ascorbic-acid and 5-hydroxymethylcytosine

5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) play a critical role in development and normal physiology. Alterations in 5-mC and 5-hmC patterns are common events in hematopoietic neoplasms. In this review, we begin by emphasizing the importance of 5-mC, 5-hmC, and their enzymatic modifiers in hematological malignancies. Then, we discuss the functions of 5-mC and 5-hmC at distinct genic contexts, including promoter regions, gene bodies, intron-exon boundaries, alternative promoters, and intragenic microRNAs. Recent advances in technology have allowed for the study of 5-mC and 5-hmC independently and specifically permitting distinction between the bases that show them to have transcriptional effects that vary by their location relative to gene structure. We extend these observations to their functions at enhancers and transcription factor binding sites. We discuss dietary influences on 5-mC and 5-hmC levels and summarize the literature on the effects of folate and vitamin C on 5-mC and 5-hmC, respectively. Finally, we discuss how these new themes in the functions of 5-mC and 5-hmC will likely influence the broader research field of epigenetics.

Topics: 5-Methylcytosine; Animals; Ascorbic Acid; Cytosine; Diet; DNA Methylation; Folic Acid; Hematologic Neoplasms; Humans; MicroRNAs; Promoter Regions, Genetic; Structure-Activity Relationship; Transcription Factors

DNA 5-hydroxymethylcytosine (5hmC) is produced by dynamic 5mC oxidation process contributing to tissue specification, and loss of 5hmC has been reported in multiple cancers including genitourinary cancers. However, 5hmC is also cell-type specific, and its variability may exist between differentiated tumor cells and cancer stem cells. Thus, cancer-associated changes in 5hmC may be contributed by distinct sets of tumor cells within the tumor tissues.. Here, we applied a sensitive immunoprecipitation-based method (hMeDIP-seq) to analyze 5hmC changes during genitourinary carcinogenesis (including prostate, urothelial and kidney). We confirmed the tissue-specific distribution of 5hmC in genitourinary tissues and identified regional gain and global loss of 5hmC coexisting in genitourinary cancers. The genes with gain of 5hmC during tumorigenesis were functionally enriched in regulating stemness and hypoxia, whereas were associated with poor clinical prognosis irrespective of their differences in tumor type. We identified that gain of 5hmC occurred in soft fibrin gel-induced 3D tumor spheres with a tumor-repopulating phenotype in two prostate cancer cell lines, 22RV1 and PC3, compared with conventional two-dimensional (2D) rigid dishes. Then, we defined a malignant signature derived from the differentially hydroxymethylated regions affected genes of cancer stem-like cells, which could predict a worse clinical outcome and identified phenotypically malignant populations of cells from prostate cancer tumors. Notably, an oxidation-resistant vitamin C derivative, ascorbyl phosphate magnesium, restored 5hmC and killed the cancer stem cell-like cells leading to apoptosis in prostate cancer cell lines.. Collectively, our study dissects the regional gain of 5hmC in maintaining cancer stem-like cells and related to poor prognosis, which provides proof of concept for an epigenetic differentiation therapy with vitamin C by 5hmC reprogramming.

Topics: 5-Methylcytosine; Ascorbic Acid; Carcinogenesis; DNA; DNA Methylation; Fibrin; Humans; Magnesium; Male; Phosphates; Prostatic Neoplasms; Urogenital Neoplasms

Pancreatic ductal adenocarcinoma (PDAC) is characterized by advanced disease stage at presentation, aggressive disease biology, and resistance to therapy, resulting in an extremely poor 5-year survival rate of <10%. PDAC is classified into transcriptional subtypes with distinct survival characteristics, although how these arise is not known. Epigenetic deregulation, rather than genetics, has been proposed to underpin progression, but exactly why is unclear and is hindered by the technical limitations of analyzing clinical samples.. We performed genome-wide epigenetic mapping of DNA modifications 5-methylcytosine and 5-hydroxymethylcytosine (5hmc) using oxidative bisulfite sequencing from formalin-embedded sections. We identified overlap with transcriptional signatures in formalin-fixed, paraffin-embedded tissue from resected patients, via bioinformatics using iCluster and mutational profiling and confirmed them in vivo.. We found that aggressive squamous-like PDAC subtypes result from epigenetic inactivation of loci, including GATA6, which promote differentiated classical pancreatic subtypes. We showed that squamous-like PDAC transcriptional subtypes are associated with greater loss of 5hmc due to reduced expression of the 5-methylcytosine hydroxylase TET2. Furthermore, we found that SMAD4 directly supports TET2 levels in classical pancreatic tumors, and loss of SMAD4 expression was associated with reduced 5hmc, GATA6, and squamous-like tumors. Importantly, enhancing TET2 stability using metformin and vitamin C/ascorbic acid restores 5hmc and GATA6 levels, reverting squamous-like tumor phenotypes and WNT-dependence in vitro and in vivo.. We identified epigenetic deregulation of pancreatic differentiation as an underpinning event behind the emergence of transcriptomic subtypes in PDAC. Our data showed that restoring epigenetic control increases biomarkers of classical pancreatic tumors that are associated with improved therapeutic responses and survival.

Topics: 5-Methylcytosine; Animals; Antineoplastic Combined Chemotherapy Protocols; Ascorbic Acid; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Cell Differentiation; Cell Line, Tumor; Dioxygenases; DNA Methylation; DNA-Binding Proteins; Epigenesis, Genetic; Epigenome; Epigenomics; GATA6 Transcription Factor; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Metformin; Mice, Nude; Mice, Transgenic; Pancreatic Neoplasms; Retrospective Studies; Smad4 Protein; Transcription, Genetic; Transcriptome; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

Vitamin C is not only an antioxidant but also a regulator of epigenetic modifications that can enhance the activity of the ten-eleven translocation (TET) family dioxygenases and promote the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Here, we investigated the effects of vitamin C in regulating DNA methylation in sheep somatic cells or embryos in an effort to improve the cloned embryo development. Vitamin C treatment of sheep foetal fibroblast cells significantly increased the 5hmC levels but did not affect the 5mC levels in cells. After nuclear transfer, vitamin C-treated donor cells could not support a higher blastocyst development rate than non-treated cells. Although combination of serum starvation and vitamin C treatment could induce significant 5mC decrease in donor cells, it failed to promote the development of resultant cloned embryos. When cloned embryos were directly treated with vitamin C, the pre-implantation development of embryos and the 5hmC levels in blastocysts were significantly improved. This beneficial role of vitamin C on embryo development was also observed in fertilized embryos. Our results suggest that vitamin C treatment of the embryos, but not the donor cells, can improve the development of cloned sheep embryos.

Topics: 5-Methylcytosine; Animals; Antioxidants; Ascorbic Acid; Blastocyst; Cloning, Organism; Culture Media; DNA Methylation; Embryonic Development; Fibroblasts; Male; Nuclear Transfer Techniques; Sheep

Major efforts are underway to identify agents that can potentiate effects of immune checkpoint inhibition. Here, we show that ascorbic acid (AA) treatment caused genomewide demethylation and enhanced expression of endogenous retroviral elements in lymphoma cells. AA also increased 5-hydroxymethylcytosine (5hmC) levels of CD8+ T cells and enhanced their cytotoxic activity in a lymphoma coculture system. High-dose AA treatment synergized with anti-PD1 therapy in a syngeneic lymphoma mouse model, resulting in marked inhibition of tumor growth compared with either agent alone. Analysis of the intratumoral epigenome revealed increased 5hmC with AA treatment, consistent with in vitro findings. Analysis of the tumor immune microenvironment revealed that AA strikingly increased intratumoral infiltration of CD8+ T cells and macrophages, suggesting enhanced tumor immune recognition. The combination treatment markedly enhanced intratumoral infiltration of macrophages and CD8+ T lymphocytes, granzyme B production by cytotoxic cells (cytotoxic T cells and natural killer cells), and interleukin 12 production by antigen-presenting cells compared with single-agent anti-PD1. These data indicate that AA potentiates anti-PD1 checkpoint inhibition through synergistic mechanisms. The study provides compelling rationale for testing combinations of high-dose AA and anti-PD1 agents in patients with aggressive B cell lymphoma as well as in preclinical models of other malignancies.

Topics: 5-Methylcytosine; Animals; Antibodies, Monoclonal; Antineoplastic Agents; Ascorbic Acid; B7-H1 Antigen; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Survival; Combined Modality Therapy; Disease Models, Animal; Drug Synergism; Female; Granzymes; Immunotherapy; Lymphoma; Mice; Mice, Inbred BALB C; Programmed Cell Death 1 Receptor; Tumor Microenvironment

Epigenetic dysregulations are closely associated with the development of pancreatic ductal adenocarcinoma (PDAC), which is one of the most aggressive malignancies and currently has limited treatment options. Vitamin C (VC), an epigenetic mediator, exerts antitumor effects on several types of cancer. However, the clinical application of VC is limited, particularly in PDAC. Thus, to investigate the antitumor effects and explore the potential clinical application of VC in PDAC, the survival of patients from The Cancer Genome Atlas database were analyzed, and proliferation, apoptosis and migration assays were performed in the present study. It was first established that high expression levels of the sodium‑dependent VC transporter 2, a critical VC transporter, predicted a good prognosis in patients with pancreatic adenocarcinoma. It was further confirmed that VC directly inhibited proliferation, induced apoptosis and suppressed migration of human pancreatic cancer cells. Global 5‑hydroxymethylcytosine (5hmC) content was significantly upregulated in pancreatic cancer cells following VC treatment, predominantly relying on ten‑eleven translocation 2. Furthermore, VC could specifically increase 5hmC levels at the promotor region on PH domain leucine‑rich repeat protein phosphatase 2 (PHLPP2) and enhance PHLPP2 expression levels. When PHLPP2 expression levels were knocked down, VC was able to partially overcome the inhibition of pancreatic cancer cells. These results illustrated a novel and precise mechanism of action of epigenetic alterations that underly the inhibition of VC in pancreatic cancer, and emphasized that PHLPP2 may be a new biomarker and epigenetic target for the clinical treatment of VC in PDAC.

Topics: 5-Methylcytosine; Ascorbic Acid; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Humans; Pancreatic Neoplasms; Phosphoprotein Phosphatases; Prognosis; Promoter Regions, Genetic; Sodium-Coupled Vitamin C Transporters; Survival Analysis; Up-Regulation

Understanding epigenetic regulation in the differentiation and maturation of dopaminergic neurons is critical to improve and develop new medications for Parkinson's disease (PD). To explore the role of ten-eleven translocation (TETs) family of dioxygenases and chromatin remodeling genes in the development of human midbrain dopaminergic (mDA) neurons, we globally analyze the epigenetic regulation of gene expression in human induced pluripotent stem cells (iPSCs) and iPSCs-derived mDA neurons. During the conversion of iPSCs into neuronal lineages of dopaminergic progenitors and mDA neurons, the expression patterns of epigenetic genes in multiple sets alter significantly. Vitamin C, an activator of TET enzymes, increases hydroxymethylcytosine (5hmC) level along with a higher yield of mDA neurons. Additionally, vitamin C treatment elevates gene expressions of TET2/3 and vitamin C transporters. Importantly, functional arrays indicate that vitamin C can promote neuronal maturation, synaptic activity, and dopamine release. Collectively, our study demonstrates that chromatin remodeling genes and the TET-5hmC pathway, which is regulated by vitamin C, are critical for the vital developmental stages of human mDA neurons.

Topics: 5-Methylcytosine; Adult; Aged; Ascorbic Acid; Cell Differentiation; Cell Line; Cell Lineage; Chromatin Assembly and Disassembly; Dioxygenases; DNA-Binding Proteins; Dopamine; Dopaminergic Neurons; Epigenesis, Genetic; Humans; Induced Pluripotent Stem Cells; Mesencephalon; Middle Aged; Models, Biological; Proto-Oncogene Proteins

Topics: 5-Methylcytosine; Animals; Ascorbic Acid; Axons; Contusions; Dioxygenases; Epigenesis, Genetic; Female; Motor Cortex; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord; Spinal Cord Injuries

Vitamin C (ascorbic acid, AA) is a well-known regulator of bone and cartilage metabolism. However, the mechanisms of AA's action in these tissues are only partly understood. In this study, we confirmed that AA contributes to bone and cartilage metabolism by showing decreased articular cartilage and trabecular bone in AA-deficient spontaneous fracture (sfx) mutant mice. In vitro, we found that AA exerts differential effects on chondrocyte and osteoblast differentiation. Since AA is known to increase levels of 5-hydroxymethylcytosine (5-hmC) and induce DNA demethylation via the ten-eleven translocases (TETs), and since prolyl hydroxylase domain-containing protein 2 (PHD2), a known mediator of AA's effects in these tissues, is part of the same enzyme family as the TETs, we next investigated whether increases in 5-hmC might mediate some of these effects. All TETs and PHDs are expressed in chondrocytes and osteoblasts, and PHD2 is localized in both the cytoplasm and nucleus of the cell, lending plausibility to the hypothesis of altered 5-hmC content in these cells. We found that AA treatment increased levels of 5-hmC in both cell types globally, notably including promoter regions of osteoblast differentiation genes. Furthermore, inhibition of PHD2 decreased 5-hmC levels in chondrocyte differentiation gene promoters, and knockdown of Phd2 in chondrocytes reduced global 5-hmC levels, suggesting for the first time that PHD2 may itself directly mediate increases in 5-hmC in chondrocyte and osteoblast genes. Further investigation of this mechanism could lead to novel therapeutic approaches to treat debilitating diseases such as osteoarthritis and osteoporosis.

Topics: 5-Methylcytosine; Animals; Ascorbic Acid; Cell Differentiation; Cells, Cultured; Chondrocytes; Gene Expression; Hypoxia-Inducible Factor-Proline Dioxygenases; Mice; Osteoblasts

Oocyte vitrification has extensively been applied in the field of embryo engineering and in the preservation of genetic resources of fine livestock. Following our previous work in oocyte vitrification and the level change of DNA methylation, here we further explored the dynamic change of three active demethylation proteins: Ten-Eleven-Translocation 1/2/3(TET1/2/3), 5-methylcytosine (5 mC) and 5-hydroxymethycytosine (5hmC) after vitrification and warming. In order to observe the active demethylation in vitrified oocytes, two small molecular regulators, i.e. Vitamin C (VC) and dimethyloxaloylglycine (DMOG) were used to adjust activity and level of the TET 3 protein. The results showed that the levels of 5 mC and 5hmC were significantly decreased after 2 h of vitrification (P < 0.01). Moreover, the level of TET3 protein was significantly increased after 2 h warming (P < 0.01). And the relative gene expression of TET2/3 did not change in the first 2 h, but significantly increased after 2 h (P < 0.01). When VC was added to vitrification and recovery medium, it could not significantly improve the level of TET3 gene expression, and affect 5 mC and 5hmC expression (P > 0.05). When the DMOG was added to the solutions of vitrification, the level of 5hmC showed significantly increase (P < 0.01). In conclusion, the oocyte vitrification procedure reduced DNA methylation and hydroxymethylation in MII oocytes, but adding VC and DMOG to vitrification medium can prevent the reduction of DNA hydroxymethylation by increasing activity of TET3 methylation protein after vitrification and warming.

Topics: 5-Methylcytosine; Amino Acids, Dicarboxylic; Animals; Ascorbic Acid; Cryopreservation; Cryoprotective Agents; Dioxygenases; DNA Methylation; DNA-Binding Proteins; Embryo Research; Female; Gene Expression; Mice; Oocytes; Proto-Oncogene Proteins; Vitrification

As the precursors of sperm and eggs, human primordial germ cells (hPGCs) emerge as early as weeks 2 to 3 of post-implantation development. Recently, robust hPGC induction models have been established in vitro with different protocols, but global 5mC/5hmC epigenetic reprogramming is not initiated in vitro. Previous studies found that vitamin C can enhance Tet (ten-eleven translocation) enzyme expression and improve 5hmC level in cells. But the effect of vitamin C supplementation on hPGC in vitro induction is still unknown.. We generated a gene-edited human embryonic stem cell (hESC) line carrying a BLIMP1-mkate2 reporter by CRISPR/Cas9 technology and used flow cytometry to optimize the PGC differentiation protocol; meanwhile, the expression of PGC genes (BLIMP1, TFAP2C, SOX17, OCT4) was evaluated by qRT-PCR. When different concentrations of vitamin C were added to the induction medium, the percentage of hPGCLCs (hPGC-like cells) was analyzed by flow cytometry; dot blot and ELISA were used to detect the levels of 5hmC and 5mC. The expression of TET enzymes was also evaluated by qRT-PCR.. We optimized the PGC differentiation protocol with the BLIMP1-mkate reporter hESCs, and the efficiency of PGC induction in vitro can be improved to 30~40%. When 50 μg/mL vitamin C was added, the derived hPGCLCs not only upregulated the expression of key genes involved in human early germ cell development such as NANOS3, TFAP2C, BLIMP1, and SOX17, but also increased the levels of 5hmC and TET enzymes.. Taken together, supplementation of vitamin C can promote the in vitro induction of hPGCLCs from hESCs, which might be related to vitamin C-mediated epigenetic regulations during the differentiation process.

Topics: 5-Methylcytosine; Ascorbic Acid; Cell Line; Epigenesis, Genetic; Genes, Reporter; Germ Cells; Human Embryonic Stem Cells; Humans; Positive Regulatory Domain I-Binding Factor 1

The TET (Ten-Eleven Translocation) proteins catalyze the oxidation of 5mC (5-methylcytosine) to 5hmC (5-hydroxymethylcytosine) and play crucial roles in embryonic development. Ascorbic acid (Vc, Vitamin C) stimulates the expression of TET proteins, whereas DMOG (dimethyloxallyl glycine) inhibits TET expression. To investigate the role of TET1, TET2, and TET3 in PA (parthenogenetic) embryonic development, Vc and DMOG treatments were administered during early embryonic development. The results showed that Vc treatment increased the blastocyst rate (20.73 ± 0.46 compared with 26.57 ± 0.53%). By contrast, DMOG reduced the blastocyst rate (20.73 ± 0.46 compared with 11.18 ± 0.13%) in PA embryos. qRT-PCR (quantitative real-time PCR) and IF (immunofluorescence) staining results revealed that TET1, TET2, and TET3 expressions were significantly lower in PA embryos compared with normal fertilized (Con) embryos. Our results revealed that Vc stimulated the expression of TET proteins in PA embryos. However, treatment with DMOG significantly inhibited the expression of TET proteins. In addition, 5hmC was increased following treatment with Vc and suppressed by DMOG in PA embryos. Taken together, these results indicate that the expression of TET proteins plays crucial roles mediated by 5hmC in PA embryonic development.

Topics: 5-Methylcytosine; Animals; Ascorbic Acid; Blastocyst; Dioxygenases; DNA-Binding Proteins; Embryonic Development; Female; Gene Expression Regulation, Developmental; Glycine; Male; Mice; Mice, Inbred C57BL; Parthenogenesis; Proto-Oncogene Proteins; Signal Transduction

Although clear cell renal cell carcinoma (ccRCC) has been shown to result in widespread aberrant cytosine methylation and loss of 5-hydroxymethylcytosine (5hmC), the prognostic impact and therapeutic targeting of this epigenetic aberrancy has not been fully explored. Analysis of 576 primary ccRCC samples demonstrated that loss of 5hmC was strongly associated with aggressive clinicopathologic features and was an independent adverse prognostic factor. Loss of 5hmC also predicted reduced progression-free survival after resection of nonmetastatic disease. The loss of 5hmC in ccRCC was not due to mutational or transcriptional inactivation of ten eleven translocation (TET) enzymes, but to their functional inactivation by l-2-hydroxyglutarate (L2HG), which was overexpressed due to the deletion and underexpression of L2HG dehydrogenase (L2HGDH). Ascorbic acid (AA) reduced methylation and restored genome-wide 5hmC levels via TET activation. Fluorescence quenching of the recombinant TET-2 protein was unaffected by L2HG in the presence of AA. Pharmacologic AA treatment led to reduced growth of ccRCC in vitro and reduced tumor growth in vivo, with increased intratumoral 5hmC. These data demonstrate that reduced 5hmC is associated with reduced survival in ccRCC and provide a preclinical rationale for exploring the therapeutic potential of high-dose AA in ccRCC.

Topics: 5-Methylcytosine; Adult; Alcohol Oxidoreductases; Animals; Ascorbic Acid; Carcinoma, Renal Cell; Cell Line, Tumor; Female; Gene Deletion; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Male; Mice

Ten-eleven translocation (TET) proteins have been shown to be abnormally expressed in different cancers. To investigate the expression pattern of TET proteins in HepG2 cells, sodium ascorbate was used to treat HepG2 cells. Our results showed that TET1, TET2 and TET3 expression was increased after sodium ascorbate treatment. The TET proteins catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thus, 5mC and 5hmC levels were examined. The results suggested that 5hmC was increased after sodium ascorbate treatment. To further determine the biological function of the TET proteins, si-TET1, si-TET2 and si-TET3 were transfected into HepG2 cells. The results showed that a knock down of TET3 expression stimulated cell proliferation of HepG2 cells. To further understand the effects of TET3 expression on cell proliferation, sodium ascorbate was added to the cells after transfection with si-TET3. The results demonstrated that sodium ascorbate could rescue TET3 expression and inhibit cell proliferation. Taken together, these results indicate that TET3 expression regulated cell proliferation, which is associated with 5hmC in HepG2 cells.

Topics: 5-Methylcytosine; Ascorbic Acid; Cell Proliferation; Dioxygenases; DNA Methylation; DNA-Binding Proteins; Hep G2 Cells; Humans; Mixed Function Oxygenases; Oxidation-Reduction; Proto-Oncogene Proteins; Transcriptome

Hypoxia resulting in hypoxia-inducible factor-1 alpha (HIF-1α) induction is known to drive scar formation during cutaneous wound healing, and may be responsible for excessive fibrosis inherent to hypertrophic scars and keloids. Because epigenetic pathways play an important role in regulation of fibrosing processes, we evaluated patient scars for DNA hydroxymethylation (5-hydroxymethylcytosine; 5-hmC) status and documented a significant decrease in scar fibroblasts. To test this finding in vitro, human fibroblasts were cultured with cobalt chloride (CoCl

Topics: 5-Methylcytosine; Ascorbic Acid; Cell Hypoxia; Cells, Cultured; Cicatrix; Dioxygenases; DNA Methylation; Epigenesis, Genetic; Fibroblasts; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression; Humans; Hypoxia-Inducible Factor 1, alpha Subunit

Genomic loss of 5-hydroxymethylcytosine (5hmC) accompanies malignant cellular transformation in breast cancer. Vitamin C serves as a cofactor for TET methylcytosine dioxygenases to increase 5hmC generation. Here we show that the transcription of SVCT2, a major vitamin C transporter, was decreased in human breast cancers (113 cases) compared to normal breast tissues from the same patients. A decreased SVCT2 expression was also observed in breast cancer cell lines. Treatment with vitamin C (100 μM) increased the 5hmC content in MDA-MB-231 breast cancer cells and markedly altered the transcriptome. The vitamin C treatment induced apoptosis in MDA-MB-231 cells, which was verified in two additional breast cancer cell lines. This pro-apoptotic effect of vitamin C appeared to be mediated by TRAIL, a known apoptosis inducer. Vitamin C upregulated TRAIL transcripts (2.3-fold increase) and increased TRAIL protein levels. The upregulation of TRAIL by vitamin C was largely abolished by siRNAs targeting TETs and anti-TRAIL antibody abrogated the induction of apoptosis. Furthermore, the apoptosis promoted by vitamin C was associated with Bax and caspases activation, Bcl-xL sequestration, and cytochrome c release. Taken together, these results suggest a potential role of physiological doses of vitamin C in breast cancer prevention and treatment.

Topics: 5-Methylcytosine; Apoptosis; Ascorbic Acid; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Receptors, TNF-Related Apoptosis-Inducing Ligand; Sodium-Coupled Vitamin C Transporters; TNF-Related Apoptosis-Inducing Ligand

Loss of 5-hydroxymethylcytosine (5hmC) occurs frequently in a wide variety of tumours, including clear-cell renal cell carcinoma (ccRCC). It remains unknown, however, whether the restoration of 5hmC patterns in tumours could have therapeutic efficacy. Here, we used sodium L-ascorbate (vitamin C, AsANa) and the oxidation-resistant form L-ascorbic acid 2-phosphate sesquimagnesium (APM) for the restoration of 5hmC patterns in ccRCC cells. At physiological concentrations, both show anti-tumour efficacy during long-term treatment

Topics: 5-Methylcytosine; Animals; Ascorbic Acid; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Dioxygenases; DNA-Binding Proteins; Enhancer Elements, Genetic; Humans; Kidney Neoplasms; Mice; Proto-Oncogene Proteins; Transcriptome; Xenograft Model Antitumor Assays

5-Hydroxymethylcytosine (5hmC) is converted from 5-methylcytosine (5mC) by a group of enzymes termed ten-eleven translocation (TET) family dioxygenases. The loss of 5hmC has been identified as a hallmark of most types of cancer and is related to tumorigenesis and progression. However, the role of 5hmC in bladder cancer is seldom investigated. Vitamin C was recently reported to induce the generation of 5hmC by acting as a cofactor for TET dioxygenases. In this study, we explored the role of 5hmC in bladder cancer and the therapeutic efficacy of vitamin C in increasing the 5hmC pattern.. 5hmC was decreased in bladder cancer samples and was related to patient overall survival. Genome-wide mapping of 5hmC in tumor tissues and vitamin C-treated bladder cancer cells revealed that 5hmC loss was enriched in cancer-related genes and that vitamin C treatment increased 5hmC levels correspondingly. Vitamin C treatment shifted the transcriptome and inhibited the malignant phenotypes associated with bladder cancer cells in both in vitro cell lines and in vivo xenografts.. This study provided mechanistic insights regarding the 5hmC loss in bladder cancer and a rationale for exploring the therapeutic use of vitamin C as a potential epigenetic treatment for bladder cancer.

Topics: 5-Methylcytosine; Aged; Animals; Ascorbic Acid; Cell Proliferation; Cell Survival; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Male; Mice; Middle Aged; Prognosis; Sequence Analysis, DNA; Sequence Analysis, RNA; Survival Analysis; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Muscle cell differentiation is a complex process that is principally governed by related myogenic regulatory factors (MRFs). DNA methylation is considered to play an important role on the expression of MRF genes and on muscle cell differentiation. However, the roles of enzymes specifically in myogenesis are not fully understood. Here, we demonstrate that Tet2, a ten-eleven translocation (Tet) methylcytosine dioxygenase, exerts a role during skeletal myoblast differentiation. By using an immunostaining method, we found that the levels of 5-hydroxymethylcytosine (5-hmC) were much higher in differentiated myotubes than in undifferentiated C2C12 myoblasts. Both Tet1 and Tet2 expression were upregulated after differentiation induction of C2C12 myoblasts. Knockdown of Tet2, but not Tet1, significantly reduced the expression of myogenin as well as Myf6 and myomaker, and impaired myoblast differentiation. DNA demethylation of myogenin and myomaker promoters was negatively influenced by Tet2 knockdown as detected by bisulfite sequencing analysis. Furthermore, although vitamin C could promote genomic 5hmC generation, myogenic gene expression and myoblast differentiation, its effect was significantly attenuated by Tet2 knockdown. Taken together, these results indicate that Tet2 is involved in myoblast differentiation through promoting DNA demethylation and myogenic gene expression.

Topics: 5-Methylcytosine; Animals; Ascorbic Acid; Cell Differentiation; Cell Line; Dioxygenases; DNA Methylation; DNA-Binding Proteins; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Mice; Muscle Development; Myoblasts, Skeletal; Promoter Regions, Genetic; Proto-Oncogene Proteins

Preeclampsia and gestational diabetes mellitus (GDM) are the most common clinical conditions in pregnancy that could result in adverse in utero environments. Fetal exposure to poor environments may raise the long-term risk of postnatal disorders, while epigenetic modifications could be involved. Recent research has implicated involvement of 5-hydroxymethylcytosine (5hmC), a DNA base derived from 5-methylcytosine, via oxidation by ten-eleven translocation (TET) enzymes, in DNA methylation-related plasticity. Here, we show that the TET2 expression and 5hmC abundance are significantly altered in the umbilical veins of GDM and preeclampsia. Genome-wide profiling of 5hmC revealed its specific reduction on intragenic regions from both GDM and preeclampsia compared to healthy controls. Gene Ontology analysis using loci bearing unique GDM- and preeclampsia-specific loss-of-5hmC indicated its impact on several critical biological pathways. Interestingly, the substantial alteration of 5hmC on several transposons and repetitive elements led to their differential expression. The alteration of TET expression, 5hmC levels and 5hmC-mediated transposon activity was further confirmed using established hypoxia cell culture model, which could be rescued by vitamin C, a known activator of TET proteins. Together, these results suggest that adverse pregnancy environments could influence 5hmC-mediated epigenetic profile and contribute to abnormal development of fetal vascular systems that may lead to postnatal diseases.

Topics: 5-Methylcytosine; Adult; Ascorbic Acid; Diabetes, Gestational; Dioxygenases; DNA Methylation; DNA Transposable Elements; DNA-Binding Proteins; Epigenesis, Genetic; Female; Gene Expression Regulation, Developmental; Humans; Pre-Eclampsia; Pregnancy; Proto-Oncogene Proteins

Dioxygenases of the TET family impact genome functions by converting 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC). Here, we identified TET2 as a crucial regulator of mast cell differentiation and proliferation. In the absence of TET2, mast cells showed disrupted gene expression and altered genome-wide 5hmC deposition, especially at enhancers and in the proximity of downregulated genes. Impaired differentiation of Tet2-ablated cells could be relieved or further exacerbated by modulating the activity of other TET family members, and mechanistically it could be linked to the dysregulated expression of C/EBP family transcription factors. Conversely, the marked increase in proliferation induced by the loss of TET2 could be rescued exclusively by re-expression of wild-type or catalytically inactive TET2. Our data indicate that, in the absence of TET2, mast cell differentiation is under the control of compensatory mechanisms mediated by other TET family members, while proliferation is strictly dependent on TET2 expression.

Topics: 5-Methylcytosine; Ascorbic Acid; Biocatalysis; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Cell Proliferation; Cytokines; Dioxygenases; DNA-Binding Proteins; Gene Deletion; Gene Expression Regulation; Gene Knockdown Techniques; Genome; HEK293 Cells; Humans; Male; Mast Cells; Proto-Oncogene Proteins; Sequence Analysis, RNA; Transcription, Genetic

The most plausible mechanism behind active demethylation of 5-methylcytosine involves TET proteins which participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine; the latter is further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil can be also generated from thymine in a TET-catalyzed process. Ascorbate was previously demonstrated to enhance generation of 5-hydroxymethylcytosine in cultured cells. The aim of this study was to determine the levels of the abovementioned TET-mediated oxidation products of 5-methylcytosine and thymine after addition of ascorbate, using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Intracellular concentration of ascorbate was determined by means of ultra-performance liquid chromatography with UV detection. Irrespective of its concentration in culture medium (10-100µM) and inside the cell, ascorbate stimulated a moderate (2- to 3-fold) albeit persistent (up to 96-h) increase in the level of 5-hydroxymethylcytosine. However, exposure of cells to higher concentrations of ascorbate (100µM or 1mM) stimulated a substantial increase in 5-formylcytosine and 5-carboxycytosine levels. Moreover, for the first time we demonstrated a spectacular (up to 18.5-fold) increase in 5-hydroxymethyluracil content what, in turn, suggests that TET enzymes contributed to the presence of the modification in cellular DNA. These findings suggest that physiological concentrations of ascorbate in human serum (10-100µM) are sufficient to maintain a stable level of 5-hydroxymethylcytosine in cellular DNA. However, markedly higher concentrations of ascorbate (ca. 100µM in the cell milieu or ca. 1mM inside the cell) were needed to obtain a sustained increase in 5-formylcytosine, 5-carboxycytosine and 5-hydroxymethyluracil levels. Such feedback to elevated concentrations of ascorbate may reflect adaptation of the cell to environmental conditions.

Topics: 5-Methylcytosine; Ascorbic Acid; Cytosine; DNA; DNA Methylation; HCT116 Cells; Humans; Mixed Function Oxygenases; Oxidation-Reduction; Pentoxyl; Proto-Oncogene Proteins; Spectrometry, Mass, Electrospray Ionization; Thymine

The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5 hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5 mC) and 5 hmC at a CCGG site within the 5' γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5 mC and 5 hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5 hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5 hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5 hmC and negatively correlated with 5 mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5 hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter.

Topics: 5-Methylcytosine; Animals; Animals, Newborn; Antineoplastic Agents; Ascorbic Acid; Azacitidine; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Cytosine; Decitabine; Dioxygenases; DNA Methylation; Erythroid Cells; gamma-Globins; Histone Demethylases; Humans; Hydroxyurea; Liver; Papio anubis; Promoter Regions, Genetic; Tranylcypromine

Tet methylcytosine dioxygenase converts 5-mC to 5-hmC in DNA.. Ascorbate significantly and specifically enhances Tet-mediated generation of 5-hmC.. Our findings suggest that ascorbate enhances 5-hmC generation, most likely by acting as a co-factor for Tet methylcytosine dioxygenase to generate 5-hmC.. The availability of ascorbate could have significant consequences for health and diseases by modulating the epigenetic control of genome activity. Ascorbate (vitamin C) is best known for its role in scurvy, in which the hydroxylation of collagen catalyzed by dioxygenases is incomplete due to ascorbate deficiency. Here, we report a novel function of ascorbate in the hydroxylation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) in DNA catalyzed by Tet (ten-eleven translocation) methylcytosine dioxygenase. The content of 5-hmC is extremely low in mouse embryonic fibroblasts cultured in ascorbate-free medium. Additions of ascorbate dose- and time-dependently enhance the generation of 5-hmC, without any effects on the expression of Tet genes. Treatment with another reducer glutathione (GSH) does not change the level of 5-hmC. Further, blocking ascorbate entry into cells by phloretin and knocking down Tet (Tet1, Tet2, and Tet3) expression by short interference RNAs (siRNA) significantly inhibit the effect of ascorbate on 5-hmC. These results suggest that ascorbate enhances 5-hmC generation, most likely by acting as a co-factor for Tet methylcytosine dioxygenase to hydroxylate 5-mC. Thus, we have uncovered a novel role for ascorbate in modulating the epigenetic control of genome activity.

Topics: 5-Methylcytosine; Animals; Ascorbic Acid; Cells, Cultured; Cytosine; Dioxygenases; DNA Methylation; DNA-Binding Proteins; Epigenesis, Genetic; Gene Knockdown Techniques; Hydroxylation; Mice; Mice, Inbred C57BL; Phloretin; Proto-Oncogene Proteins; RNA, Small Interfering

DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons. Global DNA demethylation occurs in the early embryo and the germ line, and may be mediated by Tet (ten eleven translocation) enzymes, which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Tet enzymes have been studied extensively in mouse embryonic stem (ES) cells, which are generally cultured in the absence of vitamin C, a potential cofactor for Fe(II) 2-oxoglutarate dioxygenase enzymes such as Tet enzymes. Here we report that addition of vitamin C to mouse ES cells promotes Tet activity, leading to a rapid and global increase in 5hmC. This is followed by DNA demethylation of many gene promoters and upregulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site of genes affected by vitamin C treatment. Importantly, vitamin C, but not other antioxidants, enhances the activity of recombinant Tet1 in a biochemical assay, and the vitamin-C-induced changes in 5hmC and 5mC are entirely suppressed in Tet1 and Tet2 double knockout ES cells. Vitamin C has a stronger effect on regions that gain methylation in cultured ES cells compared to blastocysts, and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A particle retroelements, which are resistant to demethylation in the early embryo, are resistant to vitamin-C-induced DNA demethylation. Collectively, the results of this study establish vitamin C as a direct regulator of Tet activity and DNA methylation fidelity in ES cells.

Topics: 5-Methylcytosine; Animals; Antioxidants; Ascorbic Acid; Blastocyst; Cell Line; Culture Media; Cytosine; Dioxygenases; DNA Methylation; DNA-Binding Proteins; Embryonic Stem Cells; Gene Expression Regulation, Developmental; Gene Knockout Techniques; Mice; Protein Binding; Proto-Oncogene Proteins; Recombinant Proteins

Tet (ten-eleven translocation) methylcytosine dioxygenases, which belong to the iron and 2-oxoglutarate (2OG)-dependent dioxygenase superfamily, convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. We recently reported that ascorbate (vitamin C) induces Tet-mediated generation of 5hmC. To initially delineate the role of ascorbate on 5hmC generation, we analyzed whether the effect of ascorbate is dependent upon the conditions of other components involved in the hydroxylation of 5mC catalyzed by Tet. We found that removing iron from the culture medium did not affect the induction of 5hmC by ascorbate (10 μM) in mouse embryonic fibroblasts (MEFs). The effect of ascorbate did not involve an increased expression of Tet1-3 or isocitrate dehydrogenases (IDH1-2), the enzymes responsible for producing 2OG. Interestingly, MEFs cultured with different concentrations of glucose, a major precursor of 2OG, exhibited nearly identical responses to ascorbate treatment. Further, blocking the uptake of the reduced form of vitamin C, ascorbic acid, through the sodium-dependent vitamin C transporters (SVCTs) inhibited the effect of ascorbate on 5hmC. However, inhibition of the facilitative glucose transporters (GLUTs), which mediate the incorporation of the oxidized form of vitamin C, dehydroascorbic acid (DHA), did not modify the ability of ascorbate to induce 5hmC generation. These results indicate that the effect of ascorbate on 5hmC is not dependent upon iron uptake, the expression of Tet and IDH, or the production of 2OG, suggesting that ascorbate may directly participate in the generation of 5hmC, most likely as a cofactor of Tet.

Topics: 5-Methylcytosine; Animals; Ascorbic Acid; Cytosine; Dioxygenases; DNA Methylation; DNA-Binding Proteins; Fibroblasts; Iron; Isocitrate Dehydrogenase; Ketoglutaric Acids; Mice; Mice, Inbred C57BL; Proto-Oncogene Proteins; Sodium-Coupled Vitamin C Transporters