Project description:In microarray analysis, combinational treatment with PJ-34 and 5-aza-dC caused dissimilar broad changes in gene expression profiles compared with their single treatments in both HCT116 and RKO cells. Profiles of reactivation of silenced genes were also different in combination of PJ-34 and 5-aza-dC and their single treatments. The results suggest that combinational use of 5-aza-dC and PARP inhibitor may be useful by causing distinct transcriptional profile changes.
Project description:To assess transcriptional regulation by DNA demethylation in SAEC, we carried out a microarray analysis of SAEC treated with a demethylating agent (5-aza-dC) and a HDAC inhibitor (TSA). We used the Agilent SurePrint G3 Human Gene Expression 8x60K v3 microarray which contains probes for 26,083 Entrez genes and 30,606 lncRNAs.
Project description:DNA hypermethylated genes undergo silencing and show re-expression in response to 5-Aza-CdR treatment. Agilent arrays were used to determine the genes that get reactivated in response to the drug treatment. To determine expression levels of genes in SW480, HCT116 and RKO.
Project description:Expression profiling the response to inhibition of DNA methylation and histone deacetylation. Comparison of expression in HepG2 cells treated with 5-aza-dC, Trichostatin A, both, or none (control) to change methylation and acetylation status. Backgroubd: DNA methylation and histone deacetylation are epigenetic mechanisms that play major roles in eukaryotic gene regulation. We hypothesize that many genes in the human hepatoma cell line HepG2 are regulated by DNA methylation and histone deacetylation. Treatment with 5-aza-2'-deoxycytidine (5-aza-dC) to inhibit DNA methylation with and/or Trichostatin A (TSA) to inhibit histone deacetylation should allow us to identify genes that are regulated epigenetically in hepatoma cells. RESULTS: 5-aza-dC had a much larger effect on gene expression in HepG2 cells than did TSA, as measured using Affymetrix HG-U133 Plus 2.0 microarrays. The expression of 1504 probe sets was affected by 5-aza-dC (at p < 0.01), 535 probe sets by TSA, and 1929 probe sets by the combination of 5-aza-dC and TSA. 5-aza-dC treatment turned on the expression of 211 probe sets that were not detectably expressed in its absence. Expression of imprinted genes regulated by DNA methylation, such as H19 and NNAT, was turned on or greatly increased in response to 5-aza-dC. Genes involved in liver processes such as xenobiotic metabolism (CYP3A4, CYP3A5, and CYP3A7) and steroid biosynthesis (CYP17A1 and CYP19A1), and genes encoding CCAAT element-binding proteins (C/EBPalpha, C/EBPbeta, and C/EBPgamma) were affected by 5-aza-dC or the combination. Many of the genes that fall within these groups are also expressed in the developing fetal liver and adult liver. Quantitative real-time RT-PCR assays confirmed selected gene expression changes seen in microarray analyses. CONCLUSION: Epigenetics play a role in regulating the expression of several genes involved in essential liver processes such as xenobiotic metabolism and steroid biosynthesis in HepG2 cells. Many genes whose expression is normally silenced in these hepatoma cells were re-expressed by 5-aza-dC treatment. DNA methylation may be a factor in restricting the expression of fetal genes during liver development and in shutting down expression in hepatoma cells
Project description:The goal of this study is to learn about the safety and tolerance of autologous TSA-DC cell and evaluate the efficacy and feasibility of the cell therapy compared to the patients’ past standard regimen. 20 gastrointestinal solid tumors subjects failed from at least one systemic therapy will be enrolled into the trial and receive a succession of treatment of TSA-DC vaccine.
Project description:Analysis of 2 cultured normal lung cell lines, Normal Human Bronchial Epithelial (NHBE) and Human Small Airway Epithelial (SAEC) cells (Lonza, Walkersville, MD), following treatment with 5-aza-dC to induce DNA demethylation. These results provide insight into the role of epigenetic alterations, specifically demethylation, in differential gene expression in various lung neoplasms. Two normal lung cell lines, NHBE and SAEC, were treated with 5uM 5-aza deoxycytidine for 72 hours and Trichostatin A for 24 hours prior to harvesting total RNA for expression array analysis using the Affymetrix Human Genome U133 Plus 2.0 expression platform. Signal intensity and statistical significance was established for each transcript using dChip version 2005. Two-fold increase based on the 90% confidence interval of the result and expression minus baseline >50 was used as the statistical cutoff value after 5Aza-dC and/or TSA treatment to identify upregulated candidate genes.
Project description:The goal of this study is to identify the gene expression changes caused by exposure of to the DNMT inhibitor 5-aza-2'-deoxycytidine (5Aza) and HDAC inhibitor Trichostatin A (TSA). We performed rRNA-depleted RNA sequencing of the untreated and drug-treated MCF7 breast cancer cell lines and carried out differential gene expression analysis. Although 5Aza caused a stranger demethylation effect than TSA, there were fewer differentially expressed gene in the 5Aza-treated MCF7 than the TSA-treated cells.
Project description:Blocking histone deacetylation with trichostatin A (TSA) or blocking cytosine methylation using 5-aza-2'-deoxycytosine (aza-dC) can derepress silenced genes in multicellular eukaryotes, including animals and plants. We questioned whether DNA methylation and histone deacetylation overlap in the regulation of endogenous plant genes by monitoring changes in expression of ~7800 Arabidopsis thaliana genes following treatment with azadC, TSA, or both chemicals together. RNA levels for ~4% of the genes were reproducibly changed 3-fold or more by at least one treatment. Distinct subsets of genes are up-regulated or down-regulated in response to aza-dC, TSA, or simultaneous treatment with both chemicals, with little overlap among subsets. Surprisingly, the microarray data indicate that TSA and aza-dC are often antagonistic rather than synergistic in their effects. Analysis of green fluorescent protein transgenic plants confirmed this finding, showing that TSA can block the up-regulation of silenced green fluorescent protein transgenes in response to aza-dC or a ddm1 (decrease in DNA methylation 1) mutation. Our results indicate that global inhibition of DNA methylation or histone deacetylation has complex, nonredundant effects for the majority of responsive genes and suggest that activation of some genes requires one or more TSA-sensitive deacetylation events in addition to cytosine demethylation.