Impact of DNA methyltransferase inhibitor 5-azacytidine on cardiac development of zebrafish in vivo and cardiomyocyte proliferation,apoptosis, and the homeostasis of gene expression in vitro

Cardiac development is a peculiar process involving coordinated cellular differentiation, migration, proliferation, and apoptosis. DNA methylation plays a key role in genomic stability, tissue-specific gene expression, cell proliferation, and apoptosis. Hypomethylation in the global genome has been reported in cardiovascular diseases. However, little is known about the impact and specific mechanism of global hypomethylation on cardiomyocytes. In the present study, we explored the impact of DNA methyltransferase inhibitors 5-azacytidine on cardiac development. In vivo experiment showed that hypomethylation of zebrafish embryos with 5-azacytidine exposure significantly reduced survival, induced malformations, and delayed general development process. Furthermore, zebrafish embryos injected with 5-azacytidine developed pericardial edema, ventricular volume reduction, looping deformity, and reduction in heart rate and ventricular shortening fraction. Cardiomyocytes treated with 5-azacytidine in vitro decreased proliferation and induced apoptosis in a concentration-dependent manner. Furthermore, 5-azacytidine treatment in cardiomyocytes resulted in 20 downregulated genes expression and two upregulated genes expression in 45 candidate genes, which indicated that DNA methylation functions as a bidirectional modulator in regulating gene expression. In conclusion, these results show the regulative effects of the epigenetic modifier 5-azacytidine in cardiac development of zebrafish embryos in vivo and cardiomyocyte proliferation and apoptosis and the homeostasis of gene expression in vitro, which offer a novel understanding of aberrant DNA methylation in the etiology of cardiovascular disease.     

Antineoplastic activity of the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine in anaplastic large cell lymphoma

DNA methylation is an epigenetic mechanism establishing long-term gene silencing during development and cell commitment, which is maintained in subsequent cell generations. Aberrant DNA methylation is found at gene promoters in most cancers and can lead to silencing of tumor suppressor genes. The DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) is able to reactivate genes silenced by DNA methylation and has been shown to be a very potent epigenetic drug in several hematological malignancies. In this report, we demonstrate that 5-aza-CdR exhibits high antineoplastic activity against anaplastic large cell lymphoma (ALCL), a rare CD30 positive non-Hodgkin lymphoma of T-cell origin. Low dose treatment of ALCL cell lines and xenografted tumors causes apoptosis and cell cycle arrest in vitro and in vivo. This is also reflected in genome-wide expression analyses, where genes related to apoptosis and cell death are amongst the most affected targets of 5-aza-CdR. Furthermore, we observed demethylation and re-expression of p16^INK4A after drug administration and senescence associated β-galactosidase activity. Thus, our data provide evidence that 5-aza-CdR is highly efficient against ALCL and warrants further clinical evaluation for future therapeutic use.     

Demethylating agent 5-aza-2-deoxycytidine enhances susceptibility of breast cancer cells to anticancer agents

DNA methylation plays an important role in regulation of gene expression and is increasingly being recognized as a determinant of chemosensitivity of human cancers. With the aim of improving the chemotherapeutic efficacy of breast carcinoma, the effect of DNA methyltransferase inhibitor, 5-Aza-2′-deoxycytidine (5-aza-CdR), on the chemosensitivity of anticancer drugs was investigated. The cytotoxicity of paclitaxel (PTX), adriamycin (ADR), and 5-fluorouracil (5-FU) was analyzed against human breast cancer cell lines, MDA MB 231 and MCF 7 cell lines using the MTT assay, and the synergy of 5-aza-CdR and these agents was determined by Drewinko’s fraction method. The effects of each single agent or the combined treatment on cell cycle arrest were analyzed by flow cytometric analysis. We also investigated the effect of each single agent or the combined treatment of anticancer drugs with 5-aza-CdR on the methylation status of the selected genes by methylation specific PCR. In MDA MB 231 cells, a synergistic antiproliferative effect was observed with a combination of 10 μM 5-aza-CdR and these three anticancer drugs, while in MCF 7 cells, a semiadditive effect was observed. Treatment with 5-aza-CdR and anticancer drug resulted in partial demethylation of a panel of genes including RARβ2, Slit2, GSTP1, and MGMT. Based on these findings, we propose that 5-aza-CdR enhances the chemosensitivity of anticancer drugs in breast cancer cells and may be a promising approach for increasing the chemotherapeutic potential of these anticancer agents for more effective management of breast carcinomas.     

Expression profiling after induction of demethylation in MCF-7 breast cancer cells identifies involvement of TNF-α mediated cancer pathways

Epigenetic methylation change is a major process that occurs during cancer development. Even though many tumor-related genes have been identified based on their relationship between methylation and expression, few studies have been conducted to investigate the relevant biological pathways involved in these changes. To identify essential pathways likely to be affected by methylation in breast cancer, we examined a pool of genes in which expression was upregulated after induction of demethylation by 5-Aza-2′-deoxycytidine (Aza) in the MCF-7 breast cancer cell line. Genome-wide demethylation was confirmed by monitoring the demethylation of a previously known gene, SULT1A1. Overall, 210 and 213 genes were found to be upregulated and downregulated (fold change ≥ 2), respectively, in common in cells treated with 5 and 10 μM of Aza. Network analysis of these 423 genes with altered expression patterns identified the involvement of a cancer related network of genes that were heavily regulated by TNF-α in breast tumorigenesis. Our results suggest that epigenetic dysregulation of cellular processes relevant to TNF-α-dependent apoptosis may be intimately involved in tumorigenesis in MCF-7 cells.     

Epigenetic changes of CXCR4 and its ligand CXCL12 as prognostic factors for sporadic breast cancer

Chemokines and their receptors are involved in the development and cancer progression. The chemokine CXCL12 interacts with its receptor, CXCR4, to promote cellular adhesion, survival, proliferation and migration. The CXCR4 gene is upregulated in several types of cancers, including skin, lung, pancreas, brain and breast tumors. In pancreatic cancer and melanoma, CXCR4 expression is regulated by DNA methylation within its promoter region. In this study we examined the role of cytosine methylation in the regulation of CXCR4 expression in breast cancer cell lines and also correlated the methylation pattern with the clinicopathological aspects of sixty-nine primary breast tumors from a cohort of Brazilian women. RT-PCR showed that the PMC-42, MCF7 and MDA-MB-436 breast tumor cell lines expressed high levels of CXCR4. Conversely, the MDA-MB-435 cell line only expressed CXCR4 after treatment with 5-Aza-CdR, which suggests that CXCR4 expression is regulated by DNA methylation. To confirm this hypothesis, a 184 bp fragment of the CXCR4 gene promoter region was cloned after sodium bisulfite DNA treatment. Sequencing data showed that cell lines that expressed CXCR4 had only 15% of methylated CpG dinucleotides, while the cell line that not have CXCR4 expression, had a high density of methylation (91%). Loss of DNA methylation in the CXCR4 promoter was detected in 67% of the breast cancer analyzed. The absence of CXCR4 methylation was associated with the tumor stage, size, histological grade, lymph node status, ESR1 methylation and CXCL12 methylation, metastasis and patient death. Kaplan-Meier curves demonstrated that patients with an unmethylated CXCR4 promoter had a poorer overall survival and disease-free survival. Furthermore, patients with both CXCL12 methylation and unmethylated CXCR4 had a shorter overall survival and disease-free survival. These findings suggest that the DNA methylation status of both CXCR4 and CXCL12 genes could be used as a biomarker for prognosis in breast cancer.     

Orally Administered Lactoperoxidase Increases Expression of the FK506 Binding Protein 5 Gene in Epithelial Cells of the Small Intestine of Mice: A DNA Microarray Study

Lactoperoxidase (LPO) is a component of milk and other external secretions. To study the influence of ingested LPO on the digestive tract, we performed DNA microarray analysis of the small intestine of mice administered LPO. LPO administration upregulated 78 genes, including genes involved in metabolism, immunity, apoptosis, and the cell cycle, and downregulated nine genes, including immunity-related genes. The most upregulated gene was FK506 binding protein 5 (FKBP5), a glucocorticoid regulating immunophilin. The upregulation of this gene was confirmed by quantitative RT-PCR in other samples. In situ hybridization revealed that expression of the FKBP5 gene in the crypt epithelial cells of the small intestine was enhanced by LPO. These results suggest that ingested LPO modulates gene expression in the small intestine and especially increases FKBP5 gene expression in the epithelial cells of the intestine.     

Orally administered lactoperoxidase increases expression of the FK506 binding protein 5 gene in epithelial cells of the small intestine of mice: a DNA microarray study

Lactoperoxidase (LPO) is a component of milk and other external secretions. To study the influence of ingested LPO on the digestive tract, we performed DNA microarray analysis of the small intestine of mice administered LPO. LPO administration upregulated 78 genes, including genes involved in metabolism, immunity, apoptosis, and the cell cycle, and downregulated nine genes, including immunity-related genes. The most upregulated gene was FK506 binding protein 5 (FKBP5), a glucocorticoid regulating immunophilin. The upregulation of this gene was confirmed by quantitative RT-PCR in other samples. In situ hybridization revealed that expression of the FKBP5 gene in the crypt epithelial cells of the small intestine was enhanced by LPO. These results suggest that ingested LPO modulates gene expression in the small intestine and especially increases FKBP5 gene expression in the epithelial cells of the intestine.     

Requirement for ERK MAP kinase in mouse preimplantation development

Preimplantation development is a crucial step for successful implantation and pregnancy. Although both compaction and blastocyst formation have been extensively studied, mechanisms regulating the early cell division stages before compaction have remained unclear. Here, we show that extracellular signal regulated kinase (ERK) mitogen-activated protein (MAP) kinase function is required for early embryonic cell division before compaction. Our analysis demonstrates that inhibition of ERK activation in late two-cell-stage embryos leads to a reversible arrest in the G2 phase at the four-cell stage. The G2-arrested four-cell-stage embryos showed weakened cell-cell adhesion as compared with control embryos. Remarkably, microarray analyses showed that most of the programmed changes of upregulated and downregulated gene expression during the four- to eight-cell stages proceeded normally in four-cell-stage-arrested embryos that were subsequently released to resume development; however, the expression profiles of a proportion of genes in these embryos closely paralleled the stages of embryonic rather than normal development. These parallel genes included the genes encoding intercellular adhesion molecules, whose expression appeared to be positively regulated by the ERK pathway. We also show that, whereas ERK inactivation in eight-cell-stage embryos did not lead to cell division arrest, it did cause this arrest when cadherin-mediated cell-cell adhesion was disrupted. These results demonstrate an essential role of ERK function in two-cell to eight-cell-stage embryos, and suggest a loose parallelism between the gene expression programs and the developmental stages before compaction.     

Effects of DNA methylation on expression of virulence genes in Streptococcus mutans

Bacteria produce a variety of enzymes capable of methylating DNA. In many species, the majority of adenine methylation is accomplished by the DNA adenine methylase Dam. In Escherichia coli the Dam methylase plays roles in the initiation of replication, mismatch repair, and gene regulation. In a number of other bacterial species, mutation or overexpression of Dam leads to attenuation of virulence. Homologues of the dam gene exist in some members of the Firmicutes, including Streptococcus mutans, a dental pathogen. An S. mutans strain inactivated in the dam gene (SMU.504; here designated damA) was engineered, and phenotypes linked to cariogenicity were examined. A prominent observation was that the damA mutant produced greater amounts of glucan than the parental strain. Real-time PCR confirmed upregulation of gtfB. To determine whether other loci were affected by the damA mutation, a microarray analysis was carried out. Seventy genes were upregulated at least 2-fold in the damA mutant, and 33 genes were downregulated at least 2-fold. In addition to gtfB (upregulated 2.6-fold; 1.7-fold when measured by real-time PCR), other upregulated virulence factors included gbpC (upregulated 2.1-fold) and loci predicted to encode bacteriocins (upregulated 2- to 7-fold). Various sugar transport operons were also upregulated, the most extreme being the cellobiose operon (upregulated nearly 40-fold). Expression of sacB, encoding fructosyltransferase, was downregulated 2.4-fold. The sequence 5'-GATC-3' appeared to constitute the recognition sequence for methylation. These results provide evidence that DNA methylation in S. mutans has a global effect on gene expression, including that of genes associated with cariogenic potential.     

Curcumin Modulates DNA Methylation in Colorectal Cancer Cells

Aim

Recent evidence suggests that several dietary polyphenols may exert their chemopreventive effect through epigenetic modifications. Curcumin is one of the most widely studied dietary chemopreventive agents for colon cancer prevention, however, its effects on epigenetic alterations, particularly DNA methylation, remain unclear. Using systematic genome-wide approaches, we aimed to elucidate the effect of curcumin on DNA methylation alterations in colorectal cancer cells.

Materials and Methods

To evaluate the effect of curcumin on DNA methylation, three CRC cell lines, HCT116, HT29 and RKO, were treated with curcumin. 5-aza-2′-deoxycytidine (5-aza-CdR) and trichostatin A treated cells were used as positive and negative controls for DNA methylation changes, respectively. Methylation status of LINE-1 repeat elements, DNA promoter methylation microarrays and gene expression arrays were used to assess global methylation and gene expression changes. Validation was performed using independent microarrays, quantitative bisulfite pyrosequencing, and qPCR.

Results

As expected, genome-wide methylation microarrays revealed significant DNA hypomethylation in 5-aza-CdR-treated cells (mean β-values of 0.12), however, non-significant changes in mean β-values were observed in curcumin-treated cells. In comparison to mock-treated cells, curcumin-induced DNA methylation alterations occurred in a time-dependent manner. In contrast to the generalized, non-specific global hypomethylation observed with 5-aza-CdR, curcumin treatment resulted in methylation changes at selected, partially-methylated loci, instead of fully-methylated CpG sites. DNA methylation alterations were supported by corresponding changes in gene expression at both up- and down-regulated genes in various CRC cell lines.

Conclusions

Our data provide previously unrecognized evidence for curcumin-mediated DNA methylation alterations as a potential mechanism of colon cancer chemoprevention. In contrast to non-specific global hypomethylation induced by 5-aza-CdR, curcumin-induced methylation changes occurred only in a subset of partially-methylated genes, which provides additional mechanistic insights into the potent chemopreventive effect of this dietary nutraceutical.     

Apert p.Ser252Trp mutation in FGFR2 alters osteogenic potential and gene expression of cranial periosteal cells

Apert syndrome (AS), a severe form of craniosynostosis, is caused by dominant gain-of-function mutations in FGFR2. Because the periosteum contribution to AS cranial pathophysiology is unknown, we tested the osteogenic potential of AS periosteal cells (p.Ser252Trp mutation) and observed that these cells are more committed toward the osteoblast lineage. To delineate the gene expression profile involved in this abnormal behavior, we performed a global gene expression analysis of coronal suture periosteal cells from seven AS patients (p.Ser252Trp), and matched controls. We identified 263 genes with significantly altered expression in AS samples (118 upregulated, 145 downregulated; SNR >or= |0.4|, P 相似文献   

5-杂氮-2′-脱氧胞苷诱导鼻咽癌细胞株Syk基因启动子去甲基化的研究

利用5-杂氮-2′-脱氧胞苷（5-aza-2′-deoxycytidine,5-aza-CdR）处理体外培养的鼻咽癌细胞株CNE-1、CNE-2及永生化非癌性人鼻咽上皮细胞株NP-69,采用BS-PCR、Q-RT-PCR及Westernblot方法分别检测经5μmol/L的5-aza-CdR处理前后,各细胞株中Syk基因启动子甲基化状况及SykmRNA和蛋白质表达情况。探讨去甲基化药物5-杂氮-2′-脱氧胞苷（5-aza-CdR）对鼻咽癌细胞株中脾酪氨酸激酶（spleen tyrosine kinase,Syk）启动子甲基化水平及其表达的影响。结果显示,Syk基因启动子甲基化水平与鼻咽癌细胞分化程度呈负相关,两种鼻咽癌细胞株的Syk mRNA和蛋白质表达水平显著低于NP-69细胞（P〈0.01）;经5-aza-CdR处理后两种鼻咽癌细胞株的Syk基因启动子甲基化水平降低,Syk mRNA及蛋白质表达升高（P〈0.05）;高分化鼻咽癌细胞株对药物敏感性高于低分化鼻咽癌细胞株（P〈0.01）。由此可见,两种鼻咽癌细胞株中存在不同程度的Syk基因启动子甲基化状态,5-aza-CdR能有效逆转鼻咽癌细胞株Syk基因启动子的甲基化状态,升高Syk mRNA及蛋白质表达,同时鼻咽癌细胞分化程度越高恢复Syk基因表达的比率越高。     

5-Aza-2′-deoxycytidine Leads to Reduced Embryo Implantation and Reduced Expression of DNA Methyltransferases and Essential Endometrial Genes

Background

The DNA demethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR) incorporates into DNA and decreases DNA methylation, sparking interest in its use as a potential therapeutic agent. We aimed to determine the effects of maternal 5-aza-CdR treatment on embryo implantation in the mouse and to evaluate whether these effects are associated with decreased levels of DNA methyltransferases (Dnmts) and three genes (estrogen receptor α [Esr1], progesterone receptor [Pgr], and homeobox A10 [Hoxa10]) that are vital for control of endometrial changes during implantation.

Methods and Principal Findings

Mice treated with 5-aza-CdR had a dose-dependent decrease in number of implantation sites, with defected endometrial decidualization and stromal cell proliferation. Western blot analysis on pseudo-pregnant day 3 (PD3) showed that 0.1 mg/kg 5-aza-CdR significantly repressed Dnmt3a protein level, and 0.5 mg/kg 5-aza-CdR significantly repressed Dnmt1, Dnmt3a, and Dnmt3b protein levels in the endometrium. On PD5, mice showed significantly decreased Dnmt3a protein level with 0.1 mg/kg 5-aza-CdR, and significantly decreased Dnmt1 and Dnmt3a with 0.5 mg/kg 5-aza-CdR. Immunohistochemical staining showed that 5-aza-CdR repressed DNMT expression in a cell type–specific fashion within the uterus, including decreased expression of Dnmt1 in luminal and/or glandular epithelium and of Dnmt3a and Dnmt3b in stroma. Furthermore, the 5′ flanking regions of the Esr1, Pgr, and Hoxa10 were hypomethylated on PD5. Interestingly, the higher (0.5 mg/kg) dose of 5-aza-CdR decreased protein expression of Esr1, Pgr, and Hoxa10 in the endometrium on PD5 in both methylation-dependent and methylation-independent manners.

Conclusions

The effects of 5-aza-CdR on embryo implantation in mice were associated with altered expression of endometrial Dnmts and genes controlling endometrial changes, suggesting that altered gene methylation, and not cytotoxicity alone, contributes to implantation defects induced by 5-aza-CdR.     

Further characterization of genes expressed during Ciona intestinalis metamorphosis

Metamorphosis of ascidians is a dynamic event by which a nonfeeding, mobile tadpole larva is transformed into a filter-feeding, fixed juvenile. This process usually begins with the settlement of the larva and is followed by a series of coordinated morphogenetic movements that rearrange organs, tissues, and cells. To identify genes that are involved in the initiation of metamorphosis, we conducted differential screening between mRNAs of swimming larvae and those of juveniles in Ciona intestinalis. This screening permitted the isolation of cDNA clones for genes whose expression is upregulated during metamorphosis, and the characterization of four such genes (Ci-meta3, Ci-meta4, Ci-meta5 and Ci-meta6) is reported here. Ci-meta3 encodes a protein with a domain found in Sp1a and the RYanodine receptor. This gene is not expressed in early swimming larvae but is expressed in the endoderm region and part of the retractile tail region in metamorphosing juveniles. The predicted proteins encoded by Ci-meta4, Ci-meta5 and Ci-meta6 do not contain any known consensus motifs, nor do they show any similarity to known proteins. Ci-meta4 and Ci-meta5 are expressed weakly in mesenchyme cells of the early larva and strongly in the metamorphosing juvenile, while Ci-meta6 is expressed in the mesenchyme in the late larva. In addition, we characterized 53 independent cDNA clones whose expression was downregulated during the period from early swimming larvae to metamorphosing juveniles by taking advantage of the Ciona intestinalis cDNA project database and BLAST searches. The expression patterns of some of these clones were changed during the larval period.