Androgen Receptor Mutations Associated with Androgen Insensitivity Syndrome: A High Content Analysis Approach Leading to Personalized Medicine

Androgen insensitivity syndrome (AIS) is a rare disease associated with inactivating mutations of AR that disrupt male sexual differentiation, and cause a spectrum of phenotypic abnormalities having as a common denominator loss of reproductive viability. No established treatment exists for these conditions, however there are sporadic reports of patients (or recapitulated mutations in cell lines) that respond to administration of supraphysiologic doses (or pulses) of testosterone or synthetic ligands. Here, we utilize a novel high content analysis (HCA) approach to study AR function at the single cell level in genital skin fibroblasts (GSF). We discuss in detail findings in GSF from three historical patients with AIS, which include identification of novel mechanisms of AR malfunction, and the potential ability to utilize HCA for personalized treatment of patients affected by this condition

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

Use of HCA in subproteome-immunization and screening of hybridoma supernatants to define distinct antibody binding patterns

Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in mass spectrometry have given us greater insights into the participating proteomes, however, monoclonal antibodies remain key to understanding the structures, functions, locations and macromolecular interactions of the involved proteins. The traditional single immunogen method to produce monoclonal antibodies using hybridoma technology are time, resource and cost intensive, limiting the number of reagents that are available. Using a high content analysis screening approach, we have developed a method in which a complex mixture of proteins (e.g., subproteome) is used to generate a panel of monoclonal antibodies specific to a subproteome located in a defined subcellular compartment such as the nucleus. The immunofluorescent images in the primary hybridoma screen are analyzed using an automated processing approach and classified using a recursive partitioning forest classification model derived from images obtained from the Human Protein Atlas. Using an ammonium sulfate purified nuclear matrix fraction as an example of reverse proteomics, we identified 866 hybridoma supernatants with a positive immunofluorescent signal. Of those, 402 produced a nuclear signal from which patterns similar to known nuclear matrix associated proteins were identified. Detailed here is our method, the analysis techniques, and a discussion of the application to further in vivo antibody production

Characterizing properties of non-estrogenic substituted bisphenol analogs using high throughput microscopy and image analysis

<div><p>Animal studies have linked the estrogenic properties of bisphenol A (BPA) to adverse effects on the endocrine system. Because of concerns for similar effects in humans, there is a desire to replace BPA in consumer products, and a search for BPA replacements that lack endocrine-disrupting bioactivity is ongoing. We used multiple cell-based models, including an established multi-parametric, high throughput microscopy-based platform that incorporates engineered HeLa cell lines with visible ERα- or ERβ-regulated transcription loci, to discriminate the estrogen-like and androgen-like properties of previously uncharacterized substituted bisphenol derivatives and hydroquinone. As expected, BPA induced 70–80% of the estrogen-like activity via ERα and ERβ compared to E2 in the HeLa prolactin array cell line. 2,2’ BPA, Bisguaiacol F, CHDM 4-hydroxybuyl acrylate, hydroquinone, and TM modified variants of BPF showed very limited estrogen-like or androgen-like activity (< 10% of that observed with the control compounds). Interestingly, TM-BFP and CHDM 4-hydroxybuyl acrylate, but not their derivatives, demonstrated evidence of anti-estrogenic and anti-androgenic activity. Our findings indicate that Bisguaiacol F, TM-BFP-ER and TM-BPF-DGE demonstrate low potential for affecting estrogenic or androgenic endocrine activity. This suggest that the tested compounds could be suitable commercially viable alternatives to BPA.</p></div

Chemical compound effect on an integrated ERE-driven luciferase reporter in the ER responsive MCF-7 cell line.

<p>MCF-7-MAR-ERE-Luc were treated with indicated compounds at 5 μM, 5 μM + 10 nM E2, or 2 μM + 10 nM E2 for 24 hours before total luciferase activity determined. The â calculated using control wells treated with E2 (10 nM) or DMSO. Dashed line indicates 10 nM E2 control response. All data represent mean of 3 experiments ± standard deviation. (*) or (**) indicates a response significantly (p < 0.05) above or below control treatment.</p

PRL-HeLa analysis of estrogenic and anti-estrogenic activity of investigational chemical compounds on ERα.

<p>(A) Experimental workflow for analysis of investigational molecules. Percentage of cells with an array in GFP-ERα:PRL-HeLa cells after 1 hour of treatment with maximal dose of compound alone or with 10 nM E2 after 1 hour (B) or 24 hours (C) of exposure. Antagonism experiments were done combining 10nM E2 with 5 μM of the compounds of interest. (D) Dose-response curves of percent of cells with arrays for E2, BPA and the seven investigational compounds. (E) Effects on SRC-3 coactivator and RNA polymerase II levels at visible arrays after 1 hour of treatment with 10 nM E2 in the presence of maximal dose of compound. (F) Effects on de novo mRNA production at the PRL array after 1 hour of treatment with compound alone or with 10 nM E2. (G) Effects on GFP-ERα expression level after 24 hours of treatment with maximal dose of compound. All data represent mean of a minimum of 4 experiments ± standard deviation. (*) or (**) indicates a response significantly (p < 0.05) above or below control treatment.</p

Chemical compound effect on an integrated ARE-containing probasin-mCherry-NLS reporter in the AR responsive LNCaP cell line.

<p>The nuclear accumulation of mCherry fluorescent protein in 2PB-mCherry-NLS:LNCaP cells treated with indicated compounds either alone at concentrations from 50 pM to 5 μM (A) or at 5μM with DHT at ranging from 0.1 nM to 30 nM (B) for 48 hours. Compounds that induced either a significant androgen-like or anti-androgen-like response in the presence of DHT are indicated. All data represent mean of 3 experiments ± standard deviation.</p

Chemical structures and identification numbers for compounds analyzed.

<p>Each compound is identified by an abbreviation, full name, and a PubChem compound identification (or CAS) number.</p

PRL-HeLa analysis of ERβ estrogenic and anti-estrogenic activity of investigational chemical compounds.

<p>Percentage of cells with an array in GFP-ERβ:PRL-HeLa cells after 1 hour of treatment with maximal dose of compound alone or with 10 nM E2 after 1 hour (A) or 24 hours (B) of exposure. (C) Dose-response curves of percent of cells with arrays for E2, BPA and the seven investigational compounds (D) Effects on GFP-ERβ expression level after 24 hours of treatment with maximal dose of compound. All data represent mean of a minimum of 4 experiments ± standard deviation. (*) or (**) indicates a response signifcantly (p < 0.05) above or below control treatment.</p