (Hcy). We aimed to investigate the role of Cyclin A in Hcy-induced EPCs dysfunction and explore its molecular mechanism. In this study, by treatment of EPCs with Hcy, we found that the expression of Cyclin A mRNA and protein were significantly downregulated in a dose-dependent manner. Knockdown of Cyclin A prominently reduced proliferation of EPCs, while over-expression of Cyclin A significantly promoted the cell proliferation, suggesting that Hcy inhibits EPCs proliferation through downregulation of Cyclin A expression. In addition, epigenetic study also demonstrated that Hcy induces DNA hypomethylation of the Cyclin A promoter in EPCs through downregulated expression of DNMT1. Moreover, we found that Hcy treatment of EPCs leads to increased SAM, SAH and MeCP2, while the ratio of SAM/SAH and MBD expression decrease. In summary, our results indicate that Hcy inhibits Cyclin A expression through hypomethylation of Cyclin A and thereby suppress EPCs proliferation. These findings demonstrate a novel mechanism of DNA methylation mediated by DNMT1 in prevention of Hcy associated cardiovascular disease.

In vitro assessment of xenogeneic complement and endothelial cell activation using a microvascular flow model

Endothelial progenitor cells (EPCs) contribute to neovasculogenesis and reendothelialization of damaged blood vessels to maintain the endothelium. Dysfunction of EPCs is implicated in the pathogenesis of vascular injury induced by homocysteine (Hcy). We aimed to investigate the role of Cyclin A in Hcy-induced EPCs dysfunction and explore its molecular mechanism. In this study, by treatment of EPCs with Hcy, we found that the expression of Cyclin A mRNA and protein were significantly downregulated in a dose-dependent manner. Knockdown of Cyclin A prominently reduced proliferation of EPCs, while over-expression of Cyclin A significantly promoted the cell proliferation, suggesting that Hcy inhibits EPCs proliferation through downregulation of Cyclin A expression. In addition, epigenetic study also demonstrated that Hcy induces DNA hypomethylation of the Cyclin A promoter in EPCs through downregulated expression of DNMT1. Moreover, we found that Hcy treatment of EPCs leads to increased SAM, SAH and MeCP2, while the ratio of SAM/SAH and MBD expression decrease. In summary, our results indicate that Hcy inhibits Cyclin A expression through hypomethylation of Cyclin A and thereby suppress EPCs proliferation. These findings demonstrate a novel mechanism of DNA methylation mediated by DNMT1 in prevention of Hcy associated cardiovascular disease.

Elevated Adenosine Induces Placental DNA Hypomethylation Independent of A2B Receptor Signaling in Preeclampsia.

Preeclampsia is a prevalent pregnancy hypertensive disease with both maternal and fetal morbidity and mortality. Emerging evidence indicates that global placental DNA hypomethylation is observed in patients with preeclampsia and is linked to altered gene expression and disease development. However, the molecular basis underlying placental epigenetic changes in preeclampsia remains unclear. Using 2 independent experimental models of preeclampsia, adenosine deaminase-deficient mice and a pathogenic autoantibody-induced mouse model of preeclampsia, we demonstrate that elevated placental adenosine not only induces hallmark features of preeclampsia but also causes placental DNA hypomethylation. The use of genetic approaches to express an adenosine deaminase minigene specifically in placentas, or adenosine deaminase enzyme replacement therapy, restored placental adenosine to normal levels, attenuated preeclampsia features, and abolished placental DNA hypomethylation in adenosine deaminase-deficient mice. Genetic deletion of CD73 (an ectonucleotidase that converts AMP to adenosine) prevented the elevation of placental adenosine in the autoantibody-induced preeclampsia mouse model and ameliorated preeclampsia features and placental DNA hypomethylation. Immunohistochemical studies revealed that elevated placental adenosine-mediated DNA hypomethylation predominantly occurs in spongiotrophoblasts and labyrinthine trophoblasts and that this effect is independent of A2B adenosine receptor activation in both preeclampsia models. Extending our mouse findings to humans, we used cultured human trophoblasts to demonstrate that adenosine functions intracellularly and induces DNA hypomethylation without A2B adenosine receptor activation. Altogether, both mouse and human studies reveal novel mechanisms underlying placental DNA hypomethylation and potential therapeutic approaches for preeclampsia.

Prenatal exposure to drinking water disinfection by-products and DNA methylation in cord blood.

Maternal exposure to drinking water disinfection by-products (DBPs) during pregnancy has been related to adverse birth outcomes. While experimental studies have shown that exposure to DBPs induce DNA hypomethylation, evidence from humans is limited. This study aimed to examine whether prenatal exposure to drinking water DBPs was associated with DNA methylation in cord blood. Maternal biomarkers of exposure to drinking water DBPs including blood trihalomethanes [THMs, including chloroform (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and bromoform (TBM)] and urinary trichloroacetic acid (TCAA) were measured during late pregnancy. DNA methylation in Alu and long interspersed nucleotide element-1 (LINE-1) repetitive elements from cord blood samples (n=115) was measured by pyrosequencing. We used multivariable linear regression to estimate the associations of DNA methylation in cord blood with maternal blood THMs and urinary TCAA. We found no statistically significant association between urinary TCAA and DNA methylation. However, we found that blood TBM was associated with decreased Alu methylation (-0.39%; 95% CI: -0.83%, 0.05% for the highest versus lowest exposure group; p for trend=0.08) and decreased LINE-1 methylation (-1.27%; 95% CI: -2.91%, 0.36% for the highest versus lowest exposure group; p for trend=0.06). Our results suggest that prenatal exposure to drinking water TBM is associated with DNA hypomethylation in cord blood. However, further studies are needed to confirm our findings.