Induction Of Progesterone Receptor Research Articles

120. Induction of progesterone receptor in fetal guinea pig uterus in organ culture

120. Induction of progesterone receptor in fetal guinea pig uterus in organ culture

Specific protein synthesis regulated by estrogen in human breast cancer

We have measured, by double-label SDS-elecirophoresis, the effect of estradiol on the relative rates of specific protein synthesis in MCF-7 human breast cancer cells. Two specific proteins, a 24,000 molecular weight (24k) cytosol protein and a 54,000 molecular weight (54k) protein located in nuclei, were found to be preferentially stimulated by estradiol. Estrogen stimulation of the 24k protein has been characterized under various experimental conditions. Synthesis was found to increase with time of estrogen incubation, reaching maximum stimulation between 4 and 6 days, was estrogen dose-dependent within the physiological range of hormone concentration and stimulation required specifically those steroids which bind to the estrogen receptor. Moreover, the magnitude of increased 24k synthesis parallels nuclear estrogen receptor processing, an event correlated with another estrogen response in MCF-7, induction of progesterone receptor. Double-label cytosols were further resolved by two-dimensional electrophoresis, which revealed two estrogen stimulated proteins in the 24k region with isoelectric points in the range of 6.7–6.9 and 6.4–6.7. The more acidic species was determined to represent a major fraction (1.6%) of newly synthesized protein. Increased 54k synthesis is detected only in nuclei and this response to estrogen has not yet been fully characterized. A 24k and a 54k protein have been synthesized in a rabbit reticulocyte translation system with MCF-7 mRNA. Adapting the double-label electrophoresis technique to this cell-free system, estradiol was found also to preferentially stimulate translatable 24k and 54k mRNA activity, suggesting that estradiol effects 24k and 54k synthesis at the transcriptional level.

Is a functional estrogen receptor always required for progesterone receptor induction in breast cancer?

Although progesterone receptors (PgR) are synthesized in response to estrogens acting through estrogen receptors (ER), in breast cancer some hormone responsive ER− but PgR + tumors are seen. We have studied a human breast cancer cell line, T47D, which may explain this paradox. These cells have low ER levels (0.5 pmol/mg DNA), which are predominantly nuclear (no cytoplasmic 8S receptor). Unlike cytoplasmic ER + cells where estradiol treatment and ensuing nuclear receptor processing lower cellular ER and increase PgR, in T47D, estradiol (10 nM) does not change ER or PgR. In contrast, the anti-estrogen nafoxidine (1 μM), which does not change total ER or PgR in ER + cells because it prevents ER processing, doubles ER in T47D. Analogous to its effect in ER − cells, in T47D cells nafoxidine does not inhibit cell growth and has only minimal effects on PgR. The cytoplasmic PgR synthetized in estradiol free medium is normal as shown by several criteria, and is present in extraordinary amounts (15–40 pmol/mg DNA). Furthermore, the PgR are functional; they can be translocated to, and quantitatively recovered from nuclei, after progesterone (0.1 μM) addition. Cytoplasmic PgR replenishment begins after 1 hr, is complete in 10–24 h and this step is also independent of estradiol and nafoxidine control. Thus, these cells are either functionally ER−, or the ER are in a permanently activated state in the nucleus; nafoxidine reverses this activated, processed state. Despite their insensitivity to estradiol or antiestrogen, PgR are not constitutively synthesized; BUdR (20 μg/ml) or butyrate (10 mM) can selectively inhibit PgR production.

Estradiol stimulates synthesis of a major intracellular protein in a human breast cancer cell line (MCF-7).

Previous studies have shown that synthesis of a 24,000 molecular weight (24k) protein (7) and the mRNA activity coding for a 24k protein (8) are selectively stimulated by estradiol in MCF-7 human breast cancer cells. Utilizing a double-label electrophoresis technique to measure the relative rates of specific protein synthesis, the present study relates further characterization of this estrogen response. The 24k protein was found to be stimulated by estradiol provided cells were preincubated with antiestrogen. Under these conditions, antiestrogens inhibit cell growth and addition of estradiol reverses growth inhibition (estrogen growth ‘rescue’). As a control experiment, specific protein synthesis was measured in another growth rescue model produced by withdrawing serum from the medium to inhibit growth and then stimulating cells by readdition of serum (serum growth ‘rescue’). This failed to stimulate synthesis of a 24k protein, implying that the effect observed following estrogen ‘rescue’ was not a non-specific result of growth stimulation. Increased 24k synthesis was found to be both time and estrogen-dose dependent and required specifically those steroid hormones which interact with the estrogen receptor. Moreover, the dose response curve for 24k stimulation paralleled closely the dose response for estrogen stimulation of nuclear estrogen receptor processing, an event correlated with another estrogenic response in MCF-7 cells, induction of progesterone receptor. These findings suggest that estradiol itself directly stimulates synthesis of the 24k protein through interaction with the estrogen receptor system. Further separation of double-label cytosols by two-dimensional electrophoresis resolved the 24k protein into two isoelectric species with pI's in the range of 6.7–6.9 and 6.4–6.7, the latter being the most abundant or rapidly labeling protein. Based on the percentage of total cytosol incorporation contained in individual two-dimensional gel spots, the major species of 24k represented in the fully stimulated cell (3H-counts) about 1.6% of newly synthesized protein. It also represented about 0.7% of newly synthesized protein in the unstimulated (14C-counts) cell, which indicates that 24k is synthesized constitutively. Because of its relative abundance, the 24k protein may be a suitable end product for investigating the mechanisms of estrogen action in human breast cancer.

Different effects of oestradiol, oestriol, oestetrol and of oestrone on human breast cancer cells (MCF-7) in long term tissue culture.

The effects of oestradiol (Oe2), oestrone (Oe1), oestriol (Oe3), oestetrol (Oe4) on the induction of the progesterone receptor (PgR) and growth of MCF-7 cells are compared. All the four oestrogens increased cell PgR concentration. Analysis of the dose-response curves shows induction by Oe2 to be 10 times and 50 times greater than Oe3 and Oe4, respectively. Oe1 and Oe2 are equally effective, even with consideration of metabolic conversion of O31 into Oe2. When compared with untreated cells, Oe2, Oe3, and Oe4 do not influence significantly the plating efficiency but all 3 hormones increase thymidine incorporation of the cells in log phase growth. Oe2, Oe3 and Oe4 are able to rescue the growth inhibition induced by antioestrogens. The respective potency compared to Oe2 is again in the range of 10 and 50 times lower for Oe3 and Oe4, respectively. On the other hand Oe1 decreases plating efficiency, thymidine incorporation and does not rescue the growth inhibition induced by antioestrogens when the metabolic conversion of Oe1 into Oe2 is prevented. Thus, Oe3 and Oe4 behave like complete Oe2 agonists whereas Oe1 has dissociated effects, agonist on PgR induction and antagonist on cell growth.

Estradiol-independent growth of a subline of MCF-7 human breast cancer cells in culture.

An estrogen-independent variant (R3) was selected by cloning wild type MCF-7 cells in soft agar in the presence of tamoxifen. R3 has a slightly slower growth rate under optimal growth conditions. Estradiol and tamoxifen have minimal effects on growth rate and thymidine incorporation as compared to wild type MCF-7 cells. R3 contains both cytoplasmic receptor and a receptor derived from crude nuclear extract with slightly higher numbers of cytosol receptor than wild type MCF-7. The same dissociation constant (Kd) and the same molecular weight (estimated by Sephadex chromatography) and sucrose density behavior for both cytoplasmic and nuclear estrogen receptor are seen in R3 and MCF-7. Estrogen receptor complexes were activated by salt and nucleotide and translocated to the nucleus equivalently in R3 and MCF-7. Relative binding affinities and extent of competition by estradiol and tamoxifen for cytoplasmic estrogen receptor are the same for MCF-7 and R3. Induction of progesterone receptor and nuclear estrogen receptor processing following treatment with estradiol in R3 is minimal compared with MCF-7. DNA affinity (as assessed by DNA cellulose chromatography) of estrogen receptor complexes from R3 and MCF-7 is different. R3 appears to represent a cell with a defect expressed in receptor function apart from the initial hormone-binding step.

Isolation and characterization of a tamoxifen-resistant cell line derived from MCF-7 human breast cancer cells.

A tamoxifen-resistant variant (R27) was selected by cloning wild type MCF-7 cells. R27 has the same growth rate as MCF-7 under optimal conditions. But tamoxifen has no effect on the growth rate (growth constant of R27 under control condition, 0.071 +/- 0.007 day-1; tamoxifen-treated cells, 0.073 +/- 0.011 day-1) and minimal effects on thymidine incorporation in R27, whereas tamoxifen has a strongly suppressive effect on the growth rate and thymidine incorporation in MCF-7. The estradiol-mediated stimulation of growth in R27 is somewhat less than in MCF-7 (growth constants of R27 and MCF-7 are 0.112 +/- 0.015 and 0.125 +/- 0.009 day-1, respectively. R27 contains unoccupied receptors in both cytoplasmic and crude nuclear extract fractions with slightly higher numbers of cytosol receptors than wild type MCF-7. The same dissociation constant and the same molecular weight estimated by Sephadex chromatography and the same sucrose density behavior were observed in R27 and MCF-7. Extent of competition with estradiol and tamoxifen for cytoplasmic estrogen receptor is the same for R27 and MCF-7. Induction of progesterone receptor following treatment with estradiol is the same for R27 and MCF-7. Estrogen receptor is activated by salt and nucleotide in both MCF-7 and R27; however, the extent of activation is much higher in MCF-7 than R27.

Nuclear Estrogen Receptor Release from Antiestrogen Suppression: Amplified Induction of Progesterone Receptor in MCF-7 Human Breast Cancer Cells*

This study describes a mechanism by which antiestrogen-suppressed MCF-7 human breast cancer cells in long term culture are rescued by estradiol. In these cells, nafoxidine (1 μM) is a potent inhibitor of cell growth, and unlike estradiol, nafoxidine fails to induce progesterone receptors at any dose tested. If in suppressed cells, nafoxidine-containing medium is replaced by medium containing estradiol (10 nM), cell growth resumes (as measured by increases in total protein and DNA), and progesterone receptor is synthesized. The average rate of progesterone receptor induction in cells treated with estradiol after nafoxidine suppression is 7-fold greater than that of cells which were given estradiol after growth in hormone-free medium. The cells rescued by estradiol from antiestrogen suppression have a 3-fold greater progesterone receptor content at the end of the study than do those cells which received estradiol without nafoxidine pretreatment. The lag period normally seen before progesterone receptor leve...

Estrogen-Induced Progesterone Receptor in the Syrian Hamster Kidney. IL Modulation by Synthetic Progestins*

The estrogen-induced progesterone receptor in the untransformed hamster kidney has been further characterized. The ability of various estrogenic hormones to induce specific renal progesterone receptor in the hamster was 17β-estradiol = estrone > estriol > 17α-estradiol. At the period of maximum progesterone receptor induction by either diethylstilbestrol (DES) or 17β-estradiol, hormone withdrawal resulted in a linear decline in progesterone receptor content to nearly castrate levels after 5 weeks. Readministration of estrogen in these hormonewithdrawn animals elicited a significant increase in progesterone receptor concentration in the kidney compared to castrate levels after only 9 days of treatment. In contrast to this anamnestic progesterone receptor response, a significant elevation in progesterone receptor concentration does not occur in previously untreated DES-primed hamsters until 19–24 days of treatment. Since progesterone had no appreciable affect on the ability of estrogen to induce progesteron...

Estrogen induction of progesterone receptor and its relationship to cell multiplication rate in the rat pituitary tumor cell line C29RAP.

The purpose of the present experiments is to compare the estrogen induction of a specific protein, cytoplasmic progesterone receptor (PR), and estrogen-stimulated cell multiplication in the C29RAP rat pituitary tumor cell line in animals and in cell culture. In tumors established by the injection of cells into isogenic rats, PR was reduced by 50% in ovariectomized rats compared to intact females carrying the same tumor cells. Estrogen replacement with 17β-estradiol (E2) Silastic implants in ovariectomized rats increased the PR concentration in the tumor cells 4–5 times above the control values in ovariectomized rats without E2 replacement. In cell cultures, PR was increased 3-to 4-fold after exposure to E2 (0.03–30 nM) for 48 h. The PR concentration was increased specifically by estrone, E2, and estriol but not by testosterone or dihydrotestosterone. The induction of PR was dose related; maximal induction was obtained at an E2 concentration of 3 nM. A latent period of more than 6 h for the induction of PR...

Assay of total estradiol receptor in tissue homogenate and tissue fractions by exchange with sodium thiocyanate at low temperature.

After injection of radioactive estradiol to ovariectomized rats, the [3H]estradiol--receptor complex transferred to the nuclei can be solubilized by low concentrations of NaSCN. The extraction by NaSCN is significantly more efficient than that obtained by KCl and is, in fact, complete; i.e., no radioactivity can be found in the nuclei after extraction. Since NaSCN also induces the exchange of receptor-bound estradiol with free hormone [Sica, V., Puca, G. A., Molinari, A. M., Buonaguro, F. M., & Bresciani, F. (1980) Biochemistry 19, 83], a simple assay method has been set up which measures receptor in tissue and tissue fractions, including nuclei and whole homogenate, at 0--4 degrees C, irrespective of whether the receptor is or is not interacting with endogenous hormone. The procedure consists of a simple incubation step at 0--4 degrees C overnight (16 h) of the nuclear fraction, cytosol, and a total homogenate in the presence of excess radioactive estradiol and 0.5 M NaSCN. This method is very easy to carry out, accurate, and precise and avoids the loss of binding sites which results from the heating procedures utilized in other methods. The ability to measure the binding in both the soluble and the particulate fractions of rat uterus permits the determination of the rate of the cytoplasmic to nuclear transfer of estrogen after injection of various hormone concentrations. No nuclear transfer was observed after administration of other nonestrogen hormones such as progesterone, testosterone, or hydrocortisone while a nonsteroid antiestrogen, tamoxifen, was able to translocate the receptor. It was found that 2 h after injection of estradiol into ovariectomized rats total receptor content of uterus shows a decrease which is proportional to the amount of hormone injected. After injection of a hyperphysiological dose of 17 beta-estradiol, a certain amount of the receptor--hormone complex remains in the cytosol for at least 4 h. The nuclear turnover of estradiol receptor related to the progesterone receptor induction has been studied. Actinomycin D and cycloheximide prevent nuclear processing.

Estrogen concentrations and effect of estradiol on progesterone receptors in the fetal and new-born guinea-pigs

Estradiol concentration in plasma of fetal guinea-pigs, determined by radioimmunoassay, varies from 9 to 40 pg/ml and the concentration of estrone between 80 to 105 pg/ml. The concentrations of both estrogens do not change significantly during fetal evolution. The total number of specific estradiol binding sites in fetal guinea-pig uterus reaches 18 to 20pmol/mg DNA at the end of gestation, and the concentrations of estradiol plus estrone is ∼1500 pg per g of this tissue which correlates well with the number of specific binding sites occupied by the endogenous estrogens (2–4 pmol/mg DNA). In the fetal uterus, estradiol receptors appear at about mid-gestation (from 34 days) but progesterone receptors are detectable only after 50–52 days of gestation. After estradiol treatment of the pregnant guinea pig (1 mg/kg/day for 3 days), the following changes take place in the fetal uterus: (1) Induction of progesterone receptor protein at an earlier period (37–42 days) when this receptor is not detectable in control animals. (2) Five to ten-fold increase in progesterone receptors at the end of gestation. (3) An increase of [ 3H]-progesterone uptake by fetal uterus (localized by autoradiography). (4) The stimulation of progesterone receptors is of long duration. Five days after treatment the values are similar to those obtained 24 h after the administration of the last dose of estradiol. (5) Similar effects were noted also in fetal ovary, but not in lungs, kidney, brain and heart or placenta. (6) The induction of progesterone receptors by estradiol is significantly lower in the uterus of new-born guinea pigs (2–7 days old). It is concluded that estradiol expresses its biological effect in the fetal compartment, increasing the levels of progesterone receptors.

Studies on mechanisms of estrogen and antiestrogen action in human breast cancer.

Studies have shown that patients whose tumors are ER (estrogen receptor) positive have at least tenfold chance of responding successfully to endocrine therapy. However, not all ER-containing tumors necessarily respond, and ERs are thus considered necessary but not sufficient markers of hormone dependence. It has been proposed that progesterone receptors (PgR) found in human breast tumors may serve as a marker of estrogen action in breast cancer, since PgR synthesis is known to be controlled by estrogens in the uterus. The authors have recently worked on a modal system which shows that PgR are under estrogen control and that PgR synthesis involves the ER. Their study concerning the effects of antiestrogens show that tamoxifen (TAM) is a potent inducer of PgR on the cells and that TAM's estrogenic property is masked at very high doses (1 mcg/M) which also inhibit cell growth. Nafoxidine (NAF), another antiestrogen, had little effect on PgR when tested over a wide dose range. It has been reported that growth-inhibitory effects of both antiestrogens can be reversed by estradiol; this indicates that the effects of these compounds are mediated through the ER system. A complex system in cells in which ER binding and translocation and turnover of nuclear receptors or their processing mediate the induction of PgR by estradiol is described.

Search for progesterone receptors in testes from various animal species

Using [ 3H]-R5020 as binding probe, we have looked for the presence of progesterone receptors in testis cytosol from a few vertebrate species ranging from the turtles to the humans. In addition, we have tested in the rat several experimental conditions potentially susceptible to induce progesterone receptors. With the exception of chicken, tfm patients and possibly the frogs, progesterone receptors could not be conclusively demonstrated in any of the other animal species tested nor could they be induced in the rat. Surprisingly, they were not present in Stanley-Grumbeck pseudohermaphrodite rats. In chicken testes, the levels of progesterone receptor were variable (3–45 fmol per mg prot.) in the adults and high (87 fmol per mg prot.) in one-day-old animals. The presence of progesterone receptor, a marker of estradiol action in many estrogen-target tissues, seems to be parallel to the capacity of chicken testes for sex reversal under the influence of estrogens. In human tfm, the presence of these receptors could also indicate that the gonads are estrogen-sensitive or that they were so during embryonic life although the nature of estrogenic action is not known.

Estrogen control of progesterone receptor in human breast cancer: role of estradiol and antiestrogen.

Estrogen antagonists are widely used in the treatment of breast cancer, and studies of their mechanism of action may provide clues to an understanding of tumor growth regulation and mechanisms of normal estrogen action. We have used human breast cancer cells in long term culture as an in vitro model to study the roles of estradiol and the antiestrogens, tamoxifen and nafoxidine, on cell growth and progesterone receptor (PgR) induction. Tamoxifen is found to have dual dose-dependent estrogenic/antiestrogenic properties. With 1 micrometer tamoxifen, cell growth and PgR induction are suppressed. These effects are reversed by estradiol. At lower doses (less than 0.1 micrometer), however, tamoxifen is a potent estrogen and rapidly induces (24--48 h) PgR, which increases 4- to 10-fold after 4--6 days and falls if tamoxifen is removed. Induction of PgR by estradiol is weaker but follows a similar time course. Tamoxifen-induced PgR is similar to that induced by estradiol; it sediments at 8S on sucrose density gradients, is a tight binder (R5020 Kd, 1.7 micrometer at 4 C and 0.87 nM at 15 C), and can be translocated to the nucleus by R5020. The dual properties of tamoxifen are not due to metabolic formation of an active antiestrogen from a prohormone precursor. In contrast, the action of the antiestrogen nafoxidine is not biphasic in MCF-7 cells; it does not induce PgR over a wide dose range and at high doses, the compound inhibits cell growth.

Nuclear mechanisms of estrogen action. Effects of estradiol and anti-estrogens on estrogen receptors and nuclear receptor processing.

This study attempted to show directly how antiestrogens (tamoxifen and nafoxidine) affect the estrogen receptor (ER) in MCF-7 human breast cancer cells. The mechanisms of estrogen action through the unfilled ERs in both cytoplasm (Rc) and nuclei (Rn) of this cell line were contrasted with the effects of estradiol (E) and antiestrogens on receptor binding nuclear processing reactions and progesterone receptor (PgR) synthesis. Within 5 minutes E bound Rc and translocated it to the nucleus; E also bound Rn directly. Beginning 30 minutes after E and continuing for 3-5 hours a progressive depletion of about 70% of filled nuclear receptors (RnE) occurred without reappearance of unfilled receptor. From that point on the RnE levels stabilized a step coincident with PgR induction. The antiestrogens also bound Rc and Rn as demonstrated by in vitro competition studies and by exchange rates of tritiated E for ER complexed to antiestrogens in whole cells. However the nuclear antiestrogen receptor complexes were aberrantly processed; 3-5 hours after tamoxifen only 30% of nuclear-bound receptors were lost before nuclear receptor levels stabilized whereas nafoxidine-bound nuclear receptors were not processed at all. In spite of the only partial ER processing after tamoxifen PgR was induced as long as lower steady-state ER levels were maintained. When tamoxifen was removed total cell ERs returned to control levels as Rc were restored. Simultaneously PgR falls. Rc binding and translocation alone however are not sufficient for PgR induction since if nuclear processing fails PgR induction is prevented (as is the case with nafoxidine). These data suggest that nuclear processing steps may be integral to the function of estrogenic compounds as inducers of PgR.

Hypothalamic and hypophysial progesterone receptors: estrogen-priming effect, differential localization, 5alpha-dihydroprogesterone binding, and nuclear receptors.

Abstract Specific progesterone receptor of 7 S which can be isolated from rat female hypothalamic and hypophysial cytosols was further investigated in terms of its induction effects by estrogen-priming, intracerebral localization, 5α-dihydroprogesterone (DHP) binding, and nuclear receptors. Estrogen-priming was necessary for the appearance of the cytosol receptors. The 7 S hypothalamic and hypophysial receptors were increased in a dose dependent fashion by estradiol injections. The maximal effective doses of estradiol benzoate for both tissues were 1 and 10 μg, respectively. The receptors were mostly localized in the median eminence, preoptic-anterior hypothalamus and anterior hypophysis of estrogen-primed immature and mature rats, but little or no 7 S binding was detected in the cerebral cortex, reticular formation, amygdaloid complex and posterior hypophysis. This differential localization of progesterone receptors resembles that of estrogen receptors, suggesting possible induction of progesterone receptors in these specific brain regions by estrogen. Progesterone receptor complexes of 5 S were isolated from “purified” nuclei of anterior pituitaries from estrogen-primed adult female rats. These results on the cytosol and nuclear receptors suggest that progesterone can directly act on the brain through its interaction with specific progesterone receptors in the hypothalamus and hypophysis. It is noteworthy that 5α-DHP can bind the progesterone receptors in both tissues, suggesting its direct feedback action on the brain through the receptors.

Estrogen control of progesterone receptor in human breast cancer. Correlation with nuclear processing of estrogen receptor.

MCF-7 cells of human breast cancer origin responded to estradiol (E) treatment with increased levels of progesterone receptor (PgR) showing that human breast cells which have undergone malignant changes can continue to synthesize a specific protein under hormone control. These results were obtained by studying the effects of addition and withdrawal of E on PgR synthesis and correlating this with estrogen receptor (ER) binding translocation nuclear processing reactions and restoration of unoccupied ER. In cells treated 1-5 days with E (.001-100 nM) basal PgR levels (.3-.7 pmol/mg of deoxyribonucleic acid) increased 3-6 fold. PgR response was dose-dependent with .1 nM E (a physiologic dose) the maximal effective dose. Growth and induction of PgR by .1 nM E was suppressed by an antiestrogen Tamoxifen at 10000-fold molar excess (1 mcgM) but was reversed by supraphysiological E (10 nM) which reduced the molar excess of Tamoxifen to only 100-fold. In this cell line ER unoccupied by hormone was not restricted to the cytoplasm (Rc) since a portion of the cellular receptor can also be found in the nucleus in unoccupied form (RN). The level of PgR induction correlated with the extent of Rc binding and translocation and also with the extent of Rn binding. At .1 nM E 85% of Rc and Rn were depleted and PgR was maximally stimulated. The levels of PgR also paralleled processing of bound ER (RnE) appearing in the nucleus. Processing during which a new steady-state level of RnE was achieved appeared to be a saturable event. At low E doses despite Rc and Rn binding of E RnE failed to accumulate and total ER decreased. The loss approached 70% at .1 nM E. But at higher E doses some nuclear receptor remained unprocessed and RnE levels increased. The processing effect was rapid beginning within 10 minutes of E addition and completed by 5 hours. Processed RnE levels were seen before PgR induction and during maintenance of induced PgR states. In estrogen withdrawn cells PgR fell in parallel with end of processing and restoration of Rc and Rn.

Progesterone receptor induction and RNA polymerase activation by estrogen derivatives

Progesterone receptor induction and RNA polymerase activation by estrogen derivatives