Control of Ornithine Decarboxylase in Chinese Hamster Ovary Cells ...

VOl. 261, No . 20, Issue of July

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc

pp. 9502-9508,1986 Printed in U.S.A.

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* (Received for publication, November 25, 1985)

Erkki Holttaz From the Department of Biochemistry, University of Helsinki, Unioninkatu 35, SF-00170 Helsinki, Finland

Pirkko Pohjanpelto From the Department of Virology, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki, Finland

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 theactivity 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 both for 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 couldbe ascribed to post-translational modifications or inhibitor interaction. Some evidence for inactivation throughornithine decarboxylase-antizyme complex formation was obtained. Gel electrophoretic determinations of the [35S]methionine-labeledornithine 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 translationof the message.

enzyme in the pathway, adenosylmethionine decarboxylase (EC 4.1.1.50) plays again a keyrolein 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 (5-11). 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 bothtranscriptional (23) andpost-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 (16-18). 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 The naturalpolyamines putrescine, spermidine, and sperm- important mechanisms of regulation of ornithine decarboxine play an important role in the control of cellular growth ylase by polyamines in the Chinese hamster ovary (CHO’) (1-4). The first and rate-controlling step in the polyamine cell line A2. The ability of these cells to grow in serum-free biosynthesis, conversion of ornithine to putrescine, is cata- medium and their marked overproduction of ornithine decarlyzed by ornithine decarboxylase (EC 4.1.1.17), which is pres- boxylase (26) make this cell line very well suited for the ent in very small amounts in mammalian cells. The second studies. In addition, the recently devised techniques for measuring the enzyme protein (9,lO) andits mRNA (27-29) have * This research was supported by grants from the Sigrid Juselius made it possible to get more direct information of the reguFoundation, the Cancer Research Fund, and the Medical Research latory mechanisms. Our results show that two factors are Council of the Academy of Finland and by Grants HD-13541 and 1 R01 CA 37695 from the National Institutes of Health. The costs of mainly responsible for the inhibition of ornithine decarboxpublication of this article were defrayed in part by the payment of ylase activity by polyamines: 1)a decrease in the synthesis of page charges. This article must therefore be hereby marked “advertisement” in’accordancewith 18U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed.

’ The abbreviations used are: CHO, Chinese hamster ovary; MEM, minimal essential medium; SDS, sodium dodecyl sulfate; dansyl, 5dimethylaminonaphthalene-l-sulfonyl.

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Regulation of Ornithine Decarboxylase by Polyamines 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). EXPERIMENTAL PROCEDURES

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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 AdenosylmeChemi~ak;-DL-[l-*~~C]ornithine (58 mCi/mmol) was purchased thionine Decarboxylases by Polyamines-Polyamines added to from Amersham International, U.K. and ~~-[5-~H]cu-difluoromethylornithine (26.5 Ci/mmol) from New England Nuclear. Putrescine the cultureof the A2 cells at a concentration of 10 PM caused was obtained from Sigma. Spermidine and spermine were from Cal- a rapid decay of the ornithinedecarboxylase activity (Fig. 1A) biochem. L-Ornithine was purchased from Fluka AG, Buchs, Switzer- and a somewhat less extensive decrease in the activity of land, and purified as previously described (31). adenosylmethionine decarboxylase(Fig. 1B). Undegraded Cell Culture-We have previously isolated from CHO cells an amines were evidently responsible for the effect, because no arginase-deficient cell line A7, which grows in serum-free medium medium. (31, 32). From these cells we subsequently obtained a mutant cell serum amineoxidases were present in the serum-free line, designated A2, that overproduces ornithine decarboxylase (26). Spermidine was more effectivethan spermine and putrescine In the current experiments a cloned strain of A2 was used. The cells in reducing the activities of both enzymes, although in the were cultured on collagen-coated Petri dishes in a 1:l mixture of case of adenosylmethionine decarboxylase there was not a minimal essential medium (MEM) and nutrient mixture F12 (without great difference betweenthe actionsof spermidine and spermputrescine) supplemented with 0.1% bovine serum albumin. of the polyamines was Determination of Enzyme Activities-The activities of ornithine ine. T o differentiate whether each effective itself or only after metabolic conversions we measand adenosylmethionine decarboxylases were measured as previously described (31), except that most of the ornithinedecarboxylase assays ured the cellular polyamine contents at different times after were now carried out with saturating ornithine concentrations (0.4 exogenous addition of the amines (Fig. 2). At the time of mM). observing the decrease in the enzyme activities a considerable Radioimmunoassay of Ornithine Decarboxyluse-Ornithine decar- portion of the putrescine taken up by the cells had already boxylase concentration was measured essentially as described by been converted to spermidine (Fig. U ) ,whereasrelatively Seely and Pegg (10) with antiserum raised in rabbits against the little of the spermidine (Fig. 2 B ) and practically none of the enzyme protein purified to homogeneity from mouse kidney (9). f5S]Methwnine Labeling and Immunoprecipitation of Ornithine spermine (Fig. 2C) takenup was converted totheother Decarboxyluse-The rate of ornithine decarboxylase synthesis in the polyamines. The results demonstrate that both spermidine absence or presence of polyamines was determined in the following and spermine alone are capable of reducing the enzyme activway. After preincubating the cells for different times in Fl2/MEM ities, while the role of putrescine is questionable. T o examine medium without or with polyamines, the medium was replaced by further theefficacy of endogenously formed polyaminesin the MEM, without or with polyamines, but lacking cold methionine. After incubation for 30 min [35S]methionine (80-150 pCi/ml) was control of the activityof the two decarboxylases the cells were added, and the incubation was continued for another 30 min. Incor- cultured in the presence of different concentrations of orniporation was stopped by washing the cells twice with MEM containing thine. Table I shows that an exposure of the cells to 1 mM cold methionine. In the degradation studies of ornithine decarboxyl- ornithine for 2 h reduced the activity of ornithine decarboxase the enzyme was prelabeled with [35S]methionineby incubating ylase to half of the control, whereas 0.1 mM ornithine was the cells in the methionine-free MEM supplemented with [=S]me- without any effect. Notably, the accumulation of putrescine thionine (80 pCi/ml) for 2 h. After washing the cells twice with MEM, of 1 mM ornithine than 0.1 was much greater in the presence the incubation was continued in methionine-containing F12IMEM medium without or with polyamines for different time periods. To mM ornithine, while no marked differences in the patternsof were found when measprepare cell extracts for immunoprecipitation, cells were homogenized formation of spermidine and spermine by sonication in 50 mM Tris-HC1 buffer, pH 7.4, containing 150 mM ured 1or 2 hafter additionof either concentrationof ornithine NaC1, 5 mM EDTA, 0.5% Nonidet P-40,and 2 mM methionine. (Table 1). That even less spermine was accumulated in the Homogenates were centrifuged a t 100,000 X g for 30 min, and the presence of the higher ornithine concentration is explainable supernatants were used for immunoprecipitation with monospecific by the fact that spermine synthase is inhibited by high puornithine decarboxylase antiserum or with normal rabbit serum (27). itself The immunoprecipitates were then subjected to SDS-polyacrylamide trescine levels (40).According to these results putrescine gel (8%) electrophoresis following the standard procedures, and the can also cause an inhibition of the ornithine decarboxylase activity. However, relatively high concentrations of putresgels were autoradiographed (4). Northern Blot Analysis of Ornithine Decarboxylase mRNA-Total cine, notnormallyencounteredinthe cells, appear to be cellular RNA was isolated by the lithium chloride/urea method (33). required for the inhibition. In the presence of 0.1 mM orniNorthern blot hybridization analysis was performed by the method thine the inhibition of ornithine decarboxylase became eviof Thomas (34), as previously detailed (26). Nick-translated plasmid dent only after 4 h, being most probably due to theaccumupODC16 (35) was used as thehybridization probe. Analysis of Polyamines-Cellular polyamines extracted with per- lations of spermidine and spermine (results not shown). The chloric acid were first dansylated, then separated by thin-layer chro- activity of adenosylmethionine decarboxylase was rather inmatography and analyzed as previously described (36). sensitive to putrescine (Table I). Ornithine DecarboxyluseAntizyme Assays-Ornithine decarboxylChanges in OrnithineDecarboxylase Polypeptide after Sperase antizyme was assayed both by the procedure developed by Heller midine Addition-To find out whether the inhibition of oret al. (37) with minor modifications (38,39) andby the newly devised nithine decarboxylase activity by polyamines was due to a method of Murakami et at. (18). In the first technique ornithine decarboxylase-antizyme complex was dissociated by adding 0.5 M decrease in the amount of enzyme protein or to some postNaCl to 0.5mlof the cytosols prepared from 8 X lo7 cells. The translational inactivation mechanisms, we analyzed the accomponents were separated on a Sephadex G-75 column (1.6 x 36 companying changes in ornithine decarboxylase polypeptide cm) equilibrated with 10 mM sodium phosphate buffer, pH 7.0, by radioimmunoassay (Table 11). Importantly, the decay of containing 0.5 M NaCl, 0.1 mM EDTA, and 0.02% Nonidet P-40. ornithine decarboxylase activity was faster after addition of Fractions of 1ml were collected, and 50 p1 was used for each assay of spermidine (Table 11) than after inhibitionof general protein enzyme and inhibitor. In thesecond technique increasing amounts of difluoromethylornithine-inactivated ornithine decarboxylase were synthesis by cycloheximide(26). This agreeswith results obtained with some other cell lines by using higher polyamine used to release active enzyme competitively from the ornithine decarboxylase-antizyme complex. Inactive ornithine decarboxylase was concentrations (18),but discrepant results also exist (24, 38). prepared by incubating the enzyme from the same ornithine decar- Table I1 shows that most of the loss of the enzyme activity

Regulation of Ornithine Decarboxylase by Polyamines

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I

1 2 TIME AFTER POLYAMINE ADDITIONS ( h )

I

3

L

FIG. 1. Effect of polyamines on the activities of ornithine and adenosylmethionine decarboxylases. The A2 cells (6 X lo6)in log phase of growth in polyamine-free F12/MEM medium were incubated in the absence M putrescine (O), spermidine (A), or spermine (0) for the indicated times. A , ornithine or presence of decarboxylase activity; B , adenosylmethionine decarboxylase activity. The values are expressed as percentages of the respective control activities and are means k S.D. for two to threeexperiments.

i"

A

1

1 2 3 4 2 3 4 TIME AFTER POLYAMINE ADDITIONS

FIG. 2. Polyamine levels in A2 cells after addition of polyamines. Polyamines were analyzed from the same cell cultures as used in Fig. 1. Putrescine (O), spermidine (A), and spermine (0)levels after additions of M putrescine ( A ) ,spermidine ( B ) ,and spermine (C). The cultures were washed twice with MEM before determining cellular polyamines. The values are means of two to three experiments.

by spermidine can be explained by the decrease in the amount of ornithine decarboxylase polypeptide. Yet the enzyme activity disappeared somewhat faster than theenzyme 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 stillremained. This suggests that about 8% of the activity was inhibited by post-translational modifications or interaction with inhibitors. Synthesis of Ornithine Decarboxylase Polypeptide i n 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 for30 min. As the half-life of the enzyme protein after the addition of spermidine (the most effective agent) was of the order of 5060 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

Regulation of Ornithine Decarboxylase by TABLE I The activities of ornithine and adenosylmethwnine decarboxylases and polyamine content in cells exposed todifferent 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. Ornithine addition

Ornithine decarboxylase

Putre'cine

100

0 0.1 1.0

'permidine

Spermine