The relationship between levels and rates of synthesis of polyamines during mammalian cell cycle

Life Sciences, Vol. 28, pp. 1497-1506 Printed in the U.S.A.

Pergamon Press

THE RELATIONSHIP B E T W E E N L E V E L S AND RATES OF SYNTHESIS OF P O L Y A M I N E S D U R I N G M A M M A L I A N C E L L C Y C L E

P r a s a d S. S u n k a r a

lW

, S e e t h a l a R a m a k r i s h n a 2, K e n j i N i s h i o k a 3 a n d Potu N. Rao I

iDepartments of Developmental Therapeutics and 3Surgery, The University of Texas System Cancer Center, ~. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030 and ~Department of Physiology and Biophysics, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, New York I179L. (Received in final form January 26, 1981) Summary The o b j e c t i v e of t h i s s t u d y was to e x a m i n e t h e r a t e of s y n t h e s i s a n d the i n t r a c e l l u l a r l e v e l s of p o l y a m i n e s as a f u n c t i o n of the HeLa c e l l c y c l e . The i n t r a c e l l u l a r l e v e l s of o r n i t h i n e , w h i c h were h i g h d u r i n g m i t o s i s a n d e a r l y G1 p h a s e , d e c r e a s e d r a p i d l y d u r i n g l a t e G1 p h a s e when the o r n i t h i n e decarboxylase a c t i v i t y was a t i t s p e a k . The a c t i v i t i e s of ornithine decarboxylase and S-adenosyl methionine decarboxylase r e a c h e d a p e a k d u r i n g G1 a n d d e c r e a s e d r a p i d l y d u r i n g the S phase. The l e v e l s of p o l y a m i n e s were m a x i m u m i n m i t o s i s a n d S phase. I n c o n s t r a s t , the r a t e of p o l y a m i n e s y n t h e s i s d u r i n g S phase w a s 5-10 fold lower than that in mitosis or G1 phase. We have also observed fluctuations in diamine-oxidase activity during the cell cycle. The enzyme activity was high during mitosis and late G1 and low during S phase. Thus, the results of this study suggest an important role for the catabolic enzymes in the regulation of polyamine levels during the m a m m a l i a n cell cycle. The stimulation of cell growth and division, both in vitro and in vivo is associated with an increase in the rate of polyamine biosy-nthesis---(l~ For example, addition of serum or insulin to plateau phase cultures (&-6), partial hepatectomy of rat liver (7,8), activation of bovine lymphocytes by lectins (9,10) and infection of cells with tumorogenic viruses (11,12) leads to an increase in the intracellular levels of polyamines. Earlier studies revealed that polyamine levels peak at two different periods in the m a m m a l i a n cell cycle; once during the late G1 period and again just before mitosis (13,1&). The enzymes involved in polyamine biosynthesis, ornithine decarboxylase and S-adenosyl methionine decarboxylase also peak at the same time during the cell cycle (15,30). These data suggest a role for polyamines in DNA synthesis and mitosis. Direct evidence for the involvement of polyamines, especially putrescine and spermidine, in DNA synthesis (13,1~, 16,17) and cytokinesis (18) of m a m m a l i a n cells has come from recent studies in which specific inhibitors of polyamine biosynthesis were used. *Present address and correspondence address : Merrell 2110 E. Galbraith Road, Cincinnati, O.H. ~5215. 0024-3205/81/131497-10502.00/0 Copyright (c) 1981 Pergamon Press Ltd.

Research

Center,

1498

Polyamine Synthesis in Cell Cycle

Vol. 28, No. 13, 1981

The r e q u i r e m e n t f o r p o l y a m i n e s f o r t h e i n i t i a t i o n of DNA s y n t h e s i s h a s been well e s t a b l i s h e d (16,19,20). I n h i b i t i o n of p o l y a m i n e b i o s y n t h e s i s has b e e n r e p o r t e d to a r r e s t n o r m a l c e l l g r o w t h d u r i n g t h e G1 p e r i o d a n d t h a t of transformed cells in th e S period (20,21). However, the levels of p o l y a m i n e s a r e r e l a t i v e l y low in S p h a s e (13,1~). It is not c l e a r w h e t h e r t h e c e l l s h a v e a h i g h r a t e of t u r n o v e r of p o l y a m i n e s a t t h i s p o i n t in t h e cell cycle. Presently, l i t t l e is k n o wn a b o u t t h e p a t t e r n of u t i l i z a t i o n ( c a t a b o l i s m ) of p o l y a m i n e s d u r i n g t h e d i v i s i o n c y c l e of m a m m a l i a n c e l l s . Hence, t h e o b j e c t i v e of t h i s s t u d y w a s to e x a m i n e not o n l y t h e a n a b o l i s m b u t a l s o t h e c a t a b o l i s m of p o l y a m i n e s d u r i n g t h e HeLa c e l l c y c l e a n d to r e l a t e t h e m to p o l y a m i n e l e v e l s a n d to d i f f e r e n t c e l l u l l a r f u n c t i o n s g o i n g on a t t h a t t i m e.

The r e s u l t s of t h i s s t u d y i n d i c a t e t h a t the l e v e l s of p o l y a m i n e s d u r i n g t h e S p h a s e a r e not s i g n i f i c a n t l y d i f f e r e n t from t h o s e of G1 c e l l s e v e n t h o u g h t h e r a t e of s y n t h e s i s a n d t h e a c t i v i t i e s of t h e b i o s y n t h e t i c e n z y m e s a r e v e r y low d u r i n g S phase. These d a t a s u g g e s t t h a t the c a t a b o l i c enzymes, such as d i a m i n e o x i d a s e and p o l y a m i n e o x i d a s e , may p l a y a regulatory r o l e in t h e m a i n t e n a n c e of p o l y a m i n e l e v e l s d u r i n g t h e c e l l cycle.

Materials

Cells

and

Cell Synchrony

and

Methods

:

HeLa c e i l s w e r e g r o w n in s p i n n e r c u l t u r e s a t 37 ° c in E a g l e ' s m i n i m a l e s s e n t i a l m e d i u m (MEM) s u p p l e m e n t e d w i t h n o n e s s e n t i a l a m i n o a c i d s , h e a t inactivated fetal calf serum (10%), sodium pyruvate, glutamine, and penicillin-streptomycin mixture. T h e s e c e l l s h a v e a c e l l - c y c l e t i m e of 22 h r consisting of 10.5 h r of pre-DNA s y n t h e t i c (G1) p e r i o d , 7 h r of DNA s y n t h e t i c p e r i o d , 3.5 h r of post-DNA s y n t h e t i c (G2) p e r i o d , a n d 1 h r of mitosis. To o b t a i n m i t o t i c p o p u l a t i o n s , HeLa cells were first partially s y n c h r o n i z e d by a s i n g l e d T h d b l o c k an d t h e n t h e y w e r e i n c u b a t e d a t 37°C f o r a b o u t 10 h r in a c h a m b e r f i l l e d w i t h N20 at a p r e s s u r e of 80 l b / i n 2 (5.36 atoms). The rounded and loosely attached mitotic cells were s e l e c t i v e l y d e t a c h e d by g e n t l e p i p e t t i n g , w h i c h y i e l d e d a p o p u l a t i o n w i t h a m i t o t i c i n d e x of 98% ( 2 2 ) . These mitotic ceils d i v i d e s y n c h r o n o u s l y and e n t e r i n t o G1 p h a s e w i t h i n 1.5 h r u p o n r e l e a s e of the N20 b l o c k ( 2 2 ) . The t r a v e r s e of t h e s m i t o t i c c e l l s t h r o u g h t h e c e l l c y c l e w as m o n i t o r e d by p u l s e labeling with [ H]-dThd and autoradiography.

Estimation

of O r n i t h i n e

Levels and

Uptake

durin~

Cell Cycle :

Mi t o t i c c e l l s w e r e p l a t e d in 60 mm p l a s t i c c u l t u r e d i s h e s . At e a c h s a m p l i n g t i m e , two 6 x 1 0 6 - c e l l b a t c h e s w e r e h a r v e s t e d by s c r a p i n g t h e dishes with a rubber policeman. The c e l l s w e r e w a s h e d t w i c e w i t h medium containing serum. One b a t c h w a s u s e d to d e t e r m i n e o r n i t h i n e l e v e l s by a model MM-60 G l e n c o a m i n o a c i d a n a l y z e r u s i n g P i c o - B u f f e r s y s t e m II ( P i e r c e Chemical Co.). The o t h e r b a t c h w a s u s e d f o r d e t e r m i n i n g t h e u p t a k e of [U-4C]ornithine. F o r th e u p t a k e s t u d i e s , 6 x 10 u c e i l s w e r e r e s u s p e n d e d in 0 . 5 ml m e d i u m c o n t a i n i n g 2.5 Ci [ U - ] 4 C ] - o r n t t h i n e (261 m C i / m m o l ) . The c e l l s w e r e i n c u b a t e d f o r 15 rain i n a n a t m o s p h e r e of 5% CO2-95% 02 a t 37°C. At the end of i n c u b a t i o n , t h e c e i l s a n d t h e medium w e r e s e p a r a t e d by centrifugation. The c e l l pellet was washed twice with medium t h e n

Vol. 28, No. 13, 1981

Polyamine Synthesis in Cell Cycle

1499

solubilized in 1 ml of soluene and transferred to a scintillation vial containing 10 ml of PCS (New England Nuclear) and the radioactivity was measured in a Packard Tricarb liquid scintillation counter.

Measurement of Ornithine Decarboxylase (SAM) Decarboxylase Activities :

(ODC) and S-adenosyl Methionine

Extracts of HeLa cells in various phases of the cell cycle were prepared in 50 m M Tris-HCl buffer, pH 7.6, containing 5 m M dithiothreitol and 501aM pyridoxal phosphate by freezing and thawing and removing the cell debris by centrifugation at 500,0 x g for I0 min. ODC activity was measured according to Oka and Perry (23) in the presence of 501aM unlabelled ornithine. Putrescine-dependent SAM decarboxylase was assayed by the method of Pegg and Williams-Ashman (24).

Assay of Polyamine

Levels and

Rate of Pool Labeling

:

HeLa cells were collected at various times ~fter reversal of the mitotic block with a rubber policeman. About 6 x lOVcells w~j{e resuspended in 0.5 ml regular culture m e d i u m containing 2.51aCi [U-'"C]-ornithine and incubated for 15 rain at 37 °C in CO 9 incubator. At the end of the incubation period, the cells and the med-lum were separated by centrifugation at 4°C in a refrigerated Sorvall RC5 centrifuge. The cell pellet was w a s h e d twice with cold medium without serum. These w a s h e d cells were then suspended in 0.2 ml of 5% HCIO/~, chilled and centrifuged. The acid extracts were neutralized with KOH and the precipitated KCI04 was removed by centrifugation. Polyamines were estimated in the neutralized acid extracts by dansylation. Dansyl polyamines were separated by thin-layer chromatography on silica gel G plates, developed four times with cyclohexane-ethyl acetate (2:1) as previously described (25) and quantitated with a Farrand Vis-UV chromatogram analyzer by comparing fluorescence of samples with standards on the same plate. The distribution of radioactivity in dansylated amines derived from the labeled orni~hine was determined by scraping the dansyl amines from the chromatograms, suspending in liquifluor and counting. About 80-90% of the individual radioactive polyamines were recovered as labelled dansyl derivatives from the chromatographic plates. The recoveries reported in this study have b e e n corrected for the losses of individual polyamines.

Calculation

of the Rate of Polyamine

Synthesis

:

The rates of putrescine, spermidine and spermine synthesis at each phase of the cell cycle was calculated based on the ratio of pool labeling of the polyamines and the specific activity (sp. act.) of ornithine or putrescine or spermidine respectively. pool labeling of polyamine Rate of polyamine

sp. act. of the polyamine (cpm/nmol)

Assay

of diamine

(cpm/106 cells)

synthesis =

oxidase

Cells were collected

precursor

activity : from various

phases of the cell cycle and w a s h e d

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Polyamine Synthesis in Cell Cycle

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Vol. 28, No. 13, 1981

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Cell cycle traverse of synchronized mitotic HeLa ceils after reversal of nitrous oxide block thrice at 4°C with m e d i u m containing no serum to remove excess of calf serum. Cell homogenates were obtained by freezing and thawing. To a rew cap tube containing 0.5 ml of cell homogenate, 0.1 mCi of [l,4"C]-putrescine in 0.i ml was a d d e d and the volume w a s brought to 2.0 ml with 0.I M sodium phosphate buffer, pH 7.0. After incubation at 37°C for 1 hr, the reaction was terminated by the addition of 0.2 ml of 2% Na2C03 and the reaction product, 1-pyrroline w a s extracted with I0 ml o ~ toluene scintillant (4 g of 2,5-diphenyl oxazole plus 0.i g of l,g-bis (g-methyl 5-phenyloxazol 2-yl) benzene in a liter of toluene) as described by • O Kobayashi (31). The suspension w a s shaken for 5 mln and frozen at -20 C. The organic phase w a s carefully collected into a scintillation vial and counted. The data presented is an average of two experiments and the duplicates agreed within 9%.

~

Autoradiography

:

^ For the determination of labeling indices, cells were incubated with [SH]-dThd (i ~Ci/ml) for 20 min, w a s h e d and deposited on clean slides by using a cytocentrifuge. The cells were fixed, extracted three times (for 20 min each) in cold 5% tricholoroacetic acid and processed for autoradiography.

Chemicals

:

L-[ I-Igc ]-Ornithine monohydrochloride (sp. act. 59 mCi/mmol) was purchased from A m e r s h a m ; L-[U-]4C]-ornithine (sp. act. 261 mCi/mmol);

Vol. 28, No. 13, 1981

Polyamine Synthesis in Cell Cycle

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Intracellular levels and the uptake of labelled ornithine from the m e d i u m during HeLa cell cycle. S-adenosyl L- [l-14C]-methionine (sp. act. 52.3 mCi/mmol) and [1,4-14C] putrescine dihydrochloride (sp. act. 60 mCi/mmol) were obtained from New England Nuclear. All the other chemicals used were of analytical grade.

Results

The cell cycle progression of the synchronized mitotic cells is shown in Fig. I. Within 2 hr after the reversal of the mitotic block, the cells rapidly divided and entered into the GI phase as evidenced by the sharp decrease of the mitotic index from 98% to 10%. The cells started entering into S phase after 7 hr. 12 hr after the reversal of the N20 block the majority of the population was actively synthesising DNA as seen by the 90% labeling index. At 15 hr, more than 50% of the cells had completed the S phase and entered the G2 period. By 18 hr, a majority of the cells were in the G2 period with a small percentage of them entering into the second round of mitosis. Based on these labeling and mitotic indices, we have considered the first hour to be mitosis, hours 1-9 to be GI phase, 9-15 to be the S phase, and hours 15-18 to be the G2 period. The cellular ornithine levels and the uptake of [14C]-ornithine by HeLa cells varied throughout the cell cycle (Fig. 2). Ornithine levels, which are high during mitosis, increased further during the early G1 period but decreased sharply during the late G1 and early S period. These levels remained relatively stable during the rest of the cell cycle. In contrast,

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Ornithine decarboxylase and S-adenosyl methionine decarboxylase activities during HeLa cell cycle. the uptake of [14C]-ornithine from the medium was low during mitosis and G1 phase, but increased rapidly during the S phase, and came d o w n J~ring the G2 period. The inverse relationship between the uptake of [1,4-"C]-ornithine and its cellular levels resulted in a sharp increase in the specific radio activity of the cellular ornithine during the S and G2 phases.

The ODC activity exhibited two peaks, a major peak during the GI-S transition and a minor one during G2-mitosis transition. In contrast SAM decarboxylase reached its peak activity during G1 but its level rapidly decreased during the S phase (Fig. 3). The activities of both these enzymes were quite low when the rate of DNA synthesis was at its m a x i m u m . The relationship between putrescine levels and its rate of synthesis during the cell cycle is shown in Fig. 4. Putrescine levels were highest during mitosis, but dropped rapidly during the Gl phase. After a further decrease during S period, putrescine levels started going up sharply during G2 phase. On the other hand, the total pool labeling, which was low during mitosis, increased during late G1 reaching a peak in the S phase followed by a decline during G2. However, the net putrescine synthesis (calculated by dividing the total pool labeling by the specific activity of ornithine) was high during mitosis and G1 and decreased rapidly during S and G2 phases.

Vol. 28, No. 13, 1981

Polyamine Synthesis in Cell Cycle

1503

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Levels and rates of synthesis of putrescine, spermidine and spermine during HeLa cell cycle. Similar trends were observed for spermidine and spermine during the HeLa cell cycle (Fig. g). 5permidine and spermine contents were highest in mitosis and decreased rapidly during G1 phase. Later the levels of spermidine and spermine increased slowly during 5 phase and again dropped in G2 phase. Pool labelling of spermidine and spermine increased rapidly up to the middle of S phase and declined thereafter. Two peaks for the rate of spermidine synthesis one during mitosis and another during G1 were observed. The rate of spermine synthesis was highest during mitosis and rapidly declined during S phase and then remained constant. The fact that the polyamine levels during the S phase are not significantly different from those of the G1 phase, although the activities of the biosynthetic enzymes and the rates of polyamine synthesis were the

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HOURS AFTER REVERSAL OF N 2 0 BLOCK FIGURE 5 Diamine oxidase activity during HeLa cell cycle lowest in 5 phase, suggests a minimal rate of catabolism of polyamines during this period of the cell cycle. In fact diamine oxidase activity was found to be high during mitosis and late GI and low during S pha~e w h e n there is active DNA synthesis (Fig. 5). Discussion. The results of this study indicate that the intracellular levels of ornithine are high during mitosis and the early G1 phase and rapidly decrease during the late G1 phase just before the initiation of DNA synthesis, w h e n the activity of ornithine decarboxylase is at its peak (Fig. 2 and 3). A similar increase in ornithine pools was observed in HTC cells stimulated to divide following refeeding with fresh m e d i u m (26). Our s~udies have revealed cell cycle-specific fluctuations in the uptake of [14C]- ornithine by the cells. The rate of uptake was low during mitosis and GI, but increased rapidly and reached a peak during the S phase.

The changes we have observed as a function of the HeLa cell cycle in the activities of the two biosynthetic enzymes, ODC and S A M decarboxylase and the levels of polyamines, putrescine and spermidine in particular, are in agreement with the earlier reports on Chinese hamster ovary cells (I~,15) and rat hepatoma cells ( 1 3 ) except that increase in SAM decarboxylase preceded the increase in ODC activity during the G1 period. In the present study we find that the relative rates of synthesis of the three polyamines we studied were high during mitosis and G1 phases, but

Vol. 28, No. 13, 1981

Polyamine Synthesis in Cell Cycle

1505

decreased rapidly and reached the lowest levels during the S phase (Fig. 4). In spite of such a drastic reduction in the rate of polyamine synthesis, their levels remained fairly high, particularly in the case of spermidine and spermine (Fig. ~). Since polyamines are k n o w n to play a role in DNA synthesis (15-17, 19) one would expect a high rate of polyamine synthesis during the S phase. But the observed rate of polyamine synthesis during the S phase was i/i0 of that during mitosis cr the G1 phase. These results suggest that the turnover rate of polyamines is very high during the mitosis and G1 phases and the lowest during the S phase. Further, the diamine oxidase activity was high during mitosis and late G1 and Iow during S phase of the cell cycle. These results indicate that the cell maintains its levels of polyamines that are necessary for DNA synthesis in S phase by regulation of the degradation rather than the biosynthesis of the polyamines.

T h e r e a r e two e n z y m e s i n v o l v e d i n the d e g r a d a t i o n of n a t u r a l l y o c c u r i n g p o l y a m i n e s . The enzyme d i a m i n e o × i d a s e c a t a l y z e s the c o n v e r s i o n of p u t r e s c i n e to 4 - a m i n o b u t y r a l d e h y d e , ammonia, and hydrogen peroxide (27). R e c e n t l y H~ltt~ (28) d i s c o v e r e d the e x i s t e n c e of a n o t h e r e n z y m e , polyamine oxidase. Thi.s e n z y m e c a t a l y z e s the i n t e r c o n v e r s i o n s of p o l y a m i n e s , s i n c e s p e r m i n e is c l e a v e d to s p e r m i d i n e a n d a m i n o - p r o p i o n a l d e h y d e , w h e r e a s s p e r m i d i n e is c o n v e r t e d to p u t r e s a i n e a n d a m i n o p r o p o n a l d e hyde. Recent: s t u d i e s b y Q u a s h et a l (29) i n d i c a t e a v a r i a t i o n i n the a c t i v i t i e s of d i a m i n e o x t d a s e a n d p o l y a m i n e o x i d a s e a s s o c i a t e d with the grow~:h of the n o r m a l a n d t r a n s f o r m e d c e l l s . D u r i n g e x p o n e n t i a l g r o w t h t h e r e w a s a s t e e p d e c l i n e in the a c t i v i t y of d i a m i n e o x i d a s e a n d as the c e l l s a p p r o a c h e d c o n f l u e n c y the a c t i v i t y of d i a m i n e o x i d a s e i n n o r m a l c e i l s was t w o - f o l d g r e a t e r t h a n t n a t of t r a n s f o r m e d c e i l s . The d e c r e s e d r a t e of p u t r e s c i n e d e g r a d a t i o n c o u l d a c c o u n t for the i n c r e a s e d level~.; of p u t r e s c i n e f o u n d i n a n u m b e r of t r a n s f o r m e d c e l l s . I n the p r e s e n t s t u d y we h a v e o b s e r v e d two p e a k s of a c t i v i t i e s for d i a m i n e o x i d a s e d u r i n g c e l l c y c l e . One i n m i t o s i s a n d a n o t h e r p e a k d u r i n g l a t e G1. The a c t i v i t y of the enzyme was low d u r i n g e a r l y G1 a n d S p h a s e . These r e s u l t s s u g g e s t a n i m p o r t a n t role for the c a t a b o l i s m of p o l y a m i n e s i n the r e g u l a t i o n of i n t r a c e l l u l a r c o n c e n t r a t i o n of p o l y a m i n e s d u r i n g the cell c y c l e of m a m r r a l i a n c e l l s . A_cknowledsements Thi s i n v e s t i g a t i o n was supported by g r a n t s CA-05831, CA-14528, a n d CA-23878 from the N a t i o n a l C a n c e r I n s t i t u t e , DHEW.

CA-11520,

References 1. 2. 3. 4. 5. 6. 7. 8.

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