5-8-dideazaisofolic-acid and Leukemia

The effects of 5-fluoro-2'-deoxyuridine (FdUrd) and 5,8-dideazaisofolic acid on the coordination of thymidylate synthase activity and DNA synthesis were examined in human CCRF-CEM leukemic cells following a continuous exposure to these agents. In logarithmically growing control tumor cells, the rate of in situ thymidylate synthase activity equaled the rate of DNA synthesis. However, in tumor cells incubated with growth-inhibitory concentrations of either FdUrd or 5,8-dideazaisofolic acid for 48 h, the rate of thymidylate synthase activity was between 15- and 17-fold greater than the rate of DNA synthesis. The loss in tumor cell viability of FdUrd-treated cells was temporally related to this prolonged dissociation of thymidylate biosynthesis from DNA biosynthesis. The dissociation of thymidylate from DNA biosynthesis in cells incubated with FdUrd was not closely related to thymidylate depletion. The intracellular concentrations and activities of thymidylate synthase were comparable in tumor cells incubated for 24 or 48 h with either a growth-inhibitory or non-growth-inhibitory concentration of FdUrd, indicating no direct relationship among these parameters. Indirect thymidylate depletion induced by the combination of 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine, hypoxanthine, and glycine inhibited in situ thymidylate synthase activity and DNA synthesis to an equal extent. In addition, the intracellular concentrations of all four deoxyribonucleoside 5'-triphosphates in tumor cells incubated with FdUrd for 48 h were between 1.3- and 3.1-fold greater than their respective concentrations in control cells, reflecting their decreased utilization in DNA synthesis in FdUrd-treated cells. These data indicated that inhibition of CCRF-CEM cell growth and DNA synthesis following a continuous exposure to cytostatic concentrations of either FdUrd or 5,8-dideazaisofolic acid resulted primarily from interference with thymidylate incorporation into DNA, and not simple blockade of thymidylate synthase.

Topics: Deoxyguanine Nucleotides; DNA; DNA Polymerase II; Dose-Response Relationship, Drug; Floxuridine; Folic Acid Antagonists; Humans; Leukemia; Pyrimethamine; Quinazolines; Thymidine Monophosphate; Thymidylate Synthase; Thymine Nucleotides

A series of 2-amino-4-hydroxy-quinazolines was synthesized and evaluated as inhibitors of colon adenocarcinoma and the folate-dependent enzymes, thymidylate synthase and dihydrofolate reductase. Of the quinazolines tested, 5,8-dideazaisopteroylglutamate, (IAHQ), when administered at 85 mg/kg on days 2 and 10 after tumor implantation delayed the growth of colon tumor No. 38, and resulted in 6 of 20 tumor-free animals at 90 days. In contrast, methotrexate had no effect on the growth of colon tumor No. 38 at maximally tolerated doses. IAHQ was also active against human colon adenocarcinoma cells (HCT-8) in tissue culture, requiring a concentration of 5 X 10(-7) M to inhibit cell growth 50% after 72 hours continuous exposure. Since IAHQ was an effective substrate for folylpolyglutamate synthetase, we examined the effects of IAHQ and its possible tri-gamma-glutamyl metabolite, 5,8-dideazaisoPteGLu3, on thymidylate synthase and dihydrofolate reductase. Neither IAHQ nor 5,8-dideazaisoPteGlu3 stimulated significant binding of 5-fluorodeoxyuridylate to thymidylate synthase. This was consistent with the observation that IAHQ antagonized the killing of HCT-8 cells by 5-fluorouracil. 5,8 DideazaisoPteGlu3 bound more tightly to thymidylate synthase than dihydrofolate reductase as indicated by Kis of 0.09 and 0.7 microM when deoxyuridylate and dihydropteroylglutamate, respectively, were the variable substrates. Inhibition studies also revealed that binding of IAHQ and 5,8-dideazaisoPteGlu3 to thymidylate synthase is promoted and not antagonized by deoxyuridylate. The data suggests that the biochemical basis for the antitumor effects of IAHQ is the intracellular conversion of IAHQ to poly-gamma-glutamyl metabolites, which inhibit thymidylate synthase via formation of an inhibitor-deoxyuridylate-enzyme complex.

Topics: Adenocarcinoma; Animals; Cell Line; Colonic Neoplasms; DNA Replication; Drug Resistance; Female; Humans; Kinetics; Leukemia; Methotrexate; Methyltransferases; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; Peptide Synthases; Quinazolines; Tetrahydrofolate Dehydrogenase; Thymidylate Synthase