RNA interference-mediated knockdown of DNA methyltransferase 1 leads to promoter demethylation and gene re-expression in human lung and breast cancer cells

DNA methyltransferase 1 (DNMT1) is required to maintain DNA methylation patterns in mammalian cells, and is thought to be the predominant maintenance methyltransferase gene. Recent studies indicate that inhibiting DNMT1 protein expression may be a useful approach for understanding the role of DNA methylation in tumorigenesis. To this end, we used RNA interference to specifically down-regulate DNMT1 protein expression in NCI-H1299 lung cancer and HCC1954 breast cancer cells. RNA interference-mediated knockdown of DNMT1 protein expression resulted in >80% reduction of promoter methylation in RASSF1A, p16(ink4A), and CDH1 in NCI-H1299; and RASSF1A, p16(ink4A), and HPP1 in HCC1954; and re-expression of p16(ink4A), CDH1, RASSF1A, and SEMA3B in NCI-H1299; and p16(ink4A), RASSF1A, and HPP1 in HCC1954. By contrast, promoter methylation and lack of gene expression was maintained when these cell lines were treated with control small interfering RNAs. The small interfering RNA treatment was stopped and 17 days later, all of the sequences showed promoter methylation and gene expression was again dramatically down-regulated, indicating the tumor cells still were programmed for these epigenetic changes. We saw no effects on soft agar colony formation of H1299 cells 14 days after DNMT1 knockdown indicating that either these genes are not functioning as tumor suppressors under these conditions, or that more prolonged knockdown or other factors are also required to inhibit the malignant phenotype. These results provide direct evidence that loss of DNMT1 expression abrogates tumor-associated promoter methylation and the resultant silencing of multiple genes implicated in the pathogenesis of human lung and breast cancer.     

Cigarette smoke induces demethylation of prometastatic oncogene synuclein-gamma in lung cancer cells by downregulation of DNMT3B

The prometastatic oncogene synuclein-gamma (SNCG) is not expressed in normal lung tissues, but it is highly expressed in lung tumors. Here, we show that cigarette smoke extract (CSE) has strong inducing effects on SNCG gene expression in A549 lung cancer cells through demethylation of SNCG CpG island. CSE treatment also augments the invasive capacity of A549 cells in an SNCG-dependent manner. To elucidate the mechanisms underlying the demethylating effects of CSE, we examined expression levels of DNA methyltransferases (DNMTs), 1, 3A and 3B in CSE-treated cells. We show that the mRNA expression of DNMT3B is specifically downregulated by CSE with a kinetics concurrent to SNCG reexpression. Utilizing siRNA to knockdown DNMT3B expression, we show that inhibition of DNMT3B directly increases SNCG mRNA expression. We further show that exogenous overexpression of DNMT3B in an SNCG-positive lung cancer cell line H292 suppresses SNCG mRNA and protein expression and induces de novo methylation of SNCG CpG island, whereas overexpression of DNMT1 or DNMT3A has no effects. Taken together, these new findings demonstrate that tobacco exposure induces the abnormal expression of SNCG in lung cancer cells through downregulation of DNMT3B. This work sheds light on the molecular understanding of demethylation of this oncogene during cancer progression.     

Delta DNMT3B variants regulate DNA methylation in a promoter-specific manner

DNA methyltransferase 3B (DNMT3B) is critical in de novo DNA methylation during development and tumorigenesis. We recently reported the identification of a DNMT3B subfamily, DeltaDNMT3B, which contains at least seven variants, resulting from alternative pre-mRNA splicing. DeltaDNMT3Bs are the predominant expression forms of DNMT3B in human lung cancer. A strong correlation was observed between the promoter methylation of RASSF1A gene but not p16 gene (both frequently inactivated by promoter methylation in lung cancer) and expression of DeltaDNMT3B4 in primary lung cancer, suggesting a role of DeltaDNMT3B in regulating promoter-specific methylation of common tumor suppressor genes in tumorigenesis. In this report, we provide first experimental evidence showing a direct involvement of DeltaDNMT3B4 in regulating RASSF1A promoter methylation in human lung cancer cells. Knockdown of DeltaDNMT3B4 expression by small interfering RNA resulted in a rapid demethylation of RASSF1A promoter and reexpression of RASSF1A mRNA but had no effect on p16 promoter in the lung cancer cells. Conversely, normal bronchial epithelial cells with stably transfected DeltaDNMT3B4 gained an increased DNA methylation in RASSF1A promoter but not p16 promoter. We conclude that promoter DNA methylation can be differentially regulated and DeltaDNMT3Bs are involved in regulation of such promoter-specific de novo DNA methylation.     

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. (Cancer Sci 2010)     

Epigenetic silencing of O6-methylguanine DNA methyltransferase gene in NiS-transformed cells

Nickel (Ni) compounds are potent carcinogens and can induce malignant transformation of rodent and human cells. To uncover the molecular mechanisms of nickel sulfide (NiS)-induced cell transformation, we investigated epigenetic alterations in a set of DNA repair genes. The silencing of the O(6)-methylguanine DNA methyltransferase (MGMT) gene locus and upregulation of DNA methyltransferase 1 (DNMT1) expression was specifically detected in NiS-transformed human bronchial epithelial (16HBE) cells. In addition, we noted epigenetic alterations including DNA hypermethylation, reduced histone H4 acetylation and a decrease in the ratio of Lys-9 acetylated/methylated histone H3 at the MGMT CpG island in NiS-transformed 16HBE cells. Meanwhile, we identified concurrent binding of methyl-CpG-binding protein 2, methylated DNA-binding domain protein 2 and DNMT1 to the CpG island of the MGMT promoter, demonstrating that these components collaborate to maintain MGMT methylation in NiS-transformed cells. Moreover, depletion of DNMT1 by introduction of a small hairpin RNA construct into NiS-transformed cells resulted in a 30% inhibition of cell proliferation and led to increased MGMT gene expression by reversion of the epigenetic modifications at the MGMT promoter region. MGMT suppression and hypermethylation at the CpG island of the MGMT promoter occurred 6 days after NiS treatment, indicating that epigenetic modifications of MGMT might be an early event in tumorigenesis. Taken together, these observations demonstrate that epigenetic silencing of MGMT is associated with DNA hypermethylation, histone modifications and DNMT1 upregulation, which contribute to NiS-induced malignant transformation.     

BaP诱导细胞恶性转化过程中DNA甲基化水平的变化

目的：通过分析苯并芘（BaP）诱导细胞恶性转化过程中DNA甲基化水平的变化,探讨BaP致癌的作用机制。方法：以正常人支气管上皮细胞（16HBE）为研究对象,使用梯度浓度BaP（0、10、20和40 μmol/L）染毒处理,构建不同染毒周期（1周、9周和15周）的细胞株,使用5-甲基胞嘧啶（5-mC）细胞免疫荧光检测各组细胞基因组DNA整体甲基化水平的变化,并进一步利用Western blotting和实时荧光定量-PCR技术分析不同染毒周期细胞甲基化蛋白酶（DNMT1、DNMT3a、DNMT3b、MBD2）表达的变化。结果：BaP染毒后,16HBE细胞的5-mC荧光强度表达下降,且随着染毒剂量的增加和染毒时间的延长,这种下降趋势更明显,其中40 μmol/L BaP染毒处理细胞15周时,肉眼已难以观察到可见荧光。与对照组比较,BaP染毒可下调细胞DNMT1蛋白及其mRNA的表达,并呈现明显的剂量和时间反应关系（P均 < 0.05）,但DNMT3a、DNMT3b、MBD2蛋白的表达变化不明显（P均 > 0.05）。结论：BaP可诱导16HBE细胞基因组DNA整体甲基化水平下调,DNMT1在其中可能发挥重要作用。     

DNA甲基转移酶在肿瘤发生预后治疗中的作用

DNA甲基转移酶(DNMT)在多种肿瘤细胞中呈表达上调,异常表达的DNMT通过催化肿瘤抑制基因启动子区域CpG岛甲基化,引起抑癌基因甲基化失活,从而促进正常细胞向肿瘤细胞转化.研究表明,DNMT在消化道肿瘤、乳腺癌、肺癌等肿瘤细胞中呈表达丰度异常,且与肿瘤的发生呈明显相关性,并影响肿瘤预后.药物及靶向干扰可抑制DNMT的活性,可使甲基化的基因去甲基化复活,进而抑制肿瘤细胞生长,促进细胞凋亡.     

Effects of specific DNMT gene depletion on cancer cell transformation and breast cancer cell invasion; toward selective DNMT inhibitors

A hallmark of cancer is aberrant DNA methylation, consisting of global hypomethylation and regional hypermethylation of tumor suppressor genes. DNA methyltransferase inhibitors have been recognized as promising candidate anticancer drugs. Drug development has focused on DNA methylation inhibitors with the goal of activating tumor suppressor genes silenced by DNA methylation. 5-azacytidine (5-AC; Vidaza), a global DNA methyltransferase inhibitor, was Food and Drug Administration approved to treat myelodysplastic syndromes and is clinically tested for solid tumors. In this paper, it was demonstrated that 5'-aza-2'-deoxycytidine (5-azaCdR) activated both silenced tumor suppressor genes and pro-metastatic genes by demethylation, raising the concern that it would promote metastasis. 5-AzaCdR treatment increased the invasiveness of non-invasive breast cancer cell lines MCF-7 cells and ZR-75-1 and dramatically induced pro-metastatic genes; Urokinase plasminogen activator (uPA), matrix metalloproteinase 2 (MMP2), metastasis-associated gene (H-MTS1; S100A4) and C-X-C chemokine receptor 4 (CXCR4). The hypothesis that the blocking of cellular transformation activity of DNA methyltransferase inhibitor could be separated from the pro-metastatic activity was tested using short interfering RNA (siRNA) targeted to the different DNA methyltransferase (DNMT) genes. Although depletion of DNMT1 had the strongest effect on colony growth suppression in cellular transformation assays, it did not result in demethylation and activation of uPA, S100A4, MMP2 and CXCR4 in MCF-7 cells. Depletion of DNMT1 did not induce cellular invasion in MCF-7 and ZR-75-1 non-invasive breast cancer cell lines. These data have implications on the design of new DNA methyltransferase inhibitor and on the proper utilization of current inhibitors.     

DNMT3B regulates proliferation of A549 cells through the microRNA-152-3p/NCAM1 pathway

The purpose of the present study was to examine the epigenetic mechanism by which microRNA (miR)-152-3p regulates proliferation in non-small cell lung cancer A549 cells via neural cell adhesion molecule 1 (NCAM1). Bisulfite sequencing PCR (BSP), the gold standard for methylation detection, uses bisulfite-treated DNA to determine its pattern of methylation. Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. It was conducted and demonstrated a relatively high level of methylation in the miR-152-3p promoter region. Chromatin immunoprecipitation was combined with PCR to detect the binding of DNA methyltransferase 3B (DNMT3B) protein to miR-152-3p, which tends to occur in the core region of the miR-152-3p gene in A549 cells. Luciferase assay identified NCAM1 as the target gene of miR-152-3p. MTT, colony formation and Transwell assays indicated that miR-152-3p could decrease cell proliferation and invasion and in addition to reducing the expression level of NCAM1. Overexpression of NCAM1 could attenuate the effect of miR-152-3p. DNMT3B knockdown decreased the proliferative ability of A549 cells and increased the expression of miR-152-3p, while decreased that of NCAM1. After treatment with miR-152-3p inhibitor, these effects were attenuated and the NCAM1 expression level was upregulated. The results indicated that miR-152-3p may suppress the proliferation of A549 cells by downregulating NCAM1. In addition, DNMT3B negatively regulated the expression of miR-152-3p via modulation of the methylation level in the miR-152-3p core region, thus mediating the proliferation of lung tumor cells.     

Decrease of DNA methyltransferase 1 expression relative to cell proliferation in transitional cell carcinoma

In many common cancers such as transitional cell carcinoma (TCC), specific genes are hypermethylated, whereas overall DNA methylation is diminished. Genome-wide DNA hypomethylation mostly affects repetitive sequences such as LINE-1 retrotransposons. Methylation of these sequences depends on adequate expression of DNA methyltransferase I (DNMT1) during DNA replication. Therefore, DNMT1 expression relative to proliferation was investigated in TCC cell lines and tissue as well as in renal carcinoma (RCC) cell lines, which also display hypomethylation, as indicated by decreased LINE-1 methylation. Cultured normal uroepithelial cells or normal bladder tissue served as controls. In all tumor cell lines, DNMT1 mRNA as well as protein was decreased relative to the DNA replication factor PCNA, and DNA hypomethylation was present. However, the extents of hypomethylation and DNMT1 downregulation did not correlate. Reporter gene assays showed that the differences in DNMT1 expression between normal and tumor cells were not established at the level of DNMT1 promoter regulation. Diminished DNMT1:PCNA mRNA ratios were also found in 28/45 TCC tissues but did not correlate with the extent of DNA hypomethylation. In addition, expression of the presumed de novo methyltransferases DNMT3A and DNMT3B mRNAs was investigated. DNMT3B overexpression was observed in about half of all high-stage TCC (DNMT3B vs. tumor stage, chi(2): p = 0.03), whereas overexpression of DNMT3A was rarer and less pronounced. Expression of DNMT3A and DNMT3B in most RCC lines was higher than in TCC lines. Our data indicate that DNMT1 expression does not increase adequately with cell proliferation in bladder cancer. This relative downregulation probably contributes to hypomethylation of repetitive DNA but does not determine its extent alone.