Gene expression analysis in AML systems after 6 hours of HDACi treatments
ABSTRACT: K562, U937 and NB4 AML cell lines were treated with 2 HDACi, MS-275 and SAHA, and gene expression profiles were analysed to 6-hours treatments. The analysis revealed commonly regulated genes in the 3 cell lines and by 2 HDACi. Overall design: Total RNA were obtained from 3 AML cell lines after 6 hours of treatment with SAHA and MS-275 and gene expression profiles were compared to untreated cells (control).
Project description:AML cell lines (K562, U937 and NB4) were treated with MS27-275 (MS) and SAHA for 6 hours and the gene expression analysis revealed commonly regulated genes by the 2 HDACi. AML blasts were treated with SAHA for 6 hours and gene expression profiles were compared to commonly regulated genes by MS and SAHA in AML cell lines. The analysis revealed commonly regulated genes in these systems by SAHA. Overall design: Total RNA were obtained from 3 AML cell lines and AML blasts after 6 hours of treatment with SAHA and MS and gene expression profiles were compared.
Project description:Esophageal cancers (ECs) are highly aggressive tumors with poor prognosis and few treatment options. This study investigated the possibility of treating esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) cells by inhibitors of broad and specific histone deacetylases (HDACi; SAHA, MS-275, FK228) and/or of DNMT (Azacytidine, AZA). Drug targets (HDAC1,2,3 and DNMT1) were present in non-neoplastic (HET-1A), ESCC (OE21) and EAC (OE33) cell lines. All cell lines responded to HDACi by reduced HDAC activity and increased histone acetylation as well as to AZA by up-regulation of p21. Expression of drug targets remained largely unaffected by HDACi and AZA treatment. Importantly, cell viability, apoptosis, cell cycle dynamics and DNA damage were only affected by HDACi and/or AZA in ESCC and EAC, but not the non-neoplastic cells. This was specifically seen for the combination of MS-275 and AZA, leading to enhanced cancer cell selectivity and drug efficiency. By transcriptome analyses of MS-275, AZA and MS-275/AZA treated cells, known (e.g. p21) as well as novel regulated genes significantly associated with the cellular effects post HDACi and/or AZA treatment in ESCC and EAC cells were identified. Finally, human EC tissue specimens frequently expressed the actionable drug targets HDAC1/2/3 and DNMT1. In summary, a combined HDACi (MS-275)/AZA treatment is cancer cell selective and efficient in vitro. Since the majority of ECs express the drug targets in situ, this paves the way for further investigations of HDACi/AZA treatment in esophageal cancer cells and their translation into a clinico-pathological setting. To elucidate the transcriptome response to HDAC inhibitors of normal esophageal cells and esophageal tumor cells, total RNA was isolated from non-neoplastic esophageal epithelial cells (Het1A cells) a well as from two esophageal tumor cell lines (OE21 and OE33), respectively. Cells were treated with either MS-275, Azacytidine (AZA) or in combination of both. DMSO treatment was used as control in each case. Total RNA was isolated from cells 24 h after treatment and experiments were performed in biological triplicates.
Project description:AML blasts and CD34+ normal progenitors were treated with SAHA, and gene expression profiles were compared to 6-hours treatments. The analysis revealed commonly regulated genes in these systems by HDACi. Overall design: Total RNA were obtained from AML blasts and normal progenitors after 6 hours of treatment with SAHA and gene expression profiles were compared.
Project description:This study reports the ability of WEB-2170, an antagonist of platelet-activating-factor receptor, to induce apoptosis in human acute myelogenous leukemia (AML) cells. Action mechanisms of WEB-2170 were first investigated in promyelocytic NB4 cells by DNA microarray profiling followed by morphologic, cytofluorimetric and biological analyses which were then extended to other AML cell lines including KG1, NB4-MR4, THP1 and U937, and, eventually, to blasts from patients with different AML subtypes (M0-M5).
Project description:This study reports the ability of WEB-2170, an antagonist of platelet-activating-factor receptor, to induce apoptosis in human acute myelogenous leukemia (AML) cells. Overall design: Action mechanisms of WEB-2170 were first investigated in promyelocytic NB4 cells by DNA microarray profiling followed by morphologic, cytofluorimetric and biological analyses which were then extended to other AML cell lines including KG1, NB4-MR4, THP1 and U937, and, eventually, to blasts from patients with different AML subtypes (M0-M5).
Project description:Illumina High-Throughput ChIP Sequencing profiling was performed using the H3K9K14ac antibody in NB4 cells treated with the compounds ATRA, MS-275,MC2392 (a hybrid molecule of ATRA with a 2-aminoanilide tail of the HDAC inhibitor MS-275) or solvent, DMSO. We find that MC2392 induces changes in H3 acetylation at a small subset of PML-RARα binding sites but also in regions not regulated by ATRA. Moreover, MC2392 alters expression of a number of stress-responsive and apoptotic genes. Overall design: Genome-wide ChIP-seq was performed in NB4 cells for histone3 acetylated in K9K14
Project description:Epigenetic modifying enzymes are commonly mutated in diffuse large B cell lymphoma (DLBCL). Importantly, genetics abnormalities lead to inactivation of HAT, which tilt the balance in favor of decreased protein acetylation in DLBCL cells. This suggests that protein acetylation regulation is an important factor in DLBCL pathogenesis and a potential target for therapy. We developed resistant cell lines to the histone deacetylase inhibitor (HDACi) vorinostat, in order to better define molecular mechanisms of action of HDACi in lymphoma cells. We found that cells resistant to HDACi have increased protein synthesis and proteasomal degradation. Additionally, cells resistant to HDACi have acquired increased susceptibility to proteasome inhibitors and this correlates with activation of the unfolded protein response. Importantly, using transcriptional signatures found in our resistant lymphoma cell line model, we show that tumors from DLBCL patients treated but unresponsive to HDACi therapy undergo similar changes. Together, these data show, for the first time, that HDACi may be used to prime DLBCL for targeted therapy including proteasome inhibitors. Gene expression in U937 cells after 12h exposure to 2µM vorinostat and after development of resistance to 2 µM vorinostat, with and without vorinostat in the media.
Project description:miRNAs deregulation contributes to cancer. miR-194-5p is up-regulated by the HDAC inhibitor (HDACi) SAHA, negatively modulating BCL2-associated transcription factor 1 (BCLAF1). We prove that the miR-194-5p/BCLAF1 equilibrium regulates differentiation, survival and self-renewal of normal progenitors and acute myeloid leukemia (AML) blasts. This equilibrium is perturbed in AMLs resulting in highly expressed BCLAF1, suppression of miR194-5p, consequently, locking cells into an immature, potentially ‘immortal’ state. HDACis reverse this scenario relocating BCLAF1 from the nucleus to a peri-membrane ring-like cytoplasmic structure, sensitizing the cells to differentiation or apoptosis. miR-194-5p and BCLAF1 are significantly deregulated in a cohort of 60 primary AMLs and get restored by HDACi. Our findings connect responsiveness to treatment to re-instatement of miR-194-5p/BCLAF1 balance. These findings might be exploited for (epi-based) anti-leukemia therapy. Overall design: U937 cells were transduced with constructs expressing mir-194-5p or a scrambled (sc) control. The transduced cell lines were used for DNAseI-seq analysis following previously published protocols (Neph et al., 2012)
Project description:Immune recognition of tumor-expressed antigens by cytotoxic CD8+ T lymphocytes is the foundation of adoptive T-cell therapy. However, therapy-induced selective pressure can sculpt the antigenicity of tumors resulting in the outgrowth of variants that have lost the target antigen. Interestingly, tumor relapse resulting from adoptive memory T cell transfer and boosting oncolytic viral vaccination can be prevented using Class I histone deacetylase inhibitor, MS-275. We demonstrate that concomitant drug delivery subverts the phenotype and suppressive function of tumor-infiltrating myeloid cells and reprograms them with the cytotoxic capacity to directly eliminate antigen-negative tumor cells. By enhancing the production of IFNγ within the tumor microenvironment, our data suggest that MS-275 modifies the local cytokine landscape in favor of antitumor myeloid cell polarization. Overall design: Total RNA (n=4 per group) obtained from tumors subjected to gp33-specific Tm + VSV-gp33 +/- MS-275 compared to untreated control tumors.
Project description:For a long time, the BARD1 (BRCA1-associated RING domain 1) protein has been considered as a BRCA1 (BReast Cancer susceptibility gene 1, early onset) interactor, and tumor suppressor mutated in breast and ovarian cancers. Despite its functions in a stable heterodimer with BRCA1, there is increasing evidence for BRCA1-independent functions of BARD1. Here, we investigated BARD1 expression and function in human acute myeloid leukemias and their modulation by epigenetic mechanisms and microRNA. We show that the HDACi (histone deacetylase inhibitor) Vorinostat reduces BARD1 mRNA levels by increasing miR-19a and miR-19b expression levels. Moreover, we identify specific BARD1 isoforms that might act as tumor diagnostic and prognostic markers. Two-condition experiment: untreated NB4 cells (control) vs. NB4 cells treated with 5µM SAHA (Vorinostat) for 6h. Biological replicates: 3 control, 3 treated, independently grown and harvested at 6 hours. One replicate per array.