The natural polyamines (putrescine, spermidine and spermine) are ubiquitous low-molecular aliphatic amines that play multifunctional roles in cell growth and differentiation. Recently, evidence has merging that polyamines are actively involved in cell death. Changes in polyamine homeostasis have been reported during cell death of nerve cells, in programmed cell death of embryonic cells and in various in vitro models of apoptosis. Polyamines and many of their structural analogs exert cytotoxic effects in vitro as well in vivo. Furthermore, polyamine analogs and inhibitors of the polyamine anabolic/catabolic pathways modulate processes of cell death in a cell-type specific way. Much ambiguity exists in the working mechanisms by which polyamines mediate apoptosis since they have been shown to act as promoting, modulating or protective agents in apoptosis. Nevertheless, from the studies reviewed here it can be concluded that polyamines are critically involved in cellular survival which makes them suitable targets for therapeutic intervention that is specifically directed to cell death pathways.

When Chinese hamster ovary (CHO) cells were cultured with low concentrations of putrescine (< 5 mM) their cell cycle time increased significantly and a fraction of the cells died. A cell line tolerant to the cytotoxic and growth inhibitory effects of millimolar concentrations of putrescine was developed by growing CHO cells over many months in increasing concentrations of the polyamine. A putrescine-tolerant cell line was obtained which was capable of growing in concentrations up to 25 mM putrescine and displayed growth and cell division rates similar to the original untreated/parental CHO cells. The tolerant cells grown in putrescine displayed relatively high intracellular putrescine yet the cell-associated putrescine concentration was estimated to be 10-fold less than the culture medium level. This high concentration of cellular putrescine diminished within 60 min when the cells were changed to non-putrescine-containing media. The putrescine-tolerant phenotype was further characterized in regards to the mechanisms involved in putrescine uptake, efflux, and biosynthesis. The parental and tolerant cell lines had similar or identical levels of cellular spermidine and spermine and no differences in the acetylated polyamine pools or diamine oxidase activity. The activity of ornithine decarboxylase was also similar in the two cell lines in both the presence and the absence of ornithine. The tolerant cells, however, had a decreased uptake rate for putrescine. The tolerant cell line also showed a greatly enhanced ability to export putrescine, especially when treated with ornithine, suggesting that an upregulated polyamine export system may be present in the tolerant cells which could be responsible for the increased cell survival in high putrescine concentrations. The data are discussed in regard to the potential for identifying the transport protein(s) responsible for the maintenance of nontoxic intracellular concentrations of putrescine in a tolerant cell line grown in putrescine.

1. A sensitive and specific assay for spermidine oxidase is described. The method involves the separation of [14C]spermidine (substrate) from [14C]putrescine (product) and other 14C-labelled products on a Dowex 50 cation-exchange column: 92% of the putrescine applied to the column was eluted by 2.3 M-HCl, but this treatment left 96% of the spermidine bound to the column. Unchanged spermidine could be removed from the column by elution with 6 M-HCl. 2. By means of this assay, foetal and adult bovine serum were each shown to contain spermidine oxidase activity, putrescine being a major product of the oxidation of spermidine by the serum enzymes. 3. In foetal bovine serum, spermidine oxidase activity is separable from putrescine oxidase activity by chromatography on a cadaverine-Sephadex column, by gel filtration and by ion-exchange column chromatography. Putrescine oxidase was purified 1900-fold and spermidine oxidase 130-fold by these procedures. The former oxidized putrescine but not spermidine, and spermidine oxidase exhibited no activity with putrescine as substrate.

1. Using protein-separative chromatographic procedures and assays specific for putrescine oxidase and spermidine oxidase, adult bovine serum was found to contain a single polyamine-degrading enzyme with substrate preferences for spermidine and spermine. Apparent Km values for these substrates were approx. 40 microM. The apparent Km for putrescine was 2 mM. With spermidine as substrate, the Ki values for aminoguanidine (AM) and methylglyoxal bis(guanylhydrazone)(MGBG) were 70 microM and 20 microM respectively. 2. Bovine serum spermidine oxidase degraded spermine to spermidine to putrescine and N8-acetylspermidine to N-acetylputrescine. Acrolein was produced in all these reactions and recovered in quantities equivalent to H2O2 recovery. 3. Spermidine oxidase activity was present in foetal bovine serum, but increased markedly after birth to levels in adult serum that were almost 100 times the activity in foetal bovine serum. 4. Putrescine oxidase, shown to be a separate enzyme from bovine serum spermidine oxidase, was present in foetal bovine serum but absent from bovine serum after birth. This enzyme displayed an apparent Km for putrescine of 2.6 microM. The enzyme was inhibited by AM and MGBG with Ki values of 20 nM. Putrescine, cadaverine and 1,3-diaminopropane proved excellent substrates for the enzyme compared with spermidine and spermine, and N-acetylputrescine was a superior substrate to N1- or N8-acetylspermidine.

Under the influence of a selective irreversible inhibitor of ornithine decarboxylase (ODC), DL-alpha-difluoromethylornithine (DFMO), early hematopoiesis was enhanced. In the bone marrow, the absolute number of cells that give rise to spleen colonies in lethally irradiated mice (CFU-S), granulocytic colonies in diffusion chambers in mice (CFU-DG), and granulocyte-monocyte colonies in agar in vitro (CFU-C) was increased 2-4 fold. This could be abrogated by administration of putrescine, confirming the association of the stimulatory effect with polyamine biosynthesis most likely via depression of ornithine decarboxylase activity and subsequent synthesis of putrescine. Analysis of cell cycle characteristics by 3H-TdR suicide technique demonstrated that the proportion of CFU-S, CFU-DG, and CFU-C in S-phase was significantly increased. Additionally, the stimulatory effect was reflected by enhanced colony formation in diffusion chambers implanted intraperitoneally in mice receiving DFMO. This could also be eliminated by treatment of the host animal with putrescine, again suggesting that polyamine biosynthesis plays an important role at the early stages of hematopoiesis in vivo. Effect of DFMO on colony formation in vitro (CFU-C) was inhibitory and not reversible with putrescine. It could be partially eliminated by aminoguanidine, which neutralizes diamine oxidase present in fetal calf serum used in the CFU-C assay. These data suggest that the effect of DFMO in vitro was nonspecific.

Activity of ornithine decarboxylase, the major rate limiting enzyme of polyamine biosynthesis, was determined in bloodstream trypomastigotes of Trypanosoma brucei brucei. The enzyme required pyridoxal-5'-phosphate, dithiothreitol and EDTA for optimal activity. Several properties of the enzyme were investigated and compared to the mammalian enzyme. Most notably, the parasite enzyme was greater than 60-fold more sensitive to the inhibitor DL-alpha-difluoromethylornithine than its mammalian counterpart, thus making it an attractive target for chemotherapy.

Diamine oxidase is the initial enzyme involved in the degradation of polyamines, whose metabolism has been reported as abnormal in cystic fibrosis (CF). Diamine oxidase activity, using putrescine as substrate, was measured in homogenates of human fibroblasts cultured in vitro from normal and cystic fibrosis patients. The pH optimum of the crude enzyme was 7.8. Diamine oxidase activity per microgram protein peaked during periods of greatest proliferative rate and reached its nadir when fibroblasts grew in static (confluent) phase. No difference was found between normal and CF fibroblasts in specific activity of diamine oxidase, but CF cells exhibited a slightly lower protein content during logarithmic growth when compared with normal cells.

Methylthioadenosine (MTA), a coproduct of polyamine biosynthesis, is known to inhibit proliferation in a variety of cell culture systems. In this paper, we show that while MTA inhibits the growth of the human promyelocytic cell line HL-60, it does not interfere with retinoic acid-induced granulocytic or phorbol ester-induced monocytic differentiation of these cells. MTA also inhibits proliferation induced by colony stimulating activity of normal human granulocytic precursor cells grown in suspension culture but does not suppress terminal differentiation of these cells. In contrast to the lack of effect of MTA on granulocytic differentiation which we report here, others have shown that MTA prevents terminal differentiation of murine erythroleukemia cells. That MTA is a normal cellular constituent which inhibits proliferation but not differentiation of normal granulopoietic cells and may have opposing effects on immature cells of erythroid lineage suggests a possible role for this compound in the regulation of hematopoiesis. In addition, MTA may be useful for studying the process of differentiation in the absence of cell proliferation in granulopoietic cells.

A difference was observed in the effect of difluoromethlyornithine (DFMO), a specific inhibitor of ornithine decarboxylase, on human and murine granulocyte-macrophage precursor cell (CFU-C) proliferation in vitro, in the presence of fetal bovine serum (FBS) and horse serum (HS). A dose of DFMO which almost totally abolished CFU-C colonies in cultures containing FBS had no effect or very little effect on CFU-C in cultures supplemented with HS. This effect could be reversed by aminoguanidine reacting with diamine oxidase (DAO), which is present in FBS but not in HS. The importance of DAO in the assessment of polyamine effects is also suggested by decreased colony formation in cultures containing HS and DFMO only after the addition of this enzyme. Additionally, Mo T cell line cultures containing DFMO demonstrated a substantially lower intracellular concentration of putrescine in the presence of FBS rather than HS.

We have previously reported that human vascular endothelial cells and skin fibroblasts carry out degradation of [3H]histamine by a mechanism involving two successive enzymatic steps: imidazole ring tele-methylation by the cells' endogenous methyltransferase and subsequent amine oxidation by an exogenous diamine oxidase. Both histamine and the exogenous second enzyme in the pathway associate with the cells via separate binding sites or receptors. The enzymatic degradation process results in cellular accumulation of the proximal and distal metabolites tele-methylhistamine and 1-methyl-4-imidazoleacetic acid (MIAA)(Haddock, R. C., Mack, P., Fogerty, F. J., and Baenziger, N. L.(1987) J. Biol. Chem. 262, 10220-10228). We have now demonstrated that this two-stage histamine degradative pathway is dependent on Na+ and Cl- in the extracellular environment. Accumulation of [3H] histamine-derived products is partially inhibited under conditions of Na+ deprivation and more substantially when Cl- is also withdrawn. The individual tele-methylation and amine oxidation enzymatic reactions themselves are unaffected or actually facilitated under these conditions. This indicates that it is the cellular mechanism for uptake coupled to the degradative pathway which reflects the cation and anion dependency. Restoration of degradative uptake displays a biphasic Na+ concentration curve, suggesting that the uptake process may be driven by multiple components. These findings indicate a role for both inward Na+ and Cl- ion movement in this cellular degradative uptake mechanism.

1. A sensitive and specific assay for spermidine oxidase is described. The method involves the separation of [14C]spermidine (substrate) from [14C]putrescine (product) and other 14C-labelled products on a Dowex 50 cation-exchange column: 92% of the putrescine applied to the column was eluted by 2.3 M-HCl, but this treatment left 96% of the spermidine bound to the column. Unchanged spermidine could be removed from the column by elution with 6 M-HCl. 2. By means of this assay, foetal and adult bovine serum were each shown to contain spermidine oxidase activity, putrescine being a major product of the oxidation of spermidine by the serum enzymes. 3. In foetal bovine serum, spermidine oxidase activity is separable from putrescine oxidase activity by chromatography on a cadaverine-Sephadex column, by gel filtration and by ion-exchange column chromatography. Putrescine oxidase was purified 1900-fold and spermidine oxidase 130-fold by these procedures. The former oxidized putrescine but not spermidine, and spermidine oxidase exhibited no activity with putrescine as substrate.

1. Using protein-separative chromatographic procedures and assays specific for putrescine oxidase and spermidine oxidase, adult bovine serum was found to contain a single polyamine-degrading enzyme with substrate preferences for spermidine and spermine. Apparent Km values for these substrates were approx. 40 microM. The apparent Km for putrescine was 2 mM. With spermidine as substrate, the Ki values for aminoguanidine (AM) and methylglyoxal bis(guanylhydrazone)(MGBG) were 70 microM and 20 microM respectively. 2. Bovine serum spermidine oxidase degraded spermine to spermidine to putrescine and N8-acetylspermidine to N-acetylputrescine. Acrolein was produced in all these reactions and recovered in quantities equivalent to H2O2 recovery. 3. Spermidine oxidase activity was present in foetal bovine serum, but increased markedly after birth to levels in adult serum that were almost 100 times the activity in foetal bovine serum. 4. Putrescine oxidase, shown to be a separate enzyme from bovine serum spermidine oxidase, was present in foetal bovine serum but absent from bovine serum after birth. This enzyme displayed an apparent Km for putrescine of 2.6 microM. The enzyme was inhibited by AM and MGBG with Ki values of 20 nM. Putrescine, cadaverine and 1,3-diaminopropane proved excellent substrates for the enzyme compared with spermidine and spermine, and N-acetylputrescine was a superior substrate to N1- or N8-acetylspermidine.

Diamine oxidase was previously measured in human pregnancy serum with putrescine or histamine as substrate. We have now documented the presence of spermidine oxidase activity in pregnancy serum by means of a specific radioactive assay with [14C]spermidine as substrate and Dowex 50 cation-exchange chromatography to separate products from substrate. The apparent Km of a partially purified preparation of this enzyme for spermidine was 10.9 microM and the Ki for aminoguanidine was 0.8 microM. The pH optimum (pH 9.0) and temperature optimum (55 degrees C) were identical with those for diamine oxidase. Spermidine oxidase activity and diamine oxidase activity eluted in a concerted fashion when pregnancy serum was subjected to cadaverine-Sepharose chromatography, gel filtration and ion-exchange chromatography. Spermidine oxidase became detectable in serum during pregnancy in the human approx. 8 weeks after the last menstrual period and increased with gestational age in concert with the increase in diamine oxidase activity, reaching a plateau at 20 weeks of gestation. Foetal-cord serum displayed virtually no activity of either enzyme. A 400-fold-purified preparation of diamine oxidase retained the same diamine oxidase/spermidine oxidase ratio as exhibited by crude pregnancy serum. These data suggest that in pregnancy serum, unlike foetal bovine serum, spermidine oxidase and diamine oxidase activity may be a single enzyme protein.

Diamine oxidase is the initial enzyme involved in the degradation of polyamines, whose metabolism has been reported as abnormal in cystic fibrosis (CF). Diamine oxidase activity, using putrescine as substrate, was measured in homogenates of human fibroblasts cultured in vitro from normal and cystic fibrosis patients. The pH optimum of the crude enzyme was 7.8. Diamine oxidase activity per microgram protein peaked during periods of greatest proliferative rate and reached its nadir when fibroblasts grew in static (confluent) phase. No difference was found between normal and CF fibroblasts in specific activity of diamine oxidase, but CF cells exhibited a slightly lower protein content during logarithmic growth when compared with normal cells.

The effects of spermidine and spermine at varying concentrations upon the replicative ability of human fibroblasts in cell culture have been studied. The average concentrations of spermidine causing a 50% inhibition of prolifertion (ID50) after 3 days of growth for three normal cell strains and three strains derived from patients with cystic fibrosis (CF) were 4.4 X 10(-6)+/- 1.2 M and 6.2 X 10(-6)+/- 2.1 M, respectively. The values for spermine were 2.0 X 10(-6)+/- 0.5 M for normal and 2.2 X 10(-6)+/- 0.1 M for fibroblasts from cystic fibrosis patients. No significant difference between the replicative ability of normal and CF cell strains was seen over a wide range of polyamine concentrations employed for a period of up to 3 days.

The inhibitory effect of the polyamines, spermidine and spermine, on the proliferation of human fibroblasts in culture was found to be reversed by the addition of aminoguanidine (AM), a specific and highly effective inhibitor of diamine oxidase (DAO) present in fetal calf serum (FCS). Aminoguanidine itself in concentration as high as 10(-3) M exhibited no effect upon cell proliferation nor did putrescine at similar concentrations. However, at higher concentrations of putrescine, cell proliferation was inhibited and this inhibition was unaffected by the addition of mM concentrations of AM. These studies support earlier hypotheses on the mechanisms of the toxic effects of polyamines on cell proliferation and establish further that the diamine oxidase-catalyzed metabolism of spermine and spermidine is necessary for their toxic effects in cell culture.

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