guanosine-triphosphate and Sarcoma-180

6-Methylmercaptopurine ribonucleoside (6-MMPR), an inhibitor of purine nucleotide biosynthesis de novo, was used as a model compound to evaluate the relationship between the levels of intracellular guanosine triphosphate (GTP) and the formation of cellular glycoproteins and their dolichol-oligosaccharide precursors in Sarcoma 180 cells. Previous studies using the purine antimetabolite, 6-thioguanine (6-TG), demonstrated a relationship between the drug-induced decrease in GTP levels and the incorporation of radiolabelled mannose and fucose into cellular glycoproteins; estimation of the importance of these cell-surface alterations to the cytotoxicity produced by this agent was complicated by the incorporation of 6-TG nucleotides into cellular DNA and RNA. In this report, evidence is presented to show that the toxicity of 6-MMPR to Sarcoma 180 cells is associated with the effects of this agent on the intracellular pools of purine nucleotides. GTP functions in part in the activation of the sugar mannose, a step necessary for the biosynthesis of glycoproteins from nucleotide sugar precursors. Thus, 6-MMPR, which blocks the de novo pathway of purine nucleotide biosynthesis, caused a pronounced decrease in the intracellular pools of GTP in Sarcoma 180 cells; this phenomenon was accompanied by a marked reduction in the incorporation of radiolabelled mannose into cellular glycoproteins and their dolichol-linked oligosaccharide precursors. In contrast, the incorporation of glucosamine, a sugar not metabolically activated by GTP, into glycoproteins, and of leucine into protein, were depressed only after prolonged incubation with either 6-MMPR or 6-TG. Adenine restored purine nucleotide pools depleted by 6-MMPR and partially prevented both the reduction in mannose incorporation into glycoprotein and the cytotoxic effects of this antimetabolite. Guanosine partially reversed the effects of 6-MMPR on intracellular GTP pools and mannose incorporation but not the depression of ATP pools produced by this anti-metabolite. However, guanosine did not reverse the cytotoxicity of 6-MMPR but instead enhanced its toxicity. The findings are consistent with the possibility of membrane changes being involved in the cytotoxicity of 6-MMPR, but clearly other factors are involved as well.

Topics: Adenosine Triphosphate; Animals; Cells, Cultured; Diterpenes; Dolichols; Glycoproteins; Guanosine; Guanosine Triphosphate; Inosine; Mannose; Methylthioinosine; Oligosaccharides; Sarcoma 180; Thioguanine

The effects of the inhibitors of IMP dehydrogenase, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and mycophenolic acid, on the synthesis of cellular glycoproteins were evaluated in Sarcoma 180 cells. Both tiazofurin and mycophenolic acid decreased the rate of incorporation of [2-3H]mannose and [2-3H]fucose into acid-precipitable glycoproteins within 4 hr of exposure; this inhibitory activity was concentration dependent and occurred in the absence of a significant effect on the incorporation of labeled glucosamine and leucine into acid-insoluble material. Interference with the utilization of [3H]mannose for the formation of glycoproteins was paralleled by an inhibition of [3H] mannose incorporation into their lipid-linked oligosaccharide precursors following treatment with cytotoxic concentrations of tiazofurin (100 microM) or mycophenolic acid (10 microM); these actions occurred within 3 hr of exposure to these agents, with maximal reductions being observed at 12 hr. Under these conditions, intracellular GTP levels were reduced by 80%, whereas ATP pools remained unaffected ant UTP levels were markedly increased. Guanosine (100 microM) prevented the cytotoxic actions of tiazofurin and mycophenolic acid and reversed the drug-induced decrease in GTP pools and in the incorporation of mannose and other metabolic precursors into acid-insoluble material. Inhibition of fucose and mannose incorporation into lipid-linked oligosaccharides and glycoproteins were preceded by decreases in the labeling of their respective guanosine nucleotide sugars and were followed sequentially by alterations in the plasma membrane as detected by both the binding and the rate of cell agglutination caused by the plant lectin, concanavalin A. The findings that tiazofurin and mycophenolic acid produce alterations in the utilization of [3H]mannose for the formation of glycoproteins and in membrane architecture are indicative of metabolic lesions induced by agents that selectively depress guanine nucleotide synthesis through inhibition of IMP dehydrogenase.

Topics: Animals; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Glucosamine; Glycoproteins; Guanosine; Guanosine Triphosphate; IMP Dehydrogenase; Ketone Oxidoreductases; Kinetics; Mannose; Mycophenolic Acid; Ribavirin; Ribonucleosides; Sarcoma 180; Tritium

Topics: Animals; Azaserine; Cell Division; Cell Line; Cells, Cultured; Chromatography, Thin Layer; Drug Synergism; Female; Guanine Nucleotides; Guanosine Triphosphate; Half-Life; Mercaptopurine; Mice; Nucleosides; Nucleotides; Peritoneal Cavity; Ribonucleosides; Ribonucleotides; Sarcoma 180; Sulfur Isotopes; Thioguanine; Time Factors; Uracil Nucleotides

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Carbon Isotopes; Chromatography, Paper; Drug Synergism; Female; Guanine Nucleotides; Guanosine Triphosphate; Mercaptopurine; Methylation; Mice; Nucleosides; Ribonucleosides; Sarcoma 180