Project description:RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) is a powerful new tool for analyzing gene knockdown phenotypes in living mammalian cells. To facilitate large-scale, high-throughput functional genomics studies using RNAi, we have developed a microarray-based technology for highly parallel analysis. Specifically, siRNAs in a transfection matrix were first arrayed on glass slides, overlaid with a monolayer of adherent cells, incubated to allow reverse transfection, and assessed for the effects of gene silencing by digital image analysis at a single cell level. Validation experiments with HeLa cells stably expressing GFP showed spatially confined, sequence-specific, time- and dose-dependent inhibition of green fluorescence for those cells growing directly on microspots containing siRNA targeting the GFP sequence. Microarray-based siRNA transfections analyzed with a custom-made quantitative image analysis system produced results that were identical to those from traditional well-based transfection, quantified by flow cytometry. Finally, to integrate experimental details, image analysis, data display, and data archiving, we developed a prototype information management system for high-throughput cell-based analyses. In summary, this RNAi microarray platform, together with ongoing efforts to develop large-scale human siRNA libraries, should facilitate genomic-scale cell-based analyses of gene function.
Project description:WM164 BRAF mutant Melanoma cells were treated with 500nM PLX4032 for 16 days and their gene expression was compared to parent cells kept in DMSO. WM164 treated and untreated cells were kept in triplicate and the mRNA from individual samples were isolated independently for analysis.
Project description:Adrenal corticosteroid biosynthesis dysregulation can give rise to various pathological conditions, such as Cushing's syndrome, a disorder characterized by the sustained and excessive production of cortisol. Despite the development of several classes of steroidogenesis inhibitors to treat human diseases associated with cortisol overproduction, their use is limited by insufficient efficacy, adverse effects, and/or tolerability. Recently, we identified a series of benzimidazolylurea derivatives, including the representative compound CJ28, as novel cortisol biosynthesis inhibitors [1]. They significantly inhibited both basal and stimulated production of cortisol in NCI-H295R cells, a human adrenocarcinoma cell line. The inhibitory effects were attributed to both attenuated steroidogenesis and de novo cholesterol biosynthesis. Here, we provide transcriptomic (RNA-seq) data from adrenal cell cultures in response to treatment with either CJ28 or metyrapone (MET), an inhibitor of 11β-hydroxylase). Total RNA was extracted from the cells treated with vehicle (0.1% DMSO), CJ28 (30 µM), or MET (30 µM) for 24 h. Primary sequence data were acquired using paired-end sequencing on an Illumina NovaSeq 6000 platform. The raw RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) database (GSE236435). This dataset is a useful resource for providing valuable information on the gene expression networks underlying adrenocortical steroidogenesis.
Project description:Primary aldosteronism accounts for 5%-10% of hypertension and in a third of cases is caused by autonomous aldosterone production by adenomas (APA). Somatic mutations in the potassium channel encoded by KCNJ5 have been detected in surgically removed APAs. To better understand the role of these mutations, we resequenced the KCNJ5 channel in a large Australian primary aldosteronism cohort. KCNJ5 mutations were detected in 37 APAs (45% of the cohort), including previously reported E145Q (n = 3), G151R (n = 20), and L168R (n = 13) mutations. In addition, we found a novel 12-bp in-frame insertion mutation (c.414-425dupGCTTTCCTGTTC, A139_F142dup) that duplicates the AFLF sequence in the pore helix upstream of the selectivity filter. Expressed in Xenopus oocytes, the A139_F142dup mutation depolarized the oocytes and produced a G-protein-sensitive Na(+) current with altered K(+) selectivity and loss of inward rectification but retained Ba(2+) sensitivity. Transfected into H295R cells, A139_F142dup increased basal aldosterone release 2.3-fold over the wild type. This was not increased further by incubation with angiotensin II. Although the A139_F142dup mutant trafficked to the plasma membrane of H295R cells, it showed reduced tetramer stability and surface expression compared with the wild-type channel. This study confirms the frequency of somatic KCNJ5 mutations in APAs and the novel mutation identified (A139_F142dup) extend the phenotypic range of the known KCNJ5 APA mutations. Being located in the pore helix, it is upstream of the previously reported mutations and shares some features in common with selectivity filter mutants but additionally demonstrates insensitivity to angiotensin II and decreased channel stability.
Project description:Glioblastoma multiforme is one of the most devastating cancers and presents unique challenges to therapy due to its aggressive behaviour. Cancer stem cells have been described to be the only cell population with tumorogenic capacity in glioblastoma. Therefore, effective therapeutic strategies targeting these cells may be beneficial. We have established different cultures of glioblastoma stem cells (GSCs) derived from surgical specimens and found that, after induction of differentiation, NFκB was activated, which allows intermediate tumor precursor cells to remain cycling. We also showed that blockade of NFκB signaling in differentiating GSCs by different genetic strategies or treatment with small molecule inhibitors, promoted replication arrest, progression to a mature phenotype, mainly neuronal cells, and senescence. This effect was partly mediated by downregulation of the NFκB target gene cyclin D1. Furthermore, intravenous treatment of immunodeficient mice bearing human GSC-derived tumors with a novel small-molecule inhibitor of the NFκB pathway induced senescence of tumor cells but no ultraestructural alterations of the brain parenchymal cells were detected. These findings reveal that activation of NFκB may keep differentiating GSCs from acquiring a mature postmitotic phenotype, thus allowing cell proliferation, and support the rationale for therapeutic strategies aimed at promoting premature senescence in GSCs undergoing differentiation. Gene expression in differentiated cells relative to stem cells in three different glioblastoma cultures
Project description:The OXE receptor is a GPCR activated by eicosanoids produced by the action of 5-lipoxygenase. We previously found that this membrane receptor participates in the regulation of cAMP-dependent and -independent steroidogenesis in human H295R adrenocortical carcinoma cells. In this study we analyzed the effects of the OXE receptor physiological activator 5-oxo-ETE on the growth and migration of H259R cells. While 5-oxo-ETE did not affect the growth of H295R cells, overexpression of OXE receptor caused an increase in cell proliferation, which was further increased by 5-oxo-ETE and blocked by 5-lipoxygenase inhibition. 5-oxo-ETE increased the migratory capacity of H295R cells in wound healing assays, but it did not induce the production of metalloproteases MMP-1, MMP-2, MMP-9 and MMP-10. The pro-migratory effect of 5-oxo-ETE was reduced by pharmacological inhibition of the MEK/ERK1/2, p38 and PKC pathways. 5-oxo-ETE caused significant activation of ERK and p38. ERK activation by the eicosanoid was reduced by the "pan" PKC inhibitor GF109203X but not by the classical PKC inhibitor Gö6976, suggesting the involvement of novel PKCs in this effect. Although H295R cells display detectable phosphorylation of Ser299 in PKC?, a readout for the activation of this novel PKC, treatment with 5-oxo-ETE per se was unable to induce additional PKC? activation. Our results revealed signaling effectors activated by 5-oxo-ETE in H295R cells and may have significant implications for our understanding of OXE receptor in adrenocortical cell pathophysiology.
Project description:Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2060 chemical samples on steroidogenesis via high-performance liquid chromatography followed by tandem mass spectrometry quantification of 10 steroid hormones, including progestagens, glucocorticoids, androgens, and estrogens. The study employed a 3 stage screening strategy. The first stage established the maximum tolerated concentration (MTC; ≥ 70% viability) per sample. The second stage quantified changes in hormone levels at the MTC whereas the third stage performed concentration-response (CR) on a subset of samples. At all stages, cells were prestimulated with 10 µM forskolin for 48 h to induce steroidogenesis followed by chemical treatment for 48 h. Of the 2060 chemical samples evaluated, 524 samples were selected for 6-point CR screening, based in part on significantly altering at least 4 hormones at the MTC. CR screening identified 232 chemical samples with concentration-dependent effects on 17β-estradiol and/or testosterone, with 411 chemical samples showing an effect on at least one hormone across the steroidogenesis pathway. Clustering of the concentration-dependent chemical-mediated steroid hormone effects grouped chemical samples into 5 distinct profiles generally representing putative mechanisms of action, including CYP17A1 and HSD3B inhibition. A distinct pattern was observed between imidazole and triazole fungicides suggesting potentially distinct mechanisms of action. From a chemical testing and prioritization perspective, this assay platform provides a robust model for high-throughput screening of chemicals for effects on steroidogenesis.