title: Automated high content screening for phosphoinositide 3 kinase inhibition using an AKT 1 redistribution assay.
abstract: High Content Screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modelling systems. In this work, we established a medium to high throughput HCS assay in the 384-well format to measure cellular type I phosphoinositide 3 kinase (PI3K) activity. Type I PI3K is involved in several intracellular pathways such as cell survival, growth and differentiation as well as immunological responses. As a cellular model system we used Chinese Hamster Ovary (CHO) cells that had been stably transfected with human insulin receptor (hIR) and an AKT1-enhanced green fluorescent protein (EGFP) fusion construct. Upon stimulation of the hIR with insulin-like growth factor-1 (IGF-1), PI3K was activated to phosphorylate phosphatidylinositol (PtdIns)-4,5-bisphosphate at the 3-position, resulting in the recruitment of AKT1-EGFP to the plasma membrane. The AKT1-EGFP redistribution assay was robust and displayed little day-to-day variability, the quantification of the fluorescence intensity associated with plasma membrane spots delivered good Z' statistics. A novel format of compound dose-response testing was employed using serial dilutions of test compounds across consecutive microtiter plates (MTPs). The dose response testing of a PI3K inhibitor series provided reproducible IC50 values. The profiling of the redistribution assay with isoform-selective inhibitors indicates that PI3Kalpha is the main isoform activated in the CHO host cells after IGF-1 stimulation. Toxic compound side effects could be determined using automated image analysis. We conclude that the AKT1-EGFP redistribution assay represents a solid medium/high throughput screening (MTS/HTS) format to determine the cellular activity of PI3K inhibitors under conditions of growth factor stimulation.
authors.affiliation: Department of Zoology and Endocrinology, University of Ulm, Albert Einstein Allee 11, D-89081 Ulm, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Wolff
authors.forename: Dorothea
authors.initials: D
authors.lastname: Haasen
authors.forename: Susanne
authors.initials: S
authors.lastname: Merk
authors.forename: Margareta
authors.initials: M
authors.lastname: Kroner
authors.forename: Udo
authors.initials: U
authors.lastname: Maier
authors.forename: Sandra
authors.initials: S
authors.lastname: Bordel
authors.forename: Jörg
authors.initials: J
authors.lastname: Wiedenmann
authors.forename: Gerd Ulrich
authors.initials: GU
authors.lastname: Nienhaus
authors.forename: Martin
authors.initials: M
authors.lastname: Valler
authors.forename: Ralf
authors.initials: R
authors.lastname: Heilker
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D008856:Microscopy, Fluorescence; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D049452:Green Fluorescent Proteins; D007334:Insulin-Like Growth Factor I; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D011972:Receptor, Insulin