Induction Of Progesterone Receptor Research Articles

Vinclozolin does not alter progesterone receptor (PR) function in vivo despite inhibition of PR binding by its metabolites in vitro

Vinclozolin, a dicarboximide fungicide, alters morphological sex differentiation in male rats following perinatal exposure. The occurrence of these abnormalities correlates with the in vivo formation of two antiandrogenic metabolites of vinclozolin, (i.e. 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (Ml) and 3′, 5′-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2)), which are potent inhibitors of rat androgen receptor binding. As steroid hormone receptors exhibit promiscuity in their ability to bind different ligands, the present study evaluated the ability of these vinclozolin metabolites to bind to the estrogen (ER) and progesterone (PR) receptors in vitro, and to alter ER and PR function following in vivo exposure. To this end, in vitro ligand binding assays demonstrated that both M1 and M2 can compete with endogenous ligand for binding to the PR ( K i = 400 and 60 μM, respectively). In contrast, neither metabolite exhibited the ability to bind ER. Subsequent in vivo studies to evaluate the potential of vinclozolin to alter ER or PR function demonstrate that, (1) the estrogen-dependent increases in uterine weight and PR induction were not altered by vinclozolin; (2) the distribution of nuclear and cytosolic PR was not altered following short-term vinclozolin exposure; and (3) vinclozolin did not disrupt ovulation in cycling female rats. These studies indicate that although vinclozolin metabolites can compete for binding to the PR in vitro, concentrations of these metabolites do not reach sufficient levels to disrupt female reproductive function following short-term in vivo exposure to vinclozolin. In addition, these studies demonstrate the importance of correlating in vitro receptor binding data with in vivo studies in order to understand the physiological consequences of exposure to environmental toxicants.

Interferon-beta can induce progesterone receptors in human endometrial adenocarcinoma.

The induction of estrogen and progesterone receptors (ER and PGR) has been reported in breast and endometrial cancer cells exposed to human fibroblast interferon-beta (hIFN-beta). Clinical verification of this finding might provide the rationale for new therapeutic approaches. This study was designed to evaluate whether clinical treatment with high doses of hIFN-beta induced ER and PGR in patients with endometrial adenocarcinoma. Two biopsies were obtained, 1 before and 1 after hIFN-beta treatment (3 x 10(6) i.m. every other day for 3 weeks) from 36 patients with endometrial adenocarcinoma. ER and PGR were determined with standard procedures using radiolabeled ligands. hIFN-beta treatment did not affect the proportion of ER-positive (i.e., >15 fmol/mg protein) or PGR-positive (i.e., >20 fmol/mg protein) cases. However, in patients with detectable ER and PGR at baseline, hIFN-beta raised the levels. Using a 35% difference before and after therapy as a cut-off, 72 and 79% of cases had increases in ER and PGR, respectively. The difference was highly significant for PGR. In patients with endometrial adenocarcinoma with undetectable ER or PGR, hIFN-beta did not induce the expression of these receptors. When the receptors were present they were upregulated by hIFN-beta. Whether this increase in receptor levels, particularly PGR, has therapeutic applications remains to be established.

Progesterone receptors: expression and regulation in the mammalian ovary.

The authors have briefly discussed the molecular structure, regulation, and function of progesterone receptors in the mammalian ovary. Particularly important is the contrast in the regulatory mechanisms of PR induction in the ovary (gonadotropins/membrane receptor mediated) and other well-known progesterone target tissues, such as the uterus and mammary gland (estrogen/nuclear receptor mediated). Future research will focus on how the PR gene responds to these hormonal regulatory signals in this cell-specific manner. Equally important in this discussion has been the mounting evidence indicating that PRs are an essential component of the ovulatory process. The observation that PR-/- knockout mice are incapable of undergoing ovulation, even in response to gonadotropin challenge, further supports the previous physiological evidence indicating that PRs in preovulatory follicles are induced before, and are necessary for, ovulation. Further studies are required to determine the identity of PR-regulated target genes during the periovulatory period. Although our knowledge of PR structure, regulation, and function has increased dramatically during the past decade, many exciting questions remain related to the regulation and function(s) of PRs in the ovary and other tissues.

RU486 on an estrogen background blocks the rise in serum follicle-stimulating hormone induced by antiserum to inhibin or ovariectomy.

We used passive immunization with an antiserum to the alpha-subunit of inhibin (anti-I) or acute ovariectomy to investigate the relationship between serum inhibin levels and FSH secretion in the presence of the progesterone/glucocorticoid antagonist RU486. We demonstrated previously that 1) anti-I administered at 1700 h causes serum FSH to rise on the morning of estrus, even in the presence of a GnRH antagonist, when the two treatments are delivered on proestrus; and that 2) RU486 given on proestrus (1230 h), a time when serum estradiol levels are high, not only blocks the natural secondary FSH surge, but also suppresses the anti-I-induced rise in serum FSH on the morning of estrus. We have now extended our studies of the relationship between inhibin and RU486 to investigate treatment with RU486 and anti-I on a different day of the cycle, estrus, when serum estradiol levels are low. When both RU486 and anti-I were given on estrus (1230 and 1700 h, respectively), RU486 failed to block the anti-I-induced rise in serum FSH on the next morning of metestrus, in contrast to the blockade seen with RU486 treatment on the day of proestrus. However, pretreatment with estradiol benzoate (50 microgram) on the evening of proestrus, before the RU486 and anti-I treatment on estrus, caused RU486 to suppress the effects of anti-I on serum FSH, as it does when given on proestrus. We then repeated the study, using ovariectomy on proestrus or estrus (1700 h) to raise serum FSH, and assessed the effects of RU486 treatment at proestrus and estrus and estradiol benzoate treatment on proestrus. Our results indicate that treatment with RU486 can block the postovariectomy rise in serum FSH only in the presence of high circulating estradiol levels. We conclude that the inhibitory action of RU486 on FSH secretion after a fall in serum inhibin depends on a precedent estradiol background, probably due to induction of progesterone receptors by estradiol.

Ginseng extract induces progesterone receptor in the estrogen responsive MCF-7 breast carcinoma cell line.

Ginseng extract induces progesterone receptor in the estrogen responsive MCF-7 breast carcinoma cell line.

Blockade of the estrogen induced increase in progesterone receptor caused by propylthiouracil, an anti-thyroid drug, in a transplantable pituitary tumor in rats.

It is well known that estrogen (E2) induces progesterone receptor (PR) in the uterus and the mammary gland. In MtT/F84, a pituitary tumor, which was established in our laboratory and has been maintained with in vivo passages, we investigated the PR regulation by E2 in relation to the host's thyroidal status. The PR level in the tumor had increased five fold 48 h after an E2 injection. When the host rats were treated with propylthiouracil (PTU), an anti-thyroid drug, the induction of PR after an E2 injection was completely blocked. This result is consistent with our previous findings indicating that E2 responsiveness in the tumor may be under the control of thyroid hormones. The estrogen receptor (ER) level in the tumor treated with PTU was 15% of the control. This low ER level may account for the blocking of PR induction after an E2 injection. When the host animals were continuously treated with various doses of E2, the PR level in the tumor rose in correlation with the E2 doses. PTU administration, however, did not prevent long term induction of PR by continuous E2 treatment. Our findings suggest that PTU lower the ER level and suppresses the short term estrogenic actions such as PR induction after an E2 injection.

Cis-regulatory elements conferring cyclic 3',5'-adenosine monophosphate responsiveness of the progesterone receptor gene in transfected rat granulosa cells.

We have previously shown that both pituitary gonadotropins and forskolin induce progesterone receptor (PR) messenger RNA expression at the level of transcription in granulosa cells of the rat ovary. To determine the DNA regulatory elements that are important for cAMP-induced transcription of the PR gene in the ovary, we examined the cAMP-induced activity of promoter sequences in rat granulosa cells transfected with various fusion constructs containing PRB promoter sequences linked to the luciferase reporter gene. When cells were transfected with a luciferase fusion construct containing the 1375-base pair 5'-flanking region of the rat PRB gene, forskolin treatment substantially increased luciferase activity. Analysis of a series of 5'-deletion mutants indicated that a minimal PRB promoter containing 116 base pairs of upstream sequence (-116/3) was sufficient to increase luciferase activity in response to forskolin in transfected rat granulosa cells. This promoter contains a consensus CCAAT site in reverse orientation (5'-ATTGG-3') and a consensus GC box (5'-GGGGCGGGCC-3'), but no known cAMP-responsive element. Site-specific mutation of the GC box notably decreased both basal and cAMP-induced activity of this minimal PRB promoter. In addition, site-specific mutation of the CCAAT binding site within this proximal promoter of the PRB gene substantially decreased cAMP-induced activity, but did not significantly affect the basal activity of this promoter. Either mutation alone failed to abolish cAMP inducibility. In contrast, double mutation of both the GC box and the CCAAT box completely abolished cAMP inducibility, suggesting that the GC box and the CCAAT box act together to mediate cAMP-induced transcription of the PRB gene. Gel shift analysis shows that the minimal PRB promoter sequences form multiple complexes with nuclear proteins of granulosa cells, all of which are specifically competed by oligonucleotides containing the GC box and the CCAAT box. Taken together, our results suggest a functional role for transcription factors binding the GC box and the CCAAT box in mediating cAMP-induced transcription of the rat PRB promoter in rat granulosa cells.

Effects of 12-O-Tetradecanoylphorbol-13-acetate on Estrogen Receptor Activity in MCF-7 Cells

The effects of long term treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) on estrogen receptor (ER) expression in the human breast cancer cell line, MCF-7, were studied. This study demonstrates that treatment of cells with the phorbol ester blocked estrogen receptor activity. Treatment of cells with 100 nM TPA resulted in an 80% decrease in the level of ER protein and a parallel decrease in ER mRNA and binding capacity. Following removal of TPA from the medium, the level of ER protein and mRNA returned to control values; however, the receptor failed to bind estradiol. These cells also failed to induce progesterone receptor in response to estradiol. In addition, TPA treatment blocked transcription from an estrogen response element in transient transfection assays and inhibited ER binding to its response element in a DNA mobility shift assay. The estrogen receptor in treated cells was recognized by two monoclonal anti-ER antibodies and was not quantitatively different from ER in control cells. RNase protection analysis failed to detect any qualitative changes in the ER mRNA transcript. Mixing experiments suggest that TPA induces/activates a factor which interacts with the ER to block binding of estradiol. The effects of TPA on ER levels and binding capacity were concentration-dependent. Low concentrations of TPA inhibited estradiol binding without a decrease in the level of protein, whereas higher concentrations were required to decrease the level of ER protein. The effects of TPA appear to be mediated by activation of protein kinase C since the protein kinase C inhibitors, H-7 and bryostatin, block the effects of TPA on estradiol induction of progesterone receptor. TPA treatment had no effect on the level or binding capacity of the glucocorticoid receptor, indicating that the effects are not universal for steroid receptors. These data demonstrate that activation of the protein kinase C signal transduction pathway modulates the estrogen receptor pathway. The long term effect of protein kinase C activation is to inhibit estrogen receptor function through induction/activation of a factor which interacts with the receptor.

Effects of Luteinizing Hormone and Follicle-Stimulating Hormone on the Progesterone Receptor Induction in Chicken Granulosa Cells In Vivo

Progesterone receptor (PR) is increased in the granulosa cells prior to ovulation. In order to investigate the factors that induce PR in granulosa cells, we examined whether the PR is induced in granulosa cells by the injection of chickens with ovine luteinizing hormone (oLH) or porcine follicle-stimulating hormone (pFSH) by Western blot analysis. In the oLH-stimulated birds, PR of 79 kDa and 110 kDa were recognized in the granulosa cells of the largest follicle, whereas a PR of 110 kDa was observed in those of the smaller follicles (the second and third largest follicles). No immunoreaction product was observed in the granulosa cells of both the largest and the smaller follicles in pFSH-stimulated birds. In the control birds that were injected with saline, no immunoprecipitate was recognized in the granulosa cells of the largest or smaller follicles. These results suggest that LH may be involved in the induction of PR in the granulosa cells prior to ovulation.

Inhibition of estrogen-induced progesterone receptor in MCF-7 human breast cancer cells by aryl hydrocarbon (Ah) receptor agonists

17β-Estradiol (E 2) induces progesterone receptor (PR) binding, immunoreactive protein, nuclear PR formation and PR mRNA levels in MCF-7 human breast cancer cells. Gel mobility shift analysis of nuclear extracts from E 2-treated cells also exhibited a higher intensity retarded band associated with formation of a PR complex with a consensus [ 32P]progesterone/glucocorticoid responsive element. In contrast, 1nM2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) alone did not alter or decrease these same responses in MCF-7 cells; however, in cells co-treated with 1 nM TCDD plus 1 nM E 2, TCDD significantly inhibited all the E 2-induced responses. Scatchard analysis of PR binding demonstrated that TCDD decreased the number of E 2-induced PR cellular binding sites but not the binding affinity of the PR for a radiolabeled promegestrone. In parallel studies, 3-methylcholanthrene, a prototypical polynuclear aromatic hydrocarbon, also inhibited E 2-induced PR binding and immunoreactive protein. For a series of halogenated aromatics including 2,3,7,8- and 1,2,7,8-tetrachlorodibenzofuran, 1,3,7,8-TCDD and 6-methyl-l,3,8-trichlorodibenzofuran, their rank order potency for inhibiting E 2-induced PR binding paralleled their rank order binding to the aryl hydrocarbon (Ah) receptor. These results support a role for the Ah receptor in mediating the antiestrogenic activity of polynuclear and halogenated aromatic hydrocarbons and illustrate cross-talk between the Ah and estrogen receptor signal transduction pathways.

Bivalent ligands as probes of estrogen receptor action

The estrogen receptor (ER) is a hormone-regulated transcription factor which is thought to bind to specific DNA sequences as a homodimer. In order to better understand structural requirements for dimerization and its functional role in ER action, we synthesized a series of bivalent ligands based on the non-steroidal estrogen hexestrol. These molecular probes join two hexestrol molecules of the erythro (E, active) configuration with either 4 or 8 carbon linkers (designated E-4-E and E-8-E series, respectively), or with longer linkers comprised of ethylene glycol units (E-eg-E series). Several other bi- and monovalent control compounds were prepared. The bivalent ligands binds to ER with a relative affinity 1–7% that of estradiol. While most of the ligands demonstrated normal monophasic displacement curves in competitive binding assays with [ 3H]estradiol, uncharacteristic biphasic competitive binding curves were seen for some of the ligands, indicating possible structure-specific, negative site-site interaction. In ER-deficient Chinese hamster ovary (CHO) cells transfected with an expression vector encoding ER, one series of bivalent ligands (E-4-E) had little stimulatory activity and inhibited transcription stimulated by hexestrol, as determined by a transient transfection assay using an estrogen-responsive reporter gene construct [(ERE) 2-TATA-CAT, containing two estrogen response elements linked to a TATA promoter and the chloramphenicol acetyl transferase reporter gene]. Monovalent or control bivalent ligands failed to antagonize hexestrol-stimulated activity and were as fully active as hexestrol itself. Studies performed in MCF-7 human breast cancer cells, which contain endogenous ER, yielded similar bioactivity profiles for the E-4-E bivalent inhibitory ligands, showing them to be effective estrogen antagonists, when using either induction of progesterone receptor or (ERE) 2-TATA-CAT transcriptional activation as the endpoint. The E-8-E ligand, however, acted as a partial agonist/antagonist of ERE-reporter gene transactivation and a full agonist of progesterone receptor induction in MCF-7 cells, thus showing cell- and response-specific differences in the effects of the bivalent ligand. The bivalent ligands for ER do not show enhanced potency or receptor binding affinity; however, some of them display binding properties that suggest the possibility of structure-specific negative site-site interaction, and some of them function as quite effective estrogen antagonists.

Estrogen and Progesterone Receptor Expression of Decidual Endometrium in a Postmenopausal Woman Treated with Tamoxifen and Megestrol Acetate

To the best of our knowledge, this is the first report of in vivo endometrial estrogen and progesterone receptor induction as a result of tamoxifen exposure in a postmenopausal breast cancer patient. The following observations, that the postmenopausal endometrium is sensitive to tamoxifen, that this agent can act as an estrogen-like substance, and that it may cause proliferation of the endometrium in the absence of progestin, may explain the endometrial decidual changes described herein as a protective mechanism against possible neoplastic endometrial changes.

Inhibition of estrogen stimulated mitogenesis by 3-phenylacetylamino-2,6-piperidinedione and its Para-hydroxy analog

3-Phenylactetylamino-2,6-piperidinedione (A10) inhibited estradiol stimulated cell growth in the MCF-7 (E3) human breast tumor cell line in vivo and in vitro. While high concentrations of A10 were needed to inhibit cell proliferation (IC 50 = 3 × 10 −3 M in vitro), the compound demonstrated little toxicity. The effect appeared specific since a hydrolysis product of A10, phenylacetylglutamine, demonstrated no growth inhibitory activity at similar concentrations in MCF-7 (E3) cells in vitro. A computer designed analog, p-hydroxy A10, was more potent than A10 in inhibiting activity in MCF-7 (E3) cells in vitro. The IC 50 for p-hydroxy A10 was 7 × 10 −6 M which was comparable to that of the antiestrogen, tamoxifen (IC 50 1 × 10 −7 M). All three compounds caused a decline in estrogen receptor levels in a dose-dependent fashion. A10 also inhibited estradiol induction of progesterone receptors. Examination of protein kinase activity following an acute exposure to a 10 −11 M growth stimulatory dose of estradiol revealed a 168% increase in protein kinase activity over that of untreated control cells. A10 in a dose-responsive fashion inhibited the estradiol stimulated increase in protein kinase activity. The protein kinase activity was also inhibited by p-hydroxy A10. These activities of A10 and p-hydroxy A10 coupled with the low toxicity and novelty of the basic A10 structure provide an exciting possibility of developing a new class of clinically useful antineoplastic drugs with minimal side effects.

Immunocytochemical Localization of Progesterone Receptors in Galanin Neurons in the Guinea Pig Hypothalamus

A double-label immunocytochemical technique was used to determine whether progesterone receptor-containing neurons in the female guinea-pig hypothalamus also contained galanin. Adult ovariectomized guinea-pigs were primed by estradiol to induce progesterone receptors and injected intracerebroventricularly with colchicine to visualize galanin-immunopositive neurons. A small proportion of progesterone receptor-containing perikarya in the medial preoptic area and the mediobasal hypothalamus were bound to be immunoreactive for galanin. The medial preoptic, periventricular and arcuate nuclei showed the greatest concentration of double-labelled cells. Galanin varicosities appeared in close proximity to neurons with progesterone receptor-containing nuclei. These results provide neuroanatomical evidence that a subset of hypothalamic galanin-immunoreactive neurons is directly regulated by progesterone.

Hormonal regulation, localization, and functional activity of the progesterone receptor in granulosa cells of rat preovulatory follicles.

Progesterone has been implicated to play a critical role in mediating LH induction of ovulation and possibly luteinization. The present study was undertaken to determine the effects of various agonists (LH, FSH, forskolin, and GnRH) known to stimulate ovulation on their abilities to induce progesterone receptor (PR) mRNA and protein in rat preovulatory follicles and in cultured rat granulosa cells exhibiting a preovulatory phenotype. In cultured granulosa cells, PR mRNA was induced by LH in a dose- and time-dependent manner. Transcripts of approximately 11.0, 7.2, 6.8, 6.2, 3.4, and 3.1 kilobases in size were induced by ovulatory (500 ng/ml), but not low (50 ng/ml), concentrations of LH and FSH as well as by forskolin (10 microM), GnRH (1 microM), and phorbol 12-myristate 13-acetate (200 nM). Two forms (A and B) of PR protein were also induced in an agonist- and time-dependent manner, with the shorter form A (mol wt, 83,000-85,000) appearing in greater abundance than the longer form B (mol wt, 115,000). Indirect immunofluorescent analyses verified nuclear localization of the induced receptor. Forskolin and progesterone, but not progesterone alone, were able to activate a glucocorticoid (progesterone) response element-E1b-chloramphenicol acetyltransferase reporter construct (12-fold) after transfection into cultured granulosa cells. The antiprogestins RU486 and ZK98299 did not inhibit induction of PR mRNA and protein, but effectively blocked agonist activation of glucocorticoid response element2-E1b-chloramphenicol acetyltransferase, as well as LH stimulation of luteinization in vitro. These results provide direct evidence that agonists stimulating diverse intracellular pathways can induce PR in granulosa cells, that progesterone plays a functional role in the luteinization process triggered by the LH surge, and that the effects are mediated at least in part by induction of PR.

Hormonal regulation, localization, and functional activity of the progesterone receptor in granulosa cells of rat preovulatory follicles

Progesterone has been implicated to play a critical role in mediating LH induction of ovulation and possibly luteinization. The present study was undertaken to determine the effects of various agonists (LH, FSH, forskolin, and GnRH) known to stimulate ovulation on their abilities to induce progesterone receptor (PR) mRNA and protein in rat preovulatory follicles and in cultured rat granulosa cells exhibiting a preovulatory phenotype. In cultured granulosa cells, PR mRNA was induced by LH in a dose- and time-dependent manner. Transcripts of approximately 11.0, 7.2, 6.8, 6.2, 3.4, and 3.1 kilobases in size were induced by ovulatory (500 ng/ml), but not low (50 ng/ml), concentrations of LH and FSH as well as by forskolin (10 microM), GnRH (1 microM), and phorbol 12-myristate 13-acetate (200 nM). Two forms (A and B) of PR protein were also induced in an agonist- and time-dependent manner, with the shorter form A (mol wt, 83,000-85,000) appearing in greater abundance than the longer form B (mol wt, 115,000). Indirect immunofluorescent analyses verified nuclear localization of the induced receptor. Forskolin and progesterone, but not progesterone alone, were able to activate a glucocorticoid (progesterone) response element-E1b-chloramphenicol acetyltransferase reporter construct (12-fold) after transfection into cultured granulosa cells. The antiprogestins RU486 and ZK98299 did not inhibit induction of PR mRNA and protein, but effectively blocked agonist activation of glucocorticoid response element2-E1b-chloramphenicol acetyltransferase, as well as LH stimulation of luteinization in vitro. These results provide direct evidence that agonists stimulating diverse intracellular pathways can induce PR in granulosa cells, that progesterone plays a functional role in the luteinization process triggered by the LH surge, and that the effects are mediated at least in part by induction of PR.

Influence of estrogen structure on nuclear binding and progesterone receptor induction by the receptor complex.

The relationship between steroid structure, estrogen receptor (ER) binding affinity, nuclear binding of the ER complex, and induction of progesterone receptor (PgR) have been examined. The level of ER in membrane-free homogenates of MCF-7 cells was found to be 10.0 +/- 0.5 fmol/micrograms of DNA by utilizing an enzyme immunoassay (EIA). However, only 2.5 +/- 0.2 fmol of ER complex/micrograms of DNA was bound by nuclei during maximal stimulation of PgR synthesis (2.9 +/- 0.2 fmol of PgR/micrograms of DNA; measured by EIA) following a pulse with 10(-10) M E2. Except at micromolar concentrations, estratriene was an ineffective estrogen. The addition of a hydroxyl group to either position 3 or position 17 beta of estratriene yielded ligands which were capable of causing nuclear binding and processing of ER as well as PgR induction. D-ring regioisomers of estradiol (E2) had lower affinity for receptor than E2. However, receptor complexed with these estrogens was fully capable of binding to nuclear material, undergoing processing, and inducing PgR. On the other hand, A-ring regioisomers of E2 displayed significant differences in their ability to mediate nuclear binding of receptor complex and induction of PgR. Although 1-hydroxyestratrien-17 beta-ol was weakly bound by ER, this dihydroxyestrogen was capable of bringing about nuclear binding and processing of ER and the stimulation of PgR synthesis. In contrast, 2- and 4-hydroxyestratrien-17 beta-ol, which caused extensive nuclear binding of ER (5-7 fmol/micrograms of DNA), were incapable of significant PgR induction.(ABSTRACT TRUNCATED AT 250 WORDS)

Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving.

In studies to determine whether Saccharomyces cerevisiae produced estrogens, the organism was grown in culture media prepared using distilled water autoclaved in polycarbonate flasks. The yeast-conditioned media showed the presence of a substance that competed with [3H]estradiol for binding to estrogen receptors (ER) from rat uterus. However, it soon became clear that the estrogenic substance in the conditioned media was not a product of the yeast grown in culture, but was leached out of the polycarbonate flasks during the autoclaving procedure. [3H]Estradiol displacement activity was monitored by ER RRA, and the active substance was purified from autoclaved medium using a series of HPLC steps. The final purified product was identified as bisphenol-A (BPA) by nuclear magnetic resonance spectroscopy and mass spectrometry. BPA could also be identified in distilled water autoclaved in polycarbonate flasks without the requirement of either the organism or the constituents of the culture medium. Authentic BPA was active in competitive RRAs, demonstrating an affinity approximately 1:2000 that of estradiol for ER. In functional assays, BPA (10-25 nM) induced progesterone receptors in cultured human mammary cancer cells (MCF-7) at a potency of approximately 1:5000 compared to that of estradiol. The BPA effect on PR induction was blocked by tamoxifen. In addition, BPA (25 nM) increased the rate of proliferation of MCF-7 cells assessed by [3H]thymidine incorporation. Thus, BPA exhibited estrogenic activity by both RRA and two functional bioresponse assays. Finally, MCF-7 cells grown in media prepared with water autoclaved in polycarbonate exhibited higher progesterone receptor levels than cells.grown in media prepared with water autoclaved in glass, suggesting an estrogenic effect of the water autoclaved in polycarbonate. Our findings raise the possibility that unsuspected estrogenic activity in the form of BPA may have an impact on experiments employing media autoclaved in polycarbonate flasks. It remains to be determined whether BPA derived from consumer products manufactured from polycarbonate could significantly contribute to the pool of estrogenic substances in the environment.

Restoration of estrogen-dependent progesterone receptor expression in a uterine myocyte cell line.

Although the in vivo effect of estrogen on myometrial differentiation is well documented, estrogen effects on primary myocytes in vitro have been difficult to demonstrate. To construct a stable uterine myocyte system, capable of direct estrogen responsiveness, we used a transformed hamster myometrial cell line. Since these cells expressed a low level of estrogen receptors (ERs), we have stably transfected them with a vector for the human ER. After transfection, ER concentration increased from less than 300 sites per cell to 17,000 +/- 2,000 sites per cell (mean +/- SEM). To test the functional integrity of the transfected receptors, a chloramphenicol acetyltransferase gene linked to an estrogen response element upstream of thymidine kinase promoter was transiently transfected, and the amount of chloramphenicol acetyltransferase activity, an indicator of estrogen responsiveness, was found to increase 20-fold in response to 17 beta-estradiol (1 nM for 48 h). Furthermore, we tested the ability of estrogen to activate endogenous genes by measuring progesterone receptor (PR) induction. PR concentration in the transfected cells was 3,700 +/- 800 and increased 9-fold to 33,000 +/- 6,000 with 17 beta-estradiol (2 nM). This receptor density increase was confirmed by immunoblotting. PR induction was maximal at 16 h, was concentration dependent, and was not elicited by tamoxifen or ICI 164,384. We conclude that transformed hamster myocytes transfected with an ER gene are capable of estrogen-dependent PR expression in vitro and may serve as a useful system to study estrogen effect on myocytes.

Restoration of estrogen-dependent progesterone receptor expression in a uterine myocyte cell line

Although the in vivo effect of estrogen on myometrial differentiation is well documented, estrogen effects on primary myocytes in vitro have been difficult to demonstrate. To construct a stable uterine myocyte system, capable of direct estrogen responsiveness, we used a transformed hamster myometrial cell line. Since these cells expressed a low level of estrogen receptors (ERs), we have stably transfected them with a vector for the human ER. After transfection, ER concentration increased from less than 300 sites per cell to 17,000 +/- 2,000 sites per cell (mean +/- SEM). To test the functional integrity of the transfected receptors, a chloramphenicol acetyltransferase gene linked to an estrogen response element upstream of thymidine kinase promoter was transiently transfected, and the amount of chloramphenicol acetyltransferase activity, an indicator of estrogen responsiveness, was found to increase 20-fold in response to 17 beta-estradiol (1 nM for 48 h). Furthermore, we tested the ability of estrogen to activate endogenous genes by measuring progesterone receptor (PR) induction. PR concentration in the transfected cells was 3,700 +/- 800 and increased 9-fold to 33,000 +/- 6,000 with 17 beta-estradiol (2 nM). This receptor density increase was confirmed by immunoblotting. PR induction was maximal at 16 h, was concentration dependent, and was not elicited by tamoxifen or ICI 164,384. We conclude that transformed hamster myocytes transfected with an ER gene are capable of estrogen-dependent PR expression in vitro and may serve as a useful system to study estrogen effect on myocytes.