Abstract #1262: Dual pulse labeling of S-phase population using click chemistry to measure changes in cell proliferation

The measurement of cellular proliferation is fundamental to the assessment of cell health, genotoxicity, and in the evaluation of drug efficacy. Changes in cell proliferation can give insights into mechanisms of cell growth, development, and cell cycle kinetics. A standard method for monitoring time dependant changes of cell proliferation is through the use of multiple pulse labeling of DNA based on the incorporation of nucleoside analogs. Typically, halogenated derivatives of deoxyuridine are used and detected with antibodies which have selective affinity for the specific halogen modified base. Although there are several halogen modified bases to use, antibody selectivity must be carefully evaluated to achieve correct labeling. By augmenting the antibody approach with a non-antibody method of detection using a click chemistry modified deoxyuridine analog, another specific label can be added to the tools used for examining changes in proliferation rates. Here, examples are shown combining click chemistry with traditional BrdU antibody labeling using flow cytometry in Jurkat T-cell leukemia, TF-1 erythroleukemia, and THP-1 monocytic leukemia cells. Click chemistry labeling uses 5-ethynyl 2\#8242;-deoxyuridine (EdU) incorporation and detection with copper catalyzed dye-azide to form a stable covalent triazole ring. Use with selected bromodeoxyuridine (BrdU) antibody results in unambiguous double labeling without cross-reactivity. An unanticipated benefit is observed when using these two nucleoside analogs in combination. Simultaneous addition of BrdU and EdU is shown to only incorporate BrdU into the newly synthesized DNA, while a first pulse of EdU followed by sequential addition of BrdU without prior removal of the EdU results in four population of cells: first, those labeled only with EdU during the first pulse are cells which have exited S-phase prior to BrdU addition; second, those labeled with both BrdU and EdU are cells which have remained in S-phase during the duration of the two pulses; third, those labeled only with BrdU are cells which entered S-phase after BrdU addition; and fourth, those cells which have not labeled with either EdU or BrdU indicating these cells were not synthesizing DNA during either pulse. Examples of both long and short pulses of double labeling are given and the complex labeling patterns are interpreted through the use of appropriate population gating. Rapid changes in proliferation rate induced by drugs or treatment can be monitored using this no wash pulse labeling method. This method has broad applications and demonstrates the value of click chemistry combined with other antibody based methods for measuring changes in cell proliferation. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1262.