Estrogenic activity of RU 486 (mifepristone) in rat uterus and cultured uterine myocytes

Dibbs

et al. Am J Obstet

12. Jacobson VC. The intraperitoneal transplantation of endometrial tissue. Arch Path01 1926; 1: 169-74. 13. Mann DR, Collins DC, Smith MM, Kessler MJ, Gould KG. Treatment of endometriosis in rhesus monkeys: effectiveness of a gonadotropin-releasing hormone agonist compared to treatment with a progestational steroid. J Clin Endocrinol Metab 1986;63: 127783. 14. Dizerega GS, Barber DL, Hodgen CD. Endometriosis: role of ovarian steroids in initiation, maintenance and suppression. Fertil Steril 1980;33:649-53. 15. Schenken RS, Asch RH, Williams RF, Hodgen CD. Etiology of infertility in monkeys with endometriosis: luteinized unruptured follicles, luteal phase defects, pelvis adhesions, and spontaneous abortions. Fertil Steril 1984;41:122-30. 16. D’Hooghe TM, Bambra CS, Cornillie FJ, Isahakia M, Koninckx PR. The prevalence and laparoscopic appearance of spontaneous endometriosis in the baboon (Pa@ anubis, Papio cynocephalus). Biol Reprod 1991;45:41 l-6. 17. D’Hooghe TM, Bambra CS, Isahakia M, Koninckx PR. Evolution of spontaneous endometriosis in the baboon (Papio anubis, Papio cynocephalus) over a 12-month period. Fertil Steril 1992;58:409-12. 18. Hendrickx AG, Kraemer DC. Reproduction. In: Hendrickx AG, ed. Embryology of the baboon. Chicago: University of Chicago Press, 1971:1-44. 19. American Fertility Society. Revised American Fertility So-

20. 21. 22.

23.

24.

25. 26. 27.

ciety classification of endometriosis, 1985. Fertil Steril 1985;43:351-2. Olive DL, Henderson DY. Endometriosis and mullerian anomalies. Obstet Gynecol 1987;69:412-5. Baggish MS, Baltoyannis P. Carbon dioxide laser treatment of cervical stenosis. Fertil Steril 1987;48:24-8. Ferenczy A. Anatomy and histology of the uterine corpus. In: Kurman RJ, ed. Blaustein’s pathology of the female genital tract. 3rd ed. New York: Springer-Verlag, 1987: 257-79. Nisolle M, Casanas-Roux F, Anaf V, Mine J, Donnez J. Morphometric study of the stromal vascularization in peritoneal endometriosis. Fertil Steril 1993;59:681-4. Surrey ES, Halme J. Effect of peritoneal fluid from endometriosis patients on endometrial stromal cell proliferation in vitro. Obstet Gynecol 1990;76:792-7. Fuji S. Secondary mullerian system and endometriosis. AM J OSSTET GYNECOL 1992;165:219-25. Redwine DB. Age-related evolution in color appearance of endometriosis. Fertil Steril 1987;48:1062-3. Koninckx PR, Meuleman C, Demeyere S, Lesaffre E, Cornillie FJ. Suggestive evidence that pelvic endometriosis is a progressive disorder, whereas deeply infiltrating endometriosis is associated with pelvic pain. Fertil Steril 1991;55:759-65.

Estrogenic activity of RU 486 (mifepristone) and cultured uterine myocytes Khaled I. Dibbs, MD,” Yoel Sadovsky, MD,” Xue-Jun Li, PhD,b Samuel Stuart Adler, MD, PhD,” and Anna-Riitta Fuchs, DSc” St. Louis, Missouri,

July 1995 Gynecol

S. Koide,

in rat uterus MD, PhD,b

and New York, New York

OBJECTIVE: Our purpose was to determine whether RU 466 (mifepristone) has direct estrogenic activity in uterine myocytes. STUDY DESIGN: Ovariectomized adult rats were treated with AU 466, and its effect on uterine oxytocin receptor concentration, as a marker of estrogenic activity, was measured. Results were compared with the induction by RU 466 of an estrogen-responsive reporter gene in a cultured Syrian hamster uterine myocyte cell line. RESULTS: Baseline oxytocin receptor concentration was 56.6 2 7.2 fmol/mg protein (mean ? SEM) and increased to 227 t 49 fmol/mg with 176estradiol (2.5 kg/kg) and to 145 f 16 fmol/mg after RU 466 (5 mg/kg) treatment, an effect that was inhibited by the antiestrogen ICI 162,760 (1.5 mg/kg). In the cultured Syrian hamster uterine myocyte cell line cells RU 466 (1 Om6 mol/L) caused a 2.17 + 0.17-fold increase in the expression of the reporter gene versus 113.0 r 7.4-fold with 176-estradiol (1 O-a mol/L). The estrogenic activity of RU 466 was dependent on the presence of both estrogen receptor and the promoter’s estrogen response element. CONCLUSION: RU 466 has a weak estrogen-like activity in uterine myocytes. This activity may partly explain the therapeutic effects of RU 466 on this target organ. (AM J OBSTET GYNECOL 1995; 173: 134-40.)

Key words:

RU

486,

estrogenic

activity,

myocytes,

ovariectomized

From the Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Washington University School of Medicine,” the Center for Biomedical Research, the Population Council,* and the Department of Obstetrics and Gynecoloa Cornell University Medical College.’ Received for publication August 9, 1994; revised November 16, 1994; accepted December 5, 1994. 134

rats,

oxytocin

receptor

Reprint requests: Yoel Sadovsky MD, Department of Obstetrics and Gynecology, Washington University School of Medicine, 491 I Barnes Hospital Plaza, St. Louis, MO 63110. Copyright 0 1995 by MO&-Year Book, Inc. 0002-9378/95 $3.00 + 0 6/l/62543

Volume 173, Kumber Am J Obstet Gynecol

1

The introduction of the antiprogestin mifepristone (RU 486) into clinical use has led to numerous therapeutic applications. In obstetrics and gynecology RU 486 has been effective for postcoital contraception, for medical termination of pregnancy in the first trimester and in cases of intrauterine fetal death, for cervical ripening, and for induction of labor at term.’ In addition, it has been used in the treatment of uterine leiomyomas, endometriosis, and breast cancer.’ Other uses include treatment of meningioma and, as an antiglucocorticoid, Cushing’s syndrome.’ Interestingly, RU 486 has also been reported to have progestomimetic activity in postmenopausal women.’ Recently RU 486 was shown to have estrogenic activity in MCF-7 human breast cancer cells, where it may bind directly to the human estrogen receptor.3 The estrogenic potential of steroid antagonists depends on cell type and promoter context.’ Because the effect of RU 486 in labor initiation culminates in myometrial contractions and because myometrial cells have estrogen receptors, we tested both in vivo and in cultured myocyte cell lines whether RU 486 has estrogenic activity in uterine myocytes. Material

and methods

Animal studies. Adult Sprague-Dawley rats weighing 180 to 200 gm (Charles River Co., Syracuse, N.Y.) underwent bilateral oophorectomy. After 14 days under a 14-hour-light and lo-hour-dark cycle, with free access to food and water, the rats were randomly divided into five treatment groups: (1) controls receiving vehicle (ethanol, suspended in corn oil) by mouth (gavage, 0.25 ml); (2) 17B-estradiol (Sigma, St. Louis), 0.5 pg per rat (2.5 kg/kg) subcutaneously; (3) mifepristone (RU 486, gift from Roussel-Uclaf, Romainville, France), 1 mg per rat (5 mg/kg) by mouth; (4) ICI 182,780 (gift from ICI Pharmaceuticals, Macclesfield, United Kingdom), 0.3 mg per rat (1.5 mgikg) subcutaneously; and (5) RU 486, 1 mg per rat by mouth and ICI 182,780, 0.3 mgirat subcutaneously. All drugs were given daily for 3 days. No anesthetic was given. The rats were killed 24 hours after the final dose, and uteri were quickly removed, freed of fat and adnexa, and frozen on dry ice. The specimens were kept at -80” C until prepared for receptor assays. Uteri from three to six rats were pooled for each assay, and four to five pools were used for each treatment group. Statistical analysis was performed by one-way analysis of variance, followed by Dunnett’s test to determine significance of differences from control. Statistical significance was determined atp < 0.05. The study followed the guidelines of and was approved by the Animal Care Committee. Tissue preparation and oxytocin receptor assays. Uterine tissue was prepared and assayed for oxytocin receptors as previously described.’ Briefly, the frozen

Dibbs

et al.

135

uteri were homogenized in a hypotonic Tris buffer, with five 5-second pulses with a Polytron tissue grinder (Brinkmann, Westbury, N.Y.), followed by up-and-down strokes in a glass tissue grinder. The homogenization buffer consisted of 10 mmol/L Tris-hydrochloric acid and 1.5 mmol/L ethylenediaminetetraacetic acid, 0.5 mmol/L dithiothreitol, and 0.002% phenylmethylsulfonyl fluoride, pH 7.5. A crude microsomal pellet was precipitated by centrifuging the 1OOOg supernatant at 100,OOOg for 30 minutes, resuspended in 1 ml of calcium-free Hanks’ solution (containing phenylmethylsulfonyl fluoride), and stored at - 80” C. Before being assayed, pellets were precipitated at 100,OOOg and suspended in assay buffer (50 mmol/L Tris-maleate, 5 mmol/L manganese chloride, 0.1% bovine serum albumin, and 0.002% phenylmethylsulfonyl fluoride), with a final volume of 220 l.rl. Microsomal protein concentration, measured by the Lowry et al. method with bovine serum albumin as standard, was 0.5 to 1.0 mg/ml. Binding of the specific ligand, which was tritiated oxytocin (New England Nuclear, Boston, specific activity 38 Ciimmol), was linearly correlated with protein concentration in this range. The assays, performed in duplicate, were conducted with eight different concentrations of labeled ligand (IO-‘” to 5 x 1O-8 mol/L). Nonspecific binding was determined in the presence of 1.1 pmol/L unlabeled oxytocin (Bachem, Torrance, Calif.) at each point. The incubations were performed for 60 minutes at 22” C and were terminated by the addition of 5 ml of ice-cold assay buffer. Receptor-bound oxytocin and free oxytocin were separated by filtration through Whatman GF/F filters (Whatman, Hillsboro, Ore.) with a Braendel cell harvester (Braendel, Gaithersburg, Md.). The filters were dried at 80” C, placed in 5 ml of Cytoscint (ICN, Irvine, Calif.), mixed in a vortex agitator, and counted 8 to 16 hours later. The binding data were analyzed with a nonlinear, iterative, curve-fitting program (Ligand, Biosoft, Elsevier Medical Publishers, Cambridge, United Kingdom). The program calculates acid ionization constant, dissociation constant, and maximum binding and the SDS. Cell cultures. The Syrian hamster myocyte cell line SHM,’ which is derived from uterine tissue, was maintained for at least 2 weeks before the experiments in estrogen-free medium, with phenol red-free Dulbecco’s modified Eagle medium/nutrient mixture F-12 Ham supplemented with 10% charcoal-stripped newborn calf serum (Gemini Bioproducts, Calabasas, Calif.) and 100 U/ml penicillin and 100 pg/ml streptomycin.’ Cells were cultured in a humidified environment with 95% air and 5% carbon dioxide. Reporter genes and expression vector plasmids. For transfection experiments we used the estrogenresponsive reported plasmid Vit”P36. This plasmid was constructed from the 36 bp prolactin promoter, down-

136

Dibbs et al. Am J Obstet

300

Oxytocin Receptor Concentration (fmollmg protein)

1

250

1T

200

-

I -r

150-

50

Fig. 1. Oxytocin mized rats under E.2, estradiol; RU, RU 486 and average c SEM. (three to six rats) ments. Differences and RU 486 and atp < 0.01 andp

C

RU

ICI

Rut ICI

receptor concentration in uteri of ovariectodifferent treatment conditions. C, control; RU 486; ICZ, ICI 182,780; RU + ICI, both ICI 182,780. Results are shown as Results are for assays from pooled uteri and represent four independent experibetween estradiol or RU 486 versus control ICI 182,780 versus control were significant < 0.05, respectively.

stream from two 26 bp estrogen response element sequences of the Xenopus vitellogenin A2 gene, and upstream from the firefly luciferase gene.8 The parent plasmid P36, lacking the estrogen response elements, served as a control.g Estrogen receptor was expressed in transfected cells with a plasmid that contains the RSV LTR promoter and human estrogen receptor complementary deoxyribonucleic acid (DNA). Plasmids expressed either a mutant estrogen receptor containing a valine in place of glycine at position 400 (RSV-HEVO),h the wild-type receptor (RSV-HEGO),” or, as a control, neomycin phosphotransferase II (RSV Neo).’ Transient

transfection

made of equal volumes of 0.25 mol/L calcium chloride and 2 x BES (N,N-bis[2-hydroxyethyll-2-aminoethanesulfonic acid)-buffered saline solution (2 x BBS) containing 50 mmol/L BES (pH 6.95), 280 mmol/L sodium chloride, and 1.5 mmol/L sodium phosphate, filter sterilized, and stored at -20” C. Plates were then placed overnight in 5% carbon dioxide. The next day cells were washed, fed with phenol red-free Dulbecco’s modified Eagle medium/nutrient mixture F-12 Ham with 10% charcoal-stripped serum. The steroids to be tested were added, and the cells were returned to 5% carbon dioxide. Calcium-phosphate-DNA complexes gradually form when the cells are shifted from 10% to 5% carbon dioxide overnight.” All hormones were dissolved in 100% ethanol, keeping the final concentration of ethanol in the media, including controls, at < 0.2%. One day after hormone treatment cells were harvested in 300 ul of a Triton lysis buffer containing 50 mmol/L Tris-MES (Z-[N-morpholinol-ethanesulfonic acid) (pH 7.8), 1 mmol/L DL-dithiothreitol, and 1% Triton X-100. The lysate was assayed for luciferase activity as previously described” with an Analytical Luminescence Laboratories (San Diego) Monolight 20 10 luminometer. The final

1.

100-

0 ~

July 1995 Gym01

and

luciferase

assays.

We performed transient transfections using the calcium-phosphate method as described by Chen and Okayama, ’ ’ modified for 35 mm six-well plates. Two days before transfection 100,000 cells per well were seeded in phenol red-free Dulbecco’s modified Eagle medium/nutrient mixture F-l 2 Ham with 10% charcoalstripped newborn calf serum. On the day of transfection media were changed to phenol red-free Dulbecco’s modified Eagle medium, with 10% charcoal-stripped serum, and incubated in 10% carbon dioxide. Four hours later each 2 ml well was transfected with 125 ~1 of BBS-calcium chloride solution containing 2.5 pg of DNA, which included 1.25 ug of luciferase reporter plasmid, 0.1 pg of receptor expression vector or control, and 1.15 pg of salmon sperm DNA. BBS-calcium chloride is

values

of luciferase

activity

are

expressed

as a fold

rela-

tive to control (ethanol). All paradigms were assayed in triplicate, and results represent a minimum of two independent experiments. Results are expressed as mean f SEM. For comparisons the unpaired two-tailed t test was used. Significance was determined at p < 0.05. Results RU

486

induces

the

expression

of rat

uterine

oxy

Estrogen is known to induce oxytocin receptor production in rat and rabbit myometrium.13. I4 To determine whether RU 486 has estrogenic activity, we measured oxytocin receptor concentration in rat uterus as a biologic marker of estrogen-responsive gene expression. As shown in Fig. 1, baseline oxytocin receptor concentration in ovariectomized rat uterus was 58.8 +- 7.2 fmol/mg microsomal protein and increased to 227 * 49 fmolimg after treatment with 17B-estradiol. RU 486 treatment resulted in an increase to 145 ? 18 fmolimg. This increase in oxytocin receptor concentration was inhibited when both RU 486 and the antiestrogen ICI 182,780” were administered. When used alone, ICI 182,780 did not have any effect on the oxytocin recep.tor concentration. The affinity of oxytocin to its receptor was unchanged in the different experimental groups (results not shown). tocin

RU

receptors

486

has

in

vivo.

estrogenic

activity

in cultured

uterine

To test whether the estrogen-like activity of RU 486 observed in vivo reflects a direct effect of RU 486 on estrogen receptors, we transfected a Syrian hamster myocyte cell line, SHM, with an estrogenresponsive luciferase reporter gene. This system of myocytes.

Volume 173, Number Am J Obstet Gynecol

Dibbs et al.

1

I

Luciferase Activity (fold)

3

137

3

Luciferase Activity

*

(fold) 1

i m-ha C

E2

RU

P

loeg 1o-8

1o-7

lo+

low5

RU486(M) Fig. 2. Induction of 17P-estradiol-responsive gene Vit’P36 by RU 486 in SHM cells. A, Induction by different ligands: C, control-ethanol (0.1%); E2, estradiol (10m8 mol/L); RU, RU 486 (1O-6 mol/L); P, progesterone (IO-* mol/L). B, Dose-response curve for induction of Vit’ P36 by RU 486. Results are shown as luciferase activity normalized to control (average ir SEM). Results are of triplicate determination and represent three independent experiments: Differences between 17@-estiadiol or RU 486 versus control and between 17P-estradiol and RU 486 are significant atp i 0.001 (progesterone

vs control is not significant, p = 0.86)

transient

transfection

uses

a sensitive

hormone-respon-

dual estrogen response element sequences that control the expression of firefly luciferase gene. The level of target gene expression in response to estrogenic ligands is reflected in luciferase activity in cell extracts, easily quantified by light production. Because SHM cells have a low constitutive concentration of estrogen receptor, we cotransfected cells with an expression vector for HEVO-estrogen receptor to maximize estrogenic responses. This system, which uses a modified human estrogen receptor,‘, lo is extremely sensitive to the effects of estrogen. We observed a 113.0 * 7.4-fold increase in reporter gene expression with a saturating concentration of 17@-estradiol (10m8 mol/L), whereas RU 486 at 1 pmol/L produced a 2.17 -+ 0.17%fold increase in the expression of the reporter gene (Fig. 2, A). The observed induction of reporter gene expression was concentration dependent; a 3.71 i 0.04-fold increase was obtained with 10 ymol/L RU 486 (Fig. 2, B). Similar results were obtained with the true wild-type HEGO-ER and with SHM cells stably transfected with HEVO-ER” (data not sive

promoter

containing

shown). These estrogen-like

To confirm gen

receptors,

results activity

suggest that RU on estrogen-responsive

that RU 486 interacts we

investigated

tion of RU 486 with 17p-estradiol We

could

not

use

164,384 to inhibit

the

antiestrogens

the

486

directly

has a direct genes.

with estro-

competitive

on estrogen ICI

182,780

interac-

receptors. or ICI

the RU 486 effect, because in this very

sensitive assay both antiestrogens displayed a weak agonistic activity (results not shown). However, when 17pestradiol was used in nonsaturating concentrations (lo-lo mol/L), RU 486 (10-j mol/L) inhibited the estradiol-induced reporter gene expression (Fig. 3). This finding suggests that RU 486 competes directly with 17P-estradiol in binding to estrogen receptors. The effect of RU 486 is not mediated through its antiprogestin activity, because progesterone had no significant effect on basal reporter gene expression (Fig. 2,A). To validate that the stimulatory effect of RU 486 depends on the presence of estrogen response elements and is not mediated by either other DNA sequences or posttranscriptional effects, we studied the effects of RU 486 on both Vit’P36 and the parent plasmid P36, which lacks estrogen response element sequences. RU 486 was able to induce the Vit’P36 but not P36, demonstrating the dependence of RU 486 induction on the presence of the estrogen response element (Fig. 4). Comment RU 486, a norethindrone described to have essentially nistic

activity.”

gesterone extent

It binds

with

and glucocorticoid to

specificity to chicken

Philibert

the

androgen

to its binding, or hamster

et al.”

derivative, was previously no steroid receptor agohigh

affinity

receptors receptor.”

to

the

pro-

and to a lesser There

is

species

because RU 486 does not bind progesterone

suggested

receptor.”

as early as 1985 that RU

138

Dibbs

et al.

July Am J Obstet

1995

Gym01

I -Iloo-

25

Luciferase Activity (fold)

I

Luciferase Activity

20

(fold)

15

3

10

5

0 !

Vit2P36 E2

E2+RU

Fig. 3. Competitive inhibition of 17P-estradiol-mediated induction of Vit’P36 reporter gene by RU 486 in SHM cells. E2, Induction by 17P-estradiol (10-l” mol/L); E2 +RU, induction by 17P-estradiol (IO-‘” mol/L) plus RU 486 (10-j mol/L). Results are shown as luciferase activity normalized to control (average * SEM). Results are of triplicate determination and represent two independent experiments. Difference is significant at p < 0.02.

486 may have an estrogen-like uterotropic effect in ovariectomized rats, yet it was not until recently that RU 486 was shown to have a direct estrogenic action in MCF-7 human breast cancer cells3 Studies were undertaken after recognizing the estrogenic potential of synthetic progestins,“” which are 19-nortestosterone derivatives. On the other hand, it was suggested that RU 486 at high doses may have a noncompetitive antiestrogenic effect on the endometrium in vivo, because RU 486 was thought not to bind to estrogen receptors.” We have demonstrated that RU 486 stimulates the production of oxytocin receptors in ovariectomized rat uterus. This effect reflects estrogenic action, because it is inhibited by the antiestrogen ICI 182,780. This finding was further supported by use of a Syrian hamster myocyte cell line. Transient transfection analyses with an estrogen-responsive luciferase reporter gene demonstrate that RU 486 has a weak estrogen-like action. This action depends on the presence of both estrogen receptors and estrogen response elements, consistent with the results of Jeng et al.’ This effect is not a reflection of progesterone antagonism, because RU 486 does not bind to the hamster progesterone receptor,” and progesterone had no effect on the basal activity of the estrogen-responsive gene. Nor is it likely that meta-

P36

Fig. 4. Induction by RU 486 in SHM cells of 17@estradiolresponsive gene Vit”P36 requires estrogen response element. E2, Estradiol (lo-” mol/L); RU, RU 486 (lOme mol/L). Luciferase activity normalized to control is shown for Vit’P36 and P36, the parent plasmid lacking estrogen response elements (average +- SEM). Results are of triplicate determination and represent three independent experiments. The difference between Vit’P36 and P36 for each of 17P-estradiol and RU 486 was significant (p < 0.001).

bolic products derived from RU 486 mediate the estrogenie effect, because no active RU 486 metabolites have been found in target cells.‘9 RU 486 was active at micromolar concentrations, consistent with serum levels achieved with therapeutic levels of RU 486 that range from micromo1ar’9 to 10 kmol/L.2’ It is noteworthy that in the presence of 1’7P-estradiol RU 486 at high concentrations exhibited antagonistic activity, which may represent competition for binding to estrogen receptors. This finding would explain the results of Wolf et al.,” that RU 486 at high doses blocks the proliferative action of estradiol on uterine endometrium and that it may resemble antiestrogens that function as both antagonists and partial agonists. A similar estrogenic activity with another antiprogestin, onapristone (ZK98.299), has been recently reported by Bigsby and Young,23 who used an in vivo mice system.“” Of interest in that report is RU 486 inability to induce DNA synthesis in neonatal mice uterine epithelial cells, a marker of estrogenic activity. This discrepancy from our finding may be a result of species or receptor differences and the sensitivity of the methods used in the different experimental designs. The weak estrogenic effect seen with RU 486 in cell culture (twofold to fourfold with RU 486 versus lOO-fold

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Gynecol

with 176-estradiol) may not completely account for the response noted in vivo (2.5-fold increase in oxytocin receptors with RU 486 versus 3.8fold with 17@ estradiol). It is possible that the 72-hour treatment and prolonged half-life of RU 486 in vivo (12 to 24 hours in humans)” resulted in a more prolonged exposure of the hormone-responsive gene than that achieved in cell culture over a 24-hour period. Alternatively, because progesterone is known to down-regulate oxytocin receptors 13, I4 part of the increase in the receptor concentration’ may have been mediated through blockade of progesterone receptors. However, the rats were ovariectomized 2 weeks before the experiments, making the conditions of the experiments relatively progesterone free; moreover, RU 486 was reported to act as a progesterone agonist in the relative absence of progesterone.‘. ” In addition, the inhibition of the effect of RU 486 on oxytocin receptor concentrations by ICI 182,780 argues against a progesterone-mediated effect and supports an estrogen-mediated effect. These results may also be explained by the complex interactions that may occur in vivo, because RU 486 binds to progesterone receptors, glucocorticoid receptors, and androgen receptors. It has been suggested that RU 486 may induce estrogenic effects indirectly through antiandrogenic activityzl or by increasing estrogen receptor concentrations.2s. 86 RU 486 has been found to increase plasma 17@estradiol concentration and slightly decrease the concentration of sex hormone-binding globulin in postmenopausal women,“. Pa whereas in menstruating women it caused either no levchangeZ6. ” or a decrease3” in serum 17P-estradiol els. Increased adrenal steroidogenesis of androgens peripherally converted to estrogens was suggested as a possible cause for the increased plasma 178-estradiol levels.*’ In a recent study of hypophysectomized, castrated, corticotropin-replaced rats, treatment with RU 486 resulted in decreased adrenal 3@hydroxysteroid dehydrogenase isomerase, 2 1 -hydroxylase, and 1 l-hydroxylase activities.31 This suggests the possibility that RU 486 may shift adrenal steroidogenesis toward producing more 19-norsteroid derivatives that could be converted peripherally to estrogens. Because our in vivo experiments were performed in nonadrenalectomized rats, RU 486 may have indirect estrogenic activity by influencing adrenal steroidogenesis or steroidbinding globulins, in addition to its direct estrogenic effects, through estrogen receptors and estrogen response elements. Because RU 486 does have significant antiglucocorticoid activity, there is a possibility that this may have contributed to RU 486 estrogenic activity in vivo. In the in vitro experiments, to address this issue, we used charcoal-stripped media that was steroid depleted. In ovariectomized rats dexamethasone was found to de-

crease uterine, but not anterior pituitary, nuclear estrogen receptors.” Therefore the possibility remains that the RU 486 effect on the estrogen receptor in vivo may be partly explained by its antiglucocorticoid activity. We plan to explore this possibility in the future. It is also possible that RU 486 may have been bioconverted in vivo to an estrogen agonist. This issue is of lesser concern in our in vitro experiments, because no active RU 486 metabolites have been found in target cells.‘9 In conclusion, we have demonstrated that RU 486 exhibits estrogenic activity in vivo, reflected by an increase in uterine oxytocin receptor concentration. We also demonstrated that RU 486 has a direct, weak estrogenic effect in a uterine myocyte cell line. This activity is dependent on the presence of both estrogen receptor and estrogen response element. These findings shed new light on the mechanism of action of RU 486 when it is used for pregnancy termination and for cervical ripening, especially with regard to the induction of oxytocin receptor synthesis with RU 486. We speculate that RU 486 in vivo may enhance estrogenic activity in the setting of an estrogen-deprived environment and may have minimal or even antagonistic effects in an estrogen-rich milieu. Further studies are needed to clarify the complex interactions involved in RU 486 estrogenic effects in vivo. REFERENCES

1. Spitz IM, Bardin CW. Mifepristone (RU 486)-a modulator of progestin and glucocorticoid action. N Engl J Med 1993:329:404-12. 2. Gravanis A, Schaison G, George M, et al. Endometrial and pituitary responses to the steroidal antiprogestin RU 486 in postmenopausal women. J Clin Endocrinol Metab 1985; 60:156-63. 3. Jeng M-H, Langan-Fahey SM, Jordan VC. Estrogenic actions of RU 486 in hormone-responsive MCF-7 human breast cancer cells. Endocrinoloav 1993:132:2622-30. 4. Berry M, Metzger D, Chambon ?‘I Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the antioestrogen 4-hydroxytamoxifen. EMBO J 1990;9:281 l-8. 5. Fuchs AR, Behrens 0, Helmer H, Liu C-H, Barros CM, Fields MJ. Oxytocin and vasopressin receptors in bovine endometrium and myometrium during the estrous cycle and early pregnancy. Endocrinology 1990;127:629-36. 6. Riemer RK, Sadovsky Y, Roberts JM. Myometrial characteristics of the Syrian hamster uterine smooth muscle cell line, SHM. In Vitro Cell Dev Biol 1993;29:478-80. 7. Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS. Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proc Nat1 Acad Sci U S A 1986;83:2496500. 8. Waterman ML, Adler S, Nelson C, Greene GL, Evans RM, Rosenfeld MG. A single domain of the estrogen receptor confers deoxyribonucleic acid binding and transcriptional activation of the rat prolactin gene. Mol Endocrinol 1988; 2:14-21. 9. Mangalam HJ, Albert VR, Ingraham HA, et al. A pituitary POU domain protein, Pit-l, activates both growth hormone and prolactin promoters transcriptionally. Genes Dev 1989;3:946-58. 10. Tora L, Mullick A, Metzger D, Ponglikitmongkol M, Park

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