Monitoring the Activation of the DNA Damage Response Pathway in a 3D Spheroid Model

• Published in: PloS one Vol. 10; no. 7; p. e0134411
• Main Authors: Mondesert, Odile, Frongia, Céline, Clayton, Olivia, Boizeau, Marie-Laure, Lobjois, Valérie, Ducommun, Bernard
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 30.07.2015; Public Library of Science (PLoS)
• Subjects: Activation; Automated teller machines; Biosensors; Cancer therapies; Cell cycle; Cell division; Damage; Deoxyribonucleic acid; DNA; DNA Damage; DNA repair; Evaluation; Fluorescence; HEK293 Cells; Humans; Kinases; Medical screening; Microscopy; Models, Biological; Monitoring; p53 Protein; Pancreatic cancer; Pharmacology; Proteins; Spheroids; Three dimensional models; Tumor proteins
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0134411

Monitoring the DNA-Damage Response (DDR) activated pathway in multicellular tumor spheroid models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological evaluation. Herein we present DDR-Act-FP, a fluorescent biosensor that allows detection of DDR activation through monitoring of the p21 promoter p53-dependent activation. We show that cells expressing the DDR-Act-FP biosensor efficiently report activation of the DDR pathway after DNA damage and its pharmacological manipulation using ATM kinase inhibitors. We also report the successful use of this assay to screen a small compound library in order to identify activators of the DDR response. Finally, using multicellular spheroids expressing the DDR-Act-FP we demonstrate that DDR activation and its pharmacological manipulation with inhibitory and activatory compounds can be efficiently monitored in live 3D spheroid model. This study paves the way for the development of innovative screening and preclinical evaluation assays.