Altered estrogen receptor system in estrogen-unresponsive human endometrial adenocarcinoma cells

Previous studies from our laboratories demonstrated that cells from a human endometrial adenocarcinoma cell line (Ishikawa) responded to estradiol whereas cells from another endometrial cancer line (HEC-50) did not. In an attempt to identify factors responsible for the observed estrogen insensitivity we compared the characteristics of the estradiol receptor (ER) systems in Ishikawa and HEC-50 cells. Saturation analyses of cytosolic estrogen binders were performed over a 0.1-70 nM range of [3H]estradiol concentrations. Equilibrium dissociation constants and number of binding sites were determined by graphic analysis of Scatchard plots or computed by applying Fourier-derived affinity spectrum analysis (FASA) of the binding data. No significant differences were noted in the dissociation constants (Kd approx. 0.6 nM) or number of binding sites (approx. 6-10 fmol/mg protein) for the single binder that could be evaluated by the graphic method in cytosol from the two cell lines. However, 2 binders in Ishikawa cells (Kd approx. 0.2 and 6 nM) could be detected by the FASA method; the higher affinity binder in HEC-50 cells could not be clearly demonstrated. Structural differences in the specific estrogen binders which might distinguish HEC-50 from Ishikawa cells or normal endometrial tissue were investigated by using the anti-ER monoclonal antibody JS 34/32. Interaction of the antibody with [3H]estradiol binders of estrogen-responsive cells and tissue was evident from the formation of labeled complexes that were shown to sediment faster in glycerol density gradients and could be immunoprecipitated with Protein A attached to Sepharose beads. In contrast, the antibody did not recognize labeled specific binders in the HEC-50 cells. Furthermore, [3H]estradiol receptors in Ishikawa cells could be transformed into a species that exhibited increased hydrophilicity, evident from its binding to DNA-cellulose, whereas binders from HEC-50 could not. These results indicate that the lack of responsiveness of HEC-50 cells to estrogens might be due to structural or functional alterations in the ER protein resulting in a loss of its capability to undergo estrogen-directed conformational changes required for biological activity.     

Growth-promoting effects of progesterone in a human endometrial cancer cell line (Ishikawa-Var I).

Significant growth responses to progesterone of human endometrial adenocarcinoma cells (Ishikawa-Var I) were observed under in vitro culture conditions. Progesterone affected both the rate of exponential proliferation and cell population densities after the exponential phase. In the presence of the hormone, the doubling time of exponentially proliferating cells was reduced from 44 to 35.6 h and cell densities were increased by as much as 2-3 times over those of controls during approx. 2 weeks in culture. The effects of progesterone on cell population growth were dose dependent. Estradiol (10(-8) M) and testosterone (10(-6) M) did not affect cell densities and the effects of dexamethasone (10(-6) M) were small. In contrast, both progesterone and estradiol stimulated colony formation under anchorage-independent conditions in soft agar. These results suggest the possibility that growth of sensitive cell clones in endometrial tumors could be enhanced in some patients during adjuvant progestin therapy.     

Characterization of androgen receptors in a well-differentiated endometrial adenocarcinoma cell line (Ishikawa)

Androgen receptors (AR) have been identified in the human endometrium, but their role in endometrial function and development towards endometrial receptivity remains poorly understood. In an effort to study the regulation and possible function in endometrial epithelium, we utilized the well-differentiated endometrial adenocarcinoma cell line, Ishikawa, as a model system. This cell line has proven to be stable, hormonally responsive, contains both estrogen and progesterone receptors, and has been shown to express endometrial proteins in a hormone responsive manner. In the present study, we demonstrate that Ishikawa cells also express AR, based on immunohistochemical staining, radioactive binding studies, RT-PCR and Northern blot analysis. The expression of AR is induced in Ishikawa cells by estrogens, similar to that reported for normal endometrium. Further, using an estrogen-responsive gene that has been characterized in this cell line, alkaline phosphatase, we show that androgens act as antiestrogens in diethylstilbestrol (DES) treated cells, inhibiting enzymatic activity in a dose-dependent manner. These data support a physiologic role for AR in the endometrium. Elevations in endometrial AR in certain clinical situations such as polycystic ovarian syndrome (PCOS) may amplify the effects of androgens on the endometrium leading to suspected defects in uterine receptivity, higher than expected infertility and high miscarriage rates observed in patients with this disorder.     

Estradiol metabolism in Ishikawa endometrial cancer cells

Estrogen-responsive human cells derived from a specimen of well differentiated endometrial adenocarcinoma (Ishikawa line) were incubated with [3H]estradiol (E2) at various concentrations and the medium was sampled at 3, 6 and 24 h to evaluate the kinetics of removal of the hormone and the formation of unconjugated or sulfated metabolites. The detectable products of metabolism were estrone and the conjugate estradiol-3-sulfate. The latter was identified by high pressure chromatography, before and after acetylation, oxidation, and hydrolysis. The disappearance of [3H]E2 from the medium was found to follow first order kinetics between 3 and 24 h, with half-lives increasing from 4.7 to 53 h as the initial concentrations of the hormone were raised from 10(-8) to 10(-6)M. At the lowest concentration, practically all of the [3H]E2 added to the cultures was converted to estradiol-3-sulfate in 24 h, whereas at 10(-6)M oxidation to estrone was quantitatively more important than sulfation. These results indicate the presence in Ishikawa cells of an estrogen sulfotransferase of low Michaelis constant for E2, and 17 beta-oxidoreductase activity that significantly contributes to the metabolism of E2 only at higher concentrations of substrate.     

Induction and inhibition of estradiol hydroxylase activities in MCF-7 human breast cancer cells in culture

The effects of estradiol, progesterone, and tamoxifen on the activity of estradiol 2- and 16 alpha-hydroxylases were studied in human breast cancer cell cultures using a radiometric assay. After 5 days' exposure to these compounds, incubations in the presence of either [2-3H]estradiol or [16 alpha-3H]estradiol as substrate were carried out. In MCF-7 cells, estradiol (10(-8) M), progesterone (10(-6) M) and tamoxifen (10(-6) M) significantly increased 16 alpha-hydroxylase activity (estradiol; 21% progesterone 10% to 32%; tamoxifen 21% to 31%; P less than 0.01). Synergistic effects were observed when the cells were successively exposed to tamoxifen and progesterone. Simultaneous treatment with tamoxifen plus estradiol or estradiol plus progesterone showed no change from estradiol alone. On the other hand, although estradiol had no direct effects on 2-hydroxylase activity, tamoxifen decreased this enzymatic activity significantly at 10(-6) M (23% to 37%). Progesterone acted synergistically to further decrease this reaction. Treatment with only progesterone caused an increase in 2-hydroxylation. In contrast, a subline of MCF-7 cells with low estrogen receptor levels showed only minimal enzyme-hormone responses. Likewise, treatment of the estrogen receptor-negative MDA-MB-231 human breast cancer cell line with these compounds showed no effects on either 2- or 16 alpha-hydroxylase activity. In the progesterone receptor-rich T47D cell line, estradiol decreased both activities while progesterone increased both.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Human mammary cancer cell mutants with altered hormone receptor activity

We have recently isolated retinoic acid-resistant clones U-2 and U-3 from human breast cancer cell line MCF-7 (Ueda et al. (1985) Cancer Res. 45, 3332-3338). Growth of MCF-7 cells was found to be stimulated by estradiol but that of U-2 or U-3 was not. Cytosol from U-2 or U-3 cells contained no detectable estradiol receptor activity, whereas that from the parental MCF-7 cells showed estradiol receptor activity of 32 fmol/mg cytosol protein with a Kd of 2.6 X 10(-10) M by Scatchard analysis. Sucrose gradient centrifugation analysis of the cytosol fraction confirmed the presence of estradiol receptor activity in MCF-7 but not in U-2. Cytosol from MCF-7 and U-2 cells showed progesterone receptor activities of 106 fmol/mg protein with a Kd of 7.4 X 10(-10) M and 13 fmol/mg protein with a Kd of 9.9 X 10(-10) M, respectively. Addition of estradiol to the culture medium of the cells increased the level of progesterone receptor about 2-fold in MCF-7, but not in U-2. U-2 or U-3 cells showed about 5-fold higher resistance to an antiestrogen, tamoxifen, than MCF-7, and they were also 300- to 1,000-fold more resistant to other antiestrogens, epitiostanol and medroxyprogesterone, than MCF-7. The altered cellular sensitivity of U-2 or U-3 to the hormone antagonists is discussed in relation to the absence or presence of hormone receptors.     

Recruitment of uterine NK cells: induction of CXC chemokine ligands 10 and 11 in human endometrium by estradiol and progesterone

Uterine NK (uNK) cells express a unique set of markers compared with blood NK cells. However, recent studies suggest that uNK cells may be derived from the recruitment of blood NK cells into the endometrium. In this study, we used an in vitro organ culture system to demonstrate that estradiol induces expression of chemokines CXCL10 and/or CXCL11 within human endometrium in 85% of patient samples tested. The average increase in gene expression after 10(-9) M estradiol treatment was 8.5-fold for CXCL10 and 7.7-fold for CXCL11 compared with medium alone. We observed that a specific estrogen receptor antagonist (ICI182780) was able to prevent chemokine gene induction, indicating that the effect of estradiol was receptor mediated. Moreover, our study showed that progesterone induced CXCL10 and CXCL11 expression in 83% of endometrial samples tested. We have also found that uNK cells and blood NK cells express the receptor for CXCL10 and CXCL11, CXCR3, with the highest expression found on uNK cells and CD56(bright) blood NK cells. These data indicate that sex hormones induce specific chemokines in nonpregnant human endometrium that can activate NK cell migration, and suggest that this mechanism may account for the increased NK cell numbers in endometrium during the menstrual cycle.     

The Ishikawa cells from birth to the present

More than 20 years have passed since the Ishikawa cell line, a well-differentiated human endometrial adenocarcinoma cell line, was established. Because this cell line bears estrogen and progesterone receptors, the cells have been used in numerous basic research areas such as reproductive biology and molecular science, and has been distributed to more than a hundred institutes. However, even the Ishikawa cells, after long-term culture, tend to transform into undifferentiated cells. In addition, it has been reported that estrogen and progesterone receptors disappeared from the cells that I distributed. I therefore attempted to establish well-differentiated cells from the parent Ishikawa cells and to produce a new and good quality supply of this cell line. I believe that it is very important for the investigator who established a cell line to be responsible for maintaining the quality of the cells. That is why I have not deposited this cell line in any cell bank. I would like to take this opportunity to report the history of Ishikawa cells from establishment to the present.     

Progesterone induces a secretory protein in cultured rabbit endometrial stromal cells

Secretion of newly synthesized proteins by rabbit endometrial stromal cells in culture was studied. Progesterone (P) stimulated the synthesis and secretion of a protein with a mol. wt of approx 62 K and a pI of 6.5-7.0. Induction of 62 K protein synthesis was dose dependent; addition of 10 nM P to primary cultures caused a 2-fold or greater increase in the amount of this protein in the medium while addition of 1 microM P resulted in a 4.3-fold increase. Synthesis of this 62 K protein was not induced by estradiol, dexamethasone, or testosterone, nor was progesterone stimulation of the protein modified by estradiol. Thus, induction appears to be specific for a progesterone receptor mediated effect. Synthesis and secretion of this protein in the culture system described is potentially useful as a progestin bioassay.     

Effects of various steroids on platelet-derived endothelial cell growth factor (PD-ECGF) and its mRNA expression in uterine endometrial cancer cells

Progestins diminish the estrogen-induced angiogenic potential related to basic fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) in uterine endometrial cancer cells. This led us to study the effect of various steroids on the expression of platelet-derived endothelial cell growth factor (PD-ECGF) as the other pertinent angiogenic factor in well-differentiated uterine endometrial cancer cell line Ishikawa.In Ishikawa cells, estradiol induced the expression of PD-ECGF and its mRNA. The estrogen-induced expression was increased approximately two-fold by progesterone and by its metabolite, 17alpha-hydroxyprogesterone, but not by medroxyprogesterone acetate (MPA). Therefore, progesterone and 17alpha-hydroxyprogesterone as endogenous steroids might induce PD-ECGF-related angiogenic potential in uterine endometrial cancer cells, but not MPA as a synthetic steroid. In conclusion, the failure of PD-ECGF induction by MPA might be the great merit of anti-angiogenic treatment with MPA for uterine endometrial cancers.     

Inhibitory effects of the antiprogestin, RU 486, on progesterone actions and luteinizing hormone secretion in pituitary gonadotrophs

The effects of RU 486 on the modulation of LH release by progesterone were investigated in cultured anterior pituitary cells from ovariectomized adult female rats. The inhibitory effect of progesterone on LH secretion was demonstrable in estrogen-treated pituitary cells, in which addition of 10(-6) M progesterone to cells cultured in the presence of 10(-9) M estradiol for 52 h reduced the LH response to GnRH (10(-11) to 10(-7) M). When RU 486 was superimposed upon such combined treatment with estradiol and progesterone, the suppressive effect of progesterone on GnRH-induced LH release was completely abolished. The converse (facilitatory) effect of progesterone on LH secretion was observed in pituitary cells pretreated with 10(-9) M estradiol for 48 h and then with 10(-6) M progesterone for 4 h. When RU 486 was added together with progesterone during the 4 h treatment period, the facilitatory effect of progesterone was blocked and LH release fell to below the corresponding control value. The direct effect of RU 486 on LH secretion in the absence of exogenous progesterone was evaluated in cells cultured in the absence or presence of 10(-9) M estradiol and then treated for 4 to 24 h with increasing concentrations of RU 486 (10(-12) to 10(-5) M) and stimulated with GnRH (10(-9) M) during the last 3 h of incubation. In estrogen-deficient cultures, 4 h exposure to RU 486 concentrations of 10(-6) M and above decreased the LH response to GnRH by up to 50%. In cultures pretreated with 10(-9) M estradiol, GnRH-stimulated LH responses was inhibited by much lower RU 486 concentrations, of 10(-9) M and above. After 24 h of incubation the effects of RU 486 were similar in control and estradiol-pretreated pituitary cell cultures. Thus, RU 486 alone has a significant inhibitory effect on LH secretion that is enhanced in the presence of estrogen. The antiprogestin is also a potent antagonist of both the inhibitory and the facilitatory actions of progesterone upon pituitary gonadotropin release in vitro.     

Regulation of epidermal growth factor-receptor by estrogen and antiestrogen in the human breast cancer cell line MCF-7

Regulation of breast tumor proliferation depends in a large part on a variety of hormones and growth factors. In this report we show that estrogen and antiestrogen modulate epidermal growth factor-receptor (EGF-R) level in the human breast cancer MCF-7 cells with opposite mechanisms. Although a short-term treatment (24h to 48h) with estradiol leads to a decrease in EGF-R number, the addition of hormone in cell culture for 5 days increases EGF-R level with a maximal effect observed at 10(-10) M estradiol. In contrast, when cells are treated with the antiestrogen hydroxytamoxifen, a dose-dependent decrease in EGF-R level occurs. We also report that EGF is able to induce estrogen receptors and, to a lesser extent, progesterone receptors when added to MCF-7 cell cultures. These results demonstrate an interaction between both estrogen receptor and EGF receptor growth promoting systems in target cells. The implications of such an interaction in the understanding of human breast cancer hormone responsiveness and, in the development of therapies, are discussed.     

An estradiol-responsive mouse endometrial cell strain with inducible aryl hydrocarbon hydroxylase activity

A mouse endometrial cell population has been isolated by mild tryptic digestion of the uterine lining. The cells were morphologically similar to endometrial gland cells in the intact mouse endometrial gland. The endometrial cells had a modal chromosome number of 66. The cells were adherent to glass as well as plastic and contained numerous large refractile, osmophilic, non-membrane-limited granules which stain with periodic acid-Schiff reagent but do not stain with oil red O, Sudan black, or Alcian blue. Cell growth was responsive to 17β-estradiol; cell number increased 1.34-fold in 4 days in the presence of 10^?8 M estradiol. The cells are not tumorigenic. The cells showed induction of aryl hydrocarbon hydroxylase (AHH) activity when 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was added to the growth media for 24 h. AHH activity and its induction were investigated with cells grown in the presence and absence 10^?8 M estradiol. Cells grown in media containing estradiol exhibited a 6.2-fold induction by TCDD; cells grown without estradiol gave an 8.4-fold induction of AHH activity. AHH activity and its induction by TCDD were demonstrated in cells grown with fetal calf serum that had been pretreated with dextran-coated charcoal to remove endogenous steroids. Benzanthracene failed to induce AHH activity significantly.     

Estrogen response in the hFOB 1.19 human fetal osteoblastic cell line stably transfected with the human estrogen receptor gene

The gene coding for the human wild-type estrogen receptor (ER) was stably transfected into the human fetal osteoblastic cell line hFOB 1.19, a clonal cell line which is conditionally immortilized with a temperature sensitive mutant of SV40 large T antigen (tsA58). Five subclones were obtained which express various levels of ER mRNA and protein. The subclone with the highest level of functional (nuclear bound) ER, hFOB/ER9, contained 3,931 (±1,341) 17β-estradiol molecules bound/nucleus as determined by the nuclear binding (NB) assay. Using the dextran coated charcoal (DCC) method, the level of total cytosolic ER measured was 204 (±2) fmol/mg protein. This subclone was examined further for estradiol (E₂) responsiveness. The ER expressed in hFOB/ER9 cells was shown to be functional using a transiently transfected ERE-TK-luciferase construct. Expression of luciferase from this construct increased ～25-fold in hFOB/ER9 cells following 10^?9M E₂ treatment. This effect on ERE-TK-luciferase expression was both dose and steroid dependant. Further, treatment of hFOB/ER9 cells with 10^?9M E₂ resulted in a 2.5–4.0-fold increase in endogenous progesterone receptor (PR) levels detected by steroid binding assays, and a noticeable increase in both the A and B forms of PR by western blot assay. The establishment of this estrogen responsive human osteoblastic cell line should provide an excellent model system for the study of estrogen action on osteoblast function. © 1995 Wiley-Liss, Inc.