trichostatin-a and sodium-bisulfite

The first cell differentiation event in mammalian embryogenesis segregates inner cell mass lineage from the trophectoderm at the blastocyst stage. Oct-4, a member of the POU family of transcription factors, is necessary for the pluripotency of the inner cell mass lineage. Embryonic stem (ES) cells, which contribute to all of embryonic lineages, express the Oct-4 gene. Trophoblast stem (TS) cells, which have the ability to differentiate into trophoblast lineage in vitro, never contribute to embryonic proper tissues in chimeras and differentiate only into trophoblastic cells in the placenta. Expression of the Oct-4 gene was undetectable and severely repressed in trophoblastic lineage, including the stem cells. We found that the culture of TS cells with 5-aza-2'-deoxycytidine or trichostatin A caused the activation of the Oct-4 gene. Analysis of the DNA methylation status of mouse Oct-4 gene upstream region revealed that Oct-4 enhancer/promoter region was hypomethylated in ES cells but hypermethylated in TS cells. Furthermore, in vitro methylation suppressed Oct-4 enhancer/promoter activity in reporter assay. In the placenta of Dnmt1(n/n) mutant mice, most of the CpGs in the enhancer/promoter region were unmethylated, and Oct-4 gene expression was aberrantly detected. Chromatin immunoprecipitation assay revealed that Oct-4 enhancer/promoter region was hyperacetylated in ES cells compared with TS cells, thus demonstrating that DNA methylation status is closely linked to the chromatin structure of the Oct-4 gene. Here we propose that the epigenetic mechanism, consisting of DNA methylation and chromatin remodeling, underlies the developmental stage- and cell type-specific mechanism of Oct-4 gene expression.

Topics: Animals; Azacitidine; Cell Lineage; Chromatin; Decitabine; DNA Methylation; DNA-Binding Proteins; Embryo, Mammalian; Enzyme Inhibitors; Gene Expression Regulation; Gene Silencing; Genes, Reporter; Heterozygote; Hydroxamic Acids; Liver; Luciferases; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Models, Genetic; NIH 3T3 Cells; Octamer Transcription Factor-3; Placenta; Polymerase Chain Reaction; Precipitin Tests; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Sulfites; Transcription Factors; Trophoblasts

Transcriptional silencing of cancer-related genes by DNA methylation is observed in various cancers. To identify genes controlled by methylation in prostate cancer, we used cDNA microarray analysis to investigate gene expression in prostate cancer cell lines LNCaP and DU145 treated with a methyltransferase inhibitor alone or together with a histone deacetylase inhibitor. We detected significant changes (3.4-5.7%) in gene expression in prostate cancer cell lines with the drug treatments. Among the affected genes, that for the vascular endothelial growth factor receptor 1 (VEGFR-1) was re-expressed in LNCaP and DU145 after the drug treatments. Bisulfite sequencing revealed the promoter and exon 1 of the VEGFR-1 to be hypermethylated in the cell lines. These results support the idea that methylation is associated with loss of VEGFR-1 mRNA expression in prostate cancer cell lines. Combined bisulfite restriction analysis (COBRA) showed the gene to be methylated in 24 (38.1%) of 63 primary local prostate cancer samples, while in all 13 benign prostate samples it was not. These findings indicate that methylation of VEGFR-1 is related with prostatic carcinogenesis.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Decitabine; DNA Methylation; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Promoter Regions, Genetic; Prostatic Hyperplasia; Prostatic Neoplasms; Sulfites; Tumor Cells, Cultured; Vascular Endothelial Growth Factor Receptor-1

Expression of the KAI1 gene, a metastasis-suppressor for prostate cancer, is reduced in all foci of prostatic metastasis. The altered regulatory mechanism is not strongly related to mutations or allelic losses of the KAI1 gene in prostate tumors. Since transcriptional silencing of genes has been found to be caused by epigenetic mechanisms, we have investigated the involvement of this epigenetic regulation of KAI1 expression in prostate cancers. The methylation status of the KAI1 promoter region was examined by restriction-enzyme digestion and sequencing, after amplifying a 331-bp fragment in the GC-rich promoter region from 4 human prostate cancer cell lines treated with bisulfite. The same 4 cell lines were also exposed to various concentrations of the demethylating agent, 5-aza-2'-deoxycytidine (5-AzaC) and / or the histone deacetylase inhibitor, trichostatin A (TSA). To clarify the influence of epigenetic modification on reduced KAI1 mRNA expression in the tumor cells, RT-PCR and northern-blot analyses were performed. Bisulfite-sequencing data showed a few methylated CpG islands in the promoter. RT-PCR analysis of 5-AzaC and / or TSA-treated cells indicated reversal of suppression of KAI1 transcription in two cell lines (PC-3 and DU-145), although the expression could not be detected by northern blots. From these results, it is suggested that epigenetic change is not the main mechanism of KAI1 down-regulation, though there remains a possibility that methylation in a more upstream region might be associated with this regulation.

Topics: Adenocarcinoma; Antigens, CD; Azacitidine; Base Sequence; Decitabine; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Kangai-1 Protein; Male; Membrane Glycoproteins; Molecular Sequence Data; Neoplasm Metastasis; Neoplasm Proteins; Polymerase Chain Reaction; Promoter Regions, Genetic; Prostatic Hyperplasia; Prostatic Neoplasms; Proto-Oncogene Proteins; Sulfites; Tumor Cells, Cultured