Both estradiol and tamoxifen decrease proliferation and invasiveness of cancer cells transfected with a mutated estrogen receptor

ft. Steroid Biochem. Molec. Biol. Vol. 61, No. 1/2, pp. 11-17, 1997 © 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain PII: S0960-0760(96)00255-5 0960-0760/97 $17.oo + o.oo

Pergamon

Both Estradiol and Tamoxifen Decrease Proliferation and Invasiveness of Cancer Ceils T r a n s f e c t e d w i t h a M u t a t e d E s t r o g e n Receptor Marcel Garcia, Danielle Derocq, Nadine Platet, Sandrine Bonnet, Jean-Paul Brouillet, Isabelle Touitou and Henri Rochefort* Institut National de la Sant~ et de la Recherche Mkdicale, Unitk 148 "'Hormones et Cancer" et Universitd Montpellier 1 60, rue de Navacelles, 34090 Montpellier, France

P r e v i o u s s t u d i e s h a v e s h o w n t h a t , a f t e r w i l d - t y p e e s t r o g e n r e c e p t o r (ER) t r a n s f e c t i o n in E R - n e g a tive b r e a s t c a n c e r cells, e s t r a d i o l b u t n o t t a m o x i f e n p r e v e n t s g r o w t h , i n v a s i v e n e s s a n d m e t a s t a s i s o f t h e s e cells in m i c e . B e c a u s e a n E R m u t a t i o n at p o s i t i o n 400 c o n v e r t s t h e t r i p h e n y l e t h y l e n e a n t i e s t r o g e n , O H - t a m o x i f e n i n t o a full e s t r o g e n a g o n i s t , we t r a n s f e c t e d this m u t a t e d f o r m o f h u m a n E R in a n E R - n e g a t i v e r a t c a n c e r cell line. T h i s was a i m e d a t i n d u c i n g a n i n h i b i t o r y , e s t r o g e n - l l k e r e s p o n s e o f t a m o x i f e n in t h e s e cells. In two s t a b l e E R - p o s i t i v e t r a n s f e c t a n t s , O H - t a m o x i f e n i n h i b i t e d cell g r o w t h a n d i n v a s i v e n e s s in v i t r o as e f f i c i e n t l y as e s t r a d i o l . T h e p u r e a n t i e s t r o g e n , I C I 164,384, was n o t a g o n i s t i c a l o n e a n d a n t a g o n i z e d e s t r o g e n a c t i o n . In c o n t r a s t , t h e t h r e e c o m p o u n d s w e r e i n e f f e c t i v e in c o n t r o l m o c k - t r a n s f e c t e d cells. W h e n i n j e c t e d i n t o o v a r l e c t o m i z e d n u d e m i c e , E R n e g a t i v e m o c k - t r a n s l ~ e c t e d cells f o r m e d t u m o u r s w h i c h w e r e s i g n i f i c a n t l y s t i m u l a t e d b y e s t r a d i o l and inhibited by tamoxifen treatment. This indicates that estradiol and tamoxifen altered the g r o w t h o f E R - n e g a t i v e t u m o u r s via a g e n e r a l e f f e c t o n t h e h o s t r e s p o n s e . S u r p r i s i n g l y , t h e h o r m o n e r e s p o n s i v e n e s s o f E R - p o s i t i v e t u m o u r s d e v e l o p e d f r o m E R - t r a n s f e c t e d cells d i d n o t s i g n i f i c a n t l y differ from that of ER-negative (mock-transfected) tumours. We conclude that transfection of a mutated human estrogen receptor inhibited, through an estrogenic activity of tamoxifen, the growth a n d i n v a s i v e n e s s o f t h e s e c a n c e r cells in v i t r o . H o w e v e r , t h e low e x p r e s s i o n o f E R d i d n o t a l l o w e d us to o b t a i n t h e s a m e e f f e c t o f t a m o x i f e n in v i v o . © 1997 E l s e v i e r S c i e n c e L t d J. SteroidBiochem. Molec. Biol., Vol. 61, No. 1/2, pp. 11-17, 1997

INTRODUCTION

breast cancer cells (MDA-MB-231), we have also shown that estrogen but not tamoxifen, a triphenylethylene antiestrogen, markedly reduced the invasive potential in vitro and the development of experimental metastases in athymic mice [5]. In contrast, in ERpositive breast cancer cell lines ( M C F 7 and T 4 7 D ) , E R transfection does not modify their hormonal responsiveness because the growth of transfected cells can still be stimulated by 17/3-estradiol (E2) [6]. These studies generally indicate that E R has opposite effects on the growth of cancer cells, depending on whether they are initially ER-positive or ER-negative. T h e use of E R transfection could also be an attractive new therapeutical approach for the management of hormone-independent breast cancers. However, E R transfer conditions must first be defined in order

T h e growth of breast cancer cells containing estrogen receptors (ER) is generally stimulated by estrogen both in vivo and in vitro, thus explaining the beneficial effect of antiestrogen therapy [1]. E R loss during breast cancer progression is associated with a more aggressive phenotype and an increased risk of metastasis [2]. Paradoxically, the expression of exogeneous E R through transfection into ER-negative mammalian cell lines and estrogen treatment leads to growth inhibition rather than ~7owth stimulation in vitro (reviewed in [3] and [4]). In ER-transfected h u m a n *Correspondence to Henri Rochefort. Tel: +33 67 04 37 61; Fax: +33 67 54 05 98. Received 20 Sep. 1996; accepted 21 Nov. 1996. 11

12

M. Garcia et aL

to inhibit the growth and invasiveness of ER-negative cancer cells by antiestrogen treatment rather than by estradiol treatment, which would stimulate the growth of possible ER-positive t u m o u r cells remaining after surgery. T h e h u m a n E R originally cloned from M C F 7 breast cancer cells [7] displayed, from a cloning artefact, a point mutation in its h o r m o n e binding domain which converts glycine to valine at amino acid 400 [8]. This mutation was previously shown to change the pharmacology of the triphenylethylene antiestrogen 4-hydroxytamoxifen (OH-Tam) by increasing its estrogenic activity in the M D A - M B - 2 3 1 h u m a n breast cancer cell line [9]. In this study, we transfected this m u t a t e d E R in another tumourigenic and low metastatic cell line (rat embryo cancer 3Y1A d l 2 ) and analysed the effects of estradiol and antiestrogens both in cultures and in nude mice. M o c k transfected cells were ER-negative and unresponsive to estrogen in vitro. After E R transfection, O H - t a m o x ifen and estradiol inhibited their growth and invasivehess in vitro. In athymic mice, the growth of turnouts developed from mock-transfected cells was surprisingly stimulated by estradiol, whereas tamoxifen ( T a m ) decreased their growth. We found that, contrary to cell culture results, E R transfection did not affect the t u m o u r response in vivo to E2 and T a m treatments.

MATERIAL AND METHODS

determined from cell cultures and tumours by an enzyme immunoassay as previously described [5]. Growth response studies Steroids were withdrawn from cells by 7 days culture in phenol-red free Dulbecco's minimal essential m e d i u m ( D M E M ) supplemented with 5% dextrancoated charcoal-treated serum ( F C S - D C C ) and 5 0 # g / m l gentamicin. For growth studies, steroiddeprived cells were plated in triplicate in 24-well plates at a density of 3 x 104 cells/well in m e d i u m containing 1.5% F C S - D C C . Media were changed every other day. At various times, cells were fixed with methanol, and the D N A content was determined by the diaminobenzoic acid m e t h o d [12]. Matrigel invasion assay F o r invasion assays, a 0.2 ml suspension of 105 cells was layered in the u p p e r c o m p a r t m e n t of a Transwell (Costar, Brumath, France) on a polycarbonate filter (8/~m pore size) precoated with 20/~g of Matrigel basement m e m b r a n e (Becton Dickinson, Le Pont-de-Claix, France), and incubated for 24 h at 37°C with conditioned m e d i u m from N I H 3T3 fibroblasts in the lower c o m p a r t m e n t , as described [13]. T h e n u m b e r of cells migrating to the lower side of one filter was determined, after 4',6-diamidino-2phenyl-indole (DAPI) staining, by counting cells in 18 microscopic fields (0.8 m m 2 area) randomly dispersed on the filter [5].

Animals

Tumour formation in athymic mice

Ovariectomized female athymic mice BALB/c nu/ nu, 5 - 6 weeks old, were obtained from Iffa-Credo (L'Arbresle, France) and housed under specific pathogen-free conditions.

Cells were grown to near confluence in 25 cm 2 tissue culture flasks and harvested by light-trypsin treatment for 1 rain. The cell suspension was supplemented with 10% F C S - D C C to inactivate the trypsin, centrifuged and resuspended in a final concentration of 2.107 cells/ml in PBS. T h e cell suspension (0.1 ml) was injected into right and left thoracic fat pads of athymic mice. T h e day before cell inoculation, mice were implanted subcutaneously with a 3week pellet containing 15 m g of E2, 25 m g T a m or a placebo pellet from Innovative Research, U.S.A. A second pellet was implanted after 3 weeks. Turnouts were measured each week in two dimensions using a caliper to determine the turnout area. At necropsy, uterine wet weight was determined to assay the efficacy of estrogen treatment, and tumours were frozen in liquid nitrogen until E R assay.

Cell culture and E R transfection T h e 3 Y 1 - A d l 2 rat t u m o u r cell line was obtained through transformation of 3Y1 normal rat embryo cells by adenovirus type 12 D N A [10]. This cell line is tumourigenic with low metastatic levels in athymic mice [11]. T h e expression vectors, p S G 1 - E R , containing the H E O sequence of m u t a t e d h u m a n ER [7, 8], or pSG1, were cotransfected into 3 Y 1 - A d l 2 cells with the selectable neomycin resistance vector pAG60. T w o cell lines resistant to 400 #g/ml G 4 1 8 were isolated (HEO-1 and H E O - 2 ) and selected for ER expression. A G418-resistant cell line (control-I), simultaneously isolated by cotransfection of the pSG1 expression vector without H E O c D N A was used as a control for the transfection experiment. All transfectants were maintained in m e d i u m supplemented with 5% fetal calf serum (FCS), 400 pg/ml of G418 and 5 0 # g / m l gentamicin. All experiments were performed from passages five to 20 and the stability of the E R expression was controlled throughout this period. T h e cytosolic ER concentration was

RESULTS

Isolation of stable E R transfectants T r a n s f o r m e d rat embryo cells 3Y1-Ad12 were cotransfected with the E R expression vector (pSG1H E O ) and a neomycin resistance expression vector. T w o stable transfectants H E O - 1 and H E O - 2 expres-

Estrogen Receptor Transfection into Cancer Cells

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Fig. 1. Estradiol de,;reases growth o f E R - t r a n s f e c t e d cell fines. (a) D o s e r e s p o n s e . T h e g r o w t h o f E R t r a n s f e c t a n t s (HEO-1 and H E O - 2 c l o n e s ) a n d m o c k - t r a n s f e c t a n t (control-1 clone) w a s d e t e r m i n e d in triplicate after 7 d a y s o f c u l t u r e in m e d i u m containing 1.5% F C S - D C C a n d t h e i n d i c a t e d e s t r a d i o l c o n c e n t r a t i o n as d e s c r i b e d in M a t e r i a l s a n d m e t h o d s ( m e a n + SD). The data a r e e x p r e s s e d as p e r c e n t a g e s o f cells c u l t u r e d in t h e a b s e n c e o f estr~adiol ( m e a n + SD). (b) T i m e c o u r s e . T h e g r o w t h o f E R - t r a n s f e c t e d c l o n e s ( H E O - 1 a n d HEO-2) w a s a s s a y e d at different t i m e p o i n t s in t h e a b s e n c e (control, @) or p r e s e n c e (+E2, o) o f 20 n M e s t r a diol. The m e a n + SD o f DNA c o n t e n t in triplicate w e l l s is r e p r e s e n t e d . T h e s e r e s u l t s w e r e c o n f i r m e d in another experiment.

sing 370 and 430 fmol/mg E R cytosol protein, respectively, were isolated. E R function in these clones was first screened in transient transfection experiments with the v i t - t k - C A T reporter vector, which contains the estrogen responsive element of the vitellogenin A2 gene [14]. T h e C A T activity was increased by E2 treatment by four- and six-fold in H E O - 1 and H E O - 2 transfectants, respectively (data not shown). A neomycin-resistant clone (control-l), isolated simultaneously by cotransfection of the PSG1 vector alone, was ER-negative and was used as a control for the transfection experiment.

Estradiol and antiestrogens inhibit the growth of ER transfectants in vitro T h e effect of estrogen on the proliferation of ERtransfected and control-transfected 3 Y 1 - A d l 2 cells was determined [Fig. 1 (a)]. T h e growth of ER-transfected clones (HEO-1 and H E O - 2 ) was markedly decreased (25% and 50%, respectively) after 7 days of culture, in the presence of E2 concentrations equal or superior to 5 nM. In contrast, increasing E2 concentrations up to 1/aM had no effect on the growth of mock-transfected control-1 cells. In time course experiments, the population-doubling time for H E O - 1 and H E O - 2 clones increased from 33.3 + 0.3 h and 27.7 + 1 h in the absence of estrogen to 48.6 + 1.5 h and 3 3 . 5 + 0 . 3 h in the presence of 2 0 r i m E2 [Fig. l(b)].

Antiestrogens can inhibit estrogen responses by forming complexes with the E R that are inactive in promoting the transcription of some estrogen-regulated genes [15-17]. We therefore tested the effects of two antiestrogens, 4 - O H - t a m o x i f e n ( O H - T a m ) and I C I 164,384 (a 7~-alkylamide analogue of E2) on transfectant growth in the absence or presence of E2 (Fig. 2). O H - T a m , from 10 to 1 0 0 n M concentrations, prevented cell growth of both E R transfectants by approximately two-fold, indicating full agonist activity for this antiestrogen when tested on the m u t a t e d receptor [Fig. 2(a)]. T h e growth of control-1 cells was only inhibited by 20% at the highest (1/aM) O H - T a m concentration. In addition to 20 n M E2, O H - T a m concentrations had no effect on the growth of E R transfectants, over the 1 0 - 1 ° - 1 0 - 7 M range. At concentrations above 10 - 7 M, O H - T a m inhibited the growth of transfectants regardless of their E R content [Fig. 2(c)]. I C I 164,384 alone, in contrast to O H - T a m , had no agonistic effect from 10 -1° to 1 0 - 7 M concentrations [Fig. 2b)]. T w e n t y per cent inhibition was observed at the highest concentrations (5 × 10 -7 and 10 - 6 M ) independently of the E R expression level. In the presence of E2, I C I 164,384 concentrations above 1 0 - 7 M partly reversed the estrogen-induced growth inhibition of the E R transfectants, whereas these concentrations slightly inhibited the ER-negative control-1 transfectant [Fig. 2(d)]. These data indicated that O H - T a m acts

M. Garcia et al.

14

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Fig. 2. Effects o f 4 - O H - t a m o x i f e n a n d I C I 164,384 on t h e g r o w t h o f E R - a n d m o c k - t r a n s f e c t e d cells. E R - t r a n s f e c t e d c l o n e s H E O - 1 (©) a n d H E O - 2 (e) a n d c o n t r o l - 1 m o c k - t r a n s fected cells (O) w e r e c u l t u r e d for 7 d a y s in m e d i u m c o n t a i n i n g i n c r e a s i n g c o n c e n t r a t i o n s o f O H - T a m (a) a n d I C I 164,384 (b). In c a n d d, m e d i a c o n t a i n i n g t h e i n d i c a t e d c o n c e n trations o f a n t i e s t r o g e n w e r e s u p p l e m e n t e d w i t h 20 n M E2. D N A c o n t e n t is e x p r e s s e d as a p e r c e n t a g e o f u n t r e a t e d cells (mean ± SE).

as a full estrogen agonist on this mutated receptor, whereas I C I 164,384 remains a pure antagonist.

Effects of estradiol and antiestrogens on the invasive capacity of E R transfectants in vitro T h e invasive capacities of control-1 and ER-transfected cell lines were analysed in vitro using transwell chambers coated with Matrigel, a reconstituted basement membrane matrix. In this assay, cells on top of the well became attached before penetrating the Matrigel-coated filter. T h e n u m b e r of HEO-1 and H E O - 2 cells invading the Matrigel significantly decreased to 60% and 75% with the addition of 20 n M estradiol, whereas control-1 cells were not significantly affected (Fig. 3). The antiestrogen O H T a m ( 1 0 - 7 M) was as efficient as estradiol in inhibiting the invasiveness of E R transfectants, whereas the pure antiestrogen I C I 164,384 was ineffective, Taken together, these data indicated that the mutated E R activated by estrogen or O H - T a m signifi-

Fig. 3. E s t r a d i o l i n h i b i t s cell i n v a s i o n t h r o u g h Matrigel. M a t r i g e l i n v a s i o n a s s a y s for m o c k - t r a n s f e c t e d ( c o n t r o l - l ) a n d E R - t r a n s f e c t e d ( H E O - 1 , H E O - 2 ) cells w e r e c a r r i e d o u t in t h e a b s e n c e (fight bars) o r p r e s e n c e (dark bars) o f 20 n M E2, 10-7M O H - T a m ( c r o s s - h a t c h e d bars) o r 1 0 - 6 M I C I 164,384 ( c r o s s - h a t c h e d b a r s ) . D a t a r e p r e s e n t t h e n u m b e r o f cells m i g r a t i n g to t h e l o w e r s i d e o f t h e filter ( m e a n ± SD o f t h r e e e x p e r i m e n t s p e r f o r m e d in d u p l i c a t e ) . T h e a s t e r i s k r e p r e s e n t s statistical s i g n i f i c a n c e vs c o r r e s p o n d i n g c o n t r o l (P < 0.01) u s i n g t h e S t u d e n t ' s t-test.

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Estrogen increases tumour growth in vivo The growth of stable transfected cell lines was determined after inoculation of 2 × 106 cells bilaterally in ovariectomized nude mice treated or untreated with 15 mg E2 pellets or 25 mg T a m pellets. As shown in Fig. 4, E2 significantly increased the mean area of turnouts formed either with mock-transfected cells or with E R transfectants. In all groups tumours were detectable 3 weeks after cell inoculation, but their growth was accelerated at least 3.5-fold by estrogen treatment. Moreover, E2 increased t u m o u r incidence in mice inoculated with control-1 (control, five tumours per eight injections; E2, six out of eight), H E O - 1 (control, six out of eight; E2, 10 out of 10) and H E O - 2 (control, none out of eight; E2, nine out of 10) cells. Estradiol efficiency was verified on uterine wet weight, which increased from 38 mg (+17 mg SE) in control animals to 362 m g (+13 mg SE) in estrogen-treated animals. In contrast to E2, T a m signifi-

15

Estrogen Receptor Transfection into Cancer Cells

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F i g . 4. E s t r a d i o l s t i m u l a t e s a n d t a m o x i f e n i n h i b i t s in v i v o t h e growth o f t u m o u r ceils i n d e p e n d e n t o f E R s t a t u s . In t w o s e r i e s o f e x p e r i m e n t s (left a n d r i g h t p a n e l s ) , o v a r i e c t o m i z e d n u d e m i c e w e r e i m p l a n t e d s u b c u t a n e o u s l y w i t h p l a c e b o p e l l e t s ( c o n t r o l ) o r w i t h p e l l e t s c o n t a i n i n g 15 m g e s t r a d i o l (+E2) o r 25 m g t a m o x i f e n ( + T a m ) . C o n t r o l - 1 o r E R - t r a n s f e c t e d ( H E O - 1 o r H E O - 2 ) cells w e r e i n j e c t e d I d a y l a t e r . F o u r c o n t r o l a n d five e s t r o g e n - t r e a t e d a n i m a l s w e r e i n o c u l a t e d w i t h e a c h ceil type. E a c h m o u s e w a s i n j e c t e d i n t w o s i t e s w i t h 2.106 cells/site. T u m o u r s w e r e e v a l u a t e d t w i c e a w e e k a n d t h e m e a n ± S D o f t u m o u r a r e a is r e p r e s e n t e d . T h e a s t e r i s k i n d i c a t e s s t a t i s t i c a l s i g n i f i c a n c e vs c o n t r o l ( P < 0.01).

cantly inhibited the growth of tumours developed from control-1 cells or from H E O - 2 transfectants, although not significantly inhibiting the growth of H E O - 1 t u m o u r s [Fig. 4 (right)]. T h e area of mocktransfected and H E O - 2 tumours was decreased to 33% by T a m treatment and that of H E O - 1 tumours only to 82%. T h e t u m o u r incidence was not significantly affected by T a m treatment (23 tumours out of 24 injections in control mice; 21 tumours out of 24 injections in T a m - t r e a t e d mice). T h e E R concentration in m o u s e t u m o u r cytosol was determined using an i m m u n o e n z y m a t i c assay. T h e E R was detected at a low concentration in two H E O - 1 tumours (14 ___4 fmol/mg cytosol protein) and two H E O - 2 t u m o u r s (49 + 4 fmol/mg cytosol protein) but was undetectable in the mock-transfected control. T h e E R concentration was m u c h lower in tumours than in the corresponding cell cultures (370 and 430 fmol/mg cytosol prc,tein for H E O - 1 and H E O - 2 , respectively). T h e validity of the E R assay in mouse t u m o u r cytosol was shown by mixing cytosol prepared from ER-expressing cell cultures with t u m o u r cytosol. T h e E R added from the cell culture cytosol was

exactly recovered in the t u m o u r cytosol, indicating that mouse proteins did not interfere with the i m m u noenzymatic assay (data not shown). We conclude that the growth of the mock-transfected cells was significantly increased by E2 and decreased by T a m in vivo. T h e s e hormonal responses were similar in HEO-expressing tumours, indicating that E R transfection is less efficient on the growth of embryo cancer cells in vivo than in vitro. DISCUSSION T o determine the potential of E R gene transfer in decreasing the aggressive behaviour of ER-negative cancer cells, we examined the effects of stable transfection of an E R m u t a n t in rat embryo cancer cells on their proliferative and invasive potentials, both in vitro and in athymic mice. We demonstrated that stable expression of functional E R restored the responsiveness to estrogen in vitro in two independent transfectants. Estradiol decreased the growth of these cells in the n a n o m o l a r concentration range, whereas it was totally inactive in mock-transfected parental cells. This

16

M. Garcia et al.

growth inhibition by E2 in ER-transfected cells has already been found in vitro after E R transfection in several different ER-negative cancer cell types. This effect contrasts with the growth stimulatory effect of estrogen in ER-positive cells. After transfection with the h u m a n ER m u t a t e d on codon 400, the triphenylethylene antiestrogen, O H - T a m , behaved as a full estrogen agonist whereas I C I 164,384 remained a pure estrogen antagonist. This extends to another cell type the initial in vitro observation of Jiang et al. [9], indicating the enhanced estrogenic activity of triphenylethylene antiestrogen in breast cancer cells transfected with this m u t a t e d ER. We also showed that E2 and O H - T a m decreased the invasiveness of the m u t a t e d E R transfectants through Matrigel. These data, associated with our previous results in ER-negative breast cancer cells ( M D A - M B 231) transfected with the wild-type E R [5], indicate that E R transfection could be used to inhibit not only cancer cell growth but also t u m o u r invasion. T h e study of the effect of E R activation in vivo on the growth of subcutaneous tumours highlighted two main points. First, the growth of tumours developed with ERnegative parental cells (mock transfectant) was significantly increased by estrogen treatment and decreased by T a m treatment. T h e estrogenic stimulation of ERnegative turnouts has already been observed experimentally with some cancer cell types [18-20]. T h e exact m e c h a n i s m by which estrogens act is not known. Several hypotheses have been proposed, including natural killer cell suppression [21], reduction of cancer cell loss [20], or stimulation of growth factors via endocrine or paracrine action. Moreover, this general effect of estrogens might also be implicated in the higher recurrence rate observed in patients who had their turnouts removed during the proliferative phase of the menstrual cycle, c o m pared to those who underwent surgery later in the cycle [22]. Our finding that T a m decreased the growth of ER-negative turnouts in ovariectomized mice suggests that T a m could antagonize the general estrogenic effect on the host response, probably by acting on E R located in host target cells. This indicates that T a m therapy, in addition to its well-docum e n t e d inhibition of ER-positive cancer cells, could also prevent the growth of some ER-negative cancer cells via an indirect mechanism. T h e second point highlighted by our in vivo data concerns the growth of turnouts obtained with E R transfectants. T h e turnouts formed from H E O - 1 and H E O - 2 transfectants were stimulated by E2 at least as efficiently as mock-transfected turnouts. On the other hand, the growth inhibition by T a m was not further increased when tumours were developed from H E O transfectants. In addition, the incidence and growth of liver experimental metastases developed from the intravenous injection of p H E O - 2 transfected cells

were also significantly enhanced by E2 treatment (data not shown). We assessed whether this absence of growth inhibition in vivo was caused by an instability or defect in ER expression. E R was significantly expressed in turnouts, but its concentration was lower than in cytosol prepared from the corresponding cells in culture. This apparent lower E R expression (determined per m g cytosol protein) could be partly explained by higher protein recovery in turnout cytosol as a result of the presence of adjacent normal tissue and blood contamination. Other mechanisms, such as a decreased stability of the mutated E R or a lower efficiency of the SV40 p r o m o t e r in mice, are also not excluded. Contrary to data obtained in cell culture, the activation of the m u t a t e d E R by E2 and T a m did not therefore decrease the growth of ER-transfected 3Y1A d l 2 cancer cells in nude mice. This suggests that the overall paracrine and/or endocrine effects of E2 (and T a m ) observed in the parental 3 Y 1 - A d l 2 cancer cells dominated the autocrine effects due to of E R transfection. Moreover, indirect evidence indicates that the efficiency of these paracrine/endocrine effects could depend on the cancer cell implantation site. T h e ERnegative h u m a n breast cancer cell line, M D A - M B - 2 3 1 , develops subcutaneous tumours which are growthstimulated by estrogen [20], whereas experimental lung metastases induced by the same cell line are insensitive to estrogen [5]. We have previously shown that E R transfection of breast cancer cells M D A - M B - 2 3 1 and activation by E2 in vivo led to the drastic inhibition of experimental lung metastases [5]. In the present study, the parental 3 Y 1 - A d l 2 rat cells formed subcutaneous tumours which are growth-stimulated by estrogen and growth-inhibited by T a m . T a m - i n d u c e d growth inhibition was not enhanced by the low expression of the m u t a t e d E R in turnouts. We conclude that transfection of a m u t a t e d ER, followed by activation by E2 or O H - T a m , is efficient for inhibiting the growth and invasiveness of embryo cancer cells in vitro. However, expression of the m u t a t e d E R in embryo cancer cells (at the low level recovered in vivo) is not sufficient to mediate T a m therapy. Whether the efficiency of E R transfer is dependent on the type of cancer cell (wild-type or mutated) or on the nature of the transfected E R remains to be elucidated. Further studies are required to improve our understanding of the host response to E2, and to define new m o d e s of ER transfer which would be growth-inhibitory in ER-negative cancer cells after antiestrogen treatment.

Acknowledgements--We are grateful to P. C h a m b o n (IGBMC, Illkirch, France) for supplying the ER expression vector, and to A. Wakeling (Zeneca, U.K.) for the antiestrogens. We thank J. Y. Cance for the artwork. N.P. is a recipient of a MRES fellowship. This work was supported by the "lnstitut National de la Sant~ et de la Recherche M~dicale", the "Association pour la Recherche sur le

E s t r o g e n R e c e p t o r T r a n s f e c t i o n into C a n c e r Cells Cancer", the "Ligue Nationale de Lutte contre le Cancer" and the "R6gion Languedoc Roussillon".

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