Transcriptional regulation of estrogen-responsive genes by non-steroidal estrogens: Doisynolic and allenolic acids

ft. SteroidBiochem.Molec.Biol. Vol. 62, No. 5/6, pp. 477-489, 1997

Pergamon

© 1997Publishedby ElsevierScienceLtd. All rights reserved Printed in Great Britain PII: S0960-0760(97)00063-0 0960-0760/97 $17.00+ 0.00

Transcriptional Regulation of Estrogen-responsive Genes by Non-steroidal Estrogens: Doisynolic and Allenolic Acids C a l Y. M e y e r s , ' H i s h a m

G. Lutfi' a n d S t u a r t A d l e r 2.

1Southern Illinois University, Department of Chemistry and Biochemistry, Carbondale, IL, U.S.A.; 2Department of Obstetrics and Gynecology, and Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO, U.S.A.

Estrogen receptor (ER), a m e m b e r of the nuclear receptor superfamily, exerts prominent physiological roles in both h u m a n s and other species by acting directly as a transcription factor, altering nuclear gene expression. One peculiarity o f estrogenic regulation is that it is affected by a wide variety of non-steroidal c.ompounds in addition to the natural hormone, estradiol. Doisynolic and allenolic acid c o m p o u n d s axe non-steroidal c o m p o u n d s that act as potent estrogens in animal studies, yet bind to ER extremely poorly in competitive binding assays, raising the possibility o f alternative m o l ecular m e c h a n i s m s for the observed estrogenic effects. In this work we demonstrate that (+)-Z-bisdehydrodoisynolic acid, (+)-Z-b/sdehydrodoisynolic acid 3-methyl ether, and (-) allenolic acid can interact directly with ER. These c o m p o u n d s all serve as ligands for ER in m e c h a n i s m - s p e c i f i c tissue culture-based reporter gene assays for both positive and negative gene regulation. We have also used a novel assay based on electromobility shift by ER for directly determining relative binding affinities for ER. In addition, we show cell-type-specific activity differences for (++)-Z-bisdehydrodoisynolic acid 3-methyl ether, supporting clinical observations indicating a higher potency of this c o m p o u n d in female animals than :in humans. © 1997 Published by Elsevier Science Ltd. All rights reserved

J. Steroid Biochem. Molec. Biol., Vol. 62, No. 5/6, pp. 477-489, 1997

INTRODUCTION

the breast and uterus, and can influence the risks for developing cancers [2]. ER, a m e m b e r of the nuclear receptor superfamily, One peculiarity of estrogenic regulation is that it is exerts prominent physiological roles in both humans affected by a wide variety of non-steroidal c o m p o u n d s and other species, by acting directly as a transcription in addition to estradiol, the natural ligand [2]. Some factor, altering nuclear gene expression [1]. E R regu- of these compounds, e.g. diethylstilbestrol, act as agolates female development and reproduction [2], and, nists in a variety of organ systems [2]. Others, includwith aromatized androgens as ligands, can play similar ing tamoxifen, have varying effects as agonists or roles in males [3]. In addition, E R participates in pro- antagonists, depending on the particular organ system cesses unrelated to reproduction, such as preservation or gene examined [4]. It has been clearly demonof bone mass and protection from cardiovascular strated in competitive binding assays that almost all of disease [2]. Furthermore, estrogens can support these non-steroidal c o m p o u n d s bind directly to E R growth in estrogen-responsive target tissues, including and thus act as ligands. There remain some compounds, however, that exert estrogenic effects in vivo but, paradoxically, fail to demonstrate appropriate *Correspondence to Stuart Adler, Washington University School of Medicine, Department of Obstetrics and Gynecology, Box affinity for the E R in competitive binding assays [5]. 8049, 4911 Barnes Hospital Plaza, Saint Louis, MO 63110- These compounds raised the possibility of alternative 1094, U.S.A. Tel: +1 314 362 8697; Fax: +1 314 362 0256; mechanisms or explanations for estrogenic effects [6]. emaih [email protected]. Received 19 Dec. 1996; accepted 12 May 1997. This concept has recently gained renewed interest, 477

Cal Y. Meyers et aL

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Table 1. Structures of the D/A compounds

CH3

-CH 3

.,,..-',,.,.,m CH3 _

[

1//", CO2H

A

=oSOlOj v

R:H R = CH3

(-) -Z-bisdehydrodoisynolic acid (-) -Z-bisdehydrodoisynolic acid 3-methyl ether

based on analyses of genetically engineered ER-nu11 mice [7, 8]. Doisynolic acids, named for and discovered by Doisy and co-workers, are a class of compounds produced by D-ring cleavages of steroids such as estrone and equilenin [9]. T w o such compounds are (+)-Zbisdehydrodoisynolic acid and its 3-methyl ether. Another c o m p o u n d of similar type is (-) allenolic acid [10]. Because they lack the 1,2-cyclopentanophenanthrene structure they are not true steroids (see Table 1). T h e doisynolic acids lack the D-ring, whereas the allenolic acids lack the D- and C-rings. All four doisynolic/allenolic (D/A) compounds are active as estrogens, as measured by in vivo assays, including uterotropism (uterine weight increases) and vaginal comification [5]. In marked contrast to most other steroidal and non-steroidal estrogens, they bind extremely poorly to the E R [5, 11]. This observation was made in competitive binding inhibition studies utilizing uterine cytosolic E R and 3H-estradiol [5] or 17-fluorescein estrone [11]. These compounds thus present a paradox: they act as potent estrogens in animal studies, yet bind to ER extremely poorly. These results have been expressed quantitatively as the ratio of in vivo estrogenic potency to in vitro binding affinity. If this ratio is set to 1 for E2, it is as high as 350-600 for (+)-Z-bisdehydrodoisynolic acid and 80 for (-) allenolic acid [5]. Intriguingly, although bioassays with these compounds show estrogenic activity in rabbits, mice, rats, and chicks, there are some questions regarding the estrogenicity of these compounds in human females [5]. Both (+)-Z-bisdehydrodoisynolic acid 3-methyl ether (Ciba-Geigy 'Fenocylin ®') and the 3-methyl ether of (-) allenolic acid (Searle 'Vallestril ~') (see Table 1) had been tested for use in human estrogen replacement therapy [5]. Fenocylin, however, was clearly shown to be clinically inactive in human females [12]. A related study strongly suggests that Vallestril may be similarly inactive in human females [13]. In this study we have exploited the availability of mechanism-specific tissue culture-based reporter gene

v

R= H (-) -Allenolic Acid R = CH3 (-) -Allenolic Acid 3-methyl ether

assays to determine directly the ability of D/A compounds to act as ligands and to participate in both positive and negative gene regulation by ER. In addition, by using cells and ERs derived from both human and non-human sources, we have re-evaluated the potential species-specific differences in the activity of these compounds. Finally, we have examined the ability of these compounds to bind to E R using a direct assay that does not require competition with other ligands. Our results indicate that these compounds can act as ligands for ER, and act as agonists for both gene activation and gene repression of model reporter genes. We also confirm cell-type-dependent differences in activity for the 3-methyl ether of (+)-Zbisdehydrodoisynolic acid, suggesting that the ability to utilize this c o m p o u n d as a ligand may require specific cellular modifications or involve cell-typespecific factors.

MATERIALS AND METHODS

Compounds and hormones Estradiol-17~ and estradiol-17/~ 3-methyl ether were obtained from Sigma Chemical Co. (St Louis, MO, U.S.A.). ICI 164,384 was a gift from ICI (Macclesfield, U.K.). Doisynolic and allenolic acid derivatives were obtained as previously described [5]. (+)-Z-bisdehydrodoisynolic acid 3-methyl ether ('Fenocylin ®') was obtained from Ciba-Geigy Inc. (Summit, NJ, U.S.A.) (+)-Z-bisdehydrodoisynolic acid was prepared from its 3-methyl ether. (-)-Allenolic acid was prepared from its 3-methyl ether ((-) allenolic acid 3-methyl ether, 'Vallestril ®', Searle, Skokie, IL, U.S.A.). Reporter genes and expression vectors Assays for gene activation used the Vit2-P36L luciferase reporter plasmid, which contains two copies of a 26 bp estrogen response element (ERE) from the xenopus vitellogenin A2 gene linked to a minimal 3 6 b p promoter derived from the rat prolactin

Estrogenic Activities of Doisynolic Acids gene [14]. Assays for gene repression used the P R L D/ E p G L 3 luciferase reperter plasmid, which contains a modified rat prolactin enhancer/promoter control region, in which estrogen response elements have been mutated [15]. P R L D/E p G L 3 contains the rat prolactin distal enhancer from - 1 8 3 1 to - 1 5 3 1 linked to the promoter from - 4 2 2 to +33 in a modified p G L 3 plasmid backbone (Promega, Madison, WI, U.S.A.). Modifications to the promoter consist of deletion of the region from - 1 7 2 to - 1 6 2 and mutations changing the estrogerL response element sequence d A T G T C C T to d G G G C C C G at - 1 5 7 0 [15]. T h e rat E R c D N A was the generous gift of M. Muramatsu [16]. T h e truncated human ER, E R 251, is as previously described [15, 17, 18]. T h e full length wild type h E R cDNA, (Gly 400), was obtained from P. C h a m b o n [19]. T h e RSV-Neo plasmid, expressing the neomycin phosphotransferase I! gene is as previously described [17]. Receptors were expressed in vectors containing the'. Rous sarcoma virus (RSV) promoter [17].

Cell lines and transfections

Hela, CV-1, and MDA-MB-231 (MB) cell lines were obtained from the American T y p e Culture Collection (Rockville, M D , U.S.A.). G C rat pituitary cells were as previously described [18, 20]. All cells are routinely surveyed for mycoplasma using a polymerase chain reaction (PCR) m e t h o d from Stratagene (La Jolla, CA, U.S.A.). G C cells were grown on poly-D-lysine-treated dishes for D E A E dextran-based transient transfections under estrogen-free cell culture conditions [ 15, 17, 18, 21 ]. Typically cells were maintained under 10% CO2 in phenol red-free D M E / F 1 2 , supplemented with 5% horse serum, 5% enriched calf serum (Gemini Bioproducts, Calabasas, CA, U.S.A.), 2.5% fetal bovine serum, 1.5g/1 glucose, 2.85g/1 NaHCO3, 10 ml/1 non-essential amino acids, Fungizone (0.5 ~tg/ml) and penicillin/streptomycin (20 IU/20 #g/l). For transfections 3 x 105 cells were seeded into each 35 turn well of six-well plates. On the following day, after cell attachment, media were changed to phenol red-free D M E / F 1 2 , 10% charcoal stripped newborn calf serum. Transfections were begun 4 days after seeding. T h e day of transfection, cells were refed with phenol red-free D M E , 10% charcoal stripped newborn calf serum. After 4 h, each well received 1 ml of D M E containing D E A E dextran and a total of 5/zg of D N A consisting of 2.5 #g of luciferase reporter plasmid, with the remaining 2.5 #g consisting of RSV-based expression plasmid as indicated for each experiment. After an additional 2 h , media were removed, and the cells were washed with phenol redfree D M E containing :250 pg/ml sodium heparin, followed by phenol red-free D M E , and then fed with phenol red-free D M E / F 1 2 , 10% charcoal stripped

479

newborn calf serum. T h e next day, cells were treated with hormone or compounds as indicated. Transient transfections for Hela, CV-1, and M D A MB-231 cells used a calcium-phosphate m e t h o d [22] modified for 35 m m six-well plates [14]. T w o days prior to transfection, 1.5 × 105 cells were seeded in phenol red-free D M E with 10% charcoal stripped newborn calf serum into each 35 m m well of six-well plates. On the day of transfection, 4 h after refeeding, each 2 ml well received 125 #1 of BBS-CaC12 solution containing a total of 2.5 pg DNA, which included 1.25 pg luciferase reporter plasmid, 0.2 #g receptor expression vector or control, and 1.05 #g salmon sperm DNA. Plates were then placed overnight in 5% CO2. T h e next day, cells were washed, fed with phenol red-free D M E with 10% charcoal stripped serum, treated with hormone or compounds as indicated, and returned to 10% CO2. One day after hormone treatment cells were harvested in a triton lysis buffer, containing 50 m M Tris, 50 m M 2 (N-morpholino)ethanesulfonic acid (MES) (pH 7.8), 1 m M dithiothreitol ( D T T ) and 1% triton X-100. T h e lysate was assayed for luciferase activity as previously described [23], using a Monolight 2010 luminometer (Analytical Luminescence Laboratories, San Diego, CA, U.S.A.). Binding assays - - eleetromobility/gel shift

Full-length human E R (Gly 400) was expressed by in vitro translation using the p C I T E vector (Novagen, Madison, WI, U.S.A.). Translations were performed using the T n T T 7 reticulocyte lysate system (Promega). Paired sense and antisense staggered 3 6 b p oligonucleotides were synthesized containing the palindromic Xenopus vitellogenin A2 ERE, d A G G T C A C A G T G A C C T [17]. Gel shift probes were prepared by annealing and 3'-end filling in the presence of either [~-32p]dCTP or [c~-32p]dATP, yielding blunt 46 bp duplexes. Electromobility gel shift assays were modified to reflect a dependence on ligand [24-26]. Binding reactions (30 #1) for electromobility shift analyses contained ER in reticulocyte lysate, and gel shift buffer ( 1 2 m M H E P E S - N a O H (pH 7.9), 5 0 m M KC1, 10 m M MgC12, 0.6 m M D T T , 20/~g/ml BSA, 12% glycerol), unlabelled non-specific D N A (7.5 pg poly dI.dC), and hormone, compounds, or ethanol (control). Various concentrations of the test compounds were employed. In addition, each experiment included a sample containing ethanol as a negative control, and a parallel series of concentrations of E2 to determine maximal binding. T h e solutions were incubated for 15 min at room temperature, 15 min at 37°C, and then chilled on ice for 10min. Labelled specific D N A (30 000 cpm) was added and incubation continued on ice overnight to achieve binding equilibrium. Samples were resolved on native 1/2x glycerol tolerant gel buffer ( T r i s - T a u r i n e - E D T A ,

Cal Y. Meyers et aL

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Amersham, Arlington Heights, IL, U.S.A.), 4% polyacrylamide (acrylamide:bisacrylamide, 80:1) gels. Gels were dried without fixation directly onto DE81 paper (Whatman, Maidstone, U.K.). Results were visualized using a model 425B PhosphorImager, and analysed with I m a g e Q u a n t software (Molecular Dynamics, Sunnyvale, CA, U.S.A.).

RESULTS

D/A compounds act as agonists for estrogenic gene activation Even though the D/A c o m p o u n d s produce a variety of gross, cellular estrogenic effects in animals, there have been no published molecular studies d e m o n strating that these c o m p o u n d s act via ER. We therefore wished to determine if these c o m p o u n d s can activate E R to regulate the expression of a specific estrogen-regulated model reporter gene. For co-transfection assays of estrogenic gene activation, h u m a n E R is expressed using an RSV-based expression vector, and a specific ERE-containing luciferase reporter gene is co-transfected. For these experiments we used Vit2-P36L, a luciferase reporter gene which contains two 26 bp EREs from the Xenopus vitellogenin A2 gene, linked to a minimal 36 bp prolactin promoter.

CV-1 cells are widely used as a model system for studying gene regulation by nuclear receptors. T h e y express no detectable endogenous ER. These cells are primate, but not human, in origin. Figure 1 shows the results of gene activation assays for each D/A c o m p o u n d in CV-1 cells. All are competent to mediate gene activation of the specific E R E containing reporter gene, with activities comparable to E2. Each acts as an E R agonist on this specific, defined, model gene. In addition, experiments performed with solutions containing submaximal concentrations of E2 and each test c o m p o u n d showed no evidence of antagonist activity (data not shown).

Gene activation by D/A compounds is dependent on ER and mediated through human E R T w o types of experiments were performed to demonstrate the ER-dependence of these actions of the D/A compounds. First, the activities of all three c o m p o u n d s were tested in the presence or absence of competing concentrations of an E R antagonist. I C I 164,384 is a true steroidal E R ligand that acts as an antagonist of gene activation, as demonstrated by its effects with E2 in this assay (Fig. 1). Activation by each D/A c o m p o u n d , like E2, is inhibited by compet-

I nM E2

I0 nM

[]

No ER

• •

Compound Alone Compound Plus ICI

Allenolic Acid

I 0 nM BDDA

0

5 Relative

Expression

10 (Fold)

Fig. 1. D / A c o m p o u n d s act as agonists for estrogenic gene activation. For gene activation CV-1 cells w e r e cotransfected with Vit2-P36L reporter p l a s m i d plus R S V H E G 0 for e x p r e s s i o n of E R , or RSV N e o , as a control. Concentrations for each c o m p o u n d were added as indicated. Light units are e x p r e s s e d as folds relative to the activity observed with e x p r e s s e d E R in the a b s e n c e of added c o m p o u n d s set to 1.0 (not shown). D a t a is the m e a n and S E M from two e x p e r i m e n t s . O p e n bars, activity of e a c h c o m p o u n d with e x p r e s s e d RSV Neo; H a t c h e d bars, activity of each c o m p o u n d with e x p r e s s e d ER; Filled bars, activity o f each c o m p o u n d with e x p r e s s e d E R in the p r e s e n c e of 100 n m ICI 164,384. B D D A , (_+)-Z-b/sdehydrodoisynoHc acid; B D D A - O M e , (+_)-Z-bisdehydrodoisynolic acid 3 - m e t h y l ether.

Estrogenic Activities of Doisynolic Acids

481

120' ---"U m--ll~ m..-d. 100

E2 E2-OMe BDDA

.I,

BDDA-OMe

.-(3

~

,

,

80 8 60 UJ

~.

40

20

0

. . . . . . . .

I0-Is

i

. . . . . . . .

,

. . . . . . . .

i0-14 i0-I~

,

. . . . . . . .

I0-Iz

i

. . . . . . . .

i0-11

i

. . . . . . . .

i0-Io

,

. . . . . . . .

I0-9

Concentration

,

. . . . . . . .

10"8

i

. . . . . . . .

10 -7

,

. . . . . . . .

I0-6

,

. . . . . . . .

10-s

i 0 -'i

(M)

Fig. 2. G e n e a c t i v a t i o n d o s e r e s p o n s e profiles for the D/A c o m p o u n d s . For gene activation M D A MB231N cells were cotransfected with Vit2-P36L r e p o r t e r p l a s m i d plus RSV H E G 0 for e x p r e s s i o n of ER. Concentrations for e a c h c o m p o u n d w e r e a d d e d as indicated. Light units are e x p r e s s e d relative to the m a x i m a l activation a c h i e v e d with estradiol (100%), and r e p r e s e n t the m e a n s from several e x p e r i m e n t s . D, E2, estradiol (n = 8); II, E2-OMe, estradiol m e t h y l ether (n = 2); A, B D D A , (±)-Z-b/sdehydrodoisynolic acid (n = 3); A, B D D A - O M e , (±)-Z-bisdehydrodoisynolic acid 3 - m e t h y l e t h e r (n = 7); (3, (-)-aUenolic acid (n = 2).

ing concentrations of ICI 164,384. Similar results were obtained in Hela c'ells (data not shown). Second, we have used an expression vector for neomycin phosphotransferase II (Neo), rather than ER, to demonstrate the requirement for E R (Fig. 1). In the absence of expressed ER, when the control gene Neo is expressed instead, neither estradiol, nor the D/ A compounds can effectively activate the target gene. This result demonstrates that gene activation of this ERE-containing model[ reporter gene by the D/A compounds requires functional ER.

Gene activation by D/A compounds is dose-dependent For each model c o m p o u n d dose-response profiles were determined by using a range of concentrations in these assays. These assays were performed in a h u m a n breast cell line:, M D A M B 2 3 1 N , which, like CV-1, is null for ER. As above, the h u m a n E R expression vector, RSV H E G 0 [14, 19] is co-transfected with a specific, ERE-containing luciferase reporter gene, Vit2-P36 luciferase [14]. These dose-response profiles in Fig. 2 show that in spite of differing potencies, each c o m p o u n d is capable of gene activation, and that typical receptor saturation profiles are observed.

D/A compounds repression

act as agonists for estrogenic gene

Recently it has become apparent that ligand can play a role in distinguishing the competence of E R to perform different mechanisms of gene regulation. For example, tamoxifen can be an estrogen antagonist for gene activation while acting as an agonist for ERmediated gene repression in the same cell type [18]. We therefore determined whether the D/A compounds can enable E R to repress the expression of a specific estrogen-repressed model reporter gene. Rat pituitary G C cells are like pituitary lactotrophs and somatotrophs, expressing both growth hormone and prolactin. P R L D/E, a modified prolactin gene, has been used to demonstrate negative regulation by ER[15, 18]. Figure 3 shows the results of assays using the D/A compounds. Each c o m p o u n d is competent to mediate negative regulation, repressing gene expression of this model D/E P R L reporter gene, and acting as an E R agonist on this specific, defined, negatively regulated model gene. Furthermore, as for positive regulation, solutions containing submaximal concentrations of E2 and the test compounds showed no antagonist effects on gene repression (data not shown).

482

Cal Y. M e y e r s et al.

1 nM E2

[] • •

ER 251 Compound Alone Compound Plus ICl

0.3 nM E2

t

10 nM Allenolic Acid I

10 nM BDDA

1 uM BDDA-OMe

0

20

40 Relative

60 Expression

8'0

I (~0

(%)

Fig. 3. DIA c o m p o u n d s act as a g o n l s t s for e s t r o g e n i c g e n e r e p r e s s i o n . F o r gone r e p r e s s i o n G C cells were cot r a n s f e c t e d with DIE pGL3 r e p o r t e r p l a s m i d p l u s RSV H E G 0 for e x p r e s s i o n o f ER, or RSV E R 251, a d o m i n a n t n e g a t i v e m u t a n t defective in gone r e p r e s s i o n . C o n c e n t r a t i o n s for e a c h c o m p o u n d were a d d e d as indicated. Light u n i t s a r e s h o w n as t h e m e a n a n d S E M relative to t h e activity o b s e r v e d in t h e a b s e n c e o f a d d e d c o m p o u n d s . O p e n h a t c h , activity o f e a c h c o m p o u n d with e x p r e s s e d E R 251 (n = 2); H a t c h e d b a r s , activity o f e a c h c o m p o u n d alone (n = 3 or 4); solid b a r s , activity o f e a c h c o m p o u n d in t h e p r e s e n c e o f 100 n m ICI 164,384 (n = 3 or 4). B D D A , ( ± ) - Z - b i s d e h y d r o d o i s y n o l l c acid; B D D A - O M e , ( ± ) - Z - b / s d e h y d r o d o i s y n o l i c acid 3 - m e t h y l ether.

100 [] cO

[]

\

80

~o

o

IZ X uJ

>

60

40

e~ 20

0

A

BDDA

• O

BDDA-OMe Allenolic Acid

. . . . . . . .

0-15

I

. . . . . . . .

i

. . . . . . . .

,

. . . . . . . .

10-14 10"13 I 0 - I z

i

. . . . . . . .

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. . . . . . . .

,

. . . . . . . .

10-11 10"1o I 0 - 9 Concentration

,

. . . . . . . .

10 .8

T

. . . . . . . .

10"7

i

. . . . . . . .

10 .6

i

I 0 -s

(M)

Fig. 4. G e n e r e p r e s s i o n dose r e s p o n s e profiles for t h e D/A c o m p o u n d s . F o r gone r e p r e s s i o n , G C cells were c o t r a n s f e c t e d with D/E pGL3 r e p o r t e r p l a s m i d p l u s RSV H E G 0 for e x p r e s s i o n o f ER. C o n c e n t r a t i o n s for e a c h c o m p o u n d were a d d e d as indicated. Light u n i t s a r e e x p r e s s e d relative to t h e value o b s e r v e d with n o c o m p o u n d (100%), a n d r e p r e s e n t t h e m e a n s f r o m several e x p e r i m e n t s . [], E2, estradiol ( n - - 8 ) ; m, E2-OMe, estradiol m e t h y l e t h e r (n = 4); A, B D D A , ( ± ) - Z - b / s d e h y d r o d o i s y n o l i c acid (n = 8); A , B D D A - O M e , ( ± ) - Z - b / s d e h y d r o o doisynoHc acid 3 - m e t h y l e t h e r (n = 8); ©, ( - ) - a l l e n o l l c a c i d (n = 8).

Estrogenic Activities of Doisynolic Acids ~1

None

I

•

I

[]

Hela

[]

cv-1

•

MB

,

:l'i'

'

'

M ii'

483

~i,

H

I~iilHIH~INilmlIUm|tIN|~ImlJ[~IN!I~,~Itt 1 nm E2

10 nm BDDA _

_

t

l||||~ 0.1

I

uM BDr~A-OMe

I

10 nm Allenolic Acid

6

"

2'0

"

4'0

"

6'0

Relative

'

8'0

i

1 60

1 20

1 z~0

Expression

Fig. 5. Activity of D/A c o m p o u n d s in various cell lines. For gene activation Hela cells, CV-1 cells, and M D A MB 231 cells were cotransfected with VitZ-P36L reporter plasmid plus RSV HEG0 for expression of ER. Concentrations for each c o m p o u n d were added as indicated. Light units are expressed as folds relative to the activity observed with 1 nM E2 set to 100. D a t a is the m e a n and S E M from two experiments. Grey bars, Hela cells; O p e n bars, CV-1 cells; Solid bars, M D A MB231 cells (MB). B D D A , (+)-Z-bisdehydrodoisynolic acid; B D D A - O M e , (_+)-Z-b/sdehydrodoisynolic acid 3-methyl ether.

Gene repression by D/A compounds is dependent on~and mediated through ER

Experiments were performed to demonstrate that in mediating gene repression, the D/A compounds are d e p e n d e n t on, and function via, the ER. First, the repressive activity of all three compounds was tested in the absence or presence of competing concentrations of ICI 164,384. In these assays (Fig. 3), ICI 164,384 acts as an anl:agonist of gene repression for each compound, preventing the decrease in gene expression by agonists. Se.cond, we have used expression of a dominant negative mutant of h u m a n ER, E R 251 [15, 17, 18], rather: than wild type receptor, to demonstrate the requirement for ER. In the presence of this mutant receptor, neither estradiol, nor the D/A compounds can fully repress the target gene (Fig. 3). This observation also demonstrates that repression by the D/A compounds requires functional ER. Gene repression by D/A compounds is dose-dependent

As for positive regulation, for each model compound dose-response profiles for negative regulation were determined by using a range of concentrations in these assays (Fig. 4). Again, these dose-response profiles show that, in spite of differing potencies, each c o m p o u n d is capable of activity in assays of g e n e repression. Also, profiles show a typical saturation curve, similar to that observed with E2, but differing in effective concentrations.

Gene activation by (!)-Z-bisdehydrodoisynolic acid 3methyl ether in different cell types and with ER from different species Limited clinical experience with (+)-Z-bisdehydro-

doisynolic acid 3-methyl ether suggested that the estrogenic activity of this c o m p o u n d differs b e t w e e n humans and other species [12]. We therefore compared the activity of (+)-Z-bisdehydrodoisynolic acid 3-methyl ether in human cell lines with that in nonhuman cell lines, and activity with h u m a n E R with that with rat ER. Figure 5 compares the activity of the D/A compounds to E2 in gene activation assays with h u m a n E R in each of three cell types: Hela cells and M D A MB231 cells, both of h u m a n origin, and CV-1 cells of monkey origin. Whereas, for the concentrations tested, the activities of (+)-Z-bisdehydrodoisynolic acid and allenolic acid are comparable in all three cell lines, CV-1 cells showed greater activity with (±)-Z-bisdehydrodoisynolic acid 3-methyl ether than did either of the two h u m a n cell lines. This result was confirmed by performing full dose response profiles (Fig. 6). T h e activity of (+)-Z-bisdehydrodoisynolic acid 3-methyl ether is detectable at lower concentrations, and achieves a higher level of g e n e expression in CV-1 cells than in the two h u m a n cell lines, an effect not seen with E2. Figure 7 shows that h u m a n and rat receptors function similarly in both CV-1 cells and Hela cells, and again indicate that (+_)-Z-bisdehydrodoisynolic acid 3-methyl ether is

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active at a lower concentration in CV-1 cells than in Hela cells (Fig. 7) or MB cells (not shown).

Direct binding assays Previous studies have measured the affinity of the D/A compounds using binding assays based on competition [5, 11]. We have used a direct binding assay to determine relative binding efficiency of the compounds in comparison to E2. This assay does not rely on competition with E2, does not require chemical modifications of compounds that may alter their affinity, and does not require radioactive labelling which may also require chemical modifications. T h e assay uses human ER translated in vitro in reticulocyte lysates, and a modified electromobility shift assay [2426]. This assay is specific for compounds that bind to ER, whether agonist or antagonist, steroidal, or nonsteroidal (data not shown). Results can be used to generate binding profiles for comparing binding affinity of test compounds relative to E2. Using this assay we have generated binding profiles (Fig. 8) and determined relative binding affinities (compound:E2 ratio) for each of the compounds (Table 2). For (+)-Z-bisdehydrodoisynolic acid, the binding affinity ratio was determined to be 0.01, a value indistinguishable from that determined via competitive binding [5] and transfection data for gene acti-

vation (Fig. 1 and Fig. 2). A relative activity ratio approximately 10-fold higher was obtained from negative gene regulation (Fig. 3 and Fig. 4). For (-) allenolic acid, the value of 0.01 is comparable to the value of 0.004 previously obtained by competitive binding [10]. T h e binding profile, however, is very broad, and it may indicate that allenolic acid, which has only a two-ring structure, does not fully stabilize the E R to the heat treatment which is the basis of this direct binding assay. Values for activity in negative and positive regulation compare very well with these binding results. For (+)-Z-bisdehydrodoisynolic acid 3-methyl ether, binding was not of sufficient magnitude for detection using competitive binding techniques [11]. In the direct binding assay, a value of 1 × 10 -4 was determined. This value is comparable to the values obtained in transfection experiments, although, as shown above, transfection results did vary by cell line. Also, comparing estradiol 3-methyl ether to E2, one can estimate the effect on affinity of substituting methoxy for hydroxy at the 3-position. This ratio (0.003) is very similar to that (0.01) observed comparing (+)-Z-bisdehydrodoisynolic acid 3-methyl ether to (+)-Z-bisdehydrodoisynolic acid. O f note is the fact that these assays do not explain the high activity of (+_)-Z-bisdehydrodoisynolic acid in

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vivo observed at low doses, c o m p a r e d to those for producing the same effect.

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DISCUSSION T h e results presented above demonstrate that the D/A c o m p o u n d s do, i:a fact, regulate specific estradiol-regulated target genes in living cells. Previous analyses relied on evaluation of gross, cellular-based assays, whether in viw~ by uterotropism and vaginal cornification [5], or in a cell culture system by cell proliferation [13]. We have further demonstrated a dependence on the E R for both gene activation and gene repression. All of these results make it unlikely that the observed in vivo estrogenic effects of the D/A c o m p o u n d s would exclusively utilize alternative pathways, receptors, or mechanisms. Our experiments utilized well-defined systems that allowed mechanism-specific approaches with appropriate controls. T h e s e analyses used two artificial luciferase reporter target genes, as models of gene activation and gene repression. T h e results d e m o n strate that these c o m p o u n d s can act as E R agonists for both gene activation and gene repression. Nonetheless, there are some differences in the relative

potency of these c o m p o u n d s for the two mechanisms of estrogen action (Fig. 2 and Fig. 4). Because all the c o m p o u n d s are tested using the same cells and prom o t e r constructs, cell type and p r o m o t e r effects m a y not fully explain these differences. These data m a y be similar, although less dramatic, to the results we observed with the non-steroidal mixed antagonist, tamoxifen, which presents very striking differences in activities for gene activation and gene repression [18]. For the D/A compounds, changes in the relative ability to mediate gene activation and gene repression may indicate more subtle changes in ligand-dependent receptor conformation, distinguished by these two molecular mechanisms of estrogenic gene regulation. We have not tested all the potential mechanisms for E R action, nor have we tested other types of model reporter genes. T h e effect of the D/A c o m p o u n d s on ligand-independent activation of ER, as has been described for dopamine [27], has not been investigated. Even though we have demonstrated direct binding of the D/A c o m p o u n d s to the estrogen receptor, it remains theoretically possible that the D/A c o m p o u n d s could also have effects on E R via this type of signal transduction pathway. We do not consider such a pathway a likely m e c h a n i s m for the D/A

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487

Estrogenic Activities of Doisynolic Acids

Table 2. Relative activities of D/A compounds Compound Estradiol- 17fl Estradiol 3-methyl ether (±) -Z-b/sdehydrodoisynolic acid (+)-Z-bisdehydrodoisynolic acki 3-methyl ether (-) Allenolic acid

Competitive binding* 1 0.03 [5] 0.01 [5] <0.003 [5] 0.004 [10]

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* F r o m previous studies. *Varies by cell line.

compounds. Were such a mechanism contributing to the observed activities, one might also expect activation of other steroid receptors, such as the progesterone receptor [28], by the D/A compounds; however, effects of the D/A compounds on the activities of other receptors have not been reported. Other mechanisms of gene regulation that may require interaction of E R with transcriptional co-activators or other transcription factors, including 'composite' regulation, may exhibit different results for the D/A compounds. Indeed, there is evidence that these D/A compounds do no~ act as estrogen agonists for all the actions of estradiol. It has been found that whereas estradiol causes an increase in the rate of weight gain in immature mice, both (+)-Z-bisdehydrodoisynolic acid and its 3-methyl ether actually cause these animals to gain weight slower than control animals [29]. T h e mechanisms, gene targets, and corresponding molecular basis of this difference between the D/A compounds and estradiol have yet to be determined. Also, the potential interaction of these compounds with the recently described ER /3 form [30], remains to be determined. We also present supportive data for the original observations that (+)-Z-bisdehydrodoisynolic acid 3methyl ether exhibits species-dependent effects, exhibiting lower potency in humans than in other species [5, 12]. Surprisingly, differential activity was observed in simple cell culture systems. These systems do not reflect hepatic metabolism, serum binding proteins, or the other complex processes that may occur in whole animal systems. Rather, the observed differences can only be dependent on cellular factors differing between the cell lines examined. We also observed similar results using either the h u m a n or the rat ER. These data are consistent with the observed clinical observations, suggesting differences in activity for (+)Z-bisdehydrodoisynolic acid 3-methyl ether between humans and animals [1:2]. Our data provide no support for any difference in activity based on the species of origin of the E R itself. However, our tissue culture data do not tell whether the differing effects are based on the different species of origin (human vs monkey), or organ and cell-type-specific effects (breast, cervix, or kidney). N o r do these data exclude the potential

contributions of mechanisms specific to intact animals. T h e use of a direct binding assay provides a new way to assess the affinity of compounds for ER without requiring competition with other ligands. This assay relies on the enhanced thermal stability of ER conferred by ligand binding. We have found that this assay accurately distinguishes between compounds that act as ligands, whether agonists or antagonists, and compounds that do not act as ligands (Adler, unpublished data). Although this assay requires higher concentrations of ligand than those used for gene regulation or for competition binding studies, our results, comparing compounds to estradiol and expressed as a relative binding affinity, are very similar to those obtained with competition assays. In addition, using this assay we have been able to determine a relative binding affinity for (+)-Zbisdehydrodoisynolic acid 3-methyl ether, a value that could not be determined using competition assays [11]. Recently it has been reported that some pairs of non-steroidal estrogenic compounds can act synergistically [31]. This remains controversial and others have failed to confirm these findings [32]. One possible explanation for synergistic effects is co-occupancy of the ligand binding site by two compounds. It is tempting to speculate that compounds that exhibit this type of interaction, by binding to only part of the ligand binding site, might compete poorly if estradiol is already bound and occupying the entire ligand binding site. In a direct binding assay, greater binding might be detectable, even if the partial ligand site occupancy is not competent for gene regulation. We have found no evidence for synergistic binding among the D/A compounds using the direct binding assay (Adler, unpublished data). T h e evaluation of other pairs of non-steroidal compounds remains for future investigations. It is becoming increasingly clear that non-steroidal compounds used in our industrialized society may act as estrogens or partial estrogens (see brief review by Stone [33]). These compounds include insecticides, plasticizers, dioxins and other xenobiotics [33]. T h e h u m a n health implications of these 'environmental es-

488

Cal Y. M e y e r s et al.

trogens' or endocrine disrupters are uncertain and extremely controversial. Recent clinical epidemiological studies are inconclusive as to whether these compounds have etiological significance, and contribute to the observed increases in breast cancer incidence [34, 35]. It has been argued that concerns regarding the impact of these c o m p o u n d s on h u m a n health m a y have been overstated because m a n y of these c o m p o u n d s bind to E R only weakly, because humans m a y exhibit less sensitivity to these c o m pounds than do other species, and because certain of these c o m p o u n d s m a y act as antiestrogens and balance the effects of those c o m p o u n d s that act as estrogen agonists [33]. In this work we have examined a group of non-steroidal c o m p o u n d s that were uniquely positioned to serve as models for addressing these controversial issues. T h e y are highly estrogenic in vivo, yet presented the peculiar paradox of exhibiting low binding affinity for the E R using existing in vitro competition assays. T h e y also had clinically demonstrated different potencies in animals c o m p a r e d to humans. We hope to extend these approaches and types of studies to examine other non-steroidal c o m p o u n d s with industrial uses, to determine their potential to function as endocrine disrupters in humans. Acknowledgements--We thank Mary Ann Mallon for technical assistance. This work was supported by National Institutes of Health and National Cancer Institute grant R03 CA70515.

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