Increase In Estrogen Receptor Levels Research Articles

Monocyte subsets in breast cancer patients under treatment with aromatase inhibitor and mucin-1 cancer vaccine

BackgroundMonocytes comprise subsets of classical, intermediate and non-classical monocytes with distinct anti- or pro-tumor effects in breast cancer (BC). They are modulated by estrogen, and can contribute to BC control by endocrine therapy in preclinical models.MethodsTo elucidate whether changes in monocyte subsets are associated with treatment and response, we investigated peripheral blood samples of 73 postmenopausal women with estrogen receptor (ER) positive BC, who received aromatase inhibitor therapy with or without the mucin-1 vaccine tecemotide in the ABCSG34 trial. Blood was retrieved at baseline, midterm and end of therapy, and was analyzed for the distribution and ER expression of monocyte subsets by flow cytometry.ResultsWhen 40 healthy, age-matched women were compared with BC patients before treatment start, ER levels of monocytes did not differ, yet patients presented with a higher frequency of classical and fewer non-classical monocytes. Endocrine therapy triggered a significant increase in ER levels in all monocyte subsets, without affecting subset distribution. Vaccination had no overall impact on subset frequency and ER expression. Yet, a shift from intermediate to classical monocytes during therapy correlated with changes in plasma cytokines and chemokines and was significantly associated with low residual cancer burden in vaccinated patients. Without tecemotide, baseline ER levels in classical monocytes were significantly higher in women with good response to endocrine therapy.ConclusionsThis study identified classical monocytes to be associated with ER positive BC and with patient response to neoadjuvant endocrine treatment and cancer vaccination.

Regulation of estrogen receptor (ER) levels in MCF-7 cells by progesterone metabolites

Estradiol-17β (E2) may participate in carcinoma of mammary cells containing estradiol receptors (ER) at sufficient levels. Hence, the regulation of ER levels may be important for the progression of estrogen-dependent mammary carcinomas. Our previous findings that the progesterone metabolite, 5α-pregnane-3,20-dione (5αP), exhibits marked mitogenic and metastatic properties, whereas the progesterone metabolites, 4-pregnen-3α-ol-20-one (3αHP) and 4-pregnen-20α-ol-3-one (20αHP), oppose these actions, prompted examination of the possible effects of these progesterone metabolites on ER concentration in MCF-7 breast cancer cells. Cells were exposed for 24 h to 0 (control) or 10 −10 to 10 −6 M E2, 5αP, 3αHP, 20αHP or combinations of these steroids, and ER concentrations were determined for intracellular estrogen receptors by specific binding of [ 3H]E2. The total ER number (nuclear plus cytosolic) in control samples was 2551 ± 164 per cell. E2 and 5αP resulted in significant dose-dependent increases in total ER numbers (∼1.6-fold and ∼2.2-fold at 10 −6 M, respectively). In combination, E2 + 5αP resulted in additive increases in ER numbers. Individually, 3αHP and 20αHP each resulted in dose-dependent decreases (43% and 54% at 10 −6 M, respectively) in total ER numbers and inhibited the E2- or 5αP-induced increases in ER levels. In combination, 3αHP + 20αHP resulted in dose-dependent additive suppression of ER levels. Treatment with cycloheximide or actinomycin D indicated that both transcription and translation are involved in 5αP and 3αHP action on ER numbers. Real time RT-PCR showed increases in expression of ERα transcripts due to 5αP and increases in expression of ERβ due to 3αHP; expression levels of either ERα or ERβ were not significantly altered when cells were treated with 5αP + 3αHP. The results are the first to show that the pro- and anti-cancer progesterone metabolites also have marked selective (up or down) regulatory effects on ER levels in MCF-7 breast cancer cells.

Activation of estrogen response element dependent transcription by thyroid hormone with increase in estrogen receptor levels in a rat pituitary cell line, GH3.

Interrelationships between thyroid hormone and estrogen actions have been documented with regard to a variety of physiological functions. Both hormones stimulate transcription of target genes by binding to their nuclear receptors that interact with specific responsive elements (estrogen and thyroid hormone response elements, i.e ERE and TRE, respectively) in the regulatory regions of the gene. In vitro studies have suggested that interplay between the two hormones might be due to cross-talk at hormone responsive elements, with the respective hormone receptors and ligands able to interact, although physiological relevance has yet to be proved. We have proposed a simpler mechanism for thyroid hormone effects on estrogen responses via increase in estrogen receptor alpha (ERalpha) with resultant increase in progesterone receptors, prolactin production and tumor growth. A pituitary cell line, GH3, has been widely used to investigate the function of mammo-somatotropic cells, especially regarding regulation of GH and prolactin production. In the present study, an ERE-luc reporter was transfected into GH3 cells and the responses to endogenous ERalpha were examined. We demonstrated that: (1)l -3,5,3'-triiodothyronine (T3) induces mRNA expression of ERalpha; (2) T3 alone is able to induce ERE-luc activity and this is inhibited by OH-tamoxifen; (3) T3 synergistically acts on estradiol (E2)-induced ERE responses; and (4) ERE-luc activity is enchanted by co-transfection of an ERalpha expression vector. These results support the hypothesis that estrogen responses are potentiated by T3 through up-regulation of ERalpha levels.

Effects of OK-432 (picibanil) on the estrogen receptors of MCF-7 cells and potentiation of antiproliferative effects of tamoxifen in combination with OK-432.

OK-432 (picibanil), a streptococcal preparation, has a strong biological response modifier (BRM) function and is expected to produce clinical improvement and prolongation of survival in treated cancer patients in Japan. We were interested in whether OK-432 augments estrogen receptor (ER) levels in breast cancer. To investigate the effect of the BRMs on cellular growth and the characteristics of ER and progesterone receptors (PgR) in the human breast cancer cell line MCF-7, we used OK-432, Krestin (PSK), a protein-bound polysaccharide extracted from Coriolus versicolor, and lentinan, a fungal branched (1...3)-beta-D-glycan. OK432 and PSK dose dependently inhibited DNA synthesis of MCF-7 cells, and the 50% inhibitory concentrations of OK-432 and PSK were 1.2 KE (klinische Einheit, clinical unit)/ml and 200 micrograms/ml, respectively. Lentinan showed no direct anticancer effect in vitro. We found that OK-432 induced a 2-fold increase in ER levels in MCF-7 cells at 0.005 KE/ml, but not in PgR. Lentinan and low-dose PSK did not change ER or PgR levels, but high-dose PSK decreased ER and PgR. We also studied the combined effect of OK-432 and antiestrogens, tamoxifen (TAM) and DP-TAT-59. The combined treatment with OK-432 and TAM showed an additive inhibitory effect on MCF-7 cells. These results suggest that OK-432 may augment the therapeutic effect of TAM in breast cancer.

Paradoxical production of target protein using antisense RNA expression vectors.

We used antisense RNA in a protocol designed to reduce estrogen receptor (ER) content in human breast cancer cells and observed paradoxical increases in ER levels. ER protein activity was measured using a highly sensitive reporter gene assay that relies on the ability of functional ER to bind a consensus estrogen response element (ERE) and drive the production of chloramphenicol acetyl-transferase (CAT). Upon transient transfection of ER-positive cell lines with three different vectors containing the full-length ER cDNA cloned in an antisense orientation, we observed unexpected increases in ER-driven CAT activity. To further investigate this phenomenon, expression from the antisense ER vectors was studied in an ER-negative breast tumor cell line, MDA-MB-453. ER activity was observed in these ER-negative cells upon transient transfection with each of three antisense ER vectors, but not from control vectors. Expression of ER from antisense constructs was 30-100-times less efficient than ER expression from isogenic sense constructs. The paradoxical ER activity was consistent with expected ER behavior in that it exhibited characteristic binding to the natural ligand, 17 beta-estradiol (E2), and it was inhibited by the antiestrogens, 4-hydroxy-tamoxifen (OHT) and ICI 164384 (ICI). Control vectors containing a truncated antisense ER cDNA produced no ER activity. Although the mechanism for this ER expression has not been determined, it appears likely that it is due to transcription off the opposite strand of the antisense construct.(ABSTRACT TRUNCATED AT 250 WORDS)

A new triphenylethylene derivative, TAT-59; hormone receptors; insulin-like growth factor 1; and growth suppression of hormone-dependent MCF-7 tumors in athymic mice

TAT-59 ((E)-4-[1-[4-[2-(dimethylamino)ethoxy]-phenyl]-2-(4- isopropyl)phenyl-1-butenyl]-phenyl-monophosphate) treatment was performed on hormone-dependent MCF-7 tumors in athymic mice. TAT-59 given at 1, 5, and 20 mg/kg inhibited the estrogen-stimulated growth of MCF-7 tumors in athymic mice in a dose-dependent fashion. The most clear decrease in tumor growth was shown in the TAT-59 alone group, although it was not dramatic. Average serum concentrations of DP-TAT-59((Z)-[1-[4-[2-(dimethylamino)- ethoxy]phenyl]-2-(4-isopropyl)phenyl-1-butenyl]-4-hydroxybenzene) and DM-DP-TAT-59(desmethyl-DP-TAT-59), metabolites of TAT-59, increased in a dose-dependent manner. Much higher levels of DP-TAT-59 and DM-DP-TAT-59 were shown in tumors (target tissues of estrogen) as compared with muscles (nontarget tissues of estrogen) or serum. A serum concentration of DP-TAT-59 or DM-DP-TAT-59 corresponding to the physiologic levels of serum estradiol in premenopausal women was sufficient to inhibit the estrogen-stimulated growth of MCF-7 tumors in mice. TAT-59 induced a dose-dependent increase in estrogen receptor levels in the MCF-7 tumors. In contrast, it prevented the estradiol (E2)-induced increase in progesterone receptor levels in a dose-dependent manner. Insulin-like growth factor 1 levels measured in the MCF-7 tumors significantly decreased in the TAT-59 alone group and in the no treatment group as compared with the E2 alone group. These results show the pronounced antiestrogenic action of TAT-59 on hormone-dependent MCF-7 tumors in athymic mice.

Increase in estrogen receptor levels in MNU-induced thyroid tumors in LE rats.

Estrogen receptor (ER) levels were evaluated in thyroid tumors induced by N-methyl-N-nitrosourea (MNU) and low iodine diet (LID) or propylthiouracil (PTU) in intact and estrogen (E2) loaded Long-Evans (LE) rats. MNU at 40 mg/kg body wt was injected in 50 day-old LE rats of both sexes. The animals were killed 17-22 weeks later and the thyroid tissues were subjected to ER assay. In LID-treated groups, cytosolic ER (cER) levels were 6.7 +/- 5.8 (fmol/mg protein, mean +/- SE) in females and 0.7 +/- 1.4 in males, E2 increased the ER levels. In E2-loaded LID groups, cER levels were 12.9 +/- 3.7 in females and 1.7 +/- 1.7 in males. PTU treatment produced almost comparable ER levels as LID treatment. PTU treatment as well as LID treatment increased the serum TSH levels with E2 treatment producing additional elevation. In evaluating ER levels by histological type of thyroid tumors, the level in cER plus nER showed the lowest value of 6 +/- 6.4 (fmol/mg DNA, mean +/- SE) in hyperplasia, followed by 129 +/- 52.3 in adenoma and 289 +/- 51.7 in carcinoma. The rates of BrdU incorporation in thyroid follicles indicated higher proliferation activity in the area of adenoma and carcinoma rather than in the hyperplastic area. These data suggested that E2 treatment increases the ER levels in MNU and LID/PTU-induced thyroid tumors. The level of ER was correlated to the histological type of thyroid tumors.

Construction of Cell Lines that Express High Levels of the Human Estrogen Receptor and Are Killed by Estrogens

To prepare large amounts of the human estrogen receptor (ER) for biochemical and biophysical studies we have employed the cloned ER sequences to construct Chinese hamster ovary (CHO) cell line derivatives that overexpress the receptor. We have employed an efficient expression vector (SV40 enhancer, human metallothionein IIA promoter) and a new system of gene amplification based on the human metallothionein IIA gene and stepwise selection in cadmium. Cells from the initial transfected pools, before gene amplification, had as much or more ER than human MCF7 cells and responded to the subsequent stepwise cadmium selection and amplification with increases in ER levels to about 2 million receptors/cell. Cell lines isolated from these pools are stable for human ER expression and display up to 6 million receptors/cell, or about 0.4% of the total cell protein. The CHO receptor activates a transfected reporter gene in responses to estrogen, is down-regulated in response to estrogens, displays the same electrophoretic mobility as the MCF7 receptor, and is free of degradation as initially extracted. CHO cells displaying 3 million or more human ER/cell (but not cells with lower levels) flatten and stop growing within the first 24 h after exposure to physiological estrogen concentrations. After several days in estrogen the majority of the cells lyse. The antiestrogen 4-hydroxytamoxifen also causes cell death, but another antiestrogen, ICI 164,384, is without toxic effect. The basis for these phenomena are unknown, but mutants isolated for survival of estrogen treatment have lost receptor expression, thereby confirming the role of receptor in cell death.

Estrogen receptors in the rhesus monkey brain during fetal development

Estrogen receptor (ER) levels were measured in brain tissue cytosol from fetal male and female rhesus monkeys at Days 70, 100 and 160 postconception. The brain regions which were examined included medial basal hypothalamus (MBH), amygdala (AMG), cerebral cotex (CTX) and cerebellum (CB). For comparison, brain tissues were also obtained from an adult female, and muscle (MUS) and genital tract (GEN, ovaries + uterus) ER values were measured in several Day 70 fetuses. Tissues were dissected and homogenized as previously described 24. Cytosol was passed through a microcolumn of Lipidex 1000 to remove interfering lipids and incubated with [ 3H]Moxestrol (4 nM) in the presence or absence of 500 nM Moxestrol. Incubations were carried out for 24 h at 4 °C, and free and bound ligand separated by Sephadex LH-20 gel filtration. In one case (Day 160 male fetus), saturation analysis yielded an estimate of apparent K d of 0.46 × 10 −9M and indicated that maximal specific binding was achieved at a ligand concentration of 1–2 nM. There was no sex difference at any stage of development (ANOVA). A significant age effect ( P < 0.002) was noted for the MBH and CB but not for any of the other tissues examined. In the MBH the significance of this effect was due to a progressive increase in ER levels with fetal age and into adulthood. In contrast, CB levels exhibited a progressive decline with age. These studies revealed that the ER is present during brain development. Thus any estrogens derived from the aromatization of circulating fetal androgens could potentially exert an influence upon brain differentiation, particularly upon the MBH at late gestation and adulthood. The lack of a definitive sex difference suggests that both sexes are potentially susceptible to estrogen modification. However, this susceptibility may be minimal, since receptor concentrations are considerably lower than values reported in the literature for postnatal monkeys.

Differential effects of estradiol and 16 alpha-hydroxyestrone on pituitary and preoptic estrogen receptor regulation.

16 alpha-Hydroxyestrone (16OHE1), an endogenous metabolite of estradiol (E2), binds to the estrogen receptor (ER) with low affinity, but is estrogenic in various bioassay systems. 16OHE1 binds covalently to the ER in vitro, exhibits prolonged estrogenic bioactivity in vivo, and has been implicated in several estrogen-dependent diseases. This study examined the effects of 13 days of continuous infusion of E2 or 16OHE1 on lordotic behavior, pituitary growth, and ER regulation in the cytosolic and nuclear fractions of the pituitary and preoptic area of both sexes. Finally, simultaneous pituitary nuclear exchange assays and enzyme immunoassays were performed to search for covalent 16OHE1-ER complexes in vivo. E2 induced lordosis and pituitary growth in both sexes, while 16OHE1 was only slightly less effective. While E2 treatment increased nuclear ER concentrations 2-fold vs. control values, it decreased both cytosolic and total (cytosolic plus nuclear) ER concentrations in pituitary and preoptic area by approximately 3-fold vs. control values in both sexes by exchange assay. In contrast, 16OHE1 did not decrease total pituitary ER concentrations and only minimally decreased total preoptic ER concentrations. Simultaneous exchange assay and immunoassay of pituitary nuclear extracts demonstrated proportionate increases in ER levels in female vs. male and in E2-treated vs. 16OHE1-treated rats. The ratios of (ER enzyme immunoassay divided by ER-exchange) for each rat were similar regardless of metabolite administration. The correlation of individual measurements implied that ER localized to the nuclear fraction by either E2 or 16OHE1 retained both exchangeability and immunoassayability to similar extents, but did not support the presence of 16OHE1-ER covalent complexes. The results of this study suggest that 16OHE1 has significant estrogenic bioactivity, as manifest by its effects on lordosis and pituitary growth, but, in contrast to E2, does not decrease pituitary ER concentrations and only minimally decreases preoptic ER concentrations. This property may be important in the proposed pathogenetic action of 16OHE1 in estrogen-dependent disease.

Relationship between changes of the steroid receptor and synchronization in human endometrial adenocarcinoma cells in vitro

It has been reported that the response of target cells to steroid hormone (SH) stimulation may depend on their position in the cell cycle. The DNA and RNA contents of malignant cells of the endometrium cultured in vitro were measured using flow cytometry (FCM). We also measured estrogen receptor (ER) and progesterone receptor (PR) levels of cells at different positions in the cell cycle. The G1 and S phases of the cell cycle were investigated using cells synchronized by sodium n-butyrate (G1 block), methotrexate (S block), and excess thymidine (S block). For DNA measurements, the cells were stained with propidium iodide following RNase treatment. For RNA measurements (double-stranded RNA) the cells were treated with DNase. We found that S phase synchronization by methotrexate was 136.2% of control (100%). Using the excess thymidine block and release procedure, the S phase fraction was 185.1% of control. G1 phase synchronization by sodium n-butyrate was 134% of control. The estrogen receptor level in G1 phase synchronized cells increased to 5.94 fmol/micrograms DNA in the cytosol and 12.35 fmol/micrograms DNA in the nuclear fraction. These levels represent a sevenfold total increase over that of the control estrogen receptor level. Cells in S phase showed no significant increase in estrogen receptor levels over control cells. Based on this study, the functional increase of the steroid receptor was most significant in the G1 phase.

Estrogen receptors and estrogen sensitivity of fetal thymocytes are restricted to blast lymphoid cells.

We have observed [3H]estradiol binding sites in the cytosol of lymphoid cell suspensions obtained from the fetal thymus of guinea pig. Scatchard analysis [dissociation constant (Kd), 0.5 +/- 0.02 (SE) nM], binding specificity, and diethylaminoethyl chromatography of these [3H]estradiol binding sites are similar to those described for estrogen receptors. Estrogen receptor levels in the thymic lymphoid cell population have been studied after fractionating cells by Percoll discontinuous density gradients into large and low density cells (alpha-cells) and small and high density cells (beta-cells). Estrogen receptor levels are higher in alpha-cells [1002 +/- 200 (SE) sites per cell] than beta-cells [61 +/- 6 (SE) sites per cell]. Electron microscopy shows that the ultrastructural characteristics of 95% alpha-cells are consistent with their lymphoblastoid nature, whereas 90% of the beta-cells represented here can be categorized as typical small lymphocytes. To study whether cellular estrogen receptor expression was related to blastogenesis, fetal thymocyte suspensions were cultured throughout 48 h in the presence of the mitogen Concanavalin A. A significant increase in estrogen receptor levels was observed in thymic lymphoid cells cultured in the presence of the mitogen [1500 +/- 193 (SE) sites per cell] with respect to cells cultured in the absence of the mitogen [400 +/- 54 (SE) sites per cell]. One-day and 3-day in vivo estrogen treatments decrease significantly the [3H]thymidine uptake [by 40 +/- 1% (SE) and 66 +/- 5% (SE), respectively], the mitotic index [by 82 +/- 8% (SE) and 96 +/- 2% (SE), respectively] and the frequency of lymphoblastoid cells [by 15 +/- 4% (SE) and 50 +/- 2% (SE), respectively] in fetal thymocyte suspensions. A 29 +/- 0.7% (SE), 31 +/- 0.8% (SE), and 35 +/- 2% (SE) decrease of [3H]thymidine incorporation in the DNA of alpha-cells cultured throughout 24 h, 48 h, and 72 h, respectively, in the presence of estradiol (10 nM) was observed with respect to untreated alpha-cells (P less than 0.01), while no effect of estradiol was observed on [3H]thymidine uptake by beta-cells. The mitotic index of cultured alpha-cells was significantly decreased [by 67 +/- 5% (SE)] 48 h after the addition of estradiol to the culture medium. It is concluded that: 1) estrogen receptors and estrogen sensitivity are restricted to lymphoblastoid thymic cells, and 2) estrogen receptor levels are increased after lymphoid blast transformation.