Effects of acetylated polyamines on ornithine decarboxylase in rat HTC cells

N-Monoacetylputrescine and N⁸-monoacetylspermidine, metabolites of the naturally occurring polyamines, activate the enzyme ornithine decarboxylase (ODC). When added to cultures of hepatoma (HTC) cells growing in log phase, in concentrations of 5×10^?5M and 2.5×10^?7M respectively, these substances cause a 3 to 5-fold increase in the activity of ODC with a peak effect at one hour. This previously undescribed stimulating effect is in sharp contrast to the well established suppressing effects of nonacetylated polyamines on ODC activity.     

Destabilization of ornithine decarboxylase by transfected antizyme gene expression in hepatoma tissue culture cells.

The degradation of ornithine decarboxylase (ODC) is stimulated by polyamines in a protein synthesis-dependent manner. It has been suggested that antizyme, an ODC-inhibiting protein induced by polyamines, is involved in the process of polyamine-stimulated ODC decay. In this study, we investigated the direct effect of antizyme on ODC decay in hepatoma tissue culture (HTC) cells. A truncated rat antizyme cDNA, Z1, was inserted into an expression vector at a site under the control of a glucocorticoid-inducible promoter and transfected into HTC cells. In the transfected cells dexamethasone increased the amount of Z1 mRNA and induced active antizyme in the absence of exogenous polyamines. When dexamethasone was added to cells with a high level of ODC, rapid decays of ODC activity and protein were elicited after a lag time. Cycloheximide abolished the effect of dexamethasone. These effects of dexamethasone were not observed in control HTC cells transfected with the chloramphenicol acetyltransferase gene. This study indicated that, once induced, antizyme stimulated ODC degradation independently of polyamines and strongly supported our previous hypothesis that the ODC decay-accelerating action of polyamines is mediated by antizyme.     

Replacement of natural polyamines by cadaverine and its aminopropyl derivatives in Ehrlich ascites carcinoma cells

Ehrlich ascites carcinoma cells were cultured in the presence of difluoromethyl ornithine (DFMO) and micromolar concentrations of cadaverine for several months. This treatment resulted in a complete disappearance of putrescine and spermidine and reduced spermine content to traces of its normal content. The natural polyamines were replaced by cadaverine (about 40% of total polyamines), N-(3-aminopropyl)cadaverine (about 50%) and N,N′-bis(3-aminopropyl)cadaverine (about 5%). In comparison with untreated cells or cells grown in the presence of DFMO and putrescine, the “cadaverine cells” grew definitely slower, their protein synthesis was depressed while DNA and RNA syntheses proceeded at near normal rate. In spite of the high intracellular concentrations of cadaverine and its aminopropyl derivatives, the tumor cells grown in the presence of DFMO and cadaverine, behaved exactly like cells severly depleted of putrescine and spermidine. Though exposed to DFMO, ornithine decarboxylase activity was almost 10 times higher than that in untreated cells. S-Adenosyl-L-methionine decarboxylase activity was likewise strikingly elevated, and these cells transported methylglyoxal strikingly elevated, and these cells transported methylglyoxal bis(guanylhydrazone) (MGBG) at a rate that was more than 5 times faster than that in untreated cells. Furthermore, these cells exhibited arginase activity, which was less than one fifth of that found in untreated cells.     

Polyamine-deficient Neurospora crassa mutants and synthesis of cadaverine.

The polyamine path of Neurospora crassa originates with the decarboxylation of ornithine to form putrescine (1,4-diaminobutane). Putrescine acquires one or two aminopropyl groups to form spermidine or spermine, respectively. We isolated an ornithine decarboxylase-deficient mutant and showed the mutation to be allelic with two previously isolated polyamine-requiring mutants. We here name the locus spe-1. The three spe-1 mutants form little or no polyamines and grow well on medium supplemented with putrescine, spermidine, or spermine. Cadaverine (1,5-diaminopentane), a putrescine analog, supports very slow growth of spe-1 mutants. An arginase-deficient mutant (aga) can be deprived of ornithine by growth in the presence of arginine, because arginine feedback inhibits ornithine synthesis. Like spe-1 cultures, the ornithine-deprived aga culture failed to make the normal polyamines. However, unlike spe-1 cultures, it had highly derepressed ornithine decarboxylase activity and contained cadaverine and aminopropylcadaverine (a spermidine analog), especially when lysine was added to cells. Moreover, the ornithine-deprived aga culture was capable of indefinite growth. It is likely that the continued growth is due to the presence of cadaverine and its derivatives and that ornithine decarboxylase is responsible for cadaverine synthesis from lysine. In keeping with this, an inefficient lysine decarboxylase activity (Km greater than 20 mM) was detectable in N. crassa. It varied in constant ratio with ornithine decarboxylase activity and was wholly absent in the spe-1 mutants.     

Control of ornithine decarboxylase in Chinese hamster ovary cells by polyamines. Translational inhibition of synthesis and acceleration of degradation of the enzyme by putrescine, spermidine, and spermine

We have recently isolated, without using any inhibitors, a mutant of Chinese hamster ovary cell line which greatly overproduces ornithine decarboxylase in serum-free culture. Addition of polyamines (putrescine, spermidine, or spermine, 10 microM) or ornithine (1 mM), the precursor of polyamines, to the culture medium of these cells caused a rapid and extensive decay of ornithine decarboxylase activity. At the same time the activity of S-adenosylmethionine decarboxylase showed a less pronounced decrease. Notably, the polyamine concentrations used were optimal for growth of the cells and caused no perturbation of general protein synthesis. Spermidine and spermine appeared to be the principal regulatory amines for both enzymes, but also putrescine, if accumulated at high levels in the cells, was capable of suppressing ornithine decarboxylase activity. The amount of ornithine decarboxylase protein (as measured by radioimmunoassay) declined somewhat more slowly than the enzyme activity, but no more than 10% of the loss of activity could be ascribed to post-translational modifications or inhibitor interaction. Some evidence for inactivation through ornithine decarboxylase-antizyme complex formation was obtained. Gel electrophoretic determinations of the [35S]methionine-labeled ornithine decarboxylase revealed a rapid reduction in the synthesis and acceleration in the degradation of the enzyme after polyamine additions. No decrease in the amounts of the two ornithine decarboxylase-mRNA species, hybridizable to a specific cDNA, was detected, suggesting that polyamines depressed ornithine decarboxylase synthesis by selectively inhibiting translation of the message.     

The modulation of the induction of ornithine decarboxylase by spermine,spermidine and diamines

Extremely low concentrations of putrescine, spermidine and spermine added to the extracellular medium of cultures of mammalian cells inhibit the induction of ornithine decarboxylase activity despite 100- to 1,000-fold greater intracellular polyamine concentrations. The diamines, 1,2-diaminoethane, 1,3-diaminopropane, 1,5-diaminopentane, 1,7-diaminoheptane, 1,10-diaminodecane, 1,12-diaminododecane also inhibit ornithine decarboxylase at all concentrations tested (greater than 10^?6 M). In contrast, 10^?6 M to 10 ^?3 M 1,8-diaminooctane, the alkyl analog of spermidine, enhances ornithine decarboxylase activity. The concentraton of putrescine required to inhibit the activity of ornithine decarboxylase by 50% is a characteristic of each cell line; however, it varies by as much as 1,000-fold among the five cell lines we have tested (L1210 leukemic, H35 hepatoma, N18 neuroblastoma, W256 carcinosarcoma and 3T3 fibroblasts). The antizyme to ornithine decarboxylase can be induced in all these cells by high (di)(poly)amine concentrations. Based on these and other experiments we suggest a working hypothesis: that the polyamines regulate ornithine decarboxylase activity through two different sites that may be interrelated; a sensitive membrane-mediated site that responds to minute fluctuations of extracellular polyamine levels and a coarse site which may be intracellular or membrane associated that responds to larger fluctuations of intracellular polyamine levels. The consequences of such a control mechanism operating within the whole organism are discussed.     

Functions of polyamine acetylation

Acetylation is a means to decrease the net positive charge of the polyamines and thus liberate polyamines from anionic binding sites. The acetyl derivatives can be removed from the cells by transport and catabolism. Intracellular polyamine metabolism can be formulated as a cyclic process, which explains the transformation of one polyamine into another. As a net result, this pathway metabolizes (in an energy-requiring manner) methionine to 5'-deoxy-5'-methylthioadenosine and beta-alanine, and thus appears to be futile. It is suggested that the cyclic process is necessary for the precise control of cellular polyamine concentrations, as it allows relatively rapid spermine and spermidine concentration changes, in spite of a slow basal turnover rate. For the regulation of cellular polyamine metabolism, two decarboxylases, L-ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase; the cytosolic acetyl-CoA:spermidine/spermine N1-acetyltransferase; and a polyamine transport system are required. The activity of the nuclear acetyltransferase is assumed to be the rate-limiting enzyme of nuclear polyamine turnover. The complexity and high level of sophistication of polyamine regulation is strong evidence for the important functional significance of the natural polyamines.     

Inhibition of polyamine accumulation and cell proliferation by derivatives of diaminopropane in Ehrlich ascites cells grown in culture.

1. 1,3-Diaminopropane and some of its derivatives are potent inhibitors of ornithine decarboxylase (EC 4.1.1.17) in Ehrlich ascites cells grown in suspension culture. Among the amine derivatives tested, 1,3-diamino-2-propanol most effectively prevented any accumulation of spermidine and spermine in ascites cells when the proliferation was stimulated by diluting the cells with fresh medium. 2. The effectiveness of diaminopropanol in abolishing polyamine accumulation was primarily based on a rapid decay of ornithine decarboxylase activity following the exposure of the cells to the drug. 3. The mechanism of action of diaminopropanol on ornithine decarboxylase apparently involved a formation of macromolecular inhibitors or 'antizymes' to the enzyme. 4. Even though the inhibitory effect of 1,3-diaminopropane on polyamine accumulation approached that of diaminopropanol, the former compound only marginally inhibited the incorporation of [3H]thymidine into DNA and that of [14C]leucine into protein, in contrast to the marked depression of macromolecular synthesis produced by diaminopropanol. The apparent dissociation of polyamine depletion brought about by 1,3-diaminopropane from an antiproliferative action was apparently due to the fact that diaminopropane, unlike diaminopropanol, was partially capable of taking over the function of natural polyamines. 5. The inhibition of DNA and protein synthesis as well as the prevention of increase in cell number by diaminopropanol was closely associated with polyamine depletion and was fully comparable, as regards timing and magnitude, with that achieved with difluoromethylornithine. The antiproliferative effect of diaminopropanol, however, was only partly reversed by a simultaneous addition of putrescine (or spermidine) into the culture medium. The lack of a complete reversal of the action of diaminopropanol on cell growth by natural polyamines was apparently due to the fact that it was remarkably difficult or even impossible to increase intracellular polyamine concentrations by exogenous polyamines in the presence of diaminopropanol. Nevertheless, the diaminopropanol-induced arrest of growth was reversible as judged by a rapid increase in ornithine decarboxylase activity followed by restoration of DNA synthesis.     

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.     

Regulation of thyroid ornithine decarboxylase by the polyamines. Induction of a protein inhibitor of ornithine decarboxylase by the end-products of the reaction

When spermidine, putrescine or 1,3-diaminopropane was injected (12.5 mumol/100 g body weight) into rats 1 h before thyrotropin, ornithine decarboxylase activity was increased by 75--150% over control levels. However, when greater than or equal to 75 mumol polyamine/100 g body weight was injected, thyrotropin-activated activity was inhibited by 70--95%. Multiple polyamine injections inhibited goitrogen-induced activity and gland weight increase by approx 35%. The polyamines also inhibited thyrotropin-activated rat thyroid ornithine decarboxylase in vitro in a dose-related fashion, with 50% inhibition occurring at 2--5 . 10(-4)M. The inhibition was not due to a direct effect on the enzyme. No stimulation was seen with low concentrations of polyamine. The polyamines had no effect on in vitro thyroid protein/RNA synthesis or glucose oxidation but had a biphasic effect on plasma membrane adenylate cyclase activity. A protein inhibitor to thyroid ornithine decarboxylase was generated in vivo by multiple injections of the polyamines into rats and in vitro by incubating bovine thyroid slices with 2--10 mM polyamine. The inhibitor was non-dialyzable, destroyed by boiling, and its formation was blocked in a dose-related fashion by cycloheximide. We conclude that: (1) thyroid ornithine decarboxylase is subject not only to positive control, but is also negatively regulated by its end-products, the polyamines, which induce a protein inhibitor to ornithine decarboxylase; (2) since gland growth is also inhibited under these conditions, the polyamine effect on thyroid ornithine decarboxylase may be biologically significant.     

Polyamine dependence of Chinese hamster ovary cells in serum-free culture is due to deficient arginase activity

We recently isolated a Chinese hamster ovary cell line which grows well without serum but requires the exogenous polyamines putrescine, spermidine or spermine for continuous replication. Here we show that these cells are defective in the arginase-catalyzed synthesis of ornithine, the precursor of polyamines, and that ornithine can replace polyamines in the medium for supporting growth of the cells. The activities of two other key enzymes of polyamine biosynthesis, ornithine decarboxylase and adenosylmethionine decarboxylase, are clearly detectable and show increase during polyamine starvation. In ornithine- and polyamine-free medium cellular putrescine and spermidine are rapidly depleted while the concentration of spermine decreases only moderately. We show further that the cells are able to grow in serum-containing medium without added ornithine or polyamines. This is explained by our finding that serum contains arginase which synthesizes ornithine from arginine in the medium. All the sera from different animal species tested contained arginase activity although in greatly varying amounts. Serum-free medium is therefore essential for expression of arginase deficiency in cells in tissue culture. The eventual importance of polyamines for serum-free cultures in general is discussed.     

Regulation of ornithine aminotransferase gene expression and activity by all-transretinoic acid in Caco-2 intestinal epithelial cells

Ornithine aminotransferase (OAT) is a crucial enzyme in the synthesis of citrulline and arginine from glutamine/glutamate and proline by enterocytes of the small intestine. However, a role for OAT in intestinal polyamine synthesis and cell growth is not known. All-transretinoic acid (RA), an active metabolite of vitamin A, regulates the activity of several metabolic enzymes related to OAT, including ornithine decarboxylase and arginase, which may influence the function of OAT through effects on substrate (ornithine) availability. The objective of the present study was to test the hypothesis that RA regulates OAT mRNA expression and enzymatic activity in intestinal epithelial cells. Caco-2 cells were cultured for 12-72 h in the presence of 0, 0.01 and 1 microM RA and then used for measurements of OAT mRNA levels and enzyme activity as well as ornithine and polyamines. Treatment with RA induced increases in OAT gene expression and enzymatic activity, which resulted in decreased intracellular concentrations of ornithine and polyamines (putrescine, spermidine and spermine) in a dose-dependent manner. These changes occurred concomitantly with a decrease in the total number of cells, and the increase in OAT activity was due to increased OAT mRNA expression. In cells treated with 1 microM RA, addition of 10 microM putrescine to culture medium restored both cellular levels of polyamines and cell numbers to the values for the control group (without addition of RA). We conclude that exposure of Caco-2 cells to RA induces OAT expression for increasing ornithine catabolism. This leads to a reduced availability of intracellular ornithine for polyamine synthesis, thereby decreasing cell proliferation. These novel findings indicate a functional role for OAT in regulating intestinal polyamine synthesis and growth.     

Ornithine decarboxylase modification and polyamine-stimulated enzyme inactivation in HTC cells.

Ornithine decarboxylase isolated from HTC cells was separated into two distinct charged states by salt-gradient elution from DEAE-Sepharose columns. This charge difference between the enzyme forms was maintained in partially purified preparations, but enzyme form II was observed to change to form I in a time-dependent polyamine-stimulated fashion in crude cell homogenates. The enzyme modification that produces this charge diversity between the alternative enzyme states was further investigated for its role in enzyme activity induction, protein stability and rapid turnover. Inhibition of new protein synthesis by cycloheximide resulted in a much more rapid loss of form I enzyme than of form II, suggesting that during normal enzyme turnover the latter enzyme state may be derived from the former. Culture conditions that favour the stabilization of this usually labile enzyme generally induced an increased proportion of the enzyme in the form II charge state. In particular, inhibitors of synthesis of spermidine and spermine induced the stabilization of cellular ornithine decarboxylase and promoted a marked accumulation in form II. Conversely, polyamines added to the cells in culture induced a very rapid loss in both forms of the enzyme, an effect that could not be attributed merely to an inhibition of new enzyme synthesis. It appears that the polyamines, but not putrescine, may be an essential part of the rapid ornithine decarboxylase inactivation process and that they may function in part by stimulating the conversion of the more stable enzyme form II into the less stable enzyme state, form I.     

Evaluation method for polyamine uptake by N 1-dansylspermine

Polyamine uptake by the polyamine transport system (PTS) in HTC cells was studied without the use of radioisotope-labeled polyamines. N¹-Dansylspermine (DNS343) was selected as a candidate probe to examine the PTS. DNS343 was incorporated into HTC cells, and its distribution in the cells was confirmed by fluorescence microscopy. The incorporation of DNS343 via PTS was confirmed by a competition study with bis(3-aminopropyl)amine, which is incorporated into cells via the PTS. In addition, the temperature dependency of DNS343 uptake and studies with inhibitors of ornithine decarboxylase and proteoglycan synthesis supported the use of DNS343 as a fluorescent probe for the PTS. The kinetics studies for HTC cells treated with or without an ornithine decarboxylase inhibitor indicated that DNS343 uptake was saturable and that the apparent Km values for the PTS were approximately 1.5 μM in both types of cells at 37°C. Thus, we developed an assay method for the PTS by high-performance liquid-chromatography with DNS343. The inhibitory effect of polyamine analogs and related compounds on DNS343 uptake was then examined and discussed.     

The dual action of the non-physiological diamines 1,3 diaminopropane and cadaverine on ornithine decarboxylase of HTC cells

In rat hepatoma tumor (HTC) cells 1,3 diaminopropane and cadaverine induced the ornithine decarboxylase antizyme as well as the end product of the ornithine decarboxylase reaction putrescine. Although at equal exogenous concentrations (10^?3M) the two non-physiological diamines penetrated the cells as effectively as putrescine; they decreased cellular ornithine decarboxylase considerably less rapidly than the naturally present diamine. Cell extracts treated with high concentrations of 1,3 diaminopropane and putrescine, and which as a result had a high specific activity of ornithine decarboxylase antizyme, were chromatographed on a superfine Sephadex G-75 column in the presence of 250 mM NaCl. No ornithine decarboxylase-antizyme complex could be detected indicating the original decrease of ornithine decarboxylase in the cells was likely due to some mechanism other than antizyme. These results indicate that 1,3 diaminopropane and cadaverine probably can act on ornithine decarboxylase, like putrescine, by two distinct regulatory mechanisms.     

Consequences of aberrant ornithine decarboxylase regulation in rat hepatoma cells

DH23A cells, an α-difluoromethylornithine (DFMO)–resistant variant of rat hepatoma tissue culture cells (HTC), contain high levels of very stable ornithine decarboxylase (ODC). In the absence of DFMO, the high ODC activity results in a large accumulation of endogenous putrescine. Concomitant with the putrescine increase is a period of cytostasis and a subsequent loss of viable cells. In contrast, HTC cells with a moderate polyamine content can be maintained in exponential growth. This suggests that a moderate polyamine concentration is necessary for both optimal cell growth and survival. The cytoxicity observed in the DH23A cells is apparently not due to byproducts of polyamine oxidation or alterations in steady state intracellular pH or free [Ca²⁺]. It is possible to mimic the effects of high levels of stable ODC by treatment of cells with exogenous putrescine in the presence of DFMO. This suggests that overaccumulation of putrescine is the causative agent in the observed cytotoxicity, although the mechanism is unclear. These data support the hypothesis that downregulation of ODC may be necessary to prevent accumulation of cytotoxic concentrations of the polyamines. © 1994 Wiley-Liss, Inc.     

Translational regulation of mammalian ornithine decarboxylase by polyamines

Ornithine decarboxylase, which catalyses the formation of putrescine, is the first and rate-limiting enzyme in the biosynthesis of polyamines in mammalian cells. The enzyme is highly regulated, as indicated by rapid changes in its mRNA and protein during cell growth. Here we report that ornithine decarboxylase is regulated at the translational level by polyamines in difluoromethylornithine-resistant mouse myeloma cells that overproduce the enzyme due to amplification of an ornithine decarboxylase gene. When such cells are exposed to putrescine or other polyamines, there is a rapid and specific decrease in the rate of synthesis of ornithine decarboxylase, assayed by pulse-labeling. Neither the cellular content of ornithine decarboxylase mRNA nor the half-life of ornithine decarboxylase protein is affected. Our results indicate that polyamines negatively regulate the translation of ornithine decarboxylase mRNA, thereby controlling their own synthesis.     

Amplification of ornithine decarboxylase gene in response to polyamine deprivation in Chinese hamster ovary cells

We have isolated from an arginase-deficient polyamine-dependent Chinese hamster ovary cell line a new mutant strain that has greatly increased ornithine decarboxylase activity. This enables the cells, in the absence of ornithine, to decarboxylate lysine into cadaverine (diaminopentane) that is further converted into N-(3-aminopropyl)cadaverine and N,N'-bis(3-aminopropyl)cadaverine. These unusual polyamines can support the growth of the cells without added polyamines derived from ornithine. Immunoreactive ornithine decarboxylase-like protein was clearly increased in the mutant cells but could not solely account for the greatly increased enzyme activity. Southern blot analysis of DNA hybridized to a plasmid carrying ornithine decarboxylase-cDNA revealed at least a 32-fold amplification of the ornithine decarboxylase gene. Ornithine decarboxylase-mRNA concentration was also highly increased in the cells. The half-life of the enzyme and the Km for ornithine were not altered from those of the parental cell line.