Abstract
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.
Highlights
Plate-based proliferation assays are a fundamental tool in oncology drug discovery for evaluating potency of compounds and sensitivity of different cell lines to specific agents
Using the direct cell count data we demonstrate that drug mechanisms of action (MoA) and cell line variation can both contribute to significant underestimation of potency and/or maximal efficacy when using ATP or MTS-reduction assays as compared to the actual number of cells present in the well
Cell cycle phase classification was achieved by DNA histogramming, maintaining a linear relationship between DNA content and integrated DNA intensity was critical
Summary
Plate-based proliferation assays are a fundamental tool in oncology drug discovery for evaluating potency of compounds and sensitivity of different cell lines to specific agents. In this study we will focus on three of the most common methods; determination of ATP in cell lysates by luciferin/luciferase-generated bioluminescence (e.g. CellTiter-Glo (Promega), ATPlite (Perkin Elmer)) [2,3,4], reduction of tetrazolium salts such as MTS and MTT to formazan by cellular dehydrogenases, (mitochondrial and otherwise) [3,5,6,7], and determination of the total amount of nucleic acid per well fluorescent dsDNA-binding cyanine dyes (e.g. CyQuant (Invitrogen), picoGreen (Invitrogen)) [8] As commonly used, these assays do not determine absolute mass amounts or molar concentrations of the analytes, but yield signals that have been demonstrated to have a wide dynamic range and linear response within relevant analyte concentrations
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