16-alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3-20-dione and Breast-Neoplasms

An investigation into certain aspects of the interaction between oestriol and oestrogen receptors (specificity and kinetics) shows the specificity of the receptors to be very similar in various target tissues-human breast tumour and myometrium, rat myometrium, and rabbit myometrium, pituitary, and thymus-and a preferential binding of oestriol to specific oestrogen receptors to be unlikely. Competition for the oestrogen receptors in the uterus of the three species was analysed in vitro under equilibrium and non-equilibrium conditions, and oestriol and epimestrol were found to compete with oestradiol-17 beta more strongly in the latter. In rat uterus a late oestrogenic effect, the synthesis of the progesterone receptor, induced by ethinyloestradiol, is shown to be inhibited by oestriol. Oestriol emerges as a short-action agonist when administered in a single dose. It is also concluded that oestriol may prevent eostradiol-17 beta from inducing a full uterotrophic response.

Topics: Animals; Binding, Competitive; Breast Neoplasms; Castration; Cells, Cultured; Epimestrol; Estradiol; Estriol; Ethinyl Estradiol; Female; Humans; Kinetics; Middle Aged; Norpregnenes; Pituitary Gland; Pregnenediones; Rabbits; Rats; Receptors, Estrogen; Receptors, Progesterone; Thymus Gland; Uterus

PFKFB (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) catalyses the synthesis and degradation of Fru-2,6-P2 (fructose-2,6-bisphosphate), a key modulator of glycolysis and gluconeogenesis. The PFKFB3 gene is extensively involved in cell proliferation owing to its key role in carbohydrate metabolism. In the present study we analyse its mechanism of regulation by progestins in breast cancer cells. We report that exposure of T47D cells to synthetic progestins (ORG2058 or norgestrel) leads to a rapid increase in Fru-2,6-P2 concentration. Our Western blot results are compatible with a short-term activation due to PFKFB3 isoenzyme phosphorylation and a long-term sustained action due to increased PFKFB3 protein levels. Transient transfection of T47D cells with deleted gene promoter constructs allowed us to identify a PRE (progesterone-response element) to which PR (progesterone receptor) binds and thus transactivates PFKFB3 gene transcription. PR expression in the PR-negative cell line MDA-MB-231 induces endogenous PFKFB3 expression in response to norgestrel. Direct binding of PR to the PRE box (-3490 nt) was confirmed by ChIP (chromatin immunoprecipiation) experiments. A dual mechanism affecting PFKFB3 protein and gene regulation operates in order to assure glycolysis in breast cancer cells. An immediate early response through the ERK (extracellular-signal-regulated kinase)/RSK (ribosomal S6 kinase) pathway leading to phosphorylation of PFKFB3 on Ser461 is followed by activation of mRNA transcription via cis-acting sequences on the PFKFB3 promoter.

Topics: Base Sequence; Breast Neoplasms; Cell Line, Tumor; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Norgestrel; Phosphofructokinase-2; Pregnenediones; Progesterone Congeners; Promoter Regions, Genetic; Receptors, Progesterone; RNA, Messenger; RNA, Neoplasm

Estrogen treatment of MCF-7 human breast cancer cells allows the reinitiation of synchronous cell cycle progression in antiestrogen-arrested cells. Here, we report that progestins also reinitiate cell cycle progression in this model. Using clonal cell lines derived from progesterone receptor (PR)-negative MCF-7M13 cells expressing wild-type or mutant forms of PRA and PRB, we show that this effect is mediated via PRB, not PRA. Cell cycle progression did not occur with a DNA-binding domain mutant of PRB but was unaffected by mutation in the NH(2)-terminal, SH3 domain interaction motif, which mediates rapid progestin activation of c-Src. Thus, the progestin-induced proliferative response in antiestrogen-inhibited cells is mediated primarily by the transcriptional activity of PRB. Analysis of selected cell cycle targets showed that progestin treatment induced levels of cyclin D1 expression and retinoblastoma protein (Rb) phosphorylation similar to those induced by estradiol. In contrast, progestin treatment resulted in only a 1.2-fold induction of c-Myc compared with a 10-fold induction by estradiol. These results support the conclusion that progestin, in a PRB-dependent manner, can overcome the growth-inhibitory effects of antiestrogens in estrogen receptor/PR-positive breast cancer cells by the induction of cyclin D1 expression. The mediation of this effect by PRB, but not PRA, further suggests a mechanism whereby abnormal regulation of the normal expression ratios of PR isoforms in breast cancer could lead to the attenuation of antiestrogen-mediated growth arrest.

Topics: Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Drug Interactions; Estradiol; Estrogen Receptor Modulators; Fulvestrant; Gene Expression Regulation, Neoplastic; Humans; Pregnenediones; Progestins; Proto-Oncogene Proteins c-myc; Receptors, Progesterone; Transcription, Genetic

Exposure of MCF-7 cells to single and/or repeated low gamma-ray doses (0.5 to 8 Gy) resulted in a decrease in the capacity of these cells to concentrate tritiated estradiol ([3H]E2) (reduction of the number of binding sites). The decrease in the [3H]E2-binding capacity was higher than the survival rate, indicating that it could not be ascribed to cell death. Moreover, such low irradiation doses failed to similarly affect the specific incorporation of [3H]ORG 2058, even when the progesterone receptor was induced by E2, a finding that rejects the hypothesis of a nonspecific effect on all steroid hormone receptors. This loss of [3H]E2 binding was reflected by the elimination of the estrogen receptor alpha (ER) when the latter was assessed by immunocytochemistry. However, additional immunochemical studies (Western blot data) performed on cell extracts under denaturing conditions failed to show any similar elimination of the ER peptide, suggesting that the loss of E2-binding capacity would be relevant to subtle changes in the ER structure and/or ER-associated proteins. The loss of binding capacity, produced by a 3-Gy irradiation, failed to decrease the sensitivity of the cells to E2, since progesterone receptor induction and growth stimulation were maintained. Insufficient ER diminution may explain this observation.

Topics: Binding Sites; Blotting, Western; Breast Neoplasms; Cell Division; Cell Survival; Coloring Agents; Dose-Response Relationship, Radiation; Electrophoresis, Polyacrylamide Gel; Estradiol; Humans; Immunohistochemistry; Pregnenediones; Protein Binding; Receptors, Estrogen; Receptors, Progesterone; Tetrazolium Salts; Thiazoles; Time Factors; Tumor Cells, Cultured

Long-term growth inhibition, arrest in G(1) phase and reduced activity of both cyclin D1-Cdk4 and cyclin E-Cdk2 are elicited by progestin treatment of breast cancer cells in culture. Decreased cyclin expression, induction of p18(INK4c) and increased association of the CDK inhibitors p21(WAF1/Cip1) and p27(Kip1) with cyclin E-Cdk2 have been implicated in these responses. To determine the role of decreased cyclin expression, T-47D human breast cancer cells constitutively expressing cyclin D1 or cyclin E were treated with the progestin ORG 2058. Overexpression of cyclin E had only a modest effect on growth inhibition. Although cyclin E expression was maintained during progestin treatment, cyclin E-Cdk2 activity decreased by approximately 60%. This was accompanied by p27(Kip1) association with cyclin E-Cdk2, indicating that both cyclin E down-regulation and p27(Kip1) recruitment contribute to the decrease in activity. In contrast, overexpression of cyclin D1 induced progestin resistance and cell proliferation continued despite decreased cyclin E-Cdk2 activity. Progestin treatment of cyclin D1-overexpressing cells was associated with increased p27(Kip1) association with cyclin E-Cdk2. Thus the ability of cyclin D1 to confer progestin resistance does not depend on sequestration of p27(Kip1) away from cyclin E-Cdk2, providing evidence for a critical function of cyclin D1 other than as a high-capacity "sink" for p27(Kip1). These data indicate that regulation of cyclin D1 is a critical element of progestin inhibition in breast cancer cells and suggest that breast cancers overexpressing cyclin D1 may respond poorly to progestin therapy.

Topics: Animals; Blotting, Western; Breast Neoplasms; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance; Drug Resistance, Neoplasm; Flow Cytometry; G1 Phase; Phosphorylation; Precipitin Tests; Pregnenediones; Progesterone Congeners; Progestins; Prognosis; Protein Binding; Protein Serine-Threonine Kinases; Retroviridae; S Phase; Time Factors; Tumor Suppressor Proteins

Enhanced expression of fatty acid synthase and other lipogenic enzymes has been observed in a subset of breast cancers with poor prognosis. This phenomenon has been related to amplification of a gene on chromosome region 11q13 encoding Spot 14, a putative regulator of lipogenic enzyme expression. In this paper we demonstrate that the induction of lipogenesis by progestins and androgens in the breast cancer cell line T47-D is accompanied by a marked increase in the expression of Spot 14. These data corroborate the correlation between Spot 14 expression and increased lipogenesis. Moreover they show that apart from gene amplification there is another steroid-regulated pathway that may enhance Spot 14 expression and lipogenesis in tumor cells.

Topics: Breast Neoplasms; Chromosomes, Human, Pair 11; Dihydrotestosterone; Female; Gene Expression; Humans; Lipids; Metribolone; Neoplasms, Hormone-Dependent; Nuclear Proteins; Pregnenediones; Progesterone; Progesterone Congeners; Proteins; Testosterone Congeners; Transcription Factors; Tumor Cells, Cultured

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. The long-term effect of progestins on T-47D breast cancer cells is inhibition of cellular proliferation. This is accompanied by decreased G(1) cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27(Kip1) among these CDK complexes, and alterations in the elution profile of cyclin E-Cdk2 upon gel filtration chromatography, such that high-molecular-weight complexes predominate. This study aimed to determine the relative contribution of CDK inhibitors to these events. Following progestin treatment, the majority of cyclin E- and D-CDK complexes were bound to p27(Kip1) and few were bound to p21(Cip1). In vitro, recombinant His(6)-p27 could quantitatively reproduce the effects on cyclin E-Cdk2 kinase activity and the shift in molecular weight observed following progestin treatment. In contrast, cyclin D-Cdk4 was not inhibited by His(6)-p27 in vitro or p27(Kip1) in vivo. However, an increase in the expression of the Cdk4/6 inhibitor p18(INK4c) and its extensive association with Cdk4 and Cdk6 were apparent following progestin treatment. Recombinant p18(INK4c) led to the reassortment of cyclin-CDK-CDK inhibitor complexes in vitro, with consequent decrease in cyclin E-Cdk2 activity. These results suggest a concerted model of progestin action whereby p27(Kip1) and p18(INK4c) cooperate to inhibit cyclin E-Cdk2 and Cdk4. Since similar models have been developed for growth inhibition by transforming growth factor beta and during adipogenesis, interaction between the Cip/Kip and INK4 families of inhibitors may be a common theme in physiological growth arrest and differentiation.

Topics: Breast Neoplasms; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p18; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Microtubule-Associated Proteins; Pregnenediones; Progestins; Recombinant Proteins; RNA, Messenger; Tumor Cells, Cultured; Tumor Suppressor Proteins

Interactions between transcription factors are an important means of regulating gene transcription, leading to modifications in the pattern of gene expression and cell fate. In this study, we report that the progesterone receptor (PR) can strongly interfere with transactivation mediated by the arylhydrocarbon receptor (AhR) in T47D breast cancer cells. This interference was not only demonstrated by induction of a transfected dioxin-responsive reporter plasmid but also on the AhR-mediated up-regulation of the endogenous cytochrome P450-1A1 activity. The interference was not mutual, as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent activator of the AhR, did not inhibit progestin-induced promoter activity. When the isoforms of the human PR, hPR-A and hPR-B, were expressed separately in HepG-2 hepatocarcinoma cells, both negatively interfered with the AhR signaling, indicating that the effect is not restricted to T47D cells. In addition, results obtained from studies with both antiprogestins and mutant receptors indicate differences in the underlying molecular mechanisms of repression for both PR isoforms. The suppression by hPR-A does not require additional gene expression or a full transcriptional competent conformation of the receptor. For the repressive effects of hPR-B, however, additional gene expression seems to be involved, as only the agonist-bound, wild-type hPR-B could clearly repress the TCDD-induced response. In conclusion, these studies highlight different mechanisms of repression for the progesterone receptor isoforms on the AhR-mediated trans-activation and underscore the importance of interactions between transcription factors of different families in the regulation of gene transcription.

Topics: Breast Neoplasms; Cytochrome P-450 CYP1A1; Female; Gene Expression Regulation, Enzymologic; Gonanes; Hormone Antagonists; Humans; Mifepristone; Polychlorinated Dibenzodioxins; Pregnenediones; Progesterone Congeners; Receptors, Aryl Hydrocarbon; Receptors, Progesterone; Recombinant Proteins; Signal Transduction; Transcriptional Activation; Transfection; Tumor Cells, Cultured; Up-Regulation

To define early molecular targets of progestin action, the differential display technique was used to identify genes with altered levels of expression in T-47D breast cancer cells treated with the synthetic progestin ORG 2058 for 3 h. PRG1 was first isolated as a 200-bp cDNA clone and its progestin regulation confirmed by Northern analysis. Cloning of the complete coding region of PRG1 revealed that it shared a high degree of amino acid sequence identity with isoforms of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from several tissues and species. Expression of PRG1 mRNA was observed in several normal breast epithelial and breast cancer cell lines and in a variety of human tissues, with highest expression in the breast, aorta, and brain. In T-47D cells, PRG1 mRNA was rapidly and transiently induced by progestins, expression peaking between 2 and 4 h and returning to control levels by 12 h. Progestin-induced increases in PRG1 mRNA were inhibited by the progestin antagonist RU 486 and occurred via the progesterone receptor. Progestin induction of PRG1 mRNA was also inhibited by actinomycin D but not by cycloheximide. PRG1 is therefore a novel human gene that is directly regulated by progestins via the progesterone receptor.

Topics: Amino Acid Sequence; Animals; Base Sequence; Breast Neoplasms; Cattle; Cell Cycle; Cloning, Molecular; DNA, Complementary; Female; Fructose-Bisphosphatase; Gene Expression Regulation; Glycolysis; Humans; Molecular Sequence Data; Phosphofructokinase-1; Phosphofructokinase-2; Pregnenediones; Progesterone Congeners; Progestins; Protein Synthesis Inhibitors; Proteins; RNA, Messenger; Sequence Alignment; Sequence Homology, Nucleic Acid; Transcription, Genetic; Tumor Cells, Cultured

In 1986 we reported the appearance of a progestin binding protein in the human breast cancer cell line Evsa-T, originally described as lacking both estrogen and progesterone receptors (ER and PR). In this report we show that PR of this cell line displays a binding affinity for [3H]ORG 2058 and a sucrose gradient sedimentation profile similar to those ascribed to PR from MCF-7 or T47D breast cancer cell lines. PR from Evsa-T cells is down-regulated by the progestin R-5020 as well as by the two antiprogestins, ZK 112.993 and ZK 98.299, but does not confer growth sensitivity to these compounds. ER remains undetectable by ligand binding assay, enzyme immunoassay and northern blotting. Our Evsa-T clone could be a valuable model for assessing the mechanisms leading the ER-/PR+ phenotype occurring occasionally in breast cancers and frequently in meningiomas.

Topics: Breast Neoplasms; Cell Division; Cytosol; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Gonanes; Hormone Antagonists; Humans; Mifepristone; Pregnenediones; Progestins; Receptors, Estrogen; Receptors, Progesterone; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

We have investigated the expression of insulin-like growth factor I receptors (IGFR) by the ZR-75-1 human breast cancer cell line and tamoxifen-resistant (ZR-75-9a1) and oestrogen-independent (ZR-PR-LT) variants. ZR-75-1 cells expressed 6633+/-953 receptors per cell,(K(d) 0.24+/-0.06 nM). IGFR expression was reduced in ZR-75-9a1 cells (1180+/-614 receptors per cell, K(d) 0.13+/-0.05) and increased in the ZR-PR-LT cell line (18 430+/-3210 receptors per cell, K(d) 0.24+/-17). A comparison of these data with previously published findings for epidermal growth factor receptor (EGFR) expression by these cell lines revealed that IGFR and EGFR expression are inversely related in the variant lines whereas ZR-75-1 cells express similar numbers of both receptors. Since the changes in IGFR expression observed are associated with changes in steroid hormone receptor status, we also investigated the effects of oestradiol, the synthetic progestin ORG 2058 and dexamethasone on IGFR expression. Oestradiol increased IGFR expression only in the ZR-75-1 cell line. Low concentrations of ORG 2058 increased IGFR levels in the two cell lines positive for progesterone receptor (ZR-75-1 and ZR-PR-LT). High concentrations of ORG 2058 increased IGFR expression in all cell lines, as did dexamethasone. These data suggest that EGFR and IGFR expression may be linked in breast cancer, and that EGFR/IGFR ratios in breast cancer may be a more sensitive prognostic indicator than EGFR expression alone. Regardless of basal IGFR expression by the cell studied, ORG 2058 increased IGFR expression, possibly via both the progesterone and glucocorticoid receptors.

Topics: Breast Neoplasms; Cell Line; Dexamethasone; ErbB Receptors; Estradiol; Female; Humans; Insulin-Like Growth Factor I; Kinetics; Pregnenediones; Progesterone Congeners; Receptor, IGF Type 1; Tumor Cells, Cultured

The expression and secretion of cathepsin D by ZR-75-1 human breast cancer cells, and tamoxifen-resistant (ZR-75-9a1) and estrogen-independent (ZR-PR-LT) variants was examined by electrophoresis of labeled proteins and Western blotting. Secreted proteins of 160 kDa, 52 kDa and 34 kDa were identified, and in ZR-75-1 cells, they were shown to be estrogen-inducible. Treatment of ZR-75-9a1 cells with 17 beta-estradiol (E2) and the progestin ORG 2058 increased secretion of the 52 kDa protein; ZR-PR-LT cells were unaffected. Western blotting showed that each cell line expressed high levels of the 52 kDa and 34 kDa forms of cathepsin D but that relatively little was being secreted. Each cell line secreted 52 kDa procathepsin D, but 34 kDa mature-cathepsin D was not detected as a secreted protein. ZR-75-1 cells expressed and secreted the highest levels of cathepsin D while ZR-75-9a1 cells expressed and secreted the least.

Topics: Breast Neoplasms; Cathepsin D; Cell Line; Clone Cells; Drug Resistance, Neoplasm; ErbB Receptors; Estradiol; Female; Gene Expression; Humans; Molecular Weight; Pregnenediones; Progesterone Congeners; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen; Tumor Cells, Cultured

Homeodomain-containing proteins are transcription factors that regulate the coordinated expression of multiple genes involved in development, differentiation and malignant transformation. To better understand the role played by these proteins in breast cancer cells, we demonstrate, using semi-quantitative RT-PCR experiments, that progestin induces HOXA1 mRNAs in MCF7 cells. This is the first evidence of regulation of a HOX gene by steroids. Moreover, we detected HOXA1 expression in a variety of human breast cancer lesions, suggesting that HOXA1 may be required for the establishment of breast cancer cells phenotype. We propose that HOXA1 gene could be one of the orchestrators that regulate breast epithelial cell differentiation and that alteration of HOXA1 expression could play a role in breast cancer progression.

Topics: Actins; Base Sequence; Breast Neoplasms; Cell Line; DNA Primers; DNA, Complementary; Female; Gene Expression; Homeodomain Proteins; Humans; Kinetics; Molecular Sequence Data; Neoplasm Proteins; Polymerase Chain Reaction; Pregnenediones; Progesterone Congeners; Transcription Factors; Tumor Cells, Cultured

Bcl-2 is a key protein involved in the control of apoptosis. Our previous studies on breast and endometrium indicated hormonal regulation of bcl-2 in these tissues. In the present work we have analyzed Bcl-2 and Bax protein expressions in MCF-7 and T47-D, 2 hormone-dependent breast-cancer cell lines, by immunoblots. Estradiol markedly increased Bcl-2 protein content, both in short- and in long-term treatments of MCF-7 cells. Two types of anti-estrogens (4-hydroxytamoxifen and RU 58668) were able to reverse this effect. Also, a synthetic progestin (ORG 2058) was able to decrease the Bcl-2 level in T47-D cells. The level of Bax protein, however, was not affected in the same conditions of hormonal treatments. The level of Bcl-2 expression was 4.5-fold higher in MCF-7 than in MDA-MB 231 (an estradiol-independent cell line). From these results, we infer the existence of hormonal regulation of Bcl-2 expression and evoke a novel role for estradiol and progestin in the genesis of breast cancer.

Topics: bcl-2-Associated X Protein; Blotting, Western; Breast Neoplasms; Estradiol; Estrogen Antagonists; Gene Expression; Humans; Immunoblotting; Pregnenediones; Progestins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tamoxifen; Tumor Cells, Cultured

Human progesterone receptor (PR) expression is controlled by two promoter regions giving rise to transcripts encoding PR A and B proteins. It is unknown whether estrogen and progesterone, the major physiological modulators of PR expression, exert their effects equally on the PR promoters. The aim of this study was to analyze estrogen and progestin effects on PR promoters, PR-encoding transcripts, and PR A and B proteins in T-47D human breast cancer cells. The progestin ORG 2058 caused a prolonged decrease in transcription of the PR gene and also abrogated estrogen stimulation of PR transcription. Estradiol (E2) treatment increased the activity of the B but not the A promoter transfected into T-47D cells. ORG 2058 had no effect on the basal or E2-stimulated activity of either promoter. E2 caused a preferential increase in transcripts derived from promoter B, whereas progestins decreased the levels of all PR transcripts. E2 preferentially increased the concentration of the PR B protein and caused a decrease in the PR A/B ratio. This demonstration that estrogen and progestin independently control the synthesis of transcripts arising from the PR promoters and that estrogen alters the cellular PR A/B ratio provides possible mechanisms underlying the cell and tissue specificity of PR regulation.

Topics: Breast Neoplasms; Cell Line; Drug Interactions; Estradiol; Female; Humans; Kinetics; Pregnenediones; Progesterone Congeners; Promoter Regions, Genetic; Receptors, Progesterone; Recombinant Proteins; RNA Probes; RNA, Antisense; RNA, Messenger; Transcription, Genetic; Transfection; Tumor Cells, Cultured

The effects of two classes of progestagens, e.g. pregnane [Org 2058, medroxyprogesterone acetate (MPA), R5020, progesterone (PROG)] and 19-nortestosterone derived progestagens [3-ketodesogestrel (KDG), levonorgestrel (LNG), gestodene (GES), norethisterone (NE), Org 30659] on proliferation of three estradiol (E2)-dependent human breast tumor MCF-7 cell lines of different origin [Van der Burg (B), Litton bionetics (L) and McGrath (M)] were studied. The pregnane derivatives hardly stimulated cell growth at 10(-6) M in MCF-7 B and L cells except for Org 2058 in B cells, whereas in M cells a statistically significant growth induction was observed except for PROG. The 19-nortestosterone derivatives induced cell growth at doses at 10(-7) M or higher in all three cell lines. NE, GES and Org 30659 were more potent stimulators than KDG and LNG at 10(-7) M. E2 already showed maximal stimulation at 10(-10) M. For all three cell lines, the effects and ranking of the individual progestagens were similar. Antiprogestagens, like RU 38486 and Org 31710 could not block these stimulatory effects while antiestrogens like 4-hydroxytamoxifen and ICI 164,384 could. This suggests that cell growth by the above-mentioned progestagens occurs via an interaction with the estrogen receptor. Indeed, displacement studies with cytosol from MCF-7 M cells revealed that at very high concentrations NE, GES and Org 30659 were able to displace 50% of the radiolabelled E2, while KDG and LNG could not. Relative binding affinities (RBAs) were 0.010, 0.025 and 0.015% for NE, GES and Org 30659, respectively. The effect of the two classes of progestagens on cell proliferation was also investigated at several dose levels in combination with E2 (10(-10) M) in the MCF-7 B cell line. This resulted in a statistically significant inhibition of cell growth with R5020, MPA and most of the 19-nortestosterone derivatives at concentrations of 10(-8) M. Org 2058 and NE did not have any influence on E2-induced growth. The inhibitory effects could not be blocked by antiprogestagens. In summary these studies with 3 subclones of MCF-7 cells show that the pregnane derived progestagens stimulate growth only in one subclone, whereas the 19-nortestosterone derived progestagens do so in all three subclones. The progestagens possess estrogenic activity only at high pharmacological doses, being 10,000 times weaker than estradiol. In combination with estrogens most progestagens gave a reduction of E2-stimulated growth in t

Topics: Adenocarcinoma; Breast Neoplasms; Cell Division; Desogestrel; Estradiol; Estrogen Antagonists; Female; Humans; Levonorgestrel; Medroxyprogesterone Acetate; Models, Statistical; Nandrolone; Norethindrone; Norpregnenes; Pregnanes; Pregnenediones; Progesterone; Promegestone; Receptors, Androgen; Receptors, Estrogen; Receptors, Progesterone; Tumor Cells, Cultured

Two classes of progestagens, e.g. pregnane [Org 2058, progesterone (PROG), R5020, medroxyprogesterone acetate (MPA)] and 19-nortestosterone derived progestagens [norethisterone (NE), levonorgestrel (LNG), 3-ketodesogestrel (KDG), gestodene (GES), Org 30659] were studied for their effect on cell growth of two human breast tumor T47D cell lines of different origin, i.e. from ATCC (A) and Sutherland (S) et al. [Sutherland et al., Cancer Res. 48 (1988) 5084-5091]. The effect of estradiol (E2) and progestagens alone as well as the combined effect of E2 (10(-10) M) and progestagens were investigated at several dose levels. Compared with E2-induced growth at 10(-10) M, pregnane and 19-nortestosterone derived progestagens at 10(-6) M alone did enhance cell growth in T47D-A cells up to 25 and 100% respectively, whereas in T47D-S cells they did not influence growth. All these progestagens at 10(-6) M did not affect E2-induced growth in T47D-A cells, whereas in T47D-S cells they completely reduced cell proliferation at doses between 10(-10) and 10(-8) M. The involvement of progestagen (PR) and estrogen (ER) receptors with respect to growth stimulation was studied by using specific antihormones. In T47D-A cells, the antiprogestagens RU 38486 and Org 31710 could not block progestagen-induced growth. Antiestrogens, like 4-hydroxytamoxifen and ICI 164,384, inhibited the 19-nortestosterone derivative-induced cell growth by approx. 50%. Remarkably, both antiprogestagens alone could also inhibit E2-induced growth in T47D-A cells by about 50%. In T47D-S cells, E2-induced cell growth was completely blocked by both antiprogestagens and antiestrogens. Both antiprogestagens in T47D-S cells were equipotent to 4-hydroxytamoxifen and 10-fold more potent than ICI 164,384. In conclusion pregnane and 19-nortestosterone-derived progestagens stimulated cell growth in T47D-A cells at high unphysiological concentrations, whereas they did not affect cell growth in T47D-S cells. The 19-nortestosterone derivative induced growth in T47D-A cells could partially be inhibited by antiestrogens. In T47D-A cells, E2-induced cell growth was not influenced by both classes of progestagens, whereas in T47D-S cells all tested progestagens, antiprogestagens, and antiestrogens inhibited E2-induced cell growth completely. These results with T47D cells as well as those obtained previously with MCF-7 cells show that subclones of cell lines may respond differently to various types of progestagens in the pres

Topics: Breast Neoplasms; Carcinoma; Cell Division; Desogestrel; Dose-Response Relationship, Drug; Estradiol; Estrenes; Estrogen Antagonists; Female; Furans; Humans; Levonorgestrel; Medroxyprogesterone Acetate; Mifepristone; Nandrolone; Norethindrone; Norpregnenes; Pregnanes; Pregnenediones; Progesterone; Promegestone; Tumor Cells, Cultured

We have used ligand blotting and Northern blotting techniques to examine the effects of progestins and antiestrogens on expression of insulin-like growth factor binding proteins (IGFBPs) by T-47D human breast cancer cells under conditions where these agents are growth inhibitory. Under basal conditions, conditioned medium from T-47D cells was found to contain IGFBPs of 39, 33, and 27 kDa. Northern blot and/or Western blot analysis have identified these as IGFBP 2, 5, and 4, respectively. Medroxyprogesterone acetate (MPA) treatment resulted in a time- and dose-dependent decrease in IGFBP 4 and 5 mRNA abundance and secretion of these proteins, while little if any effect was observed on IGFBP 2 expression. A decrease in the steady state mRNA levels for IGFBP 4 and 5 was observed with as little as 0.1 nM MPA. Using 10 nM MPA a maximum decrease in IGFBP 4 and 5 mRNA levels was observed between 12 and 24 hours. While RU 486 alone had little or no effect on IGFBP 4 expression, it inhibited the effect of MPA. However, in the same samples, IGFBP 5 expression was inhibited by RU 486, and RU 486 was unable to reverse the effects of progestins on the expression of IGFBP 5. Furthermore, another synthetic progestin, Org 2058, but not dexamethasone, inhibited IGFBP 4 and IGFBP 5 expression. The antiestrogen ICI 164384 also transiently decreased the steady state mRNA levels of both IGFBP 4 and IGFBP 5. Regulation of expression of the IGFBPs by these agents suggests a potential role for the IGFBPs in the growth response of T-47D cells to these agents.

Topics: Breast Neoplasms; Carrier Proteins; Culture Media, Conditioned; Dexamethasone; Estradiol; Estrogen Antagonists; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Protein 2; Insulin-Like Growth Factor Binding Protein 4; Insulin-Like Growth Factor Binding Protein 5; Medroxyprogesterone Acetate; Mifepristone; Neoplasm Proteins; Polyunsaturated Alkamides; Pregnenediones; Progesterone; Progestins; Tumor Cells, Cultured

Using synthetic peptides with sequences derived from specific regions of human estrogen (ER) and progesterone (PR) receptors, we have developed site-directed monoclonal and polyclonal antibodies to specific domains of these receptors. These antibodies interact specifically with the native (nondenatured) receptors and detect changes in the conformation of these proteins. Monoclonal antibody PR-AT 4.14 bound more tightly to PR-ORG 2058 complexes than to PR-7 alpha,17 alpha-dimethyl-19-nor-testosterone (7 alpha,17 alpha, DMNT; mibolerone) complexes, suggesting possible ligand-induced conformational changes in PR. In the absence of the antibody, PR-[3H]ORG 2058 complexes sedimented as 4S-5S entity on sucrose density gradients (SDG) containing 0.4 M KCl. In the presence of the antibody, PR-[3H]ORG 2058 complexes sedimented as 7-8S complexes. In contrast, at the same concentrations of antibody, PR-[3H]7 alpha,17 alpha, DMNT complexes sedimented at 4S-5S region in the absence of the antibody and as two populations in the presence of the antibody, suggesting that the antibody did not recognize all of the PR-7 alpha,17 alpha, DMNT complexes. To exclude the possibility that the inability of the antibody to recognize receptor-[3H]7 alpha,17 alpha, DMNT complexes was due to its binding to androgen receptors, unlabeled 5 alpha-dihydrotestosterone (5 alpha-DHT) (50 nM) was added to the incubation to inhibit 7 alpha,17 alpha, DMNT binding to androgen receptors. While PR-[3H]ORG 2058 complexes were immunoprecipitated in the presence of the antibody, PR-[3H]7 alpha,17 alpha, DMNT complexes were only partially immunoprecipitated, further confirming the results obtained with SDG.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Binding Sites, Antibody; Binding, Competitive; Blotting, Western; Breast Neoplasms; Cattle; Female; Humans; Nandrolone; Precipitin Tests; Pregnenediones; Protein Conformation; Rats; Rats, Sprague-Dawley; Receptors, Estrogen; Receptors, Progesterone; Uterus

Progesterone receptors (PgR) of human breast cancer T47-D cells grown in an estrogenic environment (presence of phenol red, natural estrogens of foetal calf serum and insulin) were found to be present in considerable amounts (1-3 pmol/mg protein and 20 pmol/mg DNA), and to specifically bind progestins with a high affinity characterized by a Kd around 3 nM for ORG2058, and 4 nM for nomegestrol acetate (NOM; 17 alpha-acetoxy-6-methyl-19-nor-pregna-4,6-diene-3, 20-dione), when measured under equilibrium conditions. Both compounds formed an highly stable ligand-receptor complex with a dissociation constant (k-1) around 1 x 10(-5) s-1. At high pharmacological concentrations, NOM, ORG2058 and other synthetic progestins including promegestone (R5020), medroxy-progesterone acetate and norethindrone acetate (NOR), induced a dose-dependent inhibition of cell proliferation as measured by [3H]thymidine incorporation. Dexamethasone, which did not bind to PgR, did not reproduce this inhibitory effect. NOM, R5020 and NOR treatments of T47-D cells at concentrations around Kd resulted in an 80% decrease in PgR content. Our data on NOM as compared to other progestins are consistent with their antiproliferative effects on human breast cancer cells grown in estrogenic conditions.

Topics: Breast Neoplasms; Cell Division; Humans; Megestrol; Norpregnadienes; Pregnenediones; Progesterone Congeners; Promegestone; Receptors, Progesterone; Tumor Cells, Cultured

Possible mechanisms by which the progestin antagonist RU 486 inhibits cell growth were investigated by comparing the effects of the antiprogestin with those of progestin and antiestrogen. Exposure of T-47D breast cancer cells to RU 486 caused a decline in the proportion of cells in S phase, indicative of a block to cell cycle progression in G1 phase. This was accompanied by a marked decrease in c-myc expression but no change in cyclin D1 expression. The cell kinetic data suggest that progestin antagonist inhibition of proliferation and progestin stimulation of proliferation are mediated by opposing effects on the same mechanism. Both estrogen antagonists and progestin antagonists appear to act at a similar part of G1 phase but there are clear differences in their effects on cyclin D1 expression, suggesting that the mechanisms by which these compounds inhibit proliferation are distinct.

Topics: Breast Neoplasms; Cell Cycle; Cyclin D1; Cyclins; Dose-Response Relationship, Drug; Estradiol; Humans; Mifepristone; Oncogene Proteins; Polyunsaturated Alkamides; Pregnenediones; Progesterone Congeners; Progestins; Proto-Oncogene Proteins c-myc; RNA, Messenger; S Phase; Tumor Cells, Cultured

In a human breast cancer cell line (ZR-PR-LT) we have found a poor overall correlation between affinity of progestins and anti-progestins for the progesterone receptor (PGR), concentration required for receptor down-regulation and anti-proliferative potency. Medroxyprogesterone acetate (MPA) and the anti-progestin RU 38.486, which possess glucocorticoid and antiglucocorticoid activity, respectively, cause receptor down-regulation at lower concentrations than their Kdi for [3H] ORG 2058 binding sites. In addition dexamethasone markedly down-regulates PGR at concentrations which fail to interact with PGR suggesting that heterospecific modulation of PGR occurs via the glucocorticoid receptor. In contrast the progestin ORG2058 and the anti-progestin ZK 98.299 caused 50% PGR down-regulation at a concentration (EC50) 50-fold higher than their Kdi values. ZK 112.993 was 500-fold more potent at PGR down-regulation than ZK 98.299 but had only a 5-fold higher affinity for PGR. Anti-proliferative concentrations of progestins/anti-progestins showing were generally higher than either Kdi values or EC50 values.

Topics: Binding, Competitive; Breast Neoplasms; Dexamethasone; Down-Regulation; Female; Gonanes; Humans; Medroxyprogesterone Acetate; Mifepristone; Mitosis; Pregnenediones; Progesterone Congeners; Progestins; Receptors, Glucocorticoid; Receptors, Progesterone; Tumor Cells, Cultured

Cyclins and proto-oncogenes including c-myc have been implicated in eukaryotic cell cycle control. The role of cyclins in steroidal regulation of cell proliferation is unknown, but a role for c-myc has been suggested. This study investigated the relationship between regulation of T-47D breast cancer cell cycle progression, particularly by steroids and their antagonists, and changes in the levels of expression of these genes. Sequential induction of cyclins D1 (early G1 phase), D3, E, A (late G1-early S phase), and B1 (G2 phase) was observed following insulin stimulation of cell cycle progression in serum-free medium. Transient acceleration of G1-phase cells by progestin was also accompanied by rapid induction of cyclin D1, apparent within 2 h. This early induction of cyclin D1 and the ability of delayed administration of antiprogestin to antagonize progestin-induced increases in both cyclin D1 mRNA and the proportion of cells in S phase support a central role for cyclin D1 in mediating the mitogenic response in T-47D cells. Compatible with this hypothesis, antiestrogen treatment reduced the expression of cyclin D1 approximately 8 h before changes in cell cycle phase distribution accompanying growth inhibition. In the absence of progestin, antiprogestin treatment inhibited T-47D cell cycle progression but in contrast did not decrease cyclin D1 expression. Thus, changes in cyclin D1 gene expression are often, but not invariably, associated with changes in the rate of T-47D breast cancer cell cycle progression. However, both antiestrogen and antiprogestin depleted c-myc mRNA by > 80% within 2 h. These data suggest the involvement of both cyclin D1 and c-myc in the steroidal control of breast cancer cell cycle progression.

Topics: Breast Neoplasms; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Cycle; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Cyclins; DNA, Neoplasm; Estradiol; Estrogen Antagonists; Female; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, myc; Growth Substances; Histones; Humans; Insulin; Kinetics; Mifepristone; Polyunsaturated Alkamides; Pregnenediones; Progesterone Congeners; Protein Kinases; Protein Serine-Threonine Kinases; Recombinant Proteins; RNA, Messenger; Time Factors; Transcription, Genetic; Tumor Cells, Cultured

The sex steroid hormone progesterone modulates the developmental and lactogenic activity of prolactin in the mammary gland. Regulation of the level of prolactin receptor (PRLR) provides one possible mechanism by which this may occur, prompting this investigation of the molecular mechanisms involved in progestin regulation of prolactin receptor levels. Treatment of T-47D and MCF-7 human breast cancer cells with 10 nM of the synthetic progestin ORG 2058 for 24 hr resulted in an increase in all four PRLR mRNA transcripts detected. The effect of ORG 2058 was shown in T-47D cells to be time- and concentration-dependent and resulted in an approximate two-fold increase in PRLR mRNA after 24 hr of treatment with 10 nM or 100 nM ORG 2058. Nuclear run-on assays indicated that ORG 2058 increased the rate of T-47D PRLR gene transcription at all times between 1 hr and 28 hr of treatment. The protein synthesis inhibitors cycloheximide and puromycin abrogated the induction of PRLR gene transcription at 1 hr and 2 hr, which demonstrated that on-going protein synthesis was required for the ORG 2058 effect and suggested that progestins may exert some transcriptional effects via the induction of an intermediary protein. These experiments demonstrated that progestin induced a transcriptionally based increase in PRLR gene expression and provided a mechanism by which progesterone may modulate the mitogenic activity of prolactin during mammary gland development.

Topics: Autoradiography; Blotting, Northern; Breast Neoplasms; Humans; Pregnenediones; Progesterone Congeners; Progestins; Protein Synthesis Inhibitors; Receptors, Prolactin; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

Lactogenic hormones which bind to the PRLR are likely to be growth-stimulatory in human breast-cancer cells. Oestrogen and progesterone control cellular expression of the PRLR; however, elevated androgen levels in some breast-cancer patients raised the possibility that androgens may also influence breast-cancer sensitivity to lactogenic hormones. This study investigated whether androgens could affect expression of the PRLR in the MCF-7 breast-cancer cell line. PRLR binding activity was increased approximately 2-fold by treatment for 24 hr with 10 nM R1881, TEST, DHT, MPA and ORG 2058. Northern analysis indicated that DHT also increased the level of PRLR mRNA. The antiprogesterone, RU 38486, displaced tritiated ORG 2058 binding but not tritiated DHT binding to MCF-7 cells; it completely antagonized ORG 2058 and partially antagonized R1881 induction of the PRLR, but had no effect on induction by DHT. The anti-androgen, RU 23908, displaced tritiated DHT binding but not tritiated ORG 2058 binding, and antagonized DHT and R1881 induction of PRLR but not induction of the PRLR by ORG 2058. These data indicated that ORG 2058 acting via the PR and DHT acting via the AR were able to induce PRLR expression in MCF-7 cells. In MDA-MB-453 cells, which express the AR but not the ER or PR, DHT and R1881 increased PRLR binding to 150% of control values at 0.1 nM. ORG 2058 was ineffective, demonstrating androgen induction of PRLR in the absence of PR and ER. These data indicate that PRLR can be regulated by androgens in MCF-7 and MDA-MB-453 human breast-cancer cells.

Topics: Androgens; Breast Neoplasms; Dihydrotestosterone; Female; Gene Expression Regulation; Humans; Mifepristone; Pregnenediones; Receptors, Prolactin; RNA, Messenger; Tumor Cells, Cultured

The expression of the 17 beta-hydroxysteroid dehydrogenase (17-HSD) gene in a series of human breast cancer cell lines was studied by Northern blot hybridization with a cDNA probe and by a time-resolved immunofluorometric assay using polyclonal antibodies against the enzyme protein. The 17-HSD enzyme protein concentration was measured in the 800 x g cell extract. A high concentration was measured in the BT-20 cell line, corresponding to one-fourth of the average concentration in placental tissue. Western blot analysis indicated that the antigen corresponded to a single Mr 35,000 band. In 2 other cell lines (MDA-MB-361 and T-47D), the 17-HSD protein concentration was much lower, but still measurable, whereas in the remaining 5 cell lines (HBL-100, MCF-7, MDA-MB-231, MDA-MB-468, and ZR-75-1) it was below the detection limit of the assay. Treatment of the cells for 5 days with the synthetic progestin, ORG2058, resulted in an increase of the 17-HSD protein concentration only in the T-47D cell line. By Northern blot analysis, a low level of 2.3-kilobase mRNA transcripts was detected in all 8 cell lines. In addition, a 1.3-kilobase 17-HSD mRNA was present in the samples from the 3 cell lines containing measurable amounts of 17-HSD protein in the cell extract, and the band intensities were proportional to the amount of protein measured with the immunofluorometric assay. Only in the T-47D cell line did progestin treatment correspond to an increased amount of the 17-HSD 1.3-kilobase mRNA. These results suggest that the 1.3-kilobase mRNA for 17-HSD is the form most closely associated with protein expression and is also the only form responding to the progestin induction of the 17-HSD gene.

Topics: 17-Hydroxysteroid Dehydrogenases; Breast Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Placenta; Pregnenediones; Progesterone Congeners; Receptors, Estrogen; Receptors, Progesterone; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

High performance hydrophobic interaction chromatography has been used to separate progestin receptors (PRs) from human uterus and from the T47D human breast cancer cell line. Reproducible separations of high resolution were achieved using a TSK Phenyl-5PW column and a reverse salt gradient of 400 mM to 0 mM sodium sulfate in phosphate buffer, pH 7.4. Peaks of radioactivity exhibiting hydrophobic behaviour were isolated, as well as a smaller proportion of specific bound receptors located in the void volume fraction. No differences in retention times were observed between uterine and breast cell line samples. When the technique was used in conjunction with rapid vertical tube sucrose density gradient centrifugation, the 8S sedimenting PR from fresh, low-salt cytosol always eluted with a retention time of 24 min. The natural 4S receptor chromatographed as a single peak at 29 min while the 4S receptor species from high-salt cytosol appeared as two distinct peaks of radioactivity with retention times of 29 and 33 min. While specific binding was shown to occur in the void volume of the column, the origin of these receptors were indeterminate. These results would suggest that under these conditions the 8S receptor occurs as a single hydrophobic class of protein, whereas the data provides evidence that transformed 4S receptor may be proportioned into two unequal entities as a function of exposure to salt.

Topics: Breast Neoplasms; Cell Line; Chromatography; Cytosol; Female; Humans; Pregnenediones; Progesterone Congeners; Radioligand Assay; Receptors, Progesterone; Uterus

This study documents a biphasic change in the rate of cell cycle progression and proliferation of T-47D human breast cancer cells treated with synthetic progestins, consisting of an initial transient acceleration in transit through G1, followed by cell cycle arrest and growth inhibition. Both components of the response were mediated via the progesterone receptor. The data are consistent with a model in which the action of progestins is to accelerate cells already progressing through G1, which are then arrested early in G1 after completing a round of replication, as are cells initially in other phases of the cell cycle. Such acceleration implies that progestins act on genes or gene products which are rate limiting for cell cycle progression. Increased production of epidermal growth factor and transforming growth factor alpha, putative autocrine growth factors in breast cancer cells, does not appear to account for the initial response to progestins, since although the mRNA abundance for these growth factors is rapidly induced by progestins, cells treated with epidermal growth factor or transforming growth factor alpha did not enter S phase until 5 to 6 h later than those stimulated by progestin. The proto-oncogenes c-fos and c-myc were rapidly but transiently induced by progestin treatment, paralleling the well-known response of these genes to mitogenic signals in other cell types. The progestin antagonist RU 486 inhibited progestin regulation of both cell cycle progression and c-myc expression, suggesting that this proto-oncogene may participate in growth modulation by progestins.

Topics: Blotting, Northern; Breast Neoplasms; Cell Cycle; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Kinetics; Mifepristone; Pregnenediones; Progesterone Congeners; Proto-Oncogene Mas; Proto-Oncogene Proteins c-fos; Transforming Growth Factor alpha; Tumor Cells, Cultured

Progestin antagonism of estrogen action is thought to be due, at least in part, to progestin down-regulation of the estrogen receptor (ER). The molecular mechanisms subserving this effect, and the functional consequences in terms of target cell sensitivity to estrogens, are poorly understood. The present study was undertaken to address these issues with particular emphasis on progestin regulation of ER gene expression at the mRNA level. The T-47D human breast cancer cell line was treated with the synthetic progestin, ORG 2058, and the resultant changes in ER mRNA and ER levels determined by Northern analysis and radioligand binding, respectively. Treatment of T-47D cells with ORG 2058 resulted in rapid down-regulation of ER mRNA levels to a nadir of 35-40% of control by 6 h. This fall in ER mRNA levels was accompanied by a slower but more sustained fall in ER binding to a nadir of 20% of control at 24 h. Between 12 and 24 h ER mRNA levels recovered partially while ER ligand binding continued to fall. At 48 h both ER mRNA and ER concentrations remained depressed, although the latter to a greater extent. ER mRNA half-life was determined by [3H]uridine incorporation to be approximately 60 min and was unaffected by progestin treatment during the early rapid phase of ER mRNA down-regulation. These data demonstrate that progestins cause rapid down-regulation of the ER mRNA and suggest that during the early rapid phase of this effect, reduced transcription of the ER gene rather than altered ER mRNA half-life mediate this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Breast Neoplasms; Down-Regulation; Half-Life; Humans; Pregnenediones; Progestins; Receptors, Estrogen; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

Data are presented which document the first known effect of retinoic acid on progesterone receptor (PR) gene expression. Treatment of T-47D human breast cancer cells with retinoic acid for 48 h resulted in a marked concentration-dependent decrease in the level of PR mRNA and immunoreactive protein which was similar to the known effect of progestins on these parameters. Retinoic acid, however, did not bind to PR, nor did it cause the previously demonstrated increase in PR molecular weight observed after progestin exposure. When T-47D cells were treated with retinoic acid for 6 h rather than 48 h, no reduction in the level of PR protein was noted at any retinoic acid concentration whereas the effects of retinoic acid on PR mRNA at 6 and 48 h were the same. Examination of the time course of the effects of retinoic acid revealed a rapid decrease in PR mRNA levels detectable 1 h after and maximal 6 h after treatment of T-47D cells with retinoic acid. These effects of retinoic acid contrasted with previously demonstrated progestin effects on PR mRNA which were not apparent until 3 h after and were not maximal until 12 h after treatment. As expected, the PR protein concentration was unaffected for at least 6 h but was maximally decreased 24-48 h after retinoic acid treatment. In summary, retinoic acid treatment of T-47D cells caused a decrease in the cellular PR concentration by decreasing levels of receptor mRNA and protein, suggesting that retinoic acid is capable of modulating sensitivity to progestins in human breast cancer cells.

Topics: Blotting, Northern; Blotting, Western; Breast Neoplasms; Cell Division; Dose-Response Relationship, Drug; Gene Expression; Humans; Pregnenediones; Receptors, Progesterone; RNA, Messenger; Time Factors; Tretinoin; Tumor Cells, Cultured

The effects of estradiol and two synthetic progestins (ORG2058 and R5020) on the expression of the high-affinity, metastasis-associated laminin receptor in two human breast carcinoma cell lines were examined. The T47D cell line contains estrogen and progesterone receptors, but the MDA-MB 231 cell line lacks both receptors. Treatment of T47D cells with 10(-9) M estradiol alone results in a three-fold increase (P less than or equal to .05) in the steady-state level of laminin receptor mRNA determined by RNA blot analysis as well as in cell-surface, laminin receptor expression that is evaluated by immunofluorescence. No effects of estradiol on the receptor-negative MDA-MB 231 cells were observed. Untreated and steroid-treated MDA-MB 231 cells had higher levels of laminin receptor mRNA than did untreated or estradiol-treated T47D cells. A more dramatic increase (five-fold; P less than or equal to .005) of mRNA and cell-surface expression in T47D cells was observed after treatment with estradiol plus 10(-8) M progestin or with progestin alone. Estradiol treatment also increased chemotaxis and haptotaxis of T47D cells but not of MDA-MB 231 cells to laminin; it had no effect on the attachment of these latter cells to laminin. Interestingly, treatment with estradiol plus progestin or progestin alone significantly increased the attachment of T47D cells to laminin but did not have an effect on either haptotaxis or chemotaxis to laminin. These results suggest that the various cell-laminin interactions are mediated by different mechanisms. The augmentation of laminin receptor mRNA by estrogen and progesterone treatment in hormone receptor-positive cells, but not in cells that lack these receptors, may relate functionally to the difference in the clinical aggressiveness between classes of breast cancers.

Topics: Breast Neoplasms; Cell Movement; Estradiol; Fluorescent Antibody Technique; Humans; Laminin; Pregnenediones; Progesterone Congeners; Promegestone; Receptors, Immunologic; Receptors, Laminin; RNA, Messenger; Tumor Cells, Cultured

The present study was designed to investigate whether inhibition of progesterone receptor (PR) gene transcription and/or regulation of PR mRNA half-life were involved in the progestin-mediated decrease of PR in T-47D human breast cancer cells. Cells were treated with the progestin ORG 2058 and PR mRNA measured by Northern blot analysis of total RNA. A major PR mRNA around 13.5 kilobases and minor species around the 28S ribosomal RNA subunit were decreased upon ORG 2058 treatment. The decrease was not detectable until 2-3 h after treatment and was the same at all ORG 2058 concentrations (1-100 nM) tested. The decrease in PR mRNA was unaffected by actinomycin D in the first 3 h but was inhibited thereafter. There was a partial recovery of PR mRNA levels 24 h after ORG 2058 exposure. Immunoblot analysis showed that immunoreactive PR decreased in parallel with PR mRNA. The rate of protein loss in the first 12 h after progestin treatment was related to the ORG 2058 concentration used. Nuclear run-on experiments showed that ORG 2058 caused a decrease of up to 70% in the transcription rate of the PR gene. The half-life of PR mRNA was shown to be 2-2.5 h by [3H]uridine incorporation, which was much shorter than estimates obtained using actinomycin D, and was unaffected by ORG 2058. In summary, these data have shown that the mechanism by which progestins decrease the concentration of PR includes inhibition of transcription of the PR gene.

Topics: Blotting, Northern; Breast Neoplasms; Cell Line; Dactinomycin; Down-Regulation; Electrophoresis, Agar Gel; Electrophoresis, Polyacrylamide Gel; Gene Expression; Humans; Pregnenediones; Progesterone Congeners; Receptors, Progesterone; RNA, Messenger; Transcription, Genetic

The molecular basis of the growth-inhibitory effects of progestins or antiestrogens in human breast cancer has not been fully elucidated. Both direct actions and indirect actions, where the growth inhibition results from modulation of the production of, and/or the response to, growth factors, have been proposed. In this study the ability of some growth factors to modulate progestin-induced inhibition of cell proliferation was investigated in vitro, using T-47D human breast cancer cells. When T-47D cells grown in insulin-containing medium were treated for 4 to 5 days with the synthetic progestin, ORG 2058, at a concentration of 10 nM, cell numbers were reduced to 10 to 20% of control. Simultaneous treatment with epidermal growth factor (EGF) and ORG 2058 led to a partial reversal of the growth-inhibitory effect of the progestin. The magnitude of the effect of EGF was concentration dependent, being half-maximal at 0.48 ng/ml (0.08 nM) and maximal at concentrations greater than 5 ng/ml (greater than 0.8 nM), where cell numbers were increased by 50% compared to those in the presence of ORG 2058 alone. ORG 2058 was no more potent in the absence of insulin, and, after several passages in insulin-free medium, addition of insulin failed to modulate the effect of ORG 2058. However, when maximal concentrations of insulin (5 micrograms/ml) and EGF (10 ng/ml) were administered together with ORG 2058, insulin and EGF appeared to act synergistically to reduce the ORG 2058-induced inhibition of proliferation. In similar experiments in which cells were treated with hydroxyclomiphene, a potent antiestrogen, insulin was shown to partially reverse the growth-inhibitory effects of hydroxyclomiphene. Significant increases in cell number above hydroxyclomiphene-treated controls were apparent at insulin concentrations greater than 50 ng/ml, and at 5 micrograms/ml the increase was approximately 2-fold. In contrast to the situation with progestins, simultaneous treatment with EGF and insulin had only an additive effect in reversing the growth-inhibitory effect of the antiestrogen. The results are compatible with the hypothesis that part of the growth-inhibitory effects of progestin and antiestrogen on human breast cancer cell proliferation is mediated by inhibition of autocrine growth factor production. However, they do not exclude more direct mechanisms involving modulation of progesterone and/or estrogen receptors by EGF and/or insulin.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Breast Neoplasms; Cell Division; Clomiphene; Epidermal Growth Factor; Estrogen Antagonists; Female; Growth Inhibitors; Humans; Insulin; Pregnenediones; Progestins; Tumor Cells, Cultured

The estrogen receptor (ER) and progesterone receptor (PR) content of cultured human breast carcinoma cells (MCF-7) was determined by biochemical assay, immunoblot analysis, and immunohistochemical assay under varying conditions of hormonal stimulation. The ER and PR content in cytosolic and nuclear extracts varied with steroid treatment. However, both the amount and distribution of each receptor in these extracts was virtually the same when determined by steroid binding and immunoblot analyses. Two immunocytochemical parameters (staining intensity and proportion of cells stained) correlated with the quantitative analyses of ER and PR, but not with the subcellular distribution. When MCF-7 cells were grown for 4 days in charcoal-stripped serum without phenol red, 93% of total ER was found in the cytosol (10 mM KCl), whereas short-term treatment with 5 nM estradiol resulted in the appearance of 82% of total ER in the nuclear extract (400 mM KCl). With either cell treatment only nuclear staining for ER was observed. Progesterone receptor was virtually undetectable in the same cells by any method. After 4 days of treatment by 5 nM estradiol, PR was strongly induced (50-fold) in MCF-7 cells as determined by all three methods. As observed for ER, 95% of total induced PR was found in the cytosol in the absence of a progestin. Short-term treatment with 5 nM ORG 2058, a synthetic progestin, resulted in the appearance of 42% of total PR in the nuclear extract. However, only strong nuclear staining for PR was observed in either the presence or absence of a progestin. These findings are consistent with the current view of ER and PR as nuclear receptors present in at least two forms. One of these, the unoccupied form of the receptor, is easily removed from the nucleus by hypotonic buffers during the cell homogenization process and appears in the cytosolic extract. The other form of the receptor, the steroid-occupied form, is more tightly bound to nuclear components and is removed from nuclei only under more vigorous extraction conditions.

Topics: Blotting, Western; Breast Neoplasms; Cell Nucleus; Cytosol; Estradiol; Humans; Immunoenzyme Techniques; Pregnenediones; Progesterone Congeners; Receptors, Estrogen; Receptors, Progesterone; Subcellular Fractions; Tumor Cells, Cultured

In this study of breast cancer specimens, the relationship between cytosolic estrogen (ER) and progesterone receptor (PR) content to the size of the respective growth fraction (GF) (expressed as percentage of proliferating tumor cells) was investigated. We applied the recently developed ligand-binding assay for extracts of frozen sections and Ki-67 immunocytochemistry for the assessment of the GF to adjoining serial sections of a single tissue block. If the receptor content is plotted against the percentage of Ki-67 labeled cells, an inverse relationship between receptor content and proliferation becomes obvious, meaning that a high receptor content is associated with a small GF and vice versa. If tumor specimens are grouped according to the evaluated receptor status, the mean percentage of Ki-67-positive cells is 12% for ER-positive/PR-positive (ER+/PR+), 26% for ER-positive/PR-negative (ER+/PR-), 55% for ER-negative/PR-positive (ER-/PR+), and 57% for ER-negative/PR-negative (ER-/PR-) specimens. A significant population of tumors exists, however, which exhibit a high receptor content and a high GF. The percentages of ER+/PR+ samples with a high proliferation index are 16 and 26% if the total ER+ population is considered.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Division; Cytosol; Estradiol; Female; Humans; Immunoblotting; Pregnenediones; Progesterone Congeners; Receptors, Estrogen; Receptors, Progesterone

An improved radioreceptor assay of unfixed cryostat sections of human target tissues has been developed. Sections collected on glass coverslips were immediately incubated with 5 nM concentrations of either tritiated estradiol-17 beta for estrogen receptor (ER) or ORG 2058 for progesterone receptor (PR) determination. For quantitation, receptor-bound and free hormone were separated by isoelectric focusing (IEF). The assay allows the determination of steroid hormone receptors and comparative histological examinations in immediately neighbouring serial sections of a single piece of tissue. Biochemically, the validity of the assay procedure was evidenced by Scatchard analysis, by ligand and tissue specificities, by the linear relations of receptor and protein concentrations and the number of sections per test tube. Diagnostically, we compared the routine (6 point DCC-Scatchard) procedure for breast cancer analysis with the section method. A good correlation for ER and a less pronounced correlation for PR was found. Statistically, the precision of the method was verified by low deviations of duplicate determinations, low day-to-day variations and low inter-assay variations.

Topics: Breast Neoplasms; Endometrium; Estradiol; Female; Humans; Isoelectric Focusing; Kinetics; Ovarian Neoplasms; Pregnenediones; Receptors, Estrogen; Receptors, Progesterone; Substrate Specificity

The estrogen receptor (ER)-positive human breast cancer cell line T 47D exhibited genetic instability under cell culture conditions which maintained almost continuous exponential growth. This resulted in the spontaneous generation of three ER-positive sublines with a range of DNA ploidies and distinctive phenotypes. One of these sublines, T 47D-5, exhibited resistance to the growth-inhibitory effects of the synthetic nonsteroidal antiestrogen tamoxifen and the synthetic progestin ORG 2058, in marked contrast to "wild type" T 47D cells (designated T 47D-7 in this study). T 47D-5 cells were cloned by limiting dilution and 11 clonal cell lines were tested for sensitivity to tamoxifen. Although all clones of T 47D-5 were significantly less sensitive than T 47D-7 cells, a spectrum of sensitivities was observed. Three clones, T 47D-5-13, T 47D-5-21, and T 47D-5-23, were further characterized by measuring the concentrations of receptors for estrogen, progesterone, growth hormone, and epidermal growth factor and responses to estradiol, tamoxifen, and progestin, in terms of both induction of specific proteins and effects on cellular proliferation. Although the T 47D-5 subline and clone T 47D-5-23 were insensitive to both the growth-stimulatory effects of estradiol and the inhibitory effects of tamoxifen, this was not related to the concentration of ER or its ability to induce progesterone receptor. Estrogen receptor levels were similar in resistant and sensitive clones of T 47D-5 [70,000-81,000 sites/cell] and were 2.5-fold greater than in the sensitive T 47D-7 line [32,600 +/- 5,000 (SEM) sites/cell]. Northern blots showed no difference in the size of ER mRNA transcripts between sensitive and resistant clones. Estradiol treatment increased progesterone receptor (PR) levels in all cell lines but the magnitude and sensitivity of this response were unrelated to growth responses indicating a divergence in estrogenic control of cellular proliferation and specific protein synthesis within these clones. T 47D-5, T 47D-5-13, T 47D-5-21, and T 47D-5-23 were all insensitive to the growth-inhibitory effects of ORG 2058. The progestin was also unable to increase lactogenic and epidermal growth factor receptor concentrations in these four lines in contrast to the response in T 47D-7 cells. The insensitivity to progestin in the T 47D-5 subline and its three clonal cell lines could be accounted for, in part, by a 75-80% reduction in PR levels when compared with T 47D-7 cells.(A

Topics: Breast Neoplasms; Cell Line; Clone Cells; Drug Resistance; ErbB Receptors; Estradiol; Female; Humans; Ploidies; Pregnenediones; Progesterone Congeners; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen; Tumor Cells, Cultured

Estradiol (ER), progesterone (PR), androgen (AR) and glucocorticoid receptor (GR) levels were assayed in 25 breast cancer tumors. The tissue was pulverized and homogenized in buffer, then divided into two parts: one was assayed by the standard dextran-coated charcoal method (DCC), with Scatchard plot analysis, the other was assayed by a micromethod developed in our laboratory, as described below: --incubation of the cytosol with several ligands (labelled and unlabelled) selected to avoid unwanted cross-reactions --DCC separation, followed by extraction of all receptor-bound steroids by precipitation of proteins with methanol/TCA --separation of these steroids on a high pressure liquid chromatography (HPLC) column using a methanol/water solvent --collection of the fractions of the column outlet and counting. Use of three labelled ligands and appropriate unlabelled ligands allowed assays of the four receptors. This micromethod was highly correlated with the standard method: ER = 0.985 (P less than 0.001); PR = 0.999 (P greater than 0.001); AR = 0.989 (P less than 0.001); GR = 0.867 (P less than 0.001). Thresholds of positivity were not modified. This micromethod allowed simultaneous measurement of several receptors in 40 mg biopsy specimens and can be applied to other hormone-dependent tissues.

Topics: Breast Neoplasms; Chromatography, High Pressure Liquid; Estrenes; Female; Humans; Ligands; Metribolone; Pregnenediones; Receptors, Androgen; Receptors, Estradiol; Receptors, Estrogen; Receptors, Glucocorticoid; Receptors, Progesterone

In MCF-7 cell culture, some sera endow estradiol-17 beta with strong growth promoting properties ("active" sera) while other fail to display this property ("inactive" sera). Passage from "inactive" to "active" sera are shown here to induce the appearance of a progestin binding capacity in the receptor negative line Evsa-T. Competition with various unlabeled steroids established the specificity of this binding reaction. The induction of progesterone receptor required neither estrogens, nor ER and failed to confer major growth sensitivity to hormonal steroids: only medroxyprogesterone acetate was slightly inhibitory at high concentration. These observations disclose the influence of seric factors independent of estrogens and of ER-related mechanisms on PgR induction.

Topics: Breast Neoplasms; Cell Line; Estradiol; Female; Humans; Pregnenediones; Receptors, Estrogen; Receptors, Progesterone

Estrogen (ER) and progesterone receptors (PgR) appear to be a prerequisite to elicit a biologic response by a hormone-target organ. Current methodologies for analysis of these proteins (e.g., dextran-coated charcoal, DCC) in single-label assay (SLA) require relatively large amounts of tissue material, time and laboriousness. Therefore, we have developed for breast cancer tissue an improved dual-label assay (DLA) for simultaneous titration (by DCC) and/or characterization (by sedimentation properties) of ER and PgR on the same sample, using 125I-E2 and 3H-Org 2058 as tracers. The interaction of 125I-E2 with ER and plasma proteins in comparison to 3H-E2 was studied in terms of specificity, time course, affinity binding and sedimentation pattern. 125I-E2 bound the same molecular forms displayed by 3H-E2 (9 and 3S) but with lower titers (about 1.3-fold), irrespective of the technique used, and did not bind to sex hormone-binding globulin. Simultaneous detection of 125I and 3H was achieved by use of a gamma counter plus a beta counter sequentially. ER and PgR titrations with DCC in DLA were in good agreement with those obtained with SLA, in terms of titers and Ka values. An analogous result was obtained with sucrose density gradient (SDG) analysis. Both the DLA methods were highly reproducible (CV less than 8.0%). Between the rotors available for SDG, the vertical one was preferable because of the larger number of samples processed and of less perturbation of sedimenting receptor molecules. Furthermore, a biochemical application of the method is described. In conclusion, the DLA procedure, by simplifying ER and PgR estimation, makes it possible to study, even on small tumor biopsies, the molecular properties of these proteins in relation to the clinical response of the disease.

Topics: Breast Neoplasms; Centrifugation, Density Gradient; Cytosol; Estradiol; Female; Humans; Iodine Radioisotopes; Methods; Pregnenediones; Receptors, Estrogen; Receptors, Progesterone; Scintillation Counting; Tritium

A 24 hr incubation of T-47D human breast cancer cells with R5020, a synthetic progestin, resulted in a 200-250% increase in the specific binding of human growth hormone (hGH) and epidermal growth factor (EGF) by these cells. This effect was specific for progestins in that similar responses were observed with progesterone, medroxyprogesterone acetate and ORG 2058 but no significant increases in hGH or EGF binding were observed in cells incubated with testosterone, estradiol or hydrocortisone. Increased binding was due to an increase in the concentration of receptors (hGH, control = 6,490 +/- 500, progestin treated = 13,180 +/- 3,270 sites/cell; EGF, control = 33,380 +/- 7,410, progestin treated = 67,460 +/- 20,330 sites/cell) while the affinity constants for the hormone-receptor interactions were unchanged by progestin treatment. The specific binding of insulin, calcitonin, transferrin and concanavalin A was unaffected by these treatments. It is concluded that expression of hGH and EGF receptors in this breast cancer cell line is regulated by progestins.

Topics: Breast Neoplasms; Cell Line; Epidermal Growth Factor; ErbB Receptors; Female; Growth Hormone; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; Pregnenediones; Progesterone; Progestins; Promegestone; Receptors, Cell Surface; Receptors, Somatotropin

A whole-cell assay for measuring estrogen (ER) and progesterone (PgR) receptors in monolayer culture of human breast cancer cell lines is described. It is based on the measurement of incorporated tritiated ligands during 50 min of incubation (i.e. [3H]estradiol for ER, [3H]ORG-2058 for PgR). The assay fulfills all criteria of specificity as shown by competitive studies and measurements of the dissociation constants of the binding reactions. Moreover, a subcellular fractionation of MCF-7 labeled cells revealed that the majority of incorporated steroids was associated with the nuclear fraction. This finding is consistent with the concept of nuclear location of steroid-receptor complexes. Cultures in the presence of 10(-8) M estradiol indicated that the methodology is adequate for detecting the well-known estrogenic induction of PgR synthesis. The assay proved suitable for the quantitative assessment of the receptor content of various neoplastic (MCF-7; ZR-75-1, Cama-1, Evsa-T) and non-neoplastic (HBL-100) cell lines. The methodology has the other advantages of being simple and rapid, of requiring small amounts of cells and of allowing histological examination of the latter before, during and after biochemical analysis.

Topics: Binding, Competitive; Breast Neoplasms; Cell Line; Cell Nucleus; Estradiol; Female; Humans; Methods; Pregnenediones; Progesterone Congeners; Receptors, Estrogen; Receptors, Progesterone

A method for the determination of estrogen and progesterone receptor levels in human mammary cell lines (MCF-7, Cama-1, ZR-75-1, Evsa-T and HBL-100) is described. Cells cultured as monolayers were incubated with the tritiated steroids, [3H]-17 beta-Estradiol or [3H] ORG-2058. Binding of steroids to receptors was a function of cellular uptake. Incubation periods of 50 min were sufficient to attain maximum intracellular incorporation. The binding of 17 beta-E2 and ORG-2058 to MCF-7 cells, a phenomenon which is saturable at low concentrations for the radioactive ligand, is a linear function of the number of cells assayed (Interval: 2.5 X 10(4) to 1.5 X 10(6) cells per well). Binding data and their Scatchard plot allowed for the calculation of affinity and capacity values. Thus, for ER, Kd = 2.0 +/- 0.5 X 10(-10) M and n = 3.76 +/- 0.91 Fmol/microgram DNA, and for PgR Kd = 2.0 +/- 0.2 X 10(-10) M and n = 14.02 +/- 2.30 Fmol/microgram DNA (Mean +/- SD). Binding specificity of 17 beta-Estradiol and ORG-2058 to MCF-7 cells was analysed by means of study on the inhibitory effect of increasing concentrations of unlabelled competitors: 17 beta-Estradiol, ORG-2058, Estrone, DES, R-5020, Cortisol, Androsterone and Testosterone. Only pharmacological doses of some of the mentioned molecules produce displacement of the hormonereceptor binding. This phenomenon appears to be related to the affinity of these chemical compounds for the receptor macromolecules to which estrogens and progesterone bind.

Topics: Binding, Competitive; Breast; Breast Neoplasms; Cell Line; DNA; DNA, Neoplasm; Estradiol; Female; Humans; Neoplasms, Hormone-Dependent; Pregnenediones; Receptors, Estrogen; Receptors, Progesterone

Topics: Breast Neoplasms; Carrier Proteins; Female; Humans; Norpregnenes; Pregnenediones; Receptors, Progesterone