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Results for “Progesterone Congeners”

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At least 73 records · Page 4Linked to original sources
PubMed1997

Inhibition of proliferation of T47D human breast cancer cells: alterations in progesterone receptor and p53 tumor suppressor protein.

We have investigated the influence of three structurally different but functionally related compounds [1, 10 ortho-phenanthroline (phenanthroline), Rifampicin and aurin tricarboxylic acid (ATA)] on the rate and the extent of proliferation of progesterone-responsive T47D human breast cancer cells. These compounds have previously been used in this laboratory and have been shown to modulate properties of nucleic acid binding proteins. Because p53 and the progesterone receptor (PR) are both DNA binding proteins that appear to regulate proliferation of breast cells, alterations in T47D cell p53 and PR levels were examined to determine their relevance in cell proliferation. T47D cells were grown in the absence of phenol red and in the presence of 5% fetal calf serum with or without charcoal stripping in the presence of the inhibitors. The rate of proliferation of cells grown in Rifampicin containing medium exhibited nearly 70% inhibition. Phenanthroline, a known metal chelator, was an effective inhibitor of proliferation at 3 mM reducing the cell number by more than 75%. ATA (0.24-2.4 micrograms/ml) inhibited the growth of the cells by nearly 50%. Analysis of the mechanism of action of these compounds revealed that treatment with these compounds caused specific changes in the molecular composition of T47D cell PR. Whereas ATA caused increased stability of PR isoforms, Rifampicin induced a upshift in the mobility of PR in SDS gels-a phenomenon associated with hyperphosphorylation of steroid receptors (SRs). Phenanthroline treatment (> 2 mM) caused a complete down-regulation of PR and the tumor suppressor protein, p53. The downregulation of p53 paralleled the changes in the molecular composition of PR. We propose that the inhibition of T47D cell proliferation by phenanthroline, Rifampicin and ATA results from a number of cellular changes that include regulation of p53 and PR.

Antineoplastic Agents↗
PubMed1999

Agonist and antagonist-induced qualitative and quantitative alterations of progesterone receptor from breast cancer cells.

T47D cells, cultured in medium containing serum stripped of endogenous steroids, proliferate in response to treatment with the progesterone receptor (PR) agonist, R5020 or the PR agonist/antagonist, RU486, whereas the full PR antagonist, ZK98299 has no proliferative effects. Under estrogenized conditions, all of the PR ligands tested inhibit cell growth [23]. In order to determine whether the levels or phosphorylation state of PR are reflected in the growth patterns of T47D cells, we monitored the effects of these PR ligands on the immunoblotted PR band intensities, the relative intensities of PR-A and PR-B, and their phosphorylation states that are reflected in their altered mobility during SDS-PAGE. Under conditions where the PR ligands inhibit cell proliferation, each ligand had distinctively different qualitative and quantitative effects on PR. Short term treatment of the cells with R5020 or RU486 induced a characteristic phosphorylation-dependent upshift of both PR-A and PR-B. The phosphorylated PR was stable for up to 4 days after treatment of the cells with RU486, but was down regulated between 6-24 h after treatment with R5020. No replenishment of PR in cells treated with R5020 was detected. ZK98299, at concentrations tested, had no qualitative or quantitative effects on PR. Culturing cells for 8 days in medium containing steroid-depleted serum caused a significant reduction in the PR band intensity without causing a change in the ratio of PR-A and PR-B or their phosphorylation states. This decrease in the PR band intensity was reversed by maintaining the cells in 1 nM estrogen, but was potentiated by RU486 or ZK98299. These observations support the view that decreased PR levels may play a role in the stimulatory effects of R5020 and RU486 when cells are cultured under non-estrogenized conditions.

Blotting, Western↗
PubMed2002

New human breast cancer cells to study progesterone receptor isoform ratio effects and ligand-independent gene regulation.

All known progesterone target cells coexpress two functionally different progesterone receptor (PR) isoforms: 120-kDa B-receptors (PR-B) and N-terminally truncated, 94-kDa A-receptors (PR-A). Their ratio varies in normal and malignant tissues. In human breast cancer cells, homodimers of progesterone-occupied PR-A or PR-B regulate different gene subsets. To study PR homo- and heterodimers, we constructed breast cancer cell lines in which isoform expression is controlled by an inducible system. PR-negative cells or cells that stably express one or the other isoform were used to construct five sets of cells: (i) PR-negative control cells (Y iNull), (ii) inducible PR-A cells (Y iA), (iii) inducible PR-B cells (Y iB), (iv) stable PR-B plus inducible PR-A cells (B iA), and (v) stable PR-A plus inducible PR-B cells (A iB). Expression levels of each isoform and/or the PR-A/PR-B ratios could be tightly controlled by the dose of inducer as demonstrated by immunoblotting and transcription studies. Induced PRs underwent normal progestin-dependent phosphorylation and down-regulation and regulated exogenous promoters as well as endogenous gene expression. Transcription of exogenous promoters was dependent on the PR-A/PR-B ratio, whereas transcription of endogenous genes was more complex. Finally, we have described several genes that are regulated by induced PR-A even in the absence of ligand.

Breast Neoplasms↗
PubMed1997

Transcriptional regulation of the murine multidrug resistance gene mdr1b by progesterone occurs via an indirect mechanism.

The murine multidrug resistance gene mdr1b is highly induced in the endometrium during pregnancy. Evidence suggests that induction occurs mainly as a result of progesterone action. To study the molecular mechanisms involved in this induction, 5'-flanking sequences between -540 and +97 of the mdr1b gene were fused to the reporter gene, bacterial chloramphenicol acetyltransferase (p540CAT). Unlike most progesterone-responsive genes, mdr1b is preferentially activated by the A form of the progesterone receptor. We now report that activation is not observed with a DNA-binding domain mutant of progesterone receptor A (PRA) suggesting that induction occurs at the transcriptional level. Time course experiments demonstrated that induction was first observed 12 hr after hormone addition, suggestive of a secondary (or late) response gene. Sequence comparison highlighted the region M1 (-234 to -206), which contains a partially conserved progesterone response element. Its functional significance was evaluated by expression assays and gel shift analysis. Reporter plasmids with modifications of this element were transfected into HeLa cells. Constructs containing the native M1 element, or a mutated element (M1mt) that eliminated any similarity to a progesterone response element, were induced four-fold by progesterone whereas an element containing a consensus progesterone response element (M1PRE) was induced eight-fold. In addition, by gel shift analysis, the M1 element did not bind the progesterone receptor or any other factors. This suggested that the M1 region does not participate in the response to progesterone. 5' Nested deletion analysis, used to identify other regions of the upstream regulatory region that contributed to induction by progesterone, demonstrated that enhancer sequences between -122 and -65, which contain binding sites for C/EBPbeta and NF-Y, were important. Mutations in the binding sites for these factors decreased induction by progesterone. On the basis of our studies using 540 bp of upstream sequence, mdr1b is activated transcriptionally by progesterone, in an indirect manner dependent on basal factors.

Animals↗
PubMed1990

Kinetic analysis of the binding of nomegestrol acetate to the progesterone receptors in rat uterus by competition studies.

The characteristics of binding (Kinetic and equilibrium binding analysis) of nomegestrol acetate (NOM, 17 alpha-acetoxy-6 alpha-methyl-19-nor-pregna-4.6-diene-3.20-dione) to the progesterone receptor (PgR) in rat uterine cytosolic fraction were determined in comparison to progesterone (P), to fully appreciate the amplitude and specificity of the induced biological response. Since an appropriate radio-labelled form of this steroid molecule was not available, competition studies were performed against the synthetic progestin: [3H]-Organon 2058 [( 3H]-ORG). This allowed a direct comparison between the unlabelled forms of NOM and P, the kinetic constants of which were respectively: Inhibition constant (Ki): 22.8 and 34.3 nM; Association rate constant (k1): 0.39 X 10(3) and 0.21 X 10(3) M-1.s-1; Dissociation rate constant (k-1): 1.81 X 10(-5) and 2.16 X 10(-5) s-1. These results are much more informative than the mere determination of relative binding affinities which only reflect the specificity of the PgR. It was concluded that NOM behaves like the natural hormone in the cytosol of rat uterus.

Animals↗
PubMed1998

Agonist and antagonists induce homodimerization and mixed ligand heterodimerization of human progesterone receptors in vivo by a mammalian two-hybrid assay.

This study utilizes the mammalian two-hybrid system to examine the role of ligand in the dimerization of human progesterone receptor (hPR). The GAL4 DNA-binding domain and the herpes simplex virus VP16 transactivation domain were fused to the amino terminus of full-length hPR (both the A and B isoforms) to produce chimeric proteins. PR dimerization was detected by the ability of cotransfected GAL4/PR and VP16/PR chimeras in COS cells to induce expression of a reporter gene under the control of GAL4-binding sites (pG5CAT). Hormone agonist-dependent interactions were observed between the two like isoforms of PR (A-A and B-B) and between PR-A and PR-B (A-B), indicating that hormone can stimulate the formation of the three possible dimeric forms of PR within cells. In contrast, neither type I (ZK98299) nor type II (RU486, ZK112993) progestin antagonists stimulated interaction between these same hybrid PR proteins. However, activation of the VP16/PR chimera by antagonists on a progesterone response element-controlled reporter gene (DHRE-E1b-CAT) was only a fraction (4-13%) of that stimulated by agonist R5020. One possibility for the failure to detect an induction in the two-hybrid assay is antagonist-induced repression of the activity of the VP16/PR fusion protein rather than a failure of antagonists to stimulate interaction between the hybrid proteins. To test this idea, an UP-1 carboxyl-terminal truncation mutant of PR was used to construct the two-hybrid proteins. PR-UP-1 selectively binds antagonists, but not agonists, and is fully activated in response to antagonists. Both types of progestin antagonists stimulated interactions between GAL4/PR(UP-1) and VP16/PR(UP-1) hybrid proteins, indicating that antagonists are capable of stimulating PR dimerization in cells and do not function by disrupting or preventing dimerization. To determine whether PR bound to an antagonist can dimerize in whole cells with PR bound to agonist, GAL4/PR(UP-1) was paired in the two-hybrid assay with a VP16/PR fusion protein harboring a point mutation in PR at amino acid 722 (Gly-Cys) that specifically binds progestin agonist but not antagonist. Neither R5020 nor RU486 alone stimulated interaction between these ligand-specific PR hybrid proteins. However, strong interaction was detected by addition of both agonist and antagonists, indicating the formation of mixed ligand heterodimers and that both PR partners require ligand for dimerization to occur. Based on electrophoretic gel mobility shift assays (EMSAs), these heterodimers appear to have substantially reduced DNA binding activity. Progestin antagonists inhibit agonist activation of PR at concentrations that are too low to be accounted for by a simple competition mechanism for binding to PR. We propose that antiprogestin inactivation of PR in trans by heterodimerization contributes to the biological potency of these compounds.

Animals↗
PubMed1999

Hormone-dependent interaction between the amino- and carboxyl-terminal domains of progesterone receptor in vitro and in vivo.

Full transcriptional activation by steroid hormone receptors requires functional synergy between two transcriptional activation domains (AF) located in the amino (AF-1) and carboxyl (AF-2) terminal regions. One possible mechanism for achieving this functional synergy is a physical intramolecular association between amino (N-) and carboxyl (C-) domains of the receptor. Human progesterone receptor (PR) is expressed in two forms that have distinct functional activities: full-length PR-B and the amino-terminally truncated PR-A. PR-B is generally a stronger activator than PR-A, whereas under certain conditions PR-A can act as a repressor in trans of other steroid receptors. We have analyzed whether separately expressed N- (PR-A and PR-B) and C-domains [hinge plus ligand-binding domain (hLBD)] of PR can functionally interact within cells by mammalian two-hybrid assay and whether this involves direct protein contact as determined in vitro with purified expressed domains of PR. A hormone agonist-dependent interaction between N-domains and the hLBD was observed functionally by mammalian two-hybrid assay and by direct protein-protein interaction assay in vitro. With both experimental approaches, N-C domain interactions were not induced by the progestin antagonist RU486. However, in the presence of the progestin agonist R5020, the N-domain of PR-B interacted more efficiently with the hLBD than the N-domain of PR-A. Coexpression of steroid receptor coactivator-1 (SRC-1) and the CREB binding protein (CBP), enhanced functional interaction between N- and C-domains by mammalian two-hybrid assay. However, addition of SRC-1 and CBP in vitro had no influence on direct interaction between purified N- and C-domains. These results suggest that the interaction between N- and C-domains of PR is direct and requires a hormone agonist-induced conformational change in the LBD that is not allowed by antagonists. Additionally, coactivators are not required for physical association between the N- and C-domains but are capable of enhancing a functionally productive interaction. In addition, the more efficient interaction of the hLBD with the N-domain of PR-B, compared with that of PR-A, suggests that distinct interactions between N- and C-terminal regions contribute to functional differences between PR-A and PR-B.

Animals↗
PubMed2001

Stromal PRs mediate induction of 17beta-hydroxysteroid dehydrogenase type 2 expression in human endometrial epithelium: a paracrine mechanism for inactivation of E2.

Progesterone stimulates the expression of 17beta-hydroxysteroid dehydrogenase (HSD) type 2, which catalyzes the conversion of the potent estrogen, E2, to an inactive form, estrone, in epithelial cells of human endometrial tissue. Various effects of progesterone on uterine epithelium have recently been shown to be mediated by stromal PRs in mice. We describe herein a critical paracrine mechanism whereby progesterone induction of 17beta-HSD type 2 enzyme activity, transcript levels, and promoter activity in human endometrial epithelial cells are mediated primarily by PR in endometrial stromal cells. Medium conditioned with progestin-pretreated human endometrial stromal cells robustly increased 17beta-HSD type 2 enzyme activity (2-fold) and mRNA levels (13.2-fold) in Ishikawa malignant endometrial epithelial cells. In contrast, direct progestin treatment of Ishikawa epithelial cells gave rise to much smaller increases in enzyme activity (1.2-fold) and mRNA levels (4-fold). These results suggest that progesterone- dependent paracrine factors arising from stromal cells are primarily responsible for the induction of epithelial 17beta-HSD type 2 expression in the endometrium. We transfected serial deletion mutants of the -1,244 bp 5'-flanking region of the 17beta-HSD type 2 gene into Ishikawa cells. No progesterone response elements could be identified upstream of the 17beta-HSD type 2 promoter. Stromal PR-dependent induction of the 17beta-HSD type 2 promoter was mediated by a critical regulatory region mapped to the -200/-100 bp sequence. Direct treatment of Ishikawa cells with progestin gave rise to a maximal increase in the activity of -200 bp/Luciferase construct only by 1.2-fold, whereas medium conditioned by progestin-pretreated endometrial stromal cells increased promoter activity up to 2.4-fold in a time- and concentration-dependent manner. The stimulatory effect of medium conditioned by progestin-pretreated stromal cells was enhanced strikingly by increasing stromal cell PR levels with the addition of estrogen. This epithelial-stromal interaction was specific for endometrial epithelial cells, since 17beta-HSD type 2 could not be induced in malignant breast epithelial cells by media conditioned with progestin-treated breast or endometrial stromal cells. In conclusion, progesterone regulates the conversion of biologically active E2 to estrone by inducing the 17beta-HSD type 2 enzyme in human endometrial epithelium primarily via PR in stromal cells, which secrete factors that induce transcription mediated primarily by the -200/-100 bp 5'-regulatory region of the 17beta-HSD type 2 promoter.

17-Hydroxysteroid Dehydrogenases↗
PubMed1995

Identification of a group of Ser-Pro motif hormone-inducible phosphorylation sites in the human progesterone receptor.

The human progesterone receptor (PR) is a member of the steroid/thyroid hormone superfamily of nuclear receptors. The receptor is expressed as two forms, PR-B and the shorter PR-A, which lacks the NH2-terminal 164 amino acids of PR-B; whereas PR-B seems to be predominantly a transcriptional activator, PR-A also functions as a repressor. Our previous studies of PR expressed in T47D breast cancer cells have shown that PR is a phosphoprotein whose phosphorylation is enhanced in response to hormone. There is an initial rapid (minutes) increase in phosphorylation followed by a slower, less substantial increase, which results in decreased mobility of the receptor on sodium dodecyl sulfate gels. We now report the identification of three phosphorylation sites, which are predominantly phosphorylated during the later phase of the response to hormone. These sites, Ser102, Ser294, and Ser345, are all found in Ser-Pro consensus sequences. Whereas Ser294 and Ser345 are common to PR-A and PR-B, Ser102 is unique to PR-B. Finally, we demonstrate that phosphorylation of Ser345 is associated with the altered mobility on sodium dodecyl sulfate gels.

Amino Acid Sequence↗
PubMed1978

Characterization and assay of the progesterone receptor in rat uterine cytosol.

The synthetic progestogen R5020 (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) binds with high affinity (Ka = 8.8 X 10(8) l/mol at 0 degree C) to the progesterone receptor from rat uterine cytosol. At nanomolar concentrations, equilibrium is attained in less than 90 min. R5020 has a very low affinity for other specific steroid-binding proteins (corticosteroid-binding globulin and oestrogen receptors) present in relatively high concentrations in the urine cytosol. The affinity of the receptor for the natural hormone progesterone is remarkably low (Ka = 1 X 10(8)-1.7 X 10(8) l/mol at 0 degree C) which explains the instability of progesterone-receptor complexes. Advantage may be taken of this property to remove endogenous progesterone easily by charcoal treatment at 0 degree C, a treatment which does not modify the concentration of receptors. A method based on these characteristics is described for the assay of the total number (progesteron-bound and unbound) of receptor sites in uterine cytosol. This assay may be used in various physiological situations where endogenous progesterone is present at unknown concentrations.

Animals↗
PubMedDate not supplied

An empirical basis for the competition by dexamethasone to progesterone receptors as estimated with the synthetic progestin R5020.

Normal mammary glands of mice contain both progesterone and glucocorticoid receptors and the levels of both these receptors are modulated as a function of development. Measurement of progesterone receptors using the synthetic progestin, R5020, has led to conflicting data both with regard to the presence of progesterone receptor during certain developmental stages of the mammary gland and the ability of the glucocorticoids to compete for specific R5020 binding sites. In this report we have identified experimental conditions which allow for the separate measurements of the progesterone and glucocorticoid receptors in the same cytosol. If sulfhydryl reducing agents such as dithiothreitol are excluded from the assay buffer, R5020 binds to only a single class of high affinity sites in mammary cytosol of virgin mice and these binding sites exhibit a strict steroid specificity characteristic of progesterone receptors. In contrast, if dithiothreitol is included in the buffers, R5020 binds not only to the high affinity sites but also to certain saturable lower affinity sites; these lower affinity sites for R5020 also bind glucocorticoids such as dexamethasone. These findings should facilitate more accurate quantitation of both progesterone and glucocorticoid receptors in normal and neoplastic tissues and also be applicable to studies on the mechanism(s) of progesterone action.

Animals↗
PubMed2003

Distinct functional differences of human progesterone receptors A and B on gene expression and growth regulation in two endometrial carcinoma cell lines.

To study the functional differences between the two progesterone receptor isoforms (hPRA and hPRB) in human endometrial cancer, two new endometrial carcinoma cell lines were created-one expressing hPRA and one expressing hPRB.A well-differentiated, hPR-negative Ishikawa cell line was stably transfected with either hPRA or hPRB cDNA. Transfected cells were selected, and two cell lines expressing approximately equal amounts of receptor were isolated-one expressing hPRA (PRA-14) and one expressing hPRB (PRB-59). Cell growth experiments revealed a growth-inhibitory response to progestins (MPA and R5020) in the PRB-59 cells but not in the PRA-14 cells. Differences in expression of genes targeted by the two isoforms were studied using a cDNA expression array technique. A different set of genes appeared to be progesterone regulated in the PRA-14 cells than in the PRB-59 cells. None of the genes were regulated by both hPRA and hPRB. Insulin-like growth factor binding protein 3 expression was studied in more detail as an example of a gene regulated in PRB-59 cells but not in PRA-14 cells. We established a new model to study functional differences between the two hPR isoforms in human endometrial carcinoma cells. This model revealed distinctive differences in target gene regulation between the two hPR isoforms. Moreover, antiproliferative actions of progesterone on human endometrial cancer cells could be observed only in the PRB-expressing cell line.

Cell Division↗
PubMedDate not supplied

[Influence of the structure of synthetic gestagens on their binding to progesteron receptors in the endometrium].

Chemical modification of progesterone molecule leads to changes both in the gestagenic activity of new derivatives and in their specific binding with progesterone receptors. The passage from esters (acetomepregenole, butagest) to the corresponding OH-forms such as 17a-acetoxy-3b-hydroxy-6-methyl-pregna-4,6-dien-20-one (ABMP)is accompanied by an increase in the binding with progesterone receptors in vitro. The translocation of a double bond from endocyclic (N6-N7) to exocyclic position (methylene group at N6 in ABMP) has no significant effect on the ability to binding with progesterone receptors.

17-alpha-Hydroxyprogesterone↗