Induces Promoter Hypermethylation Research Articles

DNMT1/DNMT3a-mediated promoter hypermethylation and transcription activation of ICAM5 augments thyroid carcinoma progression.

Promoter methylation is one of the most studied epigenetic modifications and it is highly relevant to the onset and progression of thyroid carcinoma (THCA). This study investigates the promoter methylation and expression pattern of intercellular adhesion molecule 5 (ICAM5) in THCA. CpG islands with aberrant methylation pattern in THCA, and the expression profiles of the corresponding genes in THCA, were analyzed using bioinformatics. ICAM5 was suggested to have a hypermethylation status, and it was highly expressed in THCA tissues and cells. Its overexpression promoted proliferation, mobility, and tumorigenic activity of THCA cells. As for the downstream signaling, ICAM5 was found to activate the MAPK/ERK and MAPK/JNK signaling pathways. Either inhibition of ERK or JNK blocked the oncogenic effects of ICAM5. DNA methyltransferases 1 (DNMT1) and DNMT3a were found to induce promoter hypermethylation of ICAM5 in THCA cells. Knockdown of DNMT1 or DNMT3a decreased the ICAM5 expression and suppressed malignant properties of THCA cells in vitro and in vivo, which were, however, restored by further artificial ICAM5 overexpression. Collectively, this study reveals that DNMT1 and DNMT3a mediates promoter hypermethylation and transcription activation of ICAM5 in THCA, which promotes malignant progression of THCA through the MAPK signaling pathway.

Hepatitis B virus X protein and hepatitis C virus core protein cooperate to repress E-cadherin expression via DNA methylation

Dual infection of hepatitis B virus (HBV) and hepatitis C virus (HCV) is closely associated with an increased risk of hepatocellular carcinoma; however, the underlying mechanism is poorly understood. In the present study, we found that HBV X protein (HBx) and HCV core protein work together to inhibit E-cadherin expression in human hepatoma cells. For this effect, they additively increased both the level and activity of enzymes, DNA methyltransferase 1, 3a, and 3b to induce promoter hypermethylation of E-cadherin in a p53-dependent fashion. Their additive effect on E-cadherin levels was reproduced in an in vitro HBV/HCV dual infection system using Huh7D-NTCP cells. As a result, HBV and HCV additively upregulated mesenchymal marker such as N-cadherin, Snail, Twist and Vimentin but cooperatively downregulated epithelial markers such as E-cadherin, Slug and Plakoglobin. In addition, the coinfected cells exhibited faster cell migration and higher invasion ability, as compared with monoinfection, which are hallmarks of epithelial-mesenchymal transition required for the initiation of metastasis in cancer progression.

Organochlorine pesticides induce promoter hypermethylation of MGMT in papillary thyroid carcinoma

BackgroundEnvironmental factors namely pesticides may contribute to the development of cancer through epigenetic mechanisms. In order to evaluate the impact of organochlorine pesticides (OCPs) on promoter methylation of O6-methylguanine-DNA methyltransferase (MGMT) in thyroid cancer, this research was designed. MethodsIn this study, DNA methylation profile of 61 papillary thyroid carcinoma (PTC) and 70 benign thyroid nodules (BTN) subjects was carried out. In order to measure MGMT gene promoter methylation, methylation-specific PCR (MSP) was used. ResultsHypermethylation of MGMT marker were found in PTC compare with BTN. Also, OCPs increased the odds ratio of methylation of MGMT (OR = 3.66, P = 0.001). Logistic regression analysis showed that OCPs enhance the methylation of MGMT. ConclusionThe results indicated that OCPs lead to PTC development through MGMT gene promoter hypermethylation. Furthermore, MGMT promoter methylation assessment could forecast PTC incidence.

TET-mediated DNA demethylation plays an important role in arsenic-induced HBE cells oxidative stress via regulating promoter methylation of OGG1 and GSTP1

Environmental exposure to arsenic remains a worldwide public health challenge. Oxidative stress and aberrant DNA methylation are both characteristics of arsenic toxicology; however, the relationship between these is not well understood. Ten-eleven translocation (TET1, TET2 and TET3), which is the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), plays a central role in the DNA demethylation process. Further, it can prevent cytosine-phosphate-guanine (CpG) islands from developing abnormal hypermethylation under oxidative stress. Here, we observed that NaAsO2 could induce oxidative stress in human bronchial epithelial (HBE) cells. This was accompanied by an inhibition of TET-mediated DNA demethylation. Subsequent results showed that TET1 and TET2 siRNA led to further inhibition of genome 5hmC and a higher level of oxidative stress in NaAsO2-treated HBE cells. Conversely, l-ascorbic acid enhanced TET proteins and effectively upregulated 5hmC, which antagonized the NaAsO2-induced oxidative stress. Additionally, the TETs positively regulated the promoter methylation of the antioxidant genes 8-oxoguanine DNA glycosylase (OGG1) and glutathione S-transferase Pi 1 (GSTP1). Taken together, the results indicate that arsenic induced the inhibition of TET-mediated DNA demethylation, which induced promoter hypermethylation, inhibiting the expression of the OGG1 and GSTP1, and increasing oxidative stress in lung cells in vitro. l-ascorbic acid effectively alleviated arsenic-induced oxidative stress by restoring TET function.

DNMT3a mediates paclitaxel-induced abnormal expression of LINE-1 by increasing the intragenic methylation.

The activation of long interspersed nuclear element-1 (LINE-1) leads to genomic instability, which promotes carcinogenesis and drug resistant. Therefore, exploring the mechanism underlying LINE-1 abnormal activation has the theoretical and clinical significance. DNA methylation is an important way to regulate gene expression. DNMT3a, one member of the DNA methyltransferase family, not only inhibits gene expression by inducing promoter hypermethylation, but also activates gene expression by increasing the intragenic DNA methylation. Our previous studies found that the expression of LINE-1 did not increase significantly in the promoter methylation in breast cancer cells treated with paclitaxel (PTX), a first-line chemotherapeutic drug for breast cancer. Here we explored whether DMNMT3a could directly mediate the drug-induced activation of LINE-1 in breast cancer cells through increasing the LINE-1 intragenic methylation. Our ChIP experiments and methyl analysis showed that treatment of breast cancer cells with PTX not only induced DNMT3a expression, but also promoted the binding of DNMT3a to the inner region of the LINE-1 gene to increase its methylation, resulting in upregulation of LINE-1 expression. Using expression vectors or RNA interference to alter the DNMT3a expression levels in the cells significantly changed the intragenic methylation degree and LINE-1 expression. Moreover, down-regulation of DNMT3a expression effectively inhibited the expression of LINE-1. These results indicate that DNMT3a-mediated intragenic methylation plays an important role in drug-induced abnormal activation of LINE-1, which provides a new idea for understanding the mechanism of abnormal activation of Line-1 induced by chemotherapy drug stress in breast cancer cells.

Fusaric acid-induced promoter methylation of DNA methyltransferases triggers DNA hypomethylation in human hepatocellular carcinoma (HepG2) cells

ABSTRACTFusaric acid (FA), a mycotoxin contaminant of maize, displays toxicity in plants and animals; however, its epigenetic mechanism is unknown. DNA methylation, an epigenetic modification that regulates gene expression, is mediated by DNA methyltransferases (DNMTs; DNMT1, DNMT3A, and DNMT3B) and demethylases (MBD2). The expression of DNMTs and demethylases are regulated by promoter methylation, microRNAs (miR-29b) and post-translational modifications (ubiquitination). Alterations in these DNA methylation modifying enzymes affect DNA methylation patterns and offer novel mechanisms of FA toxicity. We determined the effect of FA on global DNA methylation as well as a mechanism of FA-induced changes in DNA methylation by transcriptional (promoter methylation), post-transcriptional (miR-29b) and post-translational (ubiquitination) regulation of DNMTs and MBD2 in the human hepatocellular carcinoma (HepG2) cell line. FA induced global DNA hypomethylation (p < 0.0001) in HepG2 cells. FA decreased the mRNA and protein expression of DNMT1 (p < 0.0001), DNMT3A (p < 0.0001), and DNMT3B (p < 0.0001) by upregulating miR-29b (p < 0.0001) and inducing promoter hypermethylation of DNMT1 (p < 0.0001) and DNMT3B (p < 0.0001). FA decreased the ubiquitination of DNMT1 (p = 0.0753), DNMT3A (p = 0.0008), and DNMT3B (p < 0.0001) by decreasing UHRF1 (p < 0.0001) and USP7 (p < 0.0001). FA also induced MBD2 promoter hypomethylation (p < 0.0001) and increased MBD2 expression (p < 0.0001). Together these results indicate that FA induces global DNA hypomethylation by altering DNMT promoter methylation, upregulating miR-29b, and increasing MBD2 in HepG2 cells.

Hepatic cyclooxygenase-2 overexpression induced spontaneous hepatocellular carcinoma formation in mice

Cyclooxygenase (COX)-2 is upregulated in hepatocellular carcinoma (HCC). However, the direct causative effect of COX-2 in spontaneous HCC formation remains unknown. We thus investigate the role and molecular pathogenesis of COX-2 in HCC by using liver-specific COX-2 transgenic (TG) mice. We found spontaneous HCC formation with elevated inflammatory infiltrates and neovessels in male TG mice (3/21, 14.3%), but not in any of male WT mice (0/19). Reduced representation bisulfite sequencing (RRBS) and gene expression microarrays were performed in the HCC tumor and non-HCC liver tissues to investigate the molecular mechanisms of COX-2-driven HCC. By RRBS, DNA promoter hypermethylation was identified in HCC from TG mice. Induction of promoter hypermethylation was associated with reduced tet methylcytosine dioxygenase 1 (TET1) expression by COX-2. TET1 could catalyze the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC) and prevents DNA hypermethylation. In keeping with this, loss of 5hmC was demonstrated in COX-2-induced HCC. Consistently, COX-2 overexpression in human HCC cell lines could reduce both TET1 expression and 5hmc levels. Integrative analyses of DNA methylation and gene expression profiles further identified significantly downregulated genes including LTBP1, ADCY5 and PRKCZ by promoter methylation in COX-2-induced HCC. Reduced expression of LTBP1, ADCY5 and PRKCZ by promoter hypermethylation was further validated in human HCCs. Bio-functional investigation revealed that LTBP1 inhibited cell proliferation in HCC cell lines, suggesting its potential role as a tumor suppressor in HCC. Gene expression microarrays revealed that signaling cascades (AKT (protein kinase B), STK33 (Serine/Threonine kinase 33) and MTOR (mechanistic target of rapamycin) pathways) were enriched in COX-2-induced HCC. In conclusion, this study demonstrated for the first time that enhanced COX-2 expression in hepatocytes is sufficient to induce HCC through inducing promoter hypermethylation by reducing TET1, silencing tumor-suppressive genes and activating key oncogenic pathways. Inhibition of COX-2 represents a mechanism-based target for HCC prevention.

Methylated oligonucleotide (MON)-induced promoter hypermethylation is associated with repression of CDH1 expression and contributes to the migration and invasion of human trophoblast cell lines.

DNA cytosine-5 methylation plays a vital role in regulating the expression of E-cadherin, which is encoded by the CDH1 gene. In this study, we characterised the DNA methylation and expression pattern of CDH1 in an extravillous trophoblast cell line (HTR-8/SVneo) and two trophoblast cell lines -- JEG-3 and JAR. Promoter hypermethylation with reduced E-cadherin expression in HTR-8/SVneo cells and promoter hypomethylation with increased E-cadherin expression in JEG-3 and JAR cells were observed. Demethylation treatment significantly restored E-cadherin expression, contributing to decreases in the motility and invasiveness of HTR-8/SVneo cells. Sense-methylated oligonucleotides (MONs) labelled with Cy5 and complementary to a region of the human CDH1 promoter were designed, with the cytosines in 5'-cytosine-phosphate-guanine-3' (CpG) dinucleotides being replaced by methylated cytosines. Following MON transfection into JEG-3 cells, the level of CDH1 promoter DNA methylation as well as cell motility and invasiveness were increased and gene expression was significantly repressed. Our results indicate that MON-mediated DNA methylation of the CDH1 promoter and subsequent alterations in gene expression may contribute to trophoblast motility and invasion, suggesting a potential method for controlling the biological function of trophoblasts in vitro through epigenetic modification.

Hepatitis C virus core protein modulates pRb2/p130 expression in human hepatocellular carcinoma cell lines through promoter methylation.

BackgroundHepatitis C Virus (HCV) infection is associated with chronically evolving disease and development of hepatocellular carcinoma (HCC), albeit the mechanism of HCC induction by HCV is still controversial. The nucleocapsid (core) protein of HCV has been shown to be directly implicated in cellular transformation and immortalization, enhancing the effect of oncogenes and decreasing the one of tumor suppressor genes, as RB1 and its protein product pRB. With the aim of identifying novel molecular mechanisms of hepatocyte transformation by HCV, we examined the effect of HCV core protein on the expression of the whole Retinoblastoma (RB) family of tumor and growth suppressor factors, i.e. pRb, p107 and pRb2/p130.MethodsWe used a model system consisting of the HuH-7, HCV-free, human hepatocellular carcinoma cell line and of the HuH-7-CORE cells derived from the former and constitutively expressing the HCV core protein. We determined pRb, p107 and pRb2/p130 protein and mRNA amount of the respective genes RB1, RBL1 and RBL2, RBL2 promoter activity and methylation as well as DNA methyltransferase 1 (DNMT1) and 3b (DNMT3b) expression level. The effect of pRb2/p130 over-expression on the HCV core-expressing HuH-7-CORE cells was also evaluated.ResultsWe found that the HCV core protein expression down-regulated pRb2/p130 protein and mRNA levels in HuH-7-CORE cells by inducing promoter hyper-methylation with the concomitant up-regulation of DNMT1 and DNMT3b expression. When pRb2/p130 expression was artificially re-established in HuH-7-CORE cells, cell cycle analysis outlined an accumulation in the G0/G1 phase, as expected.ConclusionsHCV core appears indeed able to significantly down-regulate the expression and the function of two out of three RB family tumor and growth suppressor factors, i.e. pRb and pRb2/p130. The functional consequences at the level of cell cycle regulation, and possibly of more complex cell homeostatic processes, may represent a plausible molecular mechanism involved in liver transformation by HCV.

Hepatitis C virus represses E-cadherin expression via DNA methylation to induce epithelial to mesenchymal transition in human hepatocytes

Hepatitis C virus (HCV) core protein is known to induce promoter hypermethylation of tumor suppressor genes including E-cadherin to repress their expression when overexpressed in human hepatocytes; however, its actual role during HCV infection is still unknown. Here, we report that infection with HCV derived from pJFH-1 replicon system that mimics natural infection elevates protein levels of DNA methyltransferase 1 and 3b to enhance DNMT activity in human hepatocytes. As a consequence, HCV induced promoter hypermethylation of E-cadherin, resulting in repression of its expression. In addition down-regulation of E-cadherin by HCV led to epithelial–mesenchymal transition that is known to be a critical event during the late stage of tumorigenesis.

Hepatic DNA methylation modifications in early development of rats resulting from perinatal BPA exposure contribute to insulin resistance in adulthood

Perinatal exposure to bisphenol A (BPA), a widely distributed environmental endocrine disruptor, is associated with insulin resistance and diabetes in offspring. The underlying molecular mechanisms could involve epigenetics, as adverse effects induced by environmental exposure in early life are suggested through DNA methylation. In this study we sought to elucidate the relationship between perinatal BPA exposure and alteration of hepatic DNA methylation. Pregnant Wistar rats were administered BPA (50 μg/kg/day) or corn oil by oral gavage throughout gestation and lactation. Variables associated with insulin resistance and hepatic DNA methylation were examined at postnatal week 3 and week 21 in male offspring. In BPA-treated offspring, serum insulin and HOMA-insulin resistance were increased, and the insulin sensitivity index and hepatic glycogen storage were decreased compared with controls at week 21. At week 3, none of these variables were significantly changed. However, hepatic global DNA methylation was decreased, accompanied by overexpression of DNA methyltransferase 3B mRNA at week 3. Meanwhile, perinatal exposure to BPA induced promoter hypermethylation and a reduction in gene expression of hepatic glucokinase. Moreover, increased promoter hypermethylation of Gck became more pronounced in BPA-treated offspring at week 21. Abnormal DNA methylation in hepatic tissue precedes development of insulin resistance induced by perinatal BPA exposure. These findings support the potential role of epigenetics in fetal reprogramming by BPA-induced metabolic disorders.

Epigenetic, Genetic and Environmental Interactions in Esophageal Squamous Cell Carcinoma from Northeast India

BackgroundEsophageal squamous cell carcinoma (ESCC) develops as a result of complex epigenetic, genetic and environmental interactions. Epigenetic changes like, promoter hypermethylation of multiple tumour suppressor genes are frequent events in cancer, and certain habit-related carcinogens are thought to be capable of inducing aberrant methylation. However, the effects of environmental carcinogens depend upon the level of metabolism by carcinogen metabolizing enzymes. As such key interactions between habits related factors and carcinogen metabolizing gene polymorphisms towards modulating promoter methylation of genes are likely. However, this remains largely unexplored in ESCC. Here, we studied the interaction of various habits related factors and polymorphism of GSTM1/GSTT1 genes towards inducing promoter hypermethylation of multiple tumour suppressor genes.Methodology/Principal FindingsThe study included 112 ESCC cases and 130 age and gender matched controls. Conditional logistic regression was used to calculate odds ratios (OR) and multifactor dimensionality reduction (MDR) was used to explore high order interactions. Tobacco chewing and smoking were the major individual risk factors of ESCC after adjusting for all potential confounding factors. With regards to methylation status, significantly higher methylation frequencies were observed in tobacco chewers than non chewers for all the four genes under study (p<0.01). In logistic regression analysis, betel quid chewing, alcohol consumption and null GSTT1 genotypes imparted maximum risk for ESCC without promoter hypermethylation. Whereas, tobacco chewing, smoking and GSTT1 null variants were the most important risk factors for ESCC with promoter hypermethylation. MDR analysis revealed two predictor models for ESCC with promoter hypermethylation (Tobacco chewing/Smoking/Betel quid chewing/GSTT1 null) and ESCC without promoter hypermethylation (Betel quid chewing/Alcohol/GSTT1) with TBA of 0.69 and 0.75 respectively and CVC of 10/10 in both models.ConclusionOur study identified a possible interaction between tobacco consumption and carcinogen metabolizing gene polymorphisms towards modulating promoter methylation of tumour suppressor genes in ESCC.

Hepatitis C virus Core protein overcomes all-trans retinoic acid-induced cell growth arrest by inhibiting retinoic acid receptor-β2 expression via DNA methylation

Aberrant promoter methylation of tumor suppressor genes including retinoic acid receptor-β2 (RAR-β2) is frequently detected in hepatitis C virus (HCV)-associated hepatocellular carcinoma; however, the mechanism and its significance are relatively unknown. Here, we showed that HCV Core induced promoter hypermethylation of RAR-β2 to inhibit its expression via up-regulation of DNA methyltransferases 1 and 3b. Under the condition, all-trans retinoic acid (ATRA) failed to activate p16 expression and thus could not inactivate the Rb-E2F pathway. Accordingly, Core-expressing cells exhibited resistance to ATRA-induced growth inhibition. Taken together, HCV Core antagonizes ATRA, a natural anti-cancer compound, to stimulate cell growth via epigenetic down-regulation of RAR-β2.

Hepatitis C virus Core protein stimulates cell growth by down-regulating p16 expression via DNA methylation

Hepatitis C virus Core plays a vital role in the development of hepatocellular carcinoma; however, its action mechanism is still controversial. Here, we showed that Core down-regulated levels of p16, resulting in inactivation of Rb and subsequent activation of E2F1, which lead to growth stimulation of hepatocytes. For this effect, Core inhibited p16 expression by inducing promoter hypermethylation via up-regulation of DNA methyltransferase 1 (DNMT1) and DNMT3b. The growth stimulatory effect of Core was abolished when levels of p16 were restored by either exogenous complementation or treatment with 5-Aza-2′dC, indicating that the effect is critical for the stimulation of cell growth by Core.

HBx-induced androgen receptor expression in HBV-associated hepatocarcinoma is independent of the methylation status of its promoter.

A remarkable feature of HBV-associated HCC is male predominance. The cooperation of hepatitis B virus X protein (HBx) with androgen receptor (AR) signaling pathway has been documented to contribute to this dominance. HBx, a multifunctional viral regulator, has been documented to induce promoter hypermethylation and low expression of tumor suppressor genes via activation of DNA methyl-transferase (DNMT) in hepatocarcinogenesis. In prostate cancer, hypermethylation of AR promoter is associated with loss of AR expression. However, the relationship among HBx, DNMTs, the methylation status of AR and AR expression in HBV-associated HCC is still unknown. In this report, we found that HBx correlated with high levels of AR in HCC cases and induced AR expression by stimulating its transcription in liver cell lines. HBx correlated with high expression of DNMTs in HCC cases too. Both in vivo and in vitro, however, the expression of AR was not associated with its promoter methylation status, and the methylation status of AR was not regulated by DNMTs. AR expression is higher in peritumoral tissues than in tumors, as well as being higher in HBV-associated HCC than in HBV-negative cases. Therefore, HBx-induced high expression of AR plays a role during hepatocarcinogenesis, but is not involved with its promoter methylation or DNMTs. HBx-mediated DNMT deregulation is gene-specific, and the expression and methylated regulation of AR is tissue-specific.

Abstract LB-85: TGF-ß regulates its own expression and down-regulates its own receptors in prostate cancer (CaP) cells through a positive feedback pathway mediated by DNA methyltransferase

Abstract Introduction: Over expression and loss insensitivity to TGF-ß are two features which are coincident with CaP progression. In present study, we investigated whether a positive feedback pathway exists in which TGF-ß activates DNA methyltransferase (DNMT), which induces promoter hypermethylation. This hypermethylation may down-regulate TGF-ß receptors and in turn stimulate increased expression of TGF-ß. Materials and Methods: Human CaP cells (including the less aggressive lines, PC-3 and PC-3M-Pr04; and more aggressive lines, PC-3M and PC-3M-LN4) were rendered insensitive to TGF-ß using a retroviral vector containing a dominant negative TGF-ß type II receptor gene (TßRIIDN). ELISA were performed to quantify the secreted TGF-ß 1 (pg/105 cells/48 hours) in the pooled conditioned medium from CaP cells expressing T RIIDN and mock transfected. Semi-quantification western blot analyses were performed to evaluate the expression of DNA methyltransferase (DNMT), TßRI and TßRII. A [3H]-Thymidine Incorporation Assay was performed to determine cell proliferation rates in relation to TGF-ß. Results: Compared to PC-3 and PC-3M-Pr04, PC-3M and PC-3M-LN4 secreted almost 2 folds higher baseline levels of TGF-ß1 and DNMTs (1, 3α and 3ß). In contrast, TßRI and TßRII expression was significantly reduced in these same cell lines. In all cell lines, TßRIIDN expression was associated with significantly higher (∼1-2×) TGF-ß secretion, a greater than 3-fold decrease in the expression of DNMT1, DNMT3a, and DNMT 3b, and a correspondingly increased expression of both TßRI and TßRII (naïve intracellular domain). Although there was no significant difference in the original proliferation rate among PC3 variants, PC-3 and PC-3M-Pr04 cells were more sensitive to growth inhibition (&amp;gt;30%) by TGF-ß1 compared to PC-3M-LN4 and PC-3M cells (&amp;lt;7.5%). The growth inhibitory effects of TGF-ß1 disappeared when these cells were rendered insensitive to TGF-ß by infection with TßRIIDN. Conclusion: Higher expression of TGF-ß and lower expression of its own receptors are associated with more aggressive CaP phenotypes. Overexpression of tumor derived TGF-ß can activate DNMTs which will then mediate promoter methylation and down regulation of its receptors, subsequently, blockade of TGF-ß signaling may activate a feedback system which can stimulate the secretion of more TGF-ß. This novel mechanism has yet been reported, and may provide a new insight on TGF-ß regulation in prostate cancer. This study was supported in part by grants from the American Cancer Society, Illinois (#08-22), Department of Defense (W81XWH-09-1-0311), Portes Center/Institute of Medicine of Chicago (QZ), American Cancer Society Institutional Research Grant (ACS-IRG 93-037-12), American Urological Association Foundation (AUA Foundation, QZ), National Cancer Institute (P50CA090386), Illinois Department of Public Health (Contract #:83284034), a grant from the Genzyme Corporation, and a gift from Mr. Fred L. Turner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-85.

Hepatitis B virus X protein overcomes the growth-inhibitory potential of retinoic acid by downregulating retinoic acid receptor- 2 expression via DNA methylation

Aberrant promoter methylation of retinoic acid receptor-beta(2) (RAR-beta(2)) is frequently detected in hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC); however, the mechanism of methylation and its biological significance are unknown. This study showed that HBx, the principal oncogene product of HBV, induced promoter hypermethylation of RAR-beta(2) via upregulation of DNA methyltransferases 1 and 3a, resulting in downregulation of its expression in human HCC cells. In addition, HBx abolished the potential of retinoic acid (RA) to downregulate levels of G(1)-checkpoint regulators including p16, p21 and p27, resulting in activation of E2F1 in the presence of RA. As a consequence, HBx-expressing cells were less susceptible to RA-induced cell growth inhibition compared with control cells. These effects almost completely disappeared when levels of RAR-beta(2) in HBx-expressing cells were restored by treatment with a universal DNA methylation inhibitor, 5-aza-2'-deoxycytidine. As RAR-beta(2) is a major executor of the anti-tumour potential of RA, its epigenetic downregulation by HBx is likely to be an important step during HBV-mediated tumorigenesis.

Transcriptional Inactivation of ERβ Gene Is Mediated by the Induction of Promoter Hypermethylation in a Rat Colonic Epithelial Cell Model

To construct a primary rat colonic epithelial cell model for treatment with specific methylated oligonucleotides (MOs) and to determine whether the transcriptional inactivation of ERbeta mRNA is mediated by the induction of hypermethylation of the ERbeta gene promoter. Suckling rat colonic epithelial cells were cultured in DMEM. Two methylated oligonucleotides complementary to the promoter regions of ERbeta were synthesized and applied to the cultured cells to induce promoter hypermethylation of the ERbeta gene. Methylation-specific PCR (MSP) was used to determine the methylation status of the ERbeta promoter in the cultured cells. Reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the expression of ERbeta mRNA after treatment with MOs. Suckling rat colonic epithelial cells were successfully cultured in vitro. The MOs and unmethylated oligonucleotides (UMOs) we designed, synthesized, successfully transfected into the colonic epithelial cells, and assembled in the nuclei of the cells, which had extremely elevated proliferative activity. RT-PCR demonstrated that the expression of ERbeta mRNA was significantly suppressed in the cells treated with MOs, whereas its expression in the control cells treated with UMOs was not. MSP analysis showed that the promoter of ERbeta in the cells treated with MOs was hypermethylated compared with that of the control cells. The transcriptional inactivation of ERbeta mRNA in rat colonic epithelial cells may be mediated by the hypermethylation of the ERbeta gene promoter. Our model markedly simulates the epigenetic modification of the ERbeta gene in colonic cancer cells.

Epstein–Barr virus latent membrane protein 1 suppresses the growth-inhibitory effect of retinoic acid by inhibiting retinoic acid receptor-β2 expression via DNA methylation

Epigenetic alteration through DNA methylation in retinoic acid receptor-β 2 (RAR-β 2) is common in human tumors including nasopharyngeal carcinoma (NPC); however, the mechanism and its biological significance are unknown. Here, we report that the Epstein–Barr virus (EBV) oncogene product, latent membrane protein 1 (LMP1), induces promoter hypermethylation of RAR-β 2 via up-regulation of DNA methyltransferases 1, 3a, and 3b, leading to decrease in RAR-β 2 expression in NPC cells. In addition, LMP1 abolished the potentials of retinoic acid (RA) to down-regulate Cdk2 and Cdk4 and to up-regulate p16, p21, and p27, resulting in activation of E2F1 in the presence of RA. As a consequence, LMP1 could abrogate the growth-inhibitory effect of RA by releasing cell cycle arrest at G 1 phase. Considering that RAR-β 2 is a major executor of the anti-tumor potentials of retinoids, its down-regulation by LMP1 might play an important role during EBV-mediated tumorigenesis.