Analysis of ligand dependence and hormone response element synergy in transcription by estrogen receptor

ft. Steroid Biochera. Molec. Biol. Vol. 60, No. 5-6, pp. 285-294, 1997

Pergamon

PII: S0960-0760(96)00198-7

© 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0960-0760/97 $17.00 + 0.00

Analysis of Ligand Dependence and Hormone Response Element Synergy in Transcription by Estrogen Receptor Sandra Mattick, Kevin Glenn, Georgius de Haan and David J. Shapiro* Department of Biochemistry, B-4 RAL, University of Illinois, 600 S. Mathews Avenue, Urbana, IL 61801, U.S.A.

In this work we e x a m i n e d two questions: (1) Is the low, but readily detectable, ability o f estrogen receptor (ER) to actiLvate transcription in the absence o f added 17/~-estradiol caused by traces o f estrogen in the growth m e d i u m , or by a weak ligand-independent ability of ER to activate transcription? (2) D o e s the ER exhibit synergistic activation o f transcription on reporter genes containing multiple estrogen response elements (EREs)? To study these questions we developed a powerful new reporter gene, containing four EREs, which achieves inductions of up to 330-fold in the presence of liganded ER. We provided several types o f evidence indicating that under standard cell culture conditions unllganded ER is unable to activate transcription. We demonstrated that when cells are grown in serum-free m e d i u m , estrogenic c o m p o u n d s may be in the base tissue culture m e d i u m . We demonstrated a strong cell and ER-dependence in transcriptional synergy, and suggest that cooperative binding o f ER to multiple EREs can be responsible for transcriptional synergy in vivo. © 1997 Elsevier Science Ltd.

ft. Steroid Biochem. Molec. BioL, Vol. 60, No. 5-6, pp. 285-294, 1997

INTRODUCTION

The estrogen receptor (ER) is a member of the steroid/nuclear receptor superfamily of ligand-regulated transcription factors. The members of this superfamily possess common structural features with discrete protein regions responsible for ligand binding, nuclear localization, dimerization, DNA binding and transcription activation []-3]. Although these proteins share many structural and functional properties, the role played by their tmliganded forms in transcription may differ. Although unliganded forms of most members of the thyroid/re.tinoid receptor subfamily are often potent repressors of transcription [4], unliganded glucocorticoid receptors are largely cytoplasmic and appear to be devoid of the ability to activate or repress transcription [5, 6]. The function of the unliganded ER is more controversial. Tzuckerman et al. [7] reported that unliganded ER maintains substantial endogenous transcriptional ac*Correspondence to Prof. D. J. Shapiro. Tel: +1 217 333 1788; fax: +1 217 244 5858; e-mail: [email protected]. Received 15 Jul. 1996; accepted 22 Oct. 1996. 285

tivity. Numerous reports have shown a low, but readily detectable, level of ER-associated basal activity in the absence of added estrogen [8-11]. One plausible explanation for this apparent basal activity of ER is a low level activation of ER from trace amounts of 17fl-estradiol in the medium, and charcoal-dextrantreated serum used in cell culture experiments. An alternative explanation for the basal activity is that the ER is phosphorylated on both serine and tyrosine in response to signals from MAP (mitogen-activated protein) kinase, cyclic AMP (adenosine monophosphate) and other regulatory signals [12-15]. Activation of ER by protein phosphorylation could represent a parallel pathway for ER activation, or become operative only in conjunction with an estrogenic ligand bound to the ER. To identify the source of ER basal activity, we initially adapted CHO (Chinese hamster ovary) cells to serum-free growth. In CHO cells maintained in serum-free medium, we observed a surprisingly high level of basal transcription, resulting in a low-fold induction by estrogen. The fold induction was not increased when the cells were maintained in proteinfree medium lacking insulin and transferrin. The

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basal activity of E R was reduced u p o n the addition of charcoal-treated serum to the medium. This suggested that the charcoal-treated serum was binding residual estrogens in the medium. In CI-IO cells picomolar concentrations of 17#-estradiol elicited a detectable activation of transcription. We carried out transient transfections in several cell lines to assess the possibility of estrogen-independent transcription. Using serum low in endogenous estrogen and a sensitive new reporter gene containing four consensus estrogen response elements, estrogen induced transcription by 100-fold or more in human, rodent and amphibian cell lines. This suggests that under typical cell culture conditions, an estrogenic ligand is essential for activation of transcription by wild-type ER. In the course of this work we also examined transcriptional synergism in reporter genes containing one, two and four estrogen response elements (EREs). In previous studies, E R - d e p e n d e n t activation of transcription from promoters containing one or two consensus EREs had been reported either to exhibit strong synergism [16, 17] or no synergism at all [18, 19]. We found that the extent of transcriptional synergism by E R is strongly dependent on cell type and E R concentration. This strong cell type and E R concentration dependence explains the discordant data obtained in earlier studies. O u r data supports the view that in the reporter system we used, transcriptional synergism is based primarily on the ability of b o u n d E R to facilitate binding of E R to other EREs, and is not based on synergy in the formation or stabilization of the basal transcription complex.

MATERIALS AND METHODS

Plasmids In most studies, the expression of wild-type h u m a n [20] and Xenopus ER (XER) [21] was u n d e r the control of the strong cytomegalovirus (CMV) p r o m o t e r in p C M V 5 . In some experiments, X E R expression was under the control of the relatively weak thymidine kinase (TK) p r o m o t e r ( T K X E R ) [19]. T h e construction of chloramphenicol acetyl transferase (CAT) reporter constructs, 1 E R E - T A T A - C A T and 2ERET A T A - C A T , has been described previously [19]. 4 E R E - T A T A - C A T was constructed by isolating the SphI/BglII 2ERE fragment of 2 E R E - T A T A - C A T , digesting 2 E R E - T A T A - C A T with XbaI/BglII, blunt ending the SphI and BglII ends with T 4 D N A polymerase, and lastly ligating the 2ERE fragment into 2 E R E - T A T A - C A T . T h e accuracy of the insertion and junctions were confirmed by D N A sequencing. Plasmids used in transfections were purified either twice by CsCI linear gradient centrifugation or on Q I A G E N columns ( Q I A G E N Corp., Chatsworth, CA, U.S.A.).

Cell culture and transfections C H O cells were maintained in phenol-red-free D M E / F 1 2 m e d i u m (Sigma, St Louis, M O , U.S.A.) supplemented with 5% charcoal-dextran treated newborn calf serum ( C D - N B C S ) . C H O cells were also cultured in a serum-free m e d i u m of phenol-red-free D M E / F 1 2 (Sigma), 0.5x Eagle's non-essential amino acids, 2x insulin-transferrin-selenium (ITS; Collaborative Research, Bedford, MA, U.S.A.), 50000 U/1 penicillin, and 5 mg/1 streptomycin. Water used to prepare the serum-free m e d i u m was pretreated with an activated charcoal filter and then passed through a MilliQ 5 column filtration system with a pyrogen removal column (Millipore, Bedford, MA, U.S.A.). H e p G 2 cells, a h u m a n h e p a t o m a line, were maintained in Dulbecco's modified Eagle's m e d ium ( D M E M ; Gibco, G r a n d Island, NY, U.S.A.), supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA, U.S.A.) which was charcoal-dextran treated (CD-FBS). Xenopus X L 1 1 0 cells were maintained in 0.6x Higuchi's m e d i u m [22] supplemented with 10% C D - F B S and Xenopus X T C 2 cells were maintained in 0.7× D M E / F 1 2 containing 10% C D - F B S . All serum-supplemented cell lines were transfected in their appropriate growth m e d i u m using the calc i u m - p h o s p h a t e - D N A coprecipitation m e t h o d with glycerol shock. Transfections employed either 8 or 20/~g of total D N A per 60 m m or 100 m m dish, respectively. A luciferase expression vector, CMV-luciferase, was used as the internal control for transfection efficiency and P T Z 1 8 U was used as carrier D N A . T w o days before transfection, C H O cells were seeded at a density of 200 000 per 60 m m dish, and H e p G 2 cells were seeded at a density of 500 000 cells per 60 m m dish. Both cell lines were exposed to a 20% glycerol shock 1 4 - 1 6 h after crystal application. One day before transfection, X L l l 0 and X T C - 2 cells were seeded at a density of 8 - 9 × 105 cells per 100 m m dish and were exposed to a 15% glycerol shock 2 0 - 2 4 h after crystal application. For all cell lines, the indicated amounts of 17#-estradiol were added to the culture m e d i u m immediately after glycerol shock. Serum-free C H O cells were maintained for at least 60 days in the serum-free media D M E / F 1 2 . T o transfect the C H O cells in the absence of serum it was necessary to use the cationic lipopolyamine, T r a n s f e c t a m (Promega, Madison, WI, U.S.A.). C H O cells were plated at a density of 200 000 cells/60 m m plate 2 days before transfection. T h e m e d i u m was removed and replaced with a 1.0 ml mixture of D N A (0.8/~g 2 E R E - T A T A - C A T , 1.7/~g of an internal control SV-40 luciferase vector, p S L U C 2 ( A T C C # 37575), and 5 ng C M V h E R dissolved in 0.5 ml of m e d i u m ) and Transfectam (3.9/~g Transfectam//~g D N A in 0.5 ml of medium). Eight hours later the

Estrogen is Required for ER Transactivation mixture was removed, the plates were washed twice with H a n k ' s balanced salt solution, and 5 . 0 m l of media was added with either 1 0 - S M 17fl-estradiol or 0.1% E t O H . T h e cells were harvested 24 h later, and assayed as described below.

CA T and luciferase assays Transfected cells from all four s e r u m - s u p p l e m e n t e d cell lines were harvested 4 6 - 4 8 h after glycerol shock and assayed for C A T activity, essentially as previously described [19]. Briefly, extracts were prepared from both serum-containing and serum-free cells by three rounds of rapid freezing and thawing in a buffer containing 250 m M Tris/5 m M E D T A . Clarified supernatants were assayed for protein concentration using Coomassie blue reagent (Bio-Rad, Hercules, CA, U.S.A.) and for luciferase activity in a Monolight 2010 luminometer (A~alytical Luminescence Lab., Ann Arbor, MI). C A T activity was determined by our quantitative mixed phase assay [23]. RESULTS

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Table 1. Transfection of CHO cells in serum-free medium Transfection method*

Relative C A T activity1"

Polybrene Lipofectin, 10:1 Lipofectin, 20:1 Electroporation Calcium phosphate Transfectam, 4:1

>0.1 _+ 0.1 >0.1 ± 0.1 1 2 _+ 0.4 8± 1 100 _+ 7

*Transfections were carried out using standard protocols for each method. ?The data for Transfectam was set equal to 100. The data represents the mean + SEM for at least three transfections.

C H O cells maintained in serum-containing m e d i u m (data not shown), and is considerably shorter than the 18-24 h doubling times typical of m o s t m a m m a l i a n cells maintained in serum-containing medium. T h e cells were stably maintained in the serum-free m e d ium for at least 6 months with no change in doubling time, gross morphology, or sensitivity to killing by G418 (data not shown).

The role of estrogen in transfected CHO cells maintained in serum-free medium

Rapid growth of CHO ce,!ls in serum-free medium Because the charcoal-treated serum used in our standard transfections represented a potential source of estrogens, C H O cells were adapted to serum-free growth. Using D M E / F 1 2 m e d i u m , supplemented as described in Materials and Methods, we were ultimately able to grow C;HO cells with essentially the same doubling time as cells maintained in serum-containing medium. T h e average doubling time calculated from growth curves (Fig. 1) for C H O cells maintained in our serum-free medium was 14.4_+1.2 h. This is sirailar to the doubling time of

201

Although there have been n u m e r o u s reports of the successful maintenance of tissue culture cells under serum-free conditions [24-26], serum or nutritional supplements containing an undefined protein mixture are customarily added back to the cells for transfections. C H O cells in serum-free m e d i u m did not recover reproducibly when transfected by the calcium -ESTRADIOL _ 1riM E S T R A D I O L

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Fig. 1. G r o w t h c u r v e f o r C H O c e l l s m a i n t a i n e d in s e r u m - f r e e m e d i u m . C e l l c o u n t s wer, e b a s e d o n v i a b l e c e l l s a s d e t e r mined by t~pan blue dye exclusion. The data represents the m e a n ± S E M f o r at l e a s t t h r e e d e t e r m i n a t i o n s .

Fig. 2. E s t r o g e n - d e p e n d e n c e o f E R t r a n s a c d v a d o n in C H O cells m a i n t a i n e d in s e r u m - f r e e m e d i u m . C H O ceUs m a i n t a i n e d in s e r u m - f r e e m e d i u m f o r 6 d a y s o r f o r 60 d a y s , w e r e transfected with CMVhER, 2 E R E - T A T A - C A T , and the luciferase internal s t a n d a r d plasmid using T r a n s f e c t a m as described in Materials and Methods. The cells were maint a i n e d in t h e p r e s e n c e o r a b s e n c e o f a d d e d 1 7 f l - e s t r a d i o l a n d assayed f o r C A T activity. T h e d a t a r e p r e s e n t s t h e m e a n ± S E M f o r at least t h r e e s e p a r a t e t r a n s f e c t i o n s .

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p h o s p h a t e or e l e c t r o p o r a t i o n m e t h o d s . O f the several m e t h o d s we t e s t e d , o n l y t r a n s f e c t i o n with t h e c a t i o n i c l i p o s o m e , T r a n s f e c t a m , y i e l d e d r e p r o d u c i b l e results ( T a b l e 1). W h e n the C H O cells were t r a n s f e c t e d w i t h E R in the p r e s e n c e o r a b s e n c e o f estrogen, a h i g h level o f est r o g e n - i n d e p e n d e n t t r a n s c r i p t i o n o c c u r r e d (Fig. 2), r e s u l t i n g in e s t r o g e n i n d u c t i o n s o f only six- to eightfold. T h i s was n o t c a u s e d b y the p e r s i s t e n c e o f residual e s t r o g e n f r o m the cell's original s e r u m - c o n t a i n ing g r o w t h m e d i u m , as similar b a s a l activity was seen w h e t h e r the cells were m a i n t a i n e d for 6 days or 60 days in s e r u m - f r e e m e d i u m (Fig. 2). T h e s e d a t a w e r e c o n s i s t e n t w i t h several possibilities: (a) the E R c o u l d b e a c t i v a t e d b y the high levels o f insulin (10 mg/l) in the s e r u m - f r e e m e d i u m ; (b) the insulin o r t r a n s f e r r i n u s e d to s u p p l e m e n t the b a s e m e d i u m c o u l d c o n t a i n traces o f e s t r o g e n i c c o m p o u n d s ; (c) the b a s e p h e n o l red-free D M E / F 1 2 c o u l d c o n t a i n estrogens; (d) the E R c o u l d exhibit the ability to activate t r a n s c r i p t i o n in the a b s e n c e o f estrogens. T o e v a l u a t e these possibilities we c a r r i e d o u t a d d i t i o n a l t r a n s f e c t i o n s ( T a b l e 2). T h e use o f c o n d i t i o n e d m e d i u m d i d n o t i n c r e a s e t h e fold i n d u c t i o n . C o n t a m i n a t i o n o f the p o r c i n e insulin u s e d in o u r s e r u m - f r e e m e d i u m w i t h e s t r o g e n s was n o t r e s p o n sible for the l o w - f o l d i n d u c t i o n as r e c o m b i n a n t insulin d i d n o t i n c r e a s e t h e fold i n d u c t i o n . T h e s e r u m - f r e e m e d i u m we u s e d c o n t a i n s 2× I T S w h i c h a d d s 10 m g o f insulin/1 to t h e m e d i u m . T h e p o s s i b i l i t y t h a t this high level o f insulin was r e s p o n s i b l e for E R a c t i v a t i o n was t e s t e d in t r a n s f e c t i o n s with a r e d u c e d level o f I T S (0.25×) o r w i t h p r o t e i n - f r e e m e d i u m w h i c h d o e s n o t

Table 2. Transactivation by ER in serum-free cell culture medium Media supplements* 5% CD-newborn calf serum Serum-free media 2x ITS (insulinitransferrin/selenium) 2x ITS, conditioned medium 10 mg/1 recombinant insulin, 10-7 M Se 0.25x ITS (no iron + desferoxamine) Protein-free

Fold induction 53.7 + 5.3

(15)

8.7 + 1.8 8.8 + 3.6 7.4 + 3.3 8.8 6.5 + 2.4

(6) (3) (3) (1) (3)

*CHO cells were maintained in serum-free medium for over 6 months. At 6 days before the start of the experiments the medium was removed and the cells were harvested, seeded and washed three times in protein-free medium, and then maintained for 3 days in the indicated medium. The cells were harvested after 3 days, re-seeded and washed three times in protein-free medium. After another 3 days, the cells were harvested, re-seeded and transfected in the medium indicated. All transfections used the 2ERE-TATA-CAT reporter plasmid. Transfections used calcium phosphate when serum was present and Transfectam in the serum-free media. Desferoxamine is an iron chelator. The protein-free medium does not contain insulin or transferrin. The data represents the average fold induction in the presence of 17fl-estradiol for the number of transfections shown in parenthesis + SEM.

c o n t a i n a n y insulin o r transferrin. Surprisingly, t h e r e was n o increase in t h e fold i n d u c t i o n w h e n the level o f insulin was r e d u c e d (0.25× I T S ) o r in t h e p r o t e i n free m e d i u m ( T a b l e 2). T h e s e c h a n g e s r e s u l t e d in a d e c r e a s e in the fold i n d u c t i o n c a u s e d b y a large increase in the b a s a l activity a c c o m p a n i e d b y a m o d est d e c r e a s e in t h e i n d u c e d activity ( T a b l e 2). A l t h o u g h these d a t a were c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t E R exhibits e s t r o g e n - i n d e p e n d e n t t r a n scription, we believe t h a t t h e p r e s e n c e o f e s t r o g e n in o t h e r m e d i u m c o m p o n e n t s is r e s p o n s i b l e for the b a s a l activity. W h e n c h a r c o a l - t r e a t e d s e r u m was u s e d to g r o w the cells, b a s a l e s t r o g e n - i n d e p e n d e n t t r a n s c r i p t i o n was s t r o n g l y d e c r e a s e d (see below). T h i s was compatible with the idea that charcoal-dextran treatm e n t r e m o v e d the original c o m p o u n d s b o u n d at h y d r o p h o b i c sites o n s e r u m p r o t e i n s e n a b l i n g t h e m effectively to s e q u e s t e r e s t r o g e n s t h a t were p r e s e n t in medium components other than serum.

Transcriptional activation by E R is completely dependent on added 17fl-estradiol T o e v a l u a t e t h e c o n t r i b u t i o n o f e s t r o g e n s to t h e t r a n s c r i p t i o n a l activity o f E R u n d e r s t a n d a r d cell cult u r e c o n d i t i o n s , w e s e l e c t e d lots o f s e r u m w i t h low e n d o g e n o u s e s t r o g e n levels a n d m a i n t a i n e d t h e cells in p h e n o l - r e d - f r e e m e d i u m a n d c h a r c o a l - d e x t r a n - t r e a t e d s e r u m . A n e w a n d e x t r e m e l y sensitive r e p o r t e r gene c o n t a i n i n g f o u r t a n d e m E R E s was u s e d to evaluate t h e p o s s i b i l i t y t h a t u n l i g a n d e d E R c o u l d activate t r a n s c r i p t i o n . C H O a n d H e p G 2 cells were t r a n s f e c t e d with the 4 E R E - T A T A - C A T r e p o r t e r gene a n d a level o f C M V h E R e x p r e s s i o n p l a s m i d sufficient to p r o d u c e the h i g h levels o f E R n e e d e d to s a t u r a t e all f o u r E R E s was achieved. S i m i l a r t r a n s f e c t i o n s were c a r r i e d o u t with s a t u r a t i n g levels o f Xenopus E R e x p r e s s i o n p l a s m i d a n d the 4 E R E - T A T A - C A T r e p o r t e r in two Xenopus cell lines. I n all f o u r cell lines o f h u m a n , r o d e n t a n d a m p h i b i a n origin, t r a n s c r i p t i o n b y u n l i g a n d e d E R was negligible ( < 1 . 1 % o f the activity in the p r e s e n c e o f 17fl-estradiol). I n d u c t i o n s o f 100-fold or m o r e were o b t a i n e d u p o n a d d i t i o n o f s a t u r a t i n g c o n c e n t r a t i o n s o f 17fl-estradiol to the c u l t u r e m e d i u m (Fig. 3). I n H e p G 2 cells, a liver h e p a t o m a line with the ability to m e t a b o l i z e traces o f e s t r o g e n , an i n d u c tion o f o v e r 3 0 0 - f o l d . T h i s was c a u s e d b y a f u r t h e r d e c l i n e in b a s a l activity to levels well b e l o w the extrem e l y low levels seen in t h e o t h e r cell lines. A l t h o u g h this d a t a i n d i c a t e d t h a t e x o g e n o u s e s t r o gen was r e q u i r e d for E R - m e d i a t e d t r a n s c r i p t i o n , it c o u l d n o t e x p l a i n t h e E R - d e p e n d e n t t r a n s c r i p t i o n in the a b s e n c e o f a d d e d e s t r o g e n seen in m a n y p r e v i o u s studies. T o evaluate m o r e directly t h e h y p o t h e s i s t h a t e x t r e m e l y low c o n c e n t r a t i o n s o f e s t r o g e n s in the c h a r c o a l - d e x t r a n - t r e a t e d s e r u m a n d m e d i u m were r e s p o n sible for the a p p a r e n t e s t r o g e n - i n d e p e n d e n t activity o f ER, we d e t e r m i n e d the d o s e - r e s p o n s e curve for the

Estrogen is Required for ER Transactivation

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the four cell fines with either CMVhER (CHO, HepG2) or CMVXER (XLll0, XTC-2) DNA. T h e a m o u n t s o f E R e x p r e s s i o n p l a s m i d u s e d w e r e : 1 ng; 300 n g ; 1/tg; 1 pg f o r C H O , HepG2, XLll0, and XTC-2 cells, respectively. CAT activity in the extracts from transfected cells which were or were not i n d u c e d with 17fl-estradio] w e r e c o m p a r e d to calculate the fold induction. The data : r e p r e s e n t t h e mean + S E M f o r at

least five separate transfections.

a c t i v a t i o n o f t r a n s c r i p t i o n b y 17fl-estradiol in several o f t h e s e cell lines.

Low concentrations of 17fl-estradiol elicit substantial transcription by E R O u r f i n d i n g t h a t we c o u l d achieve b o t h a negligible b a s a l activity for E R a n d i n d u c t i o n s o f 100-fold o r m o r e o n t h e a d d i t i o n o f 17fl-estradiol s u g g e s t e d t h a t t h e e s t r o g e n - i n d e p e n d e n t activity o f E R o b s e r v e d in o t h e r studies was a c t u a l l y c a u s e d b y the p r e s e n c e o f e s t r o g e n in t h e c u l t u r e m e d i u m . T o a d d r e s s the q u e s t i o n o f h o w m u c h e s t r o g e n is r e q u i r e d to activate E R , we d e t e r m i n e d the 17fl-estradiol d o s e - r e s p o n s e curve for E R - d e p e n d e n t t r a n s c r i p t i o n a l activation in several cell lines. 17fl-estradiol s t o c k s o l u t i o n s r a n g i n g f r o m 10 -3 to 10 1 2 M were p r e p a r e d b y serial d i l u t i o n a n d a d d e d to dishes c o n t a i n i n g cells t h a t h a d b e e n t r a n s f e c t e d w i t h a single b a t c h o f D N A crystals. C A T activities o f t h e r e s u l t i n g cell extracts were d e t e r m i n e d a n d p l o t t e d relative to m a x i m u m C A T activity (1 ~tM estradiol). Significant C A T activity was d e t e c t e d at 0.5 p M 17fl-estradiol in C H O cells. I n c o n t r a s t , to achieve a similar leve] o f activity in H e p G 2 cells r e q u i r e d 100 p M 17fl-estradiol, a shift o f 2 0 0 - f o l d in t h e d o s e - r e s p o n s e curve (Fig. 4). T h i s is p r e s u m a b l y d u e to the ability o f H e p G 2 cells to m e t a b o l i z e traces o f e s t r o g e n in the m e d i u m . T h e d o s e - r e s p o n s e curve for t h e X E R in X T C - 2 cells was shifted a p p r o x i -

I 14 ESTRADIOL

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Fig. 3. E s t r o g e n - d e p e n d e n t i n d u c t i o n of transcription in f o u r ceil fines. Five micrograms (XLll0, XTC-2) or 2/tg (CHO, HepG2) of 4ERE-TATA-CAT D N A w a s c o t r a n s f e c t e d into

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I 12

I 10

CONCENTRATION

I 8

I 6

(-IogM)

Fig. 4. 17fl-Estradiol d o s e r e s p o n s e c u r v e s f o r transactivation r o d e n t a n d amphibian cell fines. L a r g e s e t s of DNA crystals (for six to s e v e n d i s h e s ) w e r e p r e p a r e d a t o n e time so that each of the points in a r e s p o n s e c u r v e w e r e d e r i v e d f r o m c e l l s t h a t w e r e t r a n s f e c t e d w i t h i d e n t i c a l combinations of DNA. A f t e r g l y c e r o l s h o c k e a c h member of a set of dishes received a d i f f e r e n t c o n c e n t r a t i o n of 17fl-estradiol, o r n o h o r m o n e . E s t r a d i o l s t o c k s o l u t i o n s w e r e p r e p a r e d by serial dilution in ethanol of a 10 mM master stock. CAT activity w a s set at 100% for 10-6M 17fl-estradiol in each set of transfections. The data represents the mean + S E M f o r at least f o u r s e p a r a t e t r a n s f e c t i o n s . E r r o r b a r s w h i c h a r e n o t visible are smaller than the symbols.

in human,

m a t e l y t w o - f o l d c o m p a r e d to t h e C H O cell curve, to slightly h i g h e r c o n c e n t r a t i o n s o f 17fi-estradiol. T h i s is p e r h a p s as a result o f the fact t h a t X E R m a y d i s p l a y a slightly l o w e r affinity for 17fl-estradiol t h a n h E R (k d for 17fi-estradiol is a p p r o x i m a t e l y 0.24 n M for h E R [27] a n d 0.5 n M for X E R [28]).

The level of basal transactivation is proportional to the endogenous estrogen content of the serum used in tissue culture media O n e p r o p o s e d s o u r c e o f r e s i d u a l e s t r o g e n in t h e c u l t u r e m e d i u m is the s e r u m . T o d e t e r m i n e if t h e r e was a r e l a t i o n s h i p b e t w e e n the level o f t r a n s a c t i v a t i o n b y E R in t h e a b s e n c e o f a d d e d e s t r o g e n , a n d t h e level o f e n d o g e n o u s e s t r o g e n in t h e s e r u m b e f o r e c h a r c o a l t r e a t m e n t , the e s t r o g e n c o n c e n t r a t i o n in two lots o f s e r u m was c o m p a r e d b y r a d i o i m m u n o a s s a y (RIA) [29]. T h e ability o f e a c h lot to activate t r a n s c r i p t i o n in X T C - 2 cells in t h e a b s e n c e o f a d d e d h o r m o n e was d e t e r m i n e d . C o t r a n s f e c t i o n o f X T C - 2 cells m a i n t a i n e d in these two lots o f s e r u m with 4 E R E - T A T A C A T a n d C M V X E R s u p p o r t e d t h e R I A results. Cells g r o w n in s e r u m lot 3 0 1 0 1 , which contained 6.1 + 0 . 2 p g e s t r o g e n / m l e x h i b i t e d a b o u t twice t h e C A T activity ( a n d 2 5 % o f the activity o f E 2 - t r e a t e d cells) o f cells m a i n t a i n e d in lot 4 0 0 3 E , w h i c h c o n -

S. Mattick et al.

290 tained 3.5 + 0.2 pg estrogen/ml, m a x i m u m activity.

and had

12%

of

A

6

~) Jo)

Transcriptional synergism between ERs bound at multiple EREs In the course of our studies on the E R activation of ERE-containing reporter genes in several cell lines, we obtained preliminary data suggesting that the ability of E R b o u n d at multiple EREs to exhibit synergistic activation of transcription was highly cell-specific. Because previous work in several laboratories had been quite contradictory, with some reports indicating that there was strong transcriptional synergy between ERs b o u n d at multiple EREs [16, 17], whereas others reported no synergy at all [18], it was appropriate to re-examine this question. We defined transcriptional synergy as the extent to which the activity of E R b o u n d at multiple EREs exceeds the activity to be expected by simply adding up the n u m b e r of EREs. In this formulation a two-fold synergy means that two EREs would produce four times the activity of one ERE (or two times the result predicted from simply adding the activities due to each E R E together). Ceils from the four cell lines in which we examined estrogen inducibility were transfected in the presence of saturating levels of Ez and either C M V h E R ( C H O and H e p G 2 ) or C M V X E R ( X T C - 2 and XL110). We also looked at the synergy in C H O ceils c o m p a r e d to the low levels of endogenous E R they contain. Reporter genes containing one, two or four EREs were tested in each cell line. W h e n we c o m p a r e d the transcription of reporter genes containing one and two EREs, C H O cells exhibited strong synergy (4.3fold), whereas H e p G 2 and X T C - 2 cells exhibited m u c h weaker two- and 1.5-fold synergy, respectively, and X L l l 0 cells displayed only additive activity (Fig. 5(A)). C o m p a r i s o n of the relative activities for four EREs vs one E R E yielded a fold synergy of 5.2, 3.2, 2.0, for C H O , H e p G 2 , and X T C - 2 cells, respectively, with X L 1 1 0 cells again exhibiting only additive activity (Fig. 5(A)).

Synergistic activation of transcription was facilitated by the high levels of ER expressed in transfected CHO cells, but did not require it C H O ceils appear to contain low levels of ER, which can produce modest levels of transcription from ERE-containing reporter genes in the absence of transfected ER. Although transcriptional synergy was substantially reduced c o m p a r e d to C H O cells, which were transfected with the E R expression plasmid, it was still readily detectable at 2.7- and two-fold for two and four EREs, respectively (Fig. 5(A)). These data indicated that the extent of transcriptional synergy was influenced by the level of intracellular ER. It therefore seemed possible that the apparent failure of X L 1 1 0 cells to exhibit transcriptional synergism might be caused by the expression of

'IER) ER) ER)

0 4 z

_1 oi1 2

NUMBER OF ERES

B 1 Ng CMVXER [----] 10 ng CMVXER 1 pg TKXER

.J

2

4 NUMBER OF ERES

F i g . 5. T r a n s c r i p t i o n a l s y n e r g i s m b e t w e e n a d j a c e n t c o n s e n s u s E R E s . I n p a n e l A t h e c e i l - s p e c i f i c effects o f s y n e r g i s m w e r e e x a m i n e d b y c o t r a n s f e c t l n g f o u r d i f f e r e n t cell l i n e s w i t h CAT reporter gene constructs containing one, two or four adjacent consensus EREs with enough ER expression vector to a t t a i n a h i g h l e v e l o f E R . T h e a m o u n t s w e r e : 1 n g ( C H O cells); 300 n g ( H e p G 2 cells); 1/~g ( X E R ) f o r X L l l 0 a n d X T C 2 cells, r e s p e c t i v e l y . C H O ( E n d o . ) a r e C H O ceils t r a n s f e c t e d with the reporter genes, but not with an ER expression plasmid. Relative activities were determined by dividing the CAT a c t i v i t i e s f o r t w o o r f o u r E R E s b y t h e m e a n C A T a c t i v i t y for one ERE. Fold synergies were determined by dividing the relative activities by two (2EREs) or four (4EREs). In panel B the impact of receptor concentration on synergism was e x a m i n e d b y c o t r a n s f e c t i n g X L I 1 0 cells w i t h 1 pg o r 1 0 n g o f C M V X E R o r 1/tg o f T K X E R , a n d t h e t h r e e d i f f e r e n t c o n s e n s u s E R E r e p o r t e r c o n s t r u c t s . R e l a t i v e a c t i v i t i e s a n d fold s y n e r g i e s w e r e d e t e r m i n e d as d e s c r i b e d a b o v e . T h e d a t a i n p a n e l s A a n d B r e p r e s e n t s t h e m e a n + S E M f o r at l e a s t t h r e e separate transfections.

a non-optimal level of X E R in these cells. We therefore examined transcriptional synergism in X L l l 0 cells which had been transfected with different concentrations of C M V X E R and T K X E R . T r a n scriptional synergism was readily detected in the cells transfected with the lower concentration of 10 ng of C M V X E R and with the 1/~g of T K X E R used in some of our earlier studies, but was absent in cells transfected with 1 #g of C M V X E R (Fig. 5(B)). These data indicate that there is an optimal level of E R ex-

Estrogen is Required for ER Transactivation pression for observing transcriptional synergism. T o o little or too m u c h receptor can abolish synergism.

DISCUSSION

ER activation of the tramcription of simple ERE-containing promoters is exclusively estrogen-dependent M a n y research groups have observed the estrogendependent transcription of ERE-containing receptor genes in the absence of added estrogen. T h e source of this activity has been a point of contention. T z u c k e r m a n et al. [7] concluded that it is caused by an estrogen-independent ability to activate transcription displayed by wild-type ER. A potential mechanism for estrogen-independent activity is derived from recent demonstrations tlhat the phosphorylation of Ser 118 by M A P kinase [13, 14], and of T y r 537 [12], can strongly influence E R activity. T h e s e data raise the possibility that the phosphorylation of E R mediated through cell surface signaling pathways might be able to convert E R into a conformation in which it was able to activate transcription, even in the absence of added 17fl-e~tradiol. M a n y other research groups have suggested that the apparent estrogen-independent activity of E R is caused by traces of estrogen which remained in the tissue culture m e d i u m , even after charcoal-dextran treatment [8-11,30]. T o r a et al. [10] showed that the Val 400 m u t a n t of the h E R had a significantly lower affinity for estradiol than wild-type hER. U p o n transfection of H e L a cells in the absence of added estrogen, wild-type h E R exhibited 15% of its level of activity in cells maintained in 10 m M E2, whereas the Val 400 mutant, being unable to bind the low concentration of estrogen in the m e d i u m , was completely devoid of activity. Wild-type h E R also had no basal activity when it was expressed in yeast, which are routinely grown in serum-free medium. Brown et al. [9] reported an eight- to 30-fold variation in induction of the PS-2 gene when M C F - 7 cells were grown in m e d ium supplemented with different lots of FBS. T h e y also attributed the variation in fold induction to residual estrogen in the serum. In addressing this question, we initially assumed that the charcoal-treated serum used in cell culture was the major source of estrogen in the m e d i u m . We therefore adapted C H O cells to growth in serum-free m e d i u m , and developed techniques for transfecting cultured cells in serum and protein-free medium. Surprisingly, cells grown in serum-free m e d i u m displayed substantial E R activity in the absence of added estrogen, and p o o r fold inductions when 17fl-estradiol was added (<10-fold). Because the basal estrogenindependent activity was substantially reduced on the addition of two times charcoal-dextran extracted newb o m calf serum to the culture m e d i u m , (data not shown), we concluded that low levels of estrogen in

291

media c o m p o n e n t s other than serum were responsible for the activity of E R in the absence of added 17flestradiol. T h e possibility that E R might be activated by the pharmacological concentrations of insulin (10mg/1) used in standard serum-free m e d i u m was excluded by our observation that maintaining the cells in protein-free medium, which does not contain any insulin (or transferrin), did not increase the fold induction (Table 2). Although we were aware of the possibility that a " m e m o r y effect" due to previous growth of the cells in m e d i u m containing 10 mg/ml insulin might contribute to the basal activity of the ER, this high insulin concentration is optimal for the growth of these cells [31]. High insulin concentrations are required because the cysteine in serum-free F12 m e d i u m inactivates more than 90% of the insulin within 1 h [32]. Although an extended growth of the C H O cells in insulin-free m e d i u m would have eliminated the possibility of an insulin " m e m o r y effect", this results in "reverse differentiation of the cells" with p r o n o u n c e d and well-documented alterations in cell morphology, growth and metabolism [24, 31]. T o eliminate this potential problem vee maintained the cells for 6 days in the insulin-free and reduced-insulin media before initiating the experiments. During this period the cells were harvested three times and washed a total of nine times in protein-free medium. It therefore appears unlikely that the data presented in T a b l e 2 was influenced by a " m e m o r y effect", resulting from the prior growth of the cells in insulincontaining medium. O u r observation that 17fl-estradiol concentrations as low as 0.5 p M can elicit detectable activation of E R in C H O cells (Fig. 4) is consistent with the possible presence of trace amounts of estrogen in cell culture c o m p o n e n t s other than serum. T h e unexpected reduction of this basal activity u p o n the addition of 2x C D - t r e a t e d serum led us to the surprising conclusion that charcoal treatment, which removes cholesterol, fatty acids and other hydrophobic small molecules from protein binding sites, frees m a n y of these protein sites to bind traces of estrogen in the m e d i u m and serum. This has the effect of making these estrogens less available for binding to intracellular ER. Our data are consistent with recent reports which have demonstrated the presence of environmental estrogens in a variety of materials [33, 34]. T h e recent demonstration that c o m p o u n d s which are only weakly estrogenic individually, can jointly exhibit strong synergy in their ability to bind to E R and activate transcription [35], lends further credence to the idea that residual estrogens in the serum-free m e d i u m are responsible for the apparent basal activity of the ER. In order to investigate the very low levels of activity shown by unliganded ER, we developed a new highly inducible reporter gene containing four adjacent consensus EREs upstream of a T A T A box. T h e high ac-

292

S. Mattick et al.

tivity of this plasmid enabled us to quantitate low levels of E R activity. This plasmid also exhibits strong synergy in m a m m a l i a n cells. For example, C H O cells transfected with the 4 E R E - T A T A - C A T plasmid exhibited more than 20 times the C A T activity of C H O cells transfected with 1 E R E - T A T A - C A T . Because n u m e r o u s studies report strong synergy in experiments in which weak EREs are used, we reasoned that even if unliganded ERs are only weakly active they might exhibit transcriptional synergy and show significant activity when b o u n d to adjacent EREs. Even in C H O cells, in which we observed strong synergistic activation of transcription on the 4ERE reporter gene, unliganded E R exhibited only negligible activity (Fig. 3). Cotransfection of a saturating level of E R expression vector with 4 E R E - T A T A - C A T in the presence of estradiol resulted in inductions of 100-fold or more. Transcription activation by E R b o u n d to the four EREs in the reporter gene was clearly estrogendependent as shown by the dose-response curve (Fig. 4). T h e 200-fold shift to higher estradiol concentrations in tb_g dose-response curve for H e p G 2 cells indicates that, in this liver-derived cell line, the P-450 system is still able to metabolize estrogen. Our dose response curves are in agreement with those of D a n a et al. [36] who used H e p G 2 cells to analyse the activity of unusual non-consensus EREs. T h e shift in the dose-response curve is consistent with the extremely low basal activity seen in these cells in the absence of added 17~-estradiol, and with the more than 300-fold induction observed after the addition of 17/%estradiol to the medium. In most cell lines, high level overexpression of E R is difficult because of the toxicity associated with liganded ER [37]. Our data suggest that H e p G 2 cells, maintained in charcoaltreated serum, are able to metabolize and inactivate the trace concentrations of estrogens which are toxic in other cells, perhaps making t h e m a useful cell line for the overexpression of ER. Our studies with different lots of serum also support the view that low concentrations of estrogens in the culture m e d i u m are responsible for the E R activity sometimes observed in the absence of estrogen. W h e n X T C - 2 cells were transfected with 4 E R E - T A T A C A T and X E R and maintained in two different lots of FBS, for which the relative estrogen content differed by approximately two-fold, the transfected cells maintained in the serum lot with the two-fold higher level of endogenous estrogen exhibited about twice the C A T activity of the cells maintained in the lot of serum with lower endogenous estrogen. Overall, our data strongly support the conclusion that the apparent basal activity of E R is caused by the imperfect removal of endogenous estrogen from the serum used in cell culture media, and to the presence of estrogens in the base cell culture media. Although our data support the view that, under the standard

cell culture conditions used in most transfection studies, transcription by the E R requires estrogen, under other conditions in which M A P kinase or other protein kinases are strongly activated, the E R is reported to exhibit ligand-independent transactivation. T h e failure of unliganded E R to activate transcription in these cells might be caused either by the inability of the ER to bind to the ERE, or to the presence of the E R in a conformation in which it is unable to activate transcription. We have recently used in vivo p r o m o t e r interference assays and other techniques to demonstrate that unliganded E R effectively binds to the E R E in C H O cells [38]. T h e failure of unliganded E R to activate transcription in C H O cells is therefore likely to be due to the presence of the receptor in a conformation in which the liganddependent AF2 cannot function.

Transcriptional synergy between estrogen receptors bound at multiple E R E s

Our data indicate that b o t h E R concentration and cell type can strongly influence the extent of transcriptional synergy. Differences in E R concentration and cell type probably account for the dramatic and unresolved differences in the extent of transcriptional synergy seen by several groups of researchers [16-19]. Although some workers have emphasized that multiple EREs do not function synergistically when they are close to the T A T A box [18], the EREs in our reporter genes begin only 20 nucleotides upstream of the T A T A box. Although the reason for strong celltype differences remains obscure, our data does shed light on the m e c h a n i s m of transcriptional synergism for multiple EREs located close to the T A T A box. Transcriptional synergy could be caused by the facilitation of E R binding to unoccupied EREs by ER b o u n d at adjacent EREs, or by synergistic stimulation or stabilization of formation of the basal transcription complex in the T A T A region. Transfecting a high level of X E R expression plasmid into X L l l 0 cells abolished transcriptional synergy. At these high levels of X E R expression plasmid, the X E R presumably saturated both the one and four E R E reporter genes. It therefore seems plausible that the transcription synergy seen at lower levels of X E R expression plasmid is caused by the ability of X E R b o u n d at EREs to facilitate binding of X E R to adjacent unoccupied EREs. With a plasmid containing a single E R E this synergistic binding is impossible, and synergistic activation of transcription cannot occur. It is also possible that the failure to observe transcriptional synergy at high levels of transfected X E R is a result of the presence of limiting amounts of co-activators in X L 1 1 0 cells. However, our observation that we observed only additive activity in X L l l 0 cells, not squelching, with both 2ERE and 4ERE-containing reporter genes, suggests that co-activators are not limiting.

Estrogen is Required fo~ ER Transactivation Our

data provide clear evidence that under

stan-

d a r d cell c u l t u r e c o n d i t i o n s u n l i g a n d e d E R is u n a b l e to

activate

transcription,

describe

a

powerful

new

16.

ERE-containing reporter gene, demonstrate strong cell a n d E R - d e p e n d e n c e in transcriptional synergy, and suggest that cooperative ER binding can be re-

17.

sponsible for transcriptional synergy. 18. Acknowledgements--We are grateful to Dr S. A. Ferreira of Dr G. L. Jackson's laboratory (University of Illinois, Department of Veterinary Biosciences) for providing us with the radioimmunoassay data for our serum. This research was supported by grant HD16720 from the National IrLstitute of Child Health an~t Human Development.

19.

20.

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