Selective and opposing actions of progesterone receptor isoforms in human endometrial stromal cells.

Transcriptional regulation by progesterone is mediated primarily through the two progesterone receptor (PR) isoforms, PR-A and PR-B. Primary human endometrial stromal cell cultures, in which endogenous PR expression was lost, were infected with adenovirus expressing PR-A, PR-B, or both. Global gene expression analysis was conducted on vehicle and 30 nM progesterone (P4) treated cells following 12 h treatment. Interestingly, many genes regulated by PR-B alone or upon PR-A and PR-B co-expression, did not overlap with each other or with the PR-A expression group. Although many genes known to be progestin regulated in the uterus in vivo were also regulated in this study, markedly little overlap with published P4 regulated genes from human breast cancer cells was observed. Progesterone dose response curves were generated for several genes demonstrating gene selective potency and efficacy for each PR isoform. Furthermore, the PR isoforms opposed each other in regulation of tissue factor, with PR-B increasing and PR-A decreasing both mRNA and protein levels. Our data provide a view of global gene expression by PR isoforms in human endometrial cells and a comparison with other cell types. The specific genes and regulation patterns found provide groundwork to revealing the mechanism of PR isoform selectivity, and perhaps ultimately to the tissue selective properties these receptors appear to exhibit.

Effects of 17 beta-estradiol and medroxyprogesterone acetate upon MtTW15 mammosomatotropic pituitary tumor growth and hormone production in male and female rats.

The purpose of this study was to characterize the effects of two functionally diverse steroids, 17 beta-estradiol and medroxyprogesterone acetate (MPA), on MtTW15 rat mammosomatotropic pituitary tumor growth and hormone production. Steroid responsiveness, as well as the hormonally autonomous nature of the tumor, was studied by treating both male and female tumor-bearing rats for 7 weeks with weekly injections of either 17 beta-estradiol (600 ng/g body weight/week) or MPA (200 microgram/g body weight/week) and, subsequently, comparing both the tumor weights and the in vivo production of growth hormone (GH) and prolactin (PRL) among the treatment groups. Large tumors (6 to 20 gm) were obtained in all treatment groups, indicating hormonal autonomy; however, tumors were markedly smaller, on the average, in untreated males an ovariectomized females. Treatment of such rats with 17 beta-estradiol stimulated tumor growth. Radioimmunoassay of tumor and serum GH and PRL levels in all treatment groups indicated the following: (a) tumors from untreated male or female hosts did not favor the production of one hormone over the other to any great extent; (b) MPA, however, promoted significant increases (p less than 0.05) in GH production in both male and female tumor-bearing rats while having little effect on the production of PRL; and (c) 17 beta-estradiol significantly inhibited (p less than 0.05) GH production and promoted PRL production by tumors borne by either sex. Selected studies utilizing multiple doses of MPA (1 to 500 microgram per gm body weight per week) and 17 beta-estradiol (10 to 800 ng per gm body weight per week) were accomplished and demonstrated that hormone production can be influenced in a dose-related manner. These results indicated that the estrogen-induced MtTW15 rat pituitary tumor is hormonally autonomous, yet divergently responsive to two different classes of steroidal compounds, thus making this tumor line an appropriate model for the study of hormonally responsive pituitary tumor cells.

PIP: The purpose of this study was to characterize the effects of 2 functionally diverse steriods, 17beta-estradiol and (MPA) medroxyprogesterone acetate on MtTW15 rat mammosomatotropic pituitary tumor growth and hormone production. Steroid responsiveness, as well as the hormonally autonomous nature of the tumor, was studied by treating both male and female tumor-bearing rats for 7 weeks with weekly injections of either 17beta-estradiol (600 ng/g body weight/week) or MPA (200 mcg/g body weight/week) and, subsequently, comparing both the tumor weights and the in vivo production of (GH) growth hormone and (PRL) prolactin among the treatment groups. Large tumors (6 to 20 gm) were obtained in all treatment groups, indicating hormonal autonomy; however, tumors were markedly smaller, on the average, in untreated males and ovariectomized females. Treatment of such rats with 17beta-estradiol stimulated tumor growth. Radioimmunoassay of tumor and serum GH and PRL levels in all treatment groups indicated the following: (a) tumors from untreated male or female hosts did not favor the production of 1 hormone over the other to any great extent; (b) MPA, however, promoted significant increases (p 0.05) in GH production in both male and female tumor-bearing rats while having little effect on the production of PRL; and (c) 17-estradiol significantly inhibited (p 0.05) GH production and promoted PRL production by tumors borne by either sex. Selected studies utilizing multiple doses of MPA (1 to 500 mcg/gm body weight/week) and 17 beta-estradiol (10 to 800 ng/gm body weight/week) were accomplished and demonstrated that hormone production can be influenced in a dose-related manner. There results indicated that the estrogen-induced MtTW15 rat pituitary tumor is hormonally autonomous, yet divergently responsive to 2 different classes of steroidal compounds, thus making this tumor line an appropriate model for the study of hormonally responsive pituitary tumor cells.

Estrogenic stimulation of the antiestrogen specific binding site in rat uterus and liver.

Tamoxifen and other antiestrogens bind to the estrogen receptor and to a specific antiestrogen binding site (AEBS). We have confirmed the existence of an AEBS by saturation and competitive binding analyses with [3H]tamoxifen as the radiolabeled ligand. The quantities of AEBS (pmol/g tissue) present in a low speed cytosol (25,000 X g X 30 min) of the liver and uterus vary with species and physiological state: liver, mature female rat, 82.7 +/- 6.7; ovariectomized mature rat, 46.1 +/- 2.9; immature female rat, 40.0 +/- 1.8; mature male rat, 57.3 +/- 2.7; and mature female mouse, 63.0 +/- 5.0; uterus, mature rat, 17.9 +/- 1.7; ovariectomized mature rat, 13.3 +/- 0.5; immature rat, 8.2 +/- 0.8; and mature mouse, 49.0 +/- 2.1. The dissociation constants did not differ significantly in any of the cases and were 1.5 and 2.5 nM for the liver and uterus, respectively. Developmental studies demonstrated that the liver AEBS gradually increases in both sexes from 21.0 pmol/g at birth to their respective adult levels with the only divergence occurring approximately at the time of puberty. The AEBS concentration also varied with the stage of the estrous cycle [highest at estrus (20.6 +/- 1.8) and metestrus (91.7 +/- 5.9) for the uterus and liver, respectively, and lowest at diestrus (uterus, 15.9 +/- 0.2; liver, 60.2 +/- 4.1)]. Furthermore, estradiol treatment of ovariectomized rats significantly increased both liver and uterine AEBS levels, whereas tamoxifen increased uterine AEBS concentration although having no significant effect on the level of these sites in liver. These results demonstrate the presence of a cytosolic AEBS in the liver and uterus which is distinct from the estrogen receptor and appears to be under estrogenic regulation.

Glucocorticoid-like actions of medroxyprogesterone acetate upon MtTW15 rat mammosomatotropic pituitary tumors.

We have shown recently that the MtTW15 experimental rat pituitary tumor responds to medroxyprogesterone acetate (MPA) by increasing GH production. In this study, using a single saturating dose assay and dextran-coated charcoal separation, the data indicate that the MtTW15 tumor contains cytosolic MPA binding sites. The concentration of sites (approximately 4 pmol/g tumor) was similar for tumors derived from male or female hosts but was significantly reduced in tumors from MPA- or estradiol-treated rats. The MPA binder sedimented to 4S on low salt sucrose density gradients and was of high affinity (Kd = 5.0 +/- 0.4 X 10(-9) M). However, binding specificity studies showed glucocorticoids to be better ligands than MPA. MtTw15 tumors were also analyzed for cytosolic progestin ([3H]R5020) and glucocorticoid ([3H]dexamethasone) binding sites. Only low levels of an estradiol-inducible progestin binder were found. In contrast, the concentration of glucocorticoid binding sites was similar to that observed for MPA, approximately 4 pmol/g tumor, as were the characteristics of the binding, i.e. 5.1 +/- 0.2S, Kd = 4.1 +/- 0.5 X 10(-9) M, and similar binding specificities. Both MPA and estradiol treatment of tumor-bearing rats decreased the concentration of both MPA and glucocorticoid binding sites. Furthermore, studies to determine if glucocorticoids would mimic the in vivo effect of MPA upon MtTW15 tumors, i.e. altered tumor hormone production, supported such a hypothesis. We conclude that the MtTW15 rat pituitary tumor contains a cytosolic glucocorticoid receptor and that MPA can interact with this receptor. The glucocorticoid receptor may be responsible for the MPA- and glucocorticoid-induced alterations in GH production.

Cyr61, a member of the CCN family, is required for MCF-7 cell proliferation: regulation by 17beta-estradiol and overexpression in human breast cancer.

Cyr61, a member of the CCN (CTGF/Cyr61/NOV) family of growth regulators, is a secreted cysteine-rich proangiogenic factor that has been implicated in tumorigenesis. Previous studies have also demonstrated that Cyr61 is regulated by 17beta-estradiol (E(2)) in the uterus. Therefore, we hypothesized that hormonal regulation of Cyr61 may be important in estrogen-dependent pathogenic processes such as breast tumorigenesis. Our study demonstrates that both Cyr61 messenger RNA and protein are induced by E(2) in MCF-7 mammary adenocarcinoma cells that primarily overexpress estrogen receptor alpha (ERalpha) in a dose-dependent and immediate early fashion. Cyr61 gene induction by E(2) is transcriptionally regulated by ERalpha as the antiestrogen, ICI 182,780, and actinomycin D blocked induction completely. In addition, Cyr61 is up-regulated in MCF-7 cells by epidermal growth factor (EGF) in an immediate early fashion as well. The functional relevance of steroid induction of Cyr61 in breast cancer cell growth is demonstrated by anti-Cyr61 neutralizing antibodies, which diminished E(2) and EGF-dependent DNA synthesis and dramatically reduced E(2)-driven cell proliferation by more than 70%. Most importantly, Cyr61 is overexpressed in 70% (28 of 40) of breast cancer patients with infiltrating ductal carcinoma and is localized exclusively to hyperplastic ductal epithelial cells. Moreover, the levels of Cyr61 protein are higher in breast tumors that are ER(+)/EGF receptor(+) than those that are ER(-)/EGF receptor(+), suggesting that estrogens may mediate Cyr61 expression in vivo. Collectively, our data suggest that Cyr61 may play a critical role in estrogen- as well as growth factor-dependent breast tumor growth.

Characterization of a triphenylethylene-antiestrogen-binding site on rat serum low density lipoprotein.

Several laboratories have reported the presence of an intracellular, high affinity [dissociation constant (Kd), 1-2 nM] antiestrogen-specific binding site in estrogen target tissues. In this report, we describe the binding properties and characteristics of an additional triphenylethylene antiestrogen-binding site in the serum of rats. By using saturation and competitive binding analyses with [3H]tamoxifen as the radio-labeled ligand, we have determined that rat serum contains a relatively high affinity (Kd, 28 nM), protease-sensitive binding site that is specific for antiestrogens of the triphenylethylene type (clomiphene and nafoxidine). Since this serum site has little affinity for the benzythiophene antiestrogens (LY 117018 and 156758), we have chosen the name of triphenylethylene-antiestrogen binding site (TABS). The concentration of serum TABS (picomoles of [3H]tamoxifen bound per ml serum) is roughly the same in males and females but is significantly greater in both sexes at days 5, 10, and 15 of age (400-500 pmol/ml) than in newborn or adult animals (100-200 pmol/ml). Preliminary characterization studies indicated that the serum TABS is probably a serum lipoprotein. Separation of rat serum lipoproteins on potassium bromide density gradients revealed that the serum TABS migrated with rat low density lipoprotein (LDL). Furthermore, rat LDL purified by density gradient centrifugation has an affinity and specificity for [3H]tamoxifen that is similar to the TABS found in whole serum. In contrast, the rat liver intracellular TABS does not have these density gradient characteristics. These data suggest that the rat serum TABS is LDL; however, the role of this serum lipoprotein in the mechanism of action of antiestrogens remains to be determined.

Antiandrogens do not reverse androgen-induced inhibition of sex hormone-binding globulin levels in adult female rhesus monkeys.

Studies were done to determine if changes in plasma sex hormone-binding globulin (SHBG) levels could serve as a specific marker of androgenic and antiandrogenic activities in rhesus monkeys. Treatment of adult female monkeys for 11 days with 17 alpha-methyltestosterone (MeT) produced dose and time-dependent reductions in SHBG levels. However, the non-steroidal antiandrogen flutamide (80 mg/monkey.day) did not inhibit the reduction in SHBG levels when coadministered with MeT, nor did it have an effect on SHBG levels when given alone. In contrast, the steroidal antiandrogens Win 49596 (100 mg/monkey.day) and cyproterone acetate (80 mg/monkey.day) significantly (P less than 0.01) reduced SHBG plasma concentration to about 50% of pretreatment control values whether given alone or in combination with MeT. Furthermore, Win 49596 reduced SHBG levels at doses as low as 4 mg/monkey, whereas cortico-steroid-binding globulin levels were not affected. In ovariectomized monkeys, MeT treatment (4 mg/monkey.day for 15 days) reduced plasma SHBG levels to 42% of pretreatment values and delayed the onset of withdrawal menstrual bleeding compared to that in controls. When administered concurrently with MeT, flutamide (100 mg/monkey.day) antagonized the effect on withdrawal bleeding, but was without effect on SHBG levels. Therefore, plasma SHBG levels cannot be used as a specific indicator of androgenic or antiandrogenic activity and may not be regulated through the classical androgen receptor.

Characterization of the ovariectomized rat model for the evaluation of estrogen effects on plasma cholesterol levels.

Estrogens protect against cardiovascular disease in women through effects on the vascular wall and liver. Here we further characterize the rat as a model for the evaluation of estrogenic effects on plasma lipid levels vs. uterine wet weight. In adult ovariectomized female rats treated for 4 days s.c., 17alpha-ethinyl estradiol (EE) was the most potent agent to lower plasma total and high density lipoprotein cholesterol levels, followed by 17beta-estradiol and 17alpha-estradiol. However, 17alpha-estradiol had the greatest separation of uterotropic vs. cholesterol-lowering effects. EE had the same lipid-lowering potency whether administered s.c. or orally to adult rats. It had no effect on cholesterol levels in immature rats, even though the uterotropic response was dramatic. Testosterone propionate, dexamethasone, and progesterone did not significantly lower cholesterol levels. The antiestrogens tamoxifen and raloxifene lowered cholesterol levels, but with less efficacy and potency than the estrogens. ICI 182780 had no effect on cholesterol levels. When coadministered with EE, ICI 182780 inhibited the cholesterol-lowering and uterotropic activities of EE, suggesting that the estrogen receptor pathway is involved. In conclusion, although the information from the rat is limited as a model of the low density lipoprotein-lowering effects of estrogens in humans, it can be used to study the effects and mechanism of action of estrogen and antiestrogens on plasma cholesterol levels.

An endogenous ligand for the triphenylethylene antiestrogen binding site.

We have isolated an endogenous ligand which acts as a competitive inhibitor of the binding of 3H-tamoxifen to triphenylethylene antiestrogen-binding sites (TABS) prepared from liver and from serum low density lipoproteins (LDL). This ligand is present in boiled ethanol extracts of rat liver and may represent an "endogenous antiestrogen". "Endogenous antiestrogen" is used here as an operational term, since it has not been shown that TABS are involved in the mechanism of action of the triphenylethylene antiestrogens.

The effect of the steroidal androgen receptor antagonist, Win 49,596, on the prostate and testis of beagle dogs.

The effect of the steroidal androgen receptor antagonist Win 49,596 on the prostate and testis was studied in beagle dogs and was compared to the effects of the nonsteroidal androgen receptor antagonist ICI 176,334 and the steroidal 5 alpha-reductase inhibitor MK-906. Win 49,596 was shown to bind to the androgen receptor from normal canine prostate with a Ki of 2.2 microM. After 16 weeks of treatment, prostate size, as estimated by transrectal ultrasonography, was unchanged in intact controls and was 26% of the initial size in castrate controls. Oral doses of Win 49,596 from 0.625-40 mg/kg.day for 16 weeks caused dose-dependent prostatic regression and a dose-related increase in both the incidence and severity of glandular atrophy of the prostate. Prostatic secretory function was also inhibited by Win 49,596 treatment. The effects of Win 49,596 at 40 mg/kg.day on prostatic weight, total DNA, histomorphology, and secretory function were similar to those of castration, while the effects of Win 49,596 at 10 mg/kg.day were similar to those of ICI 176,334 at 0.25 mg/kg.day and MK-906 at 1.0 mg/kg.day. No effects on testicular weight, daily sperm production, or spermatogenesis were observed; however, mild Leydig cell hyperplasia was observed in two dogs treated with 40 mg/kg.day Win 49,596. In addition, at 10 and 40 mg/kg.day Win 49,596, moderate but variable increases in serum testosterone levels were observed. In summary, Win 49,596 caused regression of the hypertrophic canine prostate without effects on spermatogenesis and/or sexual function, supporting its possible use in the treatment of human benign prostatic hypertrophy/hyperplasia.

Aberrant expression of Cyr61, a member of the CCN (CTGF/Cyr61/Cef10/NOVH) family, and dysregulation by 17 beta-estradiol and basic fibroblast growth factor in human uterine leiomyomas.

Uterine leiomyomas are the most common tumors of the reproductive tract, afflicting women between the ages of 30--55 yr. Although considered to be the leading cause of hysterectomies in the United States, little is known of the etiology and mechanisms of pathogenesis in leiomyomas. Accordingly, rapid analysis of differential expression (RADE) was employed to identify genes that are abnormally expressed in leiomyomas. Of the several genes identified, Cyr61, a member of the CCN family of growth and angiogenic regulators, was shown to be markedly down-regulated at the messenger ribonucleic acid (mRNA) and protein levels in leiomyoma tumors compared with the matched uterine myometrial controls (n = 38). In addition, in situ hybridization experiments corroborated the lack of Cyr61 expression in leiomyoma cells, whereas abundant transcript levels were identified in adjacent myometrial smooth muscle cells. To elucidate the mechanisms of Cyr61 gene regulation in leiomyomas, we determined the effects of ovarian steroids, basic fibroblast growth factor (bFGF), and serum, on Cyr61 expression using an ex vivo culture system. Treatment of human myometrial explants with 17 beta-estradiol and bFGF up-regulated Cyr61 transcripts, whereas the progesterone receptor agonist, R5020 (alone or in combination with 17 beta-estradiol), had no effect. Paradoxically, neither 17 beta-estradiol nor bFGF was capable of up-regulating Cyr61 mRNA in leiomyoma explants despite elevated levels of ER alpha mRNA, suggesting a possible defect in steroid and growth factor regulation. Thus, dysregulation of Cyr61 by estrogen and bFGF may contribute to down-regulation of Cyr61 in leiomyomas, which, in turn, may predispose uterine smooth muscle cells toward sustained growth.

A novel, nonsteroidal inhibitor of androgen binding to the rat androgen binding protein: diethyl [[[3-(2,6-dimethyl-4-pyridinyl)-4-fluorophenyl]amino]methylene] propanedioate.

In vitro binding studies demonstrate the binding specificity of a series of 4-aryl-2,6-dimethylpyridines for the rat epididymal androgen binding protein (rABP). The compounds bound competitively to rABP but have very weak or no demonstrable affinity for rat ventral prostate androgen receptor and human sex hormone binding globulin. In particular, compound 11, diethyl [[[3-(2,6-dimethyl-4-pyridinyl)-4-fluorphenyl]amino]methylene] propanedioate, bound with high affinity to rABP (binding affinity about 1/3 that of the endogenous ligand 5 alpha-dihydrotestosterone). However, additional in vitro binding studies indicated that 11 did not bind to testicular or epididymal ABP from rabbit, rhesus monkey, and human. Nevertheless, the specificity and relatively high affinity of these nonsteroidal compounds make them unique and potentially ideal agents for the study of the role of ABP in spermatogenesis and sperm maturation in the rat.

Non-steroidal glucocorticoid-like substances: receptor binding and in vivo activity.

Compounds of general structure I, prepared by a Diels-Alder reaction with diene 3, are relatives of the known potent glucocorticoid II but possess a markedly modified C- and D-ring environment. Despite these structural changes, 4, 5, 9, 10, 12a, 13, and 14 bound to the glucocorticoid receptor with an affinity which approximated that of the reference standard, 6-alpha-methylprednisolone. Four of these compounds not only exhibited antiinflammatory activity in the alpha-tocopherol pouch test but also exhibited marked adrenal suppression and other typical glucocorticoid properties at doses in the same range as the effective antiinflammatory doses.

Antiandrogenic steroidal sulfonylpyrazoles.

The steroidal sulfonylpyrazole 1 bound to the rat ventral prostate androgen receptor in vitro; it inhibited testosterone propionate induced increases in ventral prostate weight in vivo in the castrated, immature male rat with an ED50 of 15 mg/kg po. Compound 1 lacked androgenic activity in vivo in contrast to the parent steroidal pyrazole 5, which was both androgenic and antiandrogenic. The 2'- and 5'-methylsulfonyl isomers 6 and 6a did not bind to the androgen receptor. Introduction of an alkylsulfonyl at the N-1'-position has served, therefore, to isolate the intrinsic antiandrogenic properties of the steroidal heterocycle free of apparent hormone agonist properties. Structure-activity relationship studies revealed that a methylsulfonyl group at N-1' together with a C-17 alpha-substituent were the optimal combination for in vitro androgen receptor binding, in vivo antiandrogenic potency, and a lack of androgenic activity.

Antiandrogenic steroidal sulfonyl heterocycles. Utility of electrostatic complementarity in defining bioisosteric sulfonyl heterocycles.

Complementarity of electrostatic potential surface maps was utilized in defining bioisosteric steroidal androgen receptor antagonists. Semiempirical and ab initio level calculations performed on a series of methanesulfonyl heterocycles indicated the requirement for a partial negative charge at the heteroatom attached to C-3 of the steroid nucleus to attain androgen receptor affinity. Synthesis and testing of six heterocycle A-ring-fused dihydroethisterone derivatives support this hypothesis, and we have identified two new androgen receptor antagonists of this class.

Serotonin 5-HT1 receptors potentiate histamine and thrombin stimulated prostaglandin synthesis in endothelial cells.

The ability of serotonin 5-HT1 receptors to increase vascular tone was previously found to be activated by vasoconstrictiors such as histamine. In this study, treatment of cultured human aortic endothelial cells (HAEC) with the 5-HT1-selective agonist 5-carboxamidotryptamine (5-CT) alone had no effect on the levels of prostaglandin F2alpha (PGF2alpha) or 6-keto-prostaglandin F1alpha (6-keto PGF1alpha). However, 5-CT potentiated the histamine and thrombin stimulated increases in prostaglandins released by HAEC. In the presence of histamine, increasing doses of 5-CT caused a steep rise in PGF2alpha levels resulting in an increase in the ratio of PGF2alpha over 6-keto PGF1alpha. The ability of 5-CT to potentiate prostaglandin production was correlated with its ability to potentiate the histamine and thrombin mediated mobilization of arachidonic acid. These results demonstrate that the ability of 5-HT1 receptors to stimulate prostaglandin production in endothelial cells is activated by histamine and thrombin.

Estrogen receptor-like macromolecule in MtTW15 rat pituitary tumors: effects of antiestrogens.

To understand how estradiol-17 beta (E2) influences MtTW15 rat pituitary tumor function, we have evaluated the cytosolic E2 binding properties of tumors derived from control and steroid treated host animals. Specific E2 binding (approximately 3 pmoles/g tumor) was observed in all groups and was steroid responsive. The E2 binding macromolecule migrated to 7S following sucrose density gradient sedimentation and was specific for estrogenic steroids. Saturation analysis of E2 binding revealed a high affinity interaction (Kd = 5.5 +/- 0.5 x 10(-10) M). Furthermore, E2 binding was temperature-sensitive and degraded by trypsin. Thus, the MtTW15 tumor contains an estrogen receptor. Accordingly, the effects of 4 antiestrogenic drugs on tumor estrogen-receptor levels, tumor growth and hormone production were evaluated. In general, these drugs reduced cytosolic estrogen receptor levels, promoted tumor growth and increased tumor growth-hormone production. It is suggested that these compounds can exert both estrogen agonist and antagonist properties in MtTW15 tumors.

Rat uterine complement C3 expression as a model for progesterone receptor modulators: characterization of the new progestin trimegestone.

Progestins have a wide variety of activities in female reproduction. There are also pharmacological applications for progestins, including hormone replacement therapy and contraception. Here we report the development and characterization of the rat uterine complement component C3 mRNA as a molecular target for the evaluation of the antiestrogenic activity of progestins in the uterus. In this assay, ethinyl estradiol (EE) is used to stimulate C3 expression and progestins are then evaluated for their ability to inhibit this expression. The three reference progestins, progesterone (P4), levonorgestrel (LNG), and medroxyprogesterone acetate (MPA) blocked the increase in C3 mRNA levels induced by EE. Dexamethasone (DEX) and 17alpha-methyl testosterone did not inhibit the estrogen induced C3 mRNA levels; in fact, DEX caused a further increase in C3 mRNA levels. Finally, the antiprogestin RU486 was able to block the MPA inhibition of C3 message. RU486, like DEX, caused an increase in C3 mRNA levels above that of estrogen treatment alone. The model was also used to evaluate trimegestone (TMG), a new steroidal progestin, that has been shown to be a potent and selective progesterone receptor agonist. The activity of TMG in the rat uterine decidualization and ovulation inhibition assays was similar to MPA. However, in the C3 model, TMG caused a dose-dependent inhibition of the EE induced C3 message and was approximately five-fold more potent in this model than MPA (EC(50) of 4.7 microg/kg and 26.5 microg/kg, respectively). Therefore, TMG was a more potent antagonist of estrogenic activity in the uterine endometrium than any of the reference progestins tested and therefore may be more effective in protecting the endometrium in hormone replacement therapy.

Studies on the mechanism of action of danazol and gestrinone (R2323) in the rat: evidence for a masked estrogen component.

The effects of androgen and estrogen receptor antagonists on the action of danazol and gestrinone (R2323) were investigated. The tropic effect of danazol and gestrinone on sex accessory tissues of castrated, immature male rats was inhibited by the antiandrogen flutamide, whereas the uterotropic action of these steroids in immature female and adult ovariectomized rats was not inhibited by flutamide. In contrast, the uterotropic effect of danazol was reduced by the antiestrogen, LY156758. The estrogen-sensitive endpoints, vaginal keratinization and uterine progesterone receptor concentration, were enhanced by treatment with a combination of flutamide and either danazol or gestrinone. These data indicate that danazol and gestrinone have estrogenic activity that is masked by the androgenic component of these drugs.

In vitro characterization of trimegestone: a new potent and selective progestin.

Trimegestone (TMG) is a novel 19-norpregnane progestin under development for hormone replacement therapy and oral contraception. The objective of the current study was to characterize the potency and steroid receptor selectivity of TMG in several in vitro assays and to compare its activity to that of medroxyprogesterone acetate (MPA). TMG and MPA had a similar competitive binding affinity for human and rabbit progesterone receptor (PR). However, TMG had a significantly higher affinity for rat PR (IC(50) = 3.3 nM) than MPA (IC(50) = 53.3 nM). In T47D cells, both compounds increased alkaline phosphatase activity and cell proliferation with comparable potencies (EC(50s) of 0.1 nM and of 0.02 nM, respectively). To further characterize the progestational activity and steroid receptor selectivity, we established an immortalized human endometrial stromal cell line (HESC-T). This cell line lacks endogenous estrogen receptor (ER) and PR but does have functional glucocorticoid receptors (GR). When ER is transiently expressed in the cells, 17beta-estradiol (E(2)) induces PR, allowing the study of PR-regulated genes. In HESC-T cells expressing exogenous ER, and therefore PR, both TMG and MPA increased HRE-tk-luciferase activity tenfold with an EC(50) of 0.2 nM. In HESC-T cells without exogenous ER, and therefore no PR, TMG did not induce HRE-tk-luciferase activity, whereas MPA induced the reporter activity with an EC(50) of about 10 nM. This MPA-induced reporter activity is believed to be mediated through GR. The steroid receptor selectivity of TMG was further evaluated using the HRE-tk-luciferase assay in the human lung carcinoma cell line A549, which contains GR but no PR. In these cells TMG had no effect on luciferase activity, whereas MPA increased the reporter activity in a dose-dependent manner with an EC(50) of approximately 30 nM. Furthermore, HRE-tk-luciferase assay in mouse fibroblast cell line L929, which expresses androgen receptor (AR) but no PR, showed that TMG had weak antiandrogenic activity whereas MPA had androgenic activity. In summary, data from several in vitro assays demonstrate that TMG is a potent progestin with a better receptor selectivity profile than MPA.