guanosine-triphosphate and lometrexol

5,10-Dideazatetrahydrofolic acid (DDATHF) is an inhibitor of glycinamide ribonucleotide transformylase, the first of two tetrahydrofolate requiring enzymes in the de novo purine nucleotide biosynthetic pathway, and is a potent inducer of the maturation of HL-60 promyelocytic leukemia cells. The inhibition of cellular growth by DDATHF was effectively prevented by adenosine or deoxyadenosine, whereas guanosine or deoxyguanosine only partially prevented the growth inhibition produced by this folate antimetabolite, implying that the depletion of both ATP and GTP, which occurs with this agent, was responsible for its growth inhibitory effects. In contrast, the induction of differentiation by DDATHF was completely abolished by the presence of guanosine or deoxyguanosine, suggesting that the depletion of intracellular guanine nucleotides by DDATHF represents the event that is essential to the induction of differentiation by this folate analog. This possibility was supported by the observation that the concentration of dGTP was not decreased in cells treated with DDATHF under the conditions employed. Both guanine nucleosides selectively restored intracellular GTP pools depleted by the treatment with DDATHF to their normal level, whereas only adenine nucleosides completely restored the levels of both ATP and GTP to their normal intracellular concentrations. The relationship between guanine nucleotide pools and the induction of HL-60 differentiation by DDATHF was further supported by the finding that maturation and the depletion of intracellular GTP by DDATHF were not reversed by guanine nucleosides in HL-60 cells deficient in hypoxanthine-guanine phosphoribosyltransferase activity. The findings provide support for the hypothesis that the terminal differentiation of these leukemic cells by DDATHF is the result of the depletion of intracellular GTP pools.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Division; Guanosine Triphosphate; Humans; Intracellular Fluid; Leukemia, Promyelocytic, Acute; Purine Nucleotides; Tetrahydrofolates; Tumor Cells, Cultured

(6R)-5,10-Dideaza-5,6,7,8-tetrahydrofolic acid [(6R)DDATHF] is a folate antimetabolite with activity specifically directed against de novo purine synthesis, primarily through inhibition of glycinamide ribonucleotide transformylase. This inhibition resulted in major changes in the size of the nucleotide pools in CCRF-CEM cells. After a 4-h incubation with 1 microM (6R)DDATHF, dramatic reductions in the ATP and GTP pools were observed, with almost no effect on CTP, UTP, and deoxyribonucleotide pools. When the incubation was continued in drug-free medium, recovery of ATP and GTP pools was protracted. ATP did not return to normal until 24-36 h, and GTP pools were only partially repleted by 48 h. The ATP and GTP pools were not affected when the initial 4-h incubation with (6R)DDATHF was conducted in the presence of 100 microM hypoxanthine. Addition of hypoxanthine to the medium after a 4-h incubation with (6R)DDATHF caused rapid recovery of the ATP and GTP pools. Similar effects were seen when the purine precursor aminoimidazole carboxamide was used in place of hypoxanthine. The effect of (6R)DDATHF on nucleotide pools and the capability of hypoxanthine or aminoimidazole carboxamide to prevent or reverse this phenomenon correlated directly with the inhibition of cell growth. Presumably as a consequence of the decrease in purine nucleotide triphosphate levels, the conversion of exogenously added uridine, thymidine, and deoxyuridine to nucleotides was markedly decreased. These effects were protracted for almost 48 h and were also reversed by hypoxanthine. Differential repletion of ATP and GTP pools after (6R)DDATHF pre-treatment demonstrated that diminished precursor phosphorylation is primarily a consequence of GTP rather than ATP starvation.

Topics: Adenosine Triphosphate; Aminoimidazole Carboxamide; Cytidine Triphosphate; DNA; Folic Acid Antagonists; Guanosine Triphosphate; Humans; Hypoxanthine; Hypoxanthines; Leukemia-Lymphoma, Adult T-Cell; RNA; Tetrahydrofolates; Thymidylate Synthase; Time Factors; Tumor Cells, Cultured; Uridine Triphosphate

The novel tetrahydrofolate, 5,10-dideazatetrahydrofolic acid (DDATHF), was designed as an inhibitor of folate metabolism at a site other than dihydrofolate reductase. DDATHF has been shown to inhibit glycinamide ribonucleotide transformylase, a folate-requiring enzyme that catalyzes the first of two one-carbon transfer reactions in the de novo purine nucleotide biosynthetic pathway. Incubation of HL-60 promyelocytic leukemia cells with 5 x 10(-8) to 10(-5) M DDATHF resulted in a marked inhibition of growth after 48 h, with a complete cessation of cellular replication by day 4. Cell cycle analyses of DDATHF-treated HL-60 cells demonstrated an initial block in early S phase by day 3 followed by an accumulation of cells in the G1 and G2 + M phases of the cell cycle. Inhibition of growth was accompanied by a concentration-dependent increase in the percentage of mature myeloid cells that expressed nitroblue tetrazolium positivity, and a small increase in nonspecific esterase activity. Induction of differentiation and inhibition of growth by DDATHF were completely prevented by hypoxanthine and 5(4)-amino-4(5)-imidazole carboxamide, suggesting that depletion of intracellular purine nucleotide pools has an important role in the biological effects of this inhibitor. This possibility was confirmed by the finding that DDATHF caused a pronounced reduction in intracellular GTP and ATP levels within 2 h, with maximum decreases being observed by 24 h, a time interval which preceded the inhibition of cellular proliferation by this agent. Pyrimidine nucleoside triphosphate levels were markedly increased under these conditions. The findings indicate the importance of purine nucleotides to both the inhibition of growth and the induction of differentiation of HL-60 leukemia cells by DDATHF.

Topics: Acyltransferases; Adenosine Triphosphate; Cell Differentiation; Cell Division; Flow Cytometry; Folic Acid Antagonists; Guanosine Triphosphate; Humans; Hydroxymethyl and Formyl Transferases; Interphase; Leukemia, Promyelocytic, Acute; Phosphoribosylglycinamide Formyltransferase; Purine Nucleotides; Tetrahydrofolates; Tumor Cells, Cultured