deoxyguanosine-triphosphate and Leukemia

Topics: Antimetabolites, Antineoplastic; Deoxyguanine Nucleotides; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Inosine Monophosphate; Leukemia; Neoplasms; Purines; Ribavirin; Signal Transduction

The catalytic mechanism for the enzyme, IMP cyclohydrolase, may involve a reaction intermediate with negative charge in the 2-position of the purine ring (Szabados, E., Hindmarsh, E., Phillips, L., Duggleby, R.G. & Christopherson, R.I. (1994) Biochemistry 33, 14237-14245). Three analogues of IMP have been synthesised where fluorine, chlorine or bromine has been substituted in the 2-position on the purine ring. These analogues with an electronegative substituent may resemble a reaction intermediate for IMP cyclohydrolase; 2-fluoro IMP is a potent inhibitor of the enzyme with a Ki value of 0.19 microM, while 2-chloro IMP has a Ki of 1.9 microM and 2-bromo IMP is not inhibitory. However, IMP cyclohydrolase is not inhibited in human CCRF-CEM leukaemia cells exposed to 2-fluoro inosine although it is toxic to these cells with an IC50 value of 4.9 microM.

Topics: Bromine; Cell Division; Chlorine; Deoxyguanine Nucleotides; Deoxyribonucleosides; Enzyme Inhibitors; Fluorine; Humans; Inosine Monophosphate; Leukemia; Nucleosides; Nucleotide Deaminases; Phosphates; Purines; Structure-Activity Relationship; Tumor Cells, Cultured

MOLT-4 (T-), RAJI (B-), and KM-3 (non-B-non-T-, common ALL) malignant lymphoblasts demonstrated significant differences in their activities of purine de novo synthesis (PDNS) and purine salvage pathway and in their cell-kinetic parameters. Incubations with concentrations of methotrexate (0.02 and 0.2 microM), which can be maintained during many hours in the oral maintenance therapy of acute lymphoblastic leukemia, indicated large differences between the three cell lines with respect to the inhibition of PDNS, depending on the concentration of methotrexate (MTX) and on the activities of the two pathways. These dose- and cell line-dependent differences corresponded to the perturbations of cell-kinetics and purine and pyrimidine (deoxy)ribonucleotide pools in the three cell lines. Exposure of MOLT-4 cells to 0.02 microM MTX resulted in an incomplete inhibition of DNA synthesis in early S phase, as shown by DNA-flow cytometry and increase of dCTP levels, which recovered spontaneously after 48 hr. Almost no impairment of RNA synthesis occurred (unbalanced growth). In RAJI cells, exposed to 0.02 microM MTX, DNA synthesis was delayed in the S phase, not arrested, and RNA synthesis was not impaired, also indicating an unbalanced growth pattern, which, however, did not recover in time. KM-3 cells were arrested in G1 phase and subsequently in early S phase after incubation with 0.02 microM MTX, and perturbations of ribonucleotides indicated a complete inhibition of RNA synthesis, resulting in a balanced growth pattern. Cytotoxicity was more pronounced in KM-3 cells. The reliability of the soft agar colony forming assay after low dose MTX treatment is discussed. Exposure of MOLT-4 and KM-3 cells to 0.2 microM MTX resulted in a complete inhibition of DNA synthesis, with cessation of cell progression through all parts of the cell cycle and arrest in G1 phase. RAJI cells showed an increasing accumulation of cells in G1 phase without complete cessation of cell cycle progression. Perturbations of ribonucleotide pools suggested an inhibition of RNA synthesis in all cell lines, indicating a balanced growth pattern in KM-3 cells and MOLT-4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Cycle; Cell Survival; Deoxyguanine Nucleotides; Humans; Leukemia; Lymphocytes; Methotrexate; Polyglutamic Acid; Purines; Pyrimidines; RNA; Thymine Nucleotides; Tumor Cells, Cultured

The toxicity of guanosine and deoxyguanosine in the presence or absence of the purine nucleoside phosphorylase inhibitor, 8-aminoguanosine, for malignant lymphoid cell lines and mitogen-stimulated peripheral blood lymphocytes has been studied. Deoxyguanosine inhibited the proliferation of lymphoid cells more strongly than guanosine. Addition of 100 microM 8-aminoguanosine neither enhanced nor diminished the toxicity of guanosine to the lymphoid cells. Only the toxicity of deoxyguanosine for the leukemic T cell line, MOLT 4, and the leukemic nonBnonT cell line, KM-3, was enhanced by the addition of 100 microM 8-aminoguanosine. These data suggest a possible role of purine nucleoside phosphorylase inhibitors in the treatment of lymphoproliferative disorders of the T-acute lymphoblastic leukemia (ALL) as well as the nonBnonT-ALL subclass.

Topics: Cells, Cultured; Deoxyguanine Nucleotides; Deoxyguanosine; Guanosine; Humans; Leukemia; Lymphocyte Activation; Lymphocytes

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

Terminal transferase (TdT) activity and antigen have been measured in 267 specimens of human bone marrow and peripheral blood by using a biochemical assay for enzymatic activity and an immunofluorescence test for antigen. Oligo p(dA)50 and dGTP were used as reagents in the biochemical assay and either rabbit anti-calf TdT or rabbit anti-human TdT was used as the primary antibody for immunofluorescence. Because both false-positive and false-negative detection of TdT antigen occurs, the biochemical assay of TdT activity is considered the standard against which immunofluorescence assays must be measured. If specimens of cells contained TdT activity, then the immunofluorescence detected antigen in 91% of cases (rabbit anti-calf TdT) and 95% of cases (rabbit anti-human TdT). When no TdT activity was detected, the immunofluorescence test was positive in 7.8% of cases (rabbit anti-calf TdT) and 5.2% of cases (rabbit anti-human TdT). When air-dried slices were shipped by air mail to a distant location before being stained for immunofluorescence, TdT antigen was detected in only 33% of matched pair cases which contained TdT activity. From this study, the authors conclude that with current methodology, immunofluorescence tests for TdT antigen must be carried out on slides prepared in the testing laboratory and that such tests are reliable in more than 90% of cases. However, because a small percentage of results are false positives and false negatives, the authors suggest that if a patient's clinical response is not consistent with the immunofluorescence TdT result, an enzymatic assay for TdT activity be carried out.

Topics: Antigens; Bone Marrow; Deoxyguanine Nucleotides; DNA Nucleotidylexotransferase; DNA Nucleotidyltransferases; Evaluation Studies as Topic; Fluorescent Antibody Technique; Humans; Leukemia; Lymphoma; Oligodeoxyribonucleotides; Specimen Handling; Tritium