Knockdown Of DNA Methyltransferase 1 Research Articles

Epigenetic Regulation of Myofibroblast Differentiation by DNA Methylation

DNA methylation, a key mechanism of repressing gene expression, is of particular relevance in controlling development and cell differentiation. We analyzed the extent and regulation of DNA methylation of the alpha-smooth muscle actin (alpha-SMA) gene to elucidate its potential role in myofibroblast differentiation. These experiments revealed the presence of three CpG islands that were methylated at different levels in fibroblasts, myofibroblasts, and alveolar epithelial type II cells. Coordinately, these cells expressed low, high, or no alpha-SMA, respectively. In addition, inhibition of DNA methyltransferase activity or knock down of DNA methyltransferase using specific small interfering RNA caused significant induction of alpha-SMA in fibroblasts. In contrast, induced overexpression of DNA methyltransferase suppressed alpha-SMA gene expression. Transforming growth factor beta induced myofibroblast differentiation was enhanced or suppressed by knockdown or overexpression of DNA methyltransferase, respectively. Finally, in vitro DNA methylation of the alpha-SMA promoter suppressed its activity. These findings suggest that DNA methylation mediated by DNA methyltransferase is an important mechanism regulating the alpha-SMA gene expression during myofibroblast differentiation.

Reduction of pancreatic cancer cell viability and induction of apoptosis mediated by siRNA targeting DNMT1 through suppression of total DNA methyltransferase activity

Aberrant methylation leads to epigenetic changes in human genes that may cause carcinogenesis. DNA methyltransferase 1 (DNMT1) plays an important role in maintaining DNA methylation patterns during genomic DNA replication. To understand the role of this protein in pancreatic cancer cell growth and apoptosis, small interfering RNA (siRNA) oligonucleotides were used to knockdown DNMT1 expression in pancreatic cancer PaTu8988 cells. We found that the DNMT1 siRNA markedly decreased DNMT1 expression and total DNA methyltransferase activity in the cells. Upon the inhibition of DNMT1 expression, the proliferation of the tumor cells was inhibited. Tumor cell growth was arrested in the S-phase of the cell cycle and cells underwent apoptosis. The expression of p21 was up-regulated and the ratio of Bax/Bcl-2 expression was increased after DNMT1 knockdown in PaTu8988 cells. Furthermore, DNMT1 siRNA caused demethylation of the tumor suppressor gene hMLH1, resulting in its re-expression in PaTu8988 cells. The results of this study suggest that DNMT1 siRNA oligonucleotides are candidates for further evaluation as therapeutic tools for the clinical control of pancreatic cancer.

DNMT1 and DNMT3b silencing sensitizes human hepatoma cells to TRAIL‐mediated apoptosis via up‐regulation of TRAIL‐R2/DR5 and caspase‐8

DNA methylation plays a critical role in chromatin remodeling and gene expression. DNA methyltransferases (DNMTs) are hypothesized to mediate cellular DNA methylation status and gene expression during mammalian development and in malignant diseases. In this study, we examined the role of DNA methyltransferase 1 (DNMT1) and DNMT3b in cell proliferation and survival of hepatocellular carcinoma (HCC) cells. Gene silencing of both DNMT1 and DNMT3b by targeted siRNA knockdown reduces cell proliferation and sensitizes the cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cell death. The proapoptotic protein caspase-8 demonstrated promoter hypermethylation in HCC cells and was up-regulated by knockdown of DNMT1 and DNMT3b both at mRNA and protein levels. In addition, death receptor TRAIL-R2/DR5 (TRAIL receptor 2/death receptor 5) did not exhibit promoter hypermethylation in HCC cells but was also up-regulated by knockdown of DNMT1 and DNMT3b both at mRNA and protein levels. Consistent with this observation, the combined transfection of DNMT1-siRNA plus DNMT3b-siRNA enhanced formation of the TRAIL-death-inducing signaling complex formation in HCC cells. In conclusion, our data suggest that DNA methylation of specific genomic regions maintained by DNMT1 and DNMT3b plays a critical role in survival of HCC cells, and a simultaneous knockdown of both DNMT1 and DNMT3b may be a novel anticancer strategy for the treatment of HCC.

Abstract LB-358: Carcinogen-induced epigenetic silencing of miR-205 and miR-200 family promotes EMT and transformation of lung epithelial cells

Abstract An in vitro carcinogen-induced transformation model was developed using immortalized bronchial epithelial cells (HBECs) to identify key molecular changes in preneoplastic lung epithelial cells that occur during the development of lung cancer in smokers. HBECs were treated for twelve weeks (one exposure per week) with low-doses of tobacco carcinogens (MNU and BPDE) that resulted in transformation from genotoxic stress (DNA damage). The HBECs displayed a morphological change consistent with an epithelial-to-mesenchymal transition (EMT) after 4 weeks of carcinogen treatment that persisted in colonies that were recovered and expanded from soft agar (transformed cell line). Expression of mesenchymal markers vimentin, fibronectin, and N-cadherin increased significantly, while the epithelial marker E-cadherin was significantly decreased. The most notable changes associated with EMT included 4 - 55 fold reduced expression of miR-200b, -200c, and -205 that correlated with 2-1500 fold increased expression of E-box binding transcription factors (SNAI1, ZEB1, ZEB2) and basic helix-loop-helix transcription factor (TWIST1). Restoring the expression of miR-200b, -200c or -205 in carcinogen transformed cells prevented growth in soft agar; meanwhile the cells maintained a fibroblast-like mesenchymal appearance. Levels of DNA methyltransferase 1 (DNMT1) protein also increased significantly during carcinogen treatment and stable knockdown of DNMT1, but not DNMT3A and DNMT3B prior to carcinogen prevented EMT and transformation. ChIP for H3K4me2 (indicative of active/open chromatin) revealed a progressive decrease of this mark at the promoters of miR-200b, -200c, and -205 over 12 weeks of carcinogen treatment and during transformation. A modest enrichment for H3K9me2 (inactive/closed chromatin mark) was observed at all time points. In contrast, H3K27me3 (indicative of inactive/closed chromatin) was enriched at the miR promoters after 4 weeks of carcinogen treatment and enrichment declined over time. Bisulfite sequencing revealed CpG DNA methylation increased from 3% to 42% in the miR-200 family and miR-205 promoters during transformation. To examine the clinical relevance of miR-200b, -200c, and -205 their expression was analyzed in lung tumors relative to distant normal lung tissue from 24 patients. Reduced expression of at least one miR was observed in 50% of lung adenocarcinomas. These findings implicate a major role for epigenetic regulation of EMT that extends beyond that of cancer metastasis to causality for development of premalignant lung cancer. Supported by ES008801. 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-358.

56 EFFECTS OF KNOCKDOWN OF DNA METHYLTRANSFERASE 1 BY RNA INTERFERENCE ON IN VITRO DEVELOPMENT AND DNA METHYLATION STATE IN BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Reprogramming of epigenetic modification is a necessary process during mammalian development, which is aberrant in somatic cell nuclear transfer (SCNT) embryos. Previous study has demonstrated that an abnormal state of genomic hypermethylation is consistently observed in SCNT embryos (Kang et al. 2001 Nat. Genet. 28, 173–177). On the other hand, small interfering RNAs (siRNAs) are identified molecules shown to silence genes via targeted mRNA degradation and are widely used in molecular and cellar research (Hannon GJ 2002 Nature 418, 244–251). Thus, knockdown of the expression of genes related to epigenetic modifications by siRNA may be used to alter epigenetic modifications in SCNT embryos. In the present study, we investigated the effects of knockdown of DNA methyltransferase 1 by siRNA on in vitro development, gene expression, and DNA methylation state of bovine SCNT embryos. In vitro matured oocytes were enucleated, fused with bovine fibroblasts and then activated, the resultant SCNT embryos were divided into three groups; control, non-treated group; sham-NT, H2O injected group; and siRNA-NT, siRNA injected group. The siRNA corresponding to DNA methyltransferases 1, which is the enzyme responsible for maintaining DNA methylation patterns, was designed and injected into the cytoplasm of SCNT embryos. All embryos were cultured in CR1aa + 5% FCS and assessed the rates of cleavage and blastocyst formation on Days 2 and 8, respectively. All data were obtained from more than 5 replicates. Developmental percentage data were analyzed by chi-square tests (P < 0.05). Other data were analyzed with ANOVA followed by Fisher’s protected least significant difference (P < 0.05). The developmental rate to blastcysts in siRNA-NT group (38.7%; 111/287) was significantly higher (P < 0.05) than those of control (28.8%; 121/420) and sham groups (30.5%; 92/302). To estimate the effect of siRNA injection on gene expression, we sampled embryos at 48 h after culture and measured the amount of DNA methyltransferase 1 mRNA expression by real-time PCR. The amount of DNA methyltransferase 1 mRNA was significantly less (P < 0.05) than those of control and sham-NT groups. Finally, the levels of DNA methylation at satellite I region were analyzed by COBRA method in blastosyst stage embryos. The level of DNA methylation of blastocysts in siRNA-NT groups was significantly less (P < 0.05) than those of control and sham-NT and also similar to that of IVF blastocysts. In the present study, we showed that gene silencing of DNA methyltransferase 1 by siRNA enhanced the in vitro development of SCNT embryos and decreased the level of DNA methylation which was equivalent to IVF embryos. These findings suggest that knockdown of specific genes related epigenetic modifications by RNA interference may alter abnormal epigenetic reprogramming with the resultant improvement for subsequent development of SCNT embryos.

HDAC Inhibitors Act with 5-aza-2′-Deoxycytidine to Inhibit Cell Proliferation by Suppressing Removal of Incorporated Abases in Lung Cancer Cells

5-aza-2′-deoxycytidine (5-aza-CdR) is used extensively as a demethylating agent and acts in concert with histone deacetylase inhibitors (HDACI) to induce apoptosis or inhibition of cell proliferation in human cancer cells. Whether the action of 5-aza-CdR in this synergistic effect results from demethylation by this agent is not yet clear. In this study we found that inhibition of cell proliferation was not observed when cells with knockdown of DNA methyltransferase 1 (DNMT1), or double knock down of DNMT1-DNMT3A or DNMT1-DNMT3B were treated with HDACI, implying that the demethylating function of 5-aza-CdR may be not involved in this synergistic effect. Further study showed that there was a causal relationship between 5-aza-CdR induced DNA damage and the amount of [3H]-5-aza-CdR incorporated in DNA. However, incorporated [3H]-5-aza-CdR gradually decreased when cells were incubated in [3H]-5-aza-CdR free medium, indicating that 5-aza-CdR, which is an abnormal base, may be excluded by the cell repair system. It was of interest that HDACI significantly postponed the removal of the incorporated [3H]-5-aza-CdR from DNA. Moreover, HDAC inhibitor showed selective synergy with nucleoside analog-induced DNA damage to inhibit cell proliferation, but showed no such effect with other DNA damage stresses such as γ-ray and UV, etoposide or cisplatin. This study demonstrates that HDACI synergistically inhibits cell proliferation with nucleoside analogs by suppressing removal of incorporated harmful nucleotide analogs from DNA.

Depletion of DNA methyltransferase 1 and/or DNA methyltransferase 3b mediates growth arrest and apoptosis in lung and esophageal cancer and malignant pleural mesothelioma cells

DNA methyltransferase (DNMT)1, DNMT3b, or both, facilitate malignant transformation through chromatin remodeling mechanisms. The present study was undertaken to examine the effects of antisense-mediated inhibition of DNMT expression in cultured thoracic malignancies. CALU-6 and A549 lung cancer, SKGT5 and BIC esophageal adenocarcinoma, and H2373 and H2052 malignant pleural mesothelioma (MPM) cells, as well as normal human bronchial epithelial (NHBE) cells, were transfected with phosphorothioate-modified antisense oligos targeting DNMT1, DNMT3b, or both, or mismatch oligos. Quantitative reverse transcription-polymerase chain reaction, Western blotting, trypan blue exclusion, and ApoBrdU techniques were used to evaluate DNMT expression, proliferation, and apoptosis after antisense oligo transfections. Gene expression profiles were assessed by using long-oligo array techniques. Antisense oligos mediated specific and dose-dependent depletion of DNMT1 and DNMT3b, resulting in pronounced inhibition of proliferation of all thoracic cancer lines, but not NHBE cells. Depletion of DNMT1 or DNMT3b coincided with dramatic, caspase-dependent, p53-independent apoptosis in 4 of the 6 thoracic cancer lines. The antiproliferative effects of the antisense oligos were not attributable to induction of RASSF1A, p16, or p21 tumor suppressor genes, and did not coincide with demethylation of genes encoding cancer-testis antigens. DNA methyltransferase knockdown mediated induction of numerous genes regulating response to genotoxic stress. Gene expression profiles after DNMT1, DNMT3b, or combined DNMT1/3b depletion were remarkably similar, yet distinctly different from expression profiles mediated by 5 aza 2' deoxycytidine. Antisense oligos targeting DNMT1 and DNMT3b induce genomic stress, and mediate potent growth inhibition in lung and esophageal cancer and MPM cells. These findings support further evaluation of DNMT knockdown strategies for cancer therapy.

STAT3 Mediates Epigenetic Silencing

Methylation of DNA is a heritable mechanism for silencing gene expression. In some cancers, including certain types of lymphomas, aberrant methylation of the promoters of negative regulators of signaling may contribute to malignancy. Zhang et al. provide evidence that persistent activation of STAT3 (signal transducer and activator of transcription 3) contributes to methylation of the promoter of the gene encoding SHP-1 tyrosine phosphatase, a key negative regulator of T cell signaling. Analysis of the mRNA and protein abundance for DNA methyl transferase 1 (DNMT1), histone deacetylase 1 (HDAC1), and SHP-1 showed an inverse relationship, with SHP-1 low in cells that had high levels of DNMT and HDAC. Furthermore, normal peripheral blood mononuclear cells (PBMC) had low or undetectable amounts of DNMT1 and HDAC1 and no methylation of the SHP-1 promoter, whereas malignant T cells had abundant amounts of these DNA-modifying enzymes and complete or partial methylation of the SHP-1 promoter. The SHP-1 promoter had four potential binding sites for STAT3, and chromatin immunoprecipitation (ChIP) experiments indicated that, in malignant T cells, STAT3, DNMT1, and HDAC1 were bound to the SHP-1 promoter. In cells in which SHP-1 was abundant, little or no binding of these three proteins to the promoter was detectable. Knockdown of DNMT1 or STAT3 with oligonucleotides or silencing RNA (siRNA) led to demethylation of the SHP-1 promoter and the appearance of SHP-1 protein. Thus, STAT3, which is known to be activated in malignant T cells, can promote the epigenetic silencing of SHP-1 through DNA methylation. Why phosphorylated STAT3 is capable of promoting epigenetic silencing of SHP-1 in some cells, but not others, for example mitogen-activated T cells and certain populations of malignant T cells, remains unknown. Q. Zhang, H. Y. Wang, M. Marzec, P. N. Raghunath, T. Nagasawa, M. A. Wasik, STAT3- and DNA methyltransferase 1-mediated epigenetic silencing of SHP-1 tyrosine phosphatase tumor suppressor gene in malignant T lymphocytes. Proc. Nat. Acad. Sci. U.S.A. 102 , 6948-6953 (2005). [Abstract] [Full Text]

Epigenomic Stress Response: KNOCKDOWN OF DNA METHYLTRANSFERASE 1 TRIGGERS AN INTRA-S-PHASE ARREST OF DNA REPLICATION AND INDUCTION OF STRESS RESPONSE GENES

The DNA methylation pattern is an important component of the epigenome that regulates and maintains gene expression programs. In this paper, we test the hypothesis that vertebrate cells possess mechanisms protecting them from epigenomic stress similar to DNA damage checkpoints. We show that knockdown of DNMT1 (DNA methyltransferase 1) by an antisense oligonucleotide triggers an intra-S-phase arrest of DNA replication that is not observed with control oligonucleotide. The cells are arrested at different positions throughout the S-phase of the cell cycle, suggesting that this response is not specific to distinct classes of origins of replication. The intra-S-phase arrest of DNA replication is proposed to protect the genome from extensive DNA demethylation that could come about by replication in the absence of DNMT1. This protective mechanism is not induced by 5-aza-2'-deoxycytidine, a nucleoside analog that inhibits DNA methylation by trapping DNMT1 in the progressing replication fork, but does not reduce de novo synthesis of DNMT1. Our data therefore suggest that the intra-S-phase arrest is triggered by a reduction in DNMT1 and not by demethylation of DNA. DNMT1 knockdown also leads to an induction of a set of genes that are implicated in genotoxic stress response such as NF-kappaB, JunB, ATF-3, and GADD45beta (growth arrest DNA damage 45beta gene). Based on these data, we suggest that this stress response mechanism evolved to guard against buildup of DNA methylation errors and to coordinate inheritance of genomic and epigenomic information.