Androgen Receptor Functional Analyses by High Throughput Imaging: Determination of Ligand, Cell Cycle, and Mutation-Specific Effects

Understanding how androgen receptor (AR) function is modulated by exposure to steroids, growth factors or small molecules can have important mechanistic implications for AR-related disease therapies (e.g., prostate cancer, androgen insensitivity syndrome, AIS), and in the analysis of environmental endocrine disruptors.We report the development of a high throughput (HT) image-based assay that quantifies AR subcellular and subnuclear distribution, and transcriptional reporter gene activity on a cell-by-cell basis. Furthermore, simultaneous analysis of DNA content allowed determination of cell cycle position and permitted the analysis of cell cycle dependent changes in AR function in unsynchronized cell populations. Assay quality for EC50 coefficients of variation were 5–24%, with Z' values reaching 0.91. This was achieved by the selective analysis of cells expressing physiological levels of AR, important because minor over-expression resulted in elevated nuclear speckling and decreased transcriptional reporter gene activity. A small screen of AR-binding ligands, including known agonists, antagonists, and endocrine disruptors, demonstrated that nuclear translocation and nuclear “speckling” were linked with transcriptional output, and specific ligands were noted to differentially affect measurements for wild type versus mutant AR, suggesting differing mechanisms of action. HT imaging of patient-derived AIS mutations demonstrated a proof-of-principle personalized medicine approach to rapidly identify ligands capable of restoring multiple AR functions.HT imaging-based multiplex screening will provide a rapid, systems-level analysis of compounds/RNAi that may differentially affect wild type AR or clinically relevant AR mutations

Enzymes involved in lipid digestion

International audienceLipid digestion is a complex process that takes place at the lipid-water interface and involves various lipolytic enzymes present predominantly in the stomach and the small intestine [34]. These enzymes catalyse the hydrolysis of a variety of dietary lipids from animal and plant sources, such as triacylglycerols (TAGs), phospholipids, galactolipids, cholesterol and vitamin esters. They include gastric lipase, colipase-dependent pancreatic lipase, pancreatic lipase-related proteins 2 (PLRP2), carboxyl ester hydrolase or bile salt-stimulated lipase (CEH, BSSL), and pancreatic phospholipase A2. A debate still exist about the existence of a lingual lipase in human [30, 86, 140], an enzyme that has been demonstrated to be present and active in rat and mice tongue only and which is the product of a gene ortholog [53] to the gene of gastric lipase [24] in humans and many other species. Bakala N’Goma et al. [12] have reviewed the key findings that support the existence of lingual or gastric lipases in several species in term of gene expression, enzyme immunocytolocalization and lipase activity. So far, no supporter of the existence of a lingual lipase in humans has been able to provide similar data