mitoguazone and alpha-methylornithine

Canavanine, diaminopropane, alpha-methylornithine and methylglyoxal bis(guanylhydrazone) decreased the intracellular polyamine concentrations in growing baby hamster kidney cells. Each of the inhibitors also prevented polyamine efflux into the extracellular medium. Concomitant with the decrease in polyamine excretion was a change in the distribution of polyamines in the extracellular medium. In each case there was a decrease in the amount of radioactivity present as free spermidine and an increase in that found as acetyl polyamines. The magnitude of this shift correlated with the degree of inhibition of excretion. It may be that acetyl polyamines play a role in the regulation of polyamine excretion.

Topics: Acetylation; Animals; Canavanine; Cell Line; Cricetinae; DNA; Kidney; Mitoguazone; Ornithine; Polyamines; Proteins; Pyruvaldehyde; RNA

Phytohemagglutinin (PHA) and a tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) act synergistically to induce interleukin 2 (IL2) mRNA in human lymphocytes in vitro. The induction was inhibited by a potent inhibitor of protein kinase C (C-kinase), 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) at less than 10 microM. H-7 inhibited C-kinase activity itself in lymphocytes at the same range of the concentration but did not interfere with the translocation of C-kinase from the cytosol to the membrane fraction of the lymphocytes induced by TPA. H-7 is also known to inhibit cAMP-dependent protein kinase (A-kinase) and cGMP-dependent protein kinase (G-kinase). However, the lymphocytes cultured with dibutyryl cAMP or dibutyryl cGMP could not be activated to produce IL2 mRNA. These results show that activation of C-kinase but not A-kinase and G-kinase is necessary in signal transduction for IL2 gene expression. Prostaglandin E2, which is known to elevate intracellular cAMP level, also inhibited IL2 mRNA induction in the lymphocytes stimulated with PHA and TPA. Addition of alpha-methylornithine and methylglyoxal bis (guanyl hydrazone), which inhibit polyamine synthesis, did not affect the induction of IL2 mRNA in the lymphocytes stimulated with PHA and TPA, indicating that polyamine synthesis is not necessary for IL2 mRNA induction.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Bucladesine; Cyclic AMP; Cyclic GMP; Dibutyryl Cyclic GMP; Dinoprostone; Enzyme Activation; Gene Expression Regulation; Humans; Interleukin-2; Isoquinolines; Lymphocytes; Mitoguazone; Ornithine; Phytohemagglutinins; Piperazines; Prostaglandins E; Protein Kinase C; RNA, Messenger; Tetradecanoylphorbol Acetate; Thymidine

In-vitro treatment of preimplantation mouse embryos with spermine and spermidine biosynthesis inhibitor, methylglyoxal-bis-(guanylhydrazone) (MGBG), arrested embryo development at the 8-cell or morula stage. In addition, the embryo DNA synthetic rate, as measured by [3H]thymidine incorporation, was strongly inhibited. The inhibition of blastocyst formation and DNA synthesis by MGBG was readily reversible by an exogenous supply of spermine and/or spermidine to the culture medium. DL-alpha-Methylornithine or DL-alpha-difluoromethylornithine (alpha-DFMO), inhibitors of putrescine biosynthesis, had no effect on embryos cultured for 1 or 2 days, but on the 3rd day embryo DNA synthesis was significantly depressed in the presence of alpha-DFMO. These observations suggest that, during early development of the preimplantation mouse embryo, spermine and spermidine are involved in regulation of embryo growth and DNA synthesis. They may also indicate a role of putrescine at a later stage of mouse embryo development.

Topics: Animals; Blastocyst; Culture Media; DNA; Eflornithine; Embryonic Development; Female; In Vitro Techniques; Mice; Mitoguazone; Ornithine; Polyamines; Pregnancy; Spermine

Mammalian fibroblasts were cultured in the presence of alpha-methylornithine and/or methylglyoxal bis(guanylhydrazone), which inhibit the synthesis of polyamines. This led to a decrease in the cellular content of the polyamines spermine and spermidine by up to 60% when the cells were grown in the presence of both drugs together. The activity of the chromatin-associated enzyme ADP-ribosyltransferase was enhanced 2-3-fold in the drug-treated cells when measured in cells subsequently rendered permeable to exogenous NAD+, the substrate for the transferase. This is a novel and surprising observation, since the transferase is invariably activated by the addition of polyamines to a suitable incubation system such as permeabilized cells, isolated nuclei or the purified enzyme. We found no evidence that the activation was due to the appearance of DNA strand breaks, by using a variety of procedures including both neutral [the 'nucleoid' technique of Cook & Brazell [(1975) J. Cell Sci. 19, 261-279; (1976) J. Cell Sci. 22, 287-302]] and alkaline sucrose-gradient centrifugation and gel electrophoresis, suggesting that this therefore may not be the only means of regulating the activity of ADP-ribosyltransferase and that polyamines may have a role to play in this regard in vivo.

Topics: Adenosine Diphosphate Ribose; Animals; Cells, Cultured; Centrifugation, Density Gradient; Chromatography; Cricetinae; DNA; Enzyme Activation; Fibroblasts; Kidney; Mitoguazone; Nucleotidyltransferases; Ornithine; Poly(ADP-ribose) Polymerases; Spermidine; Spermine

Topics: Animals; Antineoplastic Agents; Cell Cycle; DNA Repair; Drug Therapy, Combination; Eflornithine; Humans; Mitoguazone; Neoplasms; Ornithine; Ornithine Decarboxylase Inhibitors; Polyamines; Sister Chromatid Exchange

Biochemical events were investigated in the G1 to S phase progression induced in quiescent rodent cells by human adenovirus type 5 (Ad5) and by serum. Thymidine kinase activity increased after infection of cells with Ad5 or addition of 10% serum. These stimulations were additive. An early viral gene was responsible for induction by Ad5, but the early mutants ts36, ts37, and ts125 induced thymidine kinase at the permissive and nonpermissive temperatures. Several differences were found between cells stimulated by serum compared with Ad5. Induction of thymidine kinase was delayed in Ad5-infected cells, insensitive to 0.01 microgram/ml actinomycin D and relatively resistant to reduced Ca2+ compared with induction by serum. Ornithine decarboxylase was induced by serum, but not by Ad5, alpha-Methylornithine had little effect on the induction of thymidine kinase by Ad5, but reduced the induction of thymidine kinase by serum, suggesting that Ad5-induced entry into S phase is uncoupled from polyamine biosynthesis. Methylglyoxal bis(guanylhydrazone), however, prevented the induction of thymidine kinase by both serum and Ad5. Adenovirus infection appears to induce cellular DNA synthesis and thymidine kinase in G1-arrested cells by a mechanism different from serum, and bypasses events in the normal G1 to S phase progression.

Topics: Adenoviruses, Human; Animals; Blood; Calcium; Carboxy-Lyases; Cells, Cultured; Cricetinae; Dactinomycin; Enzyme Induction; Fibroblasts; Genes, Viral; Humans; Interphase; Mice; Mitoguazone; Ornithine; Ornithine Decarboxylase; Polyamines; Rats; Thymidine Kinase; Time Factors

Topics: Animals; Brain Neoplasms; Cell Line; Cell Survival; Cells, Cultured; DNA; DNA, Neoplasm; Eflornithine; Guanidines; Mitoguazone; Ornithine; Polyamines; Putrescine; Radiation Tolerance; Rats; Spermidine; Spermine; X-Rays

The accumulation of putrescine, spermidine and spermine in activated bovine lymphocytes was blocked by the combined action of two inhibitors of polyamine biosynthesis, methylglyoxal bis(guanylhydrazone) (MGBG) and alpha-methylornithine. Lymphocytes were cultured under three conditions: (1) alpha-methylornithine alone, (2) MGBG alone, or (3) alpha-methylornithine plus MGBG. DNA synthesis in nuclei isolated from these cells was reduced from control rates by approx. 10, 55 and 75%, respectively. In each case, the degree of inhibition was similar to that observed with the intact cells. Stimulation of nuclear DNA synthesis with the postnuclear supernatant fraction was not affected by polyamine depletion of the cells. Several experiments indicate that the reduced rate of in vitro DNA synthesis was caused by the lack of polyamines and not by alternate effects of the drugs. No inhibition was observed (1) when spermidine was added to inhibited cultures 12 h before harvest and nuclear isolation, (2) when the drugs were added after polyamines had accumulated, and (3) when the drugs were added directly to the in vitro assay. In addition, the degree of inhibition of in vitro DNA synthesis correlated with the degree of polyamine deficiency. These in vitro studies confirm the results obtained with whole cells and support the hypothesis that DNA synthesis is one cellular site of action of the naturally occurring polyamines.

Topics: Animals; Cattle; Cell Nucleus; DNA; DNA Replication; Guanidines; Kinetics; Lymphocytes; Mitoguazone; Ornithine; Putrescine; Spermidine; Spermine