A Scalable High-Content Cytotoxicity Assay Insensitive to Changes in Mitochondrial Metabolic Activity
Abstract:Quantitative assessments of cell proliferation and cytotoxicity are key components of anticancer drug discovery. Advancements in genomics and combinatorial chemistry have increased the demand for cytotoxicity measurements, and almost all assays for cell proliferation that are compatible with high-throughput screening are single-parameter, homogeneous biochemical assays. Furthermore, the most popular methods currently in use are based on measurements of mitochondrial activity and therefore provide only an indirect measure of cell growth. Here we describe a high-content screening (HCS) methodology that permits the direct quantification of cell numbers as well as multiple measurements of drug activity in individual cells without the ambiguities of previously described homogenous metabolism-based assays. Using two human tumor cell lines, we compared our HCS method with a commonly used homogenous colorimetric assay that detects mitochondrial activity and found the colorimetric assay substantially underrepresented the cytotoxic effects of two mechanistically diverse, clinically used, anticancer drugs: a DNA-damaging agent, bleomycin, and a tubulin stabilizer, paclitaxel. Simultaneous evaluation of cell numbers, mitochondrial mass, and nuclear morphology by HCS provided an explanation for the differential toxicity. Furthermore, we expanded the scope of the HCS assay to include tubulin mass measurements in paclitaxel-treated cells as an example for a specific drug target. The data illustrate the utility of HCS as a powerful analysis tool compatible with the demands of high-throughput screening, and adaptable to include fluorescence-based markers of drug activity.
Document Type: Research Article
Affiliations: Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261
Publication date: 2003-01-01
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- Formerly: Oncology Research Incorporating Anti-Cancer Drug Design
Oncology Research Featuring Preclinical and Clincal Cancer Therapeutics publishes research of the highest quality that contributes to an understanding of cancer in areas of molecular biology, cell biology, biochemistry, biophysics, genetics, biology, endocrinology, and immunology, as well as studies on the mechanism of action of carcinogens and therapeutic agents, reports dealing with cancer prevention and epidemiology, and clinical trials delineating effective new therapeutic regimens.