Dissociation of RNA synthesis from the calcium requirement for serum-increased ornithine decarboxylase activity in rat glioma cells

Biochimica et Biophysica Acta, 801 (1984) 87-98 Elsevier

87

BBA 21809

DISSOCIATION OF RNA SYNTHESIS FROM THE CALCIUM REQUIREMENT FOR SERUM-INCREASED ORNITHINE DECARBOXYLASE ACTIVITY IN RAT GLIOMA CELLS JACKSON B. GIBBS

*

and GARY BROOKER **

Department of Biochemistry, Georgetown University Medical Center, Washington, DC 20007 (U.S.A.) (Received October 24th, 1983) (Revised manuscript received April llth, 1984)

Key words: Ornithine decarboxylase; Ca 2 + dependence," RATA synthesis," (Rat glioma)

When C6-2B rat glioma cells were stimulated with calf serum in the presence of calcium, ornithine decarboxylase activity increased maximally in 6 - 8 h after an initial 2 - 3 h lag period wherein RNA synthesis occurred. The increase of ornithine decarboxylase activity in serum-stimulated C6-2B cells was prevented by the calcium chelator EGTA, but EGTA had no effect upon RNA synthesis as judged by [3H]uridine incorporation into RNA. In addition, the calcium requirement for increased ornithine decarboxylase activity was temporally distal to the lag period. EGTA appeared to inhibit the synthesis of ornithine decarboxylase, because the half-life values of ornithine decarboxylase activity were similar (37-47 min) in the presence of EGTA or protein synthesis inhibitors such as cycloheximide or emetine. Also, calcium readdition rapidly reversed EGTA inhibition of ornithine decarboxylase activity by a mechanism which could be blocked by cycloheximide.

Introduction

Ornithine decarboxylase (L-ornithine carboxylyase, EC 4.1.1.17) catalyzes the conversion of D-ornithine to putrescine, and this reaction is the first step of polyamine biosynthesis [1]. Increased polyamine biosynthesis due to increased ornithine decarboxylase activity occurs upon initiation of cell proliferation and differentiation [2,3]. In mammalian cells, the polyamine requirement for cell proliferation is associated with cell progression through the S phase [3,4]. Serum, catecholamines, steroids and a variety * Present address: Merck, Sharp and Dohme Research Laboratories, Virus and Cell Biology Research, West Point, PA 19486, U.S.A. ** To whom correspondence should be addressed. Abbreviation: DRB, 5,6-dichloro-l-fl-D-ribofuranosylbenzimidazole. 0304-4165/84/$03.00 © 1984 Elsevier Science Publishers B.V.

of other hormones and agents cause' cellular ornithine decarboxylase activity to rise dramatically [2,5,6]. Even though changes in immunoreactive ornithine decarboxylase correspond to changes in ornithine decarboxylase activity [7-10], the precise mechanisms for these fluctuations are not clearly defined [1,11]. The mechanism for increased ornithine decarboxylase activity is thought to be via enzyme induction involving RNA and protein synthesis, because RNA synthesis inhibitors, such as actinomycin D, and protein synthesis inhibitors, such as cycloheximide, prevent the rise of ornithine decarboxylase activity in cells stimulated with a wide variety of agents [5]. However, drugs such as actinomycin D and cycloheximide exert many other biological actions [12-21], and thus it is not known with certainty whether these compounds inhibit ornithine decarboxylase activity by specific effects on RNA and protein synthe-

88 sis. Regulation of ornithine decarboxylase activity by post-translational modification must also be considered, because ornithine decarboxylase activity is inhibited by phosphorylation [22], transamination [23], and interaction with 26.5 kDa protein termed 'antizyme' [24]. Ca 2+ is an obligatory requirement for increased cellular ornithine decarboxylase activity [25-32]. The calcium-dependent event(s) for increased ornithine decarboxylase activity has not been defined, but calcium flux into the cell appears to be involved, as determined by experiments using the calcium ionophore A23187 [28,29,31,32], and by those using inhibitors of calcium flux, such as lanthanum ion, verapamil and nifedipine [25,29]. One approach for identification of the calciumdependent event(s) necessary for increased ornithine decarboxylase activity involves analysis of steps which might be required for increased ornithine decarboxylase activity following stimulation of cells. Potential calcium-regulated steps could include second messenger generation, RNA synthesis and processing, protein synthesis and processing, and post-translational modifications. Previously, we showed that the calcium requirement for increased ornithine decarboxylase activity in C6-2B rat glioma cells was temporally distal to the generation of a second messenger, such as cAMP [27]. Calcium does not appear to modulate ornithine decarboxylase activity in broken ceils, because in vitro ornithine decarboxylase activity is not affected by Ca 2÷ or the calcium chelator EGTA [26,29]. Thus, it seemed possible that the calcium requirement for increased cellular ornithine decarboxylase activity might be associated with an RNA or protein synthetic event. Since it has been demonstrated that in vitro translation of proteins can occur in the presence of EGTA [33,34], we tested whether calcium chelation by EGTA inhibited whole cell ornithine decarboxylase activity by interfering with RNA synthesis. Because it is presumed that RNA synthesis is required for increased ornithine decarboxylase activity, we first determined the temporal requirement of RNA synthesis for increased ornithine decarboxylase activity in serum-stimulated C62B cells. Results in this report provide detailed evidence that RNA synthesis is required for serum-stimulated ornithine decarboxylase activity in C6-2B

cells; however, the calcium dependence for increased ornithine decarboxylase activity is not associated with [3H]uridine incorporation into RNA. Experiments with and without RNA synthesis inhibitors suggest that an RNA synthetic period exists following serum stimulation of C6-2B cells. The calcium requirement for increased ornithine decarboxylase activity is temporally distal to this RNA synthetic period. In addition, the calcium chelator EGTA inhibits increased ornithine decarboxylase activity in a manner clearly distinct from that observed with R N A synthesis inhibitors. EGTA appears to inhibit C62B cell ornithine decarboxylase activity by interfering with an event necessary for ornithine decarboxylase synthesis.

Experimental procedures Materials

L-[1-14C]Ornithine (40-60 mCi/mmol), L-[2,33H]ornithine (15-30 Ci/mmol) and phenethylamine were obtained from New England Nuclear. L-[4,5-3H]Leucine (40 Ci/mmol) and [5,6-3H] uridine (55 Ci/mmol) were from ICN. Cycloheximide, emetine, puromycin, actinomycin D, dimethyl sulfoxide (Me2SO), L-ornithine, pyridoxal 5'-phosphate, dithiothreitol, tris (hydroxymethyl) aminomethane, EDTA, EGTA and putrescine (free base) were from Sigma. DRB was purchased from Calbiochem-Behring. Trichloroactic acid was from Baker, and sodium citrate was from Mallinckrodt. Dowex 50W-X8 (100-200 mesh) was from BioRad. Cell culture

Rat glioma cells (C6-2B, passage 12-38) were grown as monolayers at 37°C in a humidified incubator (95% air/5% CO2) in Ham's F-10 medium (GIBCO) supplemented with 10% donor calf serum (Flow Labs or M.A. Bioproducts) in the absence of antibiotics. Cells were grown in 35-mm plates (Coming) and in 6-well cluster plates (35 mm wells, Costar), using 3 ml of culture medium. The medium was changed on days 3 and 7 after plating, and then on day 8. The confluent cells were washed once with serum-free Ham's F-10 medium and incubated in 2 ml Ham's F-10 medium for 15-18 h. Experiments were initiated at the end

89 of this 15-18 h incubation by adding donor calf serum and 100-fold concentrated aliquots of drugs in 0.9% NaC1. Incubations were stopped by washing the cells once with ice-cold phosphate-buffered saline (10 mM sodium phosphate/0.9% NaC1 (pH 7.3)) and twice with ice-cold ornithine decarboxylase assay buffer (25 mM Tris-HC1/1 mM E D T A / 5 mM dithiothreitol (pH 7.4) at 20°C). Cells were exposed to assay buffer for less than 2 rain, and no ornithine decarboxylase activity was detected in the hypotonic buffer after washing the cells. Cells were dislodged from the culture plates in 0.2 ml assay buffer and then lysed by freezethawing. Ornithine decarboxylase activity was determined in the supernatant fraction after centrifugation at 1700 x g for 20 min.

Ornithine decarboxylase assays Two assay procedures were used to measure ornithine decarboxylase activity. One method measured the formation of 14CO2 when L-[114C]ornithine was u s e d as substrate. During the course of this research, a sensitive and reproducible ornithine decarboxylase assay was developed in our laboratory, and by this method the formation of [3H]putrescine from L-[2,3-3H]ornithine was measured (Lacher, D.L., Gibbs, J.B., Simpson, L. and Brooker, G., unpublished data). (I) Conversion of [I- t4C]ornithine to t4COe. Reactions were done in 17 x 100 mm polypropylene tubes (Falcon) fitted with a rubber stopper (Kontes) holding a polyethylene center well (Kontes). The reaction mixture contained assay buffer, 100 # M pyridoxal 5'-phosphate, and 0.5 /~Ci L-[1-14C]ornithine (60 #M). The reaction volume was 100 #1, and assays were done for 1 h at 37 °C. The reaction was stopped by injecting 0.2 ml of 0.2 M H2SO 4 into the bottom of the reaction vessel, and liberated 14CO2 was adsorbed on filter paper in the center well containing 0.1 ml phenethylamine.

(2) Conversion of [3H]ornithine to [~H]putrescine. Reactions were done in 12 x 75 mm borosilicate tubes. The reaction mixture contained assay buffer, 100 # M pyridoxal 5'-phosphate, 60 # M L-ornithine and 0.5-1.0 #Ci L-[2,3-3H]ornithine. The reaction volume was 100 #1, and assays were done for 1 h at 37 ° C. After the incubation, 1 ml ice-cold 0.2 M sodium citrate (pH 5.8) was added,

and the mixture was transferred to a pasteur pipet column (0.5 x 2.0 cm) of Dowex 50W-X8 (100-200 mesh) cation-exchange resin. Unreacted [3H]ornithine was eluted with 10 ml 0.2 M sodium citrate (pH 5.8), and [3H]putrescine was eluted directly into scintillation vials with 2.5 ml 0.1 M N a O H plus 0.1 M putrescine. Scintillation fluid (13 ml) contained Triton X-100, Triton X-114 and xylenes in a ratio of 1 : 2 : 7, 7 g / l diphenyloxazole and 20 mM glacial acetic acid to neutralize the base in the eluate. This ornithine decarboxylase assay is readily interchangeable with the ornithine decarboxylase assay that measures the formation of 14CO2.

Incorporation of radiolabeled compounds [3H]Leucine incorporation into protein and [3H]uridine incorporation into RNA were determined by adding 2.5 #Ci of the radiolabeled compounds to the cells. After the cells were incubated, the medium was aspirated, and labeled material was precipitated with 1.5 ml ice-cold 5% trichloroacetic acid. The pellet was washed once with acid, and the precipitated material was dissolved in 1.5 ml 0.2 M NaOH. Blanks were determined by adding 2.5 #Ci of the radiolabeled compounds to plates containing trichloroacetic acid instead of culture medium. Radioactivity was determined in an aliquot of the N a O H extract, and protein was determined in another aliquot by the method of Lowry et al. [35] adapted for automated analysis. Results

Delineation of an RNA synthetic period required for serum-stimulated ornithine decarboxylase activity In C6-2B cells stimulated with 18% donor calf serum, ornithine decarboxylase activity increased dramatically over basal levels after a lag period of 2-3 h and reached a maximum after 6-8 h. It was tested whether the lag period which preceded the increase of ornithine decarboxylase activity corresponded to an RNA synthetic period. Addition of the RNA synthesis inhibitors DRB (100 #M) and actinomycin D (5 #M) at the time of serum addition effectively inhibited the rise of ornithine decarboxylase activity (Fig. 1). However, addition of DRB and actinomycin D at later times resulted in

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Fig. 1. Effect of RNA synthesis inhibitors added at different times after serum stimulation of C6-2B cells. Actinomycin D (5 /~M) (O), 100 /~M DRB (zx) or the solvent Me2SO (1%) ((3) were added to C6-2B cells 0.5, 1, 2 or 3 h after the addition of 18% donor calf serum. Ornithine decarboxylase activity was measured 6 h after the time of serum addition. Basal ornithine decarboxylase activity was less than 5 pmol/h per mg protein, and ornithine decarboxylase activity in the absence of any drugs or solvent was 2.01 nmol/h per nag protein. Data are the mean of four determinations.

a progressively diminished inhibition of the ornithine decarboxylase activity response to serum. N o inhibition of ornithine decarboxylase activity was observed when D R B and actinomycin D were a d d e d to C6-2B cells 3 h after serum stimulation (i.e., 3 h with serum and then 3 h with serum plus D R B or actinomycin D). The vehicle for D R B and actinomycin D was dimethyl sulfoxide (Me2SO), and this solvent did not affect ornithine decarboxylase activity (Fig. 1). These results suggested that R N A synthesis was necessary for serum-increased ornithine decarboxylase activity and that R N A synthesis occurred during a 3 h period following serum stimulation of C6-2B cells. Furthermore, the R N A synthesized was stable for at least 3 h, because ornithine decarboxylase activity measured 6 h after serum addition was not inhibited when D R B or actinomycin D were added 3 h after serum addi-

tion. One must qualify these conclusions with the presumption that the only cellular actions of D R B and actinomycin D are the inhibition of R N A synthesis. However, it is not k n o w n whether D R B or actinomycin D exert other cellular actions which result in inhibition of ornithine decarboxylase activity. Therefore, the concept that an R N A synthetic phase was necessary for serum-increased ornithine decarboxylase activity in C6-2B cells was further tested by experiments using the following protocol. C6-2B cells were incubated with serum for up to 3 h (the time frame of the lag period) and then washed and incubated with serum-free H a m ' s F-10 medium. These pretreated cells were rechallenged with serum, and the time required for serum to increase ornithine decarboxylase activity in these cells was c o m p a r e d to the time required for serum to increase ornithine decarboxylase activity in previously untreated C6-2B cells (naive cells). The ornithine decarboxylase activity of cells rechallenged with serum after an initial serum treatment is shown in Fig. 2 (ser,ser vs. con,ser). W h e n C6-2B cells previously treated with serum were rechallenged with serum, ornithine decarboxylase activity increased above the serum-free control within 1 h and reached a m a x i m u m within 3 - 5 h (Fig. 2). Serum stimulation of cells which were not previously treated with serum caused ornithine decarboxylase activity to increase after a 2 - 3 h lag period. If the more rapid appearance of ornithine decarboxylase activity in serum-pretreated C6-2B cells was due to R N A synthesis which occurred during the pretreatment period, then the effect should be blocked by the inclusion of an R N A synthesis inhibitor during the initial serum pulse. The R N A synthesis inhibitor D R B was used to test this hypothesis, because the inhibitory effect of D R B on R N A synthesis is readily reversed by washing cells [36], and also because D R B inhibition of C6-2B cell ornithine decarboxylase activity was found to be closely correlated with inhibition of [3H]uridine incorporation into R N A ( 1 - 1 0 0 /~M D R B , n --- 5, slope = 0.99, y intercept = - 0 . 9 6 , r = 0.994). Inclusion of 30 ~ M D R B during the initial serum pretreatment time markedly reduced the ornithine decarboxylase activity response to a second serum challenge (Fig. 2A). The times required for serum to increase ornithine decarboxyl-

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Fig. 2. Effect of DRB during the serum pretreatment on the ornithine decarboxylase activity response to a second serum treatment. C6-2B cells were incubated for 3 h in the absence of serum (O,O) or in the presence of 18% calf serum with ([],11) or without (z~,*) DRB (A, 30 #M; B, 100 /JM). All cells were washed twice with serum-free Ham's F-10 medium and then incubated in the absence of serum for less than 30 rain. Cells were then incubated for the times indicated on the abscissa in the presence (O,Q, zx) or absence (e,ll,A) of 18% calf serum. No DRB was added after the cells were washed. The abbreviations ser and con refer to serum or control (no serum) treatment. The first notation refers to the condition during the initial 3 h incubation, and the second notation refers to the condition during the times indicated on the abscissa. Data are the mean of duplicate determinations, and the results in A were obtained in a different experiment than the results in B.

ase activity a b o v e the serum-free c o n t r o l s were 2 h for s e r u m - p r e t r e a t e d cells, 3 h for cells p r e t r e a t e d with serum in the presence of 3 0 / ~ M D R B , a n d 4 h for previously u n t r e a t e d cells. D R B (30 /~M) p r e v e n t e d the transient o r n i t h i n e d e c a r b o x y l a s e activity response in cells p u l s e - t r e a t e d with serum a n d then i n c u b a t e d in the absence o f s e r u m for the r e m a i n d e r o f the experiment. However, 30 # M D R B d i d n o t c o m p l e t e l y prevent the p h e n o m e n o n o b s e r v e d with s e r u m p u l s e - t r e a t m e n t a n d rechallenge of C6-2B cells. In C6-2B cells, 3 0 / L M D R B i n h i b i t e d [3H]uridine i n c o r p o r a t i o n i n t o acid-insoluble m a t e r i a l b y 62%. W h e n the e x p e r i m e n t s h o w n in Fig. 2 A was r e p e a t e d with 100 F M D R B (which i n h i b i t e d [3H]uridine i n c o r p o r a t i o n b y 85%), o r n i t h i n e d e c a r b o x y l a s e activity in s e r u m plus 100 /~M D R B - p r e t r e a t e d cells increased a p p r o x i m a t e l y at the s a m e time as o r n i t h i n e dec a r b o x y l a s e activity in cells which were n o t pretreated with s e r u m (Fig. 2B). T h e lag p e r i o d o b served with serum plus D R B - p r e t r e a t e d cells cann o t be due to a residual effect of D R B in the cells, b e c a u s e the i n h i b i t i o n o f [3H]uridine i n c o r p o r a tion into a c i d - i n s o l u b l e m a t e r i a l b y D R B was r e a d i l y reversed. [3H]Uridine i n c o r p o r a t i o n into R N A after washing cells previously treated with 30 F M or 1 0 0 / ~ M D R B was 97 a n d 88%, respectively, of that o b s e r v e d in c o n t r o l cells (no D R B treatment). Thus, it a p p e a r s that serum p r e t r e a t m e n t of C6-2B cells p r o m o t e s R N A synthesis necessary for increased o r n i t h i n e d e c a r b o x y l a s e activity. These d a t a suggested that s e r u m caused an event to occur in C6-2B cells which required 2 - 3 h to develop, a n d was i n h i b i t e d b y D R B . Thus, the conclusions b a s e d on the d a t a in Fig. 1 were s u p p o r t e d . M o s t i m p o r t a n t , these results suggested that R N A synthesis necessary for s e r u m - s t i m u l a t e d o r n i t h i n e d e c a r b o x y l a s e activity o c c u r r e d d u r i n g the lag period, a n d that D R B a n d a c t i n o m y c i n D exerted their i n h i b i t o r y effect on o r n i t h i n e d e c a r b o x y l a s e activity b y b l o c k i n g R N A synthesis, r a t h e r than b y i n h i b i t i n g some o t h e r cellular process.

Dissociation of the calcium requirement for increased ornithine decarboxylase activity from [3H]uridine incorporation into RNA Upon

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after serum stimulation of C6-2B cells in the absence of calcium. C6-2B ceils were preincubated for 2 h with 0.4 mM EGTA before addition of calcium-free serum (18%). Calcium (0.4 mM) was readded to some cells at the time of calcium-free serum addition (O). Other cells were incubated with calcium-free serum plus 0.4 mM EGTA, and then 0.4 mM calcium was readded at 2 h (A, first arrow) or 6 h (D, second arrow). (,7) No serum; (I) calcium-free serum plus 0.4 mM EGTA. Inset: In a different experiment, C6-2B cells were pretreated with 0.4 mM EGTA for 2 h ([3) or 6 h (O) before the addition of calcium-free serum. 5 h after serum addition, 0.4 mM calcium was readded, and the rise of ornithine decarboxylase activity was compared to the maximally increased ornithine decarboxylase activity in cells which received calcium at the time of serum addition (1530 pmol/h per mg protein). Data are the mean of duplicate determinations.

r e q u i r e d for s e r u m - i n c r e a s e d o r n i t h i n e d e c a r b o x y l ase activity, it was tested w h e t h e r the c a l c i u m r e q u i r e m e n t for C6-2B cell o r n i t h i n e d e c a r b o x y l ase activity was a s s o c i a t e d with this period. In e x p e r i m e n t s involving E G T A , the calcium c o n t e n t of serum was r e d u c e d b y dialysis for 18 h a g a i n s t 100 vol. of 0.4 m M E G T A a d j u s t e d to p H 7.0-7.5 with N a O H . This serum will b e referred to as ' c a l c i u m - f r e e ' serum. I n C6-2B cells p r e t r e a t e d for 2 h with 0.4 m M E G T A b e f o r e a s e r u m challenge, o r n i t h i n e d e c a r b o x y l a s e activity d i d n o t increase in r e s p o n s e to calcium-free s e r u m plus 0.4 m M E G T A (Fig. 3) as has b e e n shown previously [27], E G T A p r e t r e a t m e n t d i d n o t affect the responsiveness of C6-2B cells, b e c a u s e s e r u m s t i m u l a t i o n in the presence of c a l c i u m c a u s e d o r n i t h i n e dec a r b o x y l a s e activity to increase after a 2 - 3 h lag p e r i o d b e f o r e a m a x i m u m was o b s e r v e d at 7 h. ' A b s e n c e of c a l c i u m ' refers to i n c u b a t i o n s in the

p r e s e n c e of 0.4 m M E G T A . H a m ' s F-10 m e d i u m c o n t a i n s 0.3 m M calcium, a n d b y using the app a r e n t E G T A - c a l c i u m association c o n s t a n t of 7.6 • 106 M -1 at p H 7.1 [37], free extracellular calcium in the presence of 0.4 m M E G T A was calculated to be 0.4 ttM. W h e n E G T A - p r e t r e a t e d cells were s t i m u l a t e d with serum for 2 h in the absence of calcium, r e a d d i t i o n of 0.4 m M calcium caused o r n i t h i n e d e c a r b o x y l a s e activity to increase gradually (Fig. 3, first arrow). T h e rise of o r n i t h i n e d e c a r b o x y l a s e activity was t e m p o r a l l y similar to the rise of o r n i t h i n e d e c a r b o x y l a s e activity in cells which received c a l c i u m at the time of serum a d d i tion. Thus, r e a d d i t i o n of c a l c i u m d u r i n g the time f r a m e of the lag p e r i o d d i d not m a r k e d l y affect the t i m e that o r n i t h i n e d e c a r b o x y l a s e activity increased. O r n i t h i n e d e c a r b o x y l a s e activity d i d not increase by a d d i t i o n of 0.4 m M m a g n e s i u m in the presence of calcium-free serum plus 0.4 m M E G T A or b y a d d i t i o n of 0.4 m M calcium in the a b s e n c e o f serum ( d a t a n o t shown), as was d e m o n s t r a t e d previously [27]. W h e n 0.4 m M c a l c i u m was re-add e d to cells which h a d been previously treated with serum for 6 h in the absence of calcium (Fig. 3, second arrow), o r n i t h i n e d e c a r b o x y l a s e activity increased m a r k e d l y within 30 rain, a n d no app a r e n t lag p e r i o d was observed. T h e m a x i m u m level o f o r n i t h i n e d e c a r b o x y l a s e activity was 85% o f the level in cells which received calcium at the time of serum addition. Since it was p o s s i b l e that a 2 h E G T A preinc u b a t i o n was not sufficient time to inhibit a c a l c i u m - d e p e n d e n t lag p e r i o d event, some cells were p r e t r e a t e d with 0.4 m M E G T A for 6 h before s e r u m s t i m u l a t i o n in the a b s e n c e of calcium. 6 h was chosen b e c a u s e 0.4 m M E G T A inhibited C62B cell o r n i t h i n e d e c a r b o x y l a s e activity greater t h a n 95% within 5 h (Ref. 27, and vide infra). W h e n cells were treated with 0.4 m M E G T A for 6 h in the absence of serum and then 5 h with serum in the absence of calcium, r e a d d i t i o n of 0.4 m M calcium caused o r n i t h i n e d e c a r b o x y l a s e activity to increase without an a p p a r e n t lag p e r i o d (Fig. 3, inset). The rate at which o r n i t h i n e d e c a r b o x y l a s e activity increased was the same as that seen in cells p r e i n c u b a t e d with E G T A for 2 h. These d a t a suggested that the c a l c i u m r e q u i r e m e n t for o r n i t h i n e d e c a r b o x y l a s e activity was not associa t e d with events such as R N A synthesis which

93 occur during the lag period following serum stimulation of C6-2B cells. Although the data in Fig. 3 implied that the calcium requirement for serum-stimulated ornithine decarboxylase activity was temporally distal to the lag period, it was possible that treatment of C6-2B cells with E G T A for 6 h did not sufficiently deplete intracellular calcium which might be involved with cellular R N A synthesis. Previously, it was demonstrated that E G T A inhibited epinephrine-stimulated ornithine decarboxylase activity in C6-2B cells greater than 50% within 2 h [27]. Thus, it was tested whether R N A synthesis inhibitors could exert an effect on ornithine decarboxylase

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Fig. 4. Time-course of EGTA, actinomycin D and DRB inhibition of serum-increased ornithine decarboxylase activity. C6-2B cells were stimulated for 6 h with 185~ calf serum before addition of 0.4 mM EGTA (©), 5 ~M actinomycin D (~) or 100 /LM DRB (zx). Ornithine decarboxylase activity was measured every hour for the next 5 h after addition of the inhibitots. After 3 h in the presence of EGTA, 0.6 mM calcium was readded to some cells (O). Me2SO (1%), the solvent for actinomycin D and DRB, did not inhibit ornithine decarboxylase activity. Ornithine decarboxylase activity in the absence of serum was less than 5% of the uninhibited serum-increased ornithine decarboxylase activity. All values are expressed as the percentage of the uninhibited response at each time point. Data with actinomycin D and DRB are the mean of triplicate determinations. Data with EGTA are the mean + S.E. of nine values determined in three experiments. * P < 0.05 when compared to the preceding time point.

activity in t h i s t i m e frame. As shown in Fig. 4, 5 /~M actinomycin D or 100 /~M D R B did not inhibit C6-2B cell ornithine decarboxylase activity for approx. 3 h; however, by 4 h, actinomycin D inhibited ornithine decarboxylase activity by 50%, and D R B inhibited ornithine decarboxylase activity by 25%. The vehicle solvent Me2SO did not inhibit ornithine decarboxylase activity, as has been demonstrated in Fig. 1. E G T A inhibited serumstimulated ornithine decarboxylase activity after a lag period of 1 h (Fig. 4). Within 2 h, 50% inhibition of ornithine decarboxylase activity was observed, and greater than 95% inhibition of ornithine decarboxylase activity occurred by 5 h. Readdition of 0.6 m M calcium reversed the E G T A effect. Ornithine decarboxylase activity increased, without an apparent lag period, to 60% of the uninhibited response within 2 h of calcium readdition. Reversal to 100% of the uninhibited response 3 h after calcium readdition was observed in some, but not all, experiments. These data demonstrate that actinomycin D and D R B inhibit increase of ornithine decarboxylase activity in a m a n n e r temporally distinct from the inhibitory effect of E G T A . Actinomycin D (1 /xM) and D R B (100 # M ) effectively inhibited [3H]uridine incorporation into acid-insoluble material by 85% within 20 min, while E G T A did not affect [3H]uridine incorporation for 1 h (data not shown). Thus, actinomycin D and D R B did not inhibit ornithine decarboxylase activity for at least 3 h, even though these drugs rapidly inhibited R N A synthesis. In contrast, E G T A did not exert an effect on R N A synthesis for at least 1 h, but inhibited ornithine decarboxylase activity by 50% within 2 h. These data provide evidence that the inhibitory effect of E G T A on C6-2B cell ornithine decarboxylase activity is not associated with [3H]uridine incorporation into R N A . However, the data do not preclude the possibility that E G T A m a y affect some aspect of R N A metabolism which occurs after R N A synthesis.

Apparent effect of EGTA on ornithine decarboxylase synthesis It was possible that E G T A might inhibit ornithine decarboxylase activity either by altering a process required for omithine decarboxylase synthesis or b y affecting a process which m o d -

94

ulated ornithine decarboxylase activity at the post-translational level. The first of these two possibilities was tested. When calcium was readded to C6-2B cells which had been stimulated with serum for 5 h in the absence of calcium, the rapid ornithine decarboxylase activity response to calcium was completely blocked by concurrent incubation with 5 /~g/ml cycloheximide (Fig. 5). Actinomycin D (1 /~M) or DRB (100 /xM) did not prevent the rapid rise of ornithine decarboxylase activity, as would be predicted from the results presented earlier in this report, In the absence of cycloheximide, ornithine decarboxylase activity increased to the maximum level observed in cells which received calcium at the time of serum addition. These results suggested that de novo protein synthesis was required for the rapid increase of ornithine decarboxylase activity after calcium readdition. The

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results also supported the concept that the calcium requirement for C6-2B cell ornithine decarboxylase activity was not associated with RNA synthesis. Thus, EGTA appeared to affect the synthesis of ornithine decarboxylase, rather than the modulation of ornithine decarboxylase activity at the post-translational level. It was necessary to test whether EGTA inhibited ornithine decarboxylase synthesis by a general intracellular mechanism. The data in Fig. 6 i

,~

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Time (hr) Fig, 5, Effect of cycloheximide, actinomycin D and DRB on the rapid increase of ornithine decarboxylase activity after calcium readdition. C6-2B cells were pretreated with 0.4 mM EGTA for 2.5 h before a 5 b incubation with calcium-free serum (18%) in the absence of calcium. Then, 0.4 mM calcium was readded to cells 10 rain after the addition of 1 ~M actinomycin D (U), 100/~M DRB (zx), 5 ~ g / m l cycloheximide (v) or 1% Me, SO (O). The star at 90 min indicates ornithine decarboxylase activity in cells which received calcium at the time of serum addition. (~) N o serum; (11) calcium-free serum plus 0.4 mM EGTA. Data are the mean of triplicate determinations.

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Time (hr) Fig. 6. Time-course of EGTA, cycloheximide and puromycin inhibition of ornithine decarboxylase activity and [3H]leucine incorporation into protein. C6-2B cells were stimulated for 6 h with 18% calf serum before addition of 0.4 mM EGTA (top) or low doses of cycloheximide (3 n g / m l , middle) or puromycin (300 n g / m l , bottom). (zx) Ornithine decarboxylase activity; ((3) [3Hlleucine incorporation into trichloroacetic acid-insoluble material during a 20 min pulse-label preceding the times indicated on the abscissa. Data re the mean+S.E, of 6-15 values (top) or six values (middle and bottom) determined in at least two experiments.

95

(top) show the time-course for EGTA inhibition of increased ornithine decarboxylase activity and [3H]leucine incorporation into acid-insoluble material. In 1 h, EGTA inhibited ornithine decarboxylase activity by 15%, but no inhibition of protein synthesis was observed. 2 h after EGTA addition, ornithine decarboxylase activity was inhibited by 50%, and [3H]leucine incorporation was inhibited by 20%. Thus, EGTA inhibition of serum-increased ornithine decarboxylase activity temporally preceded EGTA inhibition of [3H]leucine incorporation into protein. However, this observation did not preclude the possibility that EGTA inhibited ornithine decarboxylase activity by a general effect on protein synthesis, because ornithine decarboxylase is a rapidly turning-over protein with apparent half-life values of 15-48 min [24]. It was possible that small inhibitions of protein synthesis could be reflected by large inhibitions of ornithine decarboxylase activity. This possibility was pursued by using less than maximal concentrations of cycloheximide and puromycin. Low doses of cycloheximide (3 ng/ml) or puromycin (300 ng/ml) inhibited ornithine decarboxylase activity and [3H]leucine incorporation within 1 h after drug addition (Fig. 6, middle and bottom). This concentration of cycloheximide inhibited protein synthesis by 20% and ornithine decarboxylase activity by 35%. Puromycin caused a more dramatic effect. At 1 h, puromycin inhibited

protein synthesis by only 10%; yet, it inhibited ornithine decarboxylase activity by 50%. These data demonstrated that greater than 25% inhibition of ornithine decarboxylase activity could occur when less than 25% inhibition of [3H]leucine incorporation was observed. Therefore, it seemed possible that EGTA might inhibit ornithine decarboxylase activity by affecting general protein synthesis. EGTA inhibition of protein synthesis was calcium-dependent. [3H]Leucine incorporation into the protein of cells treated for 3 h with 0.4 mM EGTA, EGTA plus 0.4 mM magnesium, or EGTA plus 0.4 mM calcium was 39, 44 and 99%, respectively, of the incorporation observed in control cells (no EGTA). If EGTA inhibited the synthesis of ornithine decarboxylase, then the decay rate of ornithine decarboxylase activity in the presence of EGTA would be similar to the decay rate observed in the presence of protein synthesis inhibitors. In the presence of 0.4 mM EGTA, ornithine decarboxylase activity declined (after a 30-60 re.in lag period) with a half-time of 47 rain (Table I). Low doses of cycloheximide (0.01 #g/ml) or emetine (0.025 g/ml) or a maximal dose of cycloheximide (5 /xg/ml) caused ornithine decarboxylase activity to decrease with half-times of 46, 37 and 44 rain, respectively. The concentrations of the protein synthesis inhibitors were chosen, because these

TABLE I O R N I T H I N E D E C A R B O X Y L A S E H A L F - L I F E IN T H E PRESENCE OF E G T A O R P R O T E I N SYNTHESIS INHIBITORS C6-2B cells were stimulated with 18% calf serum for 6 h before the addition of 0.4 m M EGTA, 0.01/~g/ml or 5 / ~ g / m l cycloheximide, or 0.025 ~ g / m l emetine. Ornithine decarboxylase (ODCase) activity was measured every 30 min for the next 3 h . Ornithine decarboxylase half-life was calculated from the slope of the linear regression line of log% ornithine decarboxylase activity vs. time. Data for inhibition of omithine decarboxylase activity and [3H]leucine incorporation into protein are the values observed 3 h after inhibitor addition. Treatment

ODCase t 1/2 (min)

Inhibition of ODCase activity (%)

Inhibition of [ 3H]leucine incorporation

(%) E G T A (0.4 m M ) Cycloheximide (5 ~ g / m l ) Cycloheximide (0.01/~ g / m l ) Emetine (0.025/~g/ml)

47 44 46 37

(0.984) a (0.994) (0.959) (0.985)

85 b > 95 80 95

a Correlation coefficient (r) of line used to determine ornith/ne decarboxylase half-life. b Obtained from Figs. 4 and 6. c Obtained from Fig. 6.

40 c 98 35 25

96 doses inhibited [3H]leucine incorporation either less than or greater than the inhibition observed with EGTA. The data in Table I also show that at least 80% inhibition of ornithine decarboxylase activity can occur without maximal inhibition of [ 3H]leucine incorporation. Discussion C a 2+ clearly is an obligatory requirement for C6-2B cell ornithine decarboxylase activity. In this report, our approach to elucidate the mechanism of this calcium requirement involved delineating two phases which occur in serum-stimulated C6-2B ceils (i.e., the lag period and the phase distal to the lag period). Then, it was determined in which phase the calcium requirement could be assigned. The results show that the calcium dependency of cellular ornithine decarboxylase activity is temporally distal to the lag period and is not associated with [3H]uridine incorporation into RNA. The data suggest that EGTA inhibits cellular ornithine decarboxylase activity by interfering with an event required for ornithine decarboxylase synthesis. The results in this report provide stronger support for the concept that RNA synthesis is required for serum-stimulated ornithine decarboxylase activity. The temporal values for the RNA synthetic period and for the stability of the presumed ornithine decarboxylase mRNA obtained by serum treatment of C6-2B cells agree closely with the values obtained with the RNA synthesis inhibitors, and these findings support the idea that actinomycin D and DRB inhibit ornithine decarboxylase activity by inhibiting RNA synthesis, rather than by exerting alternate actions. The temporal value for the length of the RNA synthetic period (2-3 h) determined in this study is similar to results obtained with R N A synthesis inhibitors in other cell lines and tissues [38-43]. Post-translational inhibition of ornithine decarboxylase activity has been reported, and thus calcium modulation of these events must be considered. Inhibition of ornithine decarboxylase activity by transamination probably would not be involved with the EGTA-mediated inhibition of ornithine decarboxylase activity, because transglutaminase requires Ca 2+ for enzymatic activity [44]. The EGTA-mediated inhibition of ornithine

decarboxylase activity cannot be due to stimulation of the ornithine decarboxylase inhibitor protein antizyme, because EGTA actually decreases C6-2B cell antizyme activity by an apparent mechanism that involves inhibition of antizyme synthesis. Therefore, at least two post-translational mechanisms for inhibiting ornithine decarboxylase activity can be dissociated from EGTA inhibition of C6-2B cell ornithine decarboxylase activity. Schimke and co-workers [45,46] suggested that changes in cellular enzyme levels are related to the apparent half-life of the particular enzyme. Theoretical considerations and experimental results showed that enzymes with short half-lives respond more quickly than long half-life enzymes to enhanced rates of cellular protein synthesis. Thus, it seems possible that small inhibitions of protein synthesis, as measured by [3H]leucine incorporation into the entire cell protein pool, could be reflected by large inhibitions of enzymes with short half-lives. This idea must be considered in studies of ornithine decarboxylase, because ornithine decarboxylase appears to have a very short half-life. Therefore, even though EGTA inhibition of ornithine decarboxylase activity temporally precedes EGTA inhibition of [3H]leucine incorporation into protein (Fig. 6), the data do not prove a dissociation of the two observations. Likewise, the ability of cycloheximide, emetine and puromycin to inhibit ornithine decarboxylase activity by 50-95% with 35% or less inhibition of [3H]leucine incorporation into protein cannot necessarily be used to prove that these drugs inhibit ornithine decarboxylase activity by a mechanism independent of protein synthesis. In this study, we measured ornithine decarboxylase activity and not ornithine decarboxylase mass, and thus, rigorous proof that EGTA inhibits ornithine decarboxylase synthesis will demand measurement of immunoreactive ornithine decarboxylase. However, the idea that EGTA may inhibit ornithine decarboxylase synthesis is supported by the observation that EGTA inhibits the cAMP-mediated induction of lactate dehydrogenase in C6-2B cells (Ref. 47, and Gibbs, J.B. and Brooker, G., unpublished data), and the induction of lactate dehydrogenase in C6-2B cells and C6 cells clearly requires new enzyme synthesis [48,49]. EGTA inhibition of radiolabeled amino acid

97 i n c o r p o r a t i o n i n t o a c i d - i n s o l u b l e m a t e r i a l has b e e n o b s e r v e d i n o t h e r systems, such as isolated rat p i n e a l g l a n d s [50] a n d i s o l a t e d a d r e n a l sections [51]. I n c o n t r a s t , efficient p r o t e i n t r a n s l a t i o n occurs in the p r e s e n c e of E G T A i n m R N A - d e p e n d e n t t r a n s l a t i o n systems d e r i v e d f r o m r a b b i t r e t i c u l o c y t e s [33] a n d yeast [34]. T h i s f i n d i n g suggests that c a l c i u m is n o t r e q u i r e d for the s t a b i l i t y o f the c o m p o n e n t s o r for the activity of the 'enz y m a t i c r e a c t i o n s i n the r e t i c u l o c y t e a n d yeast i n vitro t r a n s l a t i o n systems. T h u s , c a l c i u m c h e l a t i o n b y E G T A a p p e a r s to i n t e r f e r e w i t h s o m e process r e q u i r e d for p r o t e i n synthesis i n cells w h i c h is n o t n e e d e d for p r o t e i n s y n t h e s i s in the m R N A - d e p e n d e n t t r a n s l a t i o n systems. Acknowledgements W e t h a n k C a r o l y n P e d o n e for e x p e r t l y m a i n t a i n i n g cell c u l t u r e facilities. T h i s research was s u p p o r t e d b y N I H g r a n t s H L 1 5 9 8 5 , BL28940, H L 1 9 2 4 2 a n d A M 2 2 1 2 5 , J.B.G. was s u p p o r t e d b y USPHS predoctoral training grant T32GM07055 a w a r d e d to the D e p a r t m e n t of P h a r m a c o l o g y , U n i v e r s i t y of V i r g i n i a School o f M e d i c i n e . References 1 Pegg, A.E. and Williams-Ashman, H.G. (1981) in Polyamines in Biology and Medicine (Morris, D.R. and Marton, L.J., eds.), pp. 3-42, Marcel Dekker, New York 2 Tabor, C.W. and Tabor, H. (1976) Annu. Rev. Biochem. 45, 285-306 3 Heby, O. (1981) Differentiation 19, 1-20 4 Seidenfeld, J., Gray, J.W. and Marion, L.J. (1981) Exp. Cell Res. 131,209-216 5 Russell, D.H. and Haddox, M.K. (1979) Adv. Enzyme Regul. 17, 61-87 6 Bachrach, U. (1980) in Polyamines in Biomedical Research (Gaugas, J.M., ed.), pp. 81-107, Wiley, New York 7 Holtta, E. (1975) Biochim. Biophys. Acta 399, 420-427 8 Canellakis, Z.N. and Theoharides, T.C. (1976) J. Biol. Chem. 251, 4436-4441 90benrader, M.F. and Prouty, W.F. (1977) J. Biol. Chem. 252, 2866-2872 10 Kallio, A., Lofman, M., Poso, H. and Janne, J. (1979) Biochem. J. 177, 63-69 11 Canellakis, E.S., Viceps-Madore, D., Kyriakidis, D.A. and Heller, J.S. (1979) Curr. Top. Cell Regul. 15, 155-202 12 Penman, S., Vesco, C. and Penman, M. (1968) J. Mol. Biol. 34, 49-69 13 Pastan, I. and Friedman, R.M. (1968) Science 160, 316-317 14 Laszlo, J., Miller, D.S., McCarty, K.S. and Hochstein, P. (1966) Science 151, 1007-1010

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