Arginase, an s-phase enzyme in a human cell line

33

Biochimica et Biophysica Acta, 672 (1981) 33--44

© Elsevier/North-Holland Biomedical Press

BBA

29477

ARGINASE, AN S-PHASE ENZYME IN A HUMAN CELL LINE

SVEN SKOG, VIOLA ERIKSSON and EVA ELIASSON * The Wenner-Gren Institute, University of Stockholm, Norrtullsgatan 16, S-113 45 Stockholm (Sweden)

(Received July 10th, 1980) Key words: Arginase; Growth phase; Cycloheximide; Synchronization; (Chang liver cell)

Summary Suspension cultures of 'Chang liver' cells were synchronized by preincubation in a glutamine-deficient medium or by thymidine blockade. Specific arginase activity varied in the synchronized cultures, being high when the number of S-phase cells was maximal. A relationship between high arginase activity and a high percentage of (S + G2) cells was also found when unsynchronized cells were separated by velocity sedimentation. The increase in arginase activity near the G1/S border was totally inhibited in the presence of cycloheximide. The rate of decrease in activity after addition of the drug indicated that the variations in arginase activity during the mitotic cycle were the result of variations in the rate of synthesis of the enzyme, while the rate of degradation was more or less constant, corresponding to 4 - 6 % per h. The role of arginase in cells lacking a urea cycle and the regulation of arginase activity in 'Chang liver' cells is discussed.

Introduction Previous experiments with HeLa $3 cells and with Chang liver cells, which is probably another HeLa line [1 ], have shown that arginase activity (L-arginine amidinohydrolase, EC 3.5.3.1) is highly variable in response" to changes in the composition of the growth medium [2,3,4]. In cells, temporarily deprived of single essential amino acids, arginase activity increases steeply during the first hours following the addition of the missing amino acid [5]. It seems likely that this increase in enzyme activity might be secondary to a synchronization of the cells with respect to the mitotic cycle.

* To whom

correspondence should be addressed.

34 In order to study the relationship between arginase activity and the progression through the cell cycle, arginase was determined at short time intervals after preincubation of the cells in a glutamine-deficient medium, in cultures synchronized by thymidine blockade and in unsynchronized cells separated by means of sedimentation at unit g in a Ficoll gradient. The results were in good agreement and showed a correlation between high arginase activity and a high percentage of S-phase cells. The increase in enzyme activity during a distinct period of the cell cycle could be the result of changes in the rate of synthesis of the enzyme, but it might also be related to activation or stabilization of preformed enzyme molecules. Earlier experiments in which protein synthesis was inhibited by puromycin showed that the increase in arginase activity, after a period of amino acid deprivation, was dependent on protein synthesis [5]. In the same study the development of arginase was examined in the presence and in the absence of Mn 2+, which has been shown to stabilize the enzyme in cultured cells. These experiments indicated t h a t arginase was broken down continuously at a constant rate in the absence of manganese, while the rate of synthesis was variable. The true turn-over rate of the enzyme could n o t be inferred from this study, since it was n o t known whether the enzyme was totally stable in the presence of Mn 2÷. It, therefore, seemed t h a t an interesting study would be to reexamine the rate of breakdown of the enzyme during different stages of the cell cycle. The results of the present experiments, in which protein synthesis was inhibited by the addition of cycloheximide, confirm the above-mentioned results indicating that arginase is broken down at a more or less constant rate, while the synthesis of the enzyme is variable. Both in synchronized cultures and during normal growth the enzyme seems to be synthesized during a limited period within the cell cycle, in late G~ and in S phase. Methods

Cell cultures Chang liver cells (American Type Culture Collection CCL 13) were grown as spinner cultures in Eagle's minimal medium supplemented with 10% horse serum. The cell line was originally obtained from Microbiological Assoc. Inc. Bethesda, MD. Stock cultures were kept in exponential growth with a generation time of 26 h, by dilution every second day to a cell density of 0.2 • 106 cells/ml. The absence of mucoplasma was regularly checked. Synchronisation methods For glutamine starvation the cells were suspended in a growth medium lacking glutamine but with the normal concentration of undialyzed serum. After 19 or 23 h (as stated in the description of the individual experiments} glutamine was added. For thymidine blockade of DNA synthesis 3 mM thymidine was added to the cultures. After 16 h the block was reversed by resuspending the cells in fresh medium. Cell separation by sedimentation at unit g in Ficoll gradients has been described previously [6].

35

Cell counting, biochemical determinations and incorporation of labelled thymidine into DNA Cell counting. Cells were counted in a Biircher counting chamber or in an electronic counter (Ljungberg Celloscope 101, AB Lars Ljungberg, Stockholm). Arginase activity. Activity was determined as described previously using [guanido 14C]arginine as a substrate [7]. Enzyme activity expressed as nmol urea formed per h. Protein determinations. These were carried out according to the Biuret m e t h o d [8], with a coefficient of 0.095 for 1 mg protein in 3 ml total volume, 1-cm light path, at 540 mp. DNA content. The relative DNA content of individual cells was determined in a PHYWE c y t o m e t e r after treatment with pepsin and RNAase and staining with ethidium bromide according to Haanen et al. [9]. Approx. 3000 cells in each determination. Thymidine incorporation. For thymidine incorporation 5-ml portions of the cultures were transferred to culture tubes containing 0.25/aCi [14C]thymidine. After incubation in a revolving rack at 37°C for 30 min the cells were washed twice with ice cold saline, transferred to filter paper discs and washed with 5% trichloroacetic acid and ethanol. Radioactivity was determined in Omnifluor (New England Nuclear) in an Intertechnique SL 36 scintillation spectrometer. Results

Effect o f temporary glutamine deprivation In the experiments shown in Fig. 1 and Table I, cells were incubated in a glutamine-deficient medium for 19 h. Glutamine deprivation was n o t total since 10% undialyzed serum was included in the starvation medium. However, glutamine soon became growth limiting as indicated by an 80--90% inhibition of cell multiplication (Table I). The cell number did not increase during the first 10 h after the addition of glutamine (Fig. 1). The fact that a wave of high thymidine incorporation preceded any increase in cell number indicated that a considerable part of the cell population had been arrested near the GI/S border. TABLE

I

CELL NUMBERS AND PERCENTAGE GLUTAMINE DEPRIVATION

OF CELLS

I N G 1, S A N D

G 2 PHASE

AFTER

A PERIOD

OF

Cells from an exponentially growing stock culture were incubated in a glutamine deficient medium for 1 9 h . A t t i m e s i n d i c a t e d t h e r e l a t i v e D N A c o n t e n t o f a p p r o x . 3 0 0 0 cells w a s d e t e r m i n e d in t h e f l o w cytometer. T h e p e r c e n t a g e o f cells i n t h e G 1, S a n d G 2 p h a s e w a s d e r i v e d f r o m t h e h i s t o g r a m s b y t h e m e t h o d o f B a r l o g i e e t al. [ 1 0 ] . F i v e i n d e p e n d e n t d e t e r m i n a t i o n s s h o w e d t h a t e x p o n e n t i a l l y g r o w i n g cell c u l t u r e s c o n t a i n e d 6 3 +- 4 % G l - p h a s e c e l l s , 2 2 +_ 3 % S - p h a s e c e l l s a n d 1 1 _+ 2 % G2*-phase cells. Time after glutamine

addition

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47 43 10

42 48 10

34 60 6

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Fig. 1. Arginase activity in relation to increase in cell n u m b e r , p r o t e i n growth and i n c o r p o r a t i o n of [ 1 4 C ] . after a 1 9 h p e r i o d of glutamine deprivation. Cells f r o m an e x p o n e n t i a l l y growing s t o c k culture were i n c u b a t e d i n a b l u t a m i n e deficient m e d i u m for 1 9 h . Abscissa: time after glutamine a d d i t i o n ( h ) . Ordinate: A, cells/ml 1 • 1 0 - 6 ; B, p r o t e i n ( m g / m l ) ; C, arginase activity (units/ml): D, t h y m i d i n e incorporation in 3 0 m i n b y 106 cells ( c p m 1 • 1 0 - 2 ) ; E, specific arginase activity ( u n i t s / m g protein). thymidine

This conclusion was confirmed by determinations of the relative DNA content of individual cells (Table I), showing a maximum number of S-phase cells 4 h after glutamine addition. While the total protein content of the culture increased at a more or less even rate after medium restoration, the rise in arginase activity was discontinuous with a 150% increase during the first 9 h, followed by a marked decrease between 10 and 15 h. The fluctuations in the rate of increase in enzyme activity resulted in marked variations in specific arginase activity. A comparison between curves D and E of Fig. I shows that the variations in specific arginase activity followed the fluctuations in thymidine incorporation fairly well, indicating a possible relationship between high arginase activity and a high rate of DNA synthesis.

Thymidine blockade If a correlation exists between a high number of S-phase cells and high arginase activity, it should also be possible to demonstrate this correlation in cultures synchronized by other methods. As seen in Fig. 2, a 16 h block of DNA synthesis in the presence of 3 mM thymidine resulted in an increase in the number of S-phase cells from 23 to 80% of the total cell population. Initiation of DNA duplication and a slow progression through the S-phase in the presence of high concentrations of thymidine has also been reported by others, Bostock et al. [11].

37

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r e l a t i o n t o i n c r e a s e i n cell n u m b e r , p r o t e i n g r o w t h a n d i n c o r p o r a t i o n o f [ 1 4 C ] blockade of DNA synthesis. Abscissa: time after the last block (h). Ordinate: ); t h y m i d i n e i n c o r p o r a t i o n i n 3 0 r a i n b y 1 0 6 c e l l s ( c p m 1 • 1 0 - 2 ) ( . . . . . . ); ]3, -); a r g i n a s e a c t i v i t y ( u n i t s / r o t ) ( . . . . . . ), T w o e x p e r i m e n t s : e x p o n e n t i a l l y preincubated in the presence of 3 mM thymidine for 16 h as in Fig. 2 (e) or thymidine blocks separated by a 15 h interval in normal growth medium (o).

38 6 h after the release, the majority of the cells had completed DNA duplication and at a b o u t 8 h a distinct maximum of G2-phase cells was seen, followed by a sharp increase in the number of Gl-phase nuclei. At 12 h more than 80% of the cells were in G1-phase, indicating a fairly good synchronization of the first division wave. A good synchrony of the first division after thymidine blockade of DNA synthesis was confirmed by the experiments shown in Fig. 3. During the first 4 h after the release from the block, DNA synthesis could not be measured by means of [14C]thymidine incorporation, because of an expanded intracellular pool of unlabelled thymidine. The incorporation data after that period were in agreement with the experiment shown in Fig. 2, showing a low thymidine incorporation rate between 8 and 15 h after the release. As seen in Fig. 3B the protein content of the cultures increased at a more or less even rate during the entire experimental period covering a complete division cycle. Arginase activity, on the other hand, was high during the first 6 h after the release when, according to the experiment in Fig. 2, the majority of the cells were in S phase and then declined. When after 16 h, the increase in thymidine incorporation indicated the initiation of a second S phase period, arginase activity again increased. In summary, the thymidine blockade experiments confirmed that arginase activity increased and decreased during distinct periods of the cell cycle, and clearly indicated a correspondence between an increase in arginase activity and the entwance into the S-phase.

Sedimentation fractionation of exponentially growing cell populations Certain objections could be raised to the conclusions drawn from the synchronization experiments, since the fluctuations in arginase activity could be the result of an unbalanced growth pattern arising as a result of the synchronization procedures. During glutamine deprivation specific arginase activity decreased by 30--50%, while during thymidine blockade enzyme activity increased. An overshooting regulation of enzyme activity back to 'normal' might give rise to oscillations in both cases. It, therefore, seemed important to know whether arginase activity varied also within an undisturbed cell population. In a series of experiments cells from exponentially growing Chang cell cultures were separated by sedimentation at unit g in shallow Ficoll gradients as previously described [6]. Although the ceils sediment according to their size the m e t h o d does n o t allow a clear separation of cells from the successive phases of the cell cycle. The lack of success in separating the different stages was not due to contamination of the cultures by cells with a different DNA content, since the histograms based on eytometric determinations constantly showed two distinct peaks corresponding to the G1- and G2-phase nuclei and a plateau of S-phase nuclei between these peaks. The fact that different batches of cells, separated by continuous cultivation for more than 50 generations, showed identical sedimentation patterns also indicates against contamination of the cultures by cells of a different kind. It was, therefore, suggested that even during exponential growth there is a considerable heterogeneity in size of cells from the same stage of the mitotic cycle. Recently Zucker et al. [12] have drawn similar conclusions from velocity sedimentation experiments with a m o n k e y kidney cell line.

39

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Fig. 4. Arginase a c t i v i t y in e x p o n e n t i a l l y g r o w i n g cells s e p a r a t e d b y m e a n s o f v e l o c i t y s e d i m e n t a t i o n . Cells w e r e s e p a r a t e d b y s e d i m e n t a t i o n at u n i t g in 3 - - 7 % Fieoll g r a d i e n t as d e s c r i b e d p r e v i o u s l y [ 6 ] . T h e g r a d i e n t s w e r e h a r v e s t e d in 1 0 m l ( A ) o r 20 m l (B a n d C) f r a c t i o n s . Abscissa: s e d i m e n t a t i o n rate. Ordin a t e : A, n u m b e r o f cells p e r m l 1 • 10 - ~ , G l - p h a s e cells (e . . . . . . o), (S + G 2 - P h a s e ) cells ( o - - - - - - - ~ ) ; B, specific arginase a c t i v i t y ( u n i t s / r a g p r o t e i n ) ( m e a n o f five i n d e p e n d e n t s e p a r a t i o n s -+S.D.); C, p r o t e i n p e r cell (rig) (o . . . . . . o); arginase a c t i v i t y p e r cell ( u n i t s 1 • 1 0 6 ) (e -') ( o n e of the e x p e r i m e n t s s h o w n inB).

In the experiments shown in Fig. 4 arginase activity was determined in the different fractions of cells after sedimentation in Ficoll gradients. In spite of imperfect separation of cells belonging to the different stages of the cell cycle (Fig. 4A) some conclusions can be drawn from these experiments. It is obvious that if arginase activity always increased at the same relative ra~e as the total protein content, specific arginase activity would remain unchanged during the cellular growth cycle. This is evidently n o t the case (Fig. 4B). The slowly sedi-

40 menting cell fractions, containing nearly pure G l-phase cells, had a low specific arginase activity. Specific activity then increased to a maximum in cells of median size and then again decreased in large cells. Fig. 3C shows the protein content per cell and arginase activity per cell in a typical experiment. In those fractions, 1 1 0 - - 1 7 0 ml from the top, where the size of the cells increased from 0.27 to 0.40 pg protein (+48%), arginase activity increased by 160%. This steep increase in arginase activity occurred within the same fractions in which cells belonging to the later stages of the cell cycle (S- + G2-phase cells) first appeared. After this rise, activity remained more or less constant in large cells. The separation experiments clearly indicate that arginase activity increases abruptly during a limited period of the cellular growth cycle near the G1/S phase border even in unsynchronized cell populations. Changes in arginase activity as a result o f a variable rate o f synthesis combined with a constant rate o f breakdown

The increase and decrease in arginase activity in cells partially synchronized by glutamine starvation offered an opportunity for investigating the role of synthesis and breakdown of the enzyme. Cycloheximide at a concentration of 20 #g/ml totally inhibited the incorporation of labelled amino acids into protein within 5 min after the addition of the drug. In the two experiments shown

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F i g . 5. A r g i n a s e a c t i v i t y d u r i n g i n h i b i t i o n o f p r o t e i n s y n t h e s i s . C e l l s w e r e p r e i n c u b a t e d in a g l u t a x n i n e d e f i c i e n t m e d i u m f o r 2 3 h . C y c l o h e x i m i d e ( 2 0 / ~ g / m l ) w a s a d d e d at 0, 3 o r 1 1 . 5 h a f t e r g l u t a m i n e a d d i tion (arrows). Parallel cultures without cycloheximide s e r v e d as c o n t r o l s . T w o i n d e p e n d e n t experiments. Cells were harvested continuously as d e s c r i b e d b y B 6 1 c s f 6 1 d i a n d E l i a s s o n [ 1 3 ] . A b s c i s s a : t i m e a f t e r

glutamine addition (h). (A parallel experiment 10 and 16 h thymidine val. C e l l d i v i s i o n s t a r t e d

O r d i n a t e : a r g i n a s e a c t i v i t y ( u n i t s / m l ) c o n t r o l s (© . . . . . . ©), c y c l o h e x i m i d e ( o e e ) . s h o w e d t h a t t h e rate o f t h y m i d i n e i n c o r p o r a t i o n h a d a m a x i m u m at 8 h. B e t w e e n incorporation was low, indicating that few ceils were in S phase during that interat about 12 h.)

41

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F i g . 6. D e c r e a s e in a r g i n a s e a c t i v i t y in t h e p r e s e n c e o f c y c l o h e x i m i d e ( s a m e e x p e r i m e n t as in Fig. 5). Using the least-squares m e t h o d e x p o n e n t i a l decay curves were c o n s t r u c t e d fitting the e x p e r i m e n t a l data. T h e c o r r e l a t i o n c o e f f i c i e n t s w e r e - - 0 . 6 8 , - - 0 . 9 7 a n d - - 0 . 9 5 f o r c y c l o h e x i m i d e a d d e d at t i m e O, 3 a n d 1 1 . 5 h, r e s p e c t i v e l y . I f a t = a o e k t ( w h e r e a t = a r g i n a s e a c t i v i t y at t i m e t, a 0 = a r g i n a s e a c t i v i t y at t h e t i m e of c y c l o h e x i m i d e a d d i t i o n , t = t i m e a f t e r c y c l o h e x i m i d e a d d i t i o n a n d k = t h e d e c a y c o n s t a n t ) t h e d e c a y c o n s t a n t s f o r t h e r e g r e s s i o n l i n e s are - - 0 . 0 5 9 , - - 0 . 0 4 5 a n d - - 0 . 0 5 1 , r e s p e c t i v e l y . A p p r o x i m a t e half-life o f t h e a c t i v i t y w a s c a l c u l a t e d a c c o r d i n g to t h e f o r m u l a T 1 / 2 = In 2 / k . A b s c i s s a : t i m e a f t e r c y c l o h e x i m i d e a d d i t i o n ; O r d i n a t e : a r g i n a s e a c t i v i t y in p e r c e n t a g e o f t h e a c t i v i t y at t h e t i m e o f c y c l o h e x i m i d e a d d i t i o n . U p p e r c u r v e , c y c l o h e x i m i d e at z e r o - t i m e . M i d d l e c u r v e , c y c l o h e x i m i d e at 3 h. B o t t o m c u r v e , c y c l o h e x i m i d e a t 11.5 h a f t e r t h e a d d i t i o n o f g l u t a m i n e .

in Fig. 5 the cells were preincubated in a glutamine-deficient medium for 23 h and cycloheximide was added at different times after the restitution of a complete growth medium. In the presence of the inhibitor arginase activity decreased exponentially (Fig. 6). T he rate of decrease corresponded to a halflife o f th e e n z y m e of 14--15 h if cycloheximide was added either at 3 h, when e n z y m e activity normally increased, or at 11.5 h when arginase activity was decreasing in the cont r ol cultures. T he slightly shorter half-life when cycloheximide was added at 0-time might n o t be significant, since the low e n z y m e activity at that time made e n z y m e determinations less accurate. The rate of degradation o f arginase seems to be m or e or less constant t h r o u g h o u t the division cycle and this rate appears to be t o o low for explaining the changes in arginase activity as a result of t e m p o r a r y stabilization of the enzyme.

42 Discussion

Arginase is synthesized at different rates during different parts o f the mitotic cycle The present experiments showed that arginase activity varied during the cell cycle in synchronized cultures as well as in 'normally growing' cells. In order to study the role of degradation of the enzyme cycloheximide was added to synchronized cultures. Although this drug has been reported to inhibit protein degradation in some animal cells under growth restriction, experiments with hepatoma cells and fibroblasts have shown, that in the presence of a complete growth medium protein degradation is not significantly affected by inhibitors of protein synthesis [14]. The constant rate of decrease in arginase activity during a 6 h period, in the presence of cycloheximide, indicates that this might be true also for 'Chang liver' cells. Therefore, the present experiments are consistent with a degradation rate of arginase of 4--6% per h and indicate that this rate is independent of the stage of the cell cycle. A constant rate of arginase breakdown is in agreement with earlier experiments in which arginase was stabilized by the addition of manganese to the culture medium [5]. It seems reasonable to conclude that the variations in arginase activity during the cell cycle are related to changes in the rate of synthesis of the enzyme and that this rate is maximal in early S phase. The termination of enzyme synthesis in cells passing out of the S-phase could be studied 7--13 h after release from thymidine blockade of DNA synthesis. The fact that arginase activity decreased at a rate comparable to the rate of decrease during total inhibition of protein synthesis (4--6% per h) indicated that very little arginase was formed during G2, M and early G1 phases. The metabolic role o f arginase in cells lacking a functioning urea cycle In mammalia most extrahepatic cells lack the enzyme ornithine carbamoyltransferase (EC 2.1.3.3). With the exception of some hepatoma cells the same is true for all established cell lines including 'Chang liver' [15]. Arginase, on the other hand, is an enzyme of wide-spread occurrence. Arginase activity is often high in rapidly growing vertebrate tissues such as in early chick embryos [16], in the m a m m a r y gland of the rat during pregnancy and lactation and in certain tumors [17]. Elevated arginase activity has also been reported in connection with epidermal hyperplasia [18,19]. One might ask what function arginase fulfills in cells lacking a functioning urea cycle. The ornithine-oxo-acid aminotransferase (EC 2.6.1.13) reaction has its equilibrium very much to the right favouring the formation of glutamate + glutamic-~,-semialdehyde [20]. Therefore, arginase seems to be the enzyme which normally supplies the cell with ornithine. In cells lacking ornithine carbamoyltransferase ornithine can be metabolized along two different pathways. Transamination leads to the formation of glutamic acid and proline via glutamic semialdehyde, a pathway recently studied by Mezl and Knox in the lactating m a m m a r y gland of the rat [21]. Decarboxylation of ornithine, on the other hand, leads to the formation of putrescine, which is the first step in the reaction chain leading to polyamines. Polyamines are known to be of importance in connection with cell proliferation [22], and R e d m o n d and Rothberg [19],

43 therefore, suggested that the elevated arginase activity in hyperplastic epidermis might be related to the increased synthesis of polyamines in these abnormal cells.

Regulation o f arginase activity in Chang liver cells Long-term experiments extended over a 3--4 day period have shown that increased arginase activities can be induced by the addition of various inhibitors of arginase or by making the supply of arginine growth limiting. This induction of enzyme increase was abolished when proline was added to the culture medium. Eliasson and Strecker [4], therefore, suggested that arginase in Chang liver cells was regulated by product repression, and that the metabolic repressor was proline or a metabolite in the pathway from ornithine to proline. If this is true, the variations in arginase activity during the cell cycle could be due to 'oscillatory repression', a mechanism which has been suggested for the regulation of various biosynthetic enzymes in microorganisms [23]. However, the fact that the addition of proline had no immediate effect on the increase in arginase activity in cells synchronized by amino acid deprivation [5], tends to suggest that there is no direct relationship between the variations in the rate of synthesis of the enzyme and the intracellular concentration of a repressor molecule related to the ornithine-proline pathway. In fact, it seems more likely that the fluctuations in arginase activity during the cell cycle are related to the synthesis of polyamines. It has been shown that the concentration of polyamines and the activity of the enzymes involved in their synthesis from ornithine vary during the cell cycle, and both ornithine decarboxylase and S-adenosyl-methionine decarboxylase (EC 4.1.1.50) show maximum activities in the S-phase, in synchronized Chinese hamster cells [24]. Recent experiments have shown that arginase activity in Chang liver cells is very sensitive to any interference with cell multiplication. Thus, prolonged treatment with high concentrations of thymidine leads to a continuous increase in arginase activity in the S-phase-arrested cells. Likewise, when Chang liver cells are grown at high cell densities most cells become arrested near the G1/S border and arginase activity increases continuously during several days (unpublished observations). The possibility cannot be excluded that what appears to be induction of the enzyme, in the presence of various inhibitors of the enzyme, might in fact be secondary to effects on the progression through the mitotic cycle. Acknowledgements We wish to thank Professor Bernhard Tribukait, Dept of Radiobiology, Karolinska Institute, Stockholm, for helping us with the cytofluorimetric determinations. This study was supported by the Swedish Cancer Society, project No 260-B76-08X. References 1 Espm~.rk, J . A . ( 1 9 7 8 ) J. Biol. S t a n d . 6, 5 9 - - 6 2 2 S c h i m k e , R . T . ( 1 9 6 4 ) N a t l . C a n c e r Inst. M o n o g r . 13, 1 9 7 - - 2 1 4 3 E l i a s s o n , E. ( 1 9 6 5 ) B i o c h i m . B i o p h y s . A c t a 9 7 , 4 4 9 - - 4 5 9

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