concanavalin-a and ethylglyoxal-bis(guanylhydrazone)

Concanavalin A-activated lymphocytes were made polyamine deficient by treatment with alpha-difluoromethylornithine and ethylglyoxal bis(guanylhydrazone). Thymidine kinase activity in polyamine-deficient cells was 17% of the level in normal cells. Thymidine kinase mRNA increased with time after concanavalin A activation and reached a maximum at 36 h after concanavalin A addition. The amount of thymidine kinase mRNA in polyamine-deficient cells was approximately 75% of that in normal cells. The transcription of thymidine kinase gene in isolated nuclei of polyamine-deficient cells was also 75% of that from normal cells. The turnover rate of thymidine kinase mRNA in both normal and polyamine-deficient cells was nearly equal. In normal cells, 95% of thymidine kinase mRNA was polysome associated, while in polyamine-deficient cells, 60% of the mRNA was polysome associated. In addition, the size of polysomes associated with thymidine kinase mRNA in polyamine-deficient cells was smaller than that in normal cells. Synthesis of thymidine kinase was stimulated approximately seven-fold by 0.3 mM spermidine in a rabbit reticulocyte polyamine-free protein synthetic system. The half-life of thymidine kinase activity in both normal and polyamine-deficient cells was nearly equal. Thymidine kinase activity was not influenced significantly by 0.3 mM spermidine. These combined results suggested that the synthesis of thymidine kinase was mainly regulated by polyamines at the level of translation.

Topics: Adenosylmethionine Decarboxylase; Animals; Base Sequence; Cattle; Cells, Cultured; Concanavalin A; Cycloheximide; DNA Polymerase II; DNA Probes; DNA Replication; Eflornithine; Kinetics; Lymphocyte Activation; Lymphocytes; Mitoguazone; Molecular Sequence Data; Polyamines; Polyribosomes; RNA, Messenger; Spermidine; Thymidine Kinase; Thymidylate Synthase; Transcription, Genetic

When bovine small lymphocytes stimulated by concanavalin A were treated with inhibitors (alpha-difluoromethylornithine (5 mM) and ethylglyoxal bis(guanylhydrazone) (100 microM)] of polyamine biosynthesis, swelling of the Golgi apparatus was observed. This was accompanied by decreases in the amount of the Golgi apparatus and of the specific activity of galactosyltransferase. Both spermidine and spermine, at physiological concentrations, stimulated galactosyltransferase activity 2-3-fold. When mice were treated with these inhibitors, the following changes were observed in the epithelial cells of small intestine: swelling of the Golgi apparatus; decrease in the amount of the Golgi apparatus; and decrease of galactosyltransferase activity.

Topics: Animals; Cattle; Cells, Cultured; Concanavalin A; Eflornithine; Epithelium; Galactosyltransferases; Golgi Apparatus; Intestinal Mucosa; Intestine, Small; Lymphocyte Activation; Lymphocytes; Male; Mice; Mice, Inbred BALB C; Microscopy, Electron; Mitoguazone; Polyamines; Spermidine; Spermine

Ethylglyoxal bis(guanylhydrazone) (EGBG) was compared as an inhibitor of polyamine biosynthesis with methylglyoxal bis(guanylhydrazone) (MGBG) in bovine small lymphocytes stimulated by concanavalin A. EGBG brought about a decrease in spermidine and spermine levels equal to that found with MGBG, but at a 5-fold lower intracellular drug concentration. Despite identical polyamine levels, the degree of inhibition of DNA and protein synthesis by EGBG was smaller than that observed with MGBG, in either the presence or absence of the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. It was found that in vitro protein synthesis and in vivo mitochondrial function were inhibited by concentrations of MGBG necessary to inhibit polyamine synthesis in cells (1 to 3 mM), but not by efficacious levels of EGBG (0.2 to 0.6 mM). These results suggest that EGBG is more suitable as a specific inhibitor of polyamine biosynthesis and that use of this drug, rather than MGBG, in combination with alpha-difluoromethylornithine may be useful for studying the physiological functions of polyamines in animal cells.

Topics: Animals; Cattle; Cells, Cultured; Concanavalin A; DNA Replication; Guanidines; Lymphocyte Activation; Lymphocytes; Microscopy, Electron; Mitoguazone; Polyamines; Protein Biosynthesis; Structure-Activity Relationship