Rapid determination of thymidylate synthase activity and its inhibition in intact L1210 leukemia cells in vitro.
A rapid and convenient tritium release assay for measuring thymidylate (dTMP) synthase activity and its inhibition within intact mammalian cells is described in detail. Short-term incubation of murine leukemia L1210 cells with an appropriately labeled substrate precursor, either deoxyuridine ([5-3H]dUrd) or deoxycytidine ([5-3H]dCyd), allowed for: (1) uptake and intracellular conversion to the substrate deoxyuridylate ([5-3H]dUMP); and (2) the obligatory displacement of tritium from [5-3H]-dUMP during the dTMP synthase catalyzed reaction. Tritium released into the aqueous environment was quantitated after a quick one-step separation of tritiated H2O from other radiolabeled materials and cell debris. The amount of tritium released was evaluated as a function of a number of variables, including the concentration of labeled substrate precursors, cell number, and incubation time. Tritium from [5-3H]dCyd was released significantly faster than from [5-3H]dUrd under a variety of conditions. Both 5-fluorodeoxyuridine (1 microM) and methotrexate (10 microM), which effectively block intracellular dTMP synthesis, completely inhibited the release of tritium from either [5-3H]dCyd or [5-3H]dUrd demonstrating that the release of tritium is mediated exclusively by the dTMP synthase catalyzed reaction. In addition, there was a good correlation between tritium release, cellular uptake, and incorporation of [2-14C]dUrd into DNA. The inhibitory effects of antifolates such as methotrexate were independent of the type of labeled precursor used. In contrast, preferential interference with the release of tritium from [5-3H]-dCyd by dCyd derivatives and from [5-3H]dUrd by dUrd derivatives was observed, suggesting that competition for uptake and/or phosphorylation may contribute to the overall effects of certain nucleoside analogues on cellular dTMP synthase activity measured using the tritium release assay.
Yalowich
J C
JC
Kalman
T I
TI
D000818:Animals; D002478:Cells, Cultured; D003841:Deoxycytidine / Q000378:metabolism; D003857:Deoxyuridine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005467:Floxuridine / Q000494:pharmacology; D007939:Leukemia L1210 / Q000201:enzymology*; D008727:Methotrexate / Q000494:pharmacology; D008780:Methyltransferases / Q000378:metabolism*; D051379:Mice; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014316:Tritium
D005467:Floxuridine; D003841:Deoxycytidine; D014316:Tritium; D008780:Methyltransferases; D013940:Thymidylate Synthase; D003857:Deoxyuridine; D008727:Methotrexate