Regulation Of Progesterone Receptor Research Articles

Progesterone receptor regulation in T47D human breast cancer cells: analysis by density labeling of progesterone receptor synthesis and degradation and their modulation by progestin.

We have examined the effect of progestin on the regulation of cellular progesterone receptor (PR) levels and have used dense amino acid-density shift experiments to determine the mechanism by which progestin markedly decreases PR. We have utilized T47D human breast cancer cells which contain high levels of PR and are progestin responsive. When these cells are exposed to the progestin R5020, there is a time- and concentration-dependent decrease in PR levels. Experiments with different concentrations of R5020 reveal that the rate and extent of PR decrease reflect the time course of receptor occupancy and the fractional saturation of receptor. With a high concentration of ligand (20 nM) that labels all receptors rapidly, reductions in PR levels (processing) occur immediately and proceed rapidly to levels that are 15-20% of the initial; at lower concentrations (5 nM), where it takes several hours to achieve full saturation of receptors, there is a delay before the maximal rate of processing develops and then continues to achieve final receptor levels that are 15-20% of the initial; with a low concentration of ligand (0.5 nM), binding is even slower and never reaches full receptor saturation, with the consequence that processing is not only delayed but also less complete. Immunochemical detection of PR with a monoclonal antibody (B39) reveals a good correspondence between the loss of immunoreactive and hormone binding PR, and analysis of the A (Mr 85,000) and B (Mr 115,000) receptor forms on Western blots demonstrates that both A and B receptor forms are reduced after exposure to R5020. Density labeling of PR by biosynthetic incorporation of 2H, 13C, 15N (dense) amino acids reveals that PR turns over with a half-life of 21 h in control cells. In cells exposed to 20 nM R5020, PR levels decline and receptor half-life is reduced to 6 h. In addition, there is also a time-dependent decrease in the rate constant of PR synthesis, k8, which decreases to less than 10% of its initial value after 24 h of R5020 exposure. Thus, the R5020-evoked reduction in PR levels in this progestin-sensitive cell line is due both to a marked increase in the rate of receptor degradation as well as a dramatic decrease in the rate of receptor synthesis.

Regulation by estradiol of the progesterone receptor in the hypothalamus and pituitary: an immunohistochemical study in the chicken.

An antibody (IgG-RB) against the chick oviduct progesterone receptor (PR) was used to study the regulation of PR by estrogen in chicken pituitary and hypothalamus. PR was revealed exclusively in cell nuclei, whatever the hormonal state, including the absence of progesterone. In immature untreated chickens, PR was not detected in the pars distalis, while the pars tuberalis, preoptic area, and infundibular region of hypothalamus showed IgG-RB-immunoreactive cells or neurons. Estrogenic stimulation induced the appearance of PR in cells of the pars distalis and increased the immunoreactivity in the hypothalamus of young chickens of both sexes. After hormonal withdrawal, PR immunostaining returned to the level in untreated immature animals. In young hens, before they laid the first egg, PR appears progressively in pars distalis cells during the pubertal period. Antibodies to pituitary hormones (LH, FSH, GH, PRL, and ACTH) were used to characterize the secretory properties of PR-containing cells. LH- and FSH-immunoreactive cells were localized throughout the pars distalis in untreated animals. After estradiol treatment of young sexually immature chickens, immunostaining of LH and FSH was strongly reduced, up to extinction, in many gonadotropic cells, and only few PR-containing cells demonstrated some immunoreactivity in the cytoplasm. In contrast, in juvenile hens, a majority of PR-containing cells were identified as LH immunoreactive, that is gonadotrophs. There are probably two reasons for the paucity of doubly reactive cells in estradiol-treated chickens. One is technical, the optimal fixation time for both LH (greater than 20 h) and PR (less than 7 h) makes it practically impossible to reveal the hormone and the receptor at the same time. The other is related to the physiology of the system, which involves the simultaneous decrease in LH immunoreactivity and the PR induction in gonadotrophs by estradiol. The presence of PR in gonadotrophs suggests a direct feedback mechanism of sex steroids on pituitary cells in addition to the indirect effect through GnRH modulation. The hormonal content of PRL-, GH-, and ACTH-producing cells was not modified by estradiol treatment, as judged by the appropriate immunoreactivity, and their nuclei did not display PR. However, PRL cells and PR-containing cells were frequently present near each other within the same cell cords.

Ligand-modulated regulation of progesterone receptor messenger ribonucleic acid and protein in human breast cancer cell lines.

We have examined the effects of estrogen and progestin agonist and antagonist ligands on regulation of progesterone receptor (PR) protein and mRNA levels in a variety of human breast cancer cell lines. By Northern blot analysis, using human PR cDNA probes, PR mRNA in T47D and MCF-7 cells appears as five species of approximately 11.4, 5.8, 5.3, 3.5, and 2.8 kilobases. PR mRNA species are not detected in the PR protein-negative breast cancer cell lines MDA-MB-231 and LY2. T47D cells contain high levels of PR mRNA and protein (detected by hormone binding assay or Western blot analysis), and the PR protein and mRNA content of T47D cells are reduced to about 10% of the control level within 48 h of treatment with 10 nM promegestone; 17, 21-dimethyl-19-nor-pregna-4,9-diene-3, 20-dione (R5020) or 16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20-dione (ORG2058), both potent progestins. In contrast, treatment of T47D cells with the antiprogestin 17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]-17 alpha-(1-propynyl)-estra- 4, 9-dien-3-one) (RU38486) reduces PR protein and mRNA levels only transiently. PR protein and mRNA are virtually undetectable in control MCF-7 cells grown in the absence of estrogens. When estradiol is administered to MCF-7 cells, the PR mRNA and protein levels increase gradually and proportionately (10- or 40-fold, respectively, in 3 days).(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone receptor regulation by 17 beta-estradiol in human endometrial carcinoma grown in nude mice.

Regulation of progesterone receptor (PR) in human endometrial carcinoma was investigated in vivo in a multisite nude mouse tumor experimental system by estrogen administration and withdrawal. The cytosolic PR concentration was low in tumors grown in the absence of 17 beta-estradiol, but increased rapidly upon estrogen administration, reaching a maximal receptor concentration of 1.4-1.6 pmol/mg cytosol protein within 7 days. Protein blot analysis using a monoclonal antibody (hPRa 1) raised against PR from EnCa 101 showed no immunoreactivity in tumors grown in the absence of estrogen. Immunoreactive proteins of mol wt 116,000 and 81,000 were detectable 8 h after estrogen administration and increased in intensity as the cytosolic PR concentration increased. Interestingly, the protein of mol wt 116,000 was composed of mol wt isoforms and was detectable as a doublet 8 h after estrogen administration and finally as a triplet. The effect of estrogen withdrawal on EnCa 101 PR concentration and structure was determined by removal of 17 beta-estradiol pellets (200 pg/ml plasma) from EnCa 101-bearing animals after achievement of maximal tumor PR concentrations. The PR concentration in tumor cytosols decreased in a biphasic manner after estrogen removal, with the initial rapid phase having a half-life of around 2 days. Cytosolic PR was still detectable 21 days after estrogen withdrawal. Protein blot analysis showed that immunoreactive proteins of mol wt 116,000 and 81,000 were also detectable up to that time. Photoaffinity labeling with [3H]R5020 demonstrated that the 81,000 mol wt protein, as well as each of the triplet proteins at mol wt 116,000, was specifically photoaffinity labeled. The 116,000-mol wt protein was detected as a triplet on protein blots until 13 days after estrogen withdrawal, when diminution in the intensity of the highest mol wt triplet protein was noted.

Estrogen responsiveness of normal mouse mammary cells in primary cell culture: association of mammary fibroblasts with estrogenic regulation of progesterone receptors.

Estrogens enhance proliferation of normal mouse mammary cells in vivo. However, when cultured alone, normal mouse mammary epithelial cells fail to exhibit a proliferative response to estrogen in vitro; the basis for this lack of in vitro responsiveness to estrogen is not known. The purpose of the present study is to determine if cultured normal mouse mammary cells possess estrogen receptors (ER) and/or progesterone receptors (PgR) and if the ER mechanism is functional, as measured by the ability of estrogens to regulate PgR. Recent findings that mammary fibroblasts can influence the behavior of mammary epithelial cells in vitro led us to investigate their effect on epithelial cell responsiveness to estrogen. In these studies, collagenase-dissociated mammary glands of midpregnant BALB/c mice were the source of mixed cultures (containing both epithelial cells and fibroblasts) and epithelial or fibroblast cultures. The purity of epithelial or fibroblast cultures was quantified immunocytochemically using antivimentin antibody as a fibroblast marker. Steroid hormone binding was quantified in intact cultured cells using [3H]R5020 and 17 beta-[3H]estradiol as the ligands. Specific high affinity binding sites for estrogen (Kd = 3.1 +/- 0.8 X 10(-10] and progestins (Kd = 3.3 +/- 1.2 X 10(-9) M) were detected in mixed cultures. To assess the possible role of mammary fibroblasts, we investigated cultures containing only fibroblasts which were derived by differential centrifugation. When 17 beta-estradiol was added to the culture medium, a significant (P less than 0.01) increase in PgR concentration was observed in mixed cultures. While mixed cultures maintain responsiveness to estrogen in vitro, as measured herein, the epithelial cultures, derived by differential centrifugation and Percoll gradient sedimentation, did not. However, estrogenic regulation of PgR appears to be specific to epithelial cells in mixed cultures, since fibroblast cultures neither contained PgR nor displayed estrogen-inducible PgR. The lack of responsiveness of epithelial cultures is not due to a loss or decrease in the ER concentration. Thus, the presence of mammary fibroblasts appears to be associated with epithelial cell responsiveness to estrogen in vitro.

Regulation of estrogen and progesterone receptor levels in mouse mammary epithelial cells grown in serum-free collagen gel cultures.

The effect of collagenase dissociation of virgin mouse mammary glands on the level of mammary epithelial cytosolic estrogen receptors (ER) and progesterone receptors (PR) was assessed. After cell dissociation, ER was present in mammary epithelial cells at concentrations similar to those found in the whole gland. However, PR appeared to be affected by the collagenase treatment. The regulation of ER and PR in mouse mammary epithelial cells isolated by collagenase dissociation and grown within collagen gels was then determined. After 7 days in culture under serum-free conditions inside a collagen gel, PR and, to a lesser extent ER, as characterized by high affinity binding and specificity, were present in the epithelial cells. Although at a low level, the ER were determined to be functional, since estradiol (E2) was able to promote nuclear accumulation of ER and to induce PR. PRL was able to increase cytosolic ER and PR concentrations. The combination of progesterone (P) and PRL was more effective than PRL or P alone in increasing PR. The induction of PR by P and PRL was inhibited when epidermal growth factor was present in the culture medium. Previous studies have shown that P, PRL, and epidermal growth factor, but not E2 (either alone or in combination with these factors) are able to stimulate cell proliferation in vitro. We conclude that the effects of E2 on protein synthesis and proliferation are dissociated in vitro. The difference between the effect of E2 and PRL or P on growth may be related either to the initial concentrations of their respective receptors or estrogen may stimulate growth indirectly.

The regulation of progesterone receptor by 17 beta estradiol and tamoxifen in the Zr-75-1 human breast cancer cell line in defined medium.

The regulation of progesterone receptor by 17 beta estradiol and tamoxifen in the ZR-75-1 human breast cancer cell line in defined medium is described. ZR-75-1 cells maintained in serum free hormone supplemented medium minus estradiol lack progesterone receptor activity. Readdition of estradiol to these cells leads to a marked stimulation of progesterone receptor activity (0 to greater than 100 fmols of specifically bound progesterone per million cells). Tamoxifen (10(-6)M-10(-8)M) does not stimulate progesterone receptor activity in this cell line. The presence of progesterone receptor activity is not directly related to growth. Withdrawal of insulin in the continued presence of estradiol has no effect on progesterone receptor concentration although net cell growth ceases. Conversely, withdrawal of estradiol in the continued presence of insulin induces a cessation of net cell growth accompanied by a loss of all progesterone receptor activity within 3-5 days.