Dnmt3a Protein Levels Research Articles

Chronic corticosterone exposure in rats induces sex-specific alterations in hypothalamic reelin fragments, MeCP2, and DNMT3a protein levels

Women are disproportionately affected by stress-related disorders like depression. In our prior research, we discovered that females exhibit lower basal hypothalamic reelin levels, and these levels are differentially influenced by chronic stress induced through repeated corticosterone (CORT) injections. Although epigenetic mechanisms involving DNA methylation and the formation of repressor complexes by DNA methyl-transferases (DNMTs) and Methyl-CpG binding protein 2 (MeCP2) have been recognized as regulators of reelin expression in vitro, there is limited understanding of the impact of stress on the epigenetic regulation of reelin in vivo and whether sex differences exist in these mechanisms. To address these questions, we conducted various biochemical analyses on hypothalamic brain samples obtained from male and female rats previously treated with either 21 days of CORT (40 mg/kg) or vehicle (0.9 % saline) subcutaneous injections. Upon chronic CORT treatment, a reduction in reelin fragment NR2 was noted in males, while the full-length molecule remained unaffected. This decrease paralleled with an elevation in MeCP2 and a reduction in DNMT3a protein levels only in males. Importantly, sex differences in baseline and CORT-induced reelin protein levels were not associated with changes in the methylation status of the Reln promoter. These findings suggest that CORT-induced reelin decreases in the hypothalamus may be a combination of alterations in downstream processes beyond gene transcription. This research brings novel insights into the sexually distinct consequences of chronic stress, an essential aspect to understand, particularly concerning its role in the development of depression.

Study of the effect of sFRP1 protein on molecules involved in the regulation of DNA methylation in CML cell line.

Wnt-signaling pathway plays a crucial role in the pathogenesis and progression of Chronic Myeloid Leukemia (CML). sFRP1 is involved in the suppression ofthe Wnt-signaling pathway and has been shown to be epigenetically silenced by promoter hypermethylation during CML progression. DNMT3A plays a crucial role in promoter hypermethylation andis responsible for establishing methylation patterns. We aimed to analyze the relationship between sFRP1 expression and DNMT3A, TET1, TET2 and TET3 proteins that are responsible for maintaining cellular methylation patterns; along with miRNAs miR144-3p and miR-767-5p that are known to be associated with these proteins. CML cell lines K562 and K562S which stably expresses sFRP1, were used to compare the changes in miR144-3p and miR-767-5p expression. DNMT3A, TET1, TET2 and TET3 protein levels were analyzed by Western blot. In K562S cells the expression of miR-144-3p and miR-767-5p were decreased along with DNMT3A and TET1 protein levels. On the contrary, TET2 protein was increased. Our results support other reports involving sFRP1 and methylation dynamics; as well as opening new avenues of exploration. Our data supports the conclusion that re-expression of sFRP1 protein alters the expression of factors that play important roles in the overall methylation patterns in the leukemic cell line K562.

The relationship between miR-21, DNA methylation, and bisphenol a in bovine COCs and granulosa cells.

Introduction: miR-21 is a critical microRNA for the regulation of various processes in oocytes and granulosa cells. It is involved in the modulation of apoptosis and can influence other epigenetic mechanisms. Among these mechanisms, DNA methylation holds significant importance, particularly during female gametogenesis. Evidence has demonstrated that microRNAs, including miR-21, can regulate DNA methylation. Bisphenol A (BPA) is a widespread chemical that disrupts oocyte maturation and granulosa cell function. Recent findings suggested that BPA can act through epigenetic pathways, including DNA methylation and microRNAs. Methods: This study uses anti-miR-21 LNAs to explore the involvement of miR-21 in the regulation of DNA methylation in bovine Cumulus-Oocyte-Complexes (COCs) and granulosa cells, in the presence and absence of BPA. This study investigated 5mC/5hmC levels as well as gene expression of various methylation enzymes using qPCR and western blotting. Results and discussion: Results reveal that BPA reduces 5mC levels in granulosa cells but not in COCs, which can be attributed to a decrease in the methylating enzymes DNMT1 and DNMT3A, and an increase in the demethylating enzyme TET2. We observed a significant increase in the protein levels of DNMT1, DNMT3A, and TET2 upon inhibition of miR-21 in both COCs and granulosa cells. These findings directly imply a strong correlation between miR-21 signaling and the regulation of DNA methylation in bovine COCs and granulosa cells under BPA exposure.

The Notch1/Hes1 signaling pathway affects autophagy by adjusting DNA methyltransferases expression in a valproic acid-induced autism spectrum disorder model

As a pervasive neurodevelopmental disease, autism spectrum disorder (ASD) is caused by both hereditary and environmental elements. Research has demonstrated the functions of the Notch pathway and DNA methylation in the etiology of ASD. DNA methyltransferases DNMT3 and DNMT1 are responsible for methylation establishment and maintenance, respectively. In this study, we aimed to explore the association of DNA methyltransferases with the Notch pathway in ASD. Our results showed Notch1 and Hes1 were upregulated, while DNMT3A and DNMT3B were downregulated at the protein level in the prefrontal cortex (PFC), hippocampus (HC) and cerebellum (CB) of VPA-induced ASD rats compared with Control (Con) group. However, the protein levels of DNMT3A and DNMT3B were augmented after treatment with 3,5-difluorophenacetyl-L-alanyl-S-phenylglycine-2-butyl ester (DAPT), suggesting that abnormal Notch pathway activation may affect the expression of DNMT3A and DNMT3B. Besides, our previous findings revealed that the Notch pathway may participate in development of ASD by influencing autophagy. Therefore, we hypothesized the Notch pathway adjusts autophagy and contributes to ASD by affecting DNA methyltransferases. Our current results showed that after receiving the DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine (5-Aza-2'dc), the VPA + DAPT+5-Aza-2'dc (V + D + Aza) group exhibited reduced social interaction ability and increased stereotyped behaviors, and decreased expression of DNMT3A, DNMT3B and autophagy-related proteins, but did not show changes in Notch1 and Hes1 protein levels. Our results indicated that the Notch1/Hes1 pathway may adjust DNMT3A and DNMT3B expression and subsequently affect autophagy in the occurrence of ASD, providing new insight into the pathogenesis of ASD.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Peripheral T-cell lymphomas (PTCL) are a rare and heterogeneous group of malignancies that share a unifying feature of epigenetic dysregulation. Recent evidence suggests that PTCL, unlike other forms of cancer or even lymphoma, exhibits a marked vulnerability to hypomethylating agents (HMAs) alone and especially in combination with HDAC inhibitors. HMAs like azacitidine (AZA) and decitabine (DAC) have been shown to reverse transcriptional repression secondary to hypermethylation. Cladribine (CLAD) is FDA-approved for the treatment of hairy cell leukemia and is postulated to inhibit both DNA and histone methylation. Here, we sought to define the mechanistic differences between different DNA HMAs in order to inform a rationale on the optimal combinations that might exploit the epigenetic vulnerabilities of PTCL. Methods: Cell viability, caspase activity assay, and western blotting (WB) were performed on a panel of T-cell lymphoma cell lines to identify the effects of AZA/DAC/CLAD on cell survival, apoptosis, and DNA methyltransferase (DNMT) levels. Liquid chromatography-based mass spectrometry (LC-MS) was done to quantify 5-methyl cytosine (5-mC) levels. Results: Cell viability analysis confirmed that the dose and time-dependent sensitivity of the six PTCL cell lines differed following exposure to AZA/DAC/CLAD, where CLAD showed the lowest IC50 value (0.1–1 µM) across all cell lines, followed by AZA (IC50: 1–10 µM), and DAC had the least effect on cell viability (IC50: >100 µM, except T-ALL). A differential threshold of caspase activation was observed across the PTCL cell lines where CLAD showed the most superior caspase activity leading to the highest apoptotic potential. However, WB analysis showed that after 24 hours of treatment, DNMT1 and DNMT3A protein levels were significantly depleted at low concentrations of DAC (<0.01 µM) compared to AZA whereas CLAD did not affect the DNMT1 and DNMT3A levels. LC-MS-based quantification of 5-mC showed a similar relationship in methylation, with initial changes observed at doses >0.01 μM of DAC and >0.1 μM of AZA/CLAD. Taking the short half-life of AZA/DAC into account, cell viability assays with daily addition of AZA/DAC showed increased cell cytotoxicity for AZA/DAC treated samples, which was comparable to CLAD. Ongoing analyses of gene expression and metabolism of the universal methyl donor S-adenosylmethionine (SAM) will help to understand the mechanistic difference between these HMAs. Conclusions: These data suggest that the HMAs have a distinct mechanism of action for hypomethylation. While AZA/DAC induces hypomethylation by depletion of DNMTs, CLAD acts in a DNMT depletion-independent pathway. A mechanistic understanding of AZA/DAC/CLAD will inform how best to use them in clinic as well as assist in the strategic development of biological correlates to further support their therapeutic application. The research was funded by: Translational Orphan Blood Cancer Research Initiative Fund and RO1 # FD-R-006814-01. Keywords: Genomics, Epigenomics, and Other -Omics, Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.

Epigallocatechin Gallate (EGCG), an Active Phenolic Compound of Green Tea, Inhibits Tumor Growth of Head and Neck Cancer Cells by Targeting DNA Hypermethylation

Head and neck cancers are among the deadliest cancers, ranked sixth globally in rates of high mortality and poor patient prognoses. The prevalence of head and neck squamous cell carcinoma (HNSCC) is associated with smoking and excessive alcohol consumption. Despite several advances in diagnostic and interventional methods, the morbidity of subjects with HNSCC has remained unchanged over the last 30 years. Epigenetic alterations, such as DNA hypermethylation, are commonly associated with several cancers, including HNSCC. Thus, epigenetic changes are considered promising therapeutic targets for chemoprevention. Here, we investigated the effect of EGCG on DNA hypermethylation and the growth of HNSCC. First, we assessed the expression levels of global DNA methylation in HNSCC cells (FaDu and SCC-1) and observed enhanced methylation levels compared with normal human bronchial epithelial cells (NHBE). Treatment of EGCG to HNSCC cells significantly inhibited global DNA hypermethylation by up to 70-80% after 6 days. Inhibition of DNA hypermethylation in HNSCC cells was confirmed by the conversion of 5-methylcytosine (5-mc) into 5-hydroxy methylcytosine (5hmC). DNA methyltransferases regulate DNA methylation. Next, we checked the effect of EGCG on the expression levels of DNA methyltransferases (DNMTs) and DNMT activity. Treatment of EGCG to HNSCC cells significantly reduced DNMT activity to 60% in SCC-1 and 80% in FaDu cells. The protein levels of DNMT3a and DNMT3b were downregulated in both cell lines after EGCG treatment. EGCG treatment to HNSCC cells reactivated tumor suppressors and caused decreased cell proliferation. Our in vivo study demonstrated that administration of EGCG (0.5%, w/w) as a supplement within an AIN76A diet resulted in inhibition of tumor growth in FaDu xenografts in nude mice (80%; p < 0.01) compared with non-EGCG-treated controls. The growth inhibitory effect of dietary EGCG on the HNSCC xenograft tumors was associated with the inhibition of DNMTs and reactivation of silenced tumor suppressors. Together, our study provides evidence that EGCG acts as a DNA demethylating agent and can reactivate epigenetically silenced tumor suppressors to inhibit the growth of HNSCC cells.

Ovaryum kriyoprezervasyonu ve yeniden transplantasyondan sonra Dnmt3a ve Dnmt3b protein ifadelerinin düşmesi bu süreçte DNA metilasyonunun bozulduğunu gösterir

Objective DNA methylation includes maintenance and de novo methylation process that are catalyzed by DNA methyltransferase (Dnmt) enzymes and these enzymes possess crucial roles in methylation of genes associated with the development of ovarian follicles. The aim of this study is to evaluate whether expression of Dnmt3a and Dnmt3b protein levels change after cryopreservation and re-transplantation of ovarian tissue in rats. Methods Four groups were designed as; fresh control (FC), frozen/thawed (FT), fresh re-transplanted (T), and frozen/thawed and re-transplanted (FTT). Slow freezing was used to cryopreserve the ovarian tissue pieces and expression of Dnmt3a and Dnmt3b proteins was assessed by immunohistochemistry. Result Expression of Dnmt3a and Dnmt3b significantly decreased in ovarian follicles in FT, T and FTT groups when compared FC. Conclusion Expression of Dnmt enzymes has been altered both after cryopreservation alone and following re-transplantation cycles which may lead to disturbed DNA methylation processes during rat folliculogenesis

Hypermethylated PODN represses the progression of osteosarcoma by inactivating the TGF-β/Smad2/3 pathway

BackgroundPODN is reported to be an promising biomarker for prognosis of osteosarcoma (OS), while the specific function of PODN has not been explored in OS. This study is designed to explore the function and underlying mechanism of PODN in OS. MethodsThe mRNA expression of PODN was determined using qRT-PCR. Protein levels of PODN, DNMT1, DNMT3A, DNMT3B, TGF-β1, Smad2/3 and p-Smad2/3 were detected using western blot. The methylation of PODN was determined with methylation-specific PCR. Moreover, CCK-8 assay and colony formation assay were used for assessing the proliferation of OS cells. Transwell assay was used to evaluate migration and invasion abilities of OS cells. Immunohistochemical staining was performed to determine the protein expression of Ki67 and PODN in tumor tissues. For constructing a xenograft tumor model, MG-63 cells were introduced into the right side of the mouse back via subcutaneous injection. ResultsPODN was lowly expressed and was hypermethylated in OS tissues and cells. PODN overexpression prevented OS cells from proliferating, migrating and invading, and inhibited tumorigenesis in xenograft mice. After PODN overexpression, protein levels of TGF-β1 and p-Smad2/3 were decreased in OS cells. Meantime, the suppressive effects of PODN overexpression on proliferation, migration and invasion of OS cells as well as mouse tumorigenesis were partly counteracted by TGF-β1 overexpression. ConclusionsPODN overexpression inactivated the TGF-β/Smad2/3 pathway to suppress OS development in vitro and in vivo.

MiR-26a-5p Suppresses Wnt/β-Catenin Signaling Pathway by Inhibiting DNMT3A-Mediated SFRP1 Methylation and Inhibits Cancer Stem Cell-Like Properties of NSCLC

Background Lung cancer is a malignant cancer which results in the most cancer incidence and mortality worldwide. There is increasing evidence that the pattern of DNA methylation affects tumorigenesis and progression. However, the molecules and mechanisms regulating DNA methylation remain unclear. Methods The expression of miR-26a-5p in NSCLC cell lines was detected by qPCR and verified in NSCLC tissues from TCGA using Limma R package. CCK-8 assay, plate clone formation assay, flow cytometry, and sphere formation assay were used to detect the cell proliferation, colony formation, cell cycle, and cancer stem cell- (CSC-) like property in NSCLC cell lines. The immunoblotting was used to detect the protein levels of DNMT3A, SFRP1, and Ki67. Global DNA methylation levels and DNA methylation levels of SFRP1 promoter were examined using ELISA and MSP-PCR assay, respectively. The distribution of β-catenin was examined using immunofluorescence (IF). Besides, xenograft mouse model was used to investigate the antitumor effects of miR-26a-5p in vivo. The pathology and protein levels were, respectively, detected by hematoxylin and eosin (H&E) and immunocytochemistry (IHC). Results The expression of miR-26a-5p was downregulated in the tumor tissues comparted to adjacent normal tissues as well as NSCLC cell lines compared to normal lung epithelial cell (BEAS2B). The overexpression of miR-26a-5p inhibited cell proliferation, colony formation, CSC-like property, and arrested cell cycle at G1 phase. DNMT3A was a target of miR-26a-5p and upregulated DNA methylation on SFRP1 promoter. Mechanistically, miR-26a-5p repressed cell proliferation, colony formation, CSC-like property, and arrested cell cycle at G1 phase by binding DNMT3A to reduce DNA methylation levels of SFRP1 then upregulated SFRP1 expression. Moreover, miR-26a-5p exerted antitumor effects in vivo. Conclusion Our results revealed that miR-26a-5p acted as a tumor suppressor through targeting DNMT3A to upregulate SFRP1 via reducing DNMT3A-dependent DNA methylation.

Dexmedetomidine reduces propofol-induced hippocampal neuron injury by modulating the miR-377-5p/Arc pathway

BackgroundPropofol and dexmedetomidine (DEX) are widely used in general anesthesia, and exert toxic and protective effects on hippocampal neurons, respectively. The study sought to investigate the molecular mechanisms of DEX-mediated neuroprotection against propofol-induced hippocampal neuron injury in mouse brains.MethodsHippocampal neurons of mice and HT22 cells were treated with propofol, DEX, and propofol+DEX. In addition, transfection of miR-377-5p mimics or inhibitors was performed in HT22 cells. Neuronal apoptosis was evaluated by a means of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) or Hochest 33,258 staining; Arc positive expression in hippocampus tissues was detected using a microscope in immunohistochemistry assays; miRNA-377-5p expression was quantified by RT-qPCR; the protein levels of Arc, DNMT3A, and DNMT3B were determined using western blot; Cell Counting Kit-8 (CCK-8) assay was used to detect the viability and apoptotic rate of the neurons; methylation analysis in the miR-377-5p promoter was performed through methylated DNA immunoprecipitation (MeDIP) assay; dual luciferase reporter assay was performed to confirm whether Arc was under targeted regulation of miR-377-5p.ResultsIn the current study, both in vitro and in vivo, propofol treatment induced hippocampal neuron apoptosis and suppressed cell viability. DNMT3A and DNMT3B expression levels were decreased following propofol treatment, resulting in lowered methylation in the miR-377-5p promoter region and then enhanced expression of miR-377-5p, leading to a decrease in the expression of downstream Arc. Conversely, the expression levels of DNMT3A and DNMT3B were increased following DEX treatment, thus methylation in miR-377-5p promoter region was improved, and miR-377-5p expression was decreased, leading to an increase in the expression of downstream Arc. Eventually, DEX pretreatment protected hippocampal neurons against propofol-induced neurotoxicity by recovering the expression levels of DNMT3A, miR-377-5p, and Arc to the normal levels. Additionally, DNMT3A knockdown improved miR-377-5p expression but reduced Arc expression, and DNMT3A overexpression exerted the opposite effects. Dual luciferase reporter assay revealed a binding target between miR-377-5p and Arc 3’UTR. The neuroprotective effect of DEX against propofol-induced neuronal apoptosis was diminished after Arc knockdown. Silencing Arc independently triggered the apoptosis of HT22 cells, which was alleviated through transfection of miR-377-5p inhibitors.ConclusionsDEX reduced propofol-induced hippocampal neuron injury via the miR-377-5p/Arc signaling pathway.

Mitochondrial DNA methyltransferases and their regulation under freezing and dehydration stresses in the freeze-tolerant wood frog, Rana sylvatica.

Wood frogs are a few vertebrate species that can survive whole-body freezing. Multiple adaptations support this, including cryoprotectant production (glucose), metabolic rate depression, and selective changes in gene and protein expression to activate pro-survival pathways. The role of DNA methylation machinery (DNA methyltransferases, DNMTs) in regulating nuclear gene expression to support freezing survival has already been established. However, a comparable role for DNMTs in the mitochondria has not been explored in wood frogs. We examined the mitochondrial protein levels of DNMT-1, DNMT-3A, DNMT-3B, and DNMT-3L as well as mitochondrial DNMT activity in the liver and heart to assess the involvement of DNMT in the survival of freezing and dehydration stresses (cellular dehydration being a component of freezing). Our results showed stress- and tissue-specific responses to mitochondrial DNMT-1 in the liver and heart, respectively. During 24 h of freezing and whole-body dehydration, we observed an overall downregulation of mitochondrial DNMT-1, a major protein involved in maintaining methylation levels related to its role in the selective transcription of mitochondrial genes as well as antioxidant response. Tissue-specific responses of protein levels of DNMT-3A, DNMT-3B, DNMT-3L, and DNMT activity in the liver suggested a preference for a higher methylation state in the liver under both freezing and dehydration stress, but not in the heart.

Downregulation of DNMT3A Attenuates the Warburg Effect, Proliferation, and Invasion via Promoting the Inhibition of miR-603 on HK2 in Ovarian Cancer.

Background: Ovarian cancer is a highly malignant gynecological cancer. Aerobic glycolysis is one of the features of cancer cell metabolism. Studying the molecular modulation of the Warburg effect in ovarian cancer is significantly valuable for understanding the progression mechanism of ovarian cancer. Materials and Methods: The expression level and prognostic significance of DNMT3A were analyzed using public databases. DNMT3A was overexpressed by plasmid transfection, and DNMT3A was interfered with specific siRNAs transfection. miR-603 was overexpressed by mimic transfection or inhibited by inhibitor transfection. The expression of the molecules was detected by qPCR or western blotting. CCK-8 and transwell assays were used to determine the cell proliferation, migration, and invasion abilities of ovarian cancer. Results: We found that the DNMT3A protein level was higher in ovarian cancer tissues than in normal ovary tissues, but the mRNA level had no significant difference in ovarian cancer tissues and normal ovary tissues. The higher the RNA level of DNMT3A, the poorer prognosis of patients. DNMT3A knocking down impeded the Warburg effect, cell proliferation, migration, and invasion of ovarian cancer cells. Further investigations discovered that DNMT3A promoted ovarian cancer cell malignancy via silencing miR-603. Conclusion: We found that patients who overexpressed DNMT3A showed a poor prognosis. DNMT3A was found to promote the Warburg effect, cell proliferation, migration, and invasion of ovarian cancer by inhibiting the expression of miR-603. As a result, the research revealed that DNMT3A/miR-603/HK2 axis contributed to the Warburg effect of ovarian cancer and DNMT3A may be a potential therapeutic target for ovarian cancer.

Ginsenoside 20(S)-Rg3 upregulates tumor suppressor VHL gene expression by suppressing DNMT3A-mediated promoter methylation in ovarian cancer cells

To explore the mechanism by which ginsenoside 20(S)-Rg3 upregulates the expression of tumor suppressor von Hippel-Lindau (VHL) gene in ovarian cancer cells. Ovarian cancer cell line SKOV3 treated with 20(S)-Rg3 were examined for mRNA and protein levels of VHL, DNMT1, DNMT3A and DNMT3B by real-time PCR and Western blotting, respectively. The changes in VHL mRNA expression in SKOV3 cells in response to treatment with 5-Aza-CdR, a DNA methyltransferase inhibitor, were detected using real-time PCR. VHL gene promoter methylation was examined with methylation-specific PCR and VHL expression levels were determined with real-time PCR and Western blotting in non-treated or 20(S)-Rg3-treated SKOV3 cells and in 20(S)-Rg3-treated DNMT3A-overexpressing SKOV3 cells. VHL and DNMT3A protein levels were detected by immunohistochemistry in subcutaneous SKOV3 cell xenografts in nude mice. Treatment of SKOV3 cells with 20(S)-Rg3 significantly upregulated VHL and downregulated DNMT3A expressions at both the mRNA and protein levels (P < 0.05) and upregulated DNMT3B expression only at the mRNA level, but did not cause significant changes in either the mRNA or protein level of DNMT1. Treatment of the cells with 2 and 5 μmol/L 5-Aza-CdR obviously increased VHL mRNA expression by by over 3 folds (P < 0.05). 20(S)-Rg3 significantly decreased the methylation level in the promoter region of VHL gene, and this effect was abrogated by DNMT3A overexpression in the cells (P < 0.05). Immunohistochemisty showed a significantly increased VHL expression but a lowered DNMT3A expression in subcutaneous SKOV3 cell xenografts in 20 (S)-Rg3-treated nude mice. Ginsenoside 20(S)-Rg3 upregulates VHL expression in ovarian cancer cells by suppressing DNMT3A-mediated DNA methylation.

Proteomic analysis of young and old mouse hematopoietic stem cells and their progenitors reveals post-transcriptional regulation in stem cells.

The balance of hematopoietic stem cell (HSC) self-renewal and differentiation is critical for a healthy blood supply; imbalances underlie hematological diseases. The importance of HSCs and their progenitors have led to their extensive characterization at genomic and transcriptomic levels. However, the proteomics of hematopoiesis remains incompletely understood. Here we report a proteomics resource from mass spectrometry of mouse young adult and old adult mouse HSCs, multipotent progenitors and oligopotent progenitors; 12 cell types in total. We validated differential protein levels, including confirmation that Dnmt3a protein levels are undetected in young adult mouse HSCs until forced into cycle. Additionally, through integrating proteomics and RNA-sequencing datasets, we identified a subset of genes with apparent post-transcriptional repression in young adult mouse HSCs. In summary, we report proteomic coverage of young and old mouse HSCs and progenitors, with broader implications for understanding mechanisms for stem cell maintenance, niche interactions and fate determination.

182 Bisphenol A, but not bisphenol S, affects key microRNAs during bovine oocyte maturation

Oocyte maturation involves crucial hormone-dependent events that are uniquely susceptible to toxic insults by endocrine disrupting chemicals (EDCs). Emerging evidence suggests that small RNAs, including microRNAs (miRNAs), may be key participants in the response to EDCs. Bisphenol A (BPA) and bisphenol S (BPS) are chemicals with detrimental health effects, with BPA negatively affecting oocyte quality. The mode of action of bisphenols at the epigenetic level is not clear. Several miRNAs have been identified as crucial regulators of gene expression during development. This study aimed to examine key miRNAs in response to BPA or BPS treatment during oocyte maturation. Primary forms (pri-miRNA) and mature forms of miR-21, miR-155, miR-34c, and miR-146a were quantified by quantitative (q)PCR in IVM bovine cumulus-oocyte complexes (COCs) and invitro cultured (IVC) cumulus cells treated with BPA and BPS at physiologically significant doses (0.05mgmL−1). Total RNA, containing both forms of microRNAs, was isolated from pools of 40 COCs on a minimum of three biological replicates. Primary miRNAs and mature miRNAs were reverse transcribed (RT) using qScript cDNA and microRNA/cDNA kits, respectively, and quantified by qPCR, with three technical replicates for each biological one. In addition, mRNA and protein quantification of the downstream target DNMT3A in IVC cumulus cells further enhanced our understanding of EDC interference in epigenetic regulations in female reproduction. Total RNA was isolated from cumulus cells, mRNA (1μg) and mature miRNAs (0.5μg) were RT separately and cDNA was quantified by qPCR, as described above. Expression values were normalized against two housekeeping genes selected by GeNorm analysis. Twenty micrograms of proteins extracted by sonication were loaded on a 8% acrylamide gel and analysed by western blotting. Densitometry analysis was performed on 3 separate blots with protein levels normalized to the loading control, β-actin. One-way ANOVA was used to determine statistical differences among treatment groups with P&amp;lt;0.05 considered statistically significant. Results showed that BPA significantly increased miR-21 in COCs (P=0.02) and cumulus cells (P=0.01), increased pri-miR-21 in oocytes (P=0.03), suppressed miR-34c in cumulus cells (P=0.02), increased miR-155 in denuded oocytes (P=0.04), and had no effect on miR-146a. No changes were observed in response to BPS. Experiments in IVC cumulus cells showed similar miRNA profiles: miR-21 and miR-155 were significantly overexpressed in BPA-treated cells (P=0.04); however, miR-34c and miR-146a were not affected. Messenger RNA levels of DNMT3A increased (P=0.02) and protein levels of DNMT3A decreased in response to BPA (P=0.005). Overall, this study presents novel findings of BPA-induced miRNA dysregulation in IVM bovine oocytes and in IVC bovine cumulus cells. We can speculate that miR-21 participates in the BPA-induced dysregulation of DNMT3A, contributing to decreased fertility. This study did not show any effect of BPS on microRNA expression, suggesting an alternative mechanistic pathway for this analogue.

Reversal of increased mammary tumorigenesis by valproic acid and hydralazine in offspring of dams fed high fat diet during pregnancy

Maternal or paternal high fat (HF) diet can modify the epigenome in germ cells and fetal somatic cells leading to an increased susceptibility among female offspring of multiple generations to develop breast cancer. We determined if combined treatment with broad spectrum DNA methyltransferase (DNMT) inhibitor hydralazine and histone deacetylase (HDAC) inhibitor valproic acid (VPA) will reverse this increased risk. C57BL/6 mouse dams were fed either a corn oil-based HF or control diet during pregnancy. Starting at age 7 weeks, female offspring were administered 3 doses of 7,12-dimethylbenz[a]anthracene (DMBA) to initiate mammary cancer. After last dose, offspring started receiving VPA/hydralazine administered via drinking water: no adverse health effects were detected. VPA/hydralazine reduced mammary tumor multiplicity and lengthened tumor latency in HF offspring when compared with non-treated HF offspring. The drug combination inhibited DNMT3a protein levels and increased expression of the tumor suppressor gene Cdkn2a/p16 in mammary tumors of HF offspring. In control mice not exposed to HF diet in utero, VPA/hydralazine increased mammary tumor incidence and burden, and elevated expression of the unfolded protein response and autophagy genes, including HIF-1α, NFkB, PERK, and SQSTM1/p62. Expression of these genes was already upregulated in HF offspring prior to VPA/hydralazine treatment. These findings suggest that breast cancer prevention strategies with HDAC/DNMT inhibitors need to be individually tailored.

Deciphering the Role of Locally Disordered DNA Methylation on CLL Development In Vivo

Large-scale DNA methylation analysis of chronic lymphocytic leukemia (CLL) has identified a pervasive genome-wide level of discordance in local methylation state in leukemic cells compared to normal B cells. This is associated with variation in gene expression, increased clonal evolution and poorer clinical outcomes. We hypothesized that locally disordered methylation could lead to dysregulation of gene expression and hence contribute to cancer development and progression. To test this, we have engineered mouse lines with B-cell restricted homozygous or heterozygous knock-out of Dnmt3a by crossing Dnmt3a-floxed mice with CD19-Cre mice. Dnmt3a is a DNA methyltransferase, catalyzing the addition of a methyl group to CpG sequences in the DNA and thereby regulating gene expression. Although DNMT3A mutations are only rarely identified in CLL, RNA sequencing and protein expression analysis reveal dysregulation of DNMT3A. We confirmed partial or complete reduction in Dnmt3a protein levels in B cells from CD19-Cre;Dnmt3a heterozygous (Dnmt3a-het) and CD19-Cre;Dnmt3a homozygous mice (Dnmt3a-hom), respectively. These mice therefore provide a unique opportunity to study B cell restricted changes in locally discordant methylation over time. We first assessed the impact of Dnmt3a deletion on normal B cell development, prior to CLL development, by characterizing splenic B cell of CD19-Cre (control) or Dnmt3a-hom mice. Flow cytometry data using B220, CD21 and CD23 markers to identify B220+CD23+CD21- follicular B cells and B220+CD23+CD21high marginal zone B cells revealed elevated levels of follicular B cells (83.1% vs 87.6%, p=0.008) and reduced levels of marginal zone B cells (9.6% vs 4.1%, p=0.001) in Dnmt3a-hom mice in comparison to control mice (n=3 mice per group). These results indicate that mice with Dnmt3a deletion present with massive changes in their B cells, even prior to overt CLL development. We next monitored both Dnmt3a-het and Dnmt3a-hom cohorts over time for CLL development. We observed that 100% Dnmt3a-hom mice developed CLL-like disease by 7 months (n=23), characterized by CD5+B220+;Igk+ expression and evident within the blood, bone marrow (BM), spleen and peritoneum, suggesting a fundamental role of altered DNMT3A expression in generation of CLL. In comparison, 75% of Dnmt3a-het mice developed CLL-like disease by 18 months (n=12), with similar expansion of CD5+C220+ expansion in the BM and spleen. By RNA-sequencing analysis of normal splenic B cells from CD19-Cre and Dnmt3a-hom mice (n=3 mice, 10 weeks old), we detected substantial changes in gene expression, including 113 upregulated genes and 39 downregulated (p&lt;0.05, FC&gt;2). To explore the development of locally disordered methylation following transformation, CLL cells from Dnmt3a-hom mice (n=3) were subjected to reduced representation bisulfite sequencing (RRBS), a high-throughput technique to analyze genome wide methylation patterns. We found that murine CLL-like cells display locally disordered methylation, which was detected in all genomic features covered by this assay, indicating that disordered methylation is broadly affecting the murine CLL cells' epigenome. Additionally, we identified a set of differentially methylated regions (DMRs) between B cells from CD19-Cre vs CLL cells from Dnmt3a-hom (n = 2,839 DMRs), with a minimum difference of 0.2 and a minimum of 10 CpGs per DMR. Interestingly, gene ontology analysis demonstrated strong association with genes hypermethylated in TCL1 mouse model, linking this model with alternative murine models for CLL. In conclusion, we have studied B cell specific deletion of Dntm3a and showed the development of CLL in 100% of the case in Dnmt3a-hom mice. Our data suggest a fundamental role for Dnmt3a in CLL development through increased locally disordered methylation and changes in associated transcriptional signatures. This mouse model provides an exciting experimental model to undertake functional in vivo studies in order to elucidate the contribution of epigenetic changes on CLL development. Disclosures Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.

Abstract 1603: Valproic acid and hydralazine reverse increased mammary cancer risk and upregulate Cdkn2a/p16 in mice exposed to a high fat diet in utero

Abstract Maternal or paternal high fat (HF) diet that modifies the epigenome in germ cells and fetal somatic cells can program increased susceptibility to breast cancer among offspring. Resulting epigenetic changes include transgenerational upregulation of DNA methyltransferases (DNMTs). We studied here if this increased breast cancer risk can be reversed by treating HF offspring with a well-tolerated drug that inhibits DNA methyl transferase (DNMT; hydralazine), combined with a histone deacetylase (HDAC) inhibitor valproic acid (VPA). Adult C57BL/6NTac mouse offspring of dams fed either corn oil -based HF or control diet during pregnancy were treated with 5 g/kg/day VPA and 5 mg/kg/day hydralazine in drinking water, starting after mice received 7,12-dimethylbenz[a]anthracene (DMBA) to initiate mammary cancer. Treatment with VPA/hydralazine prevented the increase in mammary cancer risk associated with in utero HF diet exposure: it reduced mammary tumor multiplicity and lengthened tumor latency, compared with non-treated HF offspring. These drugs downregulated DNMT3a protein levels, and reversed down-regulation of Cdkn2a/p16 mRNA in the mammary tumors of HF offspring. p16 is a tumor suppressor that is often epigenetically silenced in human breast cancers. However, in control offspring not exposed to HF diet in utero, VPA/hydralazine increased mammary tumor incidence and burden. Further, this treatment upregulated genes involved in the unfolded protein response pathway in in utero control diet exposed animals that were already upregulated in non-VPA/hydralazine treated mammary tumors from in utero HF diet exposed animals, including HIF-1α, NFkB, PERK and SQSTM1 p62. Thus, VPA and hydralazine had opposing effects on breast cancer risk in HF and control offspring, suggesting the importance of individually tailoring breast cancer prevention strategies. Citation Format: Leena A. Hilakivi-Clarke, Fabia de Oliveira Andrade, Nguyen Nguyen, Kerrie Bouker. Valproic acid and hydralazine reverse increased mammary cancer risk and upregulate Cdkn2a/p16 in mice exposed to a high fat diet in utero [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1603.

Identification of a novel quinoline-based DNA demethylating compound highly potent in cancer cells

BackgroundDNA methyltransferases (DNMTs) are epigenetic enzymes involved in embryonic development, cell differentiation, epithelial to mesenchymal transition, and control of gene expression, whose overexpression or enhanced catalytic activity has been widely reported in cancer initiation and progression. To date, two DNMT inhibitors (DNMTi), 5-azacytidine (5-AZA) and 5-aza-2′-deoxycytidine (DAC), are approved for the treatment of myelodysplastic syndromes and acute myeloid leukemia. Nevertheless, they are chemically instable and quite toxic for healthy cells; thus, the discovery of novel DNMTi is urgent.ResultsHere, we report the identification of a new quinoline-based molecule, MC3353, as a non-nucleoside inhibitor and downregulator of DNMT. This compound was able, in promoter demethylating assays, to induce enhanced green fluorescence protein (EGFP) gene expression in HCT116 cells and transcription in a cytomegalovirus (CMV) promoter-driven luciferase reporter system in KG-1 cells. Moreover, MC3353 displayed a strong antiproliferative activity when tested on HCT116 colon cancer cells after 48 h of treatment at 0.5 μM. At higher doses, this compound provided a cytotoxic effect in double DNMT knockout HCT116 cells. MC3353 was also screened on a different panel of cancer cells (KG-1 and U-937 acute myeloid leukemia, RAJI Burkitt’s lymphoma, PC-3 prostate cancer, and MDA-MB-231 breast cancer), where it arrested cell proliferation and reduced viability after 48 h of treatment with IC50 values ranging from 0.3 to 0.9 μM. Compared to healthy cell models, MC3353 induced apoptosis (e.g., U-937 and KG-1 cells) or necrosis (e.g., RAJI cells) at lower concentrations. Importantly, together with the main DNMT3A enzyme inhibition, MC3353 was also able to downregulate the DNMT3A protein level in selected HCT116 and PC-3 cell lines. Additionally, this compound provided impairment of the epithelial-to-mesenchymal transition (EMT) by inducing E-cadherin while reducing matrix metalloproteinase (MMP2) mRNA and protein levels in PC-3 and HCT116 cells. Last, tested on a panel of primary osteosarcoma cell lines, MC3353 markedly inhibited cell growth with low single-digit micromolar IC50 ranging from 1.1 to 2.4 μM. Interestingly, in Saos-2 osteosarcoma cells, MC3353 induced both expression of genes and mineralized the matrix as evidence of osteosarcoma to osteoblast differentiation.ConclusionsThe present work describes MC3353 as a novel DNMTi displaying a stronger in cell demethylating ability than both 5-AZA and DAC, providing re-activation of the silenced ubiquitin C-terminal hydrolase L1 (UCHL1) gene. MC3353 displayed dose- and time-dependent antiproliferative activity in several cancer cell types, inducing cell death and affecting EMT through E-cadherin and MMP2 modulation. In addition, this compound proved efficacy even in primary osteosarcoma cell models, through the modulation of genes involved in osteoblast differentiation.

Effects of ginsenoside Rg3 on epigenetic modification in ovarian cancer cells.

Epigenetic modifications are closely related to oncogene activation and tumor suppressor gene inactivation. The aim of this study was to determine the effects of ginsenoside Rg3 on epigenetic modification in ovarian cancer cells. Cell proliferation, metastasis, invasion and apoptosis were respectively determined using Cell Counting Kit-8 (CCK-8), wound healing, Transwell and flow cytometric assays. Methylation levels were determined using methylation specific PCR (MSP). Related-factor expression was detected by conducting real-time-qPCR (RT-qPCR) and western blotting. The results revealed that cell proliferation was inhibited by ginsenoside Rg3 (0, 25, 50, 100 and 200 µg/ml) in a time-dependent manner (12, 24 and 48 h). Ginsenoside Rg3 (50, 100 and 200 µg/ml) was selected to treat cells in various experiments. When ovarian cells were treated with ginsenoside Rg3, cell apoptosis was observed to be promoted, while cell metastasis and invasion were inhibited at 48 h. The results of the present study revealed that in the promoter regions of p53, p16 and hMLH1, the methylation levels decreased, while the mRNA and protein levels significantly increased. The activities of DNMTs and mRNA as well as protein levels of DNMT1, DNMT3a and DNMT3b were decreased by Rg3. The data also demonstrated that the mRNA and protein levels of acetyl-H3 K14/K9 and acetyl-H4 K12/K5/K16 were increased by Rg3. Hence, ginsenoside Rg3 inhibited ovarian cancer cell viability, migration and invasion as well as promoted cell apoptosis.