Expression of estrogen and progesterone receptors and estrogen metabolizing enzymes in different breast cancer cell lines

Prolonged exposure to estrogens is a significant risk factor for the development of breast cancer. Estrogens exert carcinogenic effects by stimulating cell proliferation or through oxidative metabolism that forms DNA-damaging species. In the present study, we aimed to provide a better understanding of estrogen metabolism and actions in breast cancer, and to characterize model breast cancer cell lines. We determined the expression profiles of the genes for the estrogen and progesterone receptors, and for 18 estrogen-metabolizing enzymes in eight cell lines: MCF-7, MCF-10A, T47D, SKBR3, MDA-MB-231, MDA-MB-361, Hs-578T and Hs-578Bst cells. Similar gene expression profiles of these receptors and enzymes for the formation of estradiol via the aromatase and sulfatase pathways were observed in the MCF-7 and T47D metastatic cell lines. The MDA-MB-361 cells expressed ESR1, ESR2 and PGR as well, but differed in expression of the estrogen-metabolizing enzymes. In the MDA-MB-231 and SKBR3 cells, all of these estrogen-forming enzymes were expressed, although the lack of ESR1 and the low levels of ESR2 expression suggested that the estrogens can only act via non-ER mediated pathways. In the non-tumorigenic MCF-10A cell line, the key enzymes of the aromatase pathway were not expressed, and the sulfatase pathway also had a marginal role. The comparison between gene expression profiles of the non-tumorigenic Hs-578Bst cells and the cancerous Hs-578T cells revealed that they can both form estrogens via the sulfatase pathway, while the aromatase pathway is less important in the Hs-578Bst cells. The Hs-578T cells showed low levels of ESR1, ESR2 and PGR expression, while only ESR1 and ESR2 expression was detected in the Hs-578Bst cells. Our data show that the cell lines examined provide the full range of model systems and should further be compared with the expression profiles of breast cancer specimens.     

Correlation of immunocytochemical and immunohistochemical determination of estrogen and progesterone receptors in breast cancer

OBJECTIVE: To evaluate prospectively the degree of correlation between immunocytochemical (ICC) and immunohistochemical (IHC) determination of estrogen (ER) and progesterone receptors (PR) in breast cancer. STUDY DESIGN: Fine needle aspiration cytology (FNAC) of 101 primary breast cancers were immunostained for ER and PR. They were compared with similar determinations in formalin-fixed paraffin sections of biopsies from the same patients. In cases of discrepancy, the histologic result was considered the gold standard. RESULTS: For ER a cytohistologic correlation of 94%, with a sensitivity of 96.1% and specificity of 86.9%, was found. For PR the cytohistologic correlation was 71.2%, with a sensitivity of 65.7% and specificity of 83.8%. CONCLUSION: ICC determination of hormone receptors in routinely fixed smears obtained by FNAC is a simple method that correlates adequately with the results of IHC determinations, especially for ER.     

4-Hydroxytamoxifen-induced cytotoxicity and bisphenol a: Competition for estrogen receptors in human breast cancer cell lines

Summary Increasing concerns over the effects of environmental estrogens on wildlife and humans have highlighted the need for screening systems to assess potentially estrogenic effects of test compounds. As a result, in vitro screening methods such as cell proliferation assays using the estrogen-responsive human breast cancer cell line, MCF-7, have been developed. The present study describes an alternative in vitro approach for the assessment of such xenoestrogens, based on estrogenic rescue of MCF-7 cells from antiestrogen-induced cytotoxicity. This method measures the ability of various estrogenic compounds to compete with a known estrogen-receptor-mediated antihormonal drug, 4-hydroxytamoxifen, using the 1-[4,5-dimethylthiazol-2-yl]-3,5-diphenylformazan (MTT) assay to assess mitochondrial activity. Because 4-hydroxytamoxifen treatment of cells results in a dramatic decrease in mitochondrial dehydrogenase activity which is directly related to their estrogen-receptor content, inhibition of this effect with estrogenic compounds represents an estrogen-receptor interaction, or estrogenic rescue. The estrogenic compounds tested include a weak xenoestrogen, bisphernol A (BPA), and two biological estrogens, 17α- and 17β-estradiol. Competitive inhibition of 4-hydroxytamoxifen-induced cytotoxicity by BPA was compared to that of the biological estrogens. The results indicate that the biological estrogens can successfully compete with the antiestrogen in a dose-dependent manner. In addition, the assay is sensitive enough to detect estrogenic rescue by even the very weak xenoestrogen, BPA, albeit at high BPA concentrations. This simple in vitro method could be used as an alternative or second-line screen for potential xenoestrogens.     

Cytologic diagnosis of estrogen and progesterone receptors in breast imprints

OBJECTIVE: To investigate estrogen receptor (ER) and progesterone receptor (PR) levels in imprint specimens obtained at breast surgery and to compare their correlation with that of standard methods. STUDY DESIGN: Imprint specimens for cytology were obtained from 101 mass-forming lesions in 66 patients, and specimens were frozen in liquid nitrogen for later assay. The imprint specimens were immunocytochemically (ICC) stained by monoclonal antibody to ER or PR; diaminobenzidine-stained cell nuclei in clusters were regarded as positive. Tissue specimens were assayed by the standard method of dextran-coated charcoal assay (DCC) and enzyme immunoassay. RESULTS: Forty-five primary breast cancer lesions, 2 contralateral breast cancer, 49 dissected nodes and 5 benign breast lesions were collected. The correlation between DCC and ICC was 81% (82/101) for ER and 74% (66/101) for PR. That between EIA and ICC was 88% (88/99) for ER and 80% (79/100) for PR, higher than that between DCC and ICC for ER and PR. CONCLUSION: ICC assessment of ER or PR on imprint cytology is a promising clinical test with an acceptable correlation.     

Estrogen and progesterone receptors in some human myeloma cell lines and murine hybridomas

The control of immune responses by sex hormones is well documented but the effect of sex hormones on lymphoid cell subsets is poorly understood. We have investigated the expression of receptors for androgens (AR), estradiol (ER) and progesterone (PR) by human cell lines of the B lymphocyte lineage and by murine myeloma or hybridomas. AR, ER and PR were determined by cytosol and nuclear binding assays. Eleven human lymphoblastoid cell lines obtained by in vitro infection of blood or tonsil B cells with Epstein-Barr Virus (EBV) B95, did not express AR or ER. Similarly, 10 Burkitt's lymphoma cell lines were AR, ER and PR negative with the exception of the pre-B RAJI cells which bear AR. Among 13 cell lines derived from patients with multiple myeloma none expressed AR but five were found to bear ER (20-164 fmol/mg DNA or 5-10 fmol/mg protein). Four of the latter group also bear PR (86-450 fmol/mg DNA). Two mouse hybridomas out of seven tested were ER and PR positive. The MOPC 315 myeloma expressed ER but not PR. The possible functional role of these sex hormone binding sites in cell proliferation and immunoglobulin secretion deserves further investigation.     

Immunocytochemical determination of estrogen and progesterone receptors in human endometrial adenocarcinoma cells (Ishikawa cells).

The regulation of both estrogen and progesterone receptor levels in human endometrial adenocarcinoma cells of the Ishikawa line was investigated immunocytochemically by using monoclonal antibodies. Positive staining for estrogen and progesterone receptors was observed in the nuclei of Ishikawa cells. Intercellular heterogeneity in receptor content was evident from the presence of receptor-positive or -negative cells and from differences in staining intensity of positive cells. Quantitative analysis was performed by scoring the staining intensity and the proportion of positively stained cells. The time and dose-dependent stimulatory effect of estradiol added to culture media on progesterone receptor levels was studied by applying both immunocytochemical and biochemical methods. Estradiol at 10 nM (optimal concentration) increased the intensity score for PR from an initial value of 10.1 to 78.3 after 72 h incubation, and the proportion of the positive staining cells from 6.7 to 42.7%. Promegestone (R5020) was effective at 1 microM concentration in decreasing the intensity score for ER from 31.1 to 14.6 after 72 h exposure and the proportion of positive cells from 19.0 to 11.4%.     

A secreted glycoprotein induced by estrogen in human breast cancer cell lines

Estrogen induces the synthesis of a glycoprotein of molecular weight 46,000 daltons in three estrogen receptor-positive breast cancer cell lines (MCF₇, ZR₇₅ and T47D), but not in an estrogen receptor-negative cell line (BT 20) or a nonmalignant cell line (HBL 100). The 46K protein, which accounts for 40% of ³⁵S-methionine incorporation into secreted proteins, is only induced by steroids able to interact with the estrogen receptor. The anti-estrogens tamoxifen and hydroxytamoxifen, which by themselves were inactive, suppressed the induction of this protein by estradiol. In MCF₇ cells, estradiol also induces three intracellular proteins which are resolved in two-dimensional electrophoresis. The induction of the 46K secreted protein(s) makes these cell lines excellent in vitro systems for studying the mechanism of estrogen and anti-estrogen action. This protein may also be a useful probe for studying the action of estrogen on breast cancer growth, and may be a useful marker for predicting the hormonal responsiveness of breast cancer in vivo.     

MCF-7; a human breast cancer cell line with estrogen, androgen, progesterone, and glucocorticoid receptors.

We have identified receptors for glucocorticoids, progestins, and androgens in a human breast tumor cell line (MCF-7) known to have estrogen receptor. Sucrose density gradients show that MCF-7 cytosol contains approximately 100 fm/mg protein estradiol (E2-3H) receptor, more than 300 fm/mg protein progesterone receptor (measured with R5020-3H), about 40 fm/mg protein 5alpha-dihydrotestosterone (5alpha-DHT-3H) receptor, and 800 fm/mg glucocorticoid receptor (measured with dexamethasone-3H). Dissociation constants obtained by Scatchard analyses were approximately 0.6 x 10(-10)M (E2), 1 x 10(-9)M (R5020), 2.8 x 10(-10)M (5alpha-DHT) and 8 x 10(-9)M (dexamethasone). No cross competition was found for estrogen receptor, but progestins competed for androgen and glucocorticoid binding. The androgen, but not the glucocorticoid, partially competed for R5020 binding to progesterone receptor. This first demonstration of 4 classes of steroid receptors in human breast cancer means that MCF-7 may be an excellent in vitro model for studying the mechanism of tumor response to endocrine therapy as well as the complex relationships between binding and biological actions of these hormones.     

Specific progesterone receptors in human breast cancer.

We have identified a specific progesterone receptor in 11 of 33 human breast cancer cytosols. Since progesterone itself binds to glucocorticoid receptor, to corticosteroid binding globulin (CBG), and to nonspecific components as well as to its own receptor, we have used a synthetic progestin, R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), whose binding specificity is restricted to progesterone receptor. Bound R5020 sediments at 8 S in sucrose gradients; binding is competed by excess unlabeled R5020 or progesterone. The receptor is distinct from glucocorticoid receptor and CBG as determined by competition studies using dexamethasone and hydrocortisone. The dissociation constant for R5020 obtained by Scatchard analysis of dextran-coated charcoal assays is approximately 2 times 10- minus 9 M.     

Differential cross-talk of estrogen and growth factor receptors in two human mammary tumor cell lines

Cultured human mammary MCF7 and T47D tumor cell lines were used to test the interference of the partial antiestrogen 4′-hydroxytamoxifen (4-OH-TAM) and the pure antiestrogen ZM 182780 with growth factor (IGF-I, heregulin) signaling pathways. Growth of both cell lines was stimulated by IGF-I (20 ng/ml) or heregulin (3 nM). ZM 182780 effectively blocked growth factor induced as well as basal proliferation of MCF7 cells while the compound was ineffective in interfering with growth factor mitogenic activity in T47D cells. On both cell lines the IGF-I or heregulin- induced proliferation was enhanced further by 4-OH-TAM. This synergism could be inhibited dose-dependently by ZM 182780. When cells were grown in the presence of estradiol plus growth factors, the antiestrogenic potencies of both compounds and the efficacy of ZM 182780 were unaffected, while the efficacy of 4-OH-TAM was reduced. Our data show cell type specific cross-talk between the receptor for estrogen and that for IGF-I or heregulin, which is different in MCF7 and T47D cells, respectively. In MCF7 cells with demonstrable cross-talk, a clear superiority exists for a pure antiestrogen over a partial agonist in interfering with growth factor mitogenic activity.     

Protein kinase C subspecies in estrogen receptor-positive and -negative human breast cancer cell lines

Estrogen receptor-positive (MCF7) and -negative (BT20) human breast cancer cell lines, which are frequently used for studies on cancer chemotherapy with triphenylethylene (TPE) anti-estrogens, express at least three protein kinase C subspecies. Two of them are identified as type II PKC having the beta-sequence and type III PKC having the alpha-sequence. The other one shows typical characteristics of PKC which responds to Ca2+, phosphatidylserine and diacylglycerol, but shows kinetic properties subtly different from the previously known PKC subspecies. Immunoblot analysis has shown that this enzyme does not correspond to any of the well defined subspecies with known sequence structures. All of these PKC subspecies are similarly susceptible to the TPE antiestrogens.     

Induction of progesterone receptor in an estrogen, progesterone receptor-negative breast cancer cell line

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.     

Expression of progesterone receptor isoforms A and B is differentially regulated by estrogen in different breast cancer cell lines

Progesterone action in target tissues is mediated through two progesterone receptor (PR) isoforms, PR-A and PR-B, which display different regulatory functions in target cells. Relative expression ratio of these isoforms varies depending on cell and tissue types. Here, we studied the regulation of PR isoform expression by estradiol (E(2)), insulin, IGF-1 and cAMP in different breast cancer cell lines. Although, E(2) induced PR expression in all cell lines studied, the expression ratio of PR-A/PR-B induced by E(2) was dependent on the cell line. The differential regulation of the isoforms was also seen at the mRNA level suggesting that the PR-A and PR-B promoters are differentially regulated by E(2) in different breast cancer cells. Insulin, IGF-1 or cAMP previously reported to induce PR expression however failed to alter the PR expression in our study. This is the first report describing that in different breast cancer cell lines the expression of PR-A and PR-B is regulated by E(2) in a distinct way.     

Characterization and localization of estrogen and progesterone receptors of human fallopian tube

The cellular distribution of estrogen and progesterone receptors (ER and PR) in the human fallopian tube was investigated by immunohistochemical localization with specific monoclonal antibodies. Nuclear immunostaining was observed. Intense PR immunostaining was seen in tissues obtained at mid cycle and luteal stages of the normal menstrual cycle. On the other hand, enhanced staining for ER was seen in early follicular phase and mid cycle. Menopausal tissues showed negligible staining for both ER and PR. The ER and PR were characterized for their molecular size, anatomical distribution and levels during the menstrual cycle and in menopause. ER protein was present throughout the cycle and also during menopause. Western blot analysis revealed two forms of ER approximately 66 kDa and a truncated from approximately 49 kDa in hFT. Presence of A [approximately 90 kDa] and B [approximately 120 kDa] isoforms of human PR was detected. Follicular and early luteal tissue possessed relatively high concentration of immunoreactive PR whereas it was almost undetectable in menopausal tissues. These results suggests that ER and PR are regulated by the changing ovarian steroid hormones.     

Histochemical localization of estrogen and progesterone receptors: evaluation of a method

A histochemical method for the detection of estrogen (ER) and progesterone (PR) receptors in human endometrium, using estrogen and progesterone derivatives linked to fluorochrome-labeled bovine serum albumin (E2-BSA-fluorescein isothiocyanate (FITC) and progesterone-BSA-tetramethylrhodamine isothiocyanate (TMRITC], has been evaluated. The fluorochrome-labeled steroids were bound to the cytoplasm--preferably in glandular epithelial cells but to a lesser extent also to stromal cells. The steroid specificity of the observed binding was studied by preincubating the sections with a series of unlabled steroids and nonsteroidal, hormonally active compounds (estradiol-17 beta, diethylstilbestrol, tamoxifen, 5 alpha-dihydrotestosterone and R 1881 for ER and ORG 2058, R 5020, dexamethasone, cortisol and 5 alpha-dihydrotestosterone for PR). The inhibition studies indicated that E2-BSA-FITC and progesterone-BSA-TMRITC bind to ER and PR in human endometrium with a reasonable degree of specificity. The method was reproducible and various procedural steps were tested, showing satisfactory technical stability. The method is applicable to small tissue samples, and is a valuable complement to quantitative biochemical receptor assays, as it localizes the receptors in tissue slices.     

A dispersed-whole cell method for the determination of androgen receptors in human skin fibroblasts

A method is described for the determination of binding capacity (R_o) and dissociation constant (K_d) of androgen receptors in dispersed, whole, cultured human skin fibroblasts. The cells, obtained from punch biopsies or operative specimens of skin, are grown to confluence in monolayers, harvested, washed, and dispersed in medium. Binding is assessed by incubating the cells with [³H]dihydrotestosterone³ (DHT) at 22°C for one hour followed by washing, centrifugation and counting. Non-specific binding is determined by adding radioinert methyltrienolone (R1881). Scatchard plots are constructed using 6 concentration points; binding is expressed as sites/cell. The method is precise (R_o = 12,105 ± 4305 (S.D.) sites/cell; K_d = 1.0 ± 0.7 (S.D.) × 10^?9M, n = 14 for one prepuce cell line) and independent of cell passage number. Binding is linear with respect to cell number and shows those characteristics commonly attributed to androgen receptors: high affinity, low binding capacity, saturable nuclear binding, androgen specificity, and an 8S peak in fibroblast cytosol using sucrose density gradients. Cell lines shown by other published methods to lack androgen receptors had no detectable DHT binding with this method. This assay technique has the advantages of simplicity, rapidity, and precision.     

High affinity cytosol binding site(s) for antiestrogens in two human breast cancer cell lines and in biopsy specimens devoid of estrogen receptors

It is known from previous investigations by other authors that the non-steroidal molecule Tamoxifen competes for estradiol binding sites in target tissues and displays partial agonist-antagonist effects. It is able to bind a cytosol protein distinct from ER which has been detected in target tissues, as well as in fetal target and non-target organs. The present work investigates the antiestrogen binding activity of human breast cancer cell lines and tumor biopsies. It is found that BT-20 and MDA-MB 231 cell lines devoid of ER and PGR contain a cytosol protein component able to bind antiestrogens with a high affinity (= 2 X 10(-9) M). The 3H-labeled complex prepared in hypotonic buffer sediments at 6.25 S in a sucrose gradient. A substantial amount of antiestrogen binding sites is found to be linked to the microsomal cell fraction, in agreement with previous data from other investigators. In experiments using whole cells incubated at 37 C, the complex is depleted from the cytosol compartment. A low amount of radioactivity is extracted from the purified nuclei. In the cytosol of human breast tumor biopsies the presence of antiestrogen binding sites is not exclusively associated with the presence of ER and PGR. The identity of the natural ligand which binds to these sites under physiological conditions merits investigation.