Cytofluorometric assay for the determination of thymidine uptake and phosphorylation in living cells

Thymidine kinase is a key enzyme for the application of drugs in chemotherapy and for diagnosis. Although of great interest, its regulation during cell cycle and differentiation is difficult to study, as current techniques for isolation of cells in different phases of growth are unsatisfactory. An assay that allows the determination of enzymatic activity in situ in single cells would be much faster than present methods and would elegantly avoid synchronization procedures. We synthesized different analogues of thymidine with the 5-methyl group substituted by a fluorochrome. At least three of these compounds were phosphorylated by thymidine kinase in cell free extracts and were taken up and phosphorylated by cells in culture. The cytofluorometric signal of the accumulated fluorochrome in any given cell reflected the thymidine kinase activity of this cell. Simultaneous measurement of cell-cycle dependent parameters allowed the correlation of thymidine kinase activity with the phase of growth in mixed cell populations.