Effects of external osmolality on polyamine metabolism in HeLa cells

263

Biochimica et Biophysica Acta, 411 (1975) 263--281

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27781 EFFECTS OF E X T E R N A L OSMOLALITY ON POLYAMINE METABOLISM IN HeLa CELLS

GEORGE F. MUNRO, RUTH A. MILLER, CAROL A. BELL and EVIE L. VERDERBER Division of Molecular and Cellular Biology, National Jewish Hospital and Research Center, Denver, Colo. 8 0206 and Department of Biophysics and Genetics, University o f Colorado Medical Center, Denver, Colo. 8 0220 (U.S.A.)

(Received June 23, 1975)

Summary The polyamine content of E s c h e r i c h i a coli is inversely related to the osmolality o f the growth medium. The experiments described here demonstrate that a similar p h e n o m e n o n occurs in mammalian cells. When grown in media of low NaC1 concentration, HeLa cells and h u m a n fibroblasts were found to contain high levels of putrescine, spermidine, and spermine. The putrescine content of HeLa cells was a function of the osmolality of the medium, as shown by growing cells in media containing mannitol or additional glucose. External osmolality per se had no effect on the contents of spermidine and spermine. For all media, the total cellular polyamine content could be correlated with the activity o f ornithine decarboxylase, the first enzyme in polyamine biosynthesis. Different levels o f enzyme activity appear to result solely from variations in the rate o f enzyme degradation. A sudden increase in NaC1 concentration produced rapid loss of ornithine decarboxylase activity and a gradual loss of putrescine and spermidine. A sudden decrease in NaC1 concentration led to rapid and substantial increases in ornithine decarboxylase activity and putrescine.

Introduction The polyamines occur ubiquitously in nature [ 1 - 6 ] . In mammalian cells the biosynthetic sequence involves an initial decarboxylation of ornithine to form putrescine (1,4-diaminobutane). Spermidine is formed by the union of this diamine with a propylamine m o i e t y donated by 5'
264 levels of putrescine similar to those of spermidine and spermine [7 ]. In contrast with mammalian cells, Escherichia coli contains large amounts of putrescine and spermidine, b u t no spermine. In a variety of organisms, the polyamines have been implicated in many aspects of cell metabolism including the synthesis of RNA and the maintenance of the structure and function of ribosomes and membranes [1--4,8,9 ]. Several years ago we reported that the putrescine c o n t e n t of E. coli was substantially elevated if the cells were grown in media of low osmolality [10]. The spermidine content was unchanged. E. coli was then shown to require a high internal pool of putrescine both for rapid growth [11] and for normal phospholipid turnover [8,9 ]. A sudden increase in external osmolality produced rapid loss of putrescine to the medium [10]. After a sudden reduction in external osmolality, the putrescine pool was slowly replaced, not reaching the new steady-state level for several generations [10]. Recently, the generality of these phenomena has been questioned since external osmolality was shown to have no effect on the spermidine content of Bacillus megaterium [12 ]. Since this species lacks putrescine, a possible role for putrescine in osmotic adaptation has remained moot. We decided to examine the effects of osmotic adaptation on polyamine levels in mammalian cells. Mammalian cells in culture may be successfully adapted to media of different ionic and osmotic composition [13--18], and many of these cell lines contain readily measurable quantities of putrescine, in addition to spermidine and spermine. Sudden shifts in composition o f the medium have been found to provoke marked changes in the metabolism of the polyamines, and many of these changes may be understood in terms of the regulation o f omithine decarboxylase (L-omithine carboxy-lyase, EC 4.1.1.17), the first enzyme in the polyamine biosynthetic pathway. Materials and Methods Cells and media The standard medium used for growth of HeLa cells is Eagle's minimal essential medium [19 ], modified to contain 10 times the original level of phosphate and with 0.81 mM MgSO4 substituted for 1 mM MgC12. CaC12 was omitted so that spinner and monolayer cultures could be grown in the same medium. NaC1 was omitted from the basic formulation and then added back to the desired level. The standard mixtures of vitamins and amino acids for this medium, 100 units/ml penicillin, 100 pg/ml streptomycin, and 10% calf serum were present (all from Grand Island Biological Co.). For indicated experiments human serum, obtained from the first author (G.F.M.), was sterilized b y filtration and substituted for the calf serum. Four lines of human fibroblasts were from the collection of S.I. Goodman; lines 1033 and 1034, from patients with cystic fibrosis; line 229 from a person heterozygous for methylmalonicaciduria and homocysteinuria; line 264 from a heterozygote for maple syrup urine disease. One supposedly normal line was isolated from one of us (G.F.M.). Another normal line (CRL 1147) and three lines derived from patients with c y s t i c f i b r o s i s ( C R L 1 1 3 4 , C R L 1 1 4 3 , CRL 1154) were obtained from the American T y p e Culture Collection. All cells were

265 maintained in Falcon plastic flasks in a National Appliance incubator and continuously gassed with 5% CO2. The medium used for fibroblasts was Eagle's minimal essential medium with the original level of phosphate and CaC12, b u t with MgSO4 substituted for MgC12 (vide supra). This medium was fortified with essential and non-essential amino acids, penicillin, streptomycin, and 1 pg/ml Fungizone. Calf and fetal calf sera were heated (56°C for 30 min) before addition to the medium (5% each b y vol.). The osmolality measurements were made with a vapor pressure o s m o m e t e r (model 5120, Wescor, Inc.), and K ÷ and Na ÷ concentrations were determined using a flame p h o t o m e t e r (model 343, Instrumentation Laboratory, Inc.). Photographs o f cells were taken with a Wild M40 inverted phase microscope.

Polyamine analysis Cells for polyamine analysis were grown in the absence of phenol red, since this indicator interferes with the estimation of putrescine, and harvested in early to mid-logarithmic phase o f the growth cycle. Approx. 5 • 106--10 • 106 HeLa cells from spinner cultures were centrifuged at 4000 X g and 4°C (HB-4 rotor, Sorvall RC2-B centrifuge) and washed twice b y resuspension in phosphatebuffered saline (30 mM NaC1, 2.7 mM KC1, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.4}, adjusted to iso-osmolality by the addition of extra NaC1 equal to that of the medium in which the cells had been maintained. Phosphatebuffered saline for cultures with added glucose or mannitol were made by adding sufficient extra NaC1 to compensate for the NaC1 and sugar levels in the media. After centrifugation, cell pellets were rapidly frozen in solid CO 2/acetone. Over the range o f at least +30 mM NaC1, the osmolality of the rinsing solution made no difference in the recovery of polyamines. Similar yields were obtained using temperatures of 4 and 25°C. Cells from monolayer cultures were washed in situ with phosphatebuffered saline of the proper tonicity and then scraped from the flasks into 0.5 M HC104 for immediate analysis. Cell pellets were extracted twice in 0.5 M HC104. The supernatants were neutralized with 1.0 M KOH, centrifuged at 5000 X g for 10 min, and hydrolyzed (6 M HC1, 120°C for 12 h, ref. 20). The resultant solutions were evaporated to dryness on watchglasses and then taken up in 0.2 M HC104 for dansylation and estimation by thin-layer chromatography [21] as previously described [22]. In the absence of acid hydrolysis, recovery is reduced by 30--40%. Hydrolysis may enhance recovery b y destruction of substances which interfere with the dansylation reaction, b y disruption of non-specific complexes with other tissue components, or b y release of covalently b o u n d polyamines. The individual polyamines and contaminants were separated using either solvent I (ethyl acetate/cyclohexane (2 : 3, v/v) or solvent II (ethyl acetate/ cyclohexane (1 : 1, v/v). Solvent I was utilized for determination of putrescine (and spermidine) since putrescine has an RF in this system much less than that of dansylated ammonia; solvent II was used for spermine since spermine and histamine have different RF values in this solvent system. For measurement o f extracellular polyamines, media samples were mixed with equal volumes of 1 M HC104, centrifuged, neutralized with KOH, centri-

266 fuged again, and extracted into alkaline butanol and dansylated as described above. Acid hydrolysis must be omitted since it releases enormous quantities of ammonia, probably from glutamine. As shown by recovery of standards added to fresh media, the various c o m p o n e n t s of the medium did not interfere with the determination of polyamines. Polyamines in the cell extracts for ornithine decarboxylase assay were measured b y making the extracts 0.2 M in HC104, removing precipitated protein, and then carrying o u t dansylation of the supernatants. Polyamines were separated in solvent II only; the amounts of ammonia are small, and in solvent I, another substance migrates with the same RF as putrescine. The components of the assay mixture did not interfere with the polyamine determinations, as shown by recovery of standards added to the assay mixture.

Ornithine decarboxylase Cells for these assays were always trypsinized and diluted into fresh medium the day before the experiment to insure rapid growth on the day of the experiment. Flasks containing a b o u t 106 cells were washed twice at 25°C with phosphate-buffered saline of the appropriate tonicity (cf. polyamine analysis) and then suspended with a rubber policeman in 1.0 ml of assay mixture (0.1 mM [ethylenedinitrilo]tetraacetic acid, 0.05 mM pyridoxal-5'-phosphate, 5 m M dithiothreitol, 50 mM Tris, pH 7.1, ref. 23) and rapidly frozen. At the time of analysis, the cell preparations were thawed and centrifuged at 17 400 × g for 10 min. Samples of 0.05--0.2 ml were then placed in 14 × 100 mm centrifuge tubes; the volume was increased to 0.5 ml with additional assay mixture. After a 10-min preincubation, DL-[1-~4C]ornithine was added (43.0 Ci/mol as supplied b y New England Nuclear; diluted with non-radioactive L-ornithine to 3.22 Ci/mol L-[1-14C] ornithine; final level of L-ornithine in the assay, 200 pM). The tubes were then capped with stoppers from which were suspended plastic wells (Kontes Glass Co.) containing 0.2 ml N.C.S. tissue solubilizer (Nuclear Chicago). After 1 h at 37°C, 0.25 ml of 2 M citric acid were injected through the stopper. After an additional 20 min, the wells were placed in 10 ml of Liquifluor scintillation fluid (New England Nuclear), and radioactivity was determined (74% counting efficiency, Beckman LS-100C counter). The results in cpm were corrected for blank incubations (no enzyme added), converted to pmol of CO2 liberated, and normalized to the a m o u n t of soluble protein present in the extract [24]. The temperature or precise tonicity of the wash solution was not critical; variations of +60 mM NaC1 had no effect on recovered activity. The assay mixture has been modified from that originally described [23] by a 5-fold reduction in the level of dithiothreitol. This does not affect enzyme recovery b u t considerably lowers the blank reading for the protein determination. As alternatives to freeze-thawing, cells may be broken by sonication, by homogenization (Dounce instrument), or by suspending cells in assay mixture and vortexing with 10% (by vol.) butanol, 1% toluene or 0.5% Nonidet P-40 (Shell Chemical Co.). All these means produce equivalent yields of enzyme activity. Frozen cell extracts may be stored at --20 or --70°C for a week with no loss of enzyme activity. Reactions were proportional to time and the amount of extract present. Liberation of radioactive CO2 was not affected by the presence of

267

0.01 M malonate or 0.06 M glutamic acid [25], confirming the presumption that radioactive CO2 did not arise from conversion of ornithine to glutamate with subsequent oxidation via the Krebs cycle. Equivalent blanks could be constructed using no added enzyme, boiled enzyme or by carrying o u t the reaction at 4°C.

Measurement o f cellular DNA, R N A and protein Cells from suspension cultures were centrifuged at 800 X g and 4°C and washed twice b y resuspension in phosphate-buffered saline of the appropriate tonicity. Cells from monolayer cultures were trypsinized into phosphatebuffered saline and treated as above. Cell pellets were heated in 0.5 M HC104 (90°C for 15 min). After centrifugation (5000 X g for 10 min), the supernatants were removed for analysis of DNA b y the Burton procedure [26] and R N A b y the orcinol reaction of Mejbaum [27], using standards of 2-deoxy-D-ribose and D-ribose (Sigma Chemical Co.). As shown b y appropriate mixtures of DNA and RNA standards (type V calf t h y m u s DNA; t y p e XI yeast RNA, Sigma Chemical Co.), cellular DNA was found to alter the estimate of R N A by only 2%; this correction was ignored. Protein was determined [24] in the HC104 precipitates after they were dissolved in 0.1 M NaOH. Incorporation o f precursors into R N A and protein Monolayer cultures on 16 mM Falcon plastic dishes received 2 ml of the appropriate media containing either 1 pCi/ml [ 5 -3 H] uridine (26 Ci/mmol, New England Nuclear Corp.) or a-[G-3H]leucine (81.3 Ci/mmol, same supplier). At various times thereafter, the medium was removed and cells were overlaid with 3 ml o f 5% trichloroacetic acid. Precipitates were suspended in the acid, heated (90°C for 10 min), collected on filters (type HA, 0.45 pm, Millipore Corp.), and washed three times with 5 ml of acid. After drying the filters, radioactivity was determined (Beckman LS-100 C counter) using Liquifluor scintillation fluid (New England Nuclear). Results

Composition o f media In the absence of any added NaC1, the medium used in these experiments is approx. 140 mosM and contains 65 mM Na ÷ and 5.9 mM K +. Obvious major sources of the endogenous Na ÷ are NaHCO3, amino acids, and serum. For simplicity, media will be identified b y the a m o u n t of NaC1 added, e.g. "50 m e d i u m " is the basal formulation with 50 mM added NaC1. Cultures containing mannitol or high levels of glucose will be specifically identified; the osmolalities of these media may be calculated as the sum of the osmolality of the basal media and the molarity of the sugar added. Media conditioned b y exposure to cells for 2--3 days were equivalent to fresh media in terms of osmolality and Na ÷ and K + concentrations. Morphology and composition o f HeLa cells HeLa cells shown in Fig. 1A are representative o f cells in media containing

F i g . 1. M o r p h o l o g y o f H e L a c e l l s i n m e d i a o f d i f f e r e n t NaC1 c o n c e n t r a t i o n s . H e L a cells w e r e m a i n t a i n e d for at least 10 generations either in 60 medium (Part A) or in 110 medium (part B). The insert in each f i g u r e is 2 0 p m i n l e n g t h . C u l t u r e s w e r e n e v e r t a k e n f o r c h e m i c ' d , e n z y m e , o r p o l y a m i n e a n a l y s i s a t cell d e n s i t i e s g r e a t e r t h a n s h o w n in p a r t B.

269 40--80 mM added NaC1. Cells in part B are characteristic of cells in media containing from 100 to 140 mM added NaCI. Cells in media o f lower NaC1 concentration are more rounded, whereas cells in media with more NaC1 are generally polygonal in shape. Cells in 50 medium with 100 mM added glucose or mannitol were similar in appearance to cells in 50 medium, with the exception that glucose-grown cells were slightly plumper. For monolayer cultures, the doubling times were about 30 h for all media with osmolalities up to 300 mosM (e.g. 110 medium), b u t were increased to about 50 h in 140 medium. In contrast, spinner cultures grew with generation times of 24--30 h in both 80 and 140 media. All cultures studied were found to have similar contents of DNA (2.8 pg of deoxyribose/106 cells, based on 21 determinations). Cells in 80 and 140 media also contained similar amounts of total protein and RNA (Table I), regardless of whether they were maintained in suspension or as monolayers, in either calf or h u m a n serum. Cells grown with h u m a n serum were, however, generally smaller than cells grown with calf serum. The presence of 120 mM mannitol had no effect on cell composition. Cells maintained in 120 mM glucose contained large amounts of both RNA and protein. In summary, NaC1 has a slight effect on cell morphology b u t no obvious effect on RNA or protein c o n t e n t or on growth rate. Mannitol has no effect on composition, morphology, or growth rate. Cells maintained with excess glucose are larger than normal but grow at normal rates. One may easily transfer HeLa cells from any of these media to any other; the D NA, RNA and protein contents of the cultures are seen to increase within hours of the change, and incorporation of radioactive leucine or uridine into trichloroacetic acid precipitates continues without interruption, at least for osmotic differences less than 100 mosM.

Polyamine contents o f HeLa cells and human fibroblasts As shown in Table I, cells in 80 medium contain considerably more putrescine than cells either in 140 medium or 80 medium with added glucose or mannitol. Thus, the putrescine c o n t e n t of HeLa cells is dependent on the osmolality of the medium. Cells grown with 140 mM NaC1 also contain less spermidine and spermine; hence, the total polyamine c o n t e n t is reduced. High levels of NaC1 had similar effects on cells maintained with h u m a n serum (Table I) or as monolayers (Fig. 2). For cells grown with 120 mM glucose or mannitol, the spermidine, spermine, and total polyamine contents were not changed (Table I). Thus the spermidine and spermine contents appear to be dependent on NaC1 concentration but n o t on the osmolality of the medium. The effects of NaC1 could conceivably be specific for HeLa cells or malignant cells in general. Therefore, nine different primary cultures of h u m a n fibroblasts were grown in 50 and 100 fibroblast media (Table II); cultures in 50 medium contained significantly more putrescine and spermidine than cultures in 100 medium. It is possible t h a t the variations in intracellular polyamine levels result from loss of these compounds to the medium. Subsequent oxidation might then occur since oxidases with the proper specificities are present in bovine sera [1,4,5]. However, we have not been able to detect polyamines in either

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271 TABLE

II

POLYAMINE

CONTENTS

OF HUMAN

FIBROBLASTS

Sources of cell lines are described under Materials and Methods. Results were normalized to protein content since in these studies the cell n u m b e r was insufficient for estimation of D N A . In other experiments the p r o t e i n / D N A ratio was determined and found to be similar for several of the cell lines.Results are given as the m e a n +-S.E. of the m e a n for nine cell lines and were analyzed statistically by a repeated-measures design [50]. There were no statistically significant differences in polyamine contents b e t w e e n any of the cell lines.

A d d e d NaCI (raM)

Polyamine content (pmol/~g cell protein)

50 100

Putrescine*

Spermidine* *

Spermine* * *

3.65 +- 0.43 1.71 -+ 0.26

2.49 +- 0.35 1.56 -+ 0.063

1.09 + 0.26 0.983 -+ 0.098

*P < 0 . 0 0 5 . **P < 0 . 0 5 . * * * n o t significant.

fresh or conditioned tissue culture media and estimate that their concentrations are less than 2 pM. Also, oxidases are not present in human sera, and y e t polyamine levels are dependent on external NaCl concentration in media containing h u m a n sera, just as they are in media containing calf serum and the oxidases (Table I).

Ornithine decarboxylase in HeLa cells The very high polyamine contents of cells in media of low NaC1 c o n t e n t could result from an increase in the activity of ornithine decarboxylase. This

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Fig. 2. Effect of added N a C I on polyamines of H e L a cells. All m e d i a contained 1 0 % calf serum. T h e m e a n values for 80 m e d i a are derived from 11 separate assays; the m e a n values for 1 4 0 m e d i a are based o n 20 assays. Putrescine and spermidine contents of cells in 80 and 1 4 0 media were significantly different (P < 0.001 b y t-test),as were the spermine contents in these m e d i a (P < 0.01). Data for other m e d i a are based on two determinations, o o, p u t r e s c i n e ; ~ ~, s p e r m i d i n e ; o o spermine. Fig. 3. Effect of the osmolality of the m e d i u m o n ornithine decarboxylase. H e L a cells were g r o w n as monolayers in m e d i a containing 1 0 % calf serum and the N a C l contents shown. Cultures in 50 m e d i u m with 100 m M mannitol (z~)or 1 0 0 m M glucose (D) axe included; these m e d i a axe osmotically equivalent to 100 medium.

272 enzyme is the first enzyme in polyamine biosynthesis and is thought to control the rate of polyamine production [5,6]. Ornithine decarboxylase is notable for two unusual properties: First, it has the most rapid rate of degradation of any mammalian enzyme thus far examined [28--30]. After addition of inhibitors of protein synthesis, enzyme activity declines with a half-life of 10--60 min. This p h e n o m e n o n has been repeated in a variety of systems, both in vivo and in culture. Second, addition of fresh medium induces a many-fold increase in enzyme activity, a result of an increased rate of enzyme synthesis which may or may not be coupled with a reduction in the rate of enzyme degradation [31,32]. For the studies to be described here, cells were always growing rapidly at the initiation of each experiment. Cells adapted to media of low NaC1 content were found to contain elevated levels of ornithine decarboxylase activity (Fig. 3). The observed levels are consistent with the levels of putrescine and spermidine in the cells (Table I, Fig. 2), implying that alterations in the level of ornithine decarboxylase could be responsible for the observed differences in polyamine content of these cultures. Enzyme activities of cultures containing mannitol are similar to the activities of cells in 50 medium, in agreement with the similar total polyamine contents of these two cultures. The enzyme levels for cells in high glucose medium are lower than would be predicted from the total polyamine content per cell, probably because the enzyme levels are normalized to soluble cell protein and cells grown on high glucose medium contain more protein per cell than do the other cell types studied. Several experiments were carried out in an a t t e m p t to define the mechanism of enzyme alteration. Mixtures of enzyme extracts from cells in 50 and 100 medium possessed the activity predicted from the separate activities of the two extracts, thus ruling out the presence of soluble activators or inhibitors. It is possible that the elevated ornithine decarboxylase activity of cells in 50 medium arises from " i n d u c t i o n " of a novel enzyme; however, the following lines of evidence are inconsistent with that possibility: First, half-lives for heat inactivation were similar for enzyme extracts from cells in 50 and 100 medium (60 min at 54°C and 10 min at 60°C). Second, increasing concentrations of KCI were equally inhibitory for the two types of preparations, with 0.2 M KCI in the assay producing an approx. 50% reduction in the activity. Third, the Km for ornithine and the K i for putrescine, a competitive inhibitor of the enzyme [33], were similar for the two enzyme preparations: Km (ornithine), 46 and 56 /aM, Ki (putrescine), 450 and 440 pM, respectively. These values for the Km are slightly lower than a previously reported value of 100 pM for HeLa cells [34]. Other possible mechanisms for stimulation of the enzyme would include an alteration in the rate of degradation, the rate of synthesis, or possibly the degree of activation of the enzyme. This last possibility is often discussed [35] but has n o t been tested experimentally. Degradation is usually measured by inhibiting protein synthesis and following subsequent decay of enzyme activity [28]; the rate of loss is then calculated assuming that decay is a first-order process [30]. Cycloheximide at 50 pg/ml was found to inhibit the rate of leucine incorporation into HeLa cell

273

T A B L E III ENZYME A C T I V I T I E S AND R A T E S OF T U R N O V E R FOR HeLa CELLS E n z y m e a c t i v i t y w a s e s t i m a t e d as t h e m e a n of five d e t e r m i n a t i o n s ; t h e half-lives are t h e a v e r a g e s of t w o e x p e r i m e n t s , k = In 2/half-life. A d d e d NaC1 (mM)

Ornithine decarboxylase (pmol CO2/pg protein p e r h)

Half-life (min)

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S ( p m o l CO2/P ~ protein per h-)

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50 20

0.83 2.1

2.9 3.1

acid-precipitable protein by at least 99% within I min. The half-lives of ornithine decarboxylase for cells in 50 and 100 medium are shown in Table III. Since the level of the enzyme has achieved a constant, steady-state value 1 day after adding fresh medium, one may estimate the rate of enzyme synthesis from the known levels of enzyme in the cell and the measured rates of degradation [36]. d P / d t = S - - kP, where S is the rate of enzyme synthesis, k is the firstorder decay constant, and P is the enzyme activity. Since d P / d t = O, one may then calculate that the rates of enzyme synthesis are similar for cells in 50 and 100 medium (Table III). Thus, the differences in enzyme levels can be explained solely on the basis of their different rates of turnover. E f f e c t s o f a s u d d e n e l e v a t i o n in NaC1 c o n c e n t r a t i o n on p o l y a m i n e m e t a b o l i s m

Partially confluent dishes of cells adapted to 80 medium were exposed to 140 medium at time zero, and samples were taken for polyamine analysis at later times. The cellular levels of putrescine and spermidine promptly fell with half-lives of about 4 h, achieving new steady-state levels at 6 h. These lower polyamine contents were maintained for at least a day. The spermine c o n t e n t remained constant throughout. It is possible t h a t osmotic change would damage the cell membrane, allowing slow release of polyamines to the medium. We have estimated that a concentration of putrescine or spermidine equal to 10% of that lost from the cell could easily be identified in the medium; however, at 2, 4 and 6 h after the shift, no polyamines were found. The morphological changes described earlier were not apparent for at least 2 days after the shift and were fully developed only after 3 or 4 days. The decrease in putrescine concentration could result from a block in synthesis, along with conversion to spermidine and other metabolites. Indeed, sudden elevation in NaCl concentration produced an immediate decline in the activity of ornithine decarboxylase (half-life, about 12 min). Enzyme activity fell to undetectable levels and remained there for 24 h. We attempted to block the rapid loss of the enzyme by pretreating cells for 15 min with 1 pg/ml actinomycin D; however, after the increase in NaC1, the half-life was still very short (14 min). Actinomycin D, added to cultures in 50 medium, did n o t reduce enzyme activity for at least 1 h. Large amounts of ornithine decarboxylase were n o t lost to the medium.

274 We estimate that 50% of the loss would be sufficient for detection in the medium, y e t none was actually found. Similar osmotic shifts using 100 mM mannitol also effected p r o m p t loss of enzyme activity; however, the activity returned to normal by 4 h after the shift. The rapid loss of ornithine decarboxylase could be caused by a general disruption of protein synthesis, as is known to occur following major osmotic shocks [37]. However, incorporation of radioactive leucine into HeLa cell protein was found to continue at the control rate for more than 1 h after shifts from 140 to 80 medium or from 100 to 50 medium. Therefore, general protein synthesis is probably not affected by the changes in NaC1 concentration used here. In summary, a sudden increase in NaC1 concentration appears to produce a specific interruption in the synthesis of ornithine decarboxylase; the enzyme then decays exponentially with resultant decrease in the synthesis of putrescine. The intracellular levels of putrescine and spermidine then fall, not as a result of loss to the medium, but from their intracellular metabolism. Shifts in external NaC1 concentration obviously may be used an an alternative technique for measuring the rate of decay of enzymatic activity and thus complement the results of experiments with cycloheximide. Since decay rates are similar using either technique, cycloheximide probably does not have a direct effect on the rate of enzyme degradation. Furthermore, since decay rates are similar in the presence or absence of cycloheximide, the rate of loss of enzymatic activity is not dependent on continuing general protein synthesis. Effects o f a sudden reduction in NaCl concentration on p o l y a m i n e metabolism As shown in Fig. 4, a sudden reduction in NaC1 concentration led to a substantial increase in the intracellular level of putrescine, with a peak at 7 to 9 h; putrescine then declined over the following day. In other experiments a lag period of about 2 h was present before the putrescine level began to increase. The magnitude of the peak was found to depend on the particular osmotic shift employed, e.g. for a shift from 140 to 80 medium, the peak was 1600 pmol putrescine/pg deoxyribose; for a shift from 100 to 50 medium (Fig. 4), the peak value was 3370 pmol. Assuming a cell volume of 10~ pl/cell and uniform distribution of putrescine within the cell water, this latter putrescine content is equivalent to an intracellular concentration of approx. 10 mM. In several experiments no consistent increase in spermidine or spermine c o n t e n t was seen. No polyamines were found in the medium. Morphological changes consistent with adaptation to the new medium were not apparent for at least 2 days. In a control experiment, cells adapted to 100 medium were exposed to fresh medium of the same composition and polyamine levels were followed for the subsequent day. The putrescine c o n t e n t rose by about 40% to a broad'peak at 10 h and then declined; spermidine and spermine were unaffected. The activity of ornithine decarboxylase undergoes a rapid and substantial increase when cells are changed from 100 to 50 medium (Fig. 5A). Enzyme activity is elevated within 30 min and reaches a peak at 5--6 h. The activity then falls with a half-life of 50 min, similar to the decay rate of a culture

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F i g . 4. E f f e c t o f a s u d d e n r e d u c t i o n in N a C I c o n c e n t r a t i o n o n p o l y a m i n e levels in H e L a cells. M o n o l a y e r c u l t u r e s a d a p t e d t o 1 0 0 m e d i u m w e r e e x p o s e d t o 50 m e d i u m at t i m e z e r o a n d p o l y a m i n e c o n t e n t s w e r e d e t e r m i n e d at t h e t i m e s s h o w n . T h e solid s y m b o l s at t h e r i g h t r e p r e s e n t v a l u e s f o r c u l t u r e s w h i c h h a v e gTown f o r m a n y g e n e r a t i o n s in 50 m e d i u m , o o, p u t r e s c i n e ; A ~, s p e r m i d i n e ; D D, spermine. Fig. 5. O r n i t h i n e d e c a x b o x y l a s e f o l l o w i n g a s u d d e n d e c r e a s e in NaC1 c o n c e n t r a t i o n . I n paxt A, c u l t u r e s a d a p t e d t o 1 0 0 m e d i u m w e r e e i t h e r e x p o s e d to 50 m e d i u m at t i m e z e r o ( a A) or t h e y w e r e p r e t r e a t e d f o r 3 0 rain w i t h 1 ~ g / m l a c t i n o m y c i n D in t h e o r i g i n a l c u l t u r e m e d i u m a n d t h e n e x p o s e d t o 50 m e d i u m c o n t a i n i n g t h e i n h i b i t o r (A A). A f t e r a d d i n g a c t i n o m y c i n D, i n c o r p o r a t i o n o f r a d i o a c t i v e u r i d i n e w a s f o u n d to fall o v e r 15 r a i n to a r e s i d u a l s y n t h e t i c r a t e o n l y 5 - - 1 0 % o f n o r m a l . T h i s level o f r e s i d u a l i n c o r p o r a t i o n w a s p r o b a b l y n o t i m p o r t a n t s i n c e t h e p a t t e r n s h o w n in p a r t A w a s d u p l i c a t e d w i t h a 5-fold h i g h e r level o f t h e d r u g . I n p a r t B, m o n o l a y e r s in 1 0 0 m e d i u m w e r e f e d w i t h 1 0 0 m e d i u m at t i m e z e r o (~ ~) a n d s a c r i f i e d f o r e n z y m e a n a l y s i s a t t h e t i m e s s h o w n . T h e c o m b i n e d d a t a in t h i s f i g u r e are a c t u a l l y f r o m t h r e e s e p a r a t e e x p e r i m e n t s b u t are r e p r e s e n t a t i v e o f all s i m i l a r e x p e r i m e n t s w h i c h w e carried out.

permanently adapted to 50 medium and treated with cycloheximide. At times later than 10 h, enzyme activity approaches the steady-state level characteristic of cells in 50 medium. It never falls to zero. The increase in enzyme activity m a y well be specific for ornithine decarboxylase since the incorporation of radioactive leucine into protein was n o t affected by the change in medium {data n o t shown). This enhancement of enzyme activity is an osmotic p h e n o m e n o n since cultures shifted from 50 medium with 100 mM mannitol to 50 medium show similar patterns of enzyme activity. The peak at 6 h is n o t due to the presence of a soluble activator, as shown by mixing experiments with 0- and 6-h samples. Similarly, mixing experiments using an extract from a permanent 50 culture and samples taken at 8, 13 and 16 h helped to rule o u t the presence of a soluble inhibitor at late times. Since putrescine is a competitive inhibitor of the enzyme (K i 440/~M), large amounts of this diamine in the enzyme extract could lower the apparent activity; however, at the dilution used for measurement of enzyme activity, extracts made from 6- and 8-h samples were found to contain 19 pM putrescine, a non-inhibitory concentration. E n z y m e could also have been lost to the medium; however, none was present in medium from the 8-h sample. Loss o f 20% of the total activity to the medium should have been easily detected.

276 The rate of enzyme degradation was studied by the addition of 50 pg/ml cycloheximide, and the half-life was found to be increased from 22 min at time zero to 91 min at 2 h (averages of three experiments each). This alteration in decay rate is unfortunately n o t sufficient to account for the quantitative rise in enzyme activity. Berlin and Schimke [36] have developed an equation to predict the effects of alterations in rates of synthesis and degradation on activity levels. Assuming a constant rate of enzyme synthesis, lengthening the half-life from 20 to 80 min would increase the enzyme activity at 4 h to 5.5 enzyme units or approx. 13% of the observed increase (data taken from Table III). Assuming that the half-life were increased to 100 h, enzyme activity at 4 h would be only 31% of that observed. Therefore, a reduced rate of turnover is insufficient to account for the total increase in enzyme activity. Enzyme synthesis must also be stimulated. The addition of fresh medium is known to stimulate the rate of enzyme synthesis in several lines of tissue culture cells [23,31]. This effect is due in part to the presence of a non
277 I

I

1

2

i

I

I

3

4

5

5O ~~ =

40

~

30

~

~E

zo

Time (h)

Fig. 6. E f f e c t o f e x o g e n o u s P u t t e s c i n e o n " i n d u c t i o n " o r orrfithine d e c a x b o x y l a s e . A t t i m e z e r o m o n o l a y e r c u l t u r e s a d a p t e d t o 1 0 0 m e d i u m w e r e e x p o s e d t o 50 m e d i u m ( c o). S o m e of t h e s e flasks r e c e i v e d 5 0 0 ~tM p u t r e s c i n e at 2 h (s o), 50 /~g]ml e y c l o h e x i m i d e at 2 . 2 5 h (~ A), or b o t h (A A). Several o t h e r flasks w e r e p r e - i n c u b a t e d w i t h 5 0 0 #M p u t x e s c i n e for 15 m i n and t h e n s h i f t e d t o 50 m e d i u m c o n t a i n i n g p u t r e s c i n e (o ~). High levels o f p u t r e s c i n e in the assay m i x t t t r e s did n o t i n t e r f e r e w i t h the e s t i m a t e o f e n z y m e a c t i v i t y . P u t r e s c i n e levels w e r e m e a s u r e d in m o s t e n z y m e preparat i o n s a n d w e r e n e v e r f o u n d to b e greater t h a n the e q u i v a l e n t o f 30 pM in t h e assay.

The peak in putrescine concentration could, however, be the signal for interruption of further enzyme synthesis. Addition of the diamine at 2 h after the shift eventually shut off the increase in ornithine decarboxylase activity (Fig. 6). This effect is specific for the enzyme in question since putrescine has no effect on the rate of incorporation of radioactive leucine into total cell protein (data n o t shown). The increase in putrescine might also be part o f the mechanism for stimulating enzyme degradation, for example, putrescine might bind to the enzyme, producing conformational changes which would make the protein molecule more sensitive to proteolytic cleavage. However, cells treated with putrescine 2 h after a shift lost enzyme activity at a very slow rate (Fig. 6). We know that putrescine entered the cell in this experiment since the appearance of new enzyme activity was blocked b y the diamine. Thus, the rate of decay of ornithine decarboxylase is probably independent of the level of putrescine within the cell. It is possible that the reappearance of rapid decay at 6--7 h would require new RNA or protein synthesis. Hence, we have tried to block enhanced decay with actinomycin D or cycloheximide added either at the peak or 1 h prior to it. However, none of these conditions significantly changed the pattern of enzyme degradation shown in Fig. 5A. Hogan [23] has reported similar experiments. Discussion

As originally reported by Eagle [13] and essentially confirmed b y Stubblefield and Mueller [ 1 5 ] , HeLa cells will grow in media containing between 60 and 150 mM NaCI, maximal growth being achieved at a b o u t 100 mM NaCI. In our experiments, HeLa cells were able to maintain rapid growth in media containing as little as 40 mM added NaC1. This discrepancy may be explained in part by the presence of 65 mM Na ÷, contributed by sources other than NaC1,

278 and possibly by strain differences which may have developed in the intervening years. The range of osmolalities studied here should have little effect on cell size and gross composition. By analogy with mouse l y m p h o m a cells [16], HeLa cells adapted to any of our media should have approximately the same volumes, sudden shifts in osmolality should produce only minor perturbations in cell volume and should be resolved within 5 min. Major rents in the cell membrane probably do not occur during our shifts since polyamines were n o t released to the medium. By analogy with mouse l y m p h o m a cells, about 70% of the total change in internal osmolality during our shifts should arise from fluctuations in intracellulax K÷; this would a m o u n t to a variation of approx. 25% in total cellular K * [ 16]. High NaC1 concentration has been shown to enhance anaerobic glycolysis in HeLa cells [15] and to increase oxygen consumption in Ehrlich ascites cell sublines [39]; however, these effects are for media hypertonic to all media studied here. Exposure to hypertonic media also causes chromosomal clumping and alterations in cellular RNA, protein, and lipid contents [15], but such changes were not observed her (Table I). Sudden exposure to hypertonic media also reduces incorporation of precursors into nucleic acid and protein [ 37 ], but our hypertonic and hypotonic shifts have no effect on general protein synthesis. Several enzyme activities axe k n o w n to be induced by adaptation to media of high or low tonicity. The alkaline phosphatase activity of KB cells is inversely related to the external NaC1 concentration [40] ; enzyme activity and external osmolality are directly related in several other cell lines, including HeLa cells [40]. Acid protease and acid phosphatase activities are elevated in LS cells grown in media of increased osmolality [41]. A variety of enzyme activities axe enhanced in sublines of Ehrlich ascites cells grown in media of high tonicity, effects which are reversible on return of these sublines to media of lower tonicity [18]. As reported here, polyamine metabolism in mammalian cells reacts in similar ways to that of E. coli, when the external NaC1 concentration is changed. Most of these similarities are, however, rather superficial. E. coli contains putrescine and spermidine, but no spermine. The putrescine c o n t e n t is inversely related to the osmolality of the medium, whether determined by NaC1 or other salts and sugars; and the spermidine c o n t e n t is not affected by any of these solutes [10]. The major polyamines of mammalian cells are spermidine and spermine; putrescine is a major c o m p o n e n t only in some tissue culture lines and some other cells which are growing and dividing rapidly [7,42,43]. As with E. coli, the putrescine c o n t e n t of HeLa cells was found to be inversely related to the osmolality of the medium. Spermidine and spermine were not affected by external osmolality, but were inversely related to the external NaC1 concentration. In E. coli, putrescine m a y be synthesized by decarboxylation of either ornithine or arginine. The activities of both pathways are increased in media of low osmolality; actual control of enzyme activity occurs primarily through changes in internal K ÷ concentration, and secondarily by small increases in the levels of the pertinent enzymes. (Munro, J.L., personal communication).

279 Unlike the bacterial model, the rate of total polyamine synthesis in mammalian cells is probably regulated by alterations in the activity of ornithine decarboxylase. Clearly, other enzyme activities are also affected by changes in external NaC1 or osmolality since growth with different solutes produces different ratios of putrescine to spermidine (Table I). Polyamine auxotrophs of E. coli grown in media o f low osmolality have a specific requirement for putrescine [ 1 1 ] . It is not known whether the high polyamine contents of mammalian cells in media of low tonicity are actually necessary for continuation of any cell functions. A sudden increase in external osmolality effects rapid loss of putrescine from both E. coli and HeLa cells. In the bacterial system, putrescine is rapidly extruded from the cell b y an energy-dependent exchange for K ÷ in the medium [ 10,44]. For HeLa cells, levels of putrescine and spermidine fall with a half-life of 4 h, b y catabolism and by intracellular conversion to spermine [45]. E. coli begin rapid growth immediately after a sudden reduction in external osmolality, b u t the cellular putrescine content increases very slowly, not reaching the new steady-state level for several generations [10]. For mammalian cells, the levels of ornithine decarboxylase and putrescine rise rapidly to peaks which are many-fold higher than the final steady-state levels. In many mammalian cell systems, polyamine levels have been shown to increase during periods of rapid growth [1,7,46]. The classic pattern following partial h e p a t e c t o m y [42] involves an initial stimulation of ornithine decarboxylase [29] and putrescine synthesis, with a delayed rise in spermidine. Since putrescine is a potent inhibitor of the conversion of spermidine to spermine, spermine levels are n o t increased [6,42]. It would seem that stimulation of rapid growth is n o t the only stimulus for enhanced polyamine synthesis since shifts in NaC1 concentration can stimulate polyamine synthesis without an obvious change in growth rate. Ornithine decarboxylase is the first enzyme in the biosynthetic pathway for polyamines, and, therefore, a likely site for control of polyamine metabolism. In the experiments reported here, control is evident at the levels of transcription, translation, and enzyme degradation. For cells fully adapted to any particular medium, the level of ornithine decarboxylase is fixed by the rate of enzyme degradation, enzyme synthesis being independent of the NaC1 c o n t e n t of the medium (Table III). Inhibition of decay contributes to the large rise in enzyme activity seen after a sudden reduction in NaC1 concentration. The reappearance of significant degradation after 6 h accounts for the rapid loss of enzyme activity. After sudden reduction in NaC1 concentration, the initial burst of synthesis of ornithine decarboxylase may come from a pool of preformed m R N A specific for the enzyme. Thus, translational controls appear to be important at this time. Similar preformed pools of m R N A have previously been observed [ 2 3 ] , b u t n o t in all systems [31,47]. Following the initial burst of enzyme synthesis, enhanced transcription of specific m R N A is required for full expression of ornithine decarboxylase since a major fraction of the increase in enzyme activity may be blocked with actinomycin D (Fig. 5). This inhibitor is assumed to exert only a minor effect on the stability of preformed m R N A .

280

What role(s) might intracellular putrescine play in the regulation of polyamine synthesis? This diamine is a competitive inhibitor of ornithine decarboxylase (Ki 440 pM, as measured here). A sudden reduction in NaC1 concentration produces a dramatic increase in putrescine (Fig. 4) which might be expected to inhibit enzyme activity; however, the increase in enzyme level swamps o u t any observable inhibitory effect, and putrescine synthesis continues (Fig. 5). In fact, putrescine synthesis probably stops only after the synthesis of enzyme is inhibited. The paradoxical failure of putrescine to inhibit ornithine decarboxylase could result from physical separation of enzyme and inhibitor within the cell. It is, however, entirely possible that the high levels of putrescine achieved during this shift would be sufficient to cut off either the transcription or translation of m R N A for the enzyme and in this unusual circumstance, would be able to reduce enzyme activity. Exogenous putrescine has been shown to decrease ornithine decarboxylase activity in KB cells [48], lymphocytes [49], and rat liver [ 3 5 ] , probably by a similar mechanism. Under more usual conditions, not involving a shift in NaC1 concentration, the intracellular level o f putrescine probably does not control transcription or translation of m R N A for the enzyme. At the least, this putative control mechanism may be easily overridden: First, intracellular levels of putrescine are quite different for cells fully adapted to either 50 or 100 medium, y e t the rates of enzyme synthesis are similar (Table III). Second, adding fresh medium leads to a burst of enzyme synthesis; if putrescine controlled the rate of synthesis of the enzyme, then no burst should be possible. Putrescine could regulate enzyme degradation by binding to it and affecting its rate of catabolism. However, exogenous putrescine was shown to have no effect on the rate of enzyme decay (Fig. 6). Clearly, there are many possible sites for regulation of the activity of this enzyme, b u t putrescine is probably not important, except when its intracellular level is greatly increased. Acknowledgements We wish to thank Dr Steven I. G o o d m a n of the Department of Pediatrics, University of Colorado Medical Center for the generous gift of many lines of human fibroblasts, Mrs Kay Fuertges of National Jewish Hospital for measurement of Na ÷ and K ÷, Dr Edward J. McGuire for careful review of the manuscript, and Dr Martin Pato, who predicted that ornithine decarboxylase is regulated by enzyme decay. This work was supported b y Grant GB-36895 from the National Science Foundation and b y Grant AM-17773 from the National Institutes of Health. It is publication no. 636 o f the Department of Biophysics and Genetics. References 1 2 3 4 5

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