trichostatin-a and hydrogen-sulfite

Epigenetic alterations are common events in cancer. Using a genome wide methylation screen (Restriction Landmark Genomic Scanning-RLGS) we identified the gene for the dopamine receptor D4 (DRD4) as tumor-specific methylated. As DRD4 is involved in early brain development and may thus be involved in developmentally dependent tumors of the CNS in children epigenetic deregulation of DRD4 and its functional consequences were analyzed in vitro. CpG methylation of DRD4 was detected in 18/24 medulloblastomas, 23/29 ependymomas, 6/6 high-grade gliomas, 7/10 CNS PNET and 8/8 cell lines by qCOBRA and bisulfite sequencing. Real-time RT-PCR demonstrated a significantly inferior expression of DRD4 in primary tumors compared to cell lines and non-malignant control tissues. Epigenetic deregulation of DRD4 was analyzed in reexpression experiments and restoration of DRD4 was observed in medulloblastoma (MB) cells treated with 5-Aza-CdR. Reexpression was not accompanied by demethylation of the DRD4 promoter but by a significant decrease of H3K27me3 and of bound enhancer of zeste homologue 2 (EZH2). Knockdown of EZH2 demonstrated DRD4 as a direct target for inhibition by EZH2. Stimulation of reexpressed DRD4 resulted in an activation of ERK1/2. Our analyses thus disclose that DRD4 is epigenetically repressed in CNS tumors of childhood. DRD4 is a direct target of EZH2 in MB cell lines. EZH2 appears to dominate over aberrant DNA methylation in the epigenetic inhibition of DRD4, which eventually leads to inhibition of a DRD4-mediated stimulation of the ERK1/2 kinase pathway.

Topics: Apoptosis; Azacitidine; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Central Nervous System Neoplasms; Child; Chromatin Assembly and Disassembly; Decitabine; Dose-Response Relationship, Drug; Epigenesis, Genetic; Female; Humans; Hydroxamic Acids; Male; Medulloblastoma; Neuroectodermal Tumors, Primitive; Receptors, Dopamine D4; Sulfites; Tumor Cells, Cultured

Human CYP1A1, CYP1A2, and CYP1B1 mRNAs were constitutively expressed in MCF-7 (human breast carcinoma) cells and were extensively (6-12-fold) induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, in HeLa (human cervical adenocarcinoma) cells, CYP1A1 and CYP1B1 were induced by TCDD by up to 2-3-fold but CYP1A2 was not detected even when the cells were treated with TCDD. In the present study, the involvement of histone deacetylation and DNA methylation in the cell-specific inducibility of the human CYP1 family was investigated. The treatment of MCF-7 cells with trichostatin A (TSA), an inhibitor of histone deacetylase, and 5-aza-2'-deoxycitidine (AzaC), an inhibitor of DNA methylase, increased the constitutive expression level of CYP1A1, CYP1A2, and CYP1B1 by 2-3-fold. However, these treatments did not affect the levels of induction by TCDD. In HeLa cells, TSA and AzaC treatment increased the constitutive expression levels of CYP1A1 and CYP1B1. The induction of CYP1A2 was enhanced to a detectable level by TSA and AzaC even when the cells were not exposed to TCDD. Interestingly, pretreatment with TSA and AzaC increased the levels of CYP1A1, CYP1A2, and CYP1B1 induced by TCDD in HeLa cells. Furthermore, it was observed that TSA and AzaC treatment increased the constitutive expression level of AhR by 2-fold only in HeLa cells. To compare the methylation status of the 5'-flanking region of the human CYP1A1 gene including five XREs and the promoter region in MCF-7 and HeLa cells, the bisulfite-modified genes were amplified and sequenced. Since there was no remarkable difference in the methylation status within a -1.4 kb region of the human CYP1A1 gene, the methylation status in the CpG sites that exist in other regions of the human CYP1A1 gene might be involved in the cell-specific induction.

Topics: 5' Flanking Region; Aryl Hydrocarbon Hydroxylases; Azacitidine; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP1B1; Cytochrome P-450 Enzyme System; Dealkylation; DNA; Environmental Pollutants; Gene Expression Regulation, Enzymologic; HeLa Cells; Histones; Humans; Hydroxamic Acids; Methylation; Polychlorinated Dibenzodioxins; Promoter Regions, Genetic; Protein Synthesis Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfites; Tumor Cells, Cultured