Antiestrogen ICI 164,384 Reduces Cellular Estrogen Receptor Content by Increasing Its Turnover SOPHIE DAUVOIS, PAUL S

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Proc. Natl. Acad. Sci. USA Vol. 89, pp. 4037-4041, May 1992 Biochemistry Antiestrogen ICI 164,384 reduces cellular estrogen receptor content by increasing its turnover SOPHIE DAUVOIS, PAUL S. DANIELIAN, ROGER WHITE, AND MALCOLM G. PARKER* Molecular Endocrinology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom Communicated by Elwood V. Jensen, December 30, 1991 (received for review August 21, 1991)

ABSTRACT The ability of estrogens to stimulate the tran- with a subsequent step required for receptor-mediated gene scriptional activity of the estrogen receptor can be inhibited by transcription (16, 17). a diverse range of estrogen antagonists. Here we show that the The variation in the ability of ICI 164,384 to inhibit the antiestrogen ICI 164,384, N-(n-butyl)-11-[3,1713-dihydroxy- DNA binding activity of the estrogen receptor might be estra-1,3,5(10)-trien-7a-yl]N-methylundecanamide, rapidly explained by differences in the stability of receptor dimers. reduces the levels of receptor protein transiently expressed in Since it may not always be possible to dissociate preformed cells without affecting receptor mRNA abundance. The reduc- dimers and inhibit DNA binding in cell-free extracts, we have tion in the levels of receptor protein is dose dependent, revers- investigated whether ICI 164,384 was able to prevent DNA ible by estradiol, and mediated by the hormone-binding do- binding in intact cells. In this paper we show that ICI 164,384 main ofthe receptor. Pulse-chase experiments indicate that the treatment causes a decrease in cellular content of estrogen half-life of the receptor is reduced from -5 hr in the presence receptor protein by markedly reducing its half-life and sug- of estradiol to <1 hr by ICI 164,384. A similar reduction in gest that this might be caused by impaired receptor dimer- estrogen receptor levels is demonstrated in human breast ization. cancer cells treated with ICI 164,384. We discuss the possibility that the increased turnover of the receptor might be a consequence of impaired receptor dimerization. MATERIALS AND METHODS Cell Culture and Transient Transfection Experiments. The Estrogens regulate cell growth and differentiation by binding expression of the MOR was analyzed by transient transfec- to specific receptors that function as transcription factors tion experiments. COS-1 cells were grown in Dulbecco's (1-3). Hormone binding is required for the dissociation of an modified Eagle's medium (DMEM) supplemented with 10% inactive oligomeric receptor complex to allow receptor dimer- (vol/vol) fetal calf serum (GIBCO). After suspension in ization and high-affinity DNA binding (4,5) and to produce the phosphate-buffered saline (PBS), cells (6 x 106 per ml) were full transcriptional activity of the receptor (6-9). Stimulation transfected by electroporation with 25 ,ug of pJ3MOR1-599 of transcription is mediated by two transcriptional activation (18), pJ3MOR1-339 (9), or pJ3f1 together with 6 gg of pJ3 functions (TAF)-namely, TAF-1 and TAF-2, the activity of luciferase (19) as an internal control for transfection effi- TAF-2 being dependent upon estrogen binding. Since estro- ciency as described (20). After 24 hr the medium was replaced gens have been found to act as mitogens in at least 30o of with the same medium containing 10 nM estradiol or 1 tM ICI breast cancers, a large number of estrogen antagonists have 164,384 or no added hormone as indicated. Whole-cell ex- been developed as therapeutic agents. One type, which in- tracts were prepared in a high-salt buffer containing 0.4 M cludes the nonsteroidal breast cancer drug tamoxifen (10), can KCI; 20 mM Hepes (pH 7.4); 1 mM dithiothreitol; 20% also act as partial agonists in a number of physiological (vol/vol) glycerol; and 0.5 mg ofbacitracin, 5,g ofaprotinin, responses; while another type, which includes ICI 164,384, 40,tg ofphenylmethylsulfonyl fluoride, 5,ug ofpepstatin, and has been reported to be devoid ofagonist activity (11, 12). ICI 5 ,g of leupeptin per ml (14). Their protein concentrations 164,384 is N-(n-butyl)-11-[3,17,3-dihydroxyestra-1,3,5(10)- were determined with a Bio-Rad protein assay reagent to trien-7a-yl]N-methylundecanamide. normalize the amount of extract examined in a gel-shift The mechanism of action of some steroid hormone antag- assay. onists is beginning to emerge. It has been shown that tamox- Human breast cancer cells, ZR-75-1, were grown in phenol ifen promotes high-affinity DNA binding of the receptor but red-free DMEM supplemented with 10% charcoal-stripped fails to induce the formation ofTAF-2 in the hormone-binding fetal calf serum containing no hormone or 10 nM estradiol or domain, which accounts for its antagonist activity (7, 9). The 1 uM ICI 164,384. Whole-cell extracts were prepared as agonist effect of tamoxifen has been proposed to be derived described above. from TAF-1 in the N-terminal domain of the receptor, which Gel-Shift Assay. DNA binding activity of estrogen recep- is constitutively active (13). The effect of the so-called pure tors was assayed by gel-shift analysis. Whole-cell extracts antiestrogen ICI 164,384 is controversial. In our studies with from transfected COS-1 cells (2 jig of protein) or from human the mouse estrogen receptor (MOR), we demonstrated that breast cancer ZR-75-1 cells (8 jig of protein) were incubated ICI 164,384 inhibited DNA binding of the receptor and with a [32P]DNA probe containing an estrogen response proposed that this may be a consequence of impaired recep- element (9) and the indicated steroids as described (15). An tor dimerization (14). This impairment might result from anti-MOR antibody, MP16 (15), or preimmune serum was steric interference produced by the large 7a-alkylamide ex- also added where indicated. tension present on ICI 164,384 (12), since we have shown that Enzyme Immunoassay of Estrogen Receptors. For immu- the steroid binding pocket is at or near the dimer interface noassays, COS-1 cell extracts were diluted 1:20, and the (15). However, other workers have found that ICI 164,384 did immunoreactive receptors were measured by using mono- not inhibit DNA binding and proposed that it also interferes Abbreviations: MOR, mouse estrogen receptor; ICI 164,384, N-(n- The publication costs of this article were defrayed in part by page charge butyl)-11-[3,17p-dihydroxyestra-1,3,5(10)-trien-7a-yl]N-methylun- payment. This article must therefore be hereby marked "advertisement" decanamide. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 4037 Downloaded by guest on September 29, 2021 4038 Biochemistry: Dauvois et al. Proc. Natl. Acad. Sci. USA 89 (1992)

clonal antibodies against the human estrogen receptor in a A commercial kit (Abbott) as instructed by the manufacturer. RNA (Northern) Blot Analysis. Total RNA was isolated from transfected COS-1 cells using phenol-chloroform (1:1, vol/vol) extraction as described (21) and separated by electrophoresis in agarose gels containing 2.2 M formaldehyde - 0e d^ (22). After transfer to a nylon membrane (Pall Biodyne), the blots were hybridized with 1 x 106 dpm of riboprobes for the wo estrogen receptor (18) and y-actin (23) per ml as described (18, 24). Radiolabeling of MORs. COS-1 cells were cotransfected with pJ3MOR1-599 (containing the wild-type receptor) and pJ3MOR1-339 (containing a deletion mutant lacking the hormone binding domain) by electroporation as described above. Cells were radiolabeled with 200,iCi (1 ACi = 37 kBq) of [35S]methionine in phenol red-free, methionine-free DMEM supplemented with 10%o dialyzed charcoal-stripped fetal calf serum for 1 hr and then incubated for increasing periods of time in medium containing nonradioactive methionine and the indicated ligands. Cells were lysed with 0.3 ml FIG. 1. Effect of pretreatment of COS-1 cells with ligand on the of high-salt buffer containing 50 mM Hepes (pH 7.0); 500 mM ability of MORs to bind DNA. Cells were pretreated with no hormone (lanes NH), 10 nM estradiol (lanes E2), or 1 ,uM ICI 164,384 NaCl; 1% Nonidet P-40; and 0.5 mg bacitracin, 5 ,ug of (lanes ICI) for 24 hr. Receptor extracts were incubated in the absence aprotinin, 40 jig of phenylmethylsulfonyl fluoride, 5 Ag of (lanes -) or presence (lanes +) of MP16 before the addition of pepstatin, and 5 ,ug of leupeptin per ml for 10 min at 0°C and radiolabeled estrogen response element and then were tested for centrifuged at 10,000 x g for 10 min; the supernatant was DNA binding activity in a gel-shift assay. (A) Cells were transfected stored at -70°C. with pJ3MOR1-599 or pJ3fl (first lane on left). (B) Cells were Immunoprecipitation. [35S]Methionine-labeled COS-1 cell transfected with pJ3MOR1-599 (lanes 1-599) or deletion mutant lysates were precleared by incubating 150 Al of lysate with 10 pJ3MOR1-339 (lanes 1-339). ,ul of preimmune serum for 40 min at 0°C, followed by a further 40-min incubation at 0°C with 20 ,ul of a 50% slurry of Moreover, the deletion mutant lacking the hormone binding protein A-Sepharose (Pharmacia). After removal ofthe Seph- domain, MOR1-339, which is able to bind to DNA with high arose beads, specific immune (MP16) or preimmune serum (5 affinity only in the presence of the anti-MOR MP16 antise- ,ul) was added, and the mixtures were incubated for a further rum, was also expressed to similar levels in the presence or 1 hr on ice. Antibody-antigen complexes were precipitated absence of ICI 164,384 (Fig. 1B). This indicates that this with 50 Al of protein A-Sepharose under agitation at 4°C. The effect of ICI 164,384 on the level of receptor protein is protein A-Sepharose pellet was washed twice with NET-gel mediated by the hormone binding domain. buffer (50 mM Tris chloride, pH 7.5/150 mM NaCl/0.1% We next examined the MOR content in cells directly by Nonidet P-40/1 mM EDTA/0.25% gelatin/0.02% sodium performing enzyme immunoassays (Table 1). We observed azide) and twice with 10 mM Tris chloride, pH 7.5/0.1% an increase in MOR content after treatment with estradiol Nonidet P-40. The pellet was resuspended in SDS/PAGE from 9.1 to 14.2 fmol/4g of protein over a period of 3 hr, loading buffer and analyzed on SDS/10o polyacrylamide whereas ICI 164,384 treatment reduced the amount of recep- gels. The half-life of the receptor was calculated from the tor to 2.8 fmol/,ug of protein within 1 hr and to 1.75 fmol/,ug amounts of radioactive protein determined by scanning the of protein after 3 hr. This reduction was confirmed by gel with an Ambis systems p scanner. performing ligand binding assays (data not shown). We also investigated the kinetics of receptor loss by measuring DNA binding activity with the gel-shift assay. The DNA binding RESULTS activity of the MOR was unaffected by estradiol treatment Effect of Pretreatment of COS-1 Cells with ICI 164,384 on (Fig. 2A) but was rapidly reduced by ICI 164,384, the the Ability of MOR To Bind DNA. Previous experiments response being maximal after a 1-hr treatment (Fig. 2B). The showed that ICI 164,384 inhibited the DNA binding activity difference in the kinetics of receptor loss observed with the of the MOR in cell-free extracts when it had been overex- two methods of analysis can be explained by the fact that a pressed in insect cells (14), but this was reported not to be the functional activity, such as DNA binding, may be lost more case in a number of other cell types (16, 25). Therefore, we rapidly than specific epitopes in the protein. investigated the effect of incubating intact cells with the To further characterize the action of ICI 164,384 on MOR antiestrogen prior to the analysis of its DNA binding activity. loss, we next studied the effect of increasing concentrations When transfected COS-1 cells were pretreated with either no of antiestrogen and observed that the half-maximal inhibition hormone or estradiol, the DNA binding activity of the occurred at 10 nM ICI 164,384 (data not shown). We also receptor was similar in the presence or absence of anti-MOR Table 1. Effect of pretreatment with estradiol or ICI 164,384 on MP16 (Fig. lA). After a 24-hr treatment with ICI 164,384, MOR cellular content there was a 90% reduction in DNA binding activity that was unaffected by the addition of MP16. Since MP16 was able to MORs, fmol/,ug of protein restore the DNA binding activity of MOR complexed with Time, hr NH Estradiol ICI 164,384 ICI 164,384 in cell-free extracts (14), these observations suggest that ICI 164,384 treatment of intact cells results in a 0 9.1 9.1 9.1 loss of receptor protein. In contrast, treatment with the 0.25 - 10.8 8.8 partial agonist tamoxifen had no effect on the amount of 1 - 11.8 2.8 receptor detected in a gel-shift assay (data not shown). This 3 9.3 14.2 1.75 effect of ICI 164,384 was not part of a general effect upon Receptor extracts from COS-1 cells pretreated with no hormone gene expression because the activity of luciferase, used as an (NH), 10 nM estradiol, or 1 ,uM ICI 164,384 for the indicated period internal control, was unaffected (S.D., unpublished results). oftime were tested for their MOR content by enzyme immunoassay. Downloaded by guest on September 29, 2021 Biochemistry: Dauvois et al. Proc. Natl. Acad. Sci. USA 89 (1992) 4039

A TIME 0 5 15 30 60 180 Pretreatment 3 h NH ICI IC I0 E2 E2 -3 h NH ICI E2 E2 ICI - - IC1 MP16 -- + + - + + -18 h - E2 ICl

, . O.". Nor a- MP16 .s + + q- -, w w _ w 0 _ w f

_-_

B TIME 0 5 15 30 60 180

MP16 - + - + - + - + - + - + wuirnww

FIG. 3. Effect of ICI 164,384 on MOR levels is reversible. COS-1 cells, transfected with pJ3MOR1-599, were pretreated for two con- secutive periods of 3 hr with or without an additional 18 hr, with no hormone (lanes NH), with 10 nM estradiol (lanes E2), or with 1 ,IM ICI 164,384 (lanes ICI). Receptor extracts were then tested for DNA binding activity in a gel-shift assay in the presence (lanes +) or absence (lanes -) of the antiserum MP16. MOR1-599 was detected in cell-free extracts by immunoprecipitation with MP16. The amount of [35S]methionine-labeled receptor (arrow) and deletion mutant MOR 1-339 (arrowhead) decreased with time (Fig. 5 A-C). By plotting the FIG. 2. Time course ofthe effect ofpretreatment with estradiol or decrease of [355]methionine-labeled receptor against time, ICI 164,384 on the DNA binding activity of MORs. COS-1 cells, we calculated that the half-life of the full-length receptor was transfected with pJ3MOR1-599, were pretreated with 10 nM estradiol 2.6 hr in the absence of ligand and 5.0 hr in the presence of (A) or 1 uM ICI 164,384 (B) for the times in minutes indicated above estradiol, whereas ICI 164,384 dramatically reduced the the lanes. Receptor extracts were then tested for DNA binding activity half-life to 0.9 hr (Fig. 5D). In contrast, the effect of the in a gel-shift assay in the presence (lanes +) or absence (lanes -) of different on the antiserum MP16. treatments the turnover of MOR 1-339 was negligible (arrowhead), with half-lives ranging from 3.1 to 4.4 have examined the reversibility of this inhibitory effect. hr. We conclude therefore that the "pure"antiestrogen ICI When COS-1 cells were pretreated with 1 ,uM ICI 164,384 for 164,384 reduces the cellular content of estrogen receptor by 3 hr to reduce their MOR content maximally and then treated increasing its turnover. with 10 nM estradiol, the level of receptor was partially Effect of ICI 164,384 on Estrogen Receptors in Human restored after 3 hr and completely restored to that found in Breast Cancer Cells. To rule out the possibility that the effects untreated cells after a further 18 hr, indicating that the effect of ICI 164,384 we observe are restricted to COS-1 cells, we of ICI 164,384 was reversible (Fig. 3). It can be seen that ICI have investigated its action in human breast cancer cells. 164,384 inhibited DNA binding activity irrespective of When ZR-75-1 cells were incubated in the absence or pres- whether cells were grown in the absence or presence of estradiol. NH ICD 62 Effect of ICI 164,384 on MOR mRNA Levels. To determine 'I., 1 3 6 24 ii 1 3 62f- if the effects of ICI 164,384 on the levels of MOR were mediated by a reduction in mRNA, we analyzed Northern blots. MOR mRNA expressed from the vector pJ3MOR (18) is -2.5 kilobases (kb) in size and contains the cDNA se- _ _ _ _ _ quences encoding the full-length receptor together with sim- oS ian virus 40 polyadenylylation and termination signals. Sim- ilar amounts of MOR mRNA were expressed in COS-1 cells irrespective of whether they were incubated with estradiol or ICI 164,384 or no hormone (Fig. 4). Slight variations of receptor mRNA were paralleled by equivalent variations in y-actin mRNA. Thus, we conclude that the effect of ICI **484 G - _ _rn ACTIN 164,384 is not on estrogen receptor mRNA but on the receptor protein itself. FIG. 4. Northern blot analysis of MOR mRNA. COS-1 cells Effect of ICI 164,384 on the Half-Life of the MOR. We next transfected with pJ3MOR or pJ3fl (fl) were incubated with no investigated the turnover of the receptor in pulse-chase hormone (lane NH), 1 ,IM ICI 164,384 (lanes IC), or 10 nM estradiol experiments. COS-1 cells that had been cotransfected with (lanes E2) for the time in hours indicated above the lanes. Total RNA pJ3MOR1-599 and the deletion mutant pJ3MOR1-339 as a was isolated, and samples (10 ,ug) were separated by electrophoresis, control were with transferred to nylon, and hybridized first with a 32P-labeled riboprobe pulse-labeled [35S]methionine for 1 hr. The corresponding to the steroid binding domain ofthe MOR and second cells were then incubated with no hormone, estradiol, or ICI with a y-actin probe. The positions of the 28S and 18S ribosomal 164,384 for periods oftime from 15 min to 6 hr, and the labeled RNAs are shown on the left. Downloaded by guest on September 29, 2021 4040 Biochemistry: Dauvois et al. Proc. Natl. Acad. Sci. USA 89 (1992) A B

_w *

C D

FIG. 5. Effect of ligand on the half-life of MORs. COS-1 cells cotransfected with pJ3MOR1-599 and pJ3MOR1-339 were labeled with [35S]methionine for 1 hr and then treated with no hormone (A) or 10 nM estradiol (B) or 1 /LM ICI 164,384 (C) for 0 hr (lanes 4), 0.25 hr (lanes 5), 0.5 hr (lanes 6), 1 hr (lanes 7), 3 hr (lanes 8), or 6 hr (lanes 9). Cell lysates were prepared, and the receptor was detected by immunoprecipitation with MP16. The positions of molecular mass markers (lane 1) are given in kilodaltons on the left. Other lanes: 2, immunoprecipitation of COS-1 cells transfected with pJ3fl; 3, immunoprecipitation with preimmune serum of the sample analyzed in lane 4; 10 in B, 2 p.l of in vitro 35S-labeled receptors translated in vitro (15) to indicate the position of MOR1-599. Arrows indicate the position of MOR1-599, while arrowheads indicate the position of MOR1-339. (D) Decrease of 35S-labeled MOR was determined by assaying MOR bands for radioactivity after the different treatments: no hormone (o), 10 nM estradiol (o), or 1 ,uM ICI 164,384 (A). Results are expressed as means + SEM of two independent experiments. When SEMs overlap with the symbol used, only the symbol is illustrated. ence of estradiol, the DNA binding activity was similar with the MOR in insect cells has allowed us to propose that ICI or without the addition of the anti-MOR antiserum MP16 164,384, whose structure resembles estradiol but contains a (Fig. 6). After a 3-hr treatment with ICI 164,384, there was a 7a-alkylamide side chain, may bind to the same region of the 90-95% reduction in DNA binding activity. As in COS-1 receptor and sterically interfere with dimerization of the cells, this reduction was not accompanied by a decrease in estrogen receptor mRNA (data not shown). Thus, we conclude that the antiestrogen ICI 164,384 also reduces the cellular content ofestrogen receptors in human breast cancer cells.

DISCUSSION The steroidal estrogen antagonist ICI 164,384 has been shown to function as a "pure" antagonist in vivo (11, 12, 26), in breast cancer cell lines (27-29), and in transiently transfected cells (13, 14). In this paper, we show that ICI 164,384 reduces steady-state levels of estrogen receptor by at least 90-95%. The turnover of the receptor is increased as a result of a reduction in the half-life of the protein from 5 hr in the presence of estradiol to <1 hr in the presence of ICI 164,384. In contrast, nonsteroidal antiestrogens have been shown to have little effect on estrogen receptor turnover (30). The half-life of the human estrogen receptor in MCF-7 human breast cancer cells was determined to be 3 hr in the presence of estradiol, comparable to that of the MOR in COS-1 cells, FIG. 6. Effect of pretreatment of ZR-75-1 human breast cancer and 3.5-5.0 hr in the presence of nafoxidine or C1628 (30). cells with ICI 164,384 on the ability of the estrogen receptor to bind Tamoxifen has also been shown to increase the expression of DNA. Cells were pretreated with no hormone (lanes NH), 10 nM the ICI estradiol (lanes E2), or 1 puM ICI 164,384 (lanes ICI) for 24 hr. estrogen receptors (31). Thus, pure antiestrogen Receptor extracts were preincubated in the absence (lanes -) or 164,384 and nonsteroidal partial agonists differ markedly in presence (lanes +) of MP16 before the addition of radiolabeled their effects on the turnover of the estrogen receptor. estrogen response element and then were tested for DNA binding In previous studies we identified residues in the hormone activity in a gel-shift assay. Arrowheads indicate the position of the binding domain that are involved in dimerization and overlap estrogen receptor complex, while NS corresponds to nonspecific a region essential for estrogen binding (15). The expression of receptor complex. Downloaded by guest on September 29, 2021 Biochemistry: Dauvois et al. Proc. Natl. Acad. Sci. USA 89 (1992) 4041 protein (14). This possibility would be consistent with the 1. Evans, R. M. (1988) Science 240, 889-895. observation that a side-chain length of 16-18 atoms was 2. Beato, M. (1989) Cell 56, 335-344. optimal for both the inhibitory effects of antiestrogens on 3. Ham, J. & Parker, M. G. (1989) Curr. Opin. Cell Biol. 1, dimerization and DNA in cell-free extracts D. 503-511. binding (N. 4. Catelli, M. G., Binart, N., Jung-Testas, I., Renoir, J. M., Arbuckle and M.G.P., unpublished results) and the pure Baulieu, E.-E., Feramisco, J. R. & Welch, W. J. (1985) EMBO antagonist activity ofthese antiestrogens in vivo (12). In view J. 4, 3131-3135. of these results we favor the suggestion that the increased 5. Kumar, V. & Chambon, P. (1988) Cell 51, 145-156. turnover of estrogen receptors in the presence of ICI 164,384 6. Kumar, V., Green, S., Stack, G., Berry, M., Jin, N. R. & may occur as a consequence of impaired receptor dimeriza- Chambon, P. (1987) Cell 51, 941-951. tion. It appears that ICI 164,384 binding is required for a 7. Webster, N. J. G., Green, S., Jin, J. R. & Chambon, P. (1988) reduction in the half-life of the protein, since the deletion Cell 54, 199-207. mutant MOR 1-339 is not significantly affected by the anti- 8. Tora, L., White, J., Brou, C., Tasset, D., Webster, N., Scheer, This form of the human E. & Chambon, P. (1989) Cell 59, 477-487. estrogen. estrogen receptor lacking 9. Lees, J. A., Fawell, S. E. & Parker, M. G. (1989) Nucleic the hormone binding domain has been shown to dimerize via Acids Res. 17, 5477-5488. a weak dimerization activity in the DNA binding domain (5) 10. Jordan, V. C. (1984) Pharmacol. Rev. 36, 245-276. and to stimulate transcription constitutively (7, 8). 11. Wakeling, A. E. & Bowler, J. (1988) J. Steroid Biochem. 30, The demonstration that ICI 164,384 can act prior to DNA 141-148. binding contrasts with the proposal that this antiestrogen is 12. Bowler, J., Lilley, T. J., Pittam, J. D. & Wakeling, A. E. (1989) more likely to inhibit a subsequent step involved in gene Steroids 54, 71-99. activation (16, 17). Evidence to support this proposal was 13. Berry, M., Metzger, D. & Chambon, P. (1990) EMBO J. 9, based on two types ofobservations. First, chimeric receptors 2811-2818. that contain the GAL4 DNA domain or 14. Fawell, S. E., White, R., Hoare, S., Sydenham, M., Page, M. heterologous binding & Parker, M. G. (1990) Proc. Natl. Acad. Sci. USA 87, the VP16 transcriptional activation domain appear to bind 6883-6887. DNA in transiently transfected cells even in the presence of 15. Fawell, S. E., Lees, J. A., White, R. & Parker, M. G. (1990) ICI 164,384 (7, 17). One explanation for these results is that Cell 60, 953-962. chimeric receptors might be processed differently from the 16. Sabbah, M., Gouilleux, F., Sola, B., Redeuilh, G. & Baulieu, normal protein, possibly due to the fact that these heterolo- E. E. (1991) Proc. Natl. Acad. Sci. USA 88, 390-394. gous proteins may contain additional dimerization functions 17. Pham, T. A., Eilliston, J. F., Nawaz, Z., McDonnell, D. P., (7, 17, 32). Second, ICI 164,384 failed to inhibit the in vitro Tsai, M. J. & O'Malley, B. W. (1991) Proc. Natl. Acad. Sci. DNA binding activity of estrogen receptors from either calf USA 88, 3125-3129. uterus or or 18. White, R., Lees, J. A., Needham, M., Ham, J. & Parker, M. G. (16) HeLa cells (24) when translated in vitro (9). (1987) Mol. Endocrinol. 1, 735-744. However, other studies have demonstrated that the DNA 19. De Wet, J. R., Wood, K. V., De Luca, M., Helinski, D. R. & binding activity of estrogen receptor isolated from human or Subramani, S. (1987) Mol. Cell. Biol. 7, 725-737. pig uterus (33) and the MOR expressed in insect cells is 20. Danielian, P. S., White, R., Lees, J. A. & Parker, M. G. (1992) inhibited by ICI 164,384 (14), suggesting that the inhibitory EMBO J. 11, 1025-1033. effect of the antiestrogen may vary depending upon the 21. Ham, J., Thomson, A., Needham, M., Webb, P. & Parker, source of the receptor protein. These discrepancies might M. G. (1988) Nucleic Acids Res. 16, 5263-5276. also be explained by differences in the specific binding 22. Lehrach, H., Diamond, D., Wozney, J. M. & Boedtker, W. conditions used in the gel-shift assays, since the DNA binding (1977) Biochemistry 16, 4743-4751. 23. Enoch, T., Zinn, K. & Maniatis, T. (1986) Mol. Cell. Biol. 6, activity of the human estrogen receptor was shown to be 801-810. affected by cation concentration and temperature (34). Since 24. Church, G. M. & Gilbert, W. (1984) Proc. Natl. Acad. Sci. estrogen receptors appear to exist as dimers in cell-free USA 8, 1991-1995. extracts (5, 15, 16), variations in the effect ofICI 164,384 may 25. Martinez, E. & Wahli, W. (1989) EMBO J. 8, 3781-3791. reflect differences in the stability of receptor dimers in cells. 26. Gottardis, M. M., Jiang, S. Y., Jeng, M. H. & Jordan, V. C. Recently it has been demonstrated that ICI 164,384 also (1989) Cancer Res. 49, 4090-4093. reduces the estrogen receptor content in mouse uterine tissue 27. Wakeling, A. E., Newboult, E. & Peters, S. W. (1989) J. Mol. (35), consistent with the observation that the antiestrogen Endocrinol. 2, 225-234. acts as a pure antiestrogen in the uterus in vivo (11, 12). 28. Thomson, E. W., Katz, D., Shima, T. B., Wakeling, A. E., ICI has Lippman, M. E. & Dickson, R. B. (1989) Cancer Res. 49, However, 164,384 been found to stimulate proges- 6929-6934. terone receptor protein levels in human endometrial Ishikawa 29. May, F. E. B., Johnson, M. D., Wakeling, A. E., Kastner, P. cancer cells in vitro (36). At the present time it is not known & Westley, B. R. (1989) J. Steroid Biochem. 33, 1035-1041. whether the partial agonist effect of ICI 164,384 is solely 30. Eckert, R. L., Mullick, A., Rorke, E. A. & Katzenellenbogen, receptor-mediated or depends on other transcription factors B. S. (1984) Endocrinology 114, 629-637. such as Jun (37). 31. Kiang, D. T., Killander, R. E., Thomas, T. & Kennedy, B. J. In conclusion, ICI 164,384 binding results in a reduction in (1989) Cancer Res. 49, 5312-5316. steady-state levels of estrogen receptor protein in transiently 32. Carey, M., Kakidani, H., Leatherwood, J., Mostashari, F. & expressing cells, human breast cancer cells, and mouse Ptashne, M. (1989) J. Mol. Biol. 209, 423-432. uterus The reduction is caused a the 33. Wilson, A. P. M., Weatherill, P. J., Nicholson, R. I., Davies, (35). by decrease in P. & Wakeling, A. E. (1990) J. Steroid Biochem. 35, 421-428. half-life of the receptor protein and is likely to be a general 34. Brown, M. & Sharp, P. A. (1990) J. Biol. Chem. 265, 11238- feature of the mechanism of action of ICI 164,384 in target 11243. cells. 35. Gibson, M. K., Nemmers, L. A., Beckman, W. C., Jr., Davis, V. L., Curtis, S. W. & Korach, K. S. (1991) Endocrinology We thank A. Wakeling (ICI Pharmaceuticals) for kindly supplying 129, 2000-2010. ICI 164,384 and K. Dingley and D. Barnes for performing the enzyme 36. Jamil, A., Croxtall, J. D. & White, J. 0. (1991) J. Mol. Endo- immunoassays. We also thank C. Dickson, J. White, and members crinol. 6, 215-221. of the Molecular Endocrinology Laboratory for advice and com- 37. Gaub, M.-P., Bellard, M., Scheuer, I., Chambon, P. & ments on the manuscript. Sassone-Corsi, P. (1990) Cell 63, 1267-1276. Downloaded by guest on September 29, 2021