mRNA Expression Of DNA Methyltransferases Research Articles

Promoter methylation, mRNA expression of goat tumor‑associated genes and mRNA expression of DNA methyltransferase in enzootic nasal tumors.

The aim of the present study was to investigate the promoter methylation status and mRNA expression of goat tumor‑associated genes, in addition to the mRNA expression of DNA methyltransferase genes in enzootic nasal tumors (ENT). Methylation‑specific polymerase chain reaction and SYBR Green reverse transcription‑quantitative polymerase chain reaction were used to detect the methylation status and the mRNA expression levels of DNA methyltransferases (DNMTs), O6‑methylguanine‑DNA methyltransferase (MGMT), the tumor suppressor genes P73, P53, GADD45G, CHFR and THBS1, the transcription factor CEBPA, the proto‑oncogenes KRAS, NRAS and C‑myc and EGFR in 24 nasal tumor tissue samples and 20 normal nasal epithelia tissue samples. The associations between promoter methylation and DNMT, and promoter methylation and mRNA expression of the genes were analyzed. The results indicated that the expression levels of DNMT1 increased by 56% compared with those in normal nasal epithelial tissues, while MGMT, DNMT3a and DNMT3b had similar expression levels in the two tissue types. The expression levels of P53 decreased by 36.8% and those of THBS1 by 43%, while C‑myc increased by 2.9‑fold and CEBPA by 2‑fold compared with that in normal nasal epithelial tissues. GADD45G, P73, CHFR and NRAS were observed to have similar expression levels in the two tissue types. However, no expression was observed for EGFR and KRAS. CHFR, GADD45G and THBS1 were identified to be methylated in tumor suppressor genes. The methylation expression rate of the CHFR gene was ~60% in the two tissue types and for THBS1 it was 100% in the nasal tumor tissues as opposed to 20% in the normal nasal epithelial tissues. The exhaustive methylation expression rate of GADD45G was 62.5% and the partial methylation expression rate was 37.5% in nasal tumor tissue, while no methylation was observed in normal nasal epithelial tissues. C‑myc was the only gene identified to be methylated amongst proto‑oncogenes. The methylation expression rate of C‑myc was 87.5% in nasal tumor tissues and 15% in normal nasal epithelial tissues. The methylation expression rate of CEBPA was 100% in nasal tumor tissues and 40% in normal nasal epithelial tissues. The methylation expression rate of the EGFR gene was ~80% in the two tissues. In summary, the present study identified abnormal methylation of the C‑myc, CEBPA, GADD45G and THBS1 genes in nasal tumor tissues. The expression levels of DNMT1, C‑myc and CEBPA were upregulated and the expression of P53 and THBSI were downregulated in nasal tumor tissues, with a significant difference between the two groups (P<0.05). Therefore, it is suggested that these six genes may be used as diagnostic marker candidates for ENT. The results may serve as a foundation for screening of tumor‑specific markers for early diagnosis of ENT and further investigate the epigenetic mechanisms of enzootic nasal tumor virus (ENTV)‑induced nasal epithelium cell carcinoma.

Expression of DNA Methyltransferase Genes and Its Significance in AML Patients

To detect the expression of DNA methyltransferases (DNMT) mRNA in the patients with acute myelogenous leukemia (AML) and to analyze the retationship between the mRNA expression of DNMT and cellular and moleculogenetic risk stratifieation in AML patients, and to evaluate the role of the DNMT mRNA expression in AML prognosis and clinical treatment. The mRNA expression of DNMT was detected by real-time PCR in 123 AML patients and 20 healthy people. the mRNA expression levels of DNMT were lower in the healthy people and higher in AML patients; the mRNA expression levels of DNMT in the patients after the consolidation therapy were lower than that in the patients of initial diagnosis and replapse; The mRNA expression levels of DNMT did not correlate with age, sex and the clinical characteristics at initial diagnosis, such as white blood cell count, FAB classification and chromosomal karyotype in AML patients. In CR patients after standard treatment, the initial mRNA expression level of DNMT3b was higher. Based on cellular and moleculogenetic risk stratificantion, the DNMT expression level in the intermediate risk AML patients was higher. The mRNA expression of DNMT may play an important role in AML pathogenesis and can serve as an index for evaluating AML prognosis and for instructing clinical treatment.

Postnatal nutritional restriction affects growth and immune function of piglets with intra-uterine growth restriction.

Postnatal rapid growth by excess intake of nutrients has been associated with an increased susceptibility to diseases in neonates with intra-uterine growth restricted (IUGR). The aim of the present study was to determine whether postnatal nutritional restriction could improve intestinal development and immune function of neonates with IUGR using piglets as model. A total of twelve pairs of normal-birth weight (NBW) and IUGR piglets (7d old) were randomly assigned to receive adequate nutrient intake or restricted nutrient intake (RNI) by artificially liquid feeding for a period of 21d. Blood samples and intestinal tissues were collected at necropsy and were analysed for morphology, digestive enzyme activities, immune cells and expression of innate immunity-related genes. The results indicated that both IUGR and postnatal nutritional restriction delayed the growth rate during the sucking period. Irrespective of nutrient intake, piglets with IUGR had a significantly lower villous height and crypt depth in the ileum than the NBW piglets. Moreover, IUGR decreased alkaline phosphatase activity while enhanced lactase activity in the jejunum and mRNA expressions of Toll-like receptor 9 (TLR-9) and DNA methyltransferase 1 (DNMT1) in the ileum of piglets. Irrespective of body weight, RNI significantly decreased the number and/or percentage of peripheral leucocytes, lymphocytes and monocytes of piglets, whereas the percentage of neutrophils and the ratio of CD4+ to CD8+ were increased. Furthermore, RNI markedly enhanced the mRNA expression of TLR-9 and DNMT1, but decreased the expression of NOD2 and TRAF-6 in the ileum of piglets. In summary, postnatal nutritional restriction led to abnormal cellular and innate immune response, as well as delayed the growth and intestinal development of IUGR piglets.

MP20-02 CONDITIONED MEDIA FROM INFECTED UROTHELIAL CELLS CONTAIN EXOSOMES WITH EPIGENETIC POTENTIAL.

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Kidney & Bladder II1 Apr 2015MP20-02 CONDITIONED MEDIA FROM INFECTED UROTHELIAL CELLS CONTAIN EXOSOMES WITH EPIGENETIC POTENTIAL. Kenneth Ting, Alaleh Samiei, Karen J. Aitken, Bryce Weber, Fadi Ibrahim, Akshita Kapila, Cornelia Tolg, and Darius Bagli Kenneth TingKenneth Ting More articles by this author , Alaleh SamieiAlaleh Samiei More articles by this author , Karen J. AitkenKaren J. Aitken More articles by this author , Bryce WeberBryce Weber More articles by this author , Fadi IbrahimFadi Ibrahim More articles by this author , Akshita KapilaAkshita Kapila More articles by this author , Cornelia TolgCornelia Tolg More articles by this author , and Darius BagliDarius Bagli More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.974AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urinary tract infection (UTI) is the second most common infection in children, and results in recurrent/chronic infection in 25–40% of patients of all ages. Chronic in vitro infection of urothelial cell lines leads to increased nuclear localization of DNA methyltransferase 1 (DNMT1) and decreased p16 expression along with a rise in CpG methylation near the first exon (Lab Invest 91:825-36). We noted that uninfected cells neighbouring infected cells upregulated DNMT1. Our objective here is to study if infection-induced secreted factors (e.g. LPS, conditioned media and exosomes) alter the regulation of epigenetic writers (DNMT 1, 3A and 3B, and EZH2) and p16 in naive cells. METHODS HTB9 or normal primary urothelial cells were innoculated with 2 moi of UT189 Escherichia coli, or FimH+/- derivatives or vehicle for 2 hrs. Cells were washed in PBS+50 ug/mL gentamycin and maintained in RPMI+10 ug/mL gentamycin+5% serum. Exosome-free serum was used for exosome isolations. Conditioned media (CM) was collected every 2 days, by centrifugation of cell debris and passing through 0.2 micron filters. Exosomes were isolated from CM using Exoquick TC, and characterized by Nanosight + transmission EM. Factors secreted by host cells (CM at 50% of total media, exosomes at a ratio of 1/2 of starting CM volume and LPS (200 ng/mL)), were added to naive urothelial cells for 2 hours, or 1, 2, 6 days. mRNA of DNMT1, 3a and 3b was examined by QPCR in response to LPS and CM. p16, DNMTs and EZH2 were localized by immunofluorescence. RESULTS CM significantly increased all mRNA expression of DNMT1,3A and 3B at more than one timepoint post-infection (p<0.001). DNMT1, but not DNMT3B, localized to the nucleus during treatment with CM from infected cells, concurrent with p16 protein downregulation. LPS increased DNMTs by immunofluorescence (p<0.0001), yet upregulated mRNA of DNMTs only at discrete time points. Nuclear EZH2 was increased by infection, infection-induced CM and infection-induced exosomes, in primary and HTB9 cells (p<0.005). Exosomes from control cells had the opposite effect, leading to downregulation of EZH2 at day 2. DNMT1 but not DNMT3B was increased by infection-induced exosomes (p<0.005). CONCLUSIONS Infection and secreted factors induced by infection broadly and persistently alter p16INK4A downregulation, alongside DNMT and EZH2 upregulation. DNMT3B is regulated by LPS alone, while EZH2 and DNMT1 are upregulated by both exosomes and CM from infected cells. Dysregulation of epigenetic machinery by secreted factors may have implications in urothelial maintenance and defence. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e223-e224 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kenneth Ting More articles by this author Alaleh Samiei More articles by this author Karen J. Aitken More articles by this author Bryce Weber More articles by this author Fadi Ibrahim More articles by this author Akshita Kapila More articles by this author Cornelia Tolg More articles by this author Darius Bagli More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Maternal alcohol intake around the time of conception causes glucose intolerance and insulin insensitivity in rat offspring, which is exacerbated by a postnatal high-fat diet.

Alcohol consumption throughout pregnancy can cause metabolic dysregulation, including glucose intolerance in progeny. This study determined if periconceptional (PC) alcohol (12% v/v in a liquid diet) (PC:EtOH) consumed exclusively around conception results in similar outcomes in Sprague-Dawley rats. Control (C) rats were given a liquid diet containing no alcohol but matched to ensure equal caloric intake. PC maternal alcohol intake (from 4 days before conception until day 4 of gestation), resulted in offspring with elevated fasting plasma glucose (∼10-25%, P < 0.05), impaired glucose tolerance (P < 0.05), and decreased insulin sensitivity (P < 0.01) at 6 months of age. This was associated with increased hepatic gluconeogenesis and sex-specific alterations in peripheral protein kinase B (AKT) signaling. These changes were accompanied by increased mRNA expression of DNA methyltransferases (DNMTs) 1, 3a, and 3b (1.5- to 1.9-fold, P < 0.05) in fetal liver in late gestation, suggesting PC:EtOH may cause epigenetic changes that predispose offspring to metabolic dysfunction. Exposure to a postnatal (PN) high-fat and cholesterol diet (HFD) from 3 months of age caused hyperinsulinemia (∼2-fold increase, P < 0.001) and exacerbated the metabolic dysfunction in male offspring exposed to PC:EtOH but had no additive effects in females. Given many women may drink alcohol while planning a pregnancy, it is crucial to increase public awareness regarding the effects of alcohol consumption around conception on offspring health.

Influence of gossypol acetic acid on the growth of human adenoid cystic carcinoma ACC-M cells and the expression of DNA methyltransferase 1.

We investigated the effects of gossypol acetic acid (GAA) on the proliferation, apoptosis, and expression of DNA methyltransferase 1 (DNMT1) mRNA in human adenoid cystic carcinoma (ACC-M) cells in vitro. The proliferation and apoptosis of ACC-M cells after treatment with different concentrations of GAA were detected using Cell Counting Kit-8 and flow cytometry, respectively. DNMT1 mRNA expression was measured by real-time fluorescence quantitative polymerase chain reaction. The growth of ACC-M cells was inhibited after treatment with GAA for 24, 48, and 72 h. The apoptotic rates of ACC-M cells after treatment with GAA for 72 h were higher than those of control cells (without treatment) (P < 0.05). DNMT1 mRNA expression in ACC-M after treatment with GAA for 72 h was lower than that in control cells (P < 0.05). GAA had inhibitory effects on the proliferation and induced apoptosis of human ACC-M cells, while GAA also reduced the expression level of DNMT1 mRNA in ACC-M cells.

Effects of atrazine and chlorpyrifos on DNA methylation in the brain and gonad of the common carp

DNA methylation is known to play an important role in the regulation of gene expression in animal. The purpose of the present study was to examine the effect of atrazine (ATR), chlorpyrifos (CPF) and combined ATR/CPF exposure on DNA methylation in the brain and gonad of common carp (Cyprinus carpio L.). The carp were sampled after a 40-d exposure to CPF and ATR, individually or in combination, followed by a 40-d recovery to measure the levels of global DNA methylation and the expression of methylation enzymes (DNA methyltransferases (DNMTs) and methylcytosine binding domain 2 (MBD2)) in the brain and gonad tissues. The results revealed that a significant global DNA hypomethylation in the common carp exposed to ATR, CPF and their mixture was observed compared to the control fish. The MBD2 mRNA expression was up-regulated in the brain and gonad of the common carp exposed to ATR, CPF and their mixture, in contrast, the DNMTs mRNA expression was down-regulated. The information regarding the effects of ATR and CPF on DNA methylation status generated in this study is important for pesticides toxicology evaluation. However, the effect of ATR and CPF on the methylation status of specific genes, as well as its detailed mechanism requires further investigation.

Effects of atrazine and chlorpyrifos on DNA methylation in the liver, kidney and gill of the common carp (Cyprinus carpio L.)

Pesticide exposure has repeatedly been associated with cancers, although the molecular mechanisms behind this association are largely undetermined. Abnormal DNA methylation plays a key role in the process of some disease. However, little was known about the effect of pesticides on DNA methylation in the common carp. In this study, we investigated the mRNA levels of DNA methyltransferases (DNMTs) and methyl-CpG-binding protein DNA-binding domain protein 2 (MBD2) as well as the DNA methylation levels in the liver, kidney and gill of the common carp (Cyprinus carpio L.) after 40-d exposure to atrazine (ATR) and chlorpyrifos (CPF) alone or in combination, and a 40-d recovery period. Juvenile common carp were exposed to various concentrations of ATR (at concentrations of 4.28, 42.8 and 428μg/L), CPF (1.16, 11.6 and 116μg/L), and an ATR/CPF mixture (at concentrations of 1.13, 11.3 and 113μg/L). The results revealed that the levels of genomic DNA methylation decreased in all tissues after 40d of exposure to ATR and CPF either individually or in combination. Moreover, the mRNA expression of DNMTs was down-regulated in all treatment groups. In contrast, the mRNA expression of MBD2 was up-regulated. These results demonstrated that long-term exposure to ATR, CPF and ATR/CPF mixtures could disrupt genomic DNA. It might imply that DNA methylation is involved in the toxicity caused by ATR and CPF in the common carp.

Global DNA hypomethylation: A potential mechanism in King pigeon nerve tissue damage induced by avermectin

As an effective insecticidal and nematicidal agent, avermectin (AVM) has been widely used in agricultural production and stock farming areas. Subsequently, the residues of AVM or its active metabolites in animal manure pose a toxic threat to non-target organisms in the environment. As the most characteristic epigenetic phenomena, DNA methylation status is a useful biological signal for the toxicity assessment of environmental chemical toxicants. In this study, analyses of the overall level of genomic DNA methylation were performed, and the expression levels of DNA methyltransferases (DNMTs), as well as demethylase methyl-CpG-binding domain protein 2 (MBD2), in pigeon brain tissues after subchronic exposure (with a AVM concentration of 20mg/kg, 40mg/kg and 60mg/kg, respectively) to AVM for 30, 60 and 90days were investigated. Global DNA hypomethylation and down-regulation of DNMT mRNA expression occurred in a dose–time-dependent manner in pigeon brains. The expression level of MBD2, which functions as a demethylase, was significantly enhanced in a dose-dependent but not time-dependent manner. In addition, the elevated expression level of MBD2 had a more robust effect on genomic DNA hypomethylation compared to changes in DNMT expression. Taken together, these results suggested that subchronic dose exposures of AVM could affect the global DNA methylation status, and this mechanism is closely related to changes in the expression levels of DNMTs and MBD2.

Prenatal caffeine ingestion induces aberrant DNA methylation and histone acetylation of steroidogenic factor 1 and inhibits fetal adrenal steroidogenesis

Prenatal caffeine ingestion is one of the risk factors for intrauterine growth retardation (IUGR). Adrenal plays a pivotal role, mainly through steroidogenesis, in the regulation of intrauterine homeostasis and in fetal development and maturation. We have shown that prenatal caffeine ingestion can inhibit fetal adrenal corticosterone production, but the underlying mechanism is unknown. This study investigated the effects of prenatal caffeine ingestion on corticosterone and its associated synthesized enzymes (steroidogenic acute regulatory protein, StAR; 3β-hydroxysteroid dehydrogenase, 3β-HSD; cytochrome P450 cholesterol side chain cleavage, P450scc; P450c21; and P450c11) in the fetal adrenal in rats and further explored the underlying mechanism by analyzing the epigenetic modification and expression of the key transcription factor steroidogenic factor-1 (SF-1). The pregnant rats were intragastrically treated with 120mg/kg.d caffeine from gestational day 11–20. The results showed that the IUGR rate was 51.2% after caffeine treatment. The contents of corticosterone and the mRNA levels of StAR, P450scc, P450c21, and P450c11 were decreased significantly in the fetal adrenal. Furthermore, caffeine reduced both the protein and the mRNA expression of SF-1 in the fetal adrenal. The epigenetic analysis showed that caffeine treatment can significantly enhance the mRNA expression of DNA methyltransferase (Dnmt) 1, Dnmt3a, histone deacetylases (Hdac) 1, and Hdac2. The detection of DNA methylation by bisulfite-sequencing PCR uncovered a notably increased total methylation rate in the SF-1 promoter. The ChIP assay showed decreased acetylation levels of H3K9 and H3K14 in the SF-1 promoter. In conclusion, prenatal caffeine ingestion is able to induce aberrant DNA methylation and histone acetylation of the SF-1 promoter in the rat fetal adrenal. These effects may contribute to the inhibition of the expression of SF-1 and its associated steroidogenic enzymes and the production of corticosterone during fetal development.

Folate Deficiency and Aberrant Expression of DNA Methyltransferase 1 were Associated with Cervical Cancerization

DNA methyltransferase 1 (DNMT1) plays a significant role in maintaining DNA methylation. Aberrant DNA methylation is a recognized feature of human cancers and folate is directly involved in DNA methylation via one-carbon metabolism. Previous reports also have suggested that folate deficiency was associated with many cancers. The aim of the present study was to evaluate the effect of folate deficiency and aberrant expression of DNA methyltransferase 1 (DNMT1) on cervical cancerization. The expression of DNMT1 protein and mRNA and levels of serum folate were detected in 238 women with a diagnosis of normal cervix (NC，n = 53), cervical intraepithelial neoplasia (CIN I, n = 52; CIN II/III, n = 53), and squamous cell carcinoma of the cervix (SCC; n = 80). In addition, the expression of DNMT1 protein and mRNA was measured in cervical cancer cells (Caski and C33A) treated by different concentration of folate. Serum folate levels decreased and expression levels of DNMT1 protein and mRNA increased gradually with progressive severity of the cervix lesions (P<0.001). It was found that folate was able to reduce the viability of Caski or C33A cell (r=0.978, P=0.002; r=0.984, P<0.001) and regulated aberrant expression of DNMT1 protein (r=-0.859, P=0.01; r=-0.914, P<0.001) and mRNA (r=-0.297, P=0.159; r=0.433, P=0.034) in vitro. Our findings indicated that the low-level of serum folate and high-expression of DNMT1 protein or mRNA was significantly associated with cervical carcinogenesis. Folate deficiency and aberrant expression of DNA methyltransferase 1 had additive effect on cervical cancerization. Folate supplement and recovery of aberrant DNA methylation status may offer a new strategy for prevention and therapy of cancers.

2,4-Dichlorophenol induces global DNA hypermethylation through the increase of S-adenosylmethionine and the upregulation of DNMTs mRNA in the liver of goldfish Carassius auratus

Altered DNA methylation is associated with changes in gene expression, signal transduction and stress response after exposure to a wide range of exogenous compounds, and abnormal methylation is a major toxic effect induced by chemicals such as benzene and phenols. 2,4-Dichlorophenol (2,4-DCP), a derivative of phenol, has been classified as a priority pollutant by the US EPA due to its toxic effects on aquatic organisms. However, the effect of 2,4-DCP on DNA methylation and its potential mechanism in fish are rarely understood. The present study aims to figure out whether 2,4-DCP could impact DNA methylation and explore its potential mechanisms by measuring the global DNA methylation levels, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) contents, the mRNA expression of DNA methyltransferase1 (DNMT1) and DNA methyltransferase3 (DNMT3) in the liver of goldfish Carassius auratus. DNA methylation levels were analyzed using high performance liquid chromatography (HPLC) and MspI/HpaII ethidium bromide assay, SAM and SAH contents were determined by HPLC, the mRNA expression of DNMT1 and DNMT3 was measured by quantitative-PCR (qPCR). The results showed that 2,4-DCP caused global DNA hypermethylation, elevated the methylation levels of CpG islands, increased the SAM and SAH contents, decreased the SAM/SAH ratio, and upregulated the mRNA expression of DNMT1 and DNMT3, while depletion of SAM with Na2SeO3 and inhibition of DNMTs activity with 5-aza-2′-deoxycytidine (5AdC) impaired 2,4-DCP-induced global DNA hypermethylation, suggesting that the increase of SAM contents and upregulation of the mRNA expression of DNMT1 and DNMT3 may play important roles in 2,4-DCP-induced global DNA hypermethylation process. Our report is the first one to show that short-term 2,4-DCP exposure caused the global DNA hypermethylation via altered SAM level and DNMTs expression in fish.

Effect of DNA methyltransferase 1 and methyl-CpG-binding protein 2 abnormal expression on cervical lesions and related interaction

To explore the effect of DNA methyltransferase 1 (DNMT1) and methyl-CpG-binding protein 2 (MeCP2) on cervical cancer and cervix precancerous lesion. 74 patients with cervix squamous cell carcinoma(SCC), 52 patients with cervical intraepithelial neoplasm I (CIN I), 60 patients with cervical intraepithelial neoplasm II - II (CIN II-III)and 58 patients with histologically diagnosed cervix inflammation(CI), were included in this study. Information as demography, reproductive history, life style, HPV infection were collected. Western Blot were used to detect the expression of DNMT1 protein and MeCP2 protein. Real-time PCR was used to detect the expression of DNMT1 and MeCP2 mRNA. Levels of DNMT1 and MeCP2 protein expression increased gradually with the deterioration of cervical lesion (H = 94.33, P < 0.001;F = 21.580, P < 0.001). Along with the deterioration of cervical lesion, levels of DNMT1 and MeCP2 mRNA expression were gradually increasing( F = 4.758, P = 0.003; F = 7.804, P < 0.001). Data from Correlation analysis showed that both protein (r = 0.287, P < 0.001) and mRNA(r = 0.179, P = 0.005)were positive correlated with DNMT1 and MeCP2. of our study indicated that there was an additive interaction between high-expression of DNMT1 protein and high-expression of MeCP2 protein in SCC or CIN II-III. However, there was an additive interaction between high-expression of DNMT1 mRNA and high-expression of MeCP2 mRNA in SCC or CIN II-III. Results from our study revealed the fact that both high expression of DNMT1 protein and high expression of MeCP2 protein could increase the risk of cervix cancerization. According to our findings, there might be a synergistic action existed between DNMT1 and MeCP2 during the progression of cervix cancelation.

Role of NADPH oxidase NOX5-S, NF-κB, and DNMT1 in acid-induced p16 hypermethylation in Barrett's cells

Inactivation of tumor suppressor genes via promoter hypermethylation may play an important role in the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). We have previously shown that acid-induced p16 gene promoter hypermethylation may depend on activation of NADPH oxidase NOX5-S in BAR-T cells and OE33 EA cells. DNA methyltransferase 1 (DNMT1) is known to participate in maintaining established patterns of DNA methylation in dividing cells and may play an important role in the development of cancer. Therefore, we examined whether DNMT1 is involved in acid-induced p16 gene promoter hypermethylation in BAR-T cells. We found that the acid significantly increased p16 gene promoter methylation, decreased p16 mRNA, and increased cell proliferation, effects that may depend on activation of DNMT1 in BAR-T cells. DNMT1 is overexpressed in EA cells FLO and OE33 and EA tissues. Acid treatment upregulated DNMT1 mRNA expression and increased DNMT1 promoter activity. Acid-induced increases in DNMT1 mRNA expression and promoter activity were significantly decreased by knockdown of NOX5-S and NF-κB1 p50. Conversely, overexpression of NOX5-S, p50, or p65 significantly increased DNMT1 promoter activity. Knockdown of NOX5-S significantly decreased the acid-induced increase in luciferase activity in cells transfected with pNFκB-Luc. An NF-κB binding element GGGGTATCCC was identified in the DNMT1 gene promoter. We conclude that the acid-induced increase in p16 gene promoter methylation, downregulation of p16 mRNA, and increase in cell proliferation may depend on activation of DNMT1 in BAR-T cells. Acid-induced DNMT1 expression may depend on sequential activation of NOX5-S and NF-κB1 p50.

Ultraviolet B enhances DNA hypomethylation of CD4+ T cells in systemic lupus erythematosus via inhibiting DNMT1 catalytic activity

BackgroundCD4+ T cells DNA hypomethylation is involved in the pathogenesis of systemic lupus erythematosus (SLE). Recent studies showed that ultraviolet B (UVB, 290–320nm) might induce the exacerbation of SLE by decreasing the DNA methylation level. However, the role of DNA methyltransferase 1 (DNMT1) in the UVB-induced CD4+ T cells DNA hypomethylation remains unclear. ObjectiveTo elucidate the role of DNMT1 in lupus CD4+ T cells global DNA hypomethylation enhanced by UVB. Methods35 SLE patients and 15 healthy controls were enrolled in the study. CD4+ T cells from SLE patients and healthy controls exposed to different dosages of UVB were analyzed. The global DNA methylation measurement, real-time PCR, Western blotting and DNMT1 catalytic activity detection were employed. ResultsThe level of global DNA methylation and DNMT1 mRNA expression in CD4+ T cells from SLE patients were significantly lower than those from the control group. DNA methylation was decreased after UVB exposure in a dosage-dependent manner in SLE patients, but not in the control group. DNMT1 mRNA and protein expression level were not affected by UVB exposure in both SLE patients and healthy controls. DNMT1 catalytic activity was significantly decreased in CD4+ T cells from SLE patients after UVB exposure in a dosage-dependent manner. DNMT1 catalytic activity was lower and more sensitive to UVB exposure in CD4+ T cells from active SLE patients that from stable ones. ConclusionUVB enhanced DNA hypomethylation of CD4+ T cells in SLE via inhibiting DNMT1 catalytic activity in a dosage-dependent manner.

Abstract 5375: Androgen excess induces aberrant DNA methylation in the prostate.

Abstract Aberrant DNA methylation is deeply involved in prostate carcinogenesis. However, inducers of aberrant DNA methylation in the prostate are almost unknown. Here, we investigated whether androgen excess can induce aberrant DNA methylation or not, using a rat prostate cancer model. First, by analysis of reported data of expression microarray after treatment of a prostate cancer cell line with a demethylating agent, we isolated two marker genes (Mmp23, Nkx3.1) whose promoter CpG islands were aberrantly methylated in testosterone and 3,2′-dimethyl-4-aminobiphenyl-induced invasive adenocarcinomas. These two genes and three genes previously isolated (Aebp1, Amn1, Tgfbr2) [Yamashita et al. Cancer Res. 2008] were used as markers. Then, we analyzed temporal profiles of DNA methylation levels of the five genes in the dorsolateral lobe of rats treated with excessive androgen. Testosterone was administrated every five weeks for 15, 30, 40 and 50 weeks from six weeks old by subcutaneous implantation of a Silastic tube containing 40 mg testosterone propionate. Aberrant DNA methylation of two genes (Amn1, Mmp23) was gradually induced during the testosterone treatment while that of the other three genes was not. The former two genes had lower mRNA expression levels in the normal prostate than the latter three genes, which was in accordance with the known fact that genes with low transcription are susceptible to DNA methylation. Third, we investigated molecular mechanisms for aberrant DNA methylation induction by androgen excess. Androgen excess did not induce mRNA expression of DNA methyltransferases; Dnmt1, Dnmt3a or Dnmt3b in the rat prostrate. As for histone modifications, it did not induce significant changes in either H3K4me3 or H3K27me3 levels of the five genes methylated in rat prostate cancer. In contrast, it induced infiltration of lymphocytes and neutrophils in the prostate. Temporal profiles of expression levels of specific inflammation-related genes (Cxcl2, Il1b, Nos2 and Tnf) paralleled the methylation levels of Amn1 and Mmp23. Androgen excess did not induce bacterial infection as determined by copy number of bacterial DNA, but induced increase in Ki67-positive cells in regions with and without infiltration of inflammatory cells. The inflammatory response, known as a major mechanism for aberrant DNA methylation induction, along with cell proliferation was likely to be the cause of aberrant DNA methylation induction in the rat prostate with androgen excess. These results demonstrated that androgen excess can induce aberrant DNA methylation in the prostate, and suggested that inflammatory response underlies aberrant DNA methylation induction by androgen excess. Citation Format: Satoshi Yamashita, Satoru Takahashi, Yasunori Matsuda, Ken Gyobu, Toshikazu Ushijima. Androgen excess induces aberrant DNA methylation in the prostate. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5375. doi:10.1158/1538-7445.AM2013-5375

Expression and significance of DNMT1 in human keloid fibroblast

To investigate the expression and significance of DNA methyltransferase 1 (DNMT1) in different parts and formation time of keloid fibroblasts. Specimen were collected from fifty-three patients with keloid. The keloids were grouped according to formation time. Immunohistochemistry was used to assay the expression of DNMT1 in normal skin, invasive part, proliferative part and aged part. RT-PCR was adopted to assay the expression of DNMT1 mRNA in each group. DNMT1 is mainly distributed in the nucleus of fibroblasts. The expression of DNMT1 protein and DNMTl mRNA in keloid (72%) is higher than that in normal skin fibroblasts (8%) (P = 0.001). The expression rate of DNMT1 protein and DNMT1 mRNA in shorter formation time group (100%, Score:10.47 +/- 1.85) is higher than a longer formation time group (45%,Score: 7.71 +/- 1.80) (P = 0.007). The expression rate of DNMT1 protein and DNMT1 mRNA in the invasive part of keloid (100%, Score: 10.47 +/- 1.85) is higher than the proliferative part (78%, Score: 6.63 +/- 1.75) and aged part (38%, Score: 5.53 +/- 1.55) (P = 0.001). DNMT1 plays an important role in keloid. The expression of DNMT1 tends to decrease along with the increase of the formation time in keloid. It is also related to the part of keloid. DNMT1 plays an important role in the process of keloid growth and is closely related to infiltrative growth in keloid.

Effects of ultraviolet B exposure on DNA methylation in patients with systemic lupus erythematosus

The aim of this study was to investigate the effects of ultraviolet B (UVB) exposure on DNA methylation in patients with systemic lupus erythematosus (SLE) and its significance in the pathogenesis of SLE. T cells from 35 SLE patients and 21 healthy individuals were cultured and irradiated with UVB. The global DNA methylation profiles of the T cells obtained from the patients and controls following irradiation with UVB were assessed using specific monoclonal antibodies for 5-methylcytosine and analyzed quantitatively through flow cytometry. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the levels of DNA methyltransferase 1 (DNMT1) and methyl CpG binding domain protein 2 (MBD2) in T cells from the patients and controls following UVB irradiation. Significant global DNA hypomethylation was observed in the SLE patients compared with the controls (P<0.01). The SLE patients also had significantly lower levels of DNMT1 mRNA expression (P<0.01) and significantly higher levels of MBD2 mRNA compared with the controls (P<0.01). DNA methylation was decreased following UVB irradiation at two different dosages and the DNA methylation levels of the patients with active SLE were more sensitive to UVB. The level of DNMT1 mRNA was decreased following UVB irradiation at the higher dosage in the patients with active SLE, but no significant difference was observed in MBD2 mRNA expression. UVB exposure is able to inhibit DNA methylation and DNMT1 mRNA expression, which is subsequently involved in the epigenetic mechanism of SLE. The process by which DNA hypomethylation occurs in patients with SLE is complicated and the multiple factors that are involved in DNA methylation and demethylation events require further study.

Effect of folate in modulating the expression of DNA methyltransferase 1 and methyl-CpG-binding protein 2 in cervical cancer cell lines

To explore the effects of folate on the expression of DNA methyltransferase 1 (DNMT1) and methyl-CpG-binding protein 2 (MeCP2) in cervical cancer cell lines. Experimental study was carried out in vitro. Human cervical cancer cell lines, including C33A cell with HPV negative and Caski cell with HPV16 positive, were treated with different concentration of folate. The expression of DNMT1 and MeCP2 protein (by Western blot) and mRNA (by real-time PCR) were then detected in the two cell lines. It was found that supplement of folate was able to reduce the cell proliferation in C33A cell (r = 0.984, P < 0.001) and Caski cell (r = 0.978, P = 0.002), as well as induced the cell apoptosis (C33A: r = 0.989, P < 0.001; Caski: r = 0.994, P < 0.001). showed that the expression levels of DNMT1 protein (C33A: r = -0.914, P < 0.001; Caski: r = -0.859, P = 0.003) and MeCP2 protein (C33A: r = -0.830, P = 0.005; Caski: r = -0.981, P < 0.001) decreased gradually with the increase of folate concentrations, but the expression of DNMT1 and MeCP2 mRNA was not observed in Caski or C33A cell. When at the same levels of folate, the expression of DNMT1 protein or mRNA was higher in Caski cell than in C33A cell. However, the expression of MeCP2 protein or mRNA was higher in C33A cell than in Caski cell. Our finding indicated that adequate folate could effectively inhibit the proliferation of cervical cancer cells and facilitate their apoptosis in vitro, thus would reverse the aberration protein expression of DNMT1 and MeCP2. That there might be a synergistic action between HPV16 infection and parafunction of DNMT1 in cervical cancer, being noticed.

Significance of aberrant expression of DNA methyltransferase 1 in cervical cancerization tissue and cervical cancer cell lines

Objective To explore the effects of aberrant expression of DNA methyltransferase 1 (DNMT1) in cervical cancerization tissue and cervical cancer cells.Methods Cervical tissues were collected from 80 cases with a diagnosis of invasive cervix squamous cell carcinoma (SCC),53 cases with high-grade cervical intraepithelial neoplasia (CIN Ⅱ/Ⅲ),52 cases with low-grade cervical intraepithelial neoplasia (CIN Ⅰ)and 53 cases with normal cervix (NC).Meanwhile,Caski (HPV16-positive) and C33A (HPV-negative) cells selected from cervical cancer cell lines were cultured routinely in vitro.The expression of DNMT1 protein and mRNA were examined by Western blot analysis and real-time PCR (qRT-PCR) in the tissues and cells,respectively.Results The levels of DNMT1 protein were 1.33,1.84 and 2.28,and the Ct-ratios (DNMT1/β-actin) of DNMT1 mRNA were 1.27,1.27 and 1.26 in CIN Ⅰ,CIN Ⅱ/Ⅲand SCC group,respectively.Comparing with NC group,the expression of DNMT1 protein or mRNA was elevated in deficient cervical groups,with statistical significance (F =110.57,P ＜ 0.001,F =2.68,P =0.048).The expression levels of DNMT1 protein were increased steadily according to severity of the cervix lesions (x2tend =50.80,P ＜ 0.001),however,the expression of DNMT1 mRNA was not observed the same tendency (x2tend =3.63,P ＞ 0.05).The results from experiment in vitro showed that the levels of DNMT1 protein or mRNA were both higher in Caski cell than in C33A cell,especially for DNMT1 mRNA with significantly difference (t =7.134,P =0.002).Conclusion Aberrant expression of DNMT1 protein or mRNA could link with the risk of cervical cancerization by both transcriptional and posttranscriptional mechanisms.There would be a synergistic effect between overexpression of DNMT1 and HPV16 infection in the progression of cervix carcinogenesis. Key words: DNA methyltransferase 1; Aberrant expression; Cervical cancerization; Cervical cancer cells