Epigenetic regulation of X-linked cancer/germline antigen genes by DNMT1 and DNMT3b

We examined the function of two key DNA methyltransferase (DNMT) enzymes in epigenetic regulation of X-linked cancer/germline (CG-X) antigen genes in human cancer cells, using MAGE-A1, NY-ESO-1, and XAGE-1 as models. In HCT116 cells, genetic knockout of DNMT1 caused moderate activation of CG-X genes, DNMT3b knockout had a negligible effect, and double knockout of both enzymes caused robust gene induction. Similarly, dual DNMT knockout caused dramatic hypomethylation of the MAGE-A1 and NY-ESO-1 promoters, DNMT1 knockout showed moderate hypomethylation, and DNMT3b knockout elicited only slight methylation changes. In contrast, both single and double knockout cells showed significant hypomethylation of the XAGE-1 promoter. RNA interference (RNAi) targeting of DNMT1 in HCT116 cells validated the results seen using genetic knockout cells; however, RNAi targeting of DNMT1 in a different colorectal cancer cell line revealed a greater independent role for DNMT1 in mediating CG-X gene repression and promoter methylation in other cell types. Notably, the histone H3 modification pattern at CG-X promoters was altered following DNMT knockout. DNMT1 or DNMT3b knockout reduced dimethylated lysine-9 (diMe-H3K9) levels, but did not significantly affect dimethylated lysine-4 (diMe-H3K4) or acetylated lysine-9 (Ac-H3-K9) levels. In contrast, dual DNMT1/3b knockout reduced the level of diMe-H3K9 and dramatically increased the levels of diMe-H3K4 and Ac-H3K9 at CG-X gene loci. In summary, DNMT1 and DNMT3b were found to perform both redundant and independent functions in epigenetic regulation of CG-X antigen genes in human cancer cells.     

Genome structure-based screening identified epigenetically silenced microRNA associated with invasiveness in non-small-cell lung cancer

MicroRNA (miRNA) expression is frequently altered in human cancers. To search for epigenetically silenced miRNAs in non-small-cell lung cancer (NSCLC), we mapped human miRNAs on autosomal chromosomes and selected 55 miRNAs in silico. We treated six NSCLC cell lines with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) and determined the expressions of the 55 miRNAs. Fourteen miRNAs were decreased in the cancer cell lines and were induced after 5-aza-CdR treatment. After a detailed DNA methylation analysis, we found that mir-34b and mir-126 were silenced by DNA methylation. Mir-34b was silenced by the DNA methylation of its own promoter, whereas mir-126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir-34b and EGFL7, and H3K27me3 in EGFL7. The overexpression of mir-34b and mir-126 decreased the expression of c-Met and Crk, respectively. The 5-aza-CdR treatment of lung cancer cell line resulted in increased mir-34b expression and decreased c-Met protein. We next analyzed the DNA methylation status of these miRNAs using 99 primary NSCLCs. Mir-34b and mir-126 were methylated in 41 and 7% of all the cases, respectively. The DNA methylation of mir-34b was not associated with c-Met expression determined by immunohistochemistry, but both mir-34b methylation (p = 0.007) and c-Met expression (p = 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir-34b can be used as a biomarker for an invasive phenotype of lung cancer.     

DNA去甲基化对急性B淋巴细胞白血病细胞株中DNA甲基转移酶基因及微RNA作用的研究

目的：探索甲基化转移酶抑制剂5-杂氮-2'-脱氧胞苷（5-Aza-CdR）对急性B淋巴细胞白血病（B-ALL）细胞株NALM-6的作用以及对细胞中微RNA（miRNA）表达水平的影响。方法用不同浓度5-Aza-CdR处理NALM-6细胞,采用四甲基偶氮唑盐（MTT）法检测细胞增殖情况,采用荧光定量反转录聚合酶链反应（RT-PCR）检测5-Aza-CdR处理后细胞DNA甲基转移酶（DNMT）基因mRNA表达水平的变化,采用miScript miRNA PCR Array芯片检测去甲基化后细胞中表达量发生改变的miRNA。结果 NALM-6细胞经不同浓度5-Aza-CdR处理不同时间后,细胞生长受抑,最高抑制率达（74.163±0.381）%。5-Aza-CdR作用浓度与DNMT基因mRNA表达水平呈反比,浓度为1000μmol／L的5-Aza-CdR作用细胞72 h后,DNMT-1的相对表达量降至0.453±0.021,DNMT-3L的相对表达量为0.003±0.001, DNMT-3B的相对表达量为0.395±0.019。miScript miRNA PCR Array筛选出3个miRNA（miR-184、miR-23a-3p、miR-34a-5p）与DNA甲基化相关。结论5-Aza-CdR可下调NALM-6细胞中DNMT基因的表达,并对细胞增殖有抑制作用。miR-184、miR-23a-3p和miR-34a-5p在B-ALL的发生、发展中与DNA甲基化相关。     

Distinct effects on gene expression of chemical and genetic manipulation of the cancer epigenome revealed by a multimodality approach

We tested the hypothesis that the effects on gene expression of altered DNA methylation by 5-aza-2'-deoxycytidine (5-aza-CdR) and genetic (DNMT knockout) manipulation of DNA are similar, and distinct from Trichostatin A (TSA)-induced chromatin decondensation. Surprisingly, the effects of 5-aza-CdR were more similar to those of TSA than to DNMT1, DNMT3B, or double DNMT somatic cell knockout. Furthermore, the effects of 5-aza-CdR were similar at one and five days exposure, suggesting active demethylation or direct influence of both drugs on the stability of methylation and/or chromatin marks. Agents that induce gene activation through hypomethylation may have unintended consequences, since nearly as many genes were downregulated as upregulated after demethylation. In addition, a 75 kb cluster of metallothionein genes was coordinately regulated.     

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)     

Aberrant DNA methyltransferase expression in pancreatic ductal adenocarcinoma development and progression

Background

Altered gene methylation, regulated by DNA methyltransferases (DNMT) 1, 3a and 3b, contributes to tumorigenesis. However, the role of DNMT in pancreatic ductal adenocarcinoma (PDAC) remains unknown.

Methods

Expression of DNMT 1, 3a and 3b was detected in 88 Pancreatic ductal adenocarcinoma (PDAC) and 10 normal tissue samples by immunohistochemistry. Changes in cell viability, cell cycle distribution, and apoptosis of PDAC cell lines (Panc-1 and SW1990) were assessed after transfection with DNMT1 and 3b siRNA. Levels of CDKN1A, Bcl-2 and Bax mRNA were assessed by qRT-PCR, and methylation of the Bax gene promoter was assayed by methylation-specific PCR (MSP).

Results

DNMT1, 3a and 3b proteins were expressed in 46.6%, 23.9%, and 77.3% of PDAC tissues, respectively, but were not expressed in normal pancreatic tissues. There was a co-presence of DNMT3a and DNMT3b expression and an association of DNMT1 expression with alcohol consumption and poor overall survival. Moreover, knockdown of DNMT1 and DNMT3b expression significantly inhibited PDAC cell viability, decreased S-phase but increased G1-phase of the cell cycle, and induced apoptosis. Molecularly, expression of CDKN1A and Bax mRNA was upregulated, and the Bax gene promoter was demethylated. However, a synergistic effect of combined DNMT1 and 3b knockdown was not observed.

Conclusion

Expression of DNMT1, 3a and 3b proteins is increased in PDAC tissues, and DNMT1 expression is associated with poor prognosis of patients. Knockdown of DNMT1 and 3b expression arrests tumor cells at the G1 phase of the cell cycle and induces apoptosis. The data suggest that DNMT knockdown may be a novel treatment strategy for PDAC.     

Silencing of the metastasis suppressor RECK by RAS oncogene is mediated by DNA methyltransferase 3b-induced promoter methylation

RECK is a membrane-anchored glycoprotein that may negatively regulate matrix metalloproteinase activity to suppress tumor invasion and metastasis. Our previous study indicated that oncogenic RAS inhibited RECK expression via a histone deacetylation mechanism. In this study, we address whether DNA methyltransferases (DNMT) participate in the inhibition of RECK by RAS. Induction of Ha-RAS(Val12) oncogene increased DNMT3b, but not DNMT1 and DNMT3a, expression in 2-12 cells. In addition, induction of DNMT3b by RAS was through the extracellular signal-regulated kinase signaling pathway. Oncogenic RAS increased the binding of DNMT3b to the promoter of RECK gene and this binding induced promoter methylation, which could be reversed by 5'-azacytidine and DNMT3b small interfering RNA (siRNA). The MEK inhibitor U0126 also reversed RAS-induced DNMT3b binding and RECK promoter methylation. Treatment of 5'-azacytidine and DNMT3b siRNA restored RECK expression in 2-12 cells and potently suppressed RAS-stimulated cell invasion. In addition, the inhibitory effect of 5'-azacytidine on RAS-induced cell invasion was attenuated after knockdown of RECK by siRNA. Interestingly, human lung cancer cells harboring constitutively activated RAS exhibited lower RECK expression and higher promoter methylation of RECK gene. 5'-Azacytidine and DNMT3b siRNA restored RECK expression in these cells and effectively suppressed invasiveness. Collectively, our results suggest that RAS oncogene induces RECK gene silencing through DNMT3b-mediated promoter methylation, and DNMT inhibitors may be useful for the treatment of RAS-induced metastasis.     

Double RNA interference of DNMT3b and DNMT1 enhances DNA demethylation and gene reactivation

Small interfering RNAs (siRNAs) are newly identified molecules shown to silence genes via targeted mRNA degradation. In this study, we used specific siRNAs as a tool to probe the relationship between two DNA methyltransferase genes, DNMT3b and DNMT1, in the maintenance of DNA methylation patterns in the genome. Levels of DNMT3b or DNMT1 mRNAs and proteins were markedly decreased (up to 80%) on transfecting these siRNAs into the ovarian cancer cell line CP70. The resulting RNA interference showed differential effects on DNA demethylation and gene reactivation in the treated cells. The DNMT1 siRNA treatment led to a partial removal of DNA methylation from three inactive promoter CpG islands, TWIST, RASSF1A, and HIN-1, and restored the expression of these genes. This epigenetic alteration appeared less effective in cells transfected with DNMT3b siRNA. However, the combined treatment of DNMT3b and DNMT1 siRNAs greatly enhanced this demethylation effect, producing 7-15-fold increases in their expression. We also used a microarray approach to examine this RNA interference on 8640 CpG island loci in CP70 cells. The combined siRNA treatment had a greater demethylation effect on 241 methylated loci and selected repetitive sequences than that of the single treatment. Our data thus suggest that whereas DNMT1 plays a key role in methylation maintenance, DNMT3b may act as an accessory to support the function in CP70 cells. This study also shows that siRNA is a powerful tool for interrogating the mechanisms of DNA methylation in normal and pathological genomes.     

DNA-demethylating and anti-tumor activity of synthetic miR-29b mimics in multiple myeloma

Aberrant DNA methylation plays a relevant role in multiple myeloma (MM) pathogenesis. MicroRNAs (miRNAs) are a class of small non-coding RNAs that recently emerged as master regulator of gene expression by targeting protein-coding mRNAs. However, miRNAs involvement in the regulation of the epigenetic machinery and their potential use as therapeutics in MM remain to be investigated. Here, we provide evidence that the expression of de novo DNA methyltransferases (DNMTs) is deregulated in MM cells. Moreover, we show that miR-29b targets DNMT3A and DNMT3B mRNAs and reduces global DNA methylation in MM cells. In vitro transfection of MM cells with synthetic miR-29b mimics significantly impairs cell cycle progression and also potentiates the growth-inhibitory effects induced by the demethylating agent 5-azacitidine. Most importantly, in vivo intratumor or systemic delivery of synthetic miR-29b mimics, in two clinically relevant murine models of human MM, including the SCID-synth-hu system, induces significant anti-tumor effects. All together, our findings demonstrate that aberrant DNMTs expression is efficiently modulated by tumor suppressive synthetic miR-29b mimics, indicating that methyloma modulation is a novel matter of investigation in miRNA-based therapy of MM.     

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.     

MicroRNA profiling of novel African American and Caucasian Prostate Cancer cell lines reveals a reciprocal regulatory relationship of miR-152 and DNA methyltranferase 1

miRNA expression in African American compared to Caucasian PCa patients has not been widely explored. Herein, we probed the miRNA expression profile of novel AA and CA derived prostate cancer cell lines. We found a unique miRNA signature associated with AA cell lines, independent of tumor status. Evaluation of the most differentially expressed miRNAs showed that miR-132, miR-367b, miR-410, and miR-152 were decreased in more aggressive cells, and this was reversed after treatment of the cells with 5-aza-2′-deoxycytidine. Sequencing of the miR-152 promoter confirmed that it was highly methylated. Ectopic expression of miR-152 resulted in decreased growth, migration, and invasion. Informatics analysis of a large patient cohort showed that decreased miR-152 expression correlated with increased metastasis and a decrease in biochemical recurrence free survival. Analysis of 39 prostate cancer tissues with matched controls (20 AA and 19 CA), showed that 50% of AA patients had statistically significant lower miR-152 expression compared to only 35% of CA patients. Ectopic expression of miR-152 in LNCaP, PC-3, and MDA-PCa-2b cells down-regulated DNA (cytosine-5)-methyltransferase 1 (DNMT1) through direct binding in the DNMT1 3''UTR. There appeared to be a reciprocal regulatory relationship of miR-152/DNMT1 expression, as cells treated with siRNA DNMT1 caused miR-152 to be re-expressed in all cell lines. In summary, these results demonstrate that epigenetic regulation of miR-152/DNMT1 may play an important role in multiple events that contribute to the aggressiveness of PCa tumors, with an emphasis on AA PCa patients.     

E-cadherin expression is silenced by DNA methylation in cervical cancer cell lines and tumours

A previous study showed E-cadherin expression was lost in some cervical cancer cell lines and tumours. This study was designed to clarify the significance of DNA methylation in silencing E-cadherin expression. We examined promoter methylation of E-cadherin in five cervical cancer cell lines and 20 cervical cancer tissues using methylation-specific PCR (MSP) and bisulphite DNA sequencing. The correlation of E-cadherin methylation and expression together with methyltransferase (DNMT1) were further studied. We found that hypermethylation of E-cadherin was involved in five cervical cancer cell lines and 40% (8/20) of cervical cancer tissues. E-cadherin protein was lost in 6/8 (75%) samples and 3/5 (60%) cell lines with promoter methylation. E-cadherin methylation was significantly correlated with increased DNMT1. Using an antisense DNMT1 oligo to transfect into SiHa HeLa C33A cell line, E-cadherin protein was re-expressed. We concluded that loss of E-cadherin expression was in part correlated with DNA methylation and DNMT1 expression in cervical cancer.     

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。