Cordycepin and early effects of estradiol on the immature rat uterus

) thus o b t a i n e d was centri,fu:ged for 5 hours at 42,000 r p m (SW 50 rotor of a Spinco B e c k m a n centrifuge) on two cushions at c o n c e n t r a t i o n s of 0.5 and 1.6 M in sucrose (20 mM Tris-HC1, pH 7.5, 5 mM MgC12, 15 mM KC1, 5 mM ~-mercaptoethanoD. The t r a n s l u c i d polysome residue was treated with 500 ~1 of a d i s s o c i a t i n g solution : 1 p e r cent N-lauroyl-sarcosine, 0.03 M EDTA [23]. This solution was d i l u t e d 5 times by the a d d i t i o n of 2 ml of ¢ c o n c e n t r a t e d salt buffer >> (CSB) (5.0 rt~M TrisHC1, pH 7.5, 10 mM EDTA, 25 per cent formamide, 0.7 M Na,CI). The a b s o r b a n c e of the dissociated polysome solution was m e a s u r e d in order to s t a n d a r d i z e the p r e p a r a t i o n s c o r r e s p o n d i n g to the various treatments of the animals.

Evaluation o[ polysomal mRNA-Poly-A. This evaluation was c a r r i e d out by affinity c h r o m a t o g r a p h y on poly U-Sepharose 4.B (Phar-

Cordycepin and effects of estradiol on rat uterus. macia) a c c o r d i n g to the t e c h n i q u e described by Lin,dberg and Persson [23]. 2.2 ml of polysome s u s p e n s i o n (i.e. a m e a n of 6 units of O.D.) were filtered on a c o l u m n (0.5 em × 4 era) e q u i l i b r a t e d i n CSB buffer. The c o l u m n was w a s h e d with 4 ml of CSB. The s e p a r a t i o n of mRNA-poly A h y b r i d i z e d w i t h the poly U of the c o l u m n was effectuated u s i n g <> plus 0.5 ml o£ water.

445

d i u m (10 mM tris-HC1, pH 7.5, 10, mM EDTA, 0.2 per cent N-lauroyl sarcosine, 0.4 M NaCI). After w a s h i n g the c o l u m n w i t h 4 ml of T A buffer, the poly A r e t a i n e d was separated by elution w i t h T E m e d i u m of the following composition : 10 per cent of 10 mM tris-HCl, pH 7.5, 10 mM EDTA, 0.2 per cent N-lauroyl sarcosine, 90 per cent of formamide. The r a d i o a c t i v i t y of the fractions (0.5 ml) was d e t e r m i n e d in 12 ml of <> plus 0.5 ml of water. 5)

C O R D Y C E P I N AND T H E S P E C I F I C P R O T E I N F R A C -

TION INDUCED BY E S T R A D I O L .

4) DETERMINATION OF POLY A IN TOTAL R,NA. The t e c h n i q u e of total RNA extraction was a modification of the ¢ hot phenol-SDS >> m e t h o d r e c o m m e n d e d by Diez et al. [24] in order to obtain the m a x i m u m extraction of mRNA- poly A. 14 uteri were g r o u n d in a m o r t a r u s i n g sand a n d in 2 ml of 10 mM tri,s-HC1, pH 7.6, 2 mM EDTA. The p r o d u c t was di~luted to 4 ml by the homogeneizalion buffer an,d SDS was a d d e d ('final conc e n t r a t i o n : 0.5 p e r cent).. 4.5 ml of p h e n o l was added, followed b y h e a t i n g w i t h agitation at 55°C for 5 minutes. The aqueous phase was r e c u p e r a t e d a n d the p h e n o l i c phase reextracted w i t h 4 ml of buffer. The aqueous phases were c o m b i n e d a n d submitted to 3 a d d i t i o n a l e x t r a c t i o n s with phenol. The p h e n o l r e m a i n i n g in the aqueous phase was e l i m i n a t e d b y e x t r a c t i o n w i t h ether ; the final traces of ether w e r e e l i m i n a t e d b y air b u b b l i n g i n the solution. RNA was p r e c i p i t a t e d by the a d d i t i o n of 1/2.0 volume of 4,M NaC~ and 2 volumes of 95 p e r cent alcohol, r e c u p e r a t e d by centri,fugation, r e p l a c e d in s u s p e n s i o n in 95 p e r cent alcohol, a n d recollected by centrifugation. The t e c h n i q u e for p r e p a r a t i o n of poly A was essentially that described by J'elinek et al. [25] : extracted RNA was in solution i n 10 m,M tris-HCA, pH 7.5, 1 mM lVlgClu, 0.1 M NuC1. This solution was treated w i t h p a n c r e a t i c DNase (125 ~g/ml) for 30 m i n u t e s at 37°C. After a d d i t i o n of tSDTA (conc e n t r a t i o n of the m e d i u m : 10 raM) the solution was subjected to the action of p a n c r e a t i c r i b o n u clease A (Sigma, grade 3) ( c o n c e n t r a t i o n 5 t~g/ml) and T 1 r i b o n u c l e a s e (;Sigma) (20 u n i t s / m l ) for 30 m i n u t e s at 37°C. The so~tltion was then rendered 0.4 M in NaC1 a n d 0.2 per cent in N-lauroyl sareosine, heat at 65 ° for 2 minutes, a n d r e n d e r e d 0.1 p e r cent i n SOS. The sample was filtered on a poly U-Sepharose c o l u m n (0.5 cm × 4 era) e q u i l i b r a t e d in T A me-

BIOCHIMIE, 1976, 58, n ° 4.

a) Basis for the demonstration of IP. 2 groups of uteri were i n c u b a t e d separately i n survivo, the first h a v i n g been taken from c o n t r o l animals, the second from a n i m a l s h a v i n g previOusrly been treated w i t h an i n j e c t i o n of estradiol. The i n c u b a t i o n m e d i a c o n t a i n e d the same radioactive a m i n o acid. However, a p a r t i c u l a r isotope correspon~ded to each group of uteri. After i n c u b a tion was t e r m i n a t e d , the two groups of uteri were combined, generously r i n s e d a n d ground. After centrifugation, the o b t a i n e d s u p e r n a t a n t was frationated (by electrophoresis i n general). F o r each fraction the isotopic ratio was analyzed : [3H] (estradiot)/[14C] (control). F o r .all the fractions c o r r e s p o n d i n g to p r o t e i n s not affected by estradiol the ratio w o u l d r e m a i n c o n s t a n t and equal to R. On the other h a n d , a n y increase in p r o t e i n synthesis or a n y a p p e a r a n c e of n e w p r o t e i n s i n response to estradiot w o u l d be c h a r a c t e r i z e d by an in.crease h R of this ratio i n the c o r r e s p o n d i n g fractions. The excess of [aH] : h [aH], c o r r e s p o n d i n g to the q u a n t i t y of IP formed u n d e r the influence of estradiol, was d e t e r m i n e d for each sample i n a zone of c o n s t a n t w i d t h i n w h i c h IP appeared. The ratio

A tall] w a s determined, this R x [14C] ratio b e i n g i n d e p e n d a n t ~f a c e r t a i n n u m b e r of p a r a m e t e r s relative to the e x p e r i m e n t a l c o n d i t i o n s ( n u m b e r a n d mass of the uteri, radioactive concentrations, in,cubation time, fixation time, etc...) w h i c h p e r m i t t e d the c o m p a r i s o n of the different experiments performed. b) Treatment of animals - - Incubation o[ the

uteri. The e x p e r i m e n t s were c a r r i e d out on i m m a t u r e 1 9 - 2 0 day old Wistar rats. The a n i m a l s (3 or 4 per group) received two i n t r a p e r i t o n e a l injections of 0.2 ml, the first at one h o u r before sacrifice (control, c o r d y c e p i n ) , the second at 30 m i n u t e s before sacrifice (control, estradiol).

¢

m~

mo

rr~

mh

0"

m~

~Q aJ ~ph

°l

Cordycepin and effects of estradiol on rat uterus.

447

fig2a

fig2b

FIG. l. - - Effect of estradiol on the aspect of nucleoli of the uterine epithelium in the rat, in function of the age of the animals : r a t s 18 (a), 10 (b) a n d 5 (c) d'ay old. A. C o n t r o l r a t ; B. R a t t r e a t e d w i t h estradiol (×

20,000). U l t r a t h i n s e c t i o n s 'were s t a i n e d a c c o r d i n g to the EDTA t e c h n i q u e of B e r n h a r d .

FIG. 2. ---- Effect of cordycepin on the nucleolar stimulation by estradiol i~ the uterine epithelium of the rut : r a t s 18 (a) a n d 10 (b) d a y old. A. T r e a t m e n t vcith c o r d y c e p i n o n l y ; B. T r e a t m e n t w i t h e o r d y c e p i n and estradiol (×

20,000). U l t r a t h i n

BIOCHIMIE, 1976, 58, n ° 4.

sections w e r e s t a i n e d a c c o r d i n g to the EDTA t e c h n i q u e of B e r n h a r d .

J.-L. Borgna et coll.

448

tst injection

pit] ! ['~C] Isotope 2nd injection incubation corresponds to

NaCl (1) NaC1 NaCI NaCI NaC1 NaCI NaCl EstradIol (2) Cordycepin(3) NaCI Cordycepin Estradiol

2

3 4 5 6

p4C] [3Hl ['~C] pHI ['~C] I3H]

Control Estradiol

Estradiol ] Cordycepin

(1) 0.154 M (-}- 10 per cent Ethanol for 2 nd injection). (2) 10-~ M in NaC1 (0.154 M) d- 10 per cent Ethanol. (3) 5 my/m1 in NaC1 (0.154 M).

The animals were sacrificed by cervical luxation. Adipose tissue a d h e r e n t to the excised uteri was removed. Each group of uteri was i n c u b a t e d for 1 h o u r un,der O2/CO,z (95/5) atmosphere in 1 ml of Eagle's m i n i m a l essential m e d i u m w h i c h contained : - - odd groups : 100 ~1 of [14C] L-leucine (C.E.A., 50 taCt/rumple, con c. 0.2 m C i / m l ) - - even groups : 50 M o~f [3HJ z 4 e u c i n e (C.E.A., 2 Ci/mmote, conc. 1 m C i / m l ) or

buffer pH 8.5 (65 mM Tris, 40 mM HaBO 3, 3 mM EDTA (Na2)) at a m b i a n t t e m p e r a t u r e , at an amperage of 1 m A / t u b e , for an hour, fo.llowed by 2 m A / t u h e for five hours. The electrophoresis was then i n t e r r u p t e d , the gels removed, fixed in AcOH (7.5 per cent) for two h o u r s a n d then frozen on d r y ice and cut in slices of 2 m m thickness. Each slice was placed in a c o u n t i n g flask a n d subjected to the action of 1 i111 of soluene 350 (Packard) for 15 h o u r s at 60°C. 14 ml of s c i n t i l l a t i n g m i x t u r e were t h e n added (PPO dimethyl-l~OPOP) a n d after h o m o g e n i z a t i o n the r a d i o a c t i v i t y p r e s e n t [3H] a n d [14C] was m e a s u r e d i n a P a c k a r d Tri-Carb s c i n t i l l a t i o n spectrophotometer.

Electrophoresis on Cellulose acetate. 20 .M of the sample to be stu, died were placed on a cellogel strip. The electrcrphoresis was p e r f o r m e d i n a 40 mM Veronal-sodium veronate buffer, pH 8.6, for one h o u r in a field o,f 2'5 volts/cm. After fixation for 5 m i n u t e s in 5 per cent 3"CA, the cellogel support was .cut i n a transverse fashion i n strips of 2 m m width. Each strip was submitted to the action of 1 ml of soluene 3:50 for five hours at 60°C. T h e n 14 ml of s c i n t i l l a t i n g m i x t u r e was added. After homogenization the radioactivities were measured.

:

- - o d d groups : 100 ~1 of [14C] z-serine (Amersham R a d i o c h e m i c a l Centre, 174 m C i / m m o l e , conc. 0.1 m C i / m l ) - - e v e n groups : 50 ~1 of [aH] z-serine (Amersham R a d i o c h e m i c a l Centre, 150~) taCt/rumple, conc. 1 m C i / m l )

RESULT,S AND D,ISC,USSION. l

--

ESTRADIOL

STIMULATION

OF THE

NUCLEOLUS.

1) Electron microscopy study. a) Effect of estradiol on the nucleolus as a func-

tion of age. c) Demonslration of the induced protein frac-

tion (IP). After completion of i n c u b a t i o n , the uteri of groups 1 a n d 2, 3 a n d 4, 5 a n d 6, were c o m b i n e d , r i n s e d successively i n 3 × 50 ml of EDTA (Na z) at 0.05 per cent, then g r o u n d i n 0.5 ml of EDTA i n the p r e s e n c e of 200 mg of sand. The homogenates were centrifuged for 20 m i n u t e s at 1~,000 g a n d the s u p e r n a t a n t s obtained frozen in l i q u i d nilrogen. The three types of s u p e r n a t a n t s were fractionated by electrophoresis either on p o l y a c r y l a m i d e gel or on cellulose acetate.

Electrophoresis on polyacrylamide gel. The electrophoresis were p e r f o r m e d on 12 cm × 0.6 cm gels at 7.5 p e r cent i n a c r y l a m i d e a n d 0.2 per cent in N N ' m e t h y l e n e bis acrylamide. Aliquots (100 ul) of the p r e v i o u s l y o b t a i n e d s u p e r n a tants were m i x e d w i t h 20 ~1 of 13 per cent ficoll c o n t a i n i n g b r o m o p h e n o l blue an,d then placed on the gels. Eleetrophoresis was p e r f o r m e d i n Tris BIOCHIMIE, 1976, 58, n ° 4.

The nu,cleoli of the u t e r i n e e p i t h e l i u m were e x a m i n e d by electron m i c r o s c o p y a c c o r d i n g to B e r n h a r d ' s method [18-191 w h i c h p e r m i t s the specific coloration of r i b o n u c l e o p r o t e i n particles and thus the revelation of R NA synthesis. The control nucleoli (rats not treated w i t h estradiol) were c o m p a r e d to the uterin,e nucleoli of rats treated w i t h estr.adiol. Estra,diol i n d u c e d a net tran'sformation i n rats 18 (fig. la) or 10; (fig. l b ) day old. This t r a n s f o m n a t i o n was c h a r a c t e r i z e d by the a p p e a r a n c e of i n t e n s i v e l y colored <> d e m o n s t r a t i n g active n u c l e o l a r R N A synthesis [~01. On the other h a n d , the uter i n e nueleoli of 5 day old rats did not show this response to esiradiol (fig. le). b) Effect of cordycepin on the nucleolar stimula-

tion by estradiol. 18 a n d 10 ,day old rats received a n i n t r a p e r i t o neal i n j e c t i o n of c o r d y c e p i n (or isotonic saline for the controls) one half h o u r before the i n j e c t i o n of estradiol (or isotonic saline for the controls).

Cordycepin and effects of estradiol on rat uterus. Figure 2 shows that c o r d y c e p i n a p p a r e n t l y did not affect the nucleolar response to estradiol (fig. 2a : 18 day old rats ; fig. 2b : 10 day old rats). 2) [SH] uridine incorporation in RNA. The a n i m a l s (Wistar rats 18 day old) were subjected to the s~ame t r e a t m e n t s as those used for the electron m i c r o s c o p y studies but w i t h the injection of [aH] u r i d i n e one half h o u r before sacrifice. The specific activities of the uterus-extracted IqNA are s h o w n in table I. TABLE

I.

Effects of estradiol and co rdycepin on the incorporation of [~H] uridine in RNA in the rat uterus.

Type of treatment : successive inieetions

1

2

[JflJ uridine incorporation in total RNA (CPM/~t g RNA} Experiment Experimenl Experiment

Isotonic solution... Isotonic solution... Isotonic solution... Estradiol ..........

t

2

3

51

62

53

125

107 54

3

Cordycepin ........ Isotonic solution...

53

4

Cordycepin ........ Estradiol ..........

128

5

Actinomycin D .... Estradiol ..........

i

114

5

The animals (18 day old female rats, 5 animals per group) received an initial intraperitoneal injection of isotonic solution (groups 1 and 2), or cordycepin (groups 3 and 4, 25 mg/kg body weight), or actinomycin D (group 5, 5 mg/kg body weight), followed one half hour later by an injection of isotonic solution (groups 1 and 3) or estradiol (groups 2, 4 and 5). The animals received an injection of [8H] uridine (20 ~Ci/animal) one half hour before being sacrificed, the sacrifice t ~ i n g place 2 hours after the initial injection. The specific activities of the RNA were determined as indicated in <>.

Estradiol clearly stimulated the global synthesis of RNA a n d this s t i m u l a t i o n ~vas not notably affected by c o r d y c e p i n . This confirms the observations in electron m i c r o s c o p y since the d u r a t i o n of treatmen.t w i t h estradiot used i n our experim e n t s (1 h 1/2) implies that the s t i m u l a t i o n of RNA synthesis is for a large p a r t n u c l e o l a r i n origin [8]. It appears from these studies that c o r d y c e p i n , w h i c h a p p a r e n t l y does not affect the global syn-

BIOCHIMIE, 1976, 58,

thesis of I~NA, must have a selective roJe i n the uterus w h i c h we have tried to specify at the level of poly-A synthesis. It should be noted that the relative specific activities of RNA synthesized i n the various i n s t a n c e s does not necessarily reflect i n a rigorous m a n n e r the RNA-polymerase activities, this i n consideration of the p r o b a b l e effects of estr~diol on the pen e t r a t i o n of injected tritiated u r i d i n e o n the one h a n d [26, 27] a n d on the biosynthesis of R,NA precursors on the other [28]. The d e t e r m i n a t i o n of the specific activities of the t r i p h o s p h a t e nucleotide pools directly used b y RNA-polymerases wotfld in fact be r e q u i r e d to k n o w the actual levels of RNA synthesis s t i m u l a t i o n ; b u t these measuremerits a p p e a r to be quite difficult, or even impossible, because of c o m p a r t m e n t a l i z a t i o n p h e n o m e n a w h i c h f r e q u e n t l y occur inside the cells [29, 30]. A n y h o w it seems likely that the e n h a n c e m e n t of the specific activity of RNA is c o n n e c t e d to the s t i m u l a t i o n of RNA synthesis by estradiol [31, 32], as m o r e o v e r stated by the time-course of estradiol effects on RNA-polymerase activities [8]. ~I --

MECHANISM

IMMATURE

112

n ° 4.

449

OF ACTION OF CORDYCEPIN

ON THE

RAT UTERUS.

1) Effect of cordycepin on the appearance of mRNA-poly A in uterine polysomes. Although the existence of giant p r e c u r s o r s for mRNA has r e c e n t l y been questioned [33], it is generally a d m i t e d that cytoplasmic mRNA molecules result from p r o c e s s i n g of greater n u c l e a r molecules, HnR,NA [34, ~ ] . One of the i n i t i a l roles a t t r i b u t e d to poly A was at the level of the m a t u r a tion of nucle,ar HnRNA into cytoplasmic mRNA [13, 16]. Accordin, g to c e r t a i n a u t h o r s [14, 16], cord y c e p i n , by p r e f e r e n t i a l l y inhi~biting the postt r a n s c r i p t i o n a l a d d i t i o n of poly A, w o u l d p e r t u r b the m a t u r a t i o n process a n d t r a n s p o r t of mRNA. I n o r d e r to evaluate the v a l i d i t y of this hypothesis i n our tissue studies, we studied the effect of cord y c e p i n on the r a p i d i t y of radioactive tracer inc o r p o r a t i o n in p o l y s o m a l mRNA-p~ly A i n the imm a t u r e (18-20 days) rat uterus, p r e v i o u s l y stimulated by estradiol to synthesize more mRNA molecules. Figure 3 shows a sucrose density gradient profile ~f polysomes from rat uterus. This p r o f l e was not affected by a two h o u r c o r d y c e p i n t r e a t m e n t of animals. This is not s u r p r i s i n g since a slow t u r n - o v e r of p r e e x i s t i n g mRNA could mask an eventual effect of the i n h i b i t o r on the a p p e a r a n c e of n e w l y synthesized polysomal mRNA. It has been s h o w n that p u r e polysomal mRNA, free from c o n t a m i n a t i o n b y rRNA and tRNA can 30

J.-L. Borgna et coll.

450

b e o b t a i n e d b y t h e m e t h o d w e h a v e u s e d to filter dissociated polysomes on p o l y - U - S e p h a r o s e c o l u m n s [23].

W h i l e n o t b e i n g t h e i n i t i a l goal o f t h e s e e x p e r i m e n t s , t h e c o m p a r i s o n o.f t r i a l s 1 a n d 3 i n e x p e r i m e n t A p e r m i t t e d u s to f i n d t h e e x p e c t e d e f f e c t s of e s t r a d i o l on RNA s y n t h e s i s ( t h e m a j o r p a r t of t h e n o n - b o u n d R N A w h i c h inclu,des a m o n g s t others the poly A-free mRNA) and on the synthesis o~ m R N A - p o l y A.

OD

TOP

T h e r e s u l t s of t h e e x p e r i m e n t s c o n d u c t e d o n a n i m a l s h a v i n g b e e n s u b j e c t e d to d i v e r s e t r e a t m e n t s a r e a s s e m b l e d i n t a b l e II.

BOTTOM

T h e c o m p a r i s o n of tria~s 1 a n d 2 s h o w e d t h a t cordycepin : --

h a d no c o n s i d e r a b l e effect on the s y n l h e s i s of RNA, c o n f i r m i n g t h e p r e v i o u s l y d i s c u s s e d experixnents with uridine incorporation.

--

did not i n h i b i t the a p p e a r a n c e of n e w l y synt h e s i z e d m R N A - p o l y A i n t h e p o l y s o m e s , sin.ce the radioaetivity retained on poly U-Sepharose was not affected.

Iw

FIG. 3. Sucrose gradient profile of polysomes from rat uterus. A sample of polysomes prepared as descri~ -

bed in 34 per for 40 SW 50

Materials and Methods w a s layered on a 10cent linear sucrose gradient and centrifuged m i n u t e s at 45,000 r p m in a Spineo Beckman rotor.

T h e r e s u l t s at t h e s a m e t i m e i m p l y t h a t t h e s y n t h e s i s of H n R N A is i n s e n s i t i v e to c o r d y c e p i n , a f a c t w h i c h is u n a n i n m n s l y r e c o g n i z e d b y all c o n cerned authors.

TABLE II.

The role of cordycepin in the incorporation oI [SHj adenosine in polysomat RNA in the immature rat uterus. Poly A - - RNA: Non poly A-RNA : non retained radioactivity retained radioactivity Treatment oi animals : on poly U-Sepharose on poly U-Sephnrose successive injections CPM x 10"~/~tg polysomal CPM x 10~/,u g polysomal RNA RNA

Experiment A

Estradiol . . . . . . . . Isotonic solution.

1780

78

1640 ( - - 8 per cent versus trial 1)

75 ( - - 4 per cent versus trial 1)

Isotonic solution. Isotonic solution.

830

48

1)

Estradiol . . . . . . . . Isotonic solution.

1630

61

2)

Estradiol . . . . . . . . Cordycepin . . . . . .

1150 ( - - 27 per cent versus trial 1)

65 (-~ 6 per cent versus trial 1)

1)

2) 3)

Estradiol . . . . . . . Cordycepin . . . . . .

Experiment B

The anima}s (18-20 day old female rats, 10 a n i m a l s per group) received an initial intraperitoneal injection of isotonic solution (group 3) or estradiol (groups 1 and 2). Two hours l a t e r the a n i m a l s received a second i n j e c t i o n of isotonic solution (groups 1 and 3) or cordycepin (group 2, 25 m g / k g body weight) folio'wed by an i n j e c t i o n of [3H] adenosine (groups 1, 2 and 3), 15 minutes later. The uteri were removed 4 1/2 hours after the initial injection, qqae polysomes were prepared as indicated in <> section, dissociated arLd filtered On a poly U-Sepharose column. The trials 'were standardized by m e a s u r e m e n t of the absorbanees of the dissociated polysome solutions nvhich u n d e r w e n t filtration (mean q u a n t i t y per trial : 6 units O.D.). The values given for the effects of cordyeepin in e x p e r i m e n t s A and B represent the extreme values obtained in four identical expriments.

BIOCHIMIE, 197~6, 58, n ° 4.

451

Cordgcepin and effects of estradiol on rat uterus. It should be noted that c e r t a i n studies [14, 16] that have r e c o g n i z e d an effect of c o r d y c e p i n on the m a t u r a t i o n p r o c e s s and t r a n s p o r t of mRNA w e r e based on e x p e r i m e n t s p e r f o r m e d in the presence of a c t i n 0 m y c i n D, used to e l i m i n a t e ~he labelling of r i b o s o m a l RNA. The use of a c t i n o m y cin D h o w e v e r , a c c o r d i n g to c e r t a i n authors, should be a v o i d e d in any study c o n c e r n i n g transp o r t p h e n o m e n a E24, 36]. 2) Effect of cordycepin on the synthesis of poly A chains. The effect of e o r d y c e p i n on poly A synthesis was c h e c k e d by m e a s u r i n g the i n c o r p o r a t i o n of !3HI a d e n o s i n e in p o l y A chains isolated from TABLE I I I .

Effect of cordycepin on the synthesis of poly A in the immature rat uterus. Radioactivity retained on poly U-Sepharose after treatment with A and T~ RNases. Treatment : successive injections

Non-poly A : non-retained radioactivity on poly U-Sepharose CPM x 10"2/~ g total RNA

Poly A : retained radioactivity on poly U-Sepharose CPM x t0~/? g total RNA

Estradiol 1 ) Isotonic Solution

5290

104

Estradiol 2) Cor dycepin

4220 (-- 20 per cent versus control)(*)

( - - 65 per cent versus control)C)

36

The animals (19-20 day old female rats, 14 animals per group) received an initial intraperitoneal injection of estradiol (groups 1 and 2). 3 1/2 hours later the animals received a second injection of isotonic solution (group 1) or cordvccpin (group 2, 25 mg/kg body weight), followed 15 "minutes later by an injection of [3H] adenosine (groups 1 and 2). 30 ~Ci per animal. The uteri were excised 6 hours after the initial injection. The fragments of poly A were isolated as described in the <(Materials and Methods >> section. The trials ~¢ere standardized by measurement of the ~bsorbances of the RNA solutions subjected to filtration (a mean of 1 mg of RNA per trial). (*) The percents of cordycepin effects xvere - - 2 0 + 5 per cent for non-poly A, and - - 6 5 ~ 5 per cent for poly A, in three reproductible experiments.

total RNA extracts after digestion by A and T 1 RNases. The results of a t y p i c a l e x p e r i m e n t , w h i c h are assembled in table III, s h o w that c o r d y c e p i n has no strong effect (20 p e r cent only) on the global s y n t h e s i s of RNA, r e p r e s e n t e d by the n o n - r e t a i n e d r a d i o a c t i v i t y on po.ly U-Sepharose. On the other hand, the i n h i b i t i o n of [3H] a d e n o s i n e i n c o r p o r a tion in poly A chains ( r a d i o a c t i v i t y b o u n d to poly

BIOCHIMIE, 1976, 58, n ° 4.

U-Sepharose after the action of A and T 1 RNases on total RNA) r e a c h e s 65 p e r cent. In e x p e r i m e n t s not r e p o r t e d here, s i m i l a r effects w e r e f o u n d on p o l y A chains isolated f r o m polysomal mRNA. That l a b e l i n g of poly A - m R N A is not affected by c o r d y c e p i n (see table II) is not surprising, since the r a d i o a c t i v i t y of poly A m a y r e p r e s e n t only a small p a r t of the w h o l e r a d i o a c tivity of poly A - mP~NA and since mRNA synthesis is a c o r d y c e p i n - i n s e n s i t i v e p r o c e s s [37]. Our results also i m p l y that after c o r d y c e p i n t r e a t m e n t most of the poly A chains r e m a i n long enough to allow all t h e poty A - mRNA m o l e c u l e s to be retained on p o l y U-Sepharose, in our e x p e r i m e n t a l cond i t i o n s ; this suggests that all the p o l y A chains are r e d u c e d a p p r o x i m a t e l y to the same extent by c o r d y c e p i n , r a t h e r than some m R N A m o l e c u l e s no longer c o n t a i n p o l y A after c o r d y c e p i n treatment. Some i n f o r m a t i o n about this p r o b l e m m i g h t be obtained f r o m e l e e t r o p h o r e s i s analysis of p o l y A chains s y n t h e s i z e d in the p r e s e n c e or in the absence of c o r d y c e p i n , but such a study w o u l d c e r t a i n l y be difficult because of the small level of r a d i o a c t i v i t y associated to poly A in our e x p e r i ments. Many of the results f o u n d in the l i t e r a t u r e agree on a distinct i n h i b i t i o n of the synthesis of poly A and a negligeable or v e r y p a r t i a l i n h i b i t i o n of the synthesis of v a r i o u s classes of RNA by cordycepin, w h i c h is thus a good selective i n h i b i t o r

[12, 14, 38, 39]. III-

ConDVcEPIN

AND

THE

SPECIFIC

PROTEIN

INDUCED BY ESTRADIOL (fig. 4).

The p r e s e n c e of IP ~vas c h e c k e d by e l e c t r o p h o resis in the s u p e r n a t a n t s f r o m uteri of animals w h i c h h a v e been subnlitted to 3 types of treatm e n t s : <( c o n t r o l >> group - < gronp e c o r d y c e p i n - e s t r a d i o l >> group (for details see e Materials and Methods >>). 1) Analysis of the control. The f r a c t i o n s o b t a i n e d after e l e c t r o p h o r e s i s all p r e s e n t e d an a p p r e c i a b l y constant ratio R = [3H]/[14C], thus permittin,g the e l i m i n a t i o n o.f any effect eventually r e s u l t i n g f r o m the ulilisation of t w o distinct r a d i o a c t i v e samples. 2) Effect of estradiol. The two types of e l e c t r o p h o r e s i s p e r m i t the d e m o n s t r a t i o n of IP. It is a m a r k e d l y a c i d i c protein f r a c t i o n for w h i c h the m i g r a l i o n is 10 p e r cent g r e a t e r than BSA ( c h a r a c t e r i s t i c p r o p e r t y of IP r e p o r t e d by o t h e r a~lhors). D u r i n g the different e x p e r i m e n t s p e r f o r m e d , the i n c r e a s e ~R of

452

J.-L. B o r g n a et coll.

the ratio R varied b e t w e e n 35 a n d 65 per cent. The t r i t i u m surplus A [aH] varied, per uterus treated w i t h estradiol, between 1500 a n d 240~0 CPM (coun-

2,

of adenosine derivative pools. However, that cordyeepin only weakly inhibits adenosine incorpor a t i o n i n RNA implies that the ATP pool i n part i e u l a r is lithe affected by this i n h i b i t o r ; thus the possible actions of c o r d y c e p i n above m e n t i o n e d should not m o d i f y our i n t e r p r e t a t i o n s i n a decisive m a n n e r . Our results [40, 41] a n d those of other authors [6] agree on a strong i n h i b i t i o n of I,P synthesis by c o r d y c e p i n . I n contrast, the electron m i c r o s c o p y studies [42] and the study o'f r a d i o a c t i v e p r e c u r s o r incorp o r a t i o n i n R NA establish that the s t i m u l a t i o n of the nucleolus by estradiol is not e l i m i n a t e d by cordycepin.

lb

2'0

3o

Fro. 4. - - Effect of cordycepin on the induction of IP by estracIiol. Profiles of [3H]/[14C] ratio along eleetrophoresis aerylamide gels The soluble protein contents of uteri from animals subjected to various treatments (& control, • estradiol, • estradiol./eordycepin) have been analyzed as described in (>.

Thus, I,P synthesis i n d u c t i o n a n d nucleolar ind u c t i o n by estradiol react in a different m a n n e r to c o r d y c e p i n . This allows us to suppose that there is no direct quantitative r e l a t i o n s h i p between these two early m a n i f e s t a t i o n s of estrogenic activity.

ring output for [aH] in the v i c i n i t y of 20 per cent) a n d the ratio A [ZH]/R x [14C.J varied b e t w e e n 0.25 a n d 0.40').

The hypothesis that IP is a <> (<< KIP >>) u p o n w h i c h the n u c l e o l a r RNA p o l y m e r a s e w o u l d depen.d [4], seems r a t h e r unlikely to us, the more so as the presence of IP i n the nuclei of rat ttteri could not be d e m o n s t r a t e d [43].

n

3) Effect o[ estradiol in the presence of cordycepin (25 m g / k g ) . Our results c o n f i r m those o b t a i n e d very recently by D. Somjen et al [,6] c o n c e r n i n g the action 0f c o r d y c e p i n in vitro. 'Cordycepin i n h i b i t s the f o r m a t i o n of IP. At the doses c o n s i d e r e d this i n h i b i t i o n , if not total, is at least very considerabl.e ( > 85 p e t cent i n the different e x p e r i m e n t s performed). CONCLUSIONS. We have c o m p a r e d the action of c o r d y c e p i n on the i n d u c t i o n of two early effects of estradiol in the i m m a t u r e rat u t e r u s : nucteolar s t i m u l a t i o n and synthesis of the p r o t e i n f r a c t i o n IP. The m a i n effect of c o r d y c e p i n in the uterus, w i t h the doge we used, is to i n h i b i t the synthesis of poly A whi,ch is f o u n d at the extrem4ty of H n RNA a n d mRNA. C o r d y c e p i n m i g h t display other effects : eordyc e p i n itself or its derivatives might compete w i t h a d e n o s i n e or its metabolites for e n z y m a t i c activities such as a d e n o s i n e kinase, adenylate kinase, ATPase, adenylcyclase, phospho~diesterase, etc..., a n d the resulting i n h i b i t i o n might alter the size

BIOCHIM1E, 1 9 7 6 ,

58, n ° 4.

The reversal b y cy:cloheximide of eslradiole n h a n c e d nucleo,lar R,NA synthesis has b e e n conside.red ~by c e r l a i n authors to be a c o n s e q u e n c e of the s t o p p i n g of IP synthesis b y the drug [4]. But the effects of c y c l o h e x i m i d e are more complex [44-49] : apart from p r o t e i n synthesis i n h i b i t i o n , T i m b e r l a k e a n d Griffin have reported a direct action of c y c l o h e x i m i d e on nucleolar ILNA synthesis [48, 49]. F o r thai reason it seemed to us more suitable to use c o r d y c e p i n for our purpose. The hypothesis o:f p r e f o r m e d messenger (s), c o d i n g for IP, the e x p r e s s i o n of w h i c h w o u l d be p e r m i t t e d b y estradiol, can also be excluded. It can be c o n c l u d e d that one of the early effects of estradiol is to i n d u c e the f o r m a t i o n of Hn RNApoly A relative to IP. This effect m a y c o i n c i d e w i t h the b r i e f a n d very early (15 min) increase in the synthesis of mRN,A observed by c e r t a i n authors [8] u n d e r the effect of estradioI in the ovariectomized-rat uterus. I n effect, it is k n o w n that the synthesis of rRNA appears p r e c o c i o u s l y (30 rain) w h i l e the synthesis of mRNA does not develop to an appreciable extent u n t i l later (2 hours). Our p r e l i m i n a r y contro.] e x p e r i m e n t s concern i n g the role of cordyc~pin i n the uterus, i n i t i a l l y

Cordycepin and effects of estradiol on rat uterus. designed to interpret the mechanism of action of estradiol, permit us to perceive that the system s t u d i e d m i g h t f u r n i s h some i n f o r m a t i o n on the r o l e of p o l y A i n t h e cell, t h i s b e i n g p o o r l y u n d e r s t o o d i n s p i t e of t h e c o n s i d e r a b l e n u m b e r o f s t u d i e s d e v o t e d to t h i s p r o b l e m . T h e i n h i b i t i o n o f t h e s y n t h e s i s o f ] P s e e m s to h e r e l a t e d to t h e i n h i b i tion of the s y n t h e s i s of poly A by c o r d y c e p i n . T h e a b s e n c e of a p r o m i n e n t e f f e c t of t h i s i n h i b i t i o n o n t h e i n t e g r a t i o n of n e w l y s y n t h e s i z e d m R , N A - p o l y A i n p o l y s o m e s is n o t i n c o n s i s t e n t w i t h the positive effects of c o r d y c e p i n on poly A s y n t h e s i s a n d o n I P s y n t h e s i s I6, 40, 41]. T h e i n h i b i t i o n of I P s y n t h e s i s b y c o r d y c e p i n c o u l d b e d u e to a p o l y A d e f i c i e n c y o f its m R N A . As p r e v i o u s l y s u g g e s t e d [5~)] p o l y A c o u l d b e i n volved in the t r a n s l a t i o n of m R N A r a t h e r t h a n in its p r o c e s s i n g a n d t r a n s p o r t . More p r e c i s e future studies are n e c e s s a r y in o r d e r to e l u c i d a t e t h e r o l e of p o l y A i n t h e m a t u ration p r o c e s s of Hn RNA a n d the t r a n s l a t i o n w h i c h a r e s t i l l t h e s u b j e c t o f m u c h i n t e r e s t [51-

58]. Aeknt~wledgments. The a u t h o r s are grateful to Dr J. R. Tata for reading the m a n u s c r i p t and giving helpful advice. The a u t h o r s than& Mrs N. Berrmd and F. Michel for technical assistance. This work has received financial support f r o m the C.N.R.S., the C.E.N. and the F o n d a t i o n pour la Recherche M6dicale. R~SUM~.. L'cestradiol induit dans l'ut~rus de rate impub~re la synth6se d'une fraction prot~ique sp~cifique (IP). L'injection de cordyefipine (3' ddoxyad6nosine), irthibiteur de la synthSse du poly A, inhibe la synthbse de IP. Ce r6sultat sugg6re que Fun des effets les plus prdcoces de l'~estrog6ne est de produire la f o r m a t i o n de Hn-RNA poly A relatifs h IP. P a r ailleurs, la s t i m u l a t i o n par l'cestradiol du nucldole de l'6pith~lium d'ut~rus de rate impnbbre a 6t~ raise en dvidence par des techniques de microscopie 61ectronique. La cordyc6pine n'affeete pas de mani~re sensible cette stimulation. Des ~tudes hioehimiques (incorporation de traceurs radioaetifs dans les RNA, c h r o m a t o g r a p h i c d'affinit~ sur poly U-S~pharose) men6es parall6lement et dans des conditions eomparables h celles utilis6es pour les 6tudes de microscopic 6lcctronique m o n t r e n t que la cordyc~pine bloque la synth~se du poly A mats n'affecte pas s e n s i b l e m e n t l ' a u g m e n t a t i o n du taux de RNA ribosomal ohserv6 sous l'effet de l'~estradiol. Le hlocage de IP p a r la cordye~pine, le non effet de cet i n h i b i t e u r dans les m~mes conditions au niveau du rmcl6ole m o n t r e n t que les deux effets pr6eoees de Faction cestrog~ne darts l'ut~rus de rate i m m a t u r e ne semblent pas d i r e c t e m e n t correl6s. BIOCHIMIE, 1976, 58, n ° 4.

453

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