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.

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 WM) 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 natural polyamines putrescine, spermidine, and spermine play an important role in the control of cellular growth (1-4). The first and rate-controlling step in the polyamine biosynthesis, conversion of ornithine to putrescine, is catalyzed by ornithine decarboxylase (EC 4.1.1.17), which is present in very small amounts in mammalian cells. The second R01 CA 37695 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in'accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed. enzyme in the pathway, adenosylmethionine decarboxylase (EC 4.1.1.50) plays again a key role in the synthesis of spermidine and spermine. Regulation of ornithine decarboxylase is of special interest because of its great inducibility by a variety of growth stimuli (1-3) and its very rapid turnover rate in mammalian cells (5)(6)(7)(8)(9)(10)(11). There is increasing evidence that the activity of ornithine decarboxylase is primarily regulated by changes in the amount of enzyme protein (6-11). However, there are also reports indicating that phosphorylation (12), transglutamination (131, transitions between more and less active forms (14,15), and binding to a unique inhibitory protein called antizyme (16-18) may, at least in uitro, control the enzyme activity.
Putrescine, spermidine, and spermine seem to exert,some kind of negative feedback control on the ornithine decarboxylase activity. Both exogenously added polyamines (1-3, 16) and endogenously formed putrescine (19) have been reported to cause a decrease in the enzyme activity. Similarly, there appears to be a negative control of the activity of S-adenosylmethionine decarboxylase by spermidine (20, 21) and spermine (20,22). The mechanisms by which these decreases are brought about are not clear. There is indirect evidence for both transcriptional (23) and post-transcriptional (23, 24) regulation, as well as for a selective translational (25) and post-translational (12-15) control of ornithine decarboxylase by polyamines. Administration of polyamines also appears to induce or release the antizyme to ornithine decarboxylase in various animal tissues and cultured cells (16)(17)(18). The results concerning the regulation of the enzyme activity are thus somewhat conflicting. Moreover, in many experiments relatively high concentrations of polyamines were used making the physiological relevance of the results questionable.
We have now attempted to elucidate the physiologically important mechanisms of regulation of ornithine decarboxylase by polyamines in the Chinese hamster ovary (CHO') cell line A2. The ability of these cells to grow in serum-free medium and their marked overproduction of ornithine decarboxylase (26) make this cell line very well suited for the studies. In addition, the recently devised techniques for measuring the enzyme protein (9,lO) and its mRNA (27-29) have made it possible to get more direct information of the regulatory mechanisms. Our results show that two factors are mainly responsible for the inhibition of ornithine decarboxylase activity by polyamines: 1) a decrease in the synthesis of ' The abbreviations used are: CHO, Chinese hamster ovary; MEM, minimal essential medium; SDS, sodium dodecyl sulfate; dansyl, 5dimethylaminonaphthalene-l-sulfonyl. ornithine decarboxylase through inhibition of translation of the message and 2) an increase in the enzyme degradation, which may or may not be mediated by antizyme. An abstract of this study has been published elsewhere (30).
Cell Culture-We have previously isolated from CHO cells an arginase-deficient cell line A7, which grows in serum-free medium (31,32). From these cells we subsequently obtained a mutant cell line, designated A2, that overproduces ornithine decarboxylase (26). In the current experiments a cloned strain of A2 was used. The cells were cultured on collagen-coated Petri dishes in a 1:l mixture of minimal essential medium (MEM) and nutrient mixture F12 (without putrescine) supplemented with 0.1% bovine serum albumin.
Determination of Enzyme Activities-The activities of ornithine and adenosylmethionine decarboxylases were measured as previously described (31), except that most of the ornithine decarboxylase assays were now carried out with saturating ornithine concentrations (0.4 mM).
Radioimmunoassay of Ornithine Decarboxyluse-Ornithine decarboxylase concentration was measured essentially as described by Seely and Pegg (10) with antiserum raised in rabbits against the enzyme protein purified to homogeneity from mouse kidney (9). f5S]Methwnine Labeling and Immunoprecipitation of Ornithine Decarboxyluse-The rate of ornithine decarboxylase synthesis in the absence or presence of polyamines was determined in the following way. After preincubating the cells for different times in Fl2/MEM medium without or with polyamines, the medium was replaced by MEM, without or with polyamines, but lacking cold methionine. After incubation for 30 min [35S]methionine (80-150 pCi/ml) was added, and the incubation was continued for another 30 min. Incorporation was stopped by washing the cells twice with MEM containing cold methionine. In the degradation studies of ornithine decarboxylase the enzyme was prelabeled with [35S]methionine by incubating the cells in the methionine-free MEM supplemented with [=S]methionine (80 pCi/ml) for 2 h. After washing the cells twice with MEM, the incubation was continued in methionine-containing F12IMEM medium without or with polyamines for different time periods. To prepare cell extracts for immunoprecipitation, cells were homogenized by sonication in 50 mM Tris-HC1 buffer, pH 7.4, containing 150 mM NaC1, 5 mM EDTA, 0.5% Nonidet P-40, and 2 mM methionine. Homogenates were centrifuged at 100,000 X g for 30 min, and the supernatants were used for immunoprecipitation with monospecific ornithine decarboxylase antiserum or with normal rabbit serum (27). The immunoprecipitates were then subjected to SDS-polyacrylamide gel (8%) electrophoresis following the standard procedures, and the gels were autoradiographed (4).
Northern Blot Analysis of Ornithine Decarboxylase mRNA-Total cellular RNA was isolated by the lithium chloride/urea method (33). Northern blot hybridization analysis was performed by the method of Thomas (34), as previously detailed (26). Nick-translated plasmid pODC16 (35) was used as the hybridization probe.
Analysis of Polyamines-Cellular polyamines extracted with perchloric acid were first dansylated, then separated by thin-layer chromatography and analyzed as previously described (36).
Ornithine Decarboxyluse Antizyme Assays-Ornithine decarboxylase antizyme was assayed both by the procedure developed by Heller et al. (37) with minor modifications (38,39) and by the newly devised method of Murakami et at. (18). In the first technique ornithine decarboxylase-antizyme complex was dissociated by adding 0.5 M NaCl to 0.5 ml of the cytosols prepared from 8 X lo7 cells. The components were separated on a Sephadex G-75 column (1.6 x 36 cm) equilibrated with 10 mM sodium phosphate buffer, pH 7.0, containing 0.5 M NaCl, 0.1 mM EDTA, and 0.02% Nonidet P-40. Fractions of 1 ml were collected, and 50 p1 was used for each assay of enzyme and inhibitor. In the second technique increasing amounts of difluoromethylornithine-inactivated ornithine decarboxylase were used to release active enzyme competitively from the ornithine decarboxylase-antizyme complex. Inactive ornithine decarboxylase was prepared by incubating the enzyme from the same ornithine decar-boxylase overproducing A2 strain with 20 p M difluoromethylornithine in the assay mixture for 5 h. The preparation was then extensively dialyzed against 25 mM Tris-HC1 buffer, pH 7.5, containing 0.1 mM EDTA and 1 mM dithiothreitol.

RESULTS
Inhibition of the Activities of Ornithine and Adenosylmethionine Decarboxylases by Polyamines-Polyamines added to the culture of the A2 cells at a concentration of 10 PM caused a rapid decay of the ornithine decarboxylase activity (Fig. 1A) and a somewhat less extensive decrease in the activity of adenosylmethionine decarboxylase (Fig.  1B). Undegraded amines were evidently responsible for the effect, because no serum amine oxidases were present in the serum-free medium. Spermidine was more effective than spermine and putrescine in reducing the activities of both enzymes, although in the case of adenosylmethionine decarboxylase there was not a great difference between the actions of spermidine and spermine. T o differentiate whether each of the polyamines was effective itself or only after metabolic conversions we measured the cellular polyamine contents at different times after exogenous addition of the amines (Fig. 2). At the time of observing the decrease in the enzyme activities a considerable portion of the putrescine taken up by the cells had already been converted to spermidine (Fig. U ) , whereas relatively little of the spermidine (Fig. 2B) and practically none of the spermine ( Fig. 2C) taken up was converted to the other polyamines. The results demonstrate that both spermidine and spermine alone are capable of reducing the enzyme activities, while the role of putrescine is questionable. T o examine further the efficacy of endogenously formed polyamines in the control of the activity of the two decarboxylases the cells were cultured in the presence of different concentrations of ornithine. Table I shows that an exposure of the cells to 1 mM ornithine for 2 h reduced the activity of ornithine decarboxylase to half of the control, whereas 0.1 mM ornithine was without any effect. Notably, the accumulation of putrescine was much greater in the presence of 1 mM ornithine than 0.1 mM ornithine, while no marked differences in the patterns of formation of spermidine and spermine were found when measured 1 or 2 h after addition of either concentration of ornithine (Table 1). That even less spermine was accumulated in the presence of the higher ornithine concentration is explainable by the fact that spermine synthase is inhibited by high putrescine levels (40). According to these results putrescine itself can also cause an inhibition of the ornithine decarboxylase activity. However, relatively high concentrations of putrescine, not normally encountered in the cells, appear to be required for the inhibition. In the presence of 0.1 mM ornithine the inhibition of ornithine decarboxylase became evident only after 4 h, being most probably due to the accumulations of spermidine and spermine (results not shown). The activity of adenosylmethionine decarboxylase was rather insensitive to putrescine (Table I).
Changes in Ornithine Decarboxylase Polypeptide after Spermidine Addition-To find out whether the inhibition of ornithine decarboxylase activity by polyamines was due to a decrease in the amount of enzyme protein or to some posttranslational inactivation mechanisms, we analyzed the accompanying changes in ornithine decarboxylase polypeptide by radioimmunoassay (Table 11). Importantly, the decay of ornithine decarboxylase activity was faster after addition of spermidine (Table 11) than after inhibition of general protein synthesis by cycloheximide (26). This agrees with results obtained with some other cell lines by using higher polyamine concentrations (18), but discrepant results also exist (24, 38). Table I1 shows that most of the loss of the enzyme activity by spermidine can be explained by the decrease in the amount of ornithine decarboxylase polypeptide. Yet the enzyme activity disappeared somewhat faster than the enzyme protein. In cells exposed to spermidine for 4 h, for example, the activity of ornithine decarboxylase had declined to 0.4% of the control, while 7.5% of the enzyme protein still remained. This suggests that about 8% of the activity was inhibited by post-translational modifications or interaction with inhibitors.
Synthesis of Ornithine Decarboxylase Polypeptide in Cells Exposed to Polyamines-To study the effect of polyamines on the synthesis of ornithine decarboxylase the cells were incubated for different time periods with and without ornithine or polyamines and labeled with [3sSS]methionine for 30 min. As the half-life of the enzyme protein after the addition of spermidine (the most effective agent) was of the order of 50-60 min (see Table 11), we reasoned that the rate of enzyme synthesis could be measured by using 30-min labeling time.
The results thus obtained were confirmed by using a shorter pulse time of 10 min (results not shown). The labeled ornithine decarboxylase was immunoprecipitated with monospecific antiserum and analyzed by SDS-gel electrophoresis. A prominent band of 51 kDa corresponding to ornithine decarboxylase can be seen in the control cells (Figs. 3 and 4). Exposure of the cells to 10 @M spermidine for 1-2 h resulted  I The activities of ornithine and adenosylmethwnine decarboxylases and polyamine content in cells exposed to different concentrations of ornithine The logarithmically growing cells were incubated with the indicated concentrations of ornithine for 2 h. The activities of ornithine and adenosylmethionine decarboxylases were determined in dialyzed cell extracts. The values are means of duplicate assays from two experiments.

TABLE I1
Effect of spermidine on the activity and polypeptide content of ornithine decarboxylase The cells (7-8 X 10') in optimal growth were cultured in the absence or presence of 10 p~ spermidine for the indicated times. The activity and polypeptide content of ornithine decarboxylase in the cells were determined as described under "Experimental Procedures." The values are means of duplicate samples. in a marked reduction in the intensity of the band indicating a diminished synthesis of ornithine decarboxylase (Fig. 3). Ornithine decarboxylase formation was also effectively inhibited by putrescine, either added exogenously or synthesized from ornithine (Fig. 4). It remains, however, to be elucidated to what extent the effect of putrescine was contributed by the spermidine synthesized from putrescine. Spermine appeared to be less effective than spermidine in depressing the enzyme synthesis (Fig. 4). Inhibition of the labeling of ornithine decarboxylase by polyamines was not due to an interference with the uptake of ::%% 6.6 -;

FIG. 5. Northern blot analysis of ornithine decarboxylase-mRNA after incubation with spermidine M) for different periods of time.
Polyadenylated RNA from cells was isolated and fractionated on 1% agarose/formaldehyde gels, transferred to nitrocellulose, and hybridized to nick-translated pODC16. X DNA cleaved with restriction enzyme Hind111 was used as the molecular size marker. The quantity of RNA was 14 pg in each specimen. Kb, kilobase. methionine into total proteins was detected (results not shown).
Ornithine Decarboxylase mRNA in the Spermidine-treated Cells-To find out whether the decrease in the synthesis of ornithine decarboxylase caused by polyamines was exerted at the level of transcription or translation, we analyzed the amount of ornithine decarboxylase mRNA by Northern blotting, using a specific cDNA clone as the probe (35). Consistent with earlier studies (26,35) two distinct species of mRNA for ornithine decarboxylase were detected (Fig. 5). No decrease in the amount of either mRNA species was found at least up to 4 h after addition of spermidine. In fact, the levels of mRNA were rather increased than decreased upon exposure of the cells to spermidine (Fig. 5).
Degradation of Ornithine Decarboxylase in the Polyaminetreated Cells-The rate of degradation of ornithine decarboxylase was investigated by prelabeling ornithine decarboxylase with [35S]methionine and following then the breakdown of the labeled enzyme by SDS-gel electrophoretic runs. Fig. 6 depicts the rate of degradation of ornithine decarboxylase in cells incubated without and with spermidine. It appears that disappearance of the 51-kDa band corresponding to ornithine decarboxylase was much faster in the cells exposed to 10 ~L M spermidine than in the control cells. Degradation of the enzyme was also markedly accelerated in the presence of 10 p~ spermine and millimolar concentrations of ornithine and putrescine (Fig. 7). In most experiments several faint bands of slightly smaller molecular weights were also detected in the polyamine-treated and control cells (Figs. 6 and 7). These could represent partial degradation products of ornithine decarboxylase. However, no constant shift of the label from the 51-kDa polypeptide to any smaller band was detected in several repeated experiments.
We also tested the possibility that ornithine decarboxylase might have been trapped to some subcellular organelle for degradation. For this the 100,000 x g particulate fraction of the labeled cells was suspended in ornithine decarboxylase homogenization buffer, adjusted to contain 1% Triton X-100 and 0.5% sodium deoxycholate. After homogenization by sonication the soluble extract was subjected to immunoprecipitation and gel electrophoresis. No evidence for ornithine decarboxylase or its degradation was obtained in the particulate extracts.
Role of Antizyme in the Regulation of Ornithine Decarboxylase by Polyamines-No free antizyme appeared to be induced in the spermidine-treated cells as measurements of ornithine decarboxylase activity in the mixture of the supernatants from these and the control cells gave the expected additive enzyme activity. Two techniques were then used to investigate the possible existence of antizyme-ornithine decarboxylase complex. First we attempted to measure the amount of the complex using the recently introduced assay method where difluoromethylornithine-inactivated ornithine decarboxylase is used to displace native ornithine decarboxylase competitively from the complex (18). Addition of the inactivated ornithine decarboxylase to control cell extracts caused no or less than 5% increase in the ornithine decarboxylase activity, indicating that no significant amounts of the enzyme-antizyme complex pre-existed in untreated cells. Spermidine treatment for 1-4 h caused little accumulation of the ornithine decarboxylase-antizyme complex. Maximal reactivation of ornithine decarboxylase was detected 2 h after spermidine addition. However, in three experiments no more than 5-10% of the total decrease in the ornithine decarboxylase activity could be accounted for by reversible inhibition by antizyme.
The second assay of the ornithine decarboxylase-antizyme complex was based on dissociation of the complex in the presence of high salt concentrations and recovery of the components on Sephadex G-75 gel filtration chromatography (39). In none of the four experiments with extracts from cells exposed to spermidine for 2-4 h could we recover any extra ornithine decarboxylase activity. In fact, only about 70-80% of the input activity was recovered. No firm evidence for the antizyme was obtained either. Yet in one chromatographic run (4-h spermidine sample) a clear peak of a small molecular weight inhibitor to ornithine decarboxylase was detected. In this case the amount of the putative antizyme was sufficient for inactivating 10% of the ornithine decarboxylase activity of the control cells.

DISCUSSION
Exogenously added polyamines are known to cause a rapid decay of the cellular ornithine decarboxylase activity, but the mechanisms of the enzyme inactivation are not well understood. A major problem in studying ornithine decarboxylase is the very low amounts of the enzyme present in normal cells. T o overcome this difficulty cell strains overproducing ornithine decarboxylase have been used in the experiments. It has to be born in mind, however, that enzyme regulation in these cells may not in all respects be similar to that in normal cells. Difluoromethylornithine-resistant cell lines with augmented ornithine decarboxylase production are available (8,29,41,42); but the difficulty with these cells is that the inhibitor invalidates the activity measurements, and upon withdrawal of the inhibitor ornithine decarboxylase is depressed as polyamines are synthesized. In distinction, our A2 cells overproducing ornithine decarboxylase were isolated without any inhibitors. These cells show maximal production of ornithine decarboxylase in the absence of inhibitors and are, therefore, a very good tool in studies of regulation of this enzyme. A further advantage is that these cells grow in serum-free medium. In the absence of serum polyamines the cells can be effectively depleted of all three polyamines (putrescine, spermidine, and spermine) (26). Conversely, a rapid and extensive increase in the polyamine concentration can be obtained by supplementation of the cultures with ornithine or polyamines. This minimizes the concern of the polyamines bound to cell organelles, not participating in the enzyme regulation. It also facilitates elucidation of the functions of each polyamine separately in the control of the enzymes. Nevertheless, we still face the problem how rapidly, and what proportion of the polyamines, newly synthesized or taken up, is sequestered into the inactive state. This makes it impossible to draw definitive conclusions of the relative efficacy of individual polyamines in the regulatory processes in uiuo. *E. Holtta and P. Pohjanpelto, unpublished results.
The present study shows that all the natural polyamines, whether added exogenously or formed endogenously, are capable of reducing the ornithine decarboxylase activity. It is thus most likely that polyamines exert intracellularly some type of feedback control of the enzyme activity and not a cell surface-associated "down regulation" as also suggested (16). Spermidine and spermine appeared to be more potent inhibitors of the ornithine decarboxylase activity than putrescine. However, putrescine was able to inhibit the activity of ornithine decarboxylase almost to the same degree as spermidine and spermine, provided its intracellular concentration was raised to the same levels as those of the higher polyamines. Yet it is notable that under most physiological conditions the levels of spermidine and spermine far exceed that of putrescine. The slight inhibition of the adenosylmethionine decarboxylase by high concentrations of putrescine (see Table II), an activator of the enzyme in uitro, may even be a sign of threatening "toxicity." I t is noteworthy that the short-lived proteins like adenosylmethionine decarboxylase and ornithine decarboxylase in particular are very sensitive indicators of possible toxicity. A dose-dependent decrease of the ornithine decarboxylase activity by endogenously synthesized putrescine has also been reported in rat hepatoma cells (19), supporting the notion that at least under certain physiological conditions putrescine may also play a role in the regulation of ornithine decarboxylase activity.
In A2 cells exposed to spermidine the amount of immunoreactive ornithine decarboxylase protein decreased concomitantly with the enzyme activity, although a t a somewhat slower rate. It thus appears that the regulation of the ornithine decarboxylase activity by natural polyamines in these cells occurs mainly through changes in the amount of enzyme protein. Similar regulation has been observed in rat liver after treatment with nonphysiological 1,3-diaminopropane (43). In our cells less than 10% of the total loss of enzyme activity could be attributed to post-translational modifications or modulation by inhibitors. Some evidence was obtained that reversible inactivation of ornithine decarboxylase by antizyme might explain this minor portion of activity loss. Interestingly, in yeast the decrease in the ornithine decarboxylase activity by polyamines is not accompanied by a reduction in the enzyme protein (44). The exact post-translational regulatory mechanisms involved in yeast are still unknown, but antizyme or covalent modification by polyamines do not seem to play any role (44). Inactivation of the enzyme by phosphorylation (13) and conversion to less active forms (45) in the presence of polyamines has been suggested to occur in other lower eukaryotes. On the other hand, in Neurospora crassa polyamines have recently been shown to act by reducing both the activity of ornithine decarboxylase and the quantity of the enzyme protein (46). However, the decrease in the immunoreactive ornithine decarboxylase protein was clearly less pronounced than the loss of enzyme activity in the presence of polyamines. These results are fairly comparable to the ones presented in this paper using higher eukaryotic CHO cells.
In mammalian cells indirect evidence has been obtained that putrescine and spermidine reduce ornithine decarboxylase activity by selectively inhibiting translation (25). On the other hand, administration of the nonphysiological 1,3-diaminopropane in vivo has recently been shown to interfere in mouse kidney with general protein synthesis without causing any selective decline in ornithine decarboxylase synthesis (11). The present study again provides direct proof that putrescine, spermidine, and spermine cause a selective reduction in the synthesis of ornithine decarboxylase. Notably, the concentrations of ornithine and polyamines used in our ex-periments rather increased than decreased overall protein synthesis and cellular growth.
The observed decrease in the rate of synthesis of ornithine decarboxylase by polyamines in the A2 cells was apparently due to inhibition of translation of the enzyme message, as the amount of the two mRNAs for ornithine decarboxylase was not affected. Nucleotide sequencing of the major ornithine decarboxylase mRNA in mouse lymphoma cells (47, 48) has shown that the message has an unusually long untranslated segment at the 5'-end (48). Considering that modifications of the long 5"noncoding regions are known to affect translational efficiency (49, 50), it is tempting to speculate that polyamines might cause conformational changes, e.g. a loop formation, in the leader sequence and thus inhibit translation of the message.
Besides reducing the synthesis of ornithine decarboxylase polyamines were also found to accelerate the rate of degradation of the enzyme protein. Treatment of the HTC cells with putrescine has likewise been reported to enhance the decay of ornithine decarboxylase protein, as measured indirectly by titrating the amounts of monospecific antibody needed to reduce the ornithine decarboxylase activity to half (18). However, the latter experiments were carried out using 10 mM putrescine, i.e. a 1000-fold higher concentration than what we used, making it difficult to deduce the physiological relevance of the results. The nonphysiological diamine, 1,3diaminopropane is also known to enhance the degradation of ornithine decarboxylase in mouse kidney (11). One should, however, avoid drawing unifying conclusions of the degradation systems in these cases. In HTC cells exposed to high concentrations of putrescine the degradation of ornithine decarboxylase has been suggested to be mediated by antizyme (51). In A2 cells, exposed to low concentrations of spermidine, we found only little, if any, evidence for the formation of ornithine decarboxylase-antizyme complex as tested by the method of Murakami et al. (18). Radioimmunological titrations of ornithine decarboxylase in the 1,3-diaminopropanetreated mouse kidney have not revealed significant amounts of the enzyme-antizyme complex either (11). These results cannot, however, rule out the importance of antizyme in the regulation of ornithine decarboxylase degradation, considering the possibility that the antizyme-enzyme complex may be degraded very fast in these cells. The possibility of sequestration of the complex into subcellular organelles for degradation did not look likely, since no ornithine decarboxylase or its degradation products were found in the particulate fraction of our cells. It remains to be elucidated whether various cells differ in the induction of the antizyme or in the turnover rate of the ornithine decarboxylase-antizyme complex.
In conclusion, the loss of ornithine decarboxylase activity in the A2 cells in response to polyamine additions appears to be mostly due to a translational inhibition of the enzyme synthesis and to an enhanced enzyme degradation by an undefined mechanism. The involvement of antizyme in the degradation process of ornithine decarboxylase could neither be clearly shown nor excluded. In any case, antizyme does not seem to cause any substantial reversible inactivation of ornithine decarboxylase in the A2 cells. Significantly, all the polyamines appeared to act similarly in these cells by inhibiting ornithine decarboxylase synthesis and increasing its degradation. This contrasts the recent findings obtained with the lower eukaryote N . crassa (46). In that organism putrescine and spermidine have been suggested to play different roles in ornithine decarboxylase regulation, spermidine inhibiting the enzyme formation and putrescine promoting inactivation of the enzyme. Whether this discordance is due to the