High Levels Of Estrogen Receptors Research Articles

Mechanisms of Tumor Regression and Resistance to Estrogen Deprivation and Fulvestrant in a Model of Estrogen Receptor–Positive, HER-2/neu-Positive Breast Cancer

HER-2/neu in breast cancer is associated with tamoxifen resistance, but little data exist on its interaction with estrogen deprivation or fulvestrant. Here, we used an in vivo xenograft model of estrogen receptor (ER)-positive breast cancer with HER-2/neu overexpression (MCF7/HER-2/neu-18) to investigate mechanisms of growth inhibition and treatment resistance. MCF7/HER-2/neu-18 tumors were growth inhibited by estrogen deprivation and with fulvestrant, but resistance developed in 2 to 3 months. Inhibited tumors had reductions in ER, insulin-like growth factor-I receptor (IGF-IR), phosphorylated HER-2/neu (p-HER-2/neu), and phosphorylated p42/44 mitogen-activated protein kinase (p-MAPK). p27 was increased especially in tumors sensitive to estrogen deprivation. Tumors with acquired resistance to these therapies had complete loss of ER, increased p-HER-2/neu, increased p-MAPK, and reduced p27. In contrast, IGF-IR and phosphorylated AKT (p-AKT) levels were markedly reduced in these resistant tumors. The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib, which can block EGFR/HER-2/neu signaling, significantly delayed the emergence of resistance to both estrogen deprivation and fulvestrant. Levels of p-MAPK and p-AKT decreased with gefitinib, whereas high ER levels were restored. Eventually, however, tumors progressed in mice treated with gefitinib combined with estrogen deprivation or fulvestrant accompanied again by loss of ER and IGF-IR, increased p-HER-2/neu, high p-MAPK, and now increased p-AKT. Thus, estrogen deprivation and fulvestrant can effectively inhibit HER-2/neu-overexpressing tumors but resistance develops quickly. EGFR/HER-2/neu inhibitors can delay resistance, but reactivation of HER-2/neu and signaling through AKT leads to tumor regrowth. Combining endocrine therapy with EGFR/HER-2/neu inhibitors should be tested in clinical breast cancer, but a more complete blockade of EGFR/HER-2/neu may be optimal.

Growth regulation and transcriptional activities of estrogen and progesterone in human endometrial cancer cells

Estrogen-stimulated growth of the malignant human endometrium can be balanced by the differentiating properties of progesterone. To study the molecular basis behind this, gene expression profiling was performed using complementary DNA microarray analysis. In this study, the human endometrial cancer cell lines ECC-1 and PRAB-36 were used as models. The ECC-1 cell line, which expresses high levels of estrogen receptor α and is stimulated in growth by estrogens, was used to study estrogen regulation of gene expression. The Ishikawa sub–cell line PRAB-36, expressing both PRA and PRB, progesterone receptor isoforms, and inhibited in growth by progestagens, was used to study progesterone regulation of gene expression. Using these two well-differentiated human endometrial cancer cell lines, 148 estrogen- and 148 progesterone-regulated genes were identified. After functional classification, the estrogen- and progesterone-regulated genes could be categorized in different biologically relevant groups. Within the group of “cell growth and/or maintenance,” 81 genes were clustered, from which a number of genes could be involved in arranging the cross talk that exists between estrogen and progesterone signaling. On the basis of analysis of the current findings, it is hypothesized that cross talk between estrogen and progestagen signaling does not occur by counterregulation of single genes, but rather at the level of differential regulation of different genes within the same functional families.

Tailored therapies based upon tumor subtype biology

Breast cancer is a spectrum of diseases comprised of different tumor subtypes, each with a distinct biology and clinical behavior. To capture this diversify, we characterized the variation in gene expression across human breast tumors using DNA microarrays and identified at least five distinct tumor subtypes that are statistically significant predictors of patient overall survival [1]. Recently, we further validated these findings using a training set of 102 tumors, which was used to derive a new 'intrinsic gene set'. This gene set was then validated using a true test set of 311 tumors compiled from three different microarray studies. Our analyses demonstrate that common patterns of gene expression can be identified across different microarray platforms, that the breast tumor 'intrinsic' subtypes are reproducible across different datasets, and that this classification was a significant predictor of outcomes after correcting for standard clinical parameters such as estrogen receptor (ER), grade and node status [2]. The biology of the 'intrinsic' subtypes is rich and extensive, and many of these expression features suggest distinct therapies. The 'intrinsic' subtypes include at least two types of ER-negative tumors (Basal-like and HER2+/ER-) and at least two types of ER-positive tumors (Luminal A and Luminal B). Basal-like tumors typically show low expression of HER2 and ER, and these tumors exhibit high expression of genes characteristic of the basal epithelial cell layer, including expression of keratin 5, keratin 6, keratin 17 and four Kallikrein genes (KLK5-KLK8). The Basal-like tumors pose a challenge from the treatment perspective because they lack ER and HER2. However, we have recently shown that most are HER1-positive and/or c-KIT-positive [3], and we have initiated a clinical trial to evaluate the efficacy of HER1-inhibitors in preselected Basal-like tumor patients. HER2-positive (i.e. gene amplified) tumors fall into at least two distinct expression groups: those that are ER-negative and typically cluster near the Basal-like tumors (HER2+/ER-), and those that are ER-positive and cluster with tumors of luminal cell origin. These findings suggest that both types of HER2+ patients should receive transtuzumab, but that the ER+/HER2+ may gain a benefit from hormone therapy. Finally, the Luminal subtype A and Luminal subtype B tumors express ER, GATA3, and genes regulated by both ER and GATA3. Compared with Luminal B tumors, Luminal A tumors express higher levels of ER, BCL2 and GATA3, and they show more favorable patient outcomes. Luminal B tumors more often express HER1, HER2 and/or cyclin E1, and they show worse outcomes. Our data, when coupled with data from others [4], suggests that Luminal A patients are likely to benefit from hormone therapy and are not likely to benefit from chemotherapy, while the opposite may be true of Luminal B patients. Experiments to answer these questions in Luminal patients are underway and will be discussed.

Estrogen receptor expression in a human primitive neuroectodermal tumor cell line from the cerebral cortex: estrogen stimulates rapid ERK1/2 activation and receptor-dependent cell migration

Primitive neuroectodermal tumors (PNETs) are the most common form of pediatric brain tumor. Most often these malignant childhood brain tumors arise from neuroepithelial precursor cells in the cerebellum, and less frequently in the cerebral cortex. Because the normal PNET precursor cells from the cerebrum and cerebellum transiently express high levels of estrogen receptors (ERs), we hypothesized that the PNET cells of the cerebrocortical-derived cell line PFSK1 may also express ERs and would be responsive to estrogen. Results of immunoblot studies using ER-specific antiserum indicate that both ERα and ERβ are expressed in PFSK1 cells. The ability of estrogen to rapidly activate MAPK signaling was tested; low physiological concentrations of E2 stimulated ERK1/2 phosphorylation and nuclear translocation within 15min of exposure. Exogenously added 17β-estradiol (E2) could not stimulate PFSK1 growth, however E2 significantly increased PFSK1 cell migration, suggesting that rapid actions of E2 and ER-mediated processes might contribute to the metastatic phenotype of some PNETs.

PTEN immunohistochemical expression is suppressed in G1 endometrioid adenocarcinoma of the uterine corpus.

PTEN is a tumor suppressor gene that inhibits cell proliferation by regulating intracellular signaling pathways, and this activity can be abolished by mutations of the PTEN gene. This study was designed to examine the correlation of PTEN expression with the expression of cell cycle regulators and with clinicopathological parameters in endometrioid adenocarcinoma of the uterine corpus. Tissue samples of 117 endometrioid adenocarcinomas in addition to those of 19 normal endometria and 20 endometrial hyperplasias were used for the study. Immunohistochemical staining for PTEN protein was performed with the labeled streptavidin-biotin method on formalin-fixed and paraffin-embedded tissue samples. PTEN expression was represented as the staining score. Immunohistochemistry showed that the nuclei of cells were positive for PTEN. The PTEN staining score of normal endometrium was significantly higher in the proliferative phase than in the secretory phase. The scores of various endometrial hyperplasias were not significantly different from each other, regardless of the type of hyperplasia. The PTEN staining scores of endometrioid adenocarcinomas were 7.6+/-5.2 in G1, 9.6+/-5.2 in G2, and 11.9+/-3.7 in G3, and increased significantly as the histological grade increased. PTEN staining score was not significantly correlated with clinicopathological parameters such as FIGO stage, myometrial invasion, lymph-vascular space invasion (LVSI), lymph node metastasis or group, but was significantly correlated with labeling indices (LIs) of cell cycle regulators such as Ki-67, cdk2, cyclin A, cyclin D1, cyclin E, p27, and p53. The PTEN staining score of p53-wild cases was significantly lower than that of p53-mutant ones, but there was no significant difference of the score in cases with different PTEN gene status. PTEN expression was significantly lower in cases with both high levels of estrogen receptor and progesterone receptor. PTEN protein expression was decreased in well-differentiated and less growth-aggressive endometrial carcinoma with wild-type p53 gene and high levels of ER and PR. This suggests that disturbed PTEN expression occurs in an early phase of the tumorigenesis of well-differentiated endometrial carcinoma.

17β-Estradiol inhibits excitatory amino acid-induced activity of neurons of the nucleus tractus solitarius

The effects of 17β-estradiol (17βE2) on spontaneous and excitatory amino acid (EAA) induced nucleus tractus solitarius (NTS) neuronal activity were investigated by electrophysiological and immunohistochemical experiments in ovariectomized female Sprague–Dawley rats. Out of 62 NTS neurons tested, 42 were inhibited (68%) following iontophoretic application of 17βE2 in a current-dependent manner. The averaged firing rate decreased from 3.06±0.40 to 0.78±0.17 Hz. The inhibitory responses were rapid in onset (within 1 min) and variable in duration (2–4 min). The inhibitory effects of 17βE2 were blocked by simultaneously applied 17βE2 antagonist ICI182,780, but not by GABA antagonist, bicuculline and phaclofen. l-Glutamate, AMPA or NMDA enhanced the activity of 71, 73 or 69% of NTS cells tested, respectively. The excitatory effects of EAA were significantly inhibited in the presence of 17βE2. Fluorescent immunohistochemistry revealed that all subnuclei of the NTS contained high levels of estrogen receptors (ERs) immunoreactivity. These results suggest that 17βE2 inhibits spontaneous and EAA-induced NTS neuronal activity through 17βE2 activation of ERs.

A mechanism of drug resistance to tamoxifen in breast cancer

Drug resistance to tamoxifen (Tam) is a significant clinical problem but the mechanism through which this occurs remains elusive. We have developed a number of xenograft models of Tam-stimulated growth that model breast cancer progression using estrogen receptor positive MCF-7 or T47D breast cancer cells. When estrogen-stimulated T47D:E2 tumors are treated long term with Tam, Tam-stimulated tumors develop (T47D:Tam) that are stimulated by both estrogen and Tam. When HER-2/neu status is determined, it is clear that the T47D:Tam tumors express significantly higher levels of HER-2/neu protein by immunohistochemistry and mRNA as measured by real-time RT-PCR. The T47D:Tam tumors also express higher levels of estrogen receptor and progesterone receptor protein than their estrogen-stimulated T47D:E2 counterparts. We compared out results to the MCF-7 model of Tam-stimulated growth. The MCF-7:Tam ST (estrogen- and Tam-stimulated) and MCF-7:Tam LT (estrogen-inhibited, Tam-stimulated) were bilaterally transplanted to account for any mouse to mouse variation and characteristic growth patterns were observed. TUNEL staining was performed on MCF-7:Tam LT treated with either estrogen or Tam and it was concluded that estrogen-inhibited tumor growth was a result of increased apoptosis. Three phases of tumor progression are described that involve increases in HER-2/neu expression, de-regulation of estrogen receptor expression and increases in apoptosis which in concert determine the phenotype of drug resistance to Tam.

Estudio de las concentraciones citosolicas del antígeno polipeptídico específico tisular (TPS) en carcinomas ductales infiltrantes de mama. Relación positiva con la hormonodependencia y negativa con la proliferación celular

The tissue-specific polypeptide antigen (TPS) is an epitope of the tissue polypeptide antigen (TPA) which is defined by the M3 monoclonal antibody and is related to cytokeratin 18. Several groups have demonstrated its value as a useful parameter in the follow-up of some tumors. This work has aimed to study the TPS cytosolic levels in infiltrating ductal carcinomas of the breast (IDC) and their possible correlations with other clinical-biological parameters. The TPS was determined by means of an immunoradiometric assay (Beki Diagnostics. Sweden). Other parameters included in the study were the estrogen receptors (ER), progesterone receptors (PR), pS2, cathepsin D, tissue-type plasminogen activator (t-PA), tumor size, axillary lymph node involvement, distant metastases, histological grade, ploidy and S-phase. The TPS cytosolic levels ranged from 1.8 to 606.3 KU/mg prt. (median 110.2) and had a significant correlation with the ER (r: 0.721), PR (r: 0.287), cathepsin D (r: 0.550) and t-PA (r:0.436). The TPS positive (> 110.2 KU/mg prt.) carcinomas had higher levels of ER (p: 0.001), PR (p: 0.021), pS2 (p: 0.058), cathepsin D (p: 0.000) and t-PA (p: 0.053) than the TPS negative tumors. When the IDC were classified according to S-phase values, we observed that the positive cases (S-phase > 8.1%, which represents the median value of all carcinomas) had lower levels of TPS (p: 0.046) than the negative tumors. Likewise, the GoG1 cellular fraction correlated positively and significantly with the TPS cytosolic levels (p: 0.000). Based on our results, we suggest that there is a positive correlation between the TPS cytosolic levels and hormone-dependence parameters, as well as an inverse correlation between these and the cellular proliferation parameters. Based on the above, we consider that it is worthwhile to carry out further studies on cytosolic TPS in order to investigate its possible value as a prognostic parameter in breast carcinomas.

Sex Steroid Receptors in Hodgkin's Disease

A case report of a dramatic therapeutic response of Hodgkin's disease (HD) to diethylstilbestrol (DES) in a man who was being treated for concurrent prostate cancer suggested that there also may be a role for sex steroids in the pathogenesis of HD (I). High levels of estrogen receptors (ER) comparable to those seen in breast carcinoma cells were detected in that patient's Hodgkin's biopsy specimen. In order to determine whether this patient was unique or whether sex steroid receptors commonly are present in HD specimens. we examined expression of ER and progesterone receptors (PR) in diagnostic tissue from pediatric (n=14) and adult (n=41) patients with HD using immunohistochemistry. None of the 55 samples expressed PR. 16/55 (29%) demonstrated weak nuclear 13 positivity, which was confined to germinal center and occasional mantle zone lymphocytes and was comparable to that seen in non-malignant control lymph nodes. 4/55 (7.3%) samples exhibited moderate positivity in Reed Sternberg cells, which in one case was nuclear. ER commonly are expressed weakly in some HD tumors unrelated to clinical stage or patient sex but arc generally limited to germinal center and mantle zone lymphocytes. A rare patient displays moderate cytoplasmic or nuclear ER in Reed-Sternberg cells.

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.

Relevance of ER to the Development of Endometrial Hyperplasia and Adenocarcinoma.

Estrogen has an important role in both the etiology and treatment of hormone-dependent endometrial cancers, although the mechanism remains elusive. To definethe role of estrogen-mediated signaling we investigated the biological significance of estrogen receptors (ER) in NIH3T3 cell transformation via the [ &sup12; Val ] K-Ras mutant. This mutant enhanced the steady state level and transcriptional activity of ER. In addition, overexpression of both wild type K-Ras and ER transformed NIH3T3 cells. Co-expression of the progesterone receptor (PR) with mutant K-Ras led to suppression of tumorigenicity and inhibition of ER activation. The antisense oligomers complementary to ER suppressed proliferation and transformed phenotypes of K12V cells. These observations support the importance of ER in Ras-mediated cell transformation. To address whether ER activation is also important in the development of human endometrial cancers, we investigated ER and PR expression levels in premalignant and malignant endometrial lesions. The results suggested the implication of ER abundance in endometrial hyperplasias, though modulation of PR expression by ER was retained. G1 adenocarcinoma also expressed higher levels of ER while PR modulation by ER was abrogated. These data implied the importance of ER activitiesin endometrial hyperplasia and G1 adenocarcinoma development.

Estrogen Effects on Insulin-Like Growth Factor Gene Expression in a Human Osteoblastic Cell Line with High Levels of Estrogen Receptor

Insulin-like growth factors (IGF)-I and IGF-II are produced by osteoblasts and are important paracrine/autocrine regulators of osteoblast proliferation and differentiation. Estrogen has been reported to increase gene expression of IGF-I in rodent osteoblasts. However, because species differences have been demonstrated in expression of various aspects of the IGF system in bone cells, it is not known whether this action also occurs in human osteoblasts. Thus, we assessed the effects of estrogen treatment on IGF-I and IGF-II gene expression in vitro in a recently developed human fetal osteoblast cell line that has high levels of estrogen receptors. As assessed by a quantitative reverse transcriptase-polymerase chain reaction method, treatment of hFOB/ER9 cells with 17beta-estradiol (E2) increased steady state levels of IGF-I mRNA in a time- and dose- dependent fashion with a maximal increase of 319% +/- 33% (P < 0.01) of control occurring after treatment with 10(-7) M E2 for 48 hours. In contrast, E2 did not alter steady state levels of IGF-II mRNA. The pure (type 2) antiestrogens ICI 182,780 (10(-7) M) and ICI 164,384 (10(-6) M) blocked the E2- induced increase in IGF-I mRNA levels. Interestingly, 4-hydroxytamoxifen (10(-7) M), a documented pure antiestrogen in reproductive tissues, also increased IGF-I mRNA to levels similar to those observed in E2-treated cells. Since E2 was shown to mediate its effects on some target genes through a cAMP-dependent pathway, we studied the interaction between E2 and agents that are known to increase intracellular cAMP. Forskolin (10(-8) M) and dibutyryl cAMP (10(-3) M) increased IGF-I mRNA levels sixfold, and cotreatment with E2 did not affect these changes, consistent with a possible mediation of the estrogen effect on IGF-I gene expression by cAMP. We conclude that in human osteoblastic cells, the IGF-I gene is a target for estrogen action, suggesting that IGF-I may mediate part of the effects of estrogen in human bone.

Antiestrogen, trans-tamoxifen modulation of human breast cancer cell growth

To gain further insight into how antiestrogens modulate cell function, the effects of antiestrogen on cell proliferation were studied in human breast cancer cells. We examined the effects of trans-tamoxifen on the proliferation of three human breast cancer cell lines that differed in their estrogen receptor contents. Trans-tamoxifen (1 muM) markedly inhibited the estrogen stimulated proliferation of MCF-7 human breast cancer cells that contained high levels of estrogen receptor (1.15+/-0.03 pmole/mg protein) over that of control. In T47D cells that contained low levels of estrogen receptor (0.23+/-0.05 pmole/mg protein), trans-tamoxifen (1 muM) showed minimal inhibition of estrogen stimulated cell proliferation over that of control. MDA-MB-231 cells, that contained no detectable levels of estrogen receptors, had their growth unaffected by trans-tamoxifen treatment. These results showed their sensitivity to growth inhibition by antiestrogen correlated well with their estrogen receptor content. Also we activator activity in MCF-7 cells. Trans-tamoxifen (1 muM) showed maximal inhibition of estrogen stimulated progestrone receptor level as well as plasminogen activator activity in MCF-7 cells that were stimulated by estrogen. It is not clear whether these inhibitions of progestrone receptor and plasminogen activator activity by estrogen are related to the antiestrogen inhibition of cell proliferation of MCF-7 cells. From the results of this study, it is clearly demonstrated that trans-tamoxifen is an antiestrogen in MCF-7 human breast cancer cells. Our data suggest that the biological effectiveness of trans-tamoxifen appear to result from its affinity of interaction with the estrogen receptor.

Estrogen modulation of human breast cancer cell growth

To gain further insight into how estrogens modulate cell function, the effects of estrogen on cell proliferation were studied in human breast cancer cells. We examined the effects of estrogen on the proliferation of three human breast cancer cell lines that differed in their estrogen receptor contents. Ten nM estradiol markedly stimulated the proliferation of MCF-7 human breast cancer cells that contained high levels of estrogen receptor (1.15+/-0.03 pmole/mg protein) over that of control. In T47D cells that contained low levels of estrogen receptor (0.23+/-0.05 pmole/mg protein), Ten nM estrogen slightly stimulated the proliferation over that of control. MDA-MB-231 cells, that contained no detectable levels of estrogen receptors, had their growth unaffected by estrogen. These results showed their sensitivity to growth stimulation by estrogen correlated well with their estrogen receptor content. Also we examined the effect of estrogen on cellular progesterone receptor level as well as plasminogen activator activity in MCF-7 cells. Ten nM estradiol showed maximal stimulation of progesterone receptor level as well as plasminogen activator activity in MCF-7 cells. It is not clear whether these stimulations of progesterone receptor and plasminogen activator activity by estrogen are related to the estrogen stimulation of cell proliferation of MCF-7 cells. Studies with estrogen in human breast cancer cells in culture indicate that sensitivity to growth stimulation by estrogen correlates well with estrogen receptor contents.

Additive Estrogenic Activities of a Binary Mixture of 2′,4′,6′-Trichloro- and 2′,3′,4′,5′-Tetrachloro-4-biphenylol

The estrogenic activity of 2′,4′,6′-trichloro-4-biphenylol (HO-PCB3), 2′,3′,4′,5′-tetrachloro-4-biphenylol (HO-PCB4), and an equimolar mixture of both compounds (HO-PCB3/HO-PCB4) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 and MDA-MB-231 human breast cancer cells, HepG2 cells, and in a yeast-based reporter gene assay. Treatment of the animals with 17β-estradiol (E2) (0.02 μg/kg/day ×3) resulted in increased uterine wet weight, peroxidase activity and progesterone receptor binding. Treatment with 18, 73, 183 or 366 μmol/kg (×3) doses of HO-PCB3, HO-PCB4, or HO-PCB3/HO-PCB4 (equimolar) caused a dose-dependent increase in estrogenic activity; a maximal-induced response was not observed at any dose and the activity of the mixture was additive. Binding of E2, HO-PCB3, HO-PCB4, and HO-PCB3/HO-PCB4 to the mouse uterine estrogen receptor (ER) was determined in a competitive binding assay using [3H]E2 as the radioligand. The IC50 values were 1.1 × 10−8, 3.4 × 10−6, 9.9 × 10−7, and 4.25 × 10−6m, respectively. HO-PCB3 and HO-PCB4 maximally induced MCF-7 cell proliferation, rat creatine kinase, and human complement C3 (C3-LUC) reporter gene activity at concentrations of 10−5to 10−6m, and these compounds were 103to 104less potent than E2. The HO-PCB3/HO-PCB4 mixture was active at the high concentration (10−5m) and was additive for these responses. HO-PCB3 and HO-PCB4 also exhibited estrogenic activity in human HepG2 cells cotransfected with C3-LUC and an ER expression plasmid, and the estrogenic activity of the HO-PCB mixture was additive. Similar results were obtained in yeast transformed with the human ER and a double estrogen responsive element upstream of the β-gal reporter gene. The effects of variable ER expression on the potential synergistic interactions of HO-PCB3/HO-PCB4 were investigated in HepG2 cells cotransfected with C3-LUC (405 ng/well) and variable amounts of ER expression plasmid (270, 27, 2.7, or 0.27 ng/well). The results show that as ER levels decreased, the magnitude of the induction response by E2, HO-PCB3, HO-PCB4, and HO-PCB3/HO-PCB4 also decreased. However, the activities of the HO-PCB mixture were additive at high and low levels of ER. Similar results were obtained in MDA-MB-231 cells cotransfected with C3-LUC and variable amounts of ER expression plasmid. The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus; MCF-7, HepG2, and MDA-MBA-231 human cancer cells; and a yeast based-reporter gene assay, both HO-PCB3 and HO-PCB4 exhibited estrogenic activity. The estrogenic activity of an equimolar mixture of these compounds was additive at high and low levels of ER expression.

Gonadotrophin releasing hormone (GnRH) receptor and steroid receptors in human uterine leiomyoma, myometrium and endometrium

The present study evaluated the presence of GnRH-R in leiomyomas, in associated, non-involved uterine tissues (myometrium and endometrium) and the possible relationships between GnRH-R and the receptors for estrogen and progesterone in the same tissues. GnRH-R was found in all uterine tissues and both GnRH and the GnRH analog, goserelin, displaced its binding consistent with a single type of high affinity receptor (Kd approximate to 10(-8) M). GnRH-R were found more frequently in myometrium (81% of samples) than in endometrium (58%) or leiomyoma (42%). However, the mean receptor content was lowest in myometrium (139+/-19 fmol/mg protein) with both leiomyomas (288+/-77 fmol/mg protein) and endometrium (372+/-96 fmol/mg protein) having significantly higher values. Endometrial GnRH binding varied from 596+/-42 in uteri that were GnRH-R positive in the endothelium alone to 231+/-49 when GnRH-R was present also in the other tissues. Endometrium negative for the GnRH-R had significantly higher levels of estrogen receptor than all the other uterine samples (266+/-25 vs 61+/-7.5 fmol/mg protein, respectively). Endometrial GnRH-R seem to be dependent on its presence and/or level in other uterine tissues. Further, when GnRH-R is absent in the endometrium this tissue expresses greatly increased levels of steroid receptors.

Cellular and Molecular Effects of Growth Hormone and Estrogen on Human Bone Cells

The aim of the present thesis is to examine some aspects of the biological effects of growth hormone (GH) and estrogen on bone cells in vitro. The first part of the thesis describes characterization of model systems to study normal human osteoblasts and osteoclasts in vitro. Three culture systems for human osteoblasts have been characterized, representing different stages of osteoblasts differentiation/maturation: (1) mature osteoblasts cultured from trabecular bone explants (trabecular osteoblasts), (2) less mature osteoblasts (stromal osteoblasts) cultured from bone marrow and (3) osteoblast precursor cells cultured also from bone marrow. This classification is based on quantitative and qualitative differences in the expression of osteoblast phenotypic markers by these cells. These systems are useful in studying the regulation of hormones and growth factors of different stages of osteoblast differentiation. Further characterization of the osteoblast differentiation pathway is still needed, especially the identification of surface markers that can definitively identify intermediate stages of osteoblast differentiation. Normal human osteoclasts were cultured from bone marrow mononuclear cells and exhibited the main characteristics of osteoclast phenotype: production of tartrate-resistant acid phosphatase, presence of a ruffled border and the ability to resorb mineralized matrix. This model is useful for studying factors regulating osteoclast commitment and differentiation. The second part of the thesis deals with the effects of GH on proliferation and differentiation of trabecular and stromal osteoblasts. Effects of GH on human osteoblasts were dependent on their degree of maturation. GH stimulated cell proliferation in both trabecular and stromal osteoblasts. While it increased the functional activity of trabecular osteoblasts, these effects were absent in stromal osteoblast cultures. Human trabecular osteoblasts produce mainly IGF-II, IGFBP-3 and minute quantities of IGF-I in culture. GH does not seem to regulate the local production of IGF-II or IGFBP-3. However, IGFs and their binding proteins may exert important regulatory effects on the biological effects of GH on human osteoblasts, and this role needs to be studied. Sincer GH exerted profound effects on the biological functions of human osteoblasts in vitro, the hypothesis that either decreased production of GH or decreased sensitivity of bone cells to its action leads to bone loss and osteoporosis was examined. No differences in the basal or GH-stimulated production of IGF-I, IGF-II or IGFBP-3 were found between osteoporotic patients and age-matched normals. Similarly, The response of bone cells to in vivo and in vitro stimulation by GH was similar in the two groups. These studies do not support the hypothesis of the presence of major defects in production of GH or the presence of resistance to its effects in bone cells in patients with osteoporosis. The last part of the thesis deals with the cellular mechanisms mediating estrogen actions on bone cells. Thus, the potential role of estrogen regulation of bone-resorbing cytokines in the pathogenesis of bone loss in postmenopausal women was examined. Estrogen was found to inhibit IL-6 production and gene expression in an immortalized human osteoblastic cell line expressing high levels of estrogen receptors (hFOB/ER9 cell line). To further examine the relationship between estrogen deficiency and early postmenopausal bone loss, levels of IL-6, IL-6sR, IL-1 alpha, IL-1 beta and IL-1ra were measured in bone marrow plasma and bone marrow cultures obtained from 40 postmenopausal women, half of whom were on estrogen replacement therapy. No difference was found between the groups in any of these parameters. This suggests that neither IL-1 nor IL-6 by itself is the major mediator for increased bone loss due to estrogen deficiency in the early postmenopausal period. (ABSTRACT TRUNCATED)

Estradiol up-regulates estrogen receptor and progesterone receptor gene expression in specific ovine uterine cells.

The regulation of estrogen receptor (ER) and progesterone receptor (PR) genes is critical to estrogen and progesterone responsiveness of the uterus during the estrous cycle. A low dose of estradiol, given to ovariectomized ewes to mimic the preovulatory estrogen surge, acutely enhanced ER and PR gene expression in most uterine cells. Estradiol effects were measured at 12, 24, and 48 h post-injection (n = 6 ewes per time) with immunohistochemistry and in situ hybridization. Whereas vehicle-treated ovariectomized ewes demonstrated low to moderate ER and PR mRNA and protein expression, estradiol enhanced PR mRNA and protein expression (at 12 h and 24 h, respectively) more rapidly than ER mRNA and protein expression (at 24 h and 48 h, respectively) in most uterine cells. However, the timing and extent of the estradiol response depended partly upon cell type (epithelial, stromal, or myometrial), cell region (luminal, superficial, middle, or deep endometrial or myometrial), adjacent cells, and prior progesterone treatment. For example, PR mRNA up-regulation was prolonged in middle and deep endometrial stroma, but increases in PR protein expression were highest in superficial and middle endometrial compartments, including the luminal epithelium. The luminal epithelium and myometrium were unique in that estradiol failed to up-regulate ER gene expression within them. ER mRNA levels rose within these compartments only when estradiol followed steroid hormone treatment designed to induce an artificial estrous cycle (estradiol-progesterone-estradiol [EPE] treatment). The EPE treatment also augmented the rise in ER mRNA concentrations within stromal cells compared to estradiol treatment alone. Within uterine cell compartments, subpopulations of adjacent cells showed distinct estradiol responses, e.g., very high levels of ER and PR gene expression within stromal cells directly underlying glandular epithelial cells. Because the estradiol response did not always correlate with initial ER protein levels and was partly dependent upon cell compartment and adjacent cells, we must conclude that direct transcriptional and/or posttranscriptional actions of estradiol cooperate with other cellular and paracrine regulatory factors to regulate ER and PR gene expression and, thus, the steroid responsiveness of uterine cells.

Bcl-2, p53, and response to tamoxifen in estrogen receptor-positive metastatic breast cancer: a Southwest Oncology Group study.

To test the hypothesis that high bcl-2 expression and accumulation of p53 protein, both of which should inhibit apoptosis, are associated with a poorer tamoxifen response and a more aggressive clinical course in estrogen receptor (ER)-positive metastatic breast cancer. A total of 205 paraffin-embedded tumor blocks were evaluated for nuclear p53 (a marker of p53 inactivation) and cytoplasmic bcl-2 by immunohistochemistry (IHC). All patients received tamoxifen as initial therapy for metastatic disease. The study began in 1982 and follow-up duration of the 24 patients last known alive is 8 years. Response to tamoxifen and time to treatment failure (TTF) were not significantly associated with p53 status, although patients with higher p53 had a worse survival (P = .008; median, 36 v 20 months). Higher bcl-2 expression was associated with higher levels of ER (P = .02), better response to tamoxifen (62% v 49%; P = .07), longer TTF (median, 9 v 5 months; P = .002), and better survival (median, 40 months v 25 months; P = .009). In multivariate analyses, including ER, progesterone receptor (PgR), and p53, high bcl-2 remained significantly associated with a longer TTF (P = .007) and survival (P = .07). p53 status was a significant factor for shorter survival (P = .05), but not for TTF (P = .61). p53 status, as determined by IHC is not significantly associated with response to tamoxifen, although tumors with altered p53 protein are inherently more aggressive. Contrary to expectation, high bcl-2 identifies a relatively indolent phenotype of ER-positive metastatic breast cancer, in which patients experience a better clinical response to tamoxifen and a longer survival.

Estrogen inhibits interleukin-6 production and gene expression in a human osteoblastic cell line with high levels of estrogen receptors.

Some studies suggest that estrogen acts on bone by decreasing the production of interleukin-6 (IL-6), a cytokine that increases bone resorption, by osteoblasts or bone marrow cells. However, other studies have not confirmed this, possibly because of a low and variable number of estrogen receptors (ER) in the model systems used. Thus, we employed a recently developed human fetal osteoblast cell line with high levels of ER. Treatment (n = 4 experiments) with 0.01 to 10 nM of 17 beta-estradiol had no effect on the constitutive production of IL-6. However, stimulated production, induced by treatment with IL-1 beta plus tumor necrosis factor-alpha (TNF-alpha), was reduced in a dose-dependent manner to 74 +/- 3% (mean +/- SEM) of control (p < 0.01). This response was blocked by cotreatment with the type II antiestrogen ICI 182,780. Treatment with hydrocortisone (1 microM), a known inhibitor of IL-6 production in many cell types, reduced IL-6 production to 17 +/- 1% of control (p < 0.001). As assessed by Northern analysis, treatment (n = 3 experiments) with 0.01-10 nM of 17 beta-estradiol decreased steady-state levels of IL-6 mRNA in a dose-dependent manner. These data support the hypothesis that at least part of the antiresorptive action of estrogen in humans is mediated by decreased production of IL-6 by osteoblastic cells.