trichostatin-a and Central-Nervous-System-Neoplasms

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

Histone deacetylase inhibitors that increase histone acetylation on transformed cells are being investigated as unique anticancer drugs. The aim of this investigation was to evaluate an antiproliferative activity of the histone deacetylase inhibitors sodium butyrate (NaBT) and trichostatin A on 5 glioma cell lines, T98G, A172, U-87 MG, U-118 MG, and U-373 MG, with the examination of the altered expressions in p21 and gelsolin genes. Treatment with 5-mM NaBT and 40 ng/ml trichostatin A for 48 h caused more than a 50% growth inhibition in 5 cell lines as measured by cell proliferation assays. An increase in histone acetylation was confirmed in each cell line. After treatment with 5 mM NaBT, T98G, A172, and U118 cells undergo apoptosis as indicated by DNA ladder formation. Treatment with NaBT and trichostatin A also decreased DNA synthesis as examined by the fluorescence-activated cell sorting analysis in T98G and U87 cells. In addition to the suppression of cell growth, the up regulation of p21 and gelsolin expression was observed after treatment with NaBT, especially in T98G cells. Maximum expression of p21 and gelsolin was observed within 24 h after treatment. Results from our in vitro studies indicate that the treatment of human glioma cells with one of the histone deacetylase inhibitors suppresses cell growth with decreasing DNA synthesis and stimulates apoptosis, and that associated molecular mechanisms responsible for these effects include increased histone acetylation as well as enhanced expression of p21 and gelsolin.

Topics: Acetylation; Apoptosis; Butyrates; Cell Cycle; Cell Division; Central Nervous System Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Flow Cytometry; Gelsolin; Gene Expression Regulation, Neoplastic; Glioma; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immunoblotting; Tumor Cells, Cultured; Up-Regulation