Regulation of thymidine kinase and thymidylate synthase in intact human lymphoblast CCRF-CEM cells.
It has been reported that thymidylate synthase (TS) is a component of a multienzyme complex associated with DNA replication based on observations that enzyme activity was decreased when cells were treated with various DNA synthesis inhibitors (Plucinski, T. M., Fager, R. S., and Reddy, G. P. V. (1990) Mol. Pharmacol. 38, 114-120). The veracity of the TS assay (known as the tritium release assay) employed in these experiments may be compromised, however, because it requires the S phase-specific enzyme thymidine kinase (TK) to phosphorylate the substrate [5-3H]dUrd. In our study, this problem was further illustrated as the phosphorylated products of [14C]dCyd and [6-3H]dUrd were simultaneously quantitated to determine the activities of TS, TK, and dCyd kinase in intact CCRF-CEM cells. TS and dCyd kinase were unaffected by aphidicolin, hydroxyurea, and 1-beta-D-arabinofuranosylcytosine, whereas TK was strongly inhibited by these agents. Elevation of the cellular dTTP pool that accompanied drug treatment was not the primary mechanism affecting TK activity because incubation of cells with dCyd elevated the dTTP pool to similar levels, but did not inhibit TK to the same extent as did the drugs. Furthermore, after cells were washed from aphidicolin, [6-3H]dCyd incorporation, which primarily labels dTMP in DNA, proceeded at a linear rate, whereas a lag period of 15 min was observed before [3H]dThd was incorporated at a linear rate. These results suggest that TK activity is affected by more than one mechanism in intact cells. Because the activities of dCyd kinase and dCMP deaminase do not fluctuate as much as that of TK in response to changes in DNA synthesis activity and cell cycle, dCyd incorporation appears to be a more reliable assay of TS in intact cells than does dUrd incorporation. Our findings also imply that [3H]dThd incorporation assays may overestimate inhibition of DNA synthesis.
Xu
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Department of Clinical Investigation, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Plunkett
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D016590:Aphidicolin / Q000494:pharmacology; D002250:Carbon Radioisotopes; D002453:Cell Cycle; D003561:Cytarabine / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects; D004273:DNA, Neoplasm / Q000096:biosynthesis / Q000187:drug effects; D003841:Deoxycytidine / Q000378:metabolism; D003857:Deoxyuridine / Q000378:metabolism; D006918:Hydroxyurea / Q000494:pharmacology; D007700:Kinetics; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma; D013937:Thymidine Kinase / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000378:metabolism*; D013997:Time Factors; D014316:Tritium; D014407:Tumor Cells, Cultured
D002250:Carbon Radioisotopes; D004273:DNA, Neoplasm; D003561:Cytarabine; D003841:Deoxycytidine; D014316:Tritium; D016590:Aphidicolin; D013940:Thymidylate Synthase; D013937:Thymidine Kinase; D003857:Deoxyuridine; D006918:Hydroxyurea