pentostatin and 8-aminoguanosine

Purine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wild type and a variety of mutant host cell lines with well-defined deficiencies in purine metabolism. C. psittaci AA Mp cannot synthesize purines de novo, as assessed by its inability to incorporate exogenous glycine into nucleic acid purines. C. psittaci AA Mp can take ATP and GTP, but not dATP or dGTP, directly from the host cell. Exogenous hypoxanthine and inosine were not utilized by the parasite. In contrast, exogenous adenine, adenosine, and guanine were directly salvaged by C. psittaci AA Mp. Crude extract prepared from highly purified C. psittaci AA Mp reticulate bodies contained adenine and guanine but no hypoxanthine phosphoribosyltransferase activity. Adenosine kinase activity was detected, but guanosine kinase activity was not. There was no competition for incorporation into nucleic acid between adenine and guanine, and high-performance liquid chromatography profiles of radiolabelled nucleic acid nucleobases indicated that adenine, adenosine, and deoxyadenosine were incorporated only into adenine and that guanine, guanosine, and deoxyguanosine were incorporated only into guanine. Thus, there is no interconversion of nucleotides. Deoxyadenosine and deoxyguanosine were cleaved to adenine and guanine before being utilized, and purine (deoxy)nucleoside phosphorylase activity was present in reticulate body extract.

Topics: Adenine; Animals; Cell Line; Chlamydophila psittaci; Cricetinae; Guanine; Guanosine; Mice; Nucleic Acids; Pentostatin; Purine Nucleotides; Purines

Chlamydiae are obligate intracellular bacteria that are dependent on eukaryotic host cells for ribonucleoside triphosphates. The purpose of the present study was to determine whether Chlamydia trachomatis obtains deoxyribonucleotides from the host cell. The study was aided by the finding that host and parasite DNA synthesis activity could be distinguished by their differing sensitivities to aphidicolin and norfloxacin. Results from isotope incorporation experiments indicated that any nucleobase or ribonucleoside that could serve as a precursor for host DNA synthesis could also be utilized by C. trachomatis for DNA replication. C. trachomatis utilized only those precursors which the host cell converted to the nucleotide level. Pyrimidine deoxyribonucleotides were efficient precursors for host DNA synthesis; however, they were not used by C. trachomatis. On the other hand, purine deoxyribonucleosides are rapidly catabolized by host cells, it is necessary to regulate their metabolism to determine whether they serve as direct precursors for C. trachomatis DNA synthesis. This was partially achieved by using a hypoxanthine-guanine phosphoribosyltransferase-negative cell line and using deoxycoformycin and 8-aminoguanosine as inhibitors of (deoxy)adenosine deaminase and purine nucleoside phosphorylase, respectively. The results indicated that purine deoxyribonucleosides are efficiently utilized for host cell DNA synthesis even if degradation pathways are inhibited and salvage to ribonucleotides is minimized. In sharp contrast, the purine deoxyribonucleosides were utilized by C. trachomatis as precursors for DNA synthesis only when host catabolic pathways and salvage reactions were intact. High-pressure liquid chromatographic analysis of nucleotide pools extracted from host cells pulsed with radiolabeled precursors suggests that infected cells transport and phosphorylate all deoxynucleosides as effectively as mock-infected control cultures. In aggregate, these results show that chlamydiae do not take up deoxyribonucleotides from the host cells.

Topics: Animals; Aphidicolin; Cell Line; Chlamydia trachomatis; Cycloheximide; Deoxyribonucleosides; Diterpenes; DNA; DNA, Bacterial; Guanosine; Kinetics; L Cells; Norfloxacin; Nucleosides; Pentostatin; Purines; Pyrimidines; Ribonucleotides

The in vitro cytotoxicity of various purine nucleosides and purine enzyme inhibitors, alone or in combination, and of the alkylating agent mafosfamide (Asta Z7557), incubated for 4 and 24 h have been studied in 17 leukaemic cell lines and normal bone marrow (BM). The purine nucleosides and their analogues included: 2'chlorodeoxyadenosine (CdA), 2'deoxyadenosine (AdR), 3'deoxyadenosine (3'AdR) (cordycepin), adenosine (AR), adenine arabinoside (Ara-A), deoxyguanosine (GdR) and guanine arabinoside (Ara-G). Purine enzyme inhibitors included 2-deoxycoformycin (dCF) and 8-aminoguanosine (8-AG). Cytotoxicity was based on inhibition of (i) incorporation of 3H-leucine into cell proteins and (ii) colony forming units--granulocytic/monocytic (CFU-GM) and for mixed cell colonies (CFU-GEMM). Marked and selective inhibition of T-cell growth was shown by the combinations dCF with either AdR or Ara-A, 8-AG and GdR and by CdA or Ara-G alone; these compounds even at high concentrations produced only partial inhibition of the growth of normal bone marrow CFU-GM and CFU-GEMM except for CdA which completely inhibited the formation of CFU-GEMM colonies. The combination dCF + cordycepin and alkylating agent mafosfamide were, however, toxic to all the cell lines at the concentrations employed, as well as to CFU-GM and CFU-GEMM. The high therapeutic index of some of the purine nucleosides with a relatively short exposure time makes them candidates for selective in vitro removal of residual neoplastic cells in autologous bone marrow transplantation (ABMT) for T-ALL.

Topics: Adenosine Deaminase Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Drug Screening Assays, Antitumor; Guanosine; Humans; Leukemia; Nucleoside Deaminases; Pentostatin; Pentosyltransferases; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Tumor Cells, Cultured

The toxicity of the purine nucleoside, deoxyadenosine in the presence of the adenosine deaminase inhibitor, deoxycoformycin and of deoxyguanosine in the presence of the purine nucleoside phosphorylase inhibitor, 8-aminoguanosine was measured against two Thy-leukemic cell lines. Toxicity was assessed by the survival of clonogenic cells in a colony assay. The kill of clonogenic Thy-leukemic cells was 99.99% with both nucleoside enzyme inhibitor combinations following 4-h incubations when 50 microM concentration of nucleoside were used. With these nucleoside concentrations some reduction in toxicity was apparent when drug treated cells were cultured in the presence of deoxycytidine (50 microM), however, this reduction in toxicity was not apparent when higher nucleoside concentrations were used (100 microM). Survival of bone marrow myeloid progenitor cells (CFU.GM) was only slightly reduced by these nucleoside concentrations following 4 hour incubations. The presence of a twenty-fold excess of normal bone marrow cells reduced the cytotoxic effect but clonogenic cell incubation still ranged from 99.98 to 99.99% for deoxyguanosine and deoxyadenosine respectively. These combinations of nucleosides and enzyme inhibitors may have a therapeutic role in the elimination of malignant Thy cells from human bone marrow.

Topics: Adenosine Deaminase Inhibitors; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow; Cell Line; Cell Survival; Coformycin; Deoxyadenosines; Deoxycytidine; Deoxyguanosine; Guanosine; Hematopoietic Stem Cells; Humans; Leukemia, Lymphoid; Nucleoside Deaminases; Pentostatin; Pentosyltransferases; Purine-Nucleoside Phosphorylase; Ribonucleosides; Tumor Stem Cell Assay

Enzyme inhibitors used to simulate the inherited immunodeficiency diseases, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency, have been assessed in cultured human lymphocytes. Only 2'-deoxycoformycin (dCF) completely inhibited ADA in T and B cells at concentrations in excess of 5 microM. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and 8-amino guanosine (8-NH2GR) did not inhibit ADA or PNP completely at any concentration. Detailed metabolic experiments comparing viability and deoxynucleotide accumulation showed that B cell lines of malignant origin also accumulated high levels of dATP from 2'-deoxyadenosine (dAR), and dGTP from 2'-deoxyguanosine (dGR) as effectively as T cells--even without inhibitors, however, dAR reduced cell viability only when ADA was inhibited by dCF, whilst dGR was equally toxic with or without inhibitor, even to a line which accumulated no dGTP. These experiments indicate that cultured lymphocytes, using either EHNA or 8-NH2GR as enzyme inhibitor, are not valid models of the toxicity to the immune system in inherited ADA or PNP deficiency. They demonstrate that the ability to accumulate high levels of dATP or dGTP is not exclusive to T cells and that the in vitro toxicity of dAR or dGR could relate to the use of excess substrate and/or accumulation in different nucleotide, not deoxynucleotide pools.

Topics: Adenine; Adenosine Deaminase Inhibitors; B-Lymphocytes; Cell Line; Cell Survival; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Deoxyguanine Nucleotides; Deoxyguanosine; Dose-Response Relationship, Drug; Guanosine; Humans; Pentostatin; Purine-Nucleoside Phosphorylase; T-Lymphocytes