metribolone and Breast-Neoplasms

Breast cancer is a hormone-related carcinoma and the most commonly diagnosed malignancy in women. Although Her-2, estrogen receptor (ER), and progesterone receptor (PR) are the major diagnostic markers and therapeutic targets to breast cancer, searching for additional molecular targets remains an important issue and one of the candidates is androgen receptor (AR). AR has been shown expressed in 70% breast cancer patients and connects to low recurrence and high survival rate. Our previous study demonstrates that Ser81 phosphorylation of AR in prostate cancer cells is critical for its protein stability modulated by human epidermal growth factor receptor-2 (Her2). The aim of this study is to investigate the influence of Her2 and AR in proliferation of breast cancer cell line, MDA-MB-453. The data show that AR which was activated by synthetic androgen R1881 suppressed the proliferation of MDA-MB-453 cells. Notably, AR activation decreased the protein levels of cell growth-related proteins, including cyclin A, cyclin B, and early growth response protein 1 (Egr1), while cell-cycle inhibitor protein p27 was increased. Besides, Heregulin (HRG)-induced Her2 activation decreased the AR protein levels and its Ser81 phosphorylation. Her2 small molecular inhibitor, Lapatinib, dose-dependently suppressed cell proliferation while the levels of phospho-Ser81 AR and p27 protein were increased. Phospho-Ser81 AR was also increased after Her2 knockdown. Specifically, the influence of phospho-Ser81 AR by Lapatinib was primarily found in the nucleus of MDA-MD-453 cells, where the cell proliferation might directly be interfered. In conclusion, our findings indicate that Her2 might negatively regulate AR phosphorylation/activation and contribute to regulate the proliferation of MDA-MB 453 cells.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Humans; Lapatinib; Metribolone; Molecular Targeted Therapy; Phosphorylation; Quinazolines; Receptor, ErbB-2; Receptors, Androgen

Plasma membrane-bound carboxypeptidase-D (CPD) cleaves C-terminal arginine from extracellular substrates. In the cell, arginine is converted to nitric oxide (NO). We have reported that up-regulation of CPD mRNA/protein levels by 17β-estradiol and prolactin (PRL) in breast cancer cells, and by testosterone in prostate cancer cells, increased NO production and cell survival. The CPD promoter contains a consensus γ-interferon-activated sequence (GAS) and 3 putative androgen response elements (ARE.1, ARE.2, ARE.3) that could potentially bind PRL-activated transcription factor Stat5 (signal transducer and activator of transcription 5) and the liganded androgen receptor (AR), respectively. This study showed that synthetic androgen R1881 and PRL elevated CPD mRNA/protein levels in human MCF-7 and T47D breast cancer cells in a time-/dose-dependent manner. PRL/R1881-elevated CPD expression was blocked by actinomycin-D, and a CPD promoter construct containing these GAS and AREs was stimulated by PRL or R1881, indicating transcriptional regulation by both hormones. Luciferase reporter assays showed that GAS and the adjacent ARE.1 only were active. Mutation of GAS in the ΔGAS-CPD construct (ARE.1 intact) abolished CPD promoter activity in response to PRL and, surprisingly, to R1881 as well. ΔGAS-CPD promoter activity was restored by PRL+R1881 in combination, and enhanced by ectopic Stat5, but abolished by Stat5 gene knockdown. Chromatin immunoprecipitation analysis confirmed binding of activated Stat5 and liganded AR to GAS and ARE.1, respectively. Activated Stat5 also induced binding of unliganded AR to ARE.1, and liganded AR induced binding of unactivated Stat5 to GAS. In summary, PRL and R1881, acting through Stat5 and AR, act cooperatively to stimulate CPD gene transcription in breast cancer cells.

Topics: Androgens; Base Sequence; Breast Neoplasms; Consensus Sequence; Cycloheximide; Dactinomycin; Enzyme Activation; Enzyme Induction; Female; Gene Expression Regulation, Neoplastic; Humans; Interferon-gamma; MCF-7 Cells; Metribolone; Prolactin; Promoter Regions, Genetic; Protein Synthesis Inhibitors; Proteins; Receptors, Androgen; STAT5 Transcription Factor

Increasing interest in the role of lipids in cancer cell proliferation and resistance to drug therapies has motivated the need to develop better tools for cellular lipid analysis. Quantification of lipids in cells is typically done by destructive chromatography protocols that do not provide spatial information on lipid distribution and prevent dynamic live cell studies. Methods that allow the analysis of lipid content in live cells are therefore of great importance. Using micro-Raman spectroscopy and coherent anti-Stokes Raman scattering (CARS) microscopy, we generated a lipid profile for breast (T47D, MDA-MB-231) and prostate (LNCaP, PC3) cancer cells upon exposure to medroxyprogesterone acetate (MPA) and synthetic androgen R1881. Combining Raman spectra with CARS imaging, we can study the process of hormone-mediated lipogenesis. Our results show that hormone-treated cancer cells T47D and LNCaP have an increased number and size of intracellular lipid droplets and higher degree of saturation than untreated cells. MDA-MB-231 and PC3 cancer cells showed no significant changes upon treatment. Principal component analysis with linear discriminant analysis of the Raman spectra was able to differentiate between cancer cells that were treated with MPA, R1881, and untreated.

Topics: Breast Neoplasms; Cell Line, Tumor; Female; Humans; Lipid Droplets; Lipids; Male; Medroxyprogesterone Acetate; Metribolone; Microscopy; Molecular Imaging; Prostatic Neoplasms; Spectrum Analysis, Raman

Although a high frequency of androgen receptor (AR) expression in human breast cancers has been described, exploiting this knowledge for therapy has been challenging. This is in part because androgens can either inhibit or stimulate cell proliferation in pre-clinical models of breast cancer. In addition, many breast cancers co-express other steroid hormone receptors that can affect AR signaling, further obfuscating the effects of androgens on breast cancer cells.. To create better-defined models of AR signaling in human breast epithelial cells, we took estrogen receptor (ER)-α-negative and progesterone receptor (PR)-negative human breast epithelial cell lines, both cancerous and non-cancerous, and engineered them to express AR, thus allowing the unambiguous study of AR signaling. We cloned a full-length cDNA of human AR, and expressed this transgene in MCF-10A non-tumorigenic human breast epithelial cells and MDA-MB-231 human breast-cancer cells. We characterized the responses to AR ligand binding using various assays, and used isogenic MCF-10A p21 knock-out cell lines expressing AR to demonstrate the requirement for p21 in mediating the proliferative responses to AR signaling in human breast epithelial cells.. We found that hyperactivation of the mitogen-activated protein kinase (MAPK) pathway from both AR and epidermal growth factor receptor (EGFR) signaling resulted in a growth-inhibitory response, whereas MAPK signaling from either AR or EGFR activation resulted in cellular proliferation. Additionally, p21 gene knock-out studies confirmed that AR signaling/activation of the MAPK pathway is dependent on p21.. These studies present a new model for the analysis of AR signaling in human breast epithelial cells lacking ERα/PR expression, providing an experimental system without the potential confounding effects of ERα/PR crosstalk. Using this system, we provide a mechanistic explanation for previous observations ascribing a dual role for AR signaling in human breast cancer cells. As previous reports have shown that approximately 40% of breast cancers can lack p21 expression, our data also identify potential new caveats for exploiting AR as a target for breast cancer therapy.

Topics: Androgen Antagonists; Androgens; Anilides; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Activation; Epithelial Cells; Estrogen Receptor alpha; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression; Humans; MAP Kinase Signaling System; Metribolone; Nitriles; Receptors, Androgen; Recombinant Proteins; Tosyl Compounds; Up-Regulation

The progesterone receptor (PR) is estrogen regulated, and PR levels in breast tumors can be used to predict the success of endocrine therapies targeting the estrogen receptor (ER). Tanaproget is a nonsteroidal progestin agonist with very high PR binding affinity and excellent in vivo potency. When appropriately radiolabeled, it might be used to image PR-positive breast tumors noninvasively by positron emission tomography (PET). We describe the synthesis and PR binding affinities of a series of fluoroalkyl-substituted 6-aryl-1,4-dihydrobenzo[d][1,3]oxazine-2-thiones, analogues of Tanaproget. Some of these compounds have subnanomolar binding affinities, higher than that of either Tanaproget itself or the high affinity PR ligand R5020. Structure-binding affinity relationships can be rationalized by molecular modeling of ligand complexes with PR, and the enantioselectivity of binding has been predicted. These compounds are being further evaluated as potential diagnostic PET imaging agents for breast cancer, and enantiomerically pure materials of defined stereochemistry are being prepared.

Topics: Animals; Benzoxazines; Binding, Competitive; Breast Neoplasms; Female; Fluorine; Fluorine Radioisotopes; Models, Molecular; Promegestone; Radioligand Assay; Rats; Receptors, Progesterone; Stereoisomerism; Structure-Activity Relationship; Thermodynamics; Thiones

The purpose of this study was to understand the characteristics of prostate-derived Ets factor (PDEF) protein expression in breast and prostate cancer progression. A polyclonal antibody specific to PDEF was raised and reacted with tissue microarrays consisting of benign breast, in situ ductal, invasive ductal, and invasive lobular breast carcinomas. The antibody was also reacted with tissue microarrays, including benign prostate, prostate intraepithelial neoplasias (PINs), and prostate carcinomas. Increased expression of PDEF was identified in 18%, 50%, 46%, and 51% of benign breast tissues, intraductal, invasive ductal, and invasive lobular carcinomas, respectively. Importantly, in matched samples of benign breast vs tumor, 90% showed higher expression of PDEF in the tumor tissue. Moreover, in invasive breast carcinomas, increased PDEF expression tended to correlate with Her2/neu overexpression. Increased expression of PDEF was also found in 27%, 33%, and 40% of benign prostate tissues, PIN samples, and prostate adenocarcinomas, respectively. Again, in matching samples of cancer vs benign and cancer vs PIN, 68% and 70%, respectively, showed increased expression in the malignant tissue. Moreover, PDEF was found to be more highly expressed in tumors with intermediate or high Gleason score compared with low-grade tumors (P < .01). In addition, R1881 treatment induced PDEF expression in the LNCaP prostate tumor cell line, suggesting regulation of PDEF by androgens in vivo. Together, these results for the first time show frequent increased expression of PDEF protein in breast and prostate tumors and support a role for PDEF in breast and prostate cancer progression.

Topics: Breast; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; Male; Metribolone; Prostate; Prostatic Neoplasms; Proto-Oncogene Proteins c-ets; U937 Cells

TRPS1 mRNA is more highly expressed in androgen-dependent lymph node carcinoma of prostate-fast growing colony (LNCaP-FGC) compared with androgen-independent lymph node carcinoma of prostate-lymph node original (LNCaP-LNO) prostate cancer cell lines. Furthermore, TRPS1 mRNA expression is down-regulated by androgens in LNCaP-FGC cells, a process mediated by the androgen receptor (AR). Here, we present TRPS1 protein expression in human prostate cancer material derived from a panel of six androgen-dependent and eight androgen-independent human prostate cancer xenografts. TRPS1 protein is expressed in all androgen-dependent xenografts, which also express AR and prostate-specific antigen (PSA). Androgen withdrawal by castration resulted in an increase in TRPS1 protein in two androgen-dependent xenografts, indicating relieved repression by action of AR. TRPS1 protein is expressed in four androgen-independent xenografts and is low or absent in the other four androgen-independent xenografts. Androgen withdrawal by castration demonstrates that TRPS1 protein levels remain the same in 1 androgen-independent xenograft, most likely due to the lack of AR expression. These data show that TRPS1 protein expression is regulated by androgens via the AR in human prostate cancer xenografts. Analysis of TRPS1 mRNA expression in normal and tumour tissue of the prostate and 18 other human tissues, showed that TRPS1 had the highest mRNA expression levels in normal and tumour tissues of breast. In addition, high TRPS1 mRNA and protein expression levels were observed in four out of five human breast cancer cell lines. In conclusion, TRPS1 protein expression is down-regulated by androgens in human prostate cancer, and analysis of TRPS1 mRNA expression levels in several human tissues showed that the highest levels were observed in normal and tumour breast tissue.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Metribolone; Mice; Mice, Inbred Strains; Neoplasm Proteins; Prostatic Neoplasms; Repressor Proteins; RNA, Messenger; Testosterone Congeners; Tissue Distribution; Transcription Factors; Transplantation, Heterologous

Androgen and progesterone receptors (AR and PR) are two determining factors in gonadal differentiation that are highly expressed in developing and mature gonads. Loss of AR results in XY sex reversal and mutations causing reduced AR activity lead to varying degrees of defects in masculinization. Female PR knockout mice are infertile due to ovarian defects. While much has been discovered about positive regulation of these receptors by coactivators little is known about repression of the transcriptional activity of AR and PR in the presence of agonists. In this study we assessed the effect of SMRT and DAX-1 on AR and PR activity in the presence of both agonists and partial antagonists. We show that SMRT and DAX-1 repress agonist-dependent activity of both receptors, and the mechanism of repression includes disruption of the receptor dimer interactions rather than recruitment of histone deacetylases. We demonstrate that endogenous agonist-bound PR and DAX-1 in T47D breast cancer cells and endogenous AR and DAX-1 in LNCaP prostate cancer cells can be coimmunoprecipitated suggesting that the interaction is physiological. Surprisingly, although DAX-1 represses partial antagonist activity of AR, it was ineffective in repressing partial antagonist induced activity of PR. In contrast to most reported repressors, the expression of DAX-1 is restricted. We found that although DAX-1 is expressed in normal human prostate, its expression is strongly reduced in benign prostatic hyperplasia suggesting that DAX-1 plays a role in limiting AR activity in prostate.

Topics: Animals; Binding Sites; Breast Neoplasms; COS Cells; DAX-1 Orphan Nuclear Receptor; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Hormone Antagonists; Humans; Hydroxamic Acids; Male; Metribolone; Mifepristone; Nuclear Proteins; Nuclear Receptor Co-Repressor 1; Nuclear Receptor Co-Repressor 2; Promoter Regions, Genetic; Prostate; Prostatic Hyperplasia; Protein Structure, Tertiary; Protein Synthesis Inhibitors; Receptors, Androgen; Receptors, Calcitriol; Receptors, Interferon; Receptors, Progesterone; Receptors, Retinoic Acid; Repressor Proteins; Testosterone Congeners; Tumor Suppressor Protein p53

Genes that are regulated by androgens in the human prostate are believed to play an essential role in prostate physiology and they may also be involved in the proliferative response of prostate cancer cells to androgens. We used a cDNA subtraction approach to identify novel androgen-regulated transcripts in LNCaP cells that were exposed to 0.1 nM R1881 for 24 h. We report here that SPAK, a recently identified STE20/SPS1-related kinase that modulates p38 MAP kinase activity, exhibited increased expression in androgen-treated LNCaP cells. Androgen regulation of SPAK was both dose- and time-dependent. R1881-induced SPAK expression was completely abrogated by the antiandrogen casodex and by actinomycin D indicating that androgen induction of SPAK requires the androgen receptor and transcription. Cycloheximide caused a partial inhibition of R1881-induced SPAK expression which suggests that androgen induction of SPAK expression may require synthesis of additional proteins. Northern blot and ribonuclease protection assays demonstrated that SPAK is expressed at high levels in normal human testes and prostate, as well as in a number of breast and prostate cancer cell lines. These results identify SPAK, a member of a key cell signalling pathway, as an androgen-responsive gene in LNCaP cells. We hypothesize that SPAK may mediate androgen action in the normal and cancerous prostate gland.

Topics: Amino Acid Sequence; Base Sequence; Blotting, Northern; Breast Neoplasms; Cloning, Molecular; Cycloheximide; DNA, Complementary; Female; Gene Expression Profiling; Humans; Male; Metribolone; Molecular Sequence Data; Prostatic Neoplasms; Protein Serine-Threonine Kinases; RNA, Messenger; Testosterone Congeners; Time Factors; Tumor Cells, Cultured; Up-Regulation

The aim of the work described in this report is to develop and characterize a cell-based androgen reporter assay. For this purpose, the androgen receptor (AR) expressing human breast cancer cell line T47D was stably transfected with a luciferase gene under transcriptional control of the PB-ARE-2 androgen response element. The application of this cell line in an endogenous Androgen Receptor-mediated LUciferase eXpression assay (AR-LUX) was validated. An EC50 value of 86 pM was determined for the standard androgen R1881 with a detection limit of 46 pM. Other androgens like dihydrotestosterone, 17beta-trenbolone, and bolasterone also induced luciferase expression, while anti-androgens suppressed these responses. As expected, AR-mediated responses were also elicited by high concentrations of the steroids progesterone, 17beta-estradiol, d-aldosterone, and dexamethasone, with observed EC50 values 10 to 350,000 times higher than that for R1881. A unique feature of the AR-LUX assay is that effects on modulation of active endogenous AR-levels are reliably reflected in the luciferase induction response, as exemplified by vitamin D, all-trans-retinoic acid, epigallocatechin gallate, and forskolin. This feature is especially useful when assessing complex mixtures, e.g., environmental samples or natural compound libraries. From these data it is concluded that the AR-LUX assay is a reliable in vitro test system for the detection and quantification of AR-mediated biological effects. The 96-well plate format makes the assay particularly suitable for high-throughput screening.

Topics: Aldosterone; Androgens; Breast Neoplasms; Dexamethasone; Down-Regulation; Estradiol; Genes, Regulator; Genes, Reporter; Humans; Luciferases; Metribolone; Receptors, Androgen; Transfection; Tumor Cells, Cultured

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 mechanisms by which androgens modulate breast cancer cell growth are largely unknown. Using cultured human PMC42 breast cancer cells, we have determined effects of the androgen R1881 on the activity of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinase. R1881 did not alter JNK and p38 kinase activity, but activated ERK in a dose-dependent manner. Activation was rapid, peaking at 5 min followed by a decline to baseline after 30-60 min, and was accompanied by tyrosine phosphorylation of ERK. The androgen antagonist flutamide elevated ERK to similar levels and DNA synthesis to levels half those seen with R1881; in addition, excess flutamide lowered R1881-stimulated DNA synthesis to levels seen with flutamide alone. These findings suggest (i) that in human PMC42 breast cancer cells R1881 activates ERK through a non-genomic mechanism, (ii) that this non-genomic mechanism is equivalently activated by the androgen antagonist flutamide, and (iii) that androgen/antiandrogen effect on DNA synthesis may involve both genomic and non-genomic mechanisms. These findings may have important implications for the clinical use of such agents in breast cancer.

Topics: Androgen Antagonists; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; DNA Replication; Dose-Response Relationship, Drug; Enzyme Activation; Female; Flutamide; Humans; Metribolone; Testosterone Congeners; Tumor Cells, Cultured

mRNA differential display polymerase chain reaction analysis was used to screen systematically for novel androgen-regulated genes in the human prostatic adenocarcinoma cell line LNCaP. A 232 bp PCR fragment was found to be consistently down-regulated by the synthetic androgen R1881. Sequencing revealed complete identity with the human homologue of mouse Paternally expressed gene 3 (Peg3), an imprinted gene that plays an important role as a downstream mediator of the effects of tumor necrosis factor (TNF). The down-regulation of Peg3 mRNA by androgens was confirmed by Northern blot hybridization. The effect proved time and dose dependent with maximal repression (3.5-fold) after 24 h of treatment with 10(-8) M R1881. The steroid specificity of Peg3 mRNA regulation reflected the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells, supporting the involvement of the androgen receptor in the repression process. Basal expression of Peg3 mRNA was almost completely abolished by the protein synthesis inhibitor cycloheximide. Experiments with Actinomycin D suggested that androgens act at a transcriptional level rather than by changing the stability of Peg3 mRNA. Comparison of the expression of Peg3 mRNA in 50 different human tissues revealed ubiquitous expression, but low levels in the prostate. The highest levels were observed in endocrine tissues such as ovary, placenta, adrenal and pituitary. High levels were also noted in various parts of the brain. No detectable levels of Peg3 mRNA were observed in two other androgen receptor-positive prostate tumor cell lines (MDA PCa-2a and -2b), and in the poorly differentiated and androgen receptor-negative prostate tumor lines PC-3 and DU-145. It is concluded that both androgens and loss of differentiation may affect the expression of Peg3, a mediator of the effects of TNF. Further experiments will be required to explore whether these changes affect the responsiveness of prostate tumor cells to TNF.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cycloheximide; Dactinomycin; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Kruppel-Like Transcription Factors; Male; Metribolone; Mice; Polymerase Chain Reaction; Prostatic Neoplasms; Protein Kinases; Proteins; Receptors, Androgen; RNA, Messenger; Testosterone Congeners; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured; Zinc Fingers

Sex steroids control the proliferation of their target cells through two different pathways: 1) proliferative response (Step-1); and 2) inhibition of cell proliferation (Step-2). Mechanisms of cell proliferation regulation are incompletely understood; however, there is general agreement with the notion that sex steroid receptors play an important role in the control of the proliferation of sex steroid target cells. To test this hypothesis, a full human androgen receptor (AR) vector was transfected into human breast cancer MCF7 cells. The cloned cells that stably express the AR, called MCF7-AR1 cells, contained approximately five times more AR than the wild-type MCF7 cells from which they were derived. These AR-transfected cells retained their capacity to proliferate when estrogens were added to 10% charcoal-dextran stripped human serum but did not acquire the ability to proliferate when androgens were added to this medium. In serumless medium (ITDME), these cells proliferated maximally, as MCF7 cells did; however, natural and synthetic androgens prevented the AR-transfected cells from proliferating. Inhibition of cell proliferation occurred when physiological androgen concentrations (1 nM) were added to ITDME; this effect was almost completely reversed by Casodex, a synthetic androgen antagonist. Under the effect of androgens added to ITDME, MCF7-AR1 cells were arrested in the G0/G1 phase within 24 h. These data suggest that: 1) the androgen-induced inhibition of cell proliferation (Step-2) is AR-mediated; and 2) the AR may be necessary, but not sufficient, to mediate the androgen-induced proliferative response (Step-1).

Topics: Androgen Antagonists; Androgens; Anilides; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Division; Dihydrotestosterone; Dose-Response Relationship, Drug; Female; Flutamide; Humans; In Vitro Techniques; Metribolone; Nandrolone; Nitriles; Receptors, Androgen; Testosterone; Testosterone Congeners; Tosyl Compounds; Transfection; Tumor Cells, Cultured

The control of cell proliferation by estrogens was examined under the premises of the indirect-negative hypothesis, which states that estradiol cancels the proliferative inhibition exerted by a serum-borne protein on estrogen-target cells. Fractionation protocols resulted in the co-elution of the inhibitory activity with serum albumin. Removal of human albumin (HA) from charcoal-dextran stripped serum by hexyl-S agarose chromatography resulted in a preparation lacking inhibitory effect. HA inhibited the proliferation of human estrogen-target MCF-7 cells. Human non-estrogen-target MDA-MB231 cells were impervious to the effect of HA. MCF-7 cells were exposed to recombinant human albumin (rHA) and to its rDomain I and rDomains I + II. Inhibition of proliferation was maximal with rHA and with rDomains I + II; rDomain I was less inhibitory. The inhibitory effect of albumin was cell type and protein specific. Only estrogens cancelled the albumin inhibition; recombinant growth factors and other hormones were ineffective. These data suggest that: (a) albumin or a portion of it (most likely within Domains I and II) is the specific inhibitory signal for the proliferation of human estrogen-target, serum-sensitive cells; (b) estrogens specifically cancel this inhibition; (c) inhibitory signals prevail over putative growth factors; and (d) the default state in these cells is proliferation.

Topics: Blood Proteins; Breast Neoplasms; Cell Cycle; Cell Division; Chromatography, Affinity; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Female; Fibroblast Growth Factor 2; Fulvestrant; Humans; Kinetics; Metribolone; Peptide Fragments; Recombinant Proteins; Serum Albumin; Tumor Cells, Cultured

Amphiregulin is a heparin-binding epidermal growth factor (EGF)-related peptide that binds to the EGF receptor (EGF-R) with high affinity. In this study, we report a role for amphiregulin in androgen-stimulated regulation of prostate cancer cell growth. Androgen is known to enhance EGF-R expression in the androgen-sensitive LNCaP human prostate carcinoma cell line, and it has been suggested that androgenic stimuli may regulate proliferation, in part, through autocrine mechanisms involving the EGF-R. In this study, we demonstrate that LNCaP cells express amphiregulin mRNA and peptide and that this expression is elevated by androgenic stimulation. We also show that ligand-dependent EGF-R stimulation induces amphiregulin expression and that androgenic effects on amphiregulin synthesis are mediated through this EGF-R pathway. Parallel studies using the estrogen-responsive breast carcinoma cell line, MCF-7, suggest that regulation of amphiregulin by estrogen may also be mediated via an EGF-R pathway. In addition, heparin treatment of LNCaP cells inhibits androgen-stimulated cell growth further suggesting that amphiregulin can mediate androgen-stimulated LNCaP proliferation. Together, these results implicate an androgen-regulated autocrine loop composed of amphiregulin and its receptor in prostate cancer cell growth and suggest that the mechanism of steroid hormone regulation of amphiregulin synthesis may occur through androgen upregulation of the EGF-R and subsequent receptor-dependent pathways.

Topics: Amino Acid Sequence; Amphiregulin; Androgens; Breast Neoplasms; Cell Division; EGF Family of Proteins; ErbB Receptors; Estradiol; Female; Glycoproteins; Growth Substances; Heparin; Humans; Intercellular Signaling Peptides and Proteins; Male; Metribolone; Molecular Sequence Data; Prostatic Neoplasms; RNA, Messenger; Tumor Cells, Cultured; Up-Regulation

Androst-5-ene-3 beta,17 beta-diol (ADIOL) and 5 alpha-androstane-3 beta,17 beta-diol (5 alpha A), which are metabolites of dehydroepiandrosterone and dihydrotestosterone, are known to have estrogenic properties. This study reevaluates the estrogenic effects of ADIOL and 5 alpha A in MCF-7 cells and demonstrates additionally androgen-like inhibitory properties of these compounds in human hormone-dependent mammary cancer cells. ADIOL and 5 alpha A (10-100 nM) stimulate the proliferation of estrogen-sensitive MCF-7 cells. Binding assays with the estrogen receptor and inhibition of stimulation with the antiestrogen tamoxifen support the involvement of the estrogen receptor. On the other hand, the mammary cancer cell line MFM-223 is strongly inhibited by ADIOL and 5 alpha A in the same concentration range. This cell line is androgen receptor positive and is inhibited by androgens, but unresponsive to estrogens and progestins. The inhibitory effects of ADIOL and 5 alpha A in MFM-223 cells are mediated by the androgen receptor as demonstrated by receptor studies and competition experiments with hormone antagonists. ADIOL and 5 alpha A thus possess estrogen- and androgen-like properties and can stimulate or inhibit proliferation of human mammary cancer cells. The reactions of mammary cancer cells to these steroids depend on the receptor content and the growth properties of the individual cell line.

Topics: Androstane-3,17-diol; Androstenediol; Binding, Competitive; Breast Neoplasms; Cell Division; Female; Humans; Metribolone; Receptors, Androgen; Receptors, Estrogen; Tumor Cells, Cultured

The regulatory influence of medroxyprogesterone acetate (MPA) on estrogen and androgen receptors of the human breast cancer cell lines MCF-7 and EFM-19 was explored in conjunction with the growth-promoting properties of these steroids. In the absence of steroidal stimulation, up to 1 microM MPA had no effect on the proliferation of the MCF-7 cell strain used and of EFM-19 cells. Under stimulation with 10 nM 17 beta-estradiol or 1 microM dihydrotestosterone, dose-dependent inhibition of the cell proliferation rates by 0.1-1 microM MPA was observed. Binding of MPA to the androgen receptor (Kd = 2.1 nM) but not to the estrogen receptor was demonstrable. During incubation of MCF-7 or EFM-19 cells with 1 microM MPA for 7 days, the estrogen and androgen receptor contents were down-regulated by approximately 50% and 60%, respectively. Likewise, the number of androgen-binding sites was reduced to 35% of the untreated controls after incubation of MCF-7 cells with 1 microM synthetic progestin R5020 for 7 days. The results indicate down-regulation of estrogen and androgen receptors by progestins in the absence of stimulatory effects on the proliferation of mammary carcinoma cells.

Topics: Breast Neoplasms; Cell Division; Dihydrotestosterone; Dose-Response Relationship, Drug; Down-Regulation; Estradiol; Humans; In Vitro Techniques; Medroxyprogesterone; Metribolone; Receptors, Androgen; Receptors, Estrogen

The proliferation of three mammary carcinoma cell lines was explored for the effectiveness of dihydrotestosterone (DHT) and the antiandrogenic substances cyproterone acetate (CPA) or hydroxyflutamide. The cell growth, determined in multiple experimental cultures of the estrogen-sensitive lines MCF-7 and EFM-19, was stimulated by 10(-9) M to 10(-6) M DHT, whereas estrogen-resistant MFM-21 cells were unresponsive to the hormonal factors applied. Growth-promoting effects of 10(-8) M to 10(-6) M CPA were detected in cultures of those cell lines which were sensitive to estrogen and androgen. Competition experiments with DHT and the antiandrogens suggested involvement of the androgen receptor in the stimulation of cell growth by CPA. Participation of the estrogen receptor was excluded by lack of competition between CPA and the enhancement of proliferation by estradiol-17 beta. At the receptor level the antiandrogens were able to compete with androgen binding. The results of the study demonstrate androgenic properties of CPA in regard to the growth of human mammary carcinoma cells.

Topics: Androgen Antagonists; Androgens; Breast Neoplasms; Cell Division; Cyproterone; Cyproterone Acetate; Dihydrotestosterone; Estrenes; Estrogens; Female; Humans; Metribolone; Receptors, Androgen; Tumor Cells, Cultured

We have adapted an oil microcentrifuge assay for evaluating the binding of tritiated methyltrienolone ([3H]R1881) in metabolically active primary cultured porcine gubernaculum fibroblasts. Almost complete separation of cells with the bound hormone and medium with the unbound hormone can be achieved within 30 s of centrifugation at 10,000 g. Specific [3H]R1881 binding was found in four different gubernaculum fibroblast cultures (9,000-42,000 sites/cell) and MCF-7 human breast cancer cells (34,000 sites/cell) which were used as controls. Binding was inhibited by radio-inert R1881 and 5 alpha-dihydrotestosterone but not by estradiol and cortisol. The results indicate that androgen receptors are present in fibroblasts from the gubernaculum during both the first and second phase of testicular descent and that they may play a role in the regulation of that process.

Topics: Animals; Binding, Competitive; Breast Neoplasms; Cells, Cultured; Centrifugation; Dihydrotestosterone; Estradiol; Estrenes; Fibroblasts; Humans; Hydrocortisone; Male; Metribolone; Receptors, Androgen; Swine; Testis; 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

Quantitative and qualitative changes in estrogen receptor follow addition of estradiol to estrogen responsive MCF-7 human breast cancer cells. We asked whether similar changes would accompany treatment of these cells with physiologically relevant concentrations of androgens. Androgen receptor sites were quantified by competitive protein binding assays on whole cells or extracts at various times following hormone addition. Both direct and exchange assays were employed. The androgen receptor in all of these experiments remained in a form which is completely exchangeable and approx 85% salt extractable. Quantity of receptor was unchanged (30,000 sites/cell, Kd 0.1 nM). Responsiveness to hormone treatment was demonstrated by antagonizing the estrogen dependent augmentation of cytoplasmic progesterone receptor in the MCF-7 cells with androgens. Thus, the androgen receptor was shown to be biologically active, but no time dependent quantitative or qualitative changes were observed during the first 6 h following androgen treatment.

Topics: Androgens; Breast Neoplasms; Cell Line; Dihydrotestosterone; Estradiol; Estrenes; Female; Humans; Metribolone; Potassium Chloride; Receptors, Androgen; Receptors, Estrogen; Receptors, Progesterone; Receptors, Steroid

Topics: Breast Neoplasms; Dexamethasone; Dihydrotestosterone; Estradiol; Estrenes; Female; Humans; Metribolone; Progestins; Receptors, Androgen; Receptors, Estrogen; Receptors, Glucocorticoid; Receptors, Progesterone; Receptors, Steroid; Tritium

The synthetic radioligand [3H]-R1881 binds to both androgen and progestogen receptors; these two types of receptor activity can be separated by competition experiments with radioinert steroids of defined biological activity. Using two standard tissues, rat prostate and human uterus, which are sources of androgen and progestogen receptors respectively, the optimal conditions for the determination of each type of activity were established. For the purposes of routine assay, androgen receptors were quantified after saturation of progestogen receptor sites with 125 nM radioinert R5020 using [3H]-R1881 and increasing concentrations of radioinert R1881. Progestogen receptor activity could be identified using the same radioligand and competition with radioinert progesterone or R5020, though for routine purposes, progestogen receptors were quantified using the more specific radioligand, [3H]-R5020. The binding of [3H]-R1881 to tumour cytosol was examined in 122 human breast cancers. Seventy-two tumours (59%) showed binding. Androgen receptor activity alone was present in 16 tumours, progestogen receptor activity alone in 30 tumours and both types in 26 tumours. Tumours containing progestogen receptor activity also showed binding to the progestogen [3H]-R5020, whilst those containing androgen receptors alone did not. Androgen receptor concentration varied from 17 to 210 fmol binding sites/mg cytosol protein (mean value 68) and the mean Kd was 2.15 X 10(-9) M. Progestogen receptor concentration varied from 25 to 1350 fmol binding sites/mg cytosol protein (mean value 410) and the mean Kd was 1.35 X 10(-9) M. The biological significance of the presence of these types of receptor in human breast cancers is currently being assessed from clinical follow-up.

Topics: Animals; Binding, Competitive; Breast Neoplasms; Cytosol; Estrenes; Female; Humans; Male; Metribolone; Progesterone; Promegestone; Prostate; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, Androgen; Receptors, Progesterone; Testosterone Congeners; Uterus