Abstract |
Accurate measurement of in vitro cell growth is critical for oncology drug development, but cell counting and the most accurate indirect proliferation assays are impractical. Here, we describe a robust alternative method that monitors proliferating cell thymidine kinase 1 (TK1) activity via LC-MS/MS quantification of 3'-deoxy-3'-fluorothymidine (FLT) and its monophosphate metabolite FLT-MP. LNCaP prostate cancer cells were cultured at four densities (20,000; 10,000; 5000; and 500 cells/well) and incubated with 2000 ng/mL FLT in multi-well plates. Internal standards were FLT-d3 for FLT and d4-thymidine for FLT-MP. In culture medium, peak area ratios of FLT to FLT-d3 and FLT-MP to d4-thymidine were linear over the range 0.25-100 ng/mL (r(2)≥0.998). Accuracy for quality controls was between -7.3% and 6.3% for FLT, and from -3.3% to 1.7% for FLT-MP. Quality control precision was from 2.4% to 5.7% for FLT and 3.2% to 7.5% for FLT-MP. The limit of quantification was 0.25 ng/mL, with good control results (precision of 9.6% for FLT and 14.8% for FLT-MP). FLT-MP formation was linearly proportional to cell number from 500 to 20,000 cells/well 1 h after FLT addition. FLT-MP and ATP generation were comparable in LNCaP cells exposed to cell cycle inhibitor drugs (Spearman r=0.925, p<0.0001), demonstrating assay suitability for drug screening. This fit for purpose method is amenable to analysis of tumor tissue extracts, and should enable direct assessment of in vitro-in vivo relationships in animal models of cancer.
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Authors | Wenlin Li, Marcela Araya, Mark Elliott, Xiaolin Kang, Phillip M Gerk, Matthew S Halquist, H Thomas Karnes, Cathy Zhang, Peter J O'Brien |
Journal | Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
(J Chromatogr B Analyt Technol Biomed Life Sci)
Vol. 879
Issue 28
Pg. 2963-70
(Oct 15 2011)
ISSN: 1873-376X [Electronic] Netherlands |
PMID | 21925976
(Publication Type: Evaluation Study, Journal Article)
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Copyright | Copyright © 2011 Elsevier B.V. All rights reserved. |
Chemical References |
- Dideoxynucleosides
- Thymidine Kinase
- alovudine
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Topics |
- Cell Line, Tumor
- Cell Proliferation
- Cells
(chemistry, cytology, enzymology, metabolism)
- Chromatography, High Pressure Liquid
(methods)
- Dideoxynucleosides
(analysis, metabolism)
- Enzyme Assays
(methods)
- Humans
- Models, Biological
- Tandem Mass Spectrometry
(methods)
- Thymidine Kinase
(analysis, metabolism)
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