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P1010
The Use of Quantitative, Cell-based, High-Content Screening Assays to Detect In vitro Genotoxicity & the Effect on Cell Cycle Checkpoints
Presenter Bhaskar S. Mandavilli, Thermo Fisher Scientific, USA
Additional Authors: Suk J. Hong, Krishna M. Vattem, Richik N. Ghosh
DNA damage-induced checkpoint signaling plays an important role in the regulation of genomic integrity and the cell cycle. Chromosomal DNA damaged by either genotoxic agents or DNA replication inhibitors triggers multiple checkpoint signaling events to arrest cell cycle progression at G2/M, which allows cells to repair the damaged DNA. Specifically, DNA damage response activates phosphorylation of A™ and Chk2, resulting in p53 activation and cell cycle arrest. Phosphorylated retinoblastoma (Rb) and polo like kinase 1 (Plk1) proteins also play important roles in cell cycle arrest in G1/S and G2/M, respectively. Automated, quantitative, cell-based imaging high-content screening (HCS) assays enable multi-parametric measurements of DNA damage, activation of cell cycle checkpoints and cell cycle arrest in individual cells. Using a Thermo Scientific Cellomics® ArrayScan® HCS Reader and Thermo Scientific Cellomics HCS Reagent Kits, we measured the phosphorylation and activation of A™, Chk2, p53, Rb and Plk1 following treatment of cells with different compounds. Dose- and time-dependent responses were measured, and compounds were ranked according to their ability to activate DNA damage-induced checkpoint signaling events. These multi-parametric assays comprise an efficient platform to evaluate cell cycle arrest and checkpoint signaling events triggered by genotoxic compounds.