Estrogen Receptor Content Research Articles

Prognostic value of estrogen receptor concentrations in hepatocellular carcinoma and the surrounding liver parenchyma

The role of estrogen receptor (ER) in hepatocellular carcinoma (HCC) and the surrounding liver is not fully clarified. This study included 79 patients who had undergone curative hepatic resection between 1983 and 1991. ER concentrations were measured by a binding assay method for 79 HCCs and 49 surrounding liver tissues. The ER status in tumor and liver was retrospectively analyzed in terms of clinicopathological factors and intrahepatic recurrence (IHR) of the tumor. ERs were found in 24 (30%) of 79 HCC samples and in 26 (53%) of 49 liver samples, averaging 1.77±4.07 (range 0–25.6) and 2.51±3.57 (range 0–18.6) fmol/mgp, respectively. There were three types of changes in the tumor ER contents as compared to the surrounding liver. The tumor ER contents were unchanged in 39% (19/49) where ER was undetectable in both tumor and the surrounding liver. A decrease in the tumor ER contents was found in 45% (22/49), while the remaining 16% (8/49) had an increase in the tumor ER concentrations. There were no significant differences in the clinicopathological data between ER-positive and ERnegative HCCs. Although the patients in the last group had the poorest IHR-free survival rate, the multivariate analysis with Cox proportional hazard model could not identify ERs in HCC and the surrounding liver to be independent prognostic factors for IHR-free survival. The tumor ER contents can decrease, increase or remain unchanged. However, the majority of HCCs lack ERs. Estrogens may not be involved in tumor development in cases in which the liver has already lost ER. ERs in HCC or liver may not be a useful prognostic indicator after curative hepatic resection.

Acute effects of interferon on estrogen receptor function do not involve the extracellular signal-regulated kinases p42mapk and p44mapk.

Exposure to type I interferons (IFN) increased estrogen receptor (ER) ligand binding and induced protein kinase C (PKC) translocation within 30 min but had no effect on net incorporation of [32P] into ER in Madin Darby bovine kidney (MDBK) cells. Ligand binding was also increased within 30 min by phorbol ester and the protein phosphatase inhibitor okadaic acid. Mitogen-activated protein (MAP) kinase phosphorylation was initially inhibited between 2 and 30 min and subsequently activated between 30 and 60 min after treatment with IFN. The activatory response was blocked by the PKC inhibitor Ro 31-8220. Following transient transfection with an ERE-CAT reporter construct, IFN increased CAT expression after 6 h but decreased ER ligand binding, transcriptional activity and phosphorylation after 48 h, probably as a result of decreased ER concentrations. The results rule out rapid activation of ER ligand binding through phosphorylation at Ser118 by MAP kinase because (1) the increase in ligand binding preceded activation of MAP kinase, and (2) IFN had no short-term effect on [32P]incorporation or ER transcriptional activity. The rapid effect of IFN on ER ligand binding is postulated to reflect phosphorylation of the receptor at Tyr537 by p56lck, a member of the Src family of PKC-activated tyrosine kinases.

Lectin-binding patterns in normal, hyperplastic and neoplastic endometrium: the prognostic value of concanavalin A

Lectins are proteins and glycoproteins of non-immune origin which bind specifically to carbohydrate residues, agglutinate cells and/or precipitate complex carbohydrates. Lectin-binding patterns in normal, hyperplastic and neoplastic endometria were studied using four biotinylated lectins (Con A, LCA, e-PHA, l-PHA) and the avidin-biotin-peroxidase technique. Canavalia ensiformis agglutinin (ConA) and Lens culinaris agglutinin (LCA) reacted strongly with the luminal borders and the cytoplasm of epithelial cells but, whilst in normal and benign endometrial tissues the cytoplasmic staining was confined to the apical and the basal aspect of the cells, in endometrial carcinomas and in some atypical hyperplasias lectin binding also occurred in the lateral cytoplasm (Con-A-lat), although in differing proportions of cells. Interestingly, extensive Con-A-lat in the tumour cells was much more frequent in non-endometrioid carcinomas (P<0.05) and was significantly associated with poor histological differentiation (P<0.0001), low oestrogen and progesterone receptor content (P<0.01 and P=0.0001, respectively) and an unfavourable long-term survival (P<0.05). With Phaseolus vulgaris erythroagglutinin (e-PHA) and leucoagglutinin (l-PHA) a linear, rather inconsistent, staining at the level of the basement membranes was observed in the glands: this, also noted with LCA, appeared intact in normal and hyperplastic glands without cytological atypia, and fragmented or absent in malignant glandular structures and in most hyperplastic glands showing cytological atypia. It is concluded that changes in the distribution of lectin-binding molecules in the endometrial cells are associated with the malignant state, whilst the extent of Con-A-lat reflects the biological behaviour of the tumours.

Hormonal profiles and estrogen receptors in Egyptian female breast cancer patients.

Hormones are considered to be an important factor in the etiology of breast cancer. Serum hormonal profiles of premenopausal and postmenopausal breast cancer patients as well as estrogen receptor (ER) concentrations in breast cancer tissues were examined in an attempt to establish a possible association between hormones and breast cancer risk and to elucidate the biological features of the disease among Egyptian female patients. Levels of estradiol (E2), testosterone (T), progesterone (P), LH, FSH, prolactin, T3, T4 and TSH were measured by highly specific radioimmunoassays in the sera of women with breast cancer and compared to those of control subjects. ER concentrations in breast tumor tissues were measured using 125I-radioreceptor assay. Levels of T and prolactin showed a significant increase in both premenopausal and postmenopausal patients. E2 and P levels were significantly increased in follicular premenopausal and postmenopausal patients. Luteal E2 showed non-significant changes, whereas the luteal P level was significantly decreased. No significant alterations were found in the levels of serum LH, FSH, T3, T4 and TSH either in premenopausal or postmenopausal patients. Higher levels of ER were found in the tumors of postmenopausal than in those of premenopausal patients. A positive correlation was found between levels of ER and age of the patients (r = 0.35), whereas a negative correlation was observed between ER and serum E2 (r = -0.26). This study provides evidence of an association between high levels of serum E2 and T and increased risk of breast cancer in postmenopausal women. Abnormalities in serum P and prolactin are probably associated with a breast cancer risk and ER may be considered as a biochemical marker for breast cancer development.

Therapy of Estrogen Receptor-Positive Micrometastases in the Peritoneal Cavity with Auger Electron-Emitting Estrogens - Theoretical and Practical Considerations

Previous studies have demonstrated that Auger electron-emitting estrogens, when associated with the estrogen receptor (ER), can effect breaks in DNA and ER-dependent radiotoxicity. To evaluate the potential of [123I]-iodoestrogens, ([123I]-IE) to treat ER-positive human cancer cells, we have studied the effect of incubation of [123I]-IE with ER-positive MCF-7 breast cancer cells on cell survival in vitro and found that subnanomolar concentrations of [123I]-IE effectively reduce survival, with a mean lethal dose of about 800 decays per cell. MCF-7 cells incubated 30 min with 2 nM [123I]-IE (13 MBq/ml) showed a 2 log reduction in the ability to form tumors in immunodeficient mice. Evaluation of a mathematical model for [123I]-IE therapy for intraperitoneal micrometases in vivo in the mouse, based on variables related to the (a) specific activity of [123I]-IE; (b) its affinity for ER; (c) the characteristics of the uptake and retention of [123I]-IE by the target cells; (d) the concentration of ER in the tumor cells and (e) the tumor weight suggest that such therapy may be feasible.

4-Hydroxytamoxifen-induced cytotoxicity and bisphenol A: competition for estrogen receptors in human breast cancer cell lines.

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, bisphenol A (BPA), and two biological estrogens, 17alpha- and 17beta-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.

Morphological evidence for a subpopulation selection effect by estrogen and antiestrogen treatments in the heterogeneous MCF-7 cell line.

Recently, we developed a method to quantitatively study tumour cell heterogeneity in terms of both nuclear size and estrogen receptor (ER) content by image cytometry. The method, previously used to analyse the proliferation of the breast cancer cell line MCF‐7, was applied here to analyse the growth of this cell line under estradiol (E2), hydroxytamoxifen (OH‐TAM), and both E2 and OH‐TAM treatments. The method extracts characteristic parameters of single nuclei and features that measure the global and local organisation of the cells in their growing phase. Modifications of the heterogeneity of the cell line are emphasised through phenotypic changes and modifications of the spatial organisation of the cells. The hormone (E2) generates a very fast growth of cells with small nuclei that became ER negative in the long term. The antihormone (OH‐TAM) produces a gradual selection of ER negative or poorly positive cells with large nuclei. These modifications are reversed when E2 and OH‐TAM are simultaneously used. Moreover, estradiol induces a permissive context of proliferation, whereas hydroxytamoxifen acts only on some subpopulations. The combination of cell count, cytomorphology, and cell organisation revealed the magnitude of the potential of structuration of hormones or antihormones on in vitro growing cells.

Immunohistochemical localization of metallothionein in endometrial lesions.

Metallothioneins (MTs) are a group of ubiquitous low-molecular-weight proteins essential for the protection of cells against heavy metal ion toxicity. The immunohistochemical expression of MT was studied by immunohistochemistry using a monoclonal antibody (E9) against a conserved epitope of I and II isoforms in a series of 89 endometrial carcinomas, 34 cases of hyperplasia, and 32 samples of normal endometrium. In secretory phase endometrium, extensive MT expression was detected in most cases (92.4%). In contrast, MT immunoreactivity was confined to small foci in 22.2% of proliferative phase cases. The MT values in normal endometrium were inversely correlated with oestrogen receptor (ER) content (p<0.0001), progesterone receptor (PgR) content and with PCNA (p<0.0001) and MIB1 (p=0.001) scores. In hyperplastic lesions, MT expression was detected only in 3.3% of cases, while in the group of carcinomas it was observed in 23.1%. A statistically significant difference of MT expression was observed between carcinomas and simple hyperplasias (p=0.03). In carcinomas, MT expression was positively correlated with grade (p=0.0065), MIB1 (p=0.022), and p53 (p=0.006) expression, and inversely with PgR (p=0.03). A trend of inverse correlation between MT and ER receptor was also detected (p=0.07). These data suggest that MT expression seems to be under hormonal control in normal endometrium; that it may modify p53 expression; and that it could be used as an additional biological marker indicating aggressive behaviour in endometrial lesions.

Immunohistochemical analysis of estrogen receptors in feline mammary gland benign and malignant lesions: comparison with biochemical assay☆

Estrogen receptors (ER) were determined by both the biochemical dextran-coated charcoal (DCC-ER) and the immunohistochemical Avidin biotin- peroxidase complex (IHC-ER) methods in proliferative mammary lesions collected from 37 cats: 20 malignant tumors without metastasis at first presentation, seven malignant tumors with lung and/or lymph node metastases and 10 benign tumors and dysplasias. Total number of samples analyzed by both methods was 44. The DCC-ER method was applied to frozen tissue samples and the IHC-ER method was applied to neutral buffered formalin-fixed, paraffin wax-embedded tissue samples by using the NCL-6F11 monoclonal antibody. Biochemically, 21 (47.7%) cases had equal or more than 5 fmol/mg of protein (standard positivity threshold). Immunohistochemically, 11 (25%) cases were scored positive, the percentage of positive nuclei being statistically linked to the intensity of immunostaining. Normal mammary gland tissue (13 cases) and/or dysplastic areas (5 cases) found in the surroundings of the main lesion were IHC-ER positive in 76.9% and 40% of the cases, respectively. Concordance between DCC-ER and IHC-DCC was 72.7% and the results of the DCC and the IHC-ER methods were significantly correlated (P < 0.05) by the χ 2 test. Specificity (true negatives) and sensitivity (true positives) of the ICH-ER method were 95.6% and 47.6%, respectively. One out of eleven DCC-ER positive and IHC-ER negative discordant cases (9.09%) was a DCC-ER false positive, because the surrounding normal mammary gland tissue was IHC-ER positive. The remaining 10 cases had ER content values equal or lower than 23 fmol/mg of protein, a figure that could represent the sensitivity threshold of the immunohistochemical method employed.

The effect of estrogen usage on the subsequent hormone receptor status of primary breast cancer.

In order to determine if prior use of exogenous estrogens was related to the estrogen receptor (ER) content of primary breast cancers, a retrospective analysis was performed from 536 patients with invasive breast cancer. The patient's age, menopausal status, oral contraceptive or estrogen replacement therapy usage, and the ER and progesterone receptor (PR) content of the breast cancer were recorded for all patients. Hormone usage in premenopausal and postmenopausal patients was compared to ER and PR levels in primary breast cancers using non-parametric testing. Complete information was available from 508 (193 premenopausal and 315 postmenopausal) patients. Breast cancers were ER positive in 72% of postmenopausal patients and 57% of premenopausal patients. The majority of patients received 'Some' form of hormone therapy (111 of 193 premenopausal patients and 233 of 315 postmenopausal patients). Significantly more estrogen receptors were detected in tumors from patients receiving 'some' estrogen therapy compared to 'never' users. Postmenopausal patients 'never receiving estrogen therapy had a lower rate of ER positive tumors (62%) compared to 'some' users (75%, chi2 = 4.99, p < 0.05). The same relationship was seen for PR ('never' users 44% positive, 'some' users 58% positive, chi2 = 5.19, p < 0.05). We conclude that postmenopausal patients who received 'some' estrogen therapy are more likely to have breast cancers that are estrogen receptor and progesterone receptor positive.

Evidence for sexual dimorphism of estrogen receptors in hypothalamus and thymus of neonatal and immature Wistar rats

It is hypothesized that the rat thymus is sexually differentiated. To begin testing this, we used the 5-day-old rat, whose hypothalamus is sexually differentiated during this period. Cytosolic estrogen receptors (ER) were measured in cytosols prepared from the brain, thymus and uterus of Wistar rats at 5, 18 and 30 days post-partum. ER concentrations were significantly higher in hypothalamus in cytosols of female 5-day-old rats, and this difference had disappeared by day 18. The pattern in thymus was identical to that observed in hypothalamus, suggesting the presence in the thymus of the aromatase system that converts androgen to estrogen, and that estrogen-mediated sexual differentiation of thymus might be proceeding at 5 days. Unlike the case for hypothalamus, no experimental model exists at present for testing functional sexual differentiation in thymus. Therefore we tested the effects of aromatase inhibitors on estrogen receptor activity in thymus well after the five-day period, and before atrophy of the thymus has commenced. Male and female rats were implanted at 15 days of age with SILASTIC implants containing 5 mg of estradiol or with 25 mg of the aromatase inhibitor 4-hydroxyandrostenedione (4-OHA) and cytosolic ER prepared at 30 days and activity measured. Administration of estradiol resulted in failure to detect available receptor, suggesting that the binding components measured were ER. After 4-OHA administration, ER concentrations were significantly increased in cytosols from male but not female hypothalamus and thymus. There is therefore a basis for exploring further the hypothesis that rat thymus is sexually differentiated.

Immunohistochemical study of the proliferation index, oestrogen receptors and progesterone receptors A and B in leiomyomata and normal myometrium during the menstrual cycle and under gonadotrophin-releasing hormone agonist therapy.

The cell proliferation-associated antigen Ki 67 and the immunohistochemical content of oestrogen receptors (ER), progesterone receptors AB (PRAB) and progesterone receptors B (PRB) were evaluated in leiomyomata and adjacent myometrium during the menstrual cycle and in leiomyomata under gonadotrophin-releasing hormone agonist (GnRHa) therapy. The proliferative status of muscular cells was measured by evaluating the percentage of nuclei staining positive for Ki 67 (proliferation index). Quantitative analysis (QH-score) was carried out using advanced stereographic computer technology to investigate ER, PRAB and PRB. Leiomyoma and myometrial biopsies were taken from 30 patients undergoing hysterectomy or myomectomy because of symptomatic leiomyomata (subgroup I). Leiomyoma biopsies were taken from 10 patients suffering from symptomatic submucosal leiomyomata, after 2 month GnRH therapy (subgroup II). During the secretory phase, the proliferation index (Ki 67) was found to be higher in leiomyomata than in myometrium, but the difference was not significant. Oestrogen receptor content was significantly higher in leiomyomata than in myometrium only during the proliferative phase of the cycle. PRAB and PRB content were found to be higher in leiomyomata than in adjacent myometrium with a statistically significant dominance of PRAB over PRB. Under GnRHa therapy, a dramatic decrease was observed in PRAB and B content as well as Ki 67 but ER content remained comparable with the results obtained during the menstrual cycle. The results suggest that leiomyomata may be under the influence of progesterone which may play a major role in their growth.

Changes in uterine ornithine decarboxylase activity and steroid receptor levels during decidualization in the rat induced by CDRI-85/287.

CDRI-85/287 is an anti-estrogen and interferes with decidualization in the rat uterus. In this study, uterine estrogen receptor (ER) and progesterone receptor (PR) levels were determined during the inhibition of decidualization. The effect of 85/287 on uterine ornithine decarboxylate (ODC) activity (a marker enzyme for decidualization) was also studied, using immature ovariectomized rats divided into four different groups: control, 2.5mg/kg 85/287 only; 1 microg estradiol only; and 85/287 + estradiol. Pseudopregnant rats were administered 85/287 (2.5mg/kg p.o.) on day 3 post-coitum. Deciduoma was induced in one of the uterine horns on day 4 and animals were autopsied 18h post-traumatization. Both ERs and PRs showed an increase in traumatized horns compared with non-traumatized. In the 85/287-treated uterus, there was a reduction in cytosolic ERs in both traumatized horns. However, nuclear ER and PR levels increased in both horns under the influence of 85/287. Similarly, in a tamoxifen (0.03mg/kg)-treated group a decline was noticed in cytosolic ER with a mild increase in nuclear PR. Total ER content, expressed per 100 microg DNA, showed a decline in 85/287- or tamoxifen-treated rats. However, no significant alterations were observed in total PR levels in non-traumatized horns. In an immature rat model, 85/287 caused a significant (>50%) reduction in estradiol-induced ODC activity. These findings suggest that the decidualization inhibitory activity of 85/287 may be attributed to inhibition of certain timed biochemical events and genomic/non-genomic actions of estrogens in the rat uterus.

Growth hormone (GH) and 17beta-estradiol regulation of the expression of mouse GH receptor and GH-binding protein in cultured mouse hepatocytes.

In the present study, primary mouse hepatocytes from 8- to 10-week-old virgin female Swiss-Webster mice were perfused with collagenase (100 U/ml) using the two-step method. Isolated hepatocytes were plated in a rat tail type I collagen sandwich configuration to examine the regulation of GH receptor (GHR) and GH-binding protein (GHBP) expression by GH and 17beta-estradiol (E2). After 48 h of initial plating, hepatocytes were divided into groups of five replicates and treated for 24 h with medium containing no hormones (controls), GH (100 ng/ml), E2 (10(-9) M), E2 (10(-9) M) plus GH (100 ng/ml), or E2 plus GH and ICI 182-780 at different concentrations. Treatment of hepatocytes with GH or E2 alone did not have any effect on the cellular concentrations of GHBP and GHR. However, the combination of E2 and GH up-regulated the cellular concentrations of GHBP and GHR 2- to 3-fold. GHBP and GHR messenger RNA concentrations were also up-regulated 2- to 3-fold. ICI 182-780, a competitive inhibitor of E2 for the estrogen receptor (ER), at different concentrations inhibited the E2 and GH-induced stimulation of GHBP and GHR. Furthermore, ER concentrations increased 5- to 7-fold in hepatocytes treated with E2 and GH compared with those in untreated cells or cells treated with either E2 or GH alone. In the present study we have shown that in cultured hepatocytes from virgin female mice, GH or E2 alone did not affect the concentrations of GHBP and GHR. However, E2 and GH together significantly up-regulated GHR and GHBP expression.

Growth Hormone (GH) and 17 -Estradiol Regulation of the Expression of Mouse GH Receptor and GH-Binding Protein in Cultured Mouse Hepatocytes

In the present study, primary mouse hepatocytes from 8- to 10-week-old virgin female Swiss-Webster mice were perfused with collagenase (100 U/ml) using the two-step method. Isolated hepatocytes were plated in a rat tail type I collagen sandwich configuration to examine the regulation of GH receptor (GHR) and GH-binding protein (GHBP) expression by GH and 17β-estradiol (E2). After 48 h of initial plating, hepatocytes were divided into groups of five replicates and treated for 24 h with medium containing no hormones (controls), GH (100 ng/ml), E2 (10−9m), E2 (10−9m) plus GH (100 ng/ml), or E2 plus GH and ICI 182–780 at different concentrations. Treatment of hepatocytes with GH or E2 alone did not have any effect on the cellular concentrations of GHBP and GHR. However, the combination of E2 and GH up-regulated the cellular concentrations of GHBP and GHR 2- to 3-fold. GHBP and GHR messenger RNA concentrations were also up-regulated 2- to 3-fold. ICI 182–780, a competitive inhibitor of E2 for the estrogen receptor (ER), at different concentrations inhibited the E2 and GH-induced stimulation of GHBP and GHR. Furthermore, ER concentrations increased 5- to 7-fold in hepatocytes treated with E2 and GH compared with those in untreated cells or cells treated with either E2 or GH alone. In the present study we have shown that in cultured hepatocytes from virgin female mice, GH or E2 alone did not affect the concentrations of GHBP and GHR. However, E2 and GH together significantly up-regulated GHR and GHBP expression.

Does vascular endothelial growth factor (VEGF) predict local relapse and survival in radiotherapy-treated node-negative breast cancer?

The aim of this study was to determine the association of vascular endothelial growth factor (VEGF) content in 302 consecutive node-negative breast cancer (NNBC) patients treated with only locoregional radiotherapy to relapse free- (RFS) and overall survival (OS). VEGF content in tumour cytosols was measured by an enzymatic immunoassay for the major isoform VEGF165. The median age was 56 years, the median follow-up time 56 months. A wide range (0.01–144.79 pg μg−1 DNA) of VEGF content was found (median 1.92). Significant associations were found between VEGF and oestrogen receptor (ER) content, progesterone receptor (PR) and tumour size (P = 0.005). Univariate analysis displayed significant reduced RFS and OS for patients with higher VEGF content (P = 0.0113 and P = 0.0075 respectively). A total of 43 recurrences have been found (ten local relapses within the breast, five in the axillary or supraclavicular lymph nodes and 28 distant metastasis). There was no significant correlation between the localization of the relapse and the VEGF content. Multivariate analysis suggested VEGF as the only predictor of OS (relative risk (RR) = 3.6, 95% confidence interval (CI) = 0.97–13.37), and in patients with T1 tumours (n = 236) the multivariate analysis clearly displayed VEGF as the only independent predictor of both RFS and OS (RR = 5.1, CI = 1.07–24.59). In the sub-group with ER-positive tumours (n = 229), multivariate analysis showed VEGF as the only significant predictor of RFS and OS (RR = 10.44, CI = 1.26–86.38). The results suggest VEGF165 as a predictor of RFS and OS in NNBC patients treated with locoregional radiotherapy, comprising especially patients with favourable prognosis of T1 tumours, or ER-positive tumours. The high VEGF expression might define a radioresistant phenotype, or indicate an early distant spread which might require adjuvant systemic treatment. © 1999 Cancer Research Campaign

Role of HSP90 in mediating cross-talk between the estrogen receptor and the Ah receptor signal transduction pathways

Tetrachlorodibenzo- p-dioxin (TCDD)-mediated gene transactivation via the Ah receptor (AhR) has been shown to be dependent upon estrogen receptor (ER) expression in human breast cancer cells. We have investigated the 90-kDa heat shock protein (HSP90) as a mediator of cross-talk between the AhR and the ER signal transduction pathways. The effect of HSP90 overexpression on receptor activity was determined by transient transfection assays using a HSP90 expression vector. Ligand-inducible gene expression was inhibited when the HSP90 expression vector was cotransfected with a TCDD-responsive reporter plasmid. However, overexpression of HSP90 did not block induction of an estrogen-responsive reporter plasmid. To determine whether ER facilitates AhR signaling through its ability to squelch HSP90, two vectors expressing protein products that bind HSP90 were transfected into MDA-MB-231 cells. Introduction of (i) He11, an ER deletion mutant that does not bind DNA, and (ii) the ligand-binding domain of human AhR, both led to increased basal and TCDD-inducible CYP1A1 expression. Finally, the subcellular distribution of HSP90 was investigated in human breast cancer cell lines. These studies showed HSP90 to be primarily cytoplasmic in ER-positive cell lines, whereas in matched ER-negative cell lines HSP90 was distributed equally between the cytoplasm and nucleus. Taken together, these results demonstrate that HSP90 can regulate AhR activity in vivo, and that Ah-responsiveness is dependent upon cellular ER content through a mechanism that involves HSP90.

Detection of loss of heterozygosity at RAD51, RAD52, RAD54 and BRCA1 and BRCA2 loci in breast cancer: pathological correlations.

Loss of heterozygosity (LOH) in loci of the 15q15.1, 12p13, 1p32, 17q21 and 13q12–13 regions may collaborate in the inactivation of RAD51, RAD52, RAD54, BRCA1, BRCA2 and possibly other genes implicated in the repair of double-stranded DNA and in DNA recombination. We investigate allelic losses in microsatellites of the RAD51, RAD52, RAD54, BRCA1 and BRCA2 regions, and their correlations with nine pathologic parameters in 127 breast carcinomas. The LOH analysis was performed by amplifying DNA by PCR, using 15 markers of the 15q15.1, 12p13.3, 1p32, 17q21 and 13q12–13 regions. LOH was found in the RAD51 region in 32% of tumours, in the RAD52 region in 16%, in RAD54 in 20% and in the BRCA1 and BRCA2 regions in 49% and 44% respectively. Significant correlations between one or more regions with concomitant LOH and pathologic parameters were observed with respect to age (P = 0.008), oestrogen receptor content (P = 0.03), progesterone receptors (P = 0.003), higher grade (P = 0.001), more advanced stage (P = 0.004) and peritumoural vessel involvement (P < 0.0001). The number of cases in which LOH was observed simultaneously in two or more regions was always higher than expected on the basis of their statistical probability, and curiously, the three patients with LOH at five regions concomitantly were under the age of 30 years. These results suggest that LOH at these regions could be related to breast cancer, and probably to a poor tumour prognosis. © 1999 Cancer Research Campaign

Proteasome-mediated proteolysis of estrogen receptor: a novel component in autologous down-regulation.

Regulation of estrogen receptor (ER) concentration is a key component in limiting estrogen responsiveness in target cells. Yet the mechanisms governing ER concentration in the lactotrope cells of the anterior pituitary, a major site of estrogen action, are undetermined. In this study, we used a lactotrope cell line, PR1, to explore regulation of ER protein by estrogen. Estrogen treatment resulted in an approximate 60% decrease in ER steady state protein levels. Suprisingly, the decline in ER protein was apparent within 1 h of estrogen treatment and occurred in the absence of protein synthesis and transcription. Direct regulation of ER protein was further confirmed by pulse chase analysis, which showed that ER protein half-life was shortened from greater than 3 h to 1 h in the presence of estrogen. The estrogen-induced degradation of ER protein could be prevented by pretreatment with peptide aldehyde inhibitors of proteasome protease whereas inhibitors of calpain and lysosomal proteases were ineffective. Inhibition of proteasome activity maintained ER protein at a level equivalent to control cells not stimulated with estrogen but increased estrogen-binding activity by 1.75-fold. Proteolytic regulation of ER by the proteasome is not limited to pituitary lactotrope cells but is also operational in MCF-7 breast cancer cells, suggesting that this may be a common regulatory pathway used by estrogen. These studies describe a nongenomic action of estrogen that involves nuclear ER: rapid proteolysis of ER protein via a proteasome-mediated pathway.

Proteasome-Mediated Proteolysis of Estrogen Receptor: A Novel Component in Autologous Down-Regulation

Regulation of estrogen receptor (ER) concentration is a key component in limiting estrogen responsiveness in target cells. Yet the mechanisms governing ER concentration in the lactotrope cells of the anterior pituitary, a major site of estrogen action, are undetermined. In this study, we used a lactotrope cell line, PR1, to explore regulation of ER protein by estrogen. Estrogen treatment resulted in an approximate 60% decrease in ER steady state protein levels. Suprisingly, the decline in ER protein was apparent within 1 h of estrogen treatment and occurred in the absence of protein synthesis and transcription. Direct regulation of ER protein was further confirmed by pulse chase analysis, which showed that ER protein half-life was shortened from greater than 3 h to 1 h in the presence of estrogen. The estrogen-induced degradation of ER protein could be prevented by pretreatment with peptide aldehyde inhibitors of pro-teasome protease whereas inhibitors of calpain and lysosomal proteases were ineffective. Inhibition of proteasome activity maintained ER protein at a level equivalent to control cells not stimulated with estrogen but increased estrogen-binding activity by 1.75-fold. Proteolytic regulation of ER by the proteasome is not limited to pituitary lactotrope cells but is also operational in MCF-7 breast cancer cells, suggesting that this may be a common regulatory pathway used by estrogen. These studies describe a nongenomic action of estrogen that involves nuclear ER: rapid proteolysis of ER protein via a proteasome-mediated pathway.