title: Monitoring cellular accumulation of 3'-deoxy-3'-fluorothymidine (FLT) and its monophosphate metabolite (FLT-MP) by LC-MS/MS as a measure of cell proliferation in vitro.
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.
authors.affiliation: Department of Pharmacokinetics, Dynamics, & Metabolism, Pfizer Worldwide Research and Development, San Diego, CA 92121, USA.
authors.forename: Wenlin
authors.initials: W
authors.lastname: Li
authors.forename: Marcela
authors.initials: M
authors.lastname: Araya
authors.forename: Mark
authors.initials: M
authors.lastname: Elliott
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Kang
authors.forename: Phillip M
authors.initials: PM
authors.lastname: Gerk
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Halquist
authors.forename: H Thomas
authors.initials: HT
authors.lastname: Karnes
authors.forename: Cathy
authors.initials: C
authors.lastname: Zhang
authors.forename: Peter J
authors.initials: PJ
authors.lastname: O'Brien
mesh_terms: D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002477:Cells / Q000737:chemistry* / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015224:Dideoxynucleosides / Q000032:analysis* / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D006801:Humans; D008954:Models, Biological; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013937:Thymidine Kinase / Q000032:analysis / Q000378:metabolism
chemical_list: D015224:Dideoxynucleosides; D013937:Thymidine Kinase; C002854:alovudine