Spermidine nuclear acetylation in rat hepatocytes and in logarithmically growing rat hepatoma cells: comparison with histone acetylation.

Spermidine acetylation has been studied in nuclear homogenates and in entire nuclei from rat hepatocytes and rat hepatoma tissue culture (HTC) cells, isolated at different stages of logarithmic growth, and compared to histone acetylation. Under all experimental conditions, N8-acetylspermidine was the predominant product of the reaction (90%). Unlike histone, spermidine acetylation in HTC cell and hepatocyte entire nuclei was almost absent or strikingly reduced relative to acetylation using nuclear homogenates as the enzyme sources. This was due to the lack of a free minor pool of spermidine, most likely lost during the purification of entire nuclei. Thus, preincubation of intact nuclei in the presence of spermidine restored activities to values observed using nuclear sonicates. Spermidine acetylation in HTC cell nuclei fluctuated moderately during cell growth, being stimulated immediately after initiation of proliferation and decreasing progressively as cultures reached high cell density. This pattern corroborated that of N8-acetylspermidine intracellular accumulation induced by culturing cells in the presence of 1 mM 7-amino-2-heptanone, a competitive inhibitor of N8-acetylspermidine deacetylase. Histone acetylation during HTC cell growth was not markedly different qualitatively from that of spermidine. Moreover, spermidine and histone acetylations in hepatocyte nuclei were of the same order of magnitude as those seen in rat hepatoma cell nuclei. Finally, inhibition of deacetylation of N8-acetylspermidine had no apparent deleterious effects on cell and growth. It remains to be determined whether the acetylation step is of higher physiological importance, in particular, and as discussed in nuclear spermidine turnover.

Comparative antitumor properties in rodents of irreversible inhibitors of L-ornithine decarboxylase, used as such or as prodrugs.

The antitumor properties of (E)-2-(fluoromethyl)dehydroornithine methyl ester (delta-MFMO-ME) and of (E)-2-(fluoromethyl)dehydroornithine ethyl ester (delta-MFMO-EE), the prodrugs of delta-MFMO, an irreversible inhibitor of mammalian L-ornithine decarboxylase (ODC) 14 times more potent than alpha-difluoromethylornithine (DFMO) and equipotent to (2R,5R)-6-heptyne-2,5-diamine (MAP) in vitro, have been investigated in L1210 leukemia- and Lewis lung carcinoma-bearing mice. The anticancer properties of these esters have been compared with those of DFMO and MAP as a function of the dose, the route of administration, and the stage of the lewis lung carcinoma development in mice. The two esters, administered i.p. shortly after cell inoculation at one-fifth the dose of DFMO, prolonged the survival of mice-bearing leukemia to the same extent as DFMO and MAP. When administered orally to leukemia-bearing mice the two esters were equipotent at prolonging survival. The methyl ester appears, however, to be slightly, but not significantly, more effective than the ethyl ester against leukemia when given i.p., maximum prolongation of the mice survival (79%) occurring at 0.5 g/kg methyl ester every 12 h. The two esters achieve at one-sixth to one-twelfth the dose, antitumor effects similar to DFMO in the Lewis lung carcinoma model, the ethyl ester being slightly, but not significantly, more effective than the methyl ester when administered orally. Moreover, the ethyl ester causes greater reduction of tumor growth than DFMO (P less than 0.05) and MAP (P less than 0.01) in this model. Inhibition of tumor growth is correlated with spermidine depletion and an increase of decarboxylated-S-adenosylmethionine, the aminopropyl donor in the spermidine and spermine synthase reactions. All ODC inhibitors, however, lose most of their antitumor properties when administered at late stage of Lewis lung carcinoma development. Finally, this study demonstrates the advantage of using prodrugs of delta-MFMO, an inhibitor of ODC, since they possess longer duration of action, higher potency, and in some cases better antitumor efficiency than the parent direct inhibitor of ODC. Moreover, and as already noticed for DFMO or MAP, no sign of overt toxicity is caused by the highest effective antitumor doses of the esters.

Inhibition of mammalian spermine synthase by N-alkylated-1,3-diaminopropane derivatives in vitro and in cultured rat hepatoma cells.

A number of N-alkylated-1,3-diaminopropane derivatives [H2N-(CH2)3-NH-(CH2)nH, where n = 1-9] have been tested as potential inhibitors of partially purified rat hepatoma (HTC) cell or pure bovine spleen spermine synthase. Among the compounds described in this paper, the most potent competitive inhibitor of spermine synthase, with respect to spermidine, is N-butyl-1,3-diaminopropane with Ki values of 11.9 nM and 10.4 nM for the HTC cell and bovine spleen enzymes respectively. Inhibition of spermine synthase by this alkylated amine is selective since spermidine synthase activity is not affected up to 100 microM N-butyl-1,3-diaminopropane at a range of 5-200 microM putrescine. Added to the culture medium of growing HTC cells, N-butyl-1,3-diaminopropane causes the expected changes in the polyamine levels with a marked decrease of spermine and an increase of spermidine. Under these conditions cell growth continues unabated. Such N-alkylated-1,3-diaminopropane derivatives may have considerable potential as tools for studying the role of polyamines and in particular the functions of spermine in cell multiplication and differentiation.

Fluorinated analogues of spermidine as substrates of spermine synthase.

A series of mono- and geminal difluorinated analogues of spermidine (4-azaoctane-1,8-diamine) have been tested as potential substrates of partially purified rat hepatoma (HTC) cell or pure bovine spleen spermine synthase (EC 2.5.1.22). Substitution of the hydrogen atoms of the methylene group at position 7 by one or two fluorine atoms decreases 8-fold and 160-fold the apparent Km values for the HTC cell enzyme respectively. Similarly, the Km values of 7-monofluoro and 7,7-difluorospermidine for the pure bovine enzyme are reduced 8-fold and 100-fold respectively, in comparison with spermidine. Di-, but not monofluoro substitution, in the 6-position causes a 5-fold reduction in the affinity for the HTC cell enzyme. Gem-fluorine substitution in the 2-position abolishes substrate capability. In addition to their high affinity for spermine synthase, 7-monofluorospermidine and 7,7-difluorospermidine cause substrate inhibition. This phenomenon, which is more pronounced in the case of the difluorinated analogues is pH-dependent. These enzymatic findings are discussed with regard to the protonation sites of the spermidine analogues, determined by potentiometric titration, which vary as a function of the number and position of the fluorine substituents relative to the basic amino groups.

Immunosuppressive effects of (2R,5R)-6-heptyne-2,5-diamine, an inhibitor of polyamine synthesis: II. Beneficial effects on the development of a lupus-like disease in MRL-lpr/lpr mice.

(2R,5R)-6-heptyne-2,5-diamine (MAP; MDL 72175), a potent irreversible inhibitor of L-ornithine decarboxylase (ODC), possesses immunosuppressive activities in vitro as the result of inhibition of lymphocyte polyamine biosynthesis. The effects of MAP were now studied in vivo in MRL-lpr/lpr female mice, an animal model for human systemic lupus erythematosus (SLE). Administration of MAP (0.2% in drinking water; drug intake: 0.25-0.35 g/kg body weight/day) to female mice for 15 weeks, starting 8 weeks after birth, reduced by 47% the number of spleen cells, retarded development of lymphadenopathy and, at that time, markedly prolonged the survival of the mice. At week 23, MAP reduced plasma IgG concentrations by 50% whereas, in contrast, those of IgM were elevated 1.5-fold. No statistically significant effects of MAP were observed on plasma levels of anti-DNA autoantibodies although serum anti-RNP and anti-Sm titres tended downwards during treatment. Neither glomerular lesions nor proteinuria were improved by MAP administration. Finally chronic administration of MAP for 45 weeks prolonged the median survival time from 29.75 to 35.5 weeks.

Immunosuppressive effects of (2R,5R)-6-heptyne-2,5-diamine an inhibitor of polyamine synthesis: I. Effects on mitogen-induced immunoglobulin production in human cultured lymphocytes.

The consequences of specific inhibition of polyamine biosynthesis by (2R,5R)-6-heptyne-2,5-diamine (MAP) a potent inhibitor of L-ornithine decarboxylase (ODC), on immunoglobulin (Ig) production were studied in cultured human peripheral blood lymphocytes stimulated with pokeweed mitogen (PWM). MAP inhibits the usual PWM-induced increase of polyamine (putrescine, spermidine and spermine) concentrations and reduces concomitantly cell replication. In parallel with these biochemical effects, IgG and IgM production are diminished, a 95% decrease being observed at 100 microM MAP concentration. That the suppressive effects of the ODC inhibitor result from polyamine deficiency, and not from unrelated pharmacological effects, is demonstrated by the restoration of normal Ig production when 10 microM putrescine or spermidine are added to the culture medium. These findings established that the cellular immunological response can be affected by specific inhibition of polyamine biosynthesis and deserve further consideration both under in vitro and in vivo conditions.

Fluorine-containing polyamines: biochemistry and potential applications.

Investigations with the fluorinated spermidine analogues show clearly that these compounds have significant potential for studying the metabolism and functions of the polyamines. However, the biochemical and biological properties of these analogues are dissimilar. This is due to the influence of the fluorine substituent(s) on the basicity of the amine function proximal to the fluoromethylene group, this effect being amplified by geminal disubstitution. The monofluorinated spermidine analogues compare well with the natural amine in their ability to regulate the expression of the decarboxylase enzymes, to be substrates of spermine synthase and to support growth of polyamine-deficient cells. It is also likely that 6-monofluorospermine, formed biochemically in situ, shares with spermine similar functions. These findings raise the possibility of using these spermidine analogues to study the metabolism and pharmacology of polyamines in vivo but also to provide more insight into the regulatory role of spermidine in ODC and SAM-DC expression. Another potential application may be the use of these analogues as probes in tumor imaging and therapy control. This indication has been inferred by studies in tumor-bearing animals, using 19F-NMR spectroscopy determination of tissue fluorospermidine and fluorospermine, formed biochemically from the precursors 2-fluoro or 2,2-difluoroputrescine, and which demonstrate preferential accumulation in tumor versus normal tissue. Finally, these monofluorinated spermidine analogues may exert beneficial effects in pathological states associated with polyamine deficiency. These diseases remain however to be identified. Among the difluorinated spermidine analogues, 7,7-difluorospermidine possesses the most interesting properties. This spermidine analogue still possesses ODC and SAM-DC repressing activities although at much higher concentration than spermidine. More importantly it is a potent inhibitor of spermine synthesis both in cultured cells and in vivo due to its efficient competition with spermidine in the spermine synthase reaction. This compound not only depletes tumor cell of its spermine content but, in addition, appears to exert by itself and/or via 6,6-difluorospermine, the product of its metabolism, polyamine antagonist effects. Combined with MAP but also with DFMO, two potent irreversible inhibitors of ODC which block the synthesis of the natural endogenous polyamines, 7,7-difluorospermidine causes an immediate decrease of viability in cultured HTC cells and promotes tumor regression and stabilization in hepatoma-bearing rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Post-translational modification of the protein-synthesis initiation factor eIF-4D by spermidine in rat hepatoma cells.

The rates of synthesis and turnover of the rare amino acid hypusine [N6-(4-amino-2-hydroxybutyl)-2,6-diaminohexanoic acid] in protein were studied in relationship to polyamine metabolism and growth rates in rat hepatoma tissue-culture (HTC) cells. Hypusine is selectively formed in the eukaryotic translation initiation factor eIF-4D, by a post-translational mechanism involving spermidine [Cooper, Park, Folk, Safer & Braverman (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1854-1857]. The half-life of the hypusine-containing protein was longer than 24 h. In cells whose intracellular spermidine pools had been initially depleted, by using DL-alpha-difluoromethylornithine (DFMO), maximum synthesis rates of hypusine in protein were 5-10 times higher, on restoration of endogenous spermidine contents by exogenous addition, than those observed in untreated exponential-phase cultures. In cells pretreated with DFMO, the rate of hypusine synthesis was constant for up to 1 h after the addition of 5 microM-spermidine, whereas endogenous spermidine contents varied from less than 1 to more than 10 nmol/mg of protein. However, the overall amount of hypusine formed, during the first 1 h after the addition of various concentrations of spermidine (0.05-10 microM) to the culture medium, was markedly dependent on the final endogenous spermidine content achieved at the end of the 1 h measurement interval. Early in exponential-phase growth, protein-bound hypusine was synthesized at a rate of 1-2 pmol/h per mg of protein. This rate decreased to less than 0.5 pmol/h per mg of protein when cell growth rates decreased as cultures reached high cell densities. Analysis of the polyamine substrate specificity for hypusine formation showed that N1-acetylspermidine did not compete with spermidine in the reaction, nor did N1-(buta-2,3-dienyl)-N2-methylbutane-1,4-diamine, and irreversible inhibitor of polyamine oxidase, block the reaction. On the basis of comparative radiolabelling experiments, spermine was either a poor substrate, or not a substrate, for hypusine formation. These results confirm that spermidine is the likely precursor of the aminohydroxybutyl moiety of hypusine, and show that overall hypusine formation, but not necessarily the synthesis rate, is dependent on the endogenous spermidine concentration, especially under conditions where spermidine concentrations are initially low, as is the case after DFMO treatment, and then increase.

Polyamine depletion increases cellular ribonucleotide levels.

Depletion of the putrescine and spermidine content of Ehrlich ascites tumor cells by alpha-difluoromethylornithine (DFMO) treatment results in at least a 1 500-fold increase in the decarboxylated S-adenosylmethionine (deSAM) content. The accumulation of this adenine nucleoside occurs because of the absence of putrescine and spermidine to act as aminopropyl group acceptors in the spermidine and spermine synthase reactions and because of an increase in S-adenosylmethionine decarboxylase activity. The fact that the synthesis of deSAM continues in DFMO-treated cells makes the pathway an adenine trap. This prompted a study of the adenine nucleotide pools. High-performance liquid chromatographic analysis showed that the total adenine nucleotide pool increased, rather than decreased, as a result of DFMO treatment; the major contributors to the increase being ATP and ADP, which increased 2.6 and 1.9 times, respectively. The cellular content of other ribonucleotides increased as well, particularly that of UTP and CTP. When putrescine was added together with DFMO, the increases in cellular ribonucleotide contents were prevented, showing that they were indeed caused by polyamine depletion.

Restoration of the polyamine contents in rat hepatoma tissue-culture cells after inhibition of polyamine biosynthesis. Relationship with cell proliferation.

The restoration of the polyamine content in polyamine-deficient rat hepatoma tissue-culture (HTC) cells, after short duration of incubation in the presence of DL-alpha-difluoromethylornithine (F2MeOrn) or of (2R,5R)-6-heptyne-2,5-diamine [(2R,5R)MAP], two potent irreversible inhibitors of L-ornithine decarboxylase, has been studied in relation to cell proliferation. Both L-ornithine decarboxylase inhibitors deplete the cells of their putrescine and spermidine contents within one day after their addition to the culture medium. Thereafter, intracellular putrescine and spermidine concentrations are restored to near control values within one day when (2R,5R)MAP is removed from the medium, but remain at low levels at least for one day or longer after removal of F2MeOrn. In both conditions, spermine concentration stays at normal or above normal values and cell growth rates are unaffected. Thus, the total intracellular spermine content per culture parallels, in fact, the increase in cell number. The continuous presence of the drugs maintains the depletion of putrescine and spermidine and decreases the total intracellular spermine content of the culture to the same order of magnitude as it reduces the increase in cell numbers. These findings suggest that the antiproliferative effects of these L-ornithine decarboxylase inhibitors in HTC cells is primarily associated with the limitation of spermine biosynthesis rather than to the almost complete reduction of the putrescine and spermidine pools.

Marked and prolonged inhibition of mammalian ornithine decarboxylase in vivo by esters of (E)-2-(fluoromethyl)dehydroornithine.

(E)-2-(fluoromethyl)dehydroornithine, a new enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC) is no more effective than alpha-difluoromethylornithine (DFMO) at inhibiting polyamine biosynthesis in rat hepatoma tissue culture (HTC) cells and in rat organs even though its potency is over 15 times higher than that of DFMO in vitro. The methyl, ethyl, octyl and benzyl esters of (E)-2-(fluoromethyl)dehydroornithine were synthesized as potential prodrugs of the amino acid. When tested at concentration equivalent to the Ki value of the amino acid, they are devoid of ODC-inhibitory property. When measured 6 hr after its addition to the HTC cell culture medium, the absorption of methyl ester was 20 times higher than that of the parent amino acid or that of DFMO, and was accompanied by a more marked intracellular accumulation of (E)-2-(fluoromethyl)dehydroornithine than that achieved by the addition of the parent amino acid. The methyl ester used at 10 times lower concentrations is as effective as its parent amino acid or as DFMO at inhibiting polyamine biosynthesis in HTC cells. Similarly, the methyl and the ethyl esters of (E)-2-(fluoromethyl)dehydroornithine used at 10 times lower doses are as effective as the parent amino acid and as DFMO at inhibiting ODC in the ventral prostate of rat, 6 hr after oral administration. All the esters of (E)-2-(fluoromethyl)dehydroornithine produce a particularly long duration of ODC inhibition in the ventral prostate and in the testes. Repeated administration (25 mg/kg given once a day by gavage) of the methyl ester of (E)-2-(fluoromethyl)dehydroornithine for 8 days to rats results in a constant 80% inhibition of ODC over a 24-hr period, accompanied by a 90% decrease of putrescine and spermidine concentrations in the ventral prostate.

Effects of (2R, 5R)-6-heptyne-2,5-diamine, a potent inhibitor of L-ornithine decarboxylase, on rat hepatoma cells cultured in vitro.

DL-alpha-Difluoromethylornithine (F2MeOrn), the most widely-used inhibitor of L-ornithine decarboxylase, has been a useful tool to demonstrate that polyamine biosynthesis is required to maintain maximum rates of cell proliferation. However, in most eukaryotic cell systems, F2MeOrn exerts cytostatic rather than cytotoxic effects. This may be due to the fact that this inhibitor creates only incomplete polyamine deficiency. In particular, F2MeOrn scarcely depletes intracellular spermine levels. We now demonstrate in rat hepatoma tissue culture (HTC) cells that (2R, 5R)-6-heptyne-2,5-diamine, a more potent irreversible inhibitor of L-ornithine decarboxylase than F2MeOrn, decreases the concentrations of all polyamines including spermine. In parallel with the depletion of these amines, there is a progressive decrease in the rate of cell proliferation and in cell viability. Restoration of the intracellular polyamine content, by addition to the medium of polyamines or a high concentration of L-ornithine, the substrate of L-ornithine decarboxylase, further demonstrates that the antiproliferative effects of (2R, 5R)-6-heptyne-2,5-diamine do result from polyamine deficiency. These findings support the concept that polyamines play an essential function in the cell division processes and emphasize the vital function of spermine in mammalian cells.

Inhibition by DL-alpha-difluoromethylornithine of the pokeweed mitogen-induced immunoglobulin production in cultured human lymphocytes.

The effects of DL-alpha-difluoromethylornithine (DFMeOrn), an irreversible inhibitor of L-ornithine decarboxylase, on immunoglobulin production were studied in vitro using human peripheral blood lymphocytes stimulated with pokeweed mitogen. DFMeOrn inhibits in a concentration-dependent manner the usual pokeweed mitogen-induced increases of polyamine contents (putrescine, spermidine, spermine) and of [3H]thymidine incorporation. In parallel with the reduction of polyamine content and of thymidine incorporation, IgG and IgM productions are diminished, a 70% decrease being observed at 5 mM DFMeOrn concentration. Therefore, it appears that inhibition of polyamine biosynthesis may ultimately interfere with the cellular immunologic response by blocking cell proliferation. These findings certainly deserve further consideration both under in vitro and in vivo conditions.

Decreased protein-synthetic activity is an early consequence of spermidine depletion in rat hepatoma tissue-culture cells.

Hepatoma tissue-culture (HTC) cells were exposed to DL-alpha-difluoromethylornithine (DFMeOrn), a specific irreversible inhibitor of ornithine decarboxylase. Concomitantly with the decrease in spermidine, a decrease in the amount of ribosomes in polyribosomes was observed. Spermine concentrations remained essentially comparable with those in cells not exposed to this inhibitor. Exposure of putrescine- and spermidine-depleted HTC cells to spermidine or spermine rapidly reversed the effect of DFMeOrn on polyribosome profiles, whereas addition of putrescine to the cell culture medium had an effect only after its transformation into spermidine and spermine. The results show that the perturbation of polyribosome formation in DFMeOrn-treated HTC cells is due to spermidine deficiency and that a normal polyamine complement is required for optimal protein-synthetic activity in these cells. The results also indicate that protein synthesis is perturbed before DNA synthesis during depletion of putrescine and spermidine in HTC cells.

Metabolism of N8-monoacetylspermidine in rat hepatoma cells. Investigation of its effect on the activity of L-ornithine decarboxylase.

Recent evidence has indicated a role for the acetyl derivatives of polyamines, particularly N8-monoacetylspermidine, as activators of L-ornithine decarboxylase in rat hepatoma tissue culture (HTC) cells. This is in contrast with the well-described negative regulatory control of ornithine decarboxylase exerted by their non-acetylated counterparts. Because of the possibility of a rapid extracellular and intracellular catabolism of the acetyl derivatives of polyamines, the metabolism of N8-monoacetylspermidine and its effect on HTC cell ornithine decarboxylase have been investigated, under conditions which eliminate its extracellular catabolism. Differing from previous reports, we demonstrate that N8-monoacetylspermidine does not elevate ornithine decarboxylase activity when added at low concentrations to the culture medium of HTC cells. Higher concentrations decrease ornithine decarboxylase activity in a dose-dependent manner. This effect cannot be unambiguously attributed to the effect of the acetyl derivative itself, because of the presence in situ of a very active N8-monoacetylspermidine deacetylase, which generates spermidine intracellularly.

Accumulation of decarboxylated S-adenosyl-L-methionine in mammalian cells as a consequence of the inhibition of putrescine biosynthesis.

Biological transmethylation reactions and polyamine biosynthesis share the substrate S-adenosyl-L-methionine. Under normal conditions, decarboxylated S-adenosyl-L-methionine, the aminopropyl donor for polyamine biosynthesis, does not accumulate because of its rapid utilization in spermidine and spermine synthesis. Alteration of polyamine synthesis by DL-alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of L-ornithine decarboxylase, leads to a striking accumulation of decarboxylated S-adenosyl-L-methionine in rat hepatoma cells cultured in vitro and in rat ventral prostate. This increase is due both to lack of putrescine and spermidine for the aminopropyltransferase reactions and to the elevation of S-adenosyl-L-methionine decarboxylase activity. The biological implications of accumulation of decarboxylated S-adenosyl-L-methionine are discussed with regard to the regulation of S-adenosyl-L-methionine decarboxylase activity and to the antiproliferative effects of DL-alpha-difluoromethylornithine.

Metabolism of acetyl derivatives of polyamines in cultured polyamine-deficient rat hepatoma cells.

The acetyl derivatives of polyamines, N1-acetylspermine (N1-AcSPM) and N1-acetylspermidine (N1-AcSPD), are in vitro better substrates of tissue polyamine oxidase than the corresponding non-acetylated polyamines. Rat hepatoma tissue culture (HTC) cells, depleted of their putrescine (PUT) and spermidine (SPD) content by the use of DL-alpha-difluoromethylornithine (DFMeOrn), an irreversible inhibitor of L-ornithine decarboxylase, were used to study in situ the catabolism of these acetyl derivatives of polyamines. Normal intracellular spermidine content was restored by the addition of N1-acetylspermidine to polyamine-deficient cells. Addition of spermine (SPM) did not restore the spermidine content, although this polyamine elevated the spermine content of the cells. N1-Acetylspermidine reestablished normal spermidine levels of the cells and elevated the cellular putrescine content more efficiently and more rapidly than spermidine. Monoacetylputrescine and N1, N12-diacetylspermine (di-AcSPM) were ineffective in restoring putrescine and spermidine contents. These findings support the concept that N1-acetylspermine and N1-acetylspermidine are natural substrates of tissue polyamine oxidase and suggest poor membrane permeability of monoacetylputrescine (AcPUT) and N1, N12-diacetylspermine. Furthermore, they indicate that acetylation of polyamines by the cytosolic acetyl CoA: polyamine N1-acetyltransferase is the rate-limiting step of polyamine catabolism in rat hepatoma cells. Growth inhibition by DL-alpha-difluoromethylornithine was reversed by N1-acetylspermine and N1-acetylspermidine but not by monoacetylputrescine and N1, N12-diacetylspermine. These results suggest again that the antiproliferative effect of DL-alpha-dilfuoromethylornithine is related to inhibition of polyamine biosynthesis.

Indirect evidence for a strict negative control of S-adenosyl-L-methionine decarboxylase by spermidine in rat hepatoma cells.

1. Direct or indirect inhibitors of l-ornithine decarboxylase (EC 4.1.1.17), structurally related or unrelated to l-ornithine, including dl-alpha-difluoromethylornithine, alpha-methylornithine and 1,3-diaminopropane, used alone or in combination, decreased polyamine concentrations in rat hepatoma tissue culture (HTC) cells and increased S-adenosyl-l-methionine decarboxylase activity (EC 4.1.1.50). 2. Comparison of the catalytic properties of S-adenosyl-l-methionine from cells with elevated and normal activities revealed no apparent modification of the catalytic site as judged by affinity for the substrate, stimulation by di- and tri-amines and inhibition by methylglyoxal bis-(guanylhydrazone). 3. Actinomycin D and cycloheximide, and RNA and a proteinsynthesis inhibitor respectively, blocked the increase of S-adenosyl-l-methionine decarboxylase activity elicited by alpha-difluoromethylornithine. In polyamine-depleted cells the apparent half-life of elevated S-adenosyl-l-methionine decarboxylase activity, determined by inhibition of protein synthesis, was 2.5-fold longer than in control cells. The present results suggest that elevation of S-adenosyl-l-methionine decarboxylase activity by alpha-difluoromethylornithine is due to stabilization of the enzyme. 4. Restoration of the normal intracellular putrescine content, by addition of putrescine to the medium of polyamine-deficient cells, transiently increased S-adenosyl-l-methionine decarboxylase activity. Thereafter, intracellular conversion of putrescine into spermidine was accompanied by inactivation of the enzyme at a rate that was similar to that found on addition of spermidine itself. No relationship between total intracellular spermine content and S-adenosyl-l-methionine decarboxylase activity could be established. 5. Addition of 1mm-1,3-diaminopropane to polyamine-deficient cells did not cause a decrease in the activity of S-adenosyl-l-methionine decarboxylase, whereas addition of 1,5-diaminopentane (cadaverine) did. 1,3-Diamino-N-(3-aminopropyl)propane did not accumulate in cells treated with alpha-difluoromethylornithine and 1,3-diaminopropane, whereas addition of 1,5-diaminopentane led to the accumulation of 1,5-diamino-N-(3-aminopropyl)pentane. 1,3-Diamino-N-(3-aminopropyl)propane (10mum) was as effective as spermidine in decreasing S-adenosyl-l-methionine decarboxylase activity. Thus effectiveness of a diamine in decreasing enzyme activity is related to its capability of being converted into a closely structurally related homologue of spermidine by spermidine synthase. 6. The spermidine site of action appears to be post-translational since (a) the spermidine-induced decrease of S-adenosyl-l-methionine activity was not prevented by actinomycin D and (b) spermidine in the presence of cycloheximide led to a synergistic inactivation of the enzyme with a decay rate that progressively approached control values. Altogether these results are indirect evidence for a strict negative control of S-adenosyl-l-methionine decarboxylase by spermidine and substantiate previous findings [Mamont, Duchesne, Grove & Tardif (1978) Exp. Cell Res.115, 387-393]. Spermidine appears to act on some processes involved in denaturation and/or degradation of the enzyme protein. Putrescine appears to decrease the rate of these processes. The physiological significance of the regulatory control of S-adenosyl-l-methionine decarboxylase is discussed.

Assay of alpha-difluoromethylornithine in body fluids and tissues by automatic amino-acid analysis.

A procedure is described for the measurement of DL-alpha-difluoromethylornithine (DFMO), a selective irreversible inhibitor of ornithine decarboxylase, in biological specimens. The drug is separated from other amino acids with a commerical amino-acid analyser and detected by formation of its alkylthio-isoindole derivative with o-phthalaldehyde. DFMO concentrations of 0.1 nmol can be determined in a sample volume of 100 microliter. The assay has been used to determine the half-life of DFMO in serum of several species and the relationships between serum and tissue concentrations.