Wolff Michael, Haasen Dorothea, Merk Susanne, Kroner Margareta, Maier Udo, Bordel Sandra, Wiedenmann Jörg, Nienhaus Gerd Ulrich, Valler Martin, Heilker Ralf
Department of Zoology and Endocrinology, University of Ulm, Albert Einstein Allee 11, D-89081 Ulm, Germany.
Comb Chem High Throughput Screen. 2006 Jun;9(5):339-50. doi: 10.2174/138620706777452447.
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.
高内涵筛选（HCS）是荧光显微成像与自动图像分析的结合，已成为在细胞疾病模型系统中研究受试化合物效应的常用工具。在本研究中，我们建立了一种384孔板形式的中高通量HCS检测方法，用于测量细胞I型磷酸肌醇3激酶（PI3K）活性。I型PI3K参与多种细胞内信号通路，如细胞存活、生长、分化以及免疫反应。我们使用稳定转染了人胰岛素受体（hIR）和AKT1增强型绿色荧光蛋白（EGFP）融合构建体的中国仓鼠卵巢（CHO）细胞作为细胞模型系统。用胰岛素样生长因子-1（IGF-1）刺激hIR后，PI3K被激活，使磷脂酰肌醇（PtdIns）-4,5-二磷酸在3位磷酸化，导致AKT1-EGFP募集到质膜。AKT1-EGFP重新分布检测方法稳定，每日变化很小，与质膜斑点相关的荧光强度定量给出了良好的Z'统计值。采用了一种新型的化合物剂量反应测试形式，即跨连续微孔板（MTP）对受试化合物进行系列稀释。PI3K抑制剂系列的剂量反应测试提供了可重复的IC50值。用亚型选择性抑制剂对重新分布检测进行分析表明，PI3Kα是IGF-1刺激后CHO宿主细胞中被激活的主要亚型。可使用自动图像分析确定有毒化合物的副作用。我们得出结论，AKT1-EGFP重新分布检测代表了一种可靠的中/高通量筛选（MTS/HTS）形式，可在生长因子刺激条件下测定PI3K抑制剂的细胞活性。
