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Studies on the mechanism of in vitro estradiol-17-β induced synthesis of phosvitin in chick embryo liver cells
Cell Differentiation, 4 (1976) 441--448 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
441
STUDIES ON THE MECHANISM OF IN VITRO ESTRADIOL-17-~ INDUCED SYNTHESIS OF PHOSVITIN IN CHICK EMBRYO LIVER CELLS *
P. CARINCI, A. CARUSO, R. EVANGELISTI, E. BECCHETTI and G. STABELLINI
Institute of Histology and General Embryology, University of Ferrara, Italy Accepted 5 November 1975
The effect of estradiol-17-~ treatment on phosvitin synthesis by cultured chick embryo liver cells has been studied. Phosvitin synthesis occurs after approximately 15 hr of hormone treatment; the synthesis being blocked by actinomycin D treatment suggests that RNA synthesis is required. The life time of the newly synthesized RNA is at least 24 hr. The significance of these findings with respect to the mechanisms involved in hormone-mediated protein synthesis is discussed.
Phosvitin, a yolk protein, is normally synthesized under hormonal control in the liver of the laying hen and then secreted into the blood plasma (Heald et al., 1965; Greengard et al., 1965). Phosvitin synthesis can also be induced by treatment with estrogens in immature pullets or roosters (Haeld et al., 1964; Beuving et al., 1971a; Bergink et al., 1973; see Bergink et al., 1974, for a review). Such a system has been widely used as a tool for the studies of the mechanisms involved in steroid hormonal regulation of protein synthesis in vivo. Some relevant features of this regulation have been pointed out already, as the dependence of hormone-stimulated phosvitin synthesis on new R N A production (Greengard et al., 1964), the reduction of lag-period on sequential injections of the hormone, ' m e m o r y effect' (Jailkhani et al., 1972a; Bergink et al., 1973) and the possible correlation between protein induction and cellular multiplication (Jailkhani et al., 1972b). The estrogen-mediated phosvitin induction in vivo is accompanied by a decline in synthesis of other 'secreted' proteins (Schejde, 1963); this represents a model for investigating the hormonal influence on the production of different proteins. Since the analysis of steroid action on the whole animal is complicated by the possible interference of u n k n o w n factors, an in vitro system has been developed. In previous work (Carinci et al., 1974a), we have demonstrated by im-
* These studies were supported in part by Italian CNR Grants CT 74.001128.04.
442 munochemical and biochemical methods that de novo synthesis of phosvitin occurs also in vitro, by adding estradiol-17-fl to chick embryo liver primary cultures. A declined production of total 'secreted' proteins has been also detected (Carinci et al., 1974b). This represents an improved model for studying how a steroid h o r m o n e acts on protein synthesis in liver cells. In order to gain additional information on this in vitro system, we have now analyzed the time course of phosvitin production following hormone treatment and the actinomycin D effects on this hormone-mediated protein synthesis. Our data indicate that the in vitro estrogen-mediated phosvitin synthesis requires the synthesis of new RNA; this, however, does not immediately follow the h o r m o n e administration. In addition, the newly synthesized RNA has a life time of at least 24 hr. Finally, liver cells produce phosvitin after h o r m o n e withdrawal and maintain this ability for a few days thereafter. MATERIALS AND METHODS
Chemicals Estradiol-17-/3 was a Merck product; [3H]-uridine (specific activity 41 Ci/ mM) was purchased from Radiochemical Centre, Amersham; actinomycin D and bovine serum albumin from the Sigma Co. All other chemicals and reagents were of analytical grade.
Culture conditions Livers removed from 14-day old chick embryos were gently cut into small pieces, washed in Tyrode's solution and dissociated in 0.25% buffered trypsin (Difco) at room temperature for 25--30 min. The cells were then filtered through a nylon mesh, centrifuged (35 g, 10 min) and suspended in medium 199 (Gibco, Grand Island, N.Y.) with 20% added calf serum and penicillin (200 units/ml). 5 ml of cell suspension (4 X 106 cells/ml) were put in flasks and incubated at 37°C. 24 hr after plating, medium was replaced with nutrient containing estradiol-17-~ (50 pg/#l in propylene glycol) at the final concentration of 500 pg/culture; the controls were added with nutrient containing only propylene glycol. To investigate the time of hormonal treatment required to obtain phosvitin synthesis, parallel series of cultures were incubated in the presence of the hormone for 9, 12, 15 or 18 hr, medium was then replaced and cultures removed after additional 72 hr of in vitro maintenance. In order to examine the time course of phosvitin accumulation both treated and control cultures were incubated for 18 hr, then supplied with standard medium and subsequently removed at the following 18th, 24th, 48th or 72nd hr of in vitro maintenance. In the actinomycin D experiments, the anti-metabolite (5 pg/ml) was ad-
443 ded: 1) during the 18 hr of the hormone treatment; 2) during the first 24 hr after withdrawal of the hormone; 3) during the second 24 hr after withdrawal of the hormone. The cultures were therefore removed at different times. Cells and culture media recovery At the end of incubation, cells were detached with 0.02% EDTA (37°C, 10 min), recovered b y centrifugation (50 g, 10 min), washed with 0.9% NaC1, homogenized at 0--4°C in 10 volumes of double distilled water by means of Potter homogenizer with an equal volume of 10% TCA, centrifuged and repeatedly washed with 5% TCA. Aliquots of the pellet were used for protein, phosphorous and newly synthesized R N A determinations. Culture media were recovered by centrifugation of detached cellular suspension and precipitated with 10% TCA, repeatedly washed with 5% TCA and used for phosphorous determinations. N e w l y synthesized R N A determination The cell pellet was suspended in methanol--ethyl ether (1 : 1, v/v), dehydrated with ethyl ether, dissolved in 0.5 ml of Soluene (Packard Instrument Co. Inc.) and then added with 10 ml of a Spectrafluor PPO--POPOP scintillation fluid (Radiochemical Centre, Amersham). Radioactivity was measured in a Packard Scintillation Counter 2425. Other m e t h o d s Since preliminary data have shown a close correspondence between immunochemical and biochemical phosvitin determinations, we have used the protein phosphorous c o n t e n t to measure phosvitin concentration. The protein P was determined on TCA precipitates according to Bartlett (1959). This technique, which has been shown to be specific for phosvitin (Miienp~ et al., 1969) has been slightly modified by us. After hydrolysis with Ba(OH)2, proteins were precipitated by adding 6% HC104 and protein P was determined in the supernatant, thus avoiding the determination of non-phosvitin protein phosphorous. Proteins were determined according to L o w r y et al. (1951), with serum albumin as a standard. RESULTS Estradiol-1 7-[3 treatment and phosvitin synthesis In preliminary experiments the time of estradol-17-~ pretreatment required for subsequent phosvitin synthesis has been determined and shown to
444 TABLE I D e t e r m i n a t i o n s o f alkali-labile p r o t e i n p h o s p h o r o u s c o n t e n t in 72 hr n u t r i t i o n a l m e d i a after estradiol-17-/~ d i f f e r e n t t r e a t m e n t s . Values are t h e m e a n o f t w o i n d e p e n d e n t exp e r i m e n t s , each in triplicate. Period o f hormonal treatment (hr)
Culture m e d i u m (pg P / m l )
9 12 15 18
0 0 2.9 3.1
be between 12 and 15 hr (see Table I). The standard pretreatment of 18 hr has been used in the experiments reported in the present publication. The time course of phosvitin accumulation in nutritional media of primary liver cultures pretreated for 18 hr with estradiol-17-fl is reported in Fig. 1. At the end of hormonal treatment, alkali-labile protein P could not be detected either in the cells or in the nutritional medium. Following 12 hr from the hormone withdrawal, the protein P was detectable in definite quantities in the cells as well as in the nutritional medium; its a m o u n t increased during the following 12 hr and then diminished from the 48th to 72nd hr after the hormone withdrawal. No alkali-labile protein phosphorous was detected in the controls at any time.
e-
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e - - - - - e /~g P /
culture
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0
12
24
,
48
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72
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Fig. 1. T h e t i m e course o f p h o s v i t i n a c c u m u l a t i o n in c u l t u r e d liver cells ( e . . . . . . o) and in n u t r i t i o n a l m e d i a ( e ~). 0 indicates t h e e x p e r i m e n t a l starting p o i n t after 18 hr o f estradiol-17-~ t r e a t m e n t . T h e values are t h e m e a n o f at least 4 i n d e p e n d e n t experim e n t s , each in duplicate. T h e vertical bar gives t h e m i n i m u m and t h e m a x i m u m o f exp e r i m e n t a l values.
445 c.
;
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0
0;5 ==
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~0 4 .c_
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_¢
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0
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48
24
72
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Fig. 2. E f f e c t o f a c t i n o m y c i n D o n t h e t i m e course o f p h o s v i t i n a c c u m u l a t i o n in c u l t u r e d liver cells (o . . . . . . o) a n d in n u t r i t i o n a l m e d i a ( e e). 0 indicates t h e e x p e r i m e n t a l s t a r t i n g p o i n t a f t e r 18 h r o f estradiol-17-~ plus a c t i n o m y c i n D t r e a t m e n t . T h e values are t h e m e a n o f at least 4 i n d e p e n d e n t e x p e r i m e n t s , e a c h in d u p l i c a t e . T h e vertical bar gives t h e m i n i m u m a n d t h e m a x i m u m o f e x p e r i m e n t a l values.
Effects of actinomycin D on the phosvitin accumulation When actinomycin D was administered and removed simultaneously with the hormone (Fig. 2), Ph0svitin synthesis occurred but its maximum occurred 24 hr later than in cultures w i t h o u t actinomycin D. When actinomycin D was administered for 24 hr after hormone withdrawal (Fig. 3A), phosvitin synthesis was totally inhibited and appeared only after removal of the drug.
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4
4.5
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2
2
-E 1
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0
24
48
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0
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48
72
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Fig. 3. E f f e c t o f a c t i n o m y c i n D o n p h o s v i t i n a c c u m u l a t i o n in n u t r i t i o n a l media. A ) Actin o m y c i n D has b e e n a d m i n i s t e r e d at 0 p o i n t (~) ( a f t e r 18 h r o f estradiol-17-~ t r e a t m e n t ) a n d r e m o v e d ( t ) a f t e r o t h e r 24 h r o f c u l t u r e . B) A c t i n o m y c i n D h a s b e e n supplied at 24 h r (~) a n d r e m o v e d a f t e r o t h e r 24 h r o f c u l t u r e (1").
446 TABLE II Effect of actinomycin D on RNA synthesis. RNA * (cpm/mg protein) After 18 hr of estradiol-17-~ treatment After 18 hr of estradiol-17-~+actinomycin D treatment After 24 hr in standard nutritional medium from the hormone withdrawal After 24 hr in standard nutritional medium + actinomycin D from the hormone withdrawal
3193 380 6687 500
* Each value is the mean of two independent experiments, each in triplicate.
F i n a l l y , w h e n a c t i n o m y c i n D was a d m i n i s t e r e d f r o m the 2 4 t h hr a f t e r h o r m o n e w i t h d r a w a l (Fig. 3B), p h o s v i t i n synthesis declined f a s t e r t h a n in the controls.
Effect of actinomycin D on R N A synthesis T o v e r i f y w h e t h e r in t h e p r e s e n t c o n d i t i o n s a c t i n o m y c i n D a f f e c t e d R N A s y n t h e s i s , a p p r o p r i a t e e x p e r i m e n t s w e r e carried out. As d e m o n s t r a t e d in T a b l e II, a d r a m a t i c decrease o f t o t a l R N A s y n t h e s i s o c c u r r e d b o t h w h e n a c t i n o m y c i n D was a d m i n i s t e r e d during t h e h o r m o n a l t r e a t m e n t and w h e n the t r e a t m e n t was s t a r t e d a f t e r estradiol-17-fi w i t h d r a w a l . DISCUSSION T h e aim of t h e p r e s e n t w o r k was to collect d a t a on e s t r o g e n - i n d u c e d p h o s v i t i n s y n t h e s i s in vitro to allow f u r t h e r investigations on t h e m e c h a n i s m s involved in e s t r o g e n r e g u l a t i o n o f a specific p r o t e i n synthesis. Liver cells p r i m a r y c u l t u r e s are s t i m u l a t e d to p r o d u c e and secrete p h o s vitin i n t o t h e s u r r o u n d i n g m e d i u m w h e n t r e a t e d w i t h estradiol-17-fi. Phosvitin is d e t e c t a b l e in the n u t r i e n t a f t e r a p r o l o n g e d lag-period, first in increasing a n d t h e n in declining c o n c e n t r a t i o n . T h e lag-period is n o t d u e to a d e l a y e d ' e x p o r t ' o f t h e s y n t h e s i z e d p r o t e i n f r o m the cells; in f a c t , p h o s v i t i n is n o t f o u n d in t h e cells at the e n d o f the h o r m o n a l t r e a t m e n t . T h e observed a c c u m u l a t i o n curve results f r o m t w o o p p o s i t e processes, s y n t h e s i s a n d d e g r a d a t i o n . A t the m o m e n t we d o n o t have a n y i n f o r m a t i o n o n the kinetics o f t h e t w o processes. H o w e v e r , t h e s a m e curve p r o f i l e o f p h o s v i t i n a c c u m u l a t i o n in v i t r o o b t a i n e d in several e x p e r i m e n t s i n d i c a t e s the c o n s t a n cy o f t h e f a c t o r s i n v o l v e d b o t h in synthesis a n d d e g r a d a t i o n . I t m a y be suggested t h a t t h e n e w l y s e c r e t e d p r o t e i n is likely to u n d e r g o h y d r o l y s i s b y
447 enzymes released in the extracellular medium. Alternatively, the dephosphorylation of newly synthesized phosvitin can be postulated. Estradiol-17-~ induced phosvitin synthesis in vitro requires new R N A production as shown by the actinomycin D inhibition of phosvitin synthesis. Since the drug is effective only when added during the first 24 hr after hormone withdrawal, and since phosvitin synthesis in the controls is already detectable after 12 hr, the R N A required for phosvitin production must be synthesized in the 12 hr following the end of hormonal treatment. This R N A is therefore not synthesized during the period of hormonal treatment. Our experiments show indeed that the simultaneous addition of actinomycin D and hormone to the cultures, although effective in causing a dramatic decrease in RNA synthesis, does not interfere with phosvitin synthesis. The observation that R N A synthesis does not increase during hormonal stimulation (Evangelisti et al., 1975) agrees with the above finding. The life-time of phosvitin related R N A is at least 24 hr as indicated by the fact that actinomycin D treatment does not inhibit phosvitin synthesis when given 24 hr after estradiol-17-fi withdrawal. In all experiments with actinomycin D a shift in the time of the peak of the phosvitin concentration in the nutrient medium is observed. A similar effect has been found in vivo (Beuving et al., 1971b) and may be due to the fact that a part of DNA remains inactive in its transcriptional activity also after removal of actinomycin D. To obtain the estradiol-17-fi induced phosvitin synthesis in vitro a prolonged hormonal treatment is necessary, which cannot be reduced b e y o n d a b o u t 15 hr. During this period neither the synthesis of phosvitin nor the synthesis of R N A required for phosvitin induction occur. This fact together with the observations that phosvitin synthesis may be restored after actinomycin D pretreatment, demonstrates the presence of estradiol-17-~-dependent factor(s) which persist(s) in the cells and regulates phosvitin synthesis. The comparison of estrogen-stimulated phosvitin synthesis in vitro with that in vivo exhibits two major differences: a prolonged lag-period and a delayed R N A production. Possibly, these two events are related to one another and could depend on the embryonic material we have employed and/or on the in vitro situation. Several findings indicate a developmental temporal sequence in the ability of target cells and tissues to be affected by hormonal stimulations (Clark et al., 1970; Kaye et al., 1972; Somjen et al., 1973). Attempts to repeat experiments with liver cells removed from posthatching animals have been unsuccessful. The proposed system seems to be adequate for the elucidation of some mechanisms involved in estrogen regulation of protein synthesis; such as liver cell responsiveness to hormonal stimulation in different phases of the cell cycle; correlation between phosvitin synthesis induction and decrease of synthesis of other 'secreted' proteins, and the continued ability of liver cells to synthesize phosvitin after sequential divisions.
448 REFERENCES Bartlett, G.R.: J. Biol. Chem. 2 3 4 , 4 6 6 (1959). Bergink, E.W., H.J. Kloosterboer, M. Gruber and G. Ab: Biochim. Biophys. Acta 294, 497 (1973). Bergink, E.W., R.A. Wallace, J.A. van den Berg, E. Bos, M. Gruber and G. Ab: Amer. Zool. 14, 1177 (1974). Beuving, G. and M. Gruber: Biochim. Biophys. Acta 2 3 2 , 5 2 4 (1971a). Beuving, G. and M. Gruber: Biochim. Biophys. Acta 2 3 2 , 5 2 9 (1971b). Carinci, P., P. Locci, M.A. Bodo and A. Caruso: Experientia 30, 88 (1974a). Carinci, P., M.A. Bodo, P. Locci and R. Evangelisti: Experientia30, 1243 (1974b). Clark, J.H. and J. Gorski: Science 169, 76 (1970). Greengard, O., M. Gordon, M.A. Smith and G. Acs: J. Biol. Chem. 239, 2079 (1964). Greengard, O., A. Sentenac and G. Acs: J. Biol. Chem. 240, 1687 (1965). Evangelisti, R., A. Caruso, A. Petris, C. Calastrini and P. Carinci: I.R.C.S. Med. Sci., 3, 372 (1975). Heald, P.J. and P.M. McLachlan: Biochem. J. 92, 51 (1964). Heald, P.J. and P.M. McLachlan: Biochem. J. 94, 32 (1965). Jailkhani, B.L. and G.P. Talwar: Nature New. Biol. 236,239 (1972a). Jailkhani, B.L. and G.P. Talwar: Nature New. Biol. 239, 240 (1972b). Kaye, A.M., D. Heratzky and H.R. Linder: Biochim. Biophys. Acta 261, 475 (1972). Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall: J. Biol. Chem. 193, 265 (1951). M~/enp~, P.H. and M.R. Bernfield: Biochemistry8, 4926 ( 1969 ). Schejde, O.A. : The Chemistry of Fats and Other Lipids;eds R.T. Holman, W.O. Lundberg and T. Malkni (PergamonPress, Oxford) Vol. 6, p. 251 (1963). Sbmjen, D., G. S5mjen, R.J. King, A.M. Kaye and H.R. Linder: Biochem. J. 136, 25 (1973).
441
STUDIES ON THE MECHANISM OF IN VITRO ESTRADIOL-17-~ INDUCED SYNTHESIS OF PHOSVITIN IN CHICK EMBRYO LIVER CELLS *
P. CARINCI, A. CARUSO, R. EVANGELISTI, E. BECCHETTI and G. STABELLINI
Institute of Histology and General Embryology, University of Ferrara, Italy Accepted 5 November 1975
The effect of estradiol-17-~ treatment on phosvitin synthesis by cultured chick embryo liver cells has been studied. Phosvitin synthesis occurs after approximately 15 hr of hormone treatment; the synthesis being blocked by actinomycin D treatment suggests that RNA synthesis is required. The life time of the newly synthesized RNA is at least 24 hr. The significance of these findings with respect to the mechanisms involved in hormone-mediated protein synthesis is discussed.
Phosvitin, a yolk protein, is normally synthesized under hormonal control in the liver of the laying hen and then secreted into the blood plasma (Heald et al., 1965; Greengard et al., 1965). Phosvitin synthesis can also be induced by treatment with estrogens in immature pullets or roosters (Haeld et al., 1964; Beuving et al., 1971a; Bergink et al., 1973; see Bergink et al., 1974, for a review). Such a system has been widely used as a tool for the studies of the mechanisms involved in steroid hormonal regulation of protein synthesis in vivo. Some relevant features of this regulation have been pointed out already, as the dependence of hormone-stimulated phosvitin synthesis on new R N A production (Greengard et al., 1964), the reduction of lag-period on sequential injections of the hormone, ' m e m o r y effect' (Jailkhani et al., 1972a; Bergink et al., 1973) and the possible correlation between protein induction and cellular multiplication (Jailkhani et al., 1972b). The estrogen-mediated phosvitin induction in vivo is accompanied by a decline in synthesis of other 'secreted' proteins (Schejde, 1963); this represents a model for investigating the hormonal influence on the production of different proteins. Since the analysis of steroid action on the whole animal is complicated by the possible interference of u n k n o w n factors, an in vitro system has been developed. In previous work (Carinci et al., 1974a), we have demonstrated by im-
* These studies were supported in part by Italian CNR Grants CT 74.001128.04.
442 munochemical and biochemical methods that de novo synthesis of phosvitin occurs also in vitro, by adding estradiol-17-fl to chick embryo liver primary cultures. A declined production of total 'secreted' proteins has been also detected (Carinci et al., 1974b). This represents an improved model for studying how a steroid h o r m o n e acts on protein synthesis in liver cells. In order to gain additional information on this in vitro system, we have now analyzed the time course of phosvitin production following hormone treatment and the actinomycin D effects on this hormone-mediated protein synthesis. Our data indicate that the in vitro estrogen-mediated phosvitin synthesis requires the synthesis of new RNA; this, however, does not immediately follow the h o r m o n e administration. In addition, the newly synthesized RNA has a life time of at least 24 hr. Finally, liver cells produce phosvitin after h o r m o n e withdrawal and maintain this ability for a few days thereafter. MATERIALS AND METHODS
Chemicals Estradiol-17-/3 was a Merck product; [3H]-uridine (specific activity 41 Ci/ mM) was purchased from Radiochemical Centre, Amersham; actinomycin D and bovine serum albumin from the Sigma Co. All other chemicals and reagents were of analytical grade.
Culture conditions Livers removed from 14-day old chick embryos were gently cut into small pieces, washed in Tyrode's solution and dissociated in 0.25% buffered trypsin (Difco) at room temperature for 25--30 min. The cells were then filtered through a nylon mesh, centrifuged (35 g, 10 min) and suspended in medium 199 (Gibco, Grand Island, N.Y.) with 20% added calf serum and penicillin (200 units/ml). 5 ml of cell suspension (4 X 106 cells/ml) were put in flasks and incubated at 37°C. 24 hr after plating, medium was replaced with nutrient containing estradiol-17-~ (50 pg/#l in propylene glycol) at the final concentration of 500 pg/culture; the controls were added with nutrient containing only propylene glycol. To investigate the time of hormonal treatment required to obtain phosvitin synthesis, parallel series of cultures were incubated in the presence of the hormone for 9, 12, 15 or 18 hr, medium was then replaced and cultures removed after additional 72 hr of in vitro maintenance. In order to examine the time course of phosvitin accumulation both treated and control cultures were incubated for 18 hr, then supplied with standard medium and subsequently removed at the following 18th, 24th, 48th or 72nd hr of in vitro maintenance. In the actinomycin D experiments, the anti-metabolite (5 pg/ml) was ad-
443 ded: 1) during the 18 hr of the hormone treatment; 2) during the first 24 hr after withdrawal of the hormone; 3) during the second 24 hr after withdrawal of the hormone. The cultures were therefore removed at different times. Cells and culture media recovery At the end of incubation, cells were detached with 0.02% EDTA (37°C, 10 min), recovered b y centrifugation (50 g, 10 min), washed with 0.9% NaC1, homogenized at 0--4°C in 10 volumes of double distilled water by means of Potter homogenizer with an equal volume of 10% TCA, centrifuged and repeatedly washed with 5% TCA. Aliquots of the pellet were used for protein, phosphorous and newly synthesized R N A determinations. Culture media were recovered by centrifugation of detached cellular suspension and precipitated with 10% TCA, repeatedly washed with 5% TCA and used for phosphorous determinations. N e w l y synthesized R N A determination The cell pellet was suspended in methanol--ethyl ether (1 : 1, v/v), dehydrated with ethyl ether, dissolved in 0.5 ml of Soluene (Packard Instrument Co. Inc.) and then added with 10 ml of a Spectrafluor PPO--POPOP scintillation fluid (Radiochemical Centre, Amersham). Radioactivity was measured in a Packard Scintillation Counter 2425. Other m e t h o d s Since preliminary data have shown a close correspondence between immunochemical and biochemical phosvitin determinations, we have used the protein phosphorous c o n t e n t to measure phosvitin concentration. The protein P was determined on TCA precipitates according to Bartlett (1959). This technique, which has been shown to be specific for phosvitin (Miienp~ et al., 1969) has been slightly modified by us. After hydrolysis with Ba(OH)2, proteins were precipitated by adding 6% HC104 and protein P was determined in the supernatant, thus avoiding the determination of non-phosvitin protein phosphorous. Proteins were determined according to L o w r y et al. (1951), with serum albumin as a standard. RESULTS Estradiol-1 7-[3 treatment and phosvitin synthesis In preliminary experiments the time of estradol-17-~ pretreatment required for subsequent phosvitin synthesis has been determined and shown to
444 TABLE I D e t e r m i n a t i o n s o f alkali-labile p r o t e i n p h o s p h o r o u s c o n t e n t in 72 hr n u t r i t i o n a l m e d i a after estradiol-17-/~ d i f f e r e n t t r e a t m e n t s . Values are t h e m e a n o f t w o i n d e p e n d e n t exp e r i m e n t s , each in triplicate. Period o f hormonal treatment (hr)
Culture m e d i u m (pg P / m l )
9 12 15 18
0 0 2.9 3.1
be between 12 and 15 hr (see Table I). The standard pretreatment of 18 hr has been used in the experiments reported in the present publication. The time course of phosvitin accumulation in nutritional media of primary liver cultures pretreated for 18 hr with estradiol-17-fl is reported in Fig. 1. At the end of hormonal treatment, alkali-labile protein P could not be detected either in the cells or in the nutritional medium. Following 12 hr from the hormone withdrawal, the protein P was detectable in definite quantities in the cells as well as in the nutritional medium; its a m o u n t increased during the following 12 hr and then diminished from the 48th to 72nd hr after the hormone withdrawal. No alkali-labile protein phosphorous was detected in the controls at any time.
e-
== o
• ~g P / m l o f c u l t u r e m e d i u m
e - - - - - e /~g P /
culture
5
o
.c_
~3 ..L <
•
L
h
0
12
24
,
48
i
72
hrs
Fig. 1. T h e t i m e course o f p h o s v i t i n a c c u m u l a t i o n in c u l t u r e d liver cells ( e . . . . . . o) and in n u t r i t i o n a l m e d i a ( e ~). 0 indicates t h e e x p e r i m e n t a l starting p o i n t after 18 hr o f estradiol-17-~ t r e a t m e n t . T h e values are t h e m e a n o f at least 4 i n d e p e n d e n t experim e n t s , each in duplicate. T h e vertical bar gives t h e m i n i m u m and t h e m a x i m u m o f exp e r i m e n t a l values.
445 c.
;
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0
0;5 ==
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~3 CL
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24
72
hrs
Fig. 2. E f f e c t o f a c t i n o m y c i n D o n t h e t i m e course o f p h o s v i t i n a c c u m u l a t i o n in c u l t u r e d liver cells (o . . . . . . o) a n d in n u t r i t i o n a l m e d i a ( e e). 0 indicates t h e e x p e r i m e n t a l s t a r t i n g p o i n t a f t e r 18 h r o f estradiol-17-~ plus a c t i n o m y c i n D t r e a t m e n t . T h e values are t h e m e a n o f at least 4 i n d e p e n d e n t e x p e r i m e n t s , e a c h in d u p l i c a t e . T h e vertical bar gives t h e m i n i m u m a n d t h e m a x i m u m o f e x p e r i m e n t a l values.
Effects of actinomycin D on the phosvitin accumulation When actinomycin D was administered and removed simultaneously with the hormone (Fig. 2), Ph0svitin synthesis occurred but its maximum occurred 24 hr later than in cultures w i t h o u t actinomycin D. When actinomycin D was administered for 24 hr after hormone withdrawal (Fig. 3A), phosvitin synthesis was totally inhibited and appeared only after removal of the drug.
®
® ~
5
5
E 4
4
4.5
2k4 3
2
2
-E 1
act.~ D <
0
24
48
hrs
0
24
48
72
hrs
Fig. 3. E f f e c t o f a c t i n o m y c i n D o n p h o s v i t i n a c c u m u l a t i o n in n u t r i t i o n a l media. A ) Actin o m y c i n D has b e e n a d m i n i s t e r e d at 0 p o i n t (~) ( a f t e r 18 h r o f estradiol-17-~ t r e a t m e n t ) a n d r e m o v e d ( t ) a f t e r o t h e r 24 h r o f c u l t u r e . B) A c t i n o m y c i n D h a s b e e n supplied at 24 h r (~) a n d r e m o v e d a f t e r o t h e r 24 h r o f c u l t u r e (1").
446 TABLE II Effect of actinomycin D on RNA synthesis. RNA * (cpm/mg protein) After 18 hr of estradiol-17-~ treatment After 18 hr of estradiol-17-~+actinomycin D treatment After 24 hr in standard nutritional medium from the hormone withdrawal After 24 hr in standard nutritional medium + actinomycin D from the hormone withdrawal
3193 380 6687 500
* Each value is the mean of two independent experiments, each in triplicate.
F i n a l l y , w h e n a c t i n o m y c i n D was a d m i n i s t e r e d f r o m the 2 4 t h hr a f t e r h o r m o n e w i t h d r a w a l (Fig. 3B), p h o s v i t i n synthesis declined f a s t e r t h a n in the controls.
Effect of actinomycin D on R N A synthesis T o v e r i f y w h e t h e r in t h e p r e s e n t c o n d i t i o n s a c t i n o m y c i n D a f f e c t e d R N A s y n t h e s i s , a p p r o p r i a t e e x p e r i m e n t s w e r e carried out. As d e m o n s t r a t e d in T a b l e II, a d r a m a t i c decrease o f t o t a l R N A s y n t h e s i s o c c u r r e d b o t h w h e n a c t i n o m y c i n D was a d m i n i s t e r e d during t h e h o r m o n a l t r e a t m e n t and w h e n the t r e a t m e n t was s t a r t e d a f t e r estradiol-17-fi w i t h d r a w a l . DISCUSSION T h e aim of t h e p r e s e n t w o r k was to collect d a t a on e s t r o g e n - i n d u c e d p h o s v i t i n s y n t h e s i s in vitro to allow f u r t h e r investigations on t h e m e c h a n i s m s involved in e s t r o g e n r e g u l a t i o n o f a specific p r o t e i n synthesis. Liver cells p r i m a r y c u l t u r e s are s t i m u l a t e d to p r o d u c e and secrete p h o s vitin i n t o t h e s u r r o u n d i n g m e d i u m w h e n t r e a t e d w i t h estradiol-17-fi. Phosvitin is d e t e c t a b l e in the n u t r i e n t a f t e r a p r o l o n g e d lag-period, first in increasing a n d t h e n in declining c o n c e n t r a t i o n . T h e lag-period is n o t d u e to a d e l a y e d ' e x p o r t ' o f t h e s y n t h e s i z e d p r o t e i n f r o m the cells; in f a c t , p h o s v i t i n is n o t f o u n d in t h e cells at the e n d o f the h o r m o n a l t r e a t m e n t . T h e observed a c c u m u l a t i o n curve results f r o m t w o o p p o s i t e processes, s y n t h e s i s a n d d e g r a d a t i o n . A t the m o m e n t we d o n o t have a n y i n f o r m a t i o n o n the kinetics o f t h e t w o processes. H o w e v e r , t h e s a m e curve p r o f i l e o f p h o s v i t i n a c c u m u l a t i o n in v i t r o o b t a i n e d in several e x p e r i m e n t s i n d i c a t e s the c o n s t a n cy o f t h e f a c t o r s i n v o l v e d b o t h in synthesis a n d d e g r a d a t i o n . I t m a y be suggested t h a t t h e n e w l y s e c r e t e d p r o t e i n is likely to u n d e r g o h y d r o l y s i s b y
447 enzymes released in the extracellular medium. Alternatively, the dephosphorylation of newly synthesized phosvitin can be postulated. Estradiol-17-~ induced phosvitin synthesis in vitro requires new R N A production as shown by the actinomycin D inhibition of phosvitin synthesis. Since the drug is effective only when added during the first 24 hr after hormone withdrawal, and since phosvitin synthesis in the controls is already detectable after 12 hr, the R N A required for phosvitin production must be synthesized in the 12 hr following the end of hormonal treatment. This R N A is therefore not synthesized during the period of hormonal treatment. Our experiments show indeed that the simultaneous addition of actinomycin D and hormone to the cultures, although effective in causing a dramatic decrease in RNA synthesis, does not interfere with phosvitin synthesis. The observation that R N A synthesis does not increase during hormonal stimulation (Evangelisti et al., 1975) agrees with the above finding. The life-time of phosvitin related R N A is at least 24 hr as indicated by the fact that actinomycin D treatment does not inhibit phosvitin synthesis when given 24 hr after estradiol-17-fi withdrawal. In all experiments with actinomycin D a shift in the time of the peak of the phosvitin concentration in the nutrient medium is observed. A similar effect has been found in vivo (Beuving et al., 1971b) and may be due to the fact that a part of DNA remains inactive in its transcriptional activity also after removal of actinomycin D. To obtain the estradiol-17-fi induced phosvitin synthesis in vitro a prolonged hormonal treatment is necessary, which cannot be reduced b e y o n d a b o u t 15 hr. During this period neither the synthesis of phosvitin nor the synthesis of R N A required for phosvitin induction occur. This fact together with the observations that phosvitin synthesis may be restored after actinomycin D pretreatment, demonstrates the presence of estradiol-17-~-dependent factor(s) which persist(s) in the cells and regulates phosvitin synthesis. The comparison of estrogen-stimulated phosvitin synthesis in vitro with that in vivo exhibits two major differences: a prolonged lag-period and a delayed R N A production. Possibly, these two events are related to one another and could depend on the embryonic material we have employed and/or on the in vitro situation. Several findings indicate a developmental temporal sequence in the ability of target cells and tissues to be affected by hormonal stimulations (Clark et al., 1970; Kaye et al., 1972; Somjen et al., 1973). Attempts to repeat experiments with liver cells removed from posthatching animals have been unsuccessful. The proposed system seems to be adequate for the elucidation of some mechanisms involved in estrogen regulation of protein synthesis; such as liver cell responsiveness to hormonal stimulation in different phases of the cell cycle; correlation between phosvitin synthesis induction and decrease of synthesis of other 'secreted' proteins, and the continued ability of liver cells to synthesize phosvitin after sequential divisions.
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