tetrahydrouridine and 2--deoxycytidine-5--triphosphate

Phosphorylation of cytosine analogs by deoxycytidine kinase (dCK) and deamination by cytidine deaminase (CDA) are two important processes in the activation and elimination of these drugs. We have investigated the kinetic parameters of 2',2'-difluorodeoxycytidine (dFdC) using purified enzymes from human cells. Deoxycytidine (CdR) and dFdC had Km values of 1.5 and 4.6 microM for dCK, respectively. Feedback inhibition of dCK by deoxycytidine 5'-triphosphate (dCTP) was also studied. Our results show that dCTP produced a greater inhibition of the phosphorylation of dFdC than CdR with concentrations of dCTP ranging from 1 to 25 microM. dFdC was a good substrate for CDA. Kinetic studies with this enzyme gave Km values for CdR and dFdC of 46.3 and 95.7 microM, respectively. The effect of competitive inhibitors of CDA on the deamination of dFdC was also investigated. Diazepinone riboside was a more potent inhibitor than tetrahydrouridine using either CdR or dFdC as the substrate. Inhibitors of CDA could be useful in clinical trials in patients with cancer to increase the chemotherapeutic effectiveness of dFdC.

Topics: Antimetabolites, Antineoplastic; Azepines; Cytidine Deaminase; Deoxycytidine; Deoxycytidine Kinase; Deoxycytosine Nucleotides; Gemcitabine; Half-Life; Humans; Kinetics; Tetrahydrouridine

The interaction between 2'-deoxycytidine (dCyd) and 1-beta-D-arabinofuranosylcytosine (ara-C), administered at pharmacologically achievable concentrations, was examined in four continuously cultured human leukemia cell lines, HL-60, KG-1, K-562, and CCRF-CEM. In three of the cell lines (HL-60, K-562, and CCRF-CEM), co-administration of 20 or 50 microM dCyd with 10 microM ara-C reduced ara-CTP formation by at least 90% and incorporation of ara-C into DNA by at least 80%. In contrast, KG-1 cells exhibited substantially smaller reductions in both ara-CTP formation and incorporation of ara-C into DNA under identical conditions. KG-1 cells were distinguished by the highest activity of the enzyme cytidine deaminase of the four lines assayed, and exhibited the smallest increments in the intracellular accumulation of both dCyd and deoxycytidine triphosphate (dCTP) in response to exogenous dCyd. Co-administration of 1 mM tetrahydrouridine (THU) or 0.5 mM deoxy-tetrahydrouridine (dTHU) had little effect on the ability of dCyd to antagonize ara-C metabolism in HL-60, KG-1 and K-562 cells. In contrast, these deaminase inhibitors substantially increased the intracellular accumulation of dCTP as well as the ability of dCyd to antagonize ara-CTP formation and incorporation of ara-C into DNA in KG-1 cells. THU and dTHU also permitted dCyd to antagonize ara-C growth inhibitory effects in KG-1 cells to the extent observed in the other leukemic cell lines. These studies suggest that the intracellular deamination of exogenous deoxycytidine may influence the degree to which this nucleoside antagonizes ara-C metabolism and toxicity in some leukemic cells. They also raise the possibility that deaminase inhibitors may be employed to modulate, and perhaps to improve, the therapeutic selectivity of pharmacologically relevant concentrations of ara-C and dCyd in the treatment of acute leukemia in man.

Topics: Arabinofuranosylcytosine Triphosphate; Cell Division; Cytarabine; Cytidine Deaminase; DCMP Deaminase; Deamination; Deoxycytidine; Deoxycytosine Nucleotides; DNA, Neoplasm; Humans; Leukemia; Tetrahydrouridine; Tumor Cells, Cultured

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.

Topics: Cell Line; Cytarabine; Cytidine Diphosphate; DCMP Deaminase; Deoxycytidine; Deoxycytidine Kinase; Deoxycytosine Nucleotides; Deoxyuridine; Hydroxyurea; Kinetics; Nucleotide Deaminases; Ribonucleotide Reductases; T-Lymphocytes; Tetrahydrouridine; Thymine Nucleotides

We have examined the effect of supraphysiologic concentrations of the naturally occurring nucleoside deoxycytidine (dCyd) on the in vitro growth of normal (CFU-GM) and leukemic (L-CFU) myeloid progenitor cells. Bone marrow samples obtained from 34 consecutive patients undergoing routine diagnostic bone marrow aspirations for nonmalignant hematologic disorders exhibited nearly a twofold increment in CFU-GM when continuously cultured in the presence of 10(-4) mol/L dCyd. Higher dCyd concentrations were associated with a smaller degree of enhancement of colony formation. In contrast, the growth of leukemic blast progenitors obtained from patients with acute nonlymphocytic leukemia were not enhanced by any of the dCyd concentrations tested. Coadministration of 10(-3) mol/L tetrahydrouridine (THU), a cytidine deaminase inhibitor, failed to alter the relative inability of dCyd to enhance L-CFU colony growth. The stimulatory effect of dCyd on normal CFU-GM was not mediated by the adherent mononuclear cell population of the marrow, nor was it restricted to the subpopulation of CFU-GM in S phase at the time of initial exposure. Moreover, treatment of normal bone marrow cells with dCyd at concentrations ranging from 10(-6) to 5 X 10(-3) mol/L for 24 hours had only a minor effect on the fraction of CFU-GM in S phase. Coadministration of 10(-4) mol/L dCyd was able to reverse the inhibitory effects of several putative regulators of normal myelopoiesis, including leukemia inhibitory activity (LIA), acidic isoferritins (AIF), and prostaglandin E1 (PGE1). Leukemic myeloblasts exposed to 10(-4) mol/L dCyd exhibited substantial expansion of intracellular pools of dCyd triphosphate (dCTP), demonstrating that inability to metabolize dCyd could not be solely responsible for the absence of growth potentiation in these cells. These studies suggest that supraphysiologic concentrations of dCyd may confer a selective in vitro growth advantage upon normal v leukemic myeloid progenitor cells, and may free the former from the inhibitory effects of several potential negative regulators of myelopoiesis.

Topics: Alprostadil; Cell Cycle; Deoxycytidine; Deoxycytosine Nucleotides; Dose-Response Relationship, Drug; Ferritins; Granulocytes; Hematopoiesis; Hematopoietic Stem Cells; Histocompatibility Antigens Class II; Humans; In Vitro Techniques; Macrophages; Tetrahydrouridine