Multiplexed-Based Assessment of DNA Damage Response to Chemotherapies Using Cell Imaging Cytometry

• Published in: International journal of molecular sciences Vol. 23; no. 10; p. 5701
• Main Authors: Vezzio-Vié, Nadia, Kong-Hap, Marie-Alice, Combès, Eve, Andrade, Augusto Faria, Del Rio, Maguy, Pasero, Philippe, Theillet, Charles, Gongora, Céline, Pourquier, Philippe
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 20.05.2022; MDPI
• Subjects: anticancer drugs; Apoptosis; Automation; biomarkers; Calcein; Cancer; Cell cycle; Cell growth; Chemotherapy; CHK1 protein; CHK2 protein; Cytotoxicity; Deoxyribonucleic acid; DNA; DNA damage; DNA damage response; DNA repair; DNA-dependent protein kinase; Drugs; Evaluation; Flow cytometry; imaging cytometry; Immunofluorescence; Life Sciences; Measurement methods; Multiplexing; Ovarian cancer; oxaliplatin; p53 Protein; Phosphorylation; Propidium iodide; Prostate; Protein kinase C; Reagents; Western blotting; United States > US; ATR inhibitor; DNA damage response; biomarkers; imaging cytometry; oxaliplatin; DNA repair; anticancer drugs
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms23105701

The current methods for measuring the DNA damage response (DDR) are relatively labor-intensive and usually based on Western blotting, flow cytometry, and/or confocal immunofluorescence analyses. They require many cells and are often limited to the assessment of a single or few proteins. Here, we used the Celigo image cytometer to evaluate the cell response to DNA-damaging agents based on a panel of biomarkers associated with the main DDR signaling pathways. We investigated the cytostatic or/and the cytotoxic effects of these drugs using simultaneous propidium iodide and calcein-AM staining. We also describe new dedicated multiplexed protocols to investigate the qualitative (phosphorylation) or the quantitative changes of eleven DDR markers (H2AX, DNA-PKcs, ATR, ATM, CHK1, CHK2, 53BP1, NBS1, RAD51, P53, P21). The results of our study clearly show the advantage of using this methodology because the multiplexed-based evaluation of these markers can be performed in a single experiment using the standard 384-well plate format. The analyses of multiple DDR markers together with the cell cycle status provide valuable insights into the mechanism of action of investigational drugs that induce DNA damage in a time- and cost-effective manner due to the low amounts of antibodies and reagents required.