10:20-10:45 High-Content Screening of Combinatorial Library for Identifying Biomarkers
Hang Chang, Ph.D., Project Scientist,
Life Sciences Division, Lawrence Berkeley National Laboratory
A combinatorial fluorescence library of small molecules is used for exploring (i) differential labeling of different cell lines (e.g., do they localize differentially for a panel of cell lines) as a result of different types of treatment (e.g., radiation, drug treatment), (ii) flux analysis (e.g., what is uptake and retention of each ligand), and (iii) the relationships between structure and activity of each ligand and their corresponding subtypes. All data
and results are then registered against a database of response for bioinformatics analysis.
10:45-11:10 Targeting Cytokine Receptor-JAK-STAT Signaling Pathways - Development of an HCS Assay to Identify Selective Inhibitors of STAT-3 Phosphorylation and Nuclear Translocation
Paul A. Johnston, Ph.D., Research Associate Professor, Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine
The Signal Transducers and Activators of Transcription (STATs) are transcription factors that mediate the effects of growth factors and cytokines to regulate the expression of genes involved in cell proliferation, differentiation, inflammation, migration, and apoptosis. Activated STAT3 is an oncogene that directs tumor cells toward proliferation and survival, induces angiogenesis and alters the tumor microenvironment, and promotes tumor metastases through its effects on cell migration and invasion. In sharp contrast activated STAT1 is considered a tumor suppressor because it is a potent inhibitor of tumor growth, promotes tumor cell apoptosis, and enhances tumor immunity. Consequently, the discovery of a STAT3 selective inhibitor would be a highly desirable goal for the development of an anti-cancer drug. The development of an HCS assay to identify selective inhibitors of STAT-3 phosphorylation and nuclear translocation will be described.
11:10-11:35 Development of a Multiparametric Assay to Measure Cell Phenotypic and Pathway Parameters Indicative of ROCK Activity
Danli L. Towne, Scientist II, High-Content Screening, Lead Discovery, Abbott Laboratories
Rho kinase (ROCK) is a serine/threonine kinase which is activated by Rho GTPase. Activated ROCK phosphorylates many substrate proteins, and consequently regulates various downstream signaling pathways that control multiple cell type-specific cellular functions including smooth muscle contraction, cell migration, proliferation and neurite outgrowth. All these effects require reorganization of the actin cytoskeleton. ROCK can also increase Myosin Light Chain (MLC) phosphorylation through inhibition of Myosin Light Chain phosphatase, which may contribute to actin reorganization as well. We have developed a cell-based high-content assay using HUVEC cells to measure a combination of phenotypic (actin reorganization) and pathway (MLC phosphorylation) parameters associated with ROCK inhibition. Our results show that MLC phosphorylation and actin reorganization can be decoupled. The assay is also valuable for detecting off-target compound effects. In addition, our data suggests that the HCS assay provides a valid approach to measure ROCK inhibition in intact cells.
11:35-12:00 High-Content Cellomics Cell Cycle Analysis of the Effects of HSP90 Inhibition
Susan Lyman, Ph.D., Research Scientist, Exelixis, Inc.
Cell cycle analysis has traditionally been carried out by FACS – or more recently, by combining a measurement of DNA content with imaging of one or more phase-specific readouts, such as phospho-histone H3. We have used the latter approach to develop a novel and robust high-throughput Cellomics-based cell cycle assay that accurately reports the phase status of a cell (G1, S, G2, or M) as well as its DNA content (2n, 4n, >4n). We have applied this technique to examine the cell cycle perturbations caused by inhibition of HSP90, a molecular chaperone that enhances the stability of a wide spectrum of client proteins. HSP90 clients include cell cycle proteins such as PLK1 and CDC2 as well as cancer-associated proteins such as EGFR, ERBB2, and MET, making it an attractive cancer target. This presentation will illustrate the Cellomics cell cycle technique and will highlight the strikingly similar complex cell cycle perturbations induced by 3 different small-molecule HSP90 inhibitors in a large panel of immortalized cancer cell lines.
10:20-10:45 Using HCA to Identify a Transcription Factor Family that Regulates the Intrinsic Ability of Neurons to Extend Axons
Murray Blackmore, Ph.D., Research Assistant Professor, The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine
As neurons age, they lose the ability to extend axons, resulting in failure of regeneration after injury to the central nervous system. We used HCA to screen genes that were differentially expressed during development in retinal neurons and cortical neurons. We identified a family of transcription factors that regulate axon growth in vitro and in vivo. In the course of these studies we found that some types of primary neurons are more suitable for detecting increases in neurite growth than other types of neurons.
10:45-11:10 High-Content Analysis of Synapse Formation in Primary Neuronal Cultures for Developmental Neurotoxicity Screening
William R. Mundy, Ph.D., Integrated Systems Toxicology Division, U.S. Environmental Protection Agency
Cell-based assays can model neurodevelopmental processes including neurite growth and synaptogenesis, and may be useful for screening and evaluation of large numbers of chemicals for developmental neurotoxicity. This work describes the use of HCA to detect chemical effects on synaptogenesis in vitro. Pre-synaptic puncta were associated with the cell body and dendrites of primary cortical neurons, and increased over several weeks in culture. Several chemicals were identified that decreased synapse number during development in vitro.
11:10-11:35 Screening Neuronal Pattern Formation in vivo
Sebastian Munck, Ph.D., Coordinator, Light Microscopy and Imaging Network LiMoNe, Department of Molecular and Developmental Genetics, VIB, K.U. Leuven
We would like to describe an assay for imaging-based high-content analysis of the network formation in vivo. The neurons in the optic lobe of Drosophila melanogaster form a stereotypical pattern. In the optical system neuronal /electrical activity is believed to be a crucial part in the pattern formation process. We want to dissect the genetic and activity depended processes. The aim of the project is to identify the key players of the pattern formation and to modulate them by combining the power of a genetic screen in drosophila with the imaging based read out of the formed pattern.
11:35-12:00 Development of a Composite Primary Neuronal High-Content Screening Assay for Huntington's Disease Incorporating Non-Cell Autonomous Interactions
Linda S. Kaltenbach, Ph.D., Research Scientist, Neurobiology, Duke University
Huntington’s Disease, a fatal neurodegenerative disease caused by expansion of a polyglutamine repeat in the Huntingtin protein, incorporates obligatory non-cell autonomous pathways involving both the cortex and the striatum. We developed a fully automated high-content screen in high-density composite primary cultures including both cortical and striatal neurons as well as their supporting glial cells. Cortical and striatal neurons are transfected separately with different fluorescent protein markers such that image-based high-content analysis can be used to assay these neuronal populations separately, but still supporting their intercellular interactions within the same culture wells. The results of a screen of ~500 selected small molecule compounds and preliminary data on a neurite-based endpoint will be described.