A simple high-content cell cycle assay reveals frequent discrepancies between cell number and ATP and MTS proliferation assays

• Published in: PloS one 2013-05, Vol.8 (5), p.e63583
• Main Authors: Chan, Grace Ka Yan, Kleinheinz, Tracy L, Peterson, David, Moffat, John G
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate; Anticancer properties; Antineoplastic Agents - pharmacology; Apoptosis; Assaying; ATP; Biology; Bioluminescence; Biotechnology; Cancer; Cancer therapies; Care and treatment; Cell Count - methods; Cell cycle; Cell Cycle - drug effects; Cell Cycle - physiology; Cell division; Cell growth; Cell Line, Tumor; Cell number; Cell Proliferation - drug effects; Cell size; Cytotoxicity; Deoxyribonucleic acid; DNA; DNA biosynthesis; DNA synthesis; Dose-Response Relationship, Drug; Drug Discovery - methods; Etoposide; Fluorescence; Gemcitabine; High-Throughput Screening Assays - methods; Humans; Luminescent Measurements; Medicine; Metabolism; Microscopy; Mitochondria; Pharmacology; Phase distribution; Reduction; Tetrazolium Salts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0063583

In order to efficiently characterize both antiproliferative potency and mechanism of action of small molecules targeting the cell cycle, we developed a high-throughput image-based assay to determine cell number and cell cycle phase distribution. Using this we profiled the effects of experimental and approved anti-cancer agents with a range mechanisms of action on a set of cell lines, comparing direct cell counting versus two metabolism-based cell viability/proliferation assay formats, ATP-dependent bioluminescence, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reduction, and a whole-well DNA-binding dye fluorescence assay. We show that, depending on compound mechanisms of action, the metabolism-based proxy assays are frequently prone to 1) significant underestimation of compound potency and efficacy, and 2) non-monotonic dose-response curves due to concentration-dependent phenotypic 'switching'. In particular, potency and efficacy of DNA synthesis-targeting agents such as gemcitabine and etoposide could be profoundly underestimated by ATP and MTS-reduction assays. In the same image-based assay we showed that drug-induced increases in ATP content were associated with increased cell size and proportionate increases in mitochondrial content and respiratory flux concomitant with cell cycle arrest. Therefore, differences in compound mechanism of action and cell line-specific responses can yield significantly misleading results when using ATP or tetrazolium-reduction assays as a proxy for cell number when screening compounds for antiproliferative activity or profiling panels of cell lines for drug sensitivity.