mitoguazone and ethylglyoxal-bis(guanylhydrazone)

The role of polyamines at the G1/S boundary and in the G2/M phase of the cell cycle was studied using synchronized HeLa cells treated with thymidine or with thymidine and aphidicolin. Synchronized cells were cultured in the absence or presence of α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, plus ethylglyoxal bis(guanylhydrazone) (EGBG), an inhibitor of S-adenosylmethionine decarboxylase. When polyamine content was reduced by treatment with DFMO and EGBG, the transition from G1 to S phase was delayed. In parallel, the level of p27(Kip1) was greatly increased, so its mechanism was studied in detail. Synthesis of p27(Kip1) was stimulated at the level of translation by a decrease in polyamine levels, because of the existence of long 5'-untranslated region (5'-UTR) in p27(Kip1) mRNA. Similarly, the transition from the G2/M to the G1 phase was delayed by a reduction in polyamine levels. In parallel, the number of multinucleate cells increased by 3-fold. This was parallel with the inhibition of cytokinesis due to an unusual distribution of actin and α-tubulin at the M phase. Since an association of polyamines with chromosomes was not observed by immunofluorescence microscopy at the M phase, polyamines may have only a minor role in structural changes of chromosomes at the M phase. In general, the involvement of polyamines at the G2/M phase was smaller than that at the G1/S boundary.

Topics: Adenosylmethionine Decarboxylase; Biogenic Polyamines; Cell Division; Cyclin-Dependent Kinase Inhibitor p27; Eflornithine; Enzyme Inhibitors; G1 Phase; G2 Phase; HeLa Cells; Humans; Mitoguazone; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitors; S Phase

The significance of polyamines for the neoplastic proliferation and secretion of calcitonin (CT) and calcitonin-gene-related peptide (CGRP) by the human medullary thyroid carcinoma TT cell line was investigated. TT cells were cultured in vitro for 6 days with or without additions of pathway inhibitors of polyamine biosynthetic enzymes. Treatment of the cells with 1 mM of the specific L-ornithine decarboxylase (ODC) inhibitor DL-alpha-difluoromethylornithine (DFMO) resulted in a 97% decrease in ODC activity, lowered contents of putrescine (96%) and spermidine (85%) and cell proliferation rates (90%) along with a compensatory 15-fold increase in S-adenosyl-L-methionine decarboxylase (SAMDC) activity. DFMO treatment also led to a decrease in cellular content of CT (33%) and CGRP (26%), while the drug enhanced secretion of CT (31%) but depressed that of CGRP (26%), and elevated the ratio of CT to CGRP secreted into the medium by 74%. Ethylglyoxal bis(guanylhydrazone) (EGBG), a SAMDC inhibitor, at 100 microM evoked a similar reduction of cell proliferation and lowered the content of spermine by 81%. Furthermore, EGBG treatment caused a 34-fold increase in ODC activity and a subsequent 35-fold build-up of putrescine, but also seemed to stabilize SAMDC as evidenced by a highly enhanced SAMDC activity (approximately 200-fold) during enzyme assays in the absence of the inhibitor. EGBG exposure resulted in an increase in cellular CT content (110%) and secretion of the hormone (82%), while not affecting CGRP content or release.2+ EGBG effects were partially counteracted by DFMO.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosylmethionine Decarboxylase; Calcitonin; Calcitonin Gene-Related Peptide; Carcinoma, Medullary; Cell Division; Eflornithine; Humans; Mitoguazone; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitors; Polyamines; Putrescine; Spermidine; Spermine; Thyroid Neoplasms; Tumor Cells, Cultured

A variant cell line, termed SAM-1, which overproduced S-adenosylmethionine decarboxylase (AdoMetDC), was isolated by treatment of mouse FM3A cells with N-methyl-N'-nitro-N-nitrosoguanidine and subsequent incubation with ethylglyoxal bis(guanylhydrazone), an inhibitor of the enzyme. The cells were resistant to ethylglyoxal bis(guanylhydrazone), and showed AdoMetDC activity approximately five-times higher than control cells. The rate of AdoMetDC synthesis and the amount of AdoMetDC existing in SAM-1 cells were about five-times those in control cells. The amount of AdoMetDC mRNA existing in SAM-1 cells was five-times more than that in control cells. The amount of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine, an irreversible inhibitor of AdoMetDC, necessary to inhibit cell growth was also five-times more in SAM-1 cells than in control cells. However, the following were the same in both SAM-1 and control cells; the amount of genomic DNA for AdoMetDC, the size and nucleotide sequence of 5' untranslated region of AdoMetDC mRNA, the deduced amino acid sequence (334 residues) from the nucleotide sequence of AdoMetDC cDNA and the degradation rate (t1/2 = about 4 h) of AdoMetDC. In addition, AdoMetDC mRNA in control cells was slightly more stable than that in SAM-1 cells. The results indicate that the overproduction of AdoMetDC in SAM-1 cells was caused by the increase of AdoMetDC mRNA. The variant cell line is convenient for studying the regulation of AdoMetDC and the physiological function of polyamines.

Topics: Adenosylmethionine Decarboxylase; Animals; Base Sequence; Blotting, Northern; Blotting, Southern; Cell Division; Cell Line; DNA; Drug Resistance; Half-Life; Methylnitronitrosoguanidine; Mice; Mitoguazone; Molecular Sequence Data; Polyamines; RNA, Messenger

Trypanosoma brucei brucei contained a S-adenosyl-L-methionine decarboxylase (AdoMetDC) strongly activated by putrescine. The enzyme was also activated to a lesser extent by cadaverine and 1,3-diaminopropane. Spermidine and spermine had no effect on basal activity of the enzyme. However, they interfered with putrescine activation of trypanosomal AdoMetDC. The trypanosomal enzyme could not be precipitated with antiserum against human AdoMetDC. The trypanosomal AdoMetDC enzyme subunit was labeled by reaction with 35S-decarboxylated AdoMet in the presence of NaCNBH4, and found to have a molecular weight of 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit was readily degraded on storage to a form with a molecular weight of 26 kDa. The specificity of labeling of AdoMetDC by this procedure was confirmed by the prevention of 35S-decarboxylated S-adenosylmethionine (AdoMet) binding in the presence of specific AdoMetDC inhibitors [either methylglyoxal bis(guanylhydrazone (MGBG), a reversible inhibitor, or 5'-deoxy-5'-[(2-hydrazinoethyl)methylamino]adenosine (MHZEA), an irreversible inactivator]. As compared to human AdoMetDC, the trypanosomal enzyme showed weaker binding to a column of MGBG-Sepharose and also was significantly less sensitive to inhibition by MGBG and its congener ethylglyoxal bis(guanylhydrazone) (EGBG). Thus, the trypanosomal AdoMetDC differs significantly from its mammalian and bacterial counterparts and may therefore be exploited as a specific target for chemotherapy of trypanosomiasis.

Topics: Adenosylmethionine Decarboxylase; Animals; Biogenic Polyamines; Enzyme Activation; Female; Mammals; Mitoguazone; Molecular Weight; Precipitin Tests; Putrescine; Rats; Rats, Wistar; Sulfur Radioisotopes; Trypanosoma brucei brucei

The aim of the present study was to evaluate a new anticancer treatment for gastrointestinal cancer, using a combination of polyamine antimetabolites, an anticancer agent and a low-polyamine state. Two polyamine antimetabolites, given as either 40 mg/kg of methylglyoxal-bis-guanylhydrazone (MGBG) or ethylglyoxal-bis-guanylhydrazone (EGBG) and a normal diet (ND), or 20 mg/kg of each drug and a low polyamine diet (LPD), together with 1,000 mg/kg of alphadifluoromethylornithine (DFMO) were administered ip to nude mice for six consecutive days. Mitomycin C (MMC) at 2 mg/kg was then given ip for 3 alternate days. The combination of MGBG or EGBG with DFMO plus MMC resulted in an enhanced antitumor efficacy on LPD. However, the combination which included EGBG was much more enhanced than that which included MGBG and there was no evidence of any tumor regrowth. Weight loss was minimal or nil in the mice given the combination with EGBG, but was evident in those given the combination with MGBG. These results led to the conclusion that in mice, the combined therapy of EGBG with DFMO plus MMC and LPD is a safe and effective regimen for the treatment of gastric cancer.

Topics: Adenosylmethionine Decarboxylase; Animals; Body Weight; Cell Division; Combined Modality Therapy; Diet; DNA, Neoplasm; Drug Therapy, Combination; Eflornithine; Mice; Mice, Inbred BALB C; Mice, Nude; Mitoguazone; Mitomycin; Neoplasm Transplantation; Polyamines; Stomach Neoplasms

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

The aim of the present study was to evaluate the possible role for polyamines in the glucose regulation of the metabolism of insulin mRNA of pancreatic islet cells. For this purpose islets were prepared from adult mice and cultured for 2 days in culture medium RPMI 1640 containing 3.3 mM- or 16.7 mM-glucose with or without the addition of the inhibitors of polyamine biosynthesis difluoromethylornithine (DFMO) and ethylglyoxal bis(guanylhydrazone) (EGBG). Culture at the high glucose concentration increased the islet contents of both insulin mRNA and polyamines. The synthesis of total RNA, total islet polyamines and polyamines associated with islet nuclei was also increased. When the combination of DFMO and EGBG was added in the presence of 16.7 mM-glucose, low contents of insulin mRNA, spermine and spermidine were observed. Total islet polyamine synthesis was also depressed by DFMO + EGBG, unlike islet biosynthesis of polyamines associated with nuclei, which was not equally decreased by the polyamine-synthesis inhibitors. Total RNA synthesis and turnover was not affected by DFMO + EGBG. Finally, actinomycin D attenuated the glucose-induced enhancement of insulin mRNA, and cycloheximide counteracted the insulin-mRNA attenuation induced by inhibition of polyamine synthesis. It is concluded that the glucose-induced increase in insulin mRNA is paralleled by increased contents and rates of polyamine biosynthesis and that an attenuation of the increase in polyamines prevents the increase in insulin mRNA. In addition, the results are compatible with the view that polyamines exert their effects on insulin mRNA mainly by increasing the stability of this messenger.

Topics: Animals; Cell Nucleus; Culture Techniques; Cycloheximide; Dactinomycin; Eflornithine; Gene Expression; Glucose; Insulin; Islets of Langerhans; Male; Mice; Mitoguazone; Polyamines; RNA, Messenger; Spermidine; Spermine

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

The first systematic study on the acid-base properties of the antileukemic agents glyoxal bis(guanylhydrazone) (GBG) and methylglyoxal bis(guanylhydrazone) (MGBG) and of their non-antileukemic monoalkyl- and dialkylglyoxal analogs is reported. At physiological conditions (pH 7.4, 37 degrees C), the species distribution of GBG and MGBG differs remarkably from that of their inactive congeners, a noteworthy proportion of GBG (10.2%) and MGBG (4.0%) existing in the form of the free base while the corresponding proportion of their non-antiproliferative analogs is only 0.5% or less. Ethylglyoxal bis(guanylhydrazone) (EGBG), which has antiproliferative properties in vitro but is devoid of antileukemic activity in vivo, is intermediate between the two groups, 2.6% of it existing in the free base form. In contrast to what has been generally assumed, at physiological conditions, the predominant species of GBG, MGBG, and EGBG is the monocation form and not the dication. Considerable proportions of other congeners also exist in the monocation form. At slightly higher pH values that are of interest because of the known antimitochondrial effects of GBG and MGBG (and, in high concentrations, EGBG), the species distribution of GBG and MGBG differs even more remarkably from that of the dialkylglyoxal analogs. Thus, at pH 8.0 and 37 degrees C, as much as 36% of GBG and 19% of MGBG exist in the free base form, the corresponding proportion of EGBG being 14% and that of the other congeners studies only ca. 3-4%. On the basis of the results, it appears possible that the unusually strict structure-activity relationships of this class of antineoplastic agents may be based on the remarkable differences between the species distributions of the various congeners. The hypothesis is presented that the actual antiproliferative and antimitochondrial species of the compounds is the free base form. A compilation of pKa1 and pKa2 values, measured by potentiometric methods in 0.1 M NaCl (aq) at 25 degrees C and 37 degrees C, is given for six bis(guanylhydrazones). The species distribution curves of the compounds (at 37 degrees C) are given for the pH range 6-10.

Topics: Antineoplastic Agents; Hydrogen-Ion Concentration; Leukemia; Mitoguazone; Structure-Activity Relationship

The antitumor and antimetastatic effects of alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, combined with an inhibitor of S-adenosylmethionine decarboxylase, either methylglyoxal bis(guanylhydrazone) (MGBG) or ethylglyoxal bis(guanylhydrazone) (EGBG), were studied in mice bearing P388 leukemia or Lewis lung carcinoma. Although EGBG is a more specific inhibitor of polyamine biosynthesis than the widely used MGBG, the antitumor effect of the DFMO-EGBG combination on P388 leukemia-bearing mice was less than that of the DFMO-MGBG combination. The prolongation of survival time by the DFMO(1000 mg/kg)-MGBG(25 mg/kg) combination was 2.65-fold, while that of the DFMO(1000 mg/kg)-EGBG(50 mg/kg) combination was 1.34-fold. When mice were fed a polyamine-deficient diet, stronger antitumor effects were exerted; the prolongation of survival time by the DFMO-MGBG and the DFMO-EGBG combinations was 2.89-fold and 2.03-fold, respectively. The antitumor effect of combined use of the two polyamine antimetabolites with mice on normal and polyamine-deficient diets correlated with a decrease of polyamine charge contents in the tumor cells. The above in vivo results were confirmed clearly in the KB cell culture system. The antimetastatic activity of DFMO on Lewis lung carcinoma-bearing mice was strengthened by the addition of MGBG or EGBG. The antimetastatic activity of the DFMO-MGBG or DFMO-EGBG combination did not parallel the polyamine charge contents in the primary tumor and blood.

Topics: Adenosylmethionine Decarboxylase; Animals; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Carboxy-Lyases; Eflornithine; Female; Leukemia P388; Leukemia, Experimental; Lung Neoplasms; Mice; Mitoguazone; Neoplasm Metastasis; Polyamines; Tumor Cells, Cultured

The aim of the present study was to evaluate the role of polyamines in the metabolism and insulin production of pancreatic-islet cells. For this purpose islets were prepared from adult mice and used either immediately or after tissue culture. There was a significant decrease in the islet content of spermidine during culture, although the effect was less pronounced in a high glucose concentration. Furthermore, a stimulatory effect of a high glucose concentration, as compared with low guclose, on the content of spermine was observed. To elucidate further the role of polyamaines in beta-cell physiology, the ornithine decarboxylase inhibitors difuoromethylornithine (DFMO) and methylacetylenic putrescine (MAP) and the S-adenosylmethionine decarboxylase inhibitor ethylglyoxal bis(guanylhydrazone) (EGBG) were added to the culture media. Addition of DFMO together with MAP decreased the cellular contents of putrescine and spermidine, whereas the content of sperimine was unaffected. When EGBG was added in combination with DFMO and MAP, there was a decrease in the content of spermine also. Cell viability in the islets depleted of their polyamine contents was not impaired, as assessed by determinations of oxygen-uptake rates and ATP contents. Depletion of putescine plus spermidine by addition of DFMO+MAP was associated with decreased biosynthesis of (pro)insulin and total protein. When the content of spermine was decreased also by the further addition of EGBG, the decrease in (pro) insulin biosynthesis was more pronounced and was paralleled by a decrease in the insulin-mRNA content. Under these conditions, the glucose-stimulated insulin release, the insulin content and the rates of islet DNA synthesis were also decreased. It is concluded that putrescine and spermidine are necessary for the maintenance of normal insulin and protein biosynthesis, whereas spermine may exert a role in some other cellular processes, such as DNA replication, RNA transcription and glucose-stimulated insulin release.

Topics: Adenosine Triphosphate; Alkynes; Animals; Cells, Cultured; Diamines; DNA; Eflornithine; Glucose; Insulin; Islets of Langerhans; Male; Mice; Mitoguazone; Oxygen Consumption; Polyamines; Protein Biosynthesis; Putrescine; RNA, Messenger

Treatment of nude mice xenografted with human gastric cancer was carried out by polyamine antimetabolites combined with mitomycin C (MMC) and polyamine-free diet. Polyamine antimetabolites, alpha-difluoromethylornithine (DFMO) and ethylglyoxal-bis-guanylhydrazone (EGBG), were given ip in a daily dose of 1,000 mg/kg and 20 mg/kg, respectively, for 6 consecutive days. MMC 2.0 mg/kg was administered every other day. The polyamine-free diet was given from 4 days before start of the treatment through the end of the study. Although the tumor growth rate of the control group given polyamine-free diet was similar to that given normal diet, in the mice treated with EGBG, DFMO plus MMC, the antitumor effect in the polyamine-free diet group was superior to the normal diet group. In comparison with tumor growth suppression due to EGBG plus DFMO or MMC only, the polyamine-free diet group showed better result than the normal diet group to some extent. In mice treated with EGBG, DFMO plus MMC, tumor tissue spermine levels in the polyamine-free diet group were significantly depressed, compared to the normal diet group. Furthermore, marked suppression of DNA biosynthesis was observed in mice given EGBG, DFMO plus MMC together with the polyamine-free diet. These results suggest that combined treatments of polyamine antimetabolites and MMC revealed a marked enhancement of antitumor effects, under conditions of polyamine depletion, which may be responsible for the alteration in DNA structure.

Topics: Animals; Diet; DNA Replication; Drug Administration Schedule; Eflornithine; Humans; Mice; Mice, Inbred BALB C; Mitoguazone; Mitomycin; Mitomycins; Neoplasm Transplantation; Polyamines; Spermine; Stomach Neoplasms

Derivatives of glyoxal bis(guanylhydrazone) (GBG), such as methylglyoxal bis(guanylhydrazone) and ethylglyoxal bis(guanylhydrazone), are potent inhibitors of S-adenosylmethionine decarboxylase (EC 4.1.1.50), the key enzyme required for the synthesis of spermidine and spermine. These compounds, but not the parent compound, induce a massive accumulation of putrescine, partly by blocking the conversion of putrescine into spermidine, but also by strikingly stimulating ornithine decarboxylase (ODC; EC 4.1.1.17) activity. The mechanism of the stimulation of ODC activity and enhanced accumulation of the enzyme protein apparently involved a distinct stabilization of the enzyme against intracellular degradation. However, although the parent compound GBG also stabilized ODC, it powerfully inhibited the enzyme activity and the accumulation of immunoreactive protein in cultured L1210 leukaemia cells. Kinetic considerations indicated that, in addition to the stabilization, all three compounds, GBG in particular, inhibited the expression of ODC. It is unlikely that the decreased rate of synthesis of ODC was attributable to almost unaltered amounts of mRNA in drug-treated cells, thus supporting the view that especially GBG apparently depressed the expression of ODC at some post-transcriptional level.

Topics: Animals; Cells, Cultured; Enzyme Activation; Half-Life; Kinetics; Leukemia L1210; Mice; Mitoguazone; Ornithine Decarboxylase; Ornithine Decarboxylase Inhibitors; RNA, Messenger

Ethylglyoxal bis(guanylhydrazone), a close derivative of the known anti-cancer drug methylglyoxal bis(guanylhydrazone), is also a powerful inhibitor of S-adenosylmethionine decarboxylase (EC 4.1.1.50), the enzyme needed for the synthesis of spermidine and spermine. There were, however, marked differences between the ethyl and methyl derivatives of glyoxal bis(guanylhydrazone) when tested in cultured L1210 cells. The cellular accumulation of ethylglyoxal bis(guanylhydrazone) represented only a fraction (20-25%) of that of the methyl derivative. Moreover, polyamine depletion, which is known to strikingly stimulate the uptake of methylglyoxal bis(guanylhydrazone), decreased, if anything, the uptake of ethylglyoxal bis(guanylhydrazone) by L1210 cells. The compound produced spermidine and spermine depletion fully comparable to that achieved with methylglyoxal bis(guanylhydrazone) at micromolar concentrations. Ethylglyoxal bis(guanylhydrazone) was growth-inhibitory to L1210 cells and produced an additive antiproliferative action when used together with 2-difluoromethylornithine. Ethylglyoxal bis(guanylhydrazone) was distinctly less effective than methylglyoxal bis(guanylhydrazone) in releasing bound polyamines from isolated cell organelles in vitro. Ethylglyoxal bis(guanylhydrazone) was also devoid of the early and profound mitochondrial toxicity typical to methylglyoxal bis(guanylhydrazone). These findings may indicate that this compound is a more specific inhibitor of polyamine biosynthesis with less intracellular polyamine 'receptor-site' activity than methylglyoxal bis(guanylhydrazone).

Topics: Adenosylmethionine Decarboxylase; Animals; Cell Division; Guanidines; Leukemia L1210; Microscopy, Electron; Mitochondria; Mitoguazone; Polyamines; Receptors, Cell Surface; Spermidine; Spermine

Several congeners of methylglyoxal bis(guanylhydrazone) were tested for their ability to inhibit eukaryotic putrescine-activated S-adenosylmethionine decarboxylase (EC 4.1.1.50) and intestinal diamine oxidase (EC 1.4.3.6). All the compounds tested, namely methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone), dimethylglyoxal bis(guanylhydrazone) and the di-N"-methyl derivative of methylglyoxal bis(guanylhydrazone), were strong inhibitors of both yeast and mouse liver adenosylmethionine decarboxylase activity in vitro. The enzyme from both sources was most powerfully inhibited by ethylglyoxal bis(guanylhydrazone). All the diguanidines likewise inhibited diamine oxidase activity in vitro. The maximum intracellular concentrations of the ethyl and dimethylated analogues achieved in activated lymphocytes were only about one-fifth of that of the parent compound. However, both derivatives appeared to utilize the polyamine-carrier system, as indicated by competition experiments with spermidine.

Topics: Adenosylmethionine Decarboxylase; Amine Oxidase (Copper-Containing); Animals; Carboxy-Lyases; Cattle; Cells, Cultured; Guanidines; Intestines; Liver; Lymphocyte Activation; Lymphocytes; Mice; Mitoguazone; Saccharomyces cerevisiae

Three bis(guanylhydrazones) (those of methylglyoxal, glyoxal and ethylglyoxal) were compared for their affinity for the putative polyamine carrier and for their cellular retention in L1210 mouse leukaemia cells. All the bis(guanylhydrazones) inhibited equally effectively the uptake of spermidine by the tumour cells, indicating that the compounds had roughly equal affinity for the polyamine carrier. The fact that methylglyoxal bis(guanylhydrazone) and glyoxal bis(guanylhydrazone) were much more effectively concentrated in the animal cells than was ethylglyoxal bis(guanylhydrazone) was obviously attributable to the finding that the efflux rate of ethylglyoxal bis(guanylhydrazone) greatly exceeded that of the other bis(guanylhydrazones). The rate of efflux of the drugs was slowed down if the tumour cells were treated with 2-difluoromethylornithine before exposure to the bis(guanylhydrazones). These results suggest that intracellular binding of the bis(guanylhydrazones) determines their cellular accumulation.

Topics: Animals; Cells, Cultured; Eflornithine; Guanidines; Leukemia L1210; Mice; Mitoguazone; Ornithine; Spermidine; Time Factors

Previous results have suggested that ethylglyoxal bis(guanylhydrazone) is a more specific inhibitor of polyamine biosynthesis than the widely used methylglyoxal bis(guanylhydrazone). The physiological effects on mitogenically activated lymphocytes of polyamine depletion with ethylglyoxal bis(guanylhydrazone) were examined. In the presence of ethylglyoxal bis(guanylhydrazone) and the ornithine decarboxylase inhibitor alpha-difluoromethylornithine, the cellular contents of putrescine, spermidine, and spermine were decreased by 75 to 90, 65 to 80, and 40 to 60%, respectively, compared with control cultures. Inhibition of DNA synthesis in these polyamine-deficient cells was always greater than that of protein synthesis. Upon addition of spermidine to the deficient cells, the cellular spermidine content was restored within 4 hr, but the complete recovery of macromolecular synthesis took 10 to 20 hr. Thymidine kinase and DNA polymerase alpha activities in polyamine-deficient cells were lower than those in normal cells, whereas RNA polymerase II and leucyl transfer RNA synthase activities were nearly equal to those in normal cells. These results and studies with 2-dimensional gel electrophoresis raise the possibility that polyamines may regulate the synthesis of specific proteins. Decreased synthesis of replication proteins in polyamine-deficient cells may be one reason for the reduced synthesis of DNA.

Topics: Animals; Aphidicolin; Cattle; Cycloheximide; Diterpenes; DNA Replication; Eflornithine; Guanidines; Kinetics; Lymphocytes; Mitoguazone; Ornithine; Ornithine Decarboxylase Inhibitors; Polyamines; Protein Biosynthesis; Proteins; Spermidine

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