Estrogen and rapamycin effects on cell cycle progression in T47D breast cancer cells.

The contribution of estrogen (and progesterone) in driving cell cycle progression of hormone dependent breast cancer cells is well documented, however, the roles of the various relevant signal transduction pathways remain unclear. The immunosuppressant rapamycin is a potent inhibitor of cell cycle progression and has been used to define signal transduction pathways. In this study we have determined rapamycin's effects on cell cycle progression in estrogen dependent breast cancer cells using a novel method of inducing S-phase. In this method estradiol-17-beta alone induced S-phase without mitogen support. In our studies the T47D cells were quite sensitive to estradiol-17-beta, with half-maximal induction in the picomolar range. indicating that the estrogen can induce S-phase in the absence of mitogens such as insulin. The estrogen response does not seem to be particularly specific because estriol estrone and estradiol-17-beta-BSA were about as effective as estradiol-17-beta. R5020, a progestin also induced S-Phase, while rapamycin blocked steroid driven transition of cells from G1 to S-phase.

Heat shock proteins 70 and 90 increase calcineurin activity in vitro through calmodulin-dependent and independent mechanisms.

We have shown that heat shock proteins (HSPs) associated with steroid receptor complexes are involved in the activation of calcineurin by aldosterone and dexamethasone. To determine whether HSPs directly interact with calcineurin, we measured the effect of HSPs 90, 70 and 56 on calcineurin activity in a cell-free, in vitro system using a calcineurin-specific substrate. HSP-90 (75 or 100 nM) significantly increased calcineurin V(max) in the presence of calmodulin, while maximal stimulation by HSP-70 occurred at 50 nM. Bovine serum albumin (BSA) and actin did not change basal calcineurin activity indicating that HSP-90 and HSP-70 specifically activate calcineurin. Neither HSP-70, HSP-56, nor ATP augmented HSP-90-induced activation of calcineurin. In the absence of calmodulin, HSP-90 restored calcineurin activity to basal levels while higher concentrations (333 and 500 nM) increased calcineurin activity. In contrast, HSP-70 failed to activate calcineurin activity in the absence of calmodulin. Immunoprecipitation of HSP-90 from in vitro mixtures as well as protein extracts from LLCPK-1 cells demonstrates that calcineurin co-precipitates with HSP-90. In summary: (1) HSP-90 and 70 stimulate calcineurin V(max) in vitro; (2) non-specific protein interactions do not activate calcineurin activity; (3) HSP-70 and HSP-56 do not enhance HSP-90-induced activation of calcineurin; (4) HSP-70 and HSP-90 activate calcineurin via a calmodulin-dependent and independent pathways; (5) Calcineurin co-precipitates with HSP-90 from LLCPK-1 cells as well as cell-free in vitro preparations.

Potentiation of progesterone receptor-mediated transcription by the immunosuppressant FK506.

The nontransformed steroid receptors contain several non-steroid binding proteins, such as hsp90, hsp70, and p59. Recently, we and others have shown that p59 (FKBP59) is an immunophilin which binds two potent immunosuppressants, FK506 and rapamycin. This raises the possibility that FK506 or rapamycin may modify the function of steroid receptors. To develop this line of inquiry, we chose a yeast model system in which the human progesterone receptor form B (hPR-B) was cotransformed with a reporter gene. The reporter contains two copies of a progesterone response element/glucocorticoid response element (PRE/GRE) upstream of the CYC1 promoter which are linked to the lacZ gene of Escherichia coli. We found that FK506 potentiated the ability of progesterone in activating transcription. To gain insight into the mechanism of FK506's regulation of PR action, we questioned whether calcineurin is involved, because it has been shown that FK506 is a specific inhibitor of calcineurin, a Ca(2+)- and calmodulin-regulated phosphatase, through the formation of an FKBP12-FK506-calcineurin-calmodulin complex. We found that 15-O-desmethyl-FK520, an FK506 analogue which is an excellent ligand of FKBP12, but a poor inhibitor of calcineurin, failed to induce the same effect as FK506. We also found that calmidazolium, a calmodulin antagonist, mimicked FK506's action. Furthermore, immunoblot analysis showed that both FK506 and calmidazolium potentiated the effect of progesterone in decreasing the mobility of hPR-B upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This suggests that FK506 and calmidazolium may cooperate with progesterone in increasing the level of hPR-B phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

P59 (FK506 binding protein 59) interaction with heat shock proteins is highly conserved and may involve proteins other than steroid receptors.

P59 [also known as FK506 binding protein 59 (FKBP59) or heat shock protein 56 (hsp56)] and heat shock proteins 90 and 70 (hsp90 and hsp70) associate with steroid receptors and are believed to maintain the receptors in an inactive state. Recently, we showed that p59 purified from human lymphocytes is an immunophilin (FKBP59) which binds both FK506 and rapamycin. It was also demonstrated that immunosuppressant-FKBP59 complexes associate with hsp90, hsp70, and the glucocorticoid receptor [Tai, P.-K. K., Albers, M. W., Chang, H., Faber, L. E., & Schreiber, S. L. (1992) Science 256, 1315-1318]. Here we provide evidence that rabbit uterine p59 also binds FK506 and rapamycin and that p59 or its homologue is associated with nontransformed progesterone receptors of rabbit uterus and chicken oviduct. This suggests that the immunophilin-heat shock protein-steroid receptor interaction is ubiquitous and not limited to immune systems. A FKBP59 homologue complexed with hsp90-hsp70 was also detected in yeast, which suggests that the immunophilin-heat shock protein association has been evolutionarily conserved. In addition, we found that the FKBP59-hsp complexes are more complicated than previously thought, involving other proteins such as actin and a 63-kDa protein, p63. The association of p63 to the p59 complex was inhibited by FK506 and rapamycin, suggesting that p63 could be a potential target for the immunosuppressive actions of these two drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Rabbit FKBP59-heat shock protein binding immunophillin (HBI) is a calmodulin binding protein.

FKBP59-HBI, a heat shock protein hsp90-binding immunophilin that was originally detected in heterooligomer forms of steroid receptors, is retained on Calmodulin (CAM)-Sepharose 4B in the presence of 2 mM Ca2+ and is eluted by EGTA, demonstrating a specific p59-CAM interaction. The p59 amino acid sequence reveals the presence of two putative CAM binding sites in a helix regions of the protein, as well as PEST sequences which are generally present in CAM-binding proteins. In vitro proteolysis by calpain II (a Ca(2+)-activated neutral protease), another feature of CAM-binding proteins, generates shorter peptides revealed by the mAb EC1, but not by the pAb 173 which recognizes the C-terminal of the protein. The potential function of CAM binding by the hsp90-binding immunophilin is discussed.

Association of a 59-kilodalton immunophilin with the glucocorticoid receptor complex.

Immunophilins, a family of proteins that exhibit rotamase (peptidyl-prolyl cis-trans isomerase) activity in vitro, are expressed in many organisms and most tissues. Although some immunophilins can mediate the immunosuppressive actions of FK506, rapamycin, and cyclosporin A, the physiological role of the unligated proteins is not known. A 59-kilodalton member of the FK506- and rapamycin-binding class was found to associate in the absence of these drugs with two heat shock proteins (hsp90 and hsp70) and the glucocorticoid receptor (GR). Together, these proteins make up the inactive GR, thus biochemically linking two families of proteins proposed to be involved in protein folding and assembly as well as two potent immunosuppressive modalities.

P59, an hsp 90-binding protein. Cloning and sequencing of its cDNA and preparation of a peptide-directed polyclonal antibody.

The primary sequence of the rabbit liver cDNA coding for protein p59 has been determined. The protein binds to the 90-kDa heat shock protein (hsp 90) and is associated with it, including when hsp 90 participates in hetero-oligomeric complexes of untransformed mammalian steroid receptors that sediment at 8-10 S. The cloned cDNA codes for an open reading frame of 458 amino acids defining a yet unknown protein. However, 55% amino acid homology to peptidyl-prolyl isomerase is found between amino acids 41 and 137, suggesting rotamase activity for p59, which speculatively may apply to bound hsp 90 and thus be implied in the intracellular trafficking of hetero-oligomeric forms of steroid hormone receptors. A polyclonal antibody derived from the COOH-terminal peptide 441-458 demonstrates a good affinity for rabbit, rat, and human "p59" protein. It interacts with at least one epitope, available in 8-10 S untransformed steroid receptor complexes and different from that recognized by the monoclonal antibody KN382/EC-1.

Cytosolic and nuclear progesterone receptors in normal and luteal-phase-defect endometrial cells.

Endometrial biopsies were taken from 93 infertility patients registered in the Reproductive Endocrinology Clinic at the Medical College of Ohio. These endometrial specimens were assayed for nuclear and cytoplasmic progesterone receptors. Only the data from 53 normoprolactinemic euthyroid and nonhirsute patients were considered. Eighteen patients were found to have luteal phase defect by two consecutive out-of-phase biopsies. The remaining 35 patients had in-phase biopsies and were used as controls. Both groups were similar in age and clinical characteristics. Although we noted that the luteal-phase-defect group had fewer nuclear and cytoplasmic progesterone receptors than did the control group, the difference was not statistically significant.

The nonactivated progesterone receptor is a nuclear heterooligomer.

The discovery of the nuclear localization of estradiol and progesterone receptors in the absence of the steroid hormone has led to reconsideration of the model of cytoplasmic to nuclear translocation of these receptors upon exposure to hormone. Unoccupied nonactivated receptors are thought to be weakly bound to nuclei of target cells from which they are leaking during tissue fractionation and thus found in the cytosol fraction of homogenates in a nontransformed heterooligomeric "8-9 S" form, which includes hsp90. However, no direct biochemical evidence has yet been obtained for the presence of such heterooligomers in the target cell nucleus, possibly because it dissociates in high ionic strength medium used for extraction of the nuclear receptor. We took advantage of the combined stabilizing effects of tungstate ions and antiprogestin RU486 to extract a nuclear non-DNA binding nontransformed 8.5 S-RU486-progesterone receptor complex from estradiol-treated immature rabbit uterine explants incubated with the antagonist. As demonstrated by immunological criteria and by irreversible cross-linking with dimethylpimelimidate, the complex contained, in addition to the hormone binding unit, hsp90, and p59, another nonhormone binding protein. Control experiments carried out with the progestin R5020 yielded the expected nuclear transformed DNA binding 4.5 S-R5020-progesterone receptor complex. These results offer evidence for two distinct forms of steroid receptor in target cell nuclei. Besides the classical "4 S" agonist-receptor complex, tightly bound to the DNA-chromatin structure and in all probability able to trigger the hormonal response, we have observed in the RU486-bound state a non-DNA binding nontransformed 8.5 S form, presumably already present in the nucleus in the absence of hormone and representing the native nonactive form of the receptor.

The non-DNA-binding heterooligomeric form of mammalian steroid hormone receptors contains a hsp90-bound 59-kilodalton protein.

Untransformed cytosol receptors for progesterone (PR), androgen (AR), estrogen (ER), and glucocorticosteroid (GR) in rabbit tissues contain a 59-kDa protein (p59) (Tai, P.K.K., Maeda, Y., Nakao, K., Wakim, N.G., Duhring, J.L., and Faber, L.E. (1986) Biochemistry 25, 5269-5275) and a 90-kDa heat shock protein (hsp90). In the present study, receptors from calf uterus (PR, AR, ER, and GR) and from human breast cancer MCF7 cells (PR and GR) were also shown to be comprised of hsp90 and p59. These heterooligomer receptor complexes were stabilized both by transition metal oxyanions (molybdate and tungstate) and chemical cross-linking with dimethylpimelimidate. In 0.4 M KCl, tungstate-stabilized (but not molybdate-stabilized) PR, AR, ER, and GR retained hsp90, but lost p59. Dimethylpimelimidate cross-linking prevented p59 dissociation from hsp90-receptor complexes. Stabilization with tungstate and/or cross-linking permitted immunoaffinity purification of untransformed rabbit as well as calf PR and ER on EC1-Affi-Gel 10 column (an anti-p59 immunoadsorbant). Combined immunoaffinity purification and cross-linking experiments indicated that p59 is bound to hsp90 in the cytosol. We propose that in the nontransformed steroid receptor, p59 interacts with hsp90 rather than with the hormone binding subunit.

The 56-59-kilodalton protein identified in untransformed steroid receptor complexes is a unique protein that exists in cytosol in a complex with both the 70- and 90-kilodalton heat shock proteins.

It has previously been shown that 9S, untransformed progestin, estrogen, androgen, and glucocorticoid receptor complexes in rabbit uterine and liver cytosols contain a 59-kDa protein [Tai, P. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., & Faber, L. E. (1986) Biochemistry 25, 5269-5275]. In this work we show that the monoclonal antibody KN 382/EC1 raised against the rabbit 59-kDa protein reacts with 9S, untransformed glucocorticoid receptor complexes in cytosol prepared from human IM-9 lymphocytes but not with 4S salt-transformed receptors. The human protein recognized by the EC1 antibody is a 56-kDa protein (p56) of moderate abundance located predominantly in the cytoplasm by indirect immunofluorescence. There are at least six isomorphs of p56 by two-dimensional gel analysis. N-Terminal sequencing (20 amino acids) shows that p56 is a unique human protein. When p56 is immunoadsorbed from IM-9 cell cytosol, both the 70- and 90-kDa heat shock proteins are coadsorbed in an immune-specific manner. Neither heat shock protein reacts directly with the EC1 antibody. We conclude that p56 exists in cytosol in a higher order complex containing hsp70 and hsp90, both of which in turn have been found to be associated with untransformed steroid receptors.

Purification of unactivated progesterone receptor and identification of novel receptor-associated proteins.

Progesterone receptor complexes were purified from crude cytosol in a rapid, gentle, one-step procedure using anti-receptor monoclonal antibodies covalently attached to an agarose resin. Five nonreceptor proteins specifically co-purified with unactivated avian progesterone receptor; these proteins had molecular masses of approximately 90, 70, 54, 50, and 23 kDa. The 90- and 70-kDa proteins have been previously identified as the 90-kDa heat shock protein and a member of the 70-kDa heat shock protein family, respectively. The 54-, 50-, and 23-kDa proteins have not been previously described as associated with avian progesterone receptor. Two-dimensional gel electrophoresis revealed charge heterogeneities for all five proteins. Except for p70, each could be dissociated from receptor by salt, a process inhibited by sodium molybdate. However, molybdate was not required for protein association with receptor in low ionic strength. Following progesterone treatment in vivo p70 still co-purified with cytosolic receptor although the other affiliated proteins were reduced, suggesting hormone-dependent dissociation in conjunction with receptor activation. One of the proteins, p54, displayed in vitro hormone-dependent dissociation which was not prevented by molybdate.

Nuclear localization of two steroid receptor-associated proteins, hsp90 and p59.

It has been proposed that the unliganded nontransformed form of steroid hormone receptor is a heterooligomer comprising, in addition to the hormone-binding subunit, two associated proteins: a heat shock protein of MW 90,000 (hsp90) and another protein of MW 59,000 (p59). Using monoclonal antibodies, we demonstrate immunocytochemically the presence of both hsp90 and p59 in cell nuclei of progesterone target cells of the rabbit uterus. While steroid receptors (e.g., progesterone receptors) appear to be exclusively nuclear, we find p59 predominantly in the cell nuclei and hsp90 in both the nucleus and the cytoplasm. In addition, Western blotting of high-salt extracts of nuclear proteins detects the presence of hsp90 and p59 in the nuclei of rabbit uterus. These observations are consistent with the presence of the untransformed heterooligomeric form of steroid hormone receptors in the nuclei of target cells.

Characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin: use of chemical crosslinking and a monoclonal antibody directed against a 59-kDa protein associated with steroid receptors.

The Ah receptor regulates induction of cytochrome P450IA1 (aryl hydrocarbon hydroxylase) by "3-methylcholanthrene-type" compounds and mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons. Hepatic Ah receptor from untreated rodents is localized in the cytosol and has an apparent molecular mass of 250 to 300 kDa. This large form can be dissociated into a smaller ligand-binding subunit upon exposure to high ionic strength. The Ah receptor displays many structural similarities to the receptors for steroid hormones. Two non-ligand-binding proteins have been identified to be associated with the cytosolic forms of the steroid hormone receptors. The first is a 90-kDa heat shock protein (hsp 90); the second is a 59-kDa protein (p59) of unknown function. The cytosolic Ah receptor ligand-binding subunit previously has been shown to be associated with hsp 90. In the present study, we used a monoclonal antibody, KN 382/EC1, generated against the 59-kDa protein which is associated with rabbit steroid receptors to determine if p59 also is a component of the large cytosolic Ah receptor complex. Cytosolic forms of rabbit progesterone receptor, glucocorticoid receptor, and Ah receptor were analyzed by velocity sedimentation on sucrose gradients under low-ionic-strength conditions and in the presence of molybdate. Progesterone receptor from rabbit uterine cytosol and glucocorticoid receptor from rabbit liver each had a sedimentation coefficient of approximately 9 S. In the presence of KN 382/EC1 antibody the progesterone receptor and the glucocorticoid receptor both underwent a shift in sedimentation to a value of approximately 11 S. The increase in sedimentation velocity is an indication that the receptor-protein complexes are interacting with the antibody. Under low-ionic-strength conditions the Ah receptors from rabbit uterine cytosol and liver cytosol had a sedimentation coefficient of approximately 9 S. However, in contrast to the steroid receptors, the Ah receptor showed no change in its sedimentation properties in either tissue in the presence of KN 382/EC1, indicating that the antibody is not interacting with the Ah receptor. Multimeric Ah receptor complexes that were chemically crosslinked still did not show any interaction with KN 382/EC1. These data indicate that the 59-kDa protein either is not associated with the Ah receptor or is present in an altered form which the antibody cannot recognize.

The 56 kDa protein of human genital skin fibroblasts is identical to that radiolabelled by [3H]dihydrotestosterone 17 beta-bromoacetate.

Analysis of soluble proteins from human genital skin fibroblasts by two-dimensional polyacrylamide gel electrophoresis reveals an abundant protein doublet of mol. wt 56,000 with isoelectric points (pI) of 6.7 and 6.5. This protein is absent in non-genital skin fibroblasts as well as in genital skin fibroblasts of most patients with complete forms of androgen insensitivity. The protein specifically binds androgen. A protein of similar estimated molecular weight (58,000) from human genital skin fibroblasts has recently been found to be covalently radiolabelled by the affinity ligand dihydrotestosterone 17 beta-bromoacetate (DHT-BA). In the present study these proteins have been found to be indistinguishable on one- and two-dimensional gel electrophoresis. Antibodies raised against the 56 kDa pI 6.7/6.5 protein also recognized the protein covalently radiolabelled by DHT-BA. A third protein of estimated mol. wt 59,000 has been found to be associated with several steroid hormone receptor complexes but has no known ligand binding activity. This protein was found to be clearly separable from the 56/58 kDa protein on two-dimensional gel electrophoresis as it has a more acidic pI of approximately 5.4. Furthermore, antibodies against the 59 kDa protein do not recognize the 56 kDa species, and vice versa.

Evolving concepts in the mechanism of steroid action: current developments.

The mechanisms of steroid action remain a poorly understood enigma. Although much effort has focused on the steroid receptor as a mediator of the steroid's effect in the cell, we are only beginning to understand the structure of steroid receptors. Development of monoclonal antibodies directed against both the steroid-binding "receptors" and receptor-associated proteins has allowed novel approaches to the problem. They were important in determining the nucleotide sequences of several receptor genes and subsequently the amino acid sequence of three receptors. Surprisingly, receptors contain amino acid sequences common to v-erb-A, a potentiator of oncogenic transformation. Two receptor-associated proteins have been found and their relationship to the receptors suggests the possibility of additional functions of receptors in addition to binding deoxyribonucleic acid. Thus the role of the receptor in the mechanism of steroid action is evolving from the "two-step mechanism" to one that includes the recently discovered receptor-associated proteins.

A 59-kilodalton protein associated with progestin, estrogen, androgen, and glucocorticoid receptors.

Previous studies of the anti 8.5S progestin receptor monoclonal antibody KN 382/EC1 showed that it was specific for nontransformed progestin receptors. However, with different methods of tissue disruption and the use of protease inhibitors, we found that other nontransformed steroid receptors formed immune complexes with KN 382/EC1. Binding of the antibody to rabbit uterine estrogen, progestin, and androgen and liver glucocorticoid receptor systems was characterized by sucrose density gradient centrifugation, high-pressure liquid chromatography (HPLC), immunoadsorption, and immunoblotting. Immobilized KN 382/EC1 adsorbed both Mr 59,000 and Mr 92,000 proteins. The Mr 92,000 protein appeared to be bound to the antigenic Mr 59,000 protein, and the two proteins were present in apparently the same stoichiometric relationship in several tissues. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoadsorbed material revealed appreciable amounts of both proteins in testis, stomach, lung, liver, uterus, and kidney. Only trace amounts were found in skeletal or heart muscle, and none was found in blood serum. Cleveland digestion of isolated Mr 59,000 and 92,000 proteins revealed dissimilar peptide constituents. Immunoblots of material from uterus and liver resulted in staining of the Mr 59,000 protein but not the Mr 92,000 protein. We conclude that similar antigenic determinants reside in components of several nontransformed steroid receptors and they reside on an Mr 59,000 protein. It is likely, therefore, that there are common components present in nontransformed steroid receptors.

Isolation of dissimilar components of the 8.5S nonactivated uterine progestin receptor.

Sucrose gradient analysis of the binding of our monoclonal antibody (secreted by cell line KN 382/ECl) to [17 alpha-methyl-3H] R5020-labeled rabbit uterine progestin receptors revealed that the antibody bound only to the 8.5S form (Kd = 0.86 nM) and not to the 7S and smaller complexes. 125I-labeled antibody, on the other hand, bound to both the 8.5S complex and a component of dissociated receptor. Calculation of the relative mass (Mr) of the 125I-labeled immunoglobulin G1 (IgG1)-protein complex indicated the addition of a 60 000 Mr peptide. Electrophoretic analysis of immunoaffinity purified receptor substantiated this by revealing two protein bands (Mr approximately equal to 92 000 and Mr approximately equal to 59 000). Sequential washing of adsorbed receptor was accompanied by dissociation of the bound steroid and the Mr 92 000 peptide. The Mr approximately equal to 59 000 peptide could only be completely eluted from the immunoadsorbent under denaturing conditions. Autofluorography of receptor complexes covalently bound with [17 alpha-methyl-3H]R5020 revealed two bands, one with a Mr approximately equal to 116 000 and the second with a Mr approximately equal to 90 000. Upon immunoprecipitation both peptides precipitated with the Mr approximately equal to 92 000 and Mr approximately equal to 59 000 peptides. Gel electrophoresis demonstrated that the Mr approximately equal to 92 000 peptide and the Mr approximately equal to 90 000 did not comigrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of a monoclonal antibody to the rabbit 8.5S uterine progestin receptor.

Nonactivated (8.5S) rabbit uterine progestin receptor was enriched 10- to 30-fold by chromatography on columns of spheroidal hydroxylapatite and DEAE-cellulose. A total of approximately 25 micrograms of receptor (purity approximately 1%) was injected at multiple sites into a BALB/c mouse. After several injections, splenic lymphocytes were fused with P3x63Ag8.653 mouse myeloma cells. This fusion produced in excess of 240 hybridomas, which were screened by an enzyme-linked immunosorbent assay (ELISA), solid-phase radioimmunoassay, and sucrose gradient centrifugation. One colony (KN 382/EC1) produced a mouse immunoglobulin G1 which bound rabbit 8.5S uterine progestin receptor. The cell line has been repeatedly cloned under conditions of limiting dilution and expanded in mice as ascitic tumors. Antibody was purified by (NH4)2SO4 precipitation, DEAE-cellulose chromatography, and affinity chromatography with protein A - Sepharose CL-4B. Specificity of the antibody was determined by sucrose gradient centrifugation and solid-phase radioimmunoassay. The antibody bound to progestin receptors from rabbit uterus and MCF-7 breast cancer cells. It did not react with progestin receptors from rat uterus, guinea pig uterus, or chick oviduct, nor did it bind to estrogen receptors from any of the tissues we tested.

Tamoxifen decreases progesterone and nuclear androgen receptors in the human prostate.

Androgen (AR) and progesterone (PR) receptors were measured in resected prostate tissues of patients with benign prostatic hypertrophy. One group of patients received an anti-estrogen, tamoxifen (Tm 20 mg b.i.d.) for 10 days prior to prostate resection; a second group served as controls and were untreated. Plasma levels of Tm were 200-500 pmol/ml at the time of surgery. Statistically significant decreases (P less than 0.05) were found in cytosol PR (154 fmol/mg DNA +/- 33 SE in 14 Tm-patients vs 266 +/- 40 SE in 13 untreated patients) and in nuclear AR (103 fmol/mg DNA +/- 70 SE in 18 Tm-patients vs 257 +/- 62 SE in 17 controls). Cytosol AR was not significantly different in Tm-treated patients (257 fmol/mg DNA +/- 79 SE in 15 Tm-patients vs 346 +/- 130 SE in 17 controls, P greater than 0.6). Although receptor recycling is one of several possible explanations, these decreases in progesterone and nuclear androgen receptors in Tm-treated patients suggest that estrogen has a role in the biological regulation of steroid receptors in the human prostate.