Modulation of deoxynucleotide metabolism by the deoxycytidylate deaminase inhibitor 3,4,5,6-tetrahydrodeoxyuridine.
Tetrahydrodeoxyuridine (dTHU) inhibits deoxycytidine deaminase and, after intracellular phosphorylation to the active 5'-monophosphate, also inhibits deoxycytidylate deaminase (dCMPD). Because in vitro studies have shown that dCMPD may regulate pyrimidine deoxynucleotide metabolism, the objective of this study was to investigate the effects of dTHU on deoxynucleotide metabolism in whole cells. Nearly complete inhibition of dCMPD, measured in intact CCRF-CEM cells by incorporation of [14C]dCyd into dTTP, occurred after a 45-min incubation with 100 microM dTHU. This was accompanied by an 8-fold dCTP pool expansion, although dATP, dTTP, dGTP, and ribonucleoside triphosphate pools were unaffected. Tetrahydrouridine, which inhibits deoxycytidine deaminase exclusively, had no effect on nucleotide pools. The dCTP pool expansion was directly proportional to the dTHU concentration (3-100 microM) and reached a maximum after 2 hr. Inhibition of ribonucleotide reductase by hydroxyurea completely prevented the dTHU-induced dCTP pool expansion, indicating that the substrate of dCMPD was derived from the ribonucleotide pool and that CDP was the predominant precursor of dCTP. dTHU-mediated inhibition of dCMPD appeared reversible. Exposure of cells to 100 microM dTHU followed by washing into fresh medium resulted in a linear decrease of the dCTP pool and an increase in the dTTP pool. The increased dCTP concentration after preincubation with dTHU was associated with an inhibition of deoxycytidine kinase, as indicated by a reduced capacity of cells to phosphorylate ara-C. dTHU is a useful new tool for investigating the role of dCMPD in the regulation of deoxynucleotide metabolism in whole cells.
Heinemann
V
V
Department of Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Plunkett
W
W
D002460:Cell Line; D003561:Cytarabine / Q000378:metabolism; D003565:Cytidine Diphosphate / Q000378:metabolism; D003844:DCMP Deaminase / Q000037:antagonists & inhibitors*; D003841:Deoxycytidine / Q000378:metabolism; D003842:Deoxycytidine Kinase / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism*; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006918:Hydroxyurea / Q000494:pharmacology; D007700:Kinetics; D009709:Nucleotide Deaminases / Q000037:antagonists & inhibitors*; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors; D013601:T-Lymphocytes / Q000201:enzymology; D013767:Tetrahydrouridine / Q000494:pharmacology / Q000031:analogs & derivatives; D013942:Thymine Nucleotides / Q000378:metabolism
D003845:Deoxycytosine Nucleotides; D013942:Thymine Nucleotides; D003561:Cytarabine; D003841:Deoxycytidine; D013767:Tetrahydrouridine; C024107:2'-deoxycytidine 5'-triphosphate; C052729:2'-deoxytetrahydrouridine; D003565:Cytidine Diphosphate; D012264:Ribonucleotide Reductases; D003842:Deoxycytidine Kinase; D009709:Nucleotide Deaminases; D003844:DCMP Deaminase; C024157:thymidine 5'-triphosphate; D003857:Deoxyuridine; D006918:Hydroxyurea