trichostatin-a and 3-deazaneplanocin

We examined the consequences of 3-deazaneplanocin A (DZNep) on HACE1 expression in human Burkitt- Lymphoma-derived cells to investigate fundamental molecular mechanisms that control its expression. We treated the human Burkitt- Lymphoma-derived cells lines Ramos and Raji with DZNep and examined HACE1 mRNA expression by RT-PCR. We also studied the effect of DZNep on the methylation of lysine 9 and 27 of histone 3 (H3K27me3 and H3K9me2) associated with the CpG88 and CpG177 islands of the HACE1 promoters by chromatin immunoprecipitation and quantitative PCR. CpG88 (hypomethylated) of the HACE1 promoter was enriched for histone marks H3K27me3 and H3K9me2 whereas CpG177 (hypermethylated) was only enriched for H3K9me2. DZNep treatment increased HACE1 gene expression which was further increased by the addition of trichostatine A (TSA), a promising therapeutic compound for the treatment of human B-Lymphoma. Histone methylation (both H3K9me2 and H3K27me3) of the HACE1 promoter concomitantly decreased. Our experiments suggest that HACE1 can be downregulated by methylation of its promoter region chromatin (H3K27me3 and H3K9me2), making HACE1 a potential target for DZNep combined with TSA. These results highlight the heterogeneity of HACE1 regulation in B-lymphoma and suggest that successful drug-induced restoration of epigenetically silenced tumor suppressor genes will require accurate characterization of cell type- and locus-specific gene silencing mechanisms.

Topics: Adenosine; Burkitt Lymphoma; Cell Line, Tumor; CpG Islands; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histones; Humans; Hydroxamic Acids; Promoter Regions, Genetic; RNA, Messenger; Ubiquitin-Protein Ligases

Aberrant epigenetic histone modifications are implicated in cancer pathobiology, therefore histone modifying enzymes are emerging targets for anti-cancer therapy. There is a few evidence for deregulation of the histone modifying enzymes in glioblastomas. Glioma treatment is a clinical challenge due to its resistance to current therapies.. The effect of selected inhibitors on epigenetic modifications and viability of glioma C6 cells were studied using immunofluorescence and MTT metabolism test.. We found that VPA and TSA increase histone H4 acetylation in glioma cells, while chaetocin and BIX01294 at low concentrations reduce H3K9me3, and 3DZNep decreases H3K27me3. Long-term treatment with some epigenetic inhibitors affects viability of glioma cells.. We established the concentrations of selected inhibitors which in C6 glioma cells inhibit the enzyme activity, but do not decrease cell viability, hence allow to study the role of histone modifications in C6 glioma biology.

Topics: Acetylation; Adenosine; Animals; Azepines; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Epigenesis, Genetic; Glioma; Histones; Hydroxamic Acids; Piperazines; Quinazolines; Rats; Valproic Acid

Proangiogenic hematopoietic and endothelial progenitor cells (EPCs) contribute to postnatal neovascularization, but the mechanisms regulating differentiation to the endothelial lineage are unclear.. To elucidate the epigenetic control of endothelial gene expression in proangiogenic cells and EPCs.. Here we demonstrate that the endothelial nitric oxide synthase (eNOS) promoter is epigenetically silenced in proangiogenic cells (early EPCs), CD34(+) cells, and mesoangioblasts by DNA methylation and prominent repressive histone H3K27me3 marks. In order to reverse epigenetic silencing to facilitate endothelial commitment, we used 3-deazaneplanocin A, which inhibits the histone methyltransferase enhancer of zest homolog 2 and, thereby, reduces H3K27me3. 3-Deazaneplanocin A was not sufficient to increase eNOS expression, but the combination of 3-deazaneplanocin A and the histone deacetylase inhibitor Trichostatin A augmented eNOS expression, indicating that the concomitant inhibition of silencing histone modification and enhancement of activating histone modification facilitates eNOS expression. In ischemic tissue, hypoxia plays a role in recruiting progenitor cells. Therefore, we examined the effect of hypoxia on epigenetic modifications. Hypoxia modulated the balance of repressive to active histone marks and increased eNOS mRNA expression. The reduction of repressive H3K27me3 was associated with an increase of the histone demethylase Jmjd3. Silencing of Jmjd3 induced apoptosis and senescence in proangiogenic cells and inhibited hypoxia-mediated up-regulation of eNOS expression in mesoangioblasts.. These findings provide evidence that histone modifications epigenetically control the eNOS promoter in proangiogenic cells.

Topics: Acetylation; Adenosine; Apoptosis; Cell Hypoxia; Cell Lineage; Cells, Cultured; Cellular Senescence; DNA Methylation; Endothelial Cells; Enzyme Induction; Hematopoietic Stem Cells; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Jumonji Domain-Containing Histone Demethylases; Neovascularization, Physiologic; Nitric Oxide Synthase Type III; Promoter Regions, Genetic; Protein Processing, Post-Translational; RNA, Messenger

Polycomb protein EZH2-mediated gene silencing is implicated in breast tumorigenesis through methylation of histone H3 on Lysine 27 (H3K27). We have previously shown that S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A can modulate histone methylation and disrupt EZH2 complex. Here, we used 3-deazaneplanocin A, together with other chromatin remodeling agents, as well as RNA interference-mediated EZH2 depletion, to probe the role of EZH2 in coordination with other epigenetic components in gene regulation in breast cancer cells. Through genome-wide gene expression analysis, coupled with extensive chromatin immunoprecipitation analysis of histone modifications, we have identified a variety of gene sets that are regulated either by EZH2 alone or through the coordinated action of EZH2 with HDAC and/or DNA methylation. We further found that tumor antigen GAGEs were regulated by distinct epigenetic mechanisms in a cell context-dependent manner, possibly reflecting mechanistic heterogeneity in breast cancer. Intriguingly, we found that EZH2 regulates a remarkable cohort of genes whose functions are highly enriched in immunoresponse and autocrine inflammation network, and that their transcriptional activation upon EZH2 perturbation is cancer specific, revealing a potential novel role of EZH2 in regulating cancer immunity. These findings show the complexity and diversity of epigenetic regulation in human cancer and underscore the importance for developing combinatorial pharmacologic approaches for effective epigenetic gene reactivation.

Topics: Adenosine; Antigens, Neoplasm; Azacitidine; Breast Neoplasms; Cell Line; Cell Line, Tumor; Chromatin Immunoprecipitation; Cluster Analysis; CpG Islands; Decitabine; DNA Methylation; DNA-Binding Proteins; Enhancer of Zeste Homolog 2 Protein; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immunoblotting; Methylation; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Polycomb Repressive Complex 2; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transcription Factors

Genetic and epigenetic defects in Wnt/beta-catenin signaling play important roles in colorectal cancer progression. Here we identify DACT3, a member of the DACT (Dpr/Frodo) gene family, as a negative regulator of Wnt/beta-catenin signaling that is transcriptionally repressed in colorectal cancer. Unlike other Wnt signaling inhibitors that are silenced by DNA methylation, DACT3 repression is associated with bivalent histone modifications. Remarkably, DACT3 expression can be robustly derepressed by a pharmacological combination that simultaneously targets both histone methylation and deacetylation, leading to strong inhibition of Dishevelled (Dvl)-mediated Wnt/beta-catenin signaling and massive apoptosis of colorectal cancer cells. Our study identifies DACT3 as an important regulator of Wnt/beta-catenin signaling in colorectal cancer and suggests a potential strategy for therapeutic control of Wnt/beta-catenin signaling in colorectal cancer.

Topics: Adaptor Proteins, Signal Transducing; Adenosine; Adenosylhomocysteinase; Apoptosis; Azacitidine; beta Catenin; Colorectal Neoplasms; Decitabine; Dishevelled Proteins; DNA Methylation; DNA Modification Methylases; Down-Regulation; Enzyme Inhibitors; Epigenesis, Genetic; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; HCT116 Cells; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Oligonucleotide Array Sequence Analysis; Phosphoproteins; Promoter Regions, Genetic; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transfection; Wnt Proteins