Human endometrial adenocarcinoma transplanted into nude mice: growth regulation by estradiol.

A model for studying the growth of primary tumors of human endometrium and its regulation by 17 beta-estradiol has been developed in which ovariectomized nude mice are used as recipients. The receptors for sex steroids are maintained during serial transplantation of the tumor in this system. Although the rate of growth of receptor-negative endometrial tumors transplanted into ovariectomized nude mice is unaffected by the sustained presence or absence of estradiol, the growth of receptor-positive tumors is significantly increased by estradiol. Receptor-positive tumors treated with estradiol produced elevated concentrations of progesterone receptor. That the progesterone receptor is functional in this tumor is evident from the induction of estradiol 17 beta-dehydrogenase activity upon progestin administration. These findings are consistent with receptor-mediated regulation of growth of endometrial carcinoma.

Photoaffinity labeling of the progesterone receptor from human endometrial carcinoma.

A nude mouse model for the growth of human endometrial carcinoma and hormonal modulation of the progesterone receptor (PR) was established previously. This study describes the effect of 17 beta-estradiol and tamoxifen (TAM) on growth rate and PR concentration in a hormonally responsive human endometrial tumor (EnCa 101) grown in this experimental system and presents the first characterization of human endometrial carcinoma PR. EnCa 101 was transplanted subcutaneously into ovariectomized, BALB/c, nu/nu athymic mice and grown under 17 beta-estradiol-stimulated, TAM-stimulated, and control conditions. Both 17 beta-estradiol and TAM increased the growth rate of EnCa 101 in nude mice, and a parallel increase in the cytosol PR concentration was observed, from 130 +/- 55 (SD) fmol/mg protein to 1,311 +/- 598 fmol/mg protein and 710 +/- 310 fmol/mg protein, respectively. PR was partially purified by phosphocellulose and DEAE cellulose chromatography, and the DEAE eluate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and photoaffinity labeling with [17 alpha-methyl-3H]promegestone ([3H]R5020). Two PR-negative tumors (EnCa K and EnCa V) were also examined in parallel. Coomassie blue staining of gels revealed that the protein patterns of all of the partially purified preparations from EnCa 101, EnCa K, and EnCa V were essentially identical. In contrast, photolabeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of EnCa 101 grown in the presence of 17 beta-estradiol or TAM revealed incorporation of [3H]R5020 into proteins of molecular weight approximately 116,000 and 85,000. Labeled proteins of molecular weight 66,000, 45,000, and 35,000 were also observed. In each case, the labeling was competable with excess non-radioactive R5020. No incorporation of [3H]R5020 was observed in EnCa 101 grown in the absence of estrogen, nor was any observed in EnCa K or EnCa V.

Extracellular matrix and the patterns of differentiation of human endometrial carcinomas in vitro and in vivo.

Adenocarcinomas differ in their ability to form glandular structures, and the mechanism regulating this architectural differentiation is unknown. In the present study, the patterns of differentiation of two human endometrial carcinomas that differed with respect to their ability to form glands in their original host were studied in monolayer and three-dimensional cultures as well as in xenografts in athymic mice. A moderately differentiated adenocarcinoma of human endometrium, EnCa101, transplanted into nude mice formed tumors indistinguishable from the original neoplasm and secreted mucin. A cell line derived from this tumor, ECC-1, formed monolayers on tissue culture substratum and lost the ability to secrete mucin. However, upon culture within Matrigel, the ECC-1 cells formed glandular structures and secreted mucin. Ultrastructural examination revealed morphological polarity, as evident by intraluminal microvilli and characteristic adhesion structures composed of tight, gap, and desmosomal junctions adjacent to the lumen, and secretory activity. Whereas basal lamina was observed in vivo around glandular cells, epithelial cells were not tethered in vitro with this structure. In contrast, the epithelial cells of a poorly differentiated human endometrial adenocarcinoma, AN3, failed to form glands in nude mice or in Matrigel in vitro. These findings illustrate that gland-forming ability is an intrinsic property of well to moderately differentiated adenocarcinoma cells and that only cells with this inherent potential can be induced to form glands in response to appropriate extracellular signals.

Failure of progestins to induce estradiol dehydrogenase activity in endometrial carcinoma, in vitro.

Estradiol dehydrogenase (E2DH) is a well-known progesterone-dependent enzyme in human endometrium, and its induction has been proposed as a means to test hormonal sensitivity of endometrial carcinoma. While administration of progestins to some patients with endometrial carcinoma resulted in increased endometrial E2DH activity, efforts to induce this enzyme, in vitro, in these tumors have been unsuccessful. The reasons for such failure were investigated in the present study. Progesterone receptor (PR) concentrations and E2DH activities were simultaneously measured in proliferative and malignant endometria under organ culture conditions. Cytoplasmic PR concentrations were determined by Scatchard plot analysis of [3H]progesterone binding in fresh samples and in tissue explants incubated in nutrient medium at 37 degrees in a humidified 5% CO2 atmosphere for various periods of time. Parallel incubations of explants with and without 500 ng medroxyprogesterone acetate per ml were carried out for monitoring E2DH induction. In proliferative endometrium, the progesterone-specific binding sites remained stable during the culture periods, and the E2DH activities were stimulated severalfold by medroxyprogesterone acetate. In contrast, the PR concentrations in carcinoma explants were undetectable after a 24-hr period, and this was associated with a lack of increase in E2DH activity. These findings provide evidence that progestin-induced endometrial E2DH activity is a receptor-mediated phenomenon. In addition, these results demonstrate clearly that the ineffectiveness of progestin to induce E2DH in endometrial cancer specimens, in vitro, is related to the instability of PR under culture conditions. It is suggested that any experiment designed to follow effects of steroids on target tissues must take into account the stability of steroid receptors under in vitro conditions.

Progesterone receptor structure and protease activity in primary human endometrial carcinoma.

Monoclonal antibodies were used to investigate progesterone receptor structure (isoforms) in 33 primary human endometrial tumors. The monoclonal antibodies recognized on protein blots two progesterone receptor proteins with molecular weights of 116,000 and 81,000. The Mr 116,000 protein appeared as a triplet, while a single band was found for the Mr 81,000 protein. The triplet/singlet structure was found in all progesterone receptor-positive tumors, regardless of the degree of tumor differentiation. Protease activity, which gave rise to a false-negative pattern on protein blots, was found in approximately one-half of the tumors in which it was investigated. Inclusion of a cocktail of protease inhibitors during sample preparation resulted in the maintenance of the triplet/singlet progesterone receptor structure. Mixing experiments using a progesterone receptor-rich human endometrial carcinoma (EnCa 101), which lacks protease activity, and protease-containing primary tumor homogenates indicated that the protease was leupeptin sensitive. Interestingly, while the proteolytic activity reduced or eliminated the triplet/singlet progesterone receptor structure seen on protein blot analysis, it did not affect progesterone receptor concentration measured by Scatchard analysis. Sample preparation in the presence of protease inhibitors is therefore a requisite for structural analysis of the progesterone receptor in endometrial tumors.

Hormonal therapy of human endometrial adenocarcinoma in a nude mouse model.

The hypothesis that 17 beta-estradiol or tamoxifen (TAM) can potentiate clinical response of endometrial cancer treated with progestin was tested in an ovariectomized nude mouse system, using a sex steroid receptor-positive and a receptor-negative human endometrial carcinoma. Animals were divided into three groups: control; 17 beta-estradiol-treated; and TAM-treated. When tumors of a group reached about 1 cm in diameter, subgroups were given either 0.9% NaCl solution (saline) or medroxyprogesterone acetate (MPA). The receptor-negative tumor grew rapidly in all three groups, and several animals were dead before or during progestin treatment. The growth rate of receptor-containing carcinoma was significantly increased in TAM-treated mice compared to controls (p less than 0.02) but significantly less than that in 17 beta-estradiol-treated animals (p less than 0.01). Endometrial carcinoma in 17 beta-estradiol-saline-treated animals continued to grow rapidly, and all animals were dead by 11 weeks. The growth of tumors in the 17 beta-estradiol-progestin group was suppressed at 11 weeks, and some of these animals lived 20 weeks. Administration of progestin to TAM-exposed animals resulted in a remarkable regression of the tumor compared to TAM-saline-treated group. The growth rate of tumors in control animals (no implants) was unaffected by progestin treatment. We conclude that, in this nude mouse model, treatment with TAM and MPA is superior to MPA alone, or 17 beta-estradiol plus MPA for sex steroid receptor-positive endometrial carcinoma.

Estrogen-like effects of tamoxifen on human endometrial carcinoma transplanted into nude mice.

The effect of tamoxifen (TAM) on human endometrial carcinoma was investigated in nude mice bearing an estrogen receptor-positive or estrogen receptor-negative tumor. The receptor-negative tumor grew rapidly, and the rates of tumor growth of 17 beta-estradiol or TAM-treated animals were identical to the rate of controls. The estradiol receptor and progesterone receptor (PR) concentrations in the tumor cytosol remained undetectable under all experimental conditions. In contrast, the rate of growth of steroid receptor positive tumor was significantly accelerated in the presence of TAM compared to controls (p less than 0.02). The increased tumor growth rate was, however, significantly lower (p less than 0.01) than that observed in animals receiving 17 beta-estradiol. The PR concentration in these tumors was elevated in response to TAM treatment. That the TAM-induced PR was indeed functional was evident from (a) increased activities of the progestin-sensitive enzyme, 17 beta-estradiol hydroxysteroid dehydrogenase and (b) histological appearance of subnuclear vacuolization in these tumors after progestin administration. These studies indicate that continuous, short-term administration of TAM to nude mice results in an estrogen-like effect on endometrial carcinoma. Based on the finding that TAM induces functional PR, we predict that steroid receptor-positive endometrial carcinoma may show a greater response rate to combined, long-term treatment with TAM and progestin.

Heterogeneity of progesterone receptor distribution in human endometrial adenocarcinoma.

The clinical response of advanced endometrial adenocarcinoma to progestin therapy does not correlate perfectly with biochemically assayed progesterone receptor status of the tumor. We have previously suggested that heterogeneity of progesterone receptor at the cellular, tumor, and tissue levels, not detectable by the biochemical assay, might contribute to this discrepancy. A monoclonal antibody, hPRa-1, generated against human progesterone receptor, was used in the present study to immunohistologically define the heterogeneity of progesterone receptor distribution in primary endometrial carcinomas. Twenty-four hysterectomy specimens removed for the treatment of endometrial adenocarcinoma were examined by biochemical assay of progesterone receptor and immunohistochemistry. In two cases, in which tumor occupied almost all of the endometrial lining, more extensive sampling was performed with removal of four noncontiguous sites. Each site was subdivided for immunohistochemistry and biochemical assay of progesterone receptor. When present, progesterone receptor localization was confined to the nuclei of target cells. Variability in the distribution and intensity of staining was consistently observed within the tumors. Of 24 tumors 15 were determined to be progesterone receptor positive by biochemical assay, while 12 of 24 tumors displayed immunolocalization for progesterone receptor. The correlation of the results by the two methods was high (20 of 24 cases, 83%), and the discrepancies in three cases appeared to reflect tissue and tumor heterogeneity. Immunolocalization has demonstrated that heterogeneity is present at the tumor, tissue, and cellular level within endometrial carcinomas, and the failure of some progesterone receptor-positive (by biochemical assay) tumors to respond to progestin therapy may reflect false positive results due to contamination of progesterone receptor-negative tumors by adjacent benign endometrium or myometrium.

Contrasting actions of tamoxifen on endometrial and breast tumor growth in the athymic mouse.

The effects of the antiestrogen tamoxifen (TAM) on the growth of two hormone-sensitive human tumors have been examined in athymic mice. The endometrial tumor, EnCa101, was stimulated to grow by TAM either alone or when combined with estradiol. This contrasted with the nonstimulation of the breast tumor, MCF-7, by TAM alone and the antagonist action of TAM on estradiol-stimulated growth of MCF-7 tumors. The individual tumor responses were observed even when the two tumor types were implanted on opposite sides of the same animal. This suggests that host metabolism of TAM does not dictate tissue response. The conclusion is supported by the finding of very similar patterns of metabolites in the two tumors after administration of [ring-3H]TAM. Tissue metabolism therefore is unlikely to be involved. Progesterone receptor levels were higher in estradiol (376 +/- 35 fmol/mg cytosol protein)- or TAM (317 +/- 37 fmol/mg cytosol protein)-stimulated EnCa101 tumors than control (42 +/- 5 fmol/mg cytosol protein) and increased further with combined treatment (485 +/- 75 fmol/mg cytosol protein). Estrogen receptor levels, however, were lower in estradiol (45 +/- 11 fmol/mg cytosol protein)-treated tumors than control (92 +/- 13 fmol/mg cytosol protein) but higher than control in TAM (200 +/- 15 fmol/mg cytosol protein)-treated tumors. Tumors grown with estradiol and TAM had lower estrogen receptor levels (130 +/- 7 fmol/mg cytosol protein) than tumors grown with TAM alone. Estrogen receptor levels indicate that TAM may not be acting exactly as estradiol in the EnCa101 tumor. Overall, these findings suggest that the disparate pharmacology of TAM is a tissue-specific phenomenon.

Effect of steroidal and nonsteroidal antiestrogens on the growth of a tamoxifen-stimulated human endometrial carcinoma (EnCa101) in athymic mice.

Tamoxifen (TAM), a nonsteroidal antiestrogen, is used in the adjuvant treatment of breast cancer. Previous studies, however, have indicated that some human breast and endometrial tumors are stimulated to grow with TAM in the athymic mouse. One such TAM-stimulated tumor is the EnCa101 human endometrial adenocarcinoma. Our aim was to evaluate the ability of different doses of TAM or other nonsteroidal antiestrogens to stimulate the growth of EnCa101 tumors in athymic mice. Additionally we have evaluated less estrogenic antiestrogens (two steroidal antiestrogens, RU 39,411 and ICI 164,384, and two nonsteroidal antiestrogens, keoxifene and MER-25) for their ability to inhibit TAM-stimulated growth. All experiments were done in ovariectomized athymic mice transplanted in the axillary mammary fat with 1-mm3 pieces of EnCa101 tumor. Sustained release preparations (0.5-2.0-cm Silastic capsule or 5-mg TAM cholesterol pellet) of TAM caused similar tumor growth. The growth rate was not altered by an additional daily i.p. injection of 1 mg TAM in 0.1 ml peanut oil. A 3-mg TAM daily dose was toxic. Four weeks of treatment (100-micrograms s.c. injections, every other day) with nonsteroidal antiestrogens, trioxifene mesylate, enclomiphene, or nafoxidine stimulated tumor growth. However, keoxifene stimulated this tumor to a lesser degree than TAM and partially inhibited TAM-stimulated growth. ICI 164,384 showed no stimulatory activity (1-mg s.c. injections every other day) alone compared to controls but inhibited TAM-stimulated (0.25-cm Silastic capsule) growth. In a parallel experiment, RU 39,411 (1-mg s.c. injections every other day) stimulated EnCa101 to grow. In contrast when RU 39,411 was administered in a sustained release preparation (2.0-cm Silastic capsule) there was no stimulatory growth compared to controls. Additionally RU 39,411 inhibited TAM-stimulated growth, but the low-potency antiestrogen, MER-25, was less effective in this regard. These data suggest that less "estrogenic" antiestrogens can inhibit TAM-stimulated tumor growth in vivo. Thus these compounds or derivatives may prove useful as a second-line endocrine therapy should TAM-stimulated tumor growth occur in the clinic.

A new antiestrogen, 2-(4-hydroxy-phenyl)-3-methyl-1-[4-(2-piperidin-1-yl-ethoxy)-benzyl]-1H-indol-5-ol hydrochloride (ERA-923), inhibits the growth of tamoxifen-sensitive and -resistant tumors and is devoid of uterotropic effects in mice and rats.

PURPOSE: Tamoxifen is an antiestrogen used in women who have estrogen receptor (ER)-alpha-positive breast cancer. Unfortunately, resistance to tamoxifen is common in women with metastatic disease and side effects, including increased risk of endometrial cancer, exist. Here we describe the activity of a new selective ER modulator, ERA-923, in preclinical models focused on these limitations. EXPERIMENTAL DESIGN: The ability of ERA-923, 4-OH tamoxifen, or raloxifene to inhibit estrogen-stimulated growth was evaluated in cell-based and xenograft assays with tumor cells that are sensitive or resistant to tamoxifen. Uterine effects of selective ER modulators were compared in rodents. RESULTS: ERA-923 potently inhibits estrogen binding to ER-alpha (IC(50), 14 nM). In ER-alpha-positive human MCF-7 breast carcinoma cells, ERA-923 inhibits estrogen-stimulated growth (IC(50), 0.2 nM) associated with cytostasis. In vitro, a MCF-7 variant with inherent resistance to tamoxifen (10-fold) or 4-OH tamoxifen (>1000-fold) retains complete sensitivity to ERA-923. Partial sensitivity to ERA-923 exists in MCF-7 variants that have acquired profound tamoxifen resistance. In tumor-bearing animals, ERA-923 (10 mg/kg/day given p.o.) inhibits 17beta-estradiol-stimulated growth in human tumors derived from MCF-7, EnCa-101 endometrial, or BG-1 ovarian carcinoma cells, including a MCF-7-variant that is inherently resistant to tamoxifen. Raloxifene is inactive in the MCF-7 xenograft model. Unlike tamoxifen, droloxifene, or raloxifene, ERA-923 is not uterotropic in immature rats or ovariectomized mice. Consistent with this, tamoxifen, but not ERA-923, stimulates the growth of EnCa-101 tumors. CONCLUSIONS: In preclinical models, ERA-923 has an improved efficacy and safety compared with tamoxifen. Clinical trials with ERA-923 are in progress.

A direct effect of medroxyprogesterone acetate on 17 beta-hydroxysteroid dehydrogenase in adult rat testis.

Sixty-day-old rats were divided into four groups and treated for 30 days with either medroxyprogesterone acetate (Provera), gonadotropins (bovine LH and ovine FSH), Provera plus gonadotropins, or saline. The progestin treatment resulted in a lowering of plasma levels of testosterone, androstenedione, and LH, as well as in a reduction of epididymal sperm counts and accessory sex organ weights. The progestin-treated groups showed markedly lower levels of testicular 17 beta-hydroxysteroid dehydrogenase activity (35% of controls) and delta 5,3 beta-hydroxysteroid dehydrogenase activity (70% of controls). Rats treated with only gonadotropins exhibited reduced 17 beta-hydroxysteroid dehydrogenase but increased delta 5,3 beta-hydroxysteroid dehydrogenase activities. It was concluded from these results that progestins may affect testicular steroidogenesis and spermatogenesis not only by reducing LH secretion but also by a direct effect on the testis, as LH suppression could not account for the inhibition of 17 beta-hydroxysteroid dehydrogenase activity. Long term progestin treatment did not alter the steroidogenic response of the testis to acute administration of LH, although the testosterone to androstenedione ratio in plasma was decreased.

Distribution of progesterone receptor, estradiol dehydrogenase, and 20 alpha-dihydroprogesterone dehydrogenase activities in human endometrial glands and stroma: progestin induction of steroid dehydrogenase activities in vitro is restricted to the glandular epithelium.

Endometrial glands were separated from stromal cells by collagenase digestion of human endometrium, and the distribution of progesterone (P) receptor and the activities of P-regulated enzymes, 17 beta-estradiol dehydrogenase (E2DH) and 20 alpha-dihydroprogesterone dehydrogenase (20 alpha DH), were determined in these preparations. Concentrations of cytosolic P receptor were estimated by Scatchard plot analysis of specific [3H]P binding in intact proliferative endometrium and in glands and stromal cells isolated from this tissue. Epithelial cells were more than 10-fold enriched in high affinity, P-specific binding sites compared to stromal cells. The binding constants (Kd) for [3H]P binding were essentially similar in undissociated endometrium, glandular epithelium, and stroma, ranging from 1--5 nM. The activities of E2DH and 20 alpha DH were also 3-fold higher in the glandular epithelium than in whole tissue or stroma during both proliferative and secretory stages of the menstrual cycle. In addition, the induction of these enzyme activities by progestin in vitro in cultured explants of proliferative endometrium was restricted to the glandular epithelium. Thus, the effects of P on E2DH and 20 alpha DH are expressed in the same cells that contain receptors for P.

Progesterone receptor regulation by 17 beta-estradiol in human endometrial carcinoma grown in nude mice.

Regulation of progesterone receptor (PR) in human endometrial carcinoma was investigated in vivo in a multisite nude mouse tumor experimental system by estrogen administration and withdrawal. The cytosolic PR concentration was low in tumors grown in the absence of 17 beta-estradiol, but increased rapidly upon estrogen administration, reaching a maximal receptor concentration of 1.4-1.6 pmol/mg cytosol protein within 7 days. Protein blot analysis using a monoclonal antibody (hPRa 1) raised against PR from EnCa 101 showed no immunoreactivity in tumors grown in the absence of estrogen. Immunoreactive proteins of mol wt 116,000 and 81,000 were detectable 8 h after estrogen administration and increased in intensity as the cytosolic PR concentration increased. Interestingly, the protein of mol wt 116,000 was composed of mol wt isoforms and was detectable as a doublet 8 h after estrogen administration and finally as a triplet. The effect of estrogen withdrawal on EnCa 101 PR concentration and structure was determined by removal of 17 beta-estradiol pellets (200 pg/ml plasma) from EnCa 101-bearing animals after achievement of maximal tumor PR concentrations. The PR concentration in tumor cytosols decreased in a biphasic manner after estrogen removal, with the initial rapid phase having a half-life of around 2 days. Cytosolic PR was still detectable 21 days after estrogen withdrawal. Protein blot analysis showed that immunoreactive proteins of mol wt 116,000 and 81,000 were also detectable up to that time. Photoaffinity labeling with [3H]R5020 demonstrated that the 81,000 mol wt protein, as well as each of the triplet proteins at mol wt 116,000, was specifically photoaffinity labeled. The 116,000-mol wt protein was detected as a triplet on protein blots until 13 days after estrogen withdrawal, when diminution in the intensity of the highest mol wt triplet protein was noted.

Progestin-mediated changes in progesterone receptor forms in the normal human endometrium.

Monoclonal antibodies to the human progesterone receptor (PR) were used to detect changes in PR form during the menstrual cycle. In proliferative phase samples, two PR proteins with mol wt (Mr) of 116,000 and 81,000 were detected on protein blots probed with anti-PR monoclonal antibodies. The 116,000 Mr protein was comprised of triplet isoforms, while the 81,000 Mr protein was a singlet. By contrast, protein blot analysis of secretory phase samples revealed a doublet isoform at 116,000 Mr and an 81,000 Mr singlet. Organ culture of human endometrium was used to mimic these changes in PR form in vitro. Although the triplet to doublet conversion was not realized in organ culture, time- and dose-related changes in PR form were achieved which were similar to the in vivo state. Furthermore, these changes preceded the progestin-mediated induction of the enzyme estradiol dehydrogenase in parallel cultures, demonstrating that this is a useful system in which to study the relationship between receptor modulation and initiation and maintenance of biological response.

Differential distribution of estrogen and progesterone receptors in rabbit uterus detected by dual immunofluorescence.

The action of sex steroids on the growth and differentiation of target tissues requires the presence of specific intracellular receptors. We compared the distribution of cells containing estrogen receptor (ER) and/or progesterone receptor (PR) in rabbit uterus by immunohistochemistry using monoclonal antibodies directed against these receptors. Initial experiments using serial cryostat sections surprisingly revealed the intensity of staining for ER to be inversely proportional to that of PR, as follows: ER, luminal and glandular epithelium greater than myometrium greater than stroma; PR, stroma greater than myometrium greater than glands greater than luminal epithelium. Localization was strictly confined to the nuclei of target cells. Single and dual immunofluorescent labeling of ER and PR in cryostat sections was accomplished using fluorochromes with differing emission spectra. Individual fields of dual labeled sections were examined for red [phycoerythrin (ER)] and green [fluorescein (PR)] fluorescence, with the same distribution as noted by single antibody immunohistochemistry. Myometrial nuclei displayed fluorescence of equivalent relative intensity for both antibodies. Further, sequential exposure photomicrography (exposure first in the spectrum of phycoerythrin emission, followed by exposure in the spectrum of fluorescein emission) revealed the presence of occasional stromal cells staining only for PR and some luminal cells staining only for ER. This differential distribution of ER and PR within various cell populations of rabbit is a novel observation and challenges current concepts of receptor regulation. Dual immunofluorescent localization of both ER and PR within individual cells provides a unique perspective from which to investigate the interactive influences of these sex steroid receptors at the cellular level.

Monoclonal antibodies to human progesterone receptor: characterization by biochemical and immunohistochemical techniques.

Progesterone receptor (PR) from a human endometrial carcinoma (EnCa 101) grown in nude mice consists of two hormone-binding proteins with mol wt around 116,000 and 85,000. To generate monoclonal antibodies against this receptor, PR was partially purified from EnCa 101 and used to immunize Robertsonian mice. Immune mouse spleens were fused with HL-1 Friendly myeloma-653 cells, and hybridomas were screened by solid phase dot-blot assay and double antibody precipitation. Seven stable hybridomas were obtained, designated hPRa 1-7. Subisotyping revealed that hPRa 1 and 6 were immunoglobulin G2b, while the remainder were immunoglobulin G1. Ultracentrifugation in high salt sucrose gradients showed that six of the seven antibodies effected a shift of [3H]progestin-labeled PR from EnCa 101; only hPRa 4 was ineffective in this regard. Protein blots of EnCa 101 cytosols and DEAE eluates revealed that hPRa 1, 3, 4, 5, and 7 recognized both PR proteins equally. hPRa 2 recognized principally the 116,000 mol wt PR protein; it recognized the lower mol wt PR protein very poorly if at all, whereas hPRa 6 recognized only the 116,000 mol wt protein. Interestingly, the latter was consistently detected as a closely migrating triplet. Immunolocalization of PR by hPRa 1-7 in tissue sections was confined to nuclei of target tissues and varied in intensity: hPRa 7 greater than 3 = 5 greater than 6 = 2 greater than 1 greater than 4. In proliferative phase uterus, the intensity of staining was ranked: endometrial gland nuclei (3+) greater than myometrial cell nuclei (2-3+) greater than endometrial stromal cell nuclei (0-1+). Thus, seven monoclonal antibodies directed against human PR have been prepared, and their suitability for the study of PR by biochemical and immunohistochemical techniques has been demonstrated.

Interleukin-1 (IL-1) regulation of human endometrial function: presence of IL-1 receptor correlates with IL-1-stimulated prostaglandin E2 production.

Previous studies suggest that prostaglandin E2 (PGE2) is important in normal endometrial function and that it may be involved in certain uterine dysfunctions. In this study, the ability of the purified E. coli-derived recombinant interleukin-1 alpha (rIL-1 alpha) to regulate the production of PGE2 by the endometrial epithelium is investigated. PGE2 levels determined by the RIA consistently increase upon incubation of cultured glandular epithelial cells with rIL-1 alpha. A significant increase is obtained with 17 and 170 ng/L rIL-1 alpha. A maximal effect is obtained within 24 h of incubation with rIL-1 alpha. In the seven endometria evaluated, rIL-1 alpha increases PGE2 synthesis in all cases, but the maximal increase relative to the basal levels varies between 2- to 10-fold for a given preparation. Vibratome sections retaining the integrity of the endometrial tissue also show an increased PGE2 synthesis in response to rIL-1 alpha. rIL-1 alpha-stimulated PGE2 production is blocked by the addition of a neutralizing antibody to rIL-1 alpha. In addition, indomethacin, a cyclooxygenase inhibitor, suppresses both rIL-1 alpha-induced and basal PGE2 synthesis. Experiments using radioiodinated rIL-1 alpha reveal that the membranes prepared from human endometrial epithelium possess high affinity receptors for IL-1, suggesting that IL-1 regulates PGE2 synthesis by binding to this receptor site. The expression of IL-1 receptors and the ability to modulate PGE2 production by IL-1 in endometrial epithelium suggest that IL-1 may play a significant role in human uterine function via modulation of PGE2 production.

Antiproliferative effect of interferon-gamma in human endometrial epithelial cells in vitro: potential local growth modulatory role in endometrium.

We previously reported that recombinant interferon-gamma (IFN-gamma) induces HLA-DR (human lymphocyte antigen) molecules of the major histocompatibility complex in human endometrial epithelial cells in vitro. We now report that IFN-gamma inhibits the proliferation of human endometrial epithelial cells and a human endometrial carcinoma cell line (EnCa101AE). Human endometrial epithelial cells expressed HLA-DR molecules and underwent morphological changes when exposed to IFN. Furthermore, the proliferation of these cells, as evidenced by nuclear labeling of bromodeoxyuridine (an analog of thymidine that is incorporated into cells in S phase), was markedly reduced, in a dose-dependent manner, by IFN-gamma. IFN-gamma induced HLA-DR expression, morphological changes, shedding from the substratum, and cell death in EnCa101AE cells. In addition, cell number and the numbers of bromodeoxyuridine-, Ki-67 (a nuclear marker of proliferation)-, and MPM-2 (a marker of mitotic cells)-positive cells were markedly lower in the EnCa101AE cultures treated with IFN-gamma than those in control cultures. The cytostatic and HLA-DR-inducing effects of IFN-gamma could be abrogated by neutralization with a polyclonal antibody, and IFN-gamma effects were reversible within days after its withdrawal. These findings indicate that IFN-gamma inhibits proliferation of human endometrial epithelial cells and suggest that this factor may locally regulate the proliferation of these epithelial cells in vivo.

Modulation of HLA-DR expression in epithelial cells by interleukin 1 and estradiol-17 beta.

Both ultrapure human interleukin-1 (IL-1) and Escherichia coli derived recombinant IL-1 alpha and beta consistently induced the expression of major histocompatibility class II (HLA-DR) molecules in a human endometrial and a breast carcinoma cell line. [35S]Methionine incorporation into IL-1 induced, immunoprecipitable HLA-DR molecules demonstrated de novo synthesis of both light and heavy chains of the HLA-DR molecules. Lipopolysaccharide, recombinant interleukin-2 and recombinant interleukin-6 failed to induce HLA-DR expression in these epithelial cells. In contrast to the dramatic effect on HLA-DR expression, IL-1 had no effect on the epithelial cell proliferation. Pretreatment of T47D cells with estradiol-17 beta significantly decreased the IL-1 induced HLA-DR expression, and pretreatment of IL-1 with an IL-1 specific antibody, neutralized IL-1 action. These studies demonstrate that a cytokine (IL-1) and a sex steroid hormone estradiol-17 beta can interact to regulate the expression of HLA-DR molecules in epithelial cells.