RU486 is not an antiprogestin in the hamster

J. steroid Biochem. Vol. 28, No. 5, pp. 493-497, 1987

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RU486 IS NOT A N ANTIPROGESTIN IN THE HAMSTER GARY O. GRAY and WENDELLW. LEAV1TT* Department of Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, U.S.A. (Received 6 April 1987) Snmmary--The biological activity and progestin receptor binding activity of the synthetic steroid RU486 (RU38486; 17-fl-hydroxy-1l-fl-(4-dimethylaminophenyl)-17-ct-(1-propynl)-estra-4,9-diene-3-one)were investigated in the hamster. RU486 demonstrated no antiprogestational activity in the female hamster in that it was ineffective in blocking decidualization or interrupting early pregnancy. Competitive binding assays showed RU486 did not compete from hamster uterine progestin receptor. It is concluded that hamster uterine progestin receptor has unique steroid binding specificity.

84.4 Ci/mmol) and unlabeled R5020 were purchased from New England Nuclear. [3H]p (progesterone; 53.4-57.5Ci/mmol) was purchased from New England Nuclear. Radiochemical purity was tested by thin-layer chromatography. Unlabeled P was purchased from Sigma Chemical Co., St Louis, MO. RU486 was the generous gift of Roussel-Uelaf to WWL. (See Fig. 1 for structural formulae.)

INTRODUCTION

The synthetic steroid RU486 has been reported to block decidualization [1], induce menstruation [1-4], interrupt pregnancy maintenance [1], induce abortion [1, 3, 5, 6] and to inhibit various other progestational responses[6--8]. Further, these antiprogestational activities appear to be progestin receptor (Rp)-mediated events [6--8]. Specific binding of RU486 to Rp from human endometrium [9], rabbit uterus [3], rat uterus [6], MCF7 cells [7] and T47D cells [7, 8] has been demonstrated. Thus, RU486 appears to be a competitive antagonist of progestin action in several systems. In this study we report the unexpected finding that RU486 does not bind to hamster uterine Rp. Additionally, the antiprogestational activities of RU486 seen in other species (i.e. inhibition of decidualization and early pregnancy) are not observed. To our knowledge thisl is the first mammalian Rp with little if any binding affinity for RU486.

Other reagents Ammonium sulfate (ultra pure) was purchased from Schwarz/Mann, Cambridge, MA. Other chemical were purchased from commercial sources at reagent grade or better. Scintillation cocktail was toluene-Triton X-100 (2:1, v/v) with 5g diphenyloxazole (PPO) and 50mg 1,4-bis[2-(5-phenyloxazole)]benzene (POPOP) per liter. 5 ml of cocktail was added to each sample prior to counting. Animals Adult female golden hamsters were maintained under controlled conditions with a 14h light, 10 h dark photoperiod (lights on 0500-1900 h). The regularity of estrous cycles was established by the appearance of the postestrous vaginal discharge of cycle day 1. Animals were sacrificed by cervical dislocation on the morning of cycle day 4. Uteri were immediately removed, stripped of fat and mesentery and used immediately or frozen in liquid nitrogen and stored at -80°C. Adult female Sprague--Dawley rats were maintained under controlled conditions. Rats were

EXPERIMENTAL

Steroids [3H]ORG2058 (16-,t-ethyl-21-hydroxy- 19-nor[6,7-3H]-pregn-4-ene-3,20-dione; 40Ci/mmol) and unlabeled ORG2058 were purchased from Amersham. [aH]R5020 (promegestone; 17,21-dimethyl19-nor-4, 9-pregnadiene-3,20-dione-[ 17
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GARY O. GRAYand WENDELLW. LEAVITT

ovariectomized 2 wk prior to use and were estrogen primed 5 days prior to sacrifice, with each animal receiving a total dose of 120/~g estradiol in corn oil. All animals were sacrificed by cervical dislocation, and the uteri were used immediately.

Cytosol Cytosol was prepared at 4°C by mincing and homogenizing uteri (1 : 2, w/v) in buffer with a Polytron PT-10 (Brinkman Instruments, Westbury, NY) using 2 or 3,5-s bursts at power setting 5, cooling on ice 1 min between bursts. The homogenate was centrifuged at 150,000g for 1 h (4°C) to obtain the cytosol. Buffers used were A (50 mM Tris-HCl, 1 mM EDTA, 12 mM monothioglycerol, 30% glycerol (v/v), pH 7.5) and B (50mM Tris-HCI, I mM EDTA, 12 mM monothioglycerol, 200 mM KCI, 10 mM sodium molybdate, 10% glycerol (v/v), pH 7.5).

Ammonium sulfate precipitation Cytosol was partially purified by ammonium sulfate precipitation at 4°C. Solid ammonium sulfate (0.28 g/ml) was slowly added to undilated cytosol prepared in buffer B. After stirring for 30 min, the precipitated material was collected by centrifugation for 30 min at 150,000 g. The pellet was redissolved in buffer B, dialyzed (3 changes of 1 liter; 4°C) and diluted with buffer prior to use.

Competition assays The competitive binding activity of each steroid was measured by the dextran-coated charcoal (DCC) method described previously [10]. [3HIP, [3H]ORG2058 and [3H]R5020 were used as ligands. Five concentrations of unlabeled P, R5020, ORG2058 and RU486 between 4 x 10 10 and 4 x 10 6 M were used as competitors. Final dilutions of hamster cytosol used in the assays were 1:10-1:17 (w/v). The final dilution of rat cytosol used in the assays was 1:11 (w/v). The assays were incubated 16-22 h on ice, treated with DCC, vortexed and immediately spun 5 min at 1500 g. Aliquots from the DCC-treated supernatant from each assay tube were counted on a Beckman LS-1800 scintillation counter at 40% efficiency. The relative binding activity (RBA) of each steroid was determined at the 50% competition level [11].

Decidualization Implants containing P packed into 2.5 cm lengths of Silastic tubing were placed subcutaneously in the flank region of 3 intact hamsters on cycle day 1. This maintains serum P levels of 10 ng/ml [12,13]. Four days later, each uterine horn was traumatized by the insertion of a nylon monofilament implant and each animal received a subcutaneous injection of RU486 or vehicle. One animal received 10 mg of RU486 in 0.15 ml corn oil. The other two animals were given RU486 suspended in an ethanolic (33%)-saline solution. Injections were continued once daily for 2 days

(days 5 and 6 relative to the P implant on day I), and the animals were sacrificed on the third day. The uteri were removed, split longitudinally and examined for deciduoma formation.

Interruption of early pregnancy Five adult female hamsters were mated with fertile males on the evening of cycle day 4. Designating day 1 as the day following mating, once daily injections of RU486 were performed on days 3-7. Three animals were given 3 mg RU486 in 0.3 ml corn oil per day. Controls were given 0.3 ml corn oil per day. On day 8 the animals were sacrificed; the uteri were removed, and the number of developing implantation sites was determined. (This RU486 dosage induces 100% abortion rates in rats [5]. RESULTS

The ultraviolet absorption spectrum of the RU486 (in ethanol) utilized in this study is identical to that previously reported [8], with absorption maxima at 261 and 304 rim. The identity of this compound was further confirmed by its ability to compete for rat uterine Rp (Fig. 2A), a known RU486 binder [6]. Figure 2 shows the results of competition assays comparing the binding of RU486 to hamster Rp with RU486 binding to rat uterine Rp. As indicated in Fig. 2A, RU486 binding to rat uterine Rp is similar to that previously reported (Ka = 2 x 109 M - l ; RU486 ~ R5020 > P) [6]. Hamster uterine Rp, however, clearly does not bind RU486 under these conditions (Fig. 2B). Binding of [3H]P, [3H]R5020 or [3H]ORG2058 to hamster uterine Rp could not be displaced by 1000-fold excess of unlabeled RU486 (Table 1). The binding specificity of the other ligands tested is the same (ORG2058 > P > R5020). To test the possibility that serum contaminants (i.e. albumin, CBG) may interfere in the binding of RU486 to hamster Rp, the competition assays were repeated using ammonium sulfate-precipitated receptor. Although RU486, ORG2058 and R5020 have been shown to have low affinity for CBG [9, 14, 15], the high capacity of CBG makes it a significant second binder. Other serum components have been shown to bind most of these steroids [14]. At 48% ammonium sulfate saturation, hamster uterine Rp precipitates while albumin and CBG do not (unpublished results). Ammonium sulfate precipitated hamster Rp was prepared as described in Experimental. As indicated in Fig. 3, partial purification of hamster Rp in the presence of molybdate does not increase the affinity of RU486 for hamster uterine Rp, and no competition was observed. Similar results were obtained in buffers without molybdate. The biological activity of RU486 in the hamster was tested by determining the ability of this compound to block decidualization and early pregnancy. In the decidualization experiment, RU486 was found to be ineffective in blocking deciduoma formation.

RU486 is not antiprogestin in the hamster

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Fig. 2. Competitive binding activity of various steroids for [3H]P-bindingsites in cytosol prepared in buffer A and assayed as described in Experimental. Rat uterine cytosol (A). Hamster uterine cytosol (B). Visual inspection of the uteri of 3/3 RU486-treated animals on day 7 of the protocol showed full dccidualization. RU486 was also found to be ineffective in blocking early pregnancy. Mated control animals showed 12 to 13 developing implantation sites on day 8 after mating. Mated RU486-treated animals showed 13 to 14 developing implantation sites on day 8 after mating. DISCUSSION

The biological efficacy of RU486 as an antiprogestin has been demonstrated in the human and in a number of animals by its ability to block decidualization, induce menstruation, block implantation and interrupt early pregnancy. That these antiprogestin activities are receptor-mediated events [6-8] is substantiated by the evidence presented Table 1. *Relative binding activity of various steroids for [3H]progestin-binding sites in hamster uterine cytosol Competitor steroid

P

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in this report. Under conditions where RU486 binds to rat uterine Rp, no binding to hamster uterine Rp is observed (Fig. 2, Table 1). The preferential retention of RU486 by the unactivated form of the glucocorticoid receptor [16] and unactivated calf uterine Rp [17] has been demonstrated in some systems. This phenomenon, however, does not appear to explain our results with Rp since unactivated (molybdate-stabilized) hamster Rp was found to be incapable of binding RU486 (Fig. 3). The ligand binding specificity of the hamster uterine Rp has been shown to be distinctly different from that of the human, rabbit, and to a lesser extent the sheep Rp [10, 18, 19]. In particular, the hamster Rp does not bind well the 17-acetoxy pregnane derivates (Provera, megestrol acetate, chloromadinone acetate and 17-hydroxy progesterone acetate)[18, 19]. This may be related to the relatively low affinity of hamster Rp for R5020 (Table 1, Fig. 2B and Fig. 3). As noted here and by others [19], hamster Rp binds R5020 with essentially the same affinity as P. However, in the rat (Fig. 2A) and several other species [14, 15], Rp binds R5020 with higher affinity than P. This species difference in affinity for R5020 may be related to structural modification at the 17 position. The sensitivity of hamster Rp to 17 position modifications may also contribute to its inability to bind RU486. The 17-(prop-l-ynyl) group of RU486

496

GARY O. GRAY and WENDELL W. LEAVITT

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Fig. 3. Competitive binding activity of various steroids for [3H]P-binding sites in hamster uterine cytosol partially purified by ammonium sulfate precipitation (as described in Experimental). (Fig. 1) is relatively bulky and could reduce Rp binding affinity. Certainly with other progestins, the 16 and 11 positions are also important [19]. This study and others [19] suggest a unique sensitivity of hamster Rp to structural modification of steroidal ligands at the 11 position as well as the 17. For example, 11B-hydroxy-4-pregnen-3,20-dione (RBA = 10) and 1 Et-hydroxy-4-pregnen-2,30-dione ( R B A = 2) are bound poorly [19]. Hamster Rp is unable to bind 4-pregnen-llct-ol-3,20-dione hemisuccinate (RBA<
Grants HD06757 (GG), HD18712 and HD16069 (WWL). The authors are grateful to Mr Kevin Thompson and Mrs Sabina Rundle for their excellent technical assistance.

REFERENCES

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RU486 is not antiprogestin in the hamster

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17.

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18. Kontula K.: Progesterone-binding proteins from endometrium and myometrium of sheep uterus: a comparative study. Acta endocr, Copenh. 78 (1975) 593~03. 19. Wilks J. W., Spilman C. H. and Campbell J. A.: Steroid binding specificity of the hamster uterine progesterone receptor. Steroids 35 (1980) 697-706. 20. Moudgil V. K., Lombardo G., Hurd C., Eliezer N. and Agarwal M. K.: Evidence for separate binding sites for progesterone and RU486 in the chick oviduct. Biochim. Biophys Acta 889 (1986) 192-199. 21. Groyer A., Le Bouc Y., Joab I., Radanyi C., Renoir J. M., Robel P. and Baulieu E.-E.: Chick oviduct glucocorticoid receptor. Specific binding of the synthetic steroid RU486 and immunological studies with antibodies to chick oviduct progesterone receptor. Fur. J. Biochem. 149 (1985) 445-451.