Project description:Total RNA was extrected from HiDEP-1 (Immortalized erythroid cell line derived from human induced pluripotent stem cells; PMID: 23533656) cultured with HDAC inhibitors, Fluoro-SAHA (FS) or M344 or Valproic acid (VPA), for 24 hours using Rneasy Mini Kit (QIAGEN) by following the manufacture's protocol. After the quality/quantity determination, RNA was subjected to expression analyses using Affymetrix GeneChip® Array / Human Gene 2.0 ST Array
Project description:Analysis of gene expression change after 8hr's treatment with 5μM SAHA, which is a histone deacetylase inhibitor, in MDA-MB-231 cell lines.
Project description:Global mRNA expression profiles of murine primary PDAC cells following JQ1 or SAHA monotherapy as well as JQ1-SAHA combination therapy were collected using Affymetix mouse whole genome array (Mouse Genome 430A 2.0 Array) . Primary PDAC cells isolated from Ptf1aCre/+;Kras+/LSL-G12D;p53lox/lox (Kras;p53) mice were treated either with JQ1 (100 nM) or SAHA (2000 nM) or vehicle 10% (2-Hydroxypropyl)-β-cyclodextrin (Sigma-Aldrich) or as combination therapy with the indicated dosage for monotherapy. Total RNA isolation was performed after 6 hours of treatment. Primary PDAC cells from Ptf1aCre/+;Kras+/LSL-G12D;p53lox/lox (Kras;p53) mice treated either with JQ1, SAHA, vehicle or JQ1-SAHA combination were analyzed by global gene expression analysis.
Project description:Global mRNA expression profiles of murine primary PDAC cells following JQ1 or SAHA monotherapy as well as JQ1-SAHA combination therapy were collected using Affymetix mouse whole genome array (Mouse Genome 430A 2.0 Array) . Primary PDAC cells isolated from Ptf1aCre/+;Kras+/LSL-G12D;p53lox/lox (Kras;p53) mice were treated either with JQ1 (100 nM) or SAHA (2000 nM) or vehicle 10% (2-Hydroxypropyl)-β-cyclodextrin (Sigma-Aldrich) or as combination therapy with the indicated dosage for monotherapy. Total RNA isolation was performed after 6 hours of treatment.
Project description:While histone deacetylase (HDAC) inhibitors are thought to regulate gene expression by post-translational modification of histone as well as non-histone proteins. While histone hyperacetylation has long been considered the paradigmatic mechanism of action, recent genome-wide profiles indicate more complex interactions with the epigenome. In particular, HDAC inhibitors also induce histone deacetylation at the promoters of highly active genes, resulting in gene suppression. This was linked to the loss of histone acetyltransferase (HAT) binding. To illustrate pre-clinical utility of the HDAC inhibitor SAHA as a therapeutic, we show reversal of diabetes-associated EP300 target genes in diabetic HAECs of primary origin. These results were confirmed using SAHA, C646 (EP300/CREBBP inhibitor) or EP300 siRNA. These findings suggest the inhibition of gene expression by SAHA is mediated by EP300 function and provide a rationale for clinical trials of safety and efficacy in patients with diabetes.
Project description:We have employed mRNA microarray expression profiling as a discovery platform to identify genes with the potential to explain how small dose SAHA promoted the cell toxicity of B7-H3 CAR-T cells. B7-H3 CAR-T cells were treated by 0.5μM SAHA for 5 days, and with control samples.
Project description:Suberoylanilide hydroxamic acid (SAHA) has been assessed in clinical trials as part of a “shock and kill” strategy to cure HIV-infected patients. While it was effective at inducing expression of HIV RNA "shock" , treatment with SAHA did not result in the reduction of reservoir size "kill". We therefore utilized a systems biology approach to dissect the mechanisms of action of SAHA that may explain its limited success in “shock and kill” strategies. CD4+ T cells from HIV seronegative donors were treated with 1 uM SAHA or its solvent dimethyl sulfoxide for 24 hours. Differential protein expression and post-translational modification was measured with two-dimensional liquid chromatography - tandem mass spectrometry iTRAQ proteomics. Gene expression changes were assessed by Illumina microarrays. Using limma package in the R computing environment, we identified 185 proteins, 18 phosphorylated forms, 4 acetylated forms and 2,982 genes, whose expression was modulated by SAHA. A protein interaction network integrating these 4 data types identified the transcriptional regulator HMGA1 to be upregulated by SAHA at the transcript, protein and acetylated protein levels. HMGA1 has been shown to repress HIV transcription, which is not optimal with respect to a shock and kill strategy. Further functional category assessment of proteins and genes modulated by SAHA identified gene ontology terms related to NFB signaling, protein folding and autophagy, which are all relevant to HIV reactivation. In summary, this study identified a number of host factors that may be therapeutically targeted to achieve more potent HIV reactivation in the “shock and kill” treatment, when using SAHA, either through modification of SAHA itself or through combination with other latency reversing agents. Finally, proteome profiling highlighted a number of potential adverse effects of SAHA, which transcriptome profiling alone would not have identified.