Modulation of uterine morphology and growth by estradiol-17beta and an estrogen antagonist.

The estrogen antagonist C1628 maintains sustained hypertrophy of the uterine epithelium and the synthesis of many proteins including peroxidase. C1628 is a progestogen, inducing secretion of the protein by surface epithelial and glandular cells. C1628 is a connective tissue mitogen, inducing DNA synthesis in fibroblasts and the endothelium. C1628 and estrogen share these properties mentioned above. Estrogen, however, induced moderate growth of the mucosa within a 24-h period and massive hyperplasia of the mucosa within a 24-h period thereafter. C1628 alone, or in combination with estradiol, does not have mitogenic effect on the mucosa, and in fact blocks the mitotic response normally induced by estrogen alone.

Endogenous peroxidase: specific marker enzyme for tissues displaying growth dependency on estrogen.

Data derived from a correlated morphological and biochemical study suggest the following: (a) estradiol-17beta, diethylstilbestrol, the estrogen antagonists nafoxidine (Upjohn 11,000), and Parke Davis C1628 induce synthesis of an endogenous peroxidase in the epithelium of target tissues like the vagina, the cervix, the uterus, and in the acinar cells of the estrogen-dependent rat mammary tumor; (b) peroxidase is a "specific" secretory protein of the estrogen-sensitized uterine endometrium; (c) peroxidase synthesis is not a nonspecific response to steroid hormone action, since progesterone and testosterone do not induce its synthesis; (d) endogenous peroxidase is a possible diagnositc protein for the detection of estrogen-dependent growing tissues, including breast cancer; (e) movement of exogenous horseradish peroxidase from the interstitium to the uterine lumina is restricted by tight junctions located at the apices of epithelial cells. Estrogen and antagonists do not appear to influence the transepithelial movement of exogenous peroxidase into the lumen.

Estrogen and antagonist-induced changes in endometrial topography of immature and cycling rats.

The topographical changes of the luminal surface of the endometrium of immature and ovariectomized rats treated with estrogen, antagonists to estrogen, and progesterone. and during various stages of the estrous cycle and in pregnancy were examined by scanning electron microscopy. Massive increases in numbers and length of endometrial cell microvilli were observed at estrus, after injection of estradiol-17beta, diethylstilbestrol, estrogen plus progesterone. or the inhibitor C1628 to immature and ovariectomized rats. Withdrawal of the estrogen stimulus results in diminution of microvilli, producing a state identical to diestrus, during pregnancy, and after injection of progesterone, The estrogen antagonist appears to have both estrogenic and progestogenic properties, stimulating endometrial cell hypertrophy, secretion of protein, and production of numerous apical microvilli.

Estrogen-receptor interaction.

The interaction of estradiol with uterine cells involves the association of the hormone with an extranuclear receptor protein, followed by temperature dependent translocation of the resulting complex to the nucleus. During this process, the steroid binding unit of the protein undergoes an alteration, called "receptor transformation," that can be recognized by an increase in its sedimentation rate from 3.8S to 5.2S, and by its acquisition of the ability to bind to isolated uterine nuclei and to alleviate a tissue specific deficiency in the RNA synthesizing capacity of such nuclei. Receptor transformation can be effected in the absence of nuclei by warming uterine cytosol with estradiol. This preparation of transformed complex resembles that extracted from nuclei both in its sedimentation rate (5.3S) and in its ability to bind to uterine nuclei and augment RNA synthesis, properties that are not shown by the native complex. It is proposed that receptor transformation is an important step in estrogen action and that a principal role of the hormone is to induce conversion of the receptor protein to a biochemically functional form.

Sulfhydryl groups and estradiol-receptor interaction.

The characteristic ability of rat uteri to take up tritiated estradiol in vitro or to retain estradiol previously incorporated either in vivo or in vitro is destroyed by treating the tissue with various sulfhydryl-blocking reagents. The two radioactive estradiol-receptor complexes, observed in uterine homogenates in the supernatant fraction and in an extract of the nuclear fraction, respectively, are disrupted by brief exposure to organic mercurials in the cold. Sulfhydryl groups of uterine receptor substances apparently play a vital role in estradiol binding, perhaps indirectly through contribution to receptor conformation.

Endocrine-dependent rat mammary tumor regression: use of a gonadotropin releasing hormone analog.

Long-term administration of [D-Leu6, des-Gly-NH210, Pro-ethylamide9]-GnRH, an analog of gonadotropin releasing hormone, caused regression of neoplastic tissue in a rat bearing a spontaneous mammary adenocarcinoma and in rats in which tumors had been induced by treatment with dimethylbenzanthracene (DMBA). During two separate treatment periods with the analog, the tumor in the single animal regressed although it had previously grown spontaneously. After a third period of growth, ovariectomy also induced regression, suggesting endocrine dependency of the tumor. These observations were confirmed in the DMBA-induced tumor system, where tumor regression in the analog-treated rats was comparable to that observed in the ovariectomized rats, and in both cases the tumor regression was significant when compared to untreated controls.

Estrogen-dependent in vitro stimulation of RNA synthesis in hormone-dependent mammary tumors of the rat.

Incubation of estradiol in vitro at 25 degrees C with homogenates of carcinogen-induced mammary tumors of ovariectomized rats stimulated the magnesium-dependent RNA polymerase activity of nuclei of the hormone-dependent (HD) (regressing) tumors, but had no effect on this activity in nuclei of hormone-independent (HI) (growing) tumors. Furthermore, recombination of the nuclei and cytosol fractions of HD and HI tumors indicated that the in vitro effect of estradiol on subsequent tumor nuclear RNA synthesis required the estrogen receptor-containing cytosol but was specific to nuclei of the HD tumor. This constituted the first direct in vitro effect of estrogen on a specific biochemical process in an HD mammary tumor.

Peroxidase activity and iodide uptake in hormone-responsive and hormone-independent GR mouse mammary tumors.

Transplanted mammary tumors growing in the inbred GR/AFib mouse were assayed for peroxidase activity and ability to concentrate injected 125I. Both tumor peroxidase activity and iodide uptake were about ten times greater in the hormone-resonsive (HR) tumors than in the hormone-independent tumors. However, although peroxidases are known for their ability to participate in the iodination of proteins, over 90% of the radioactive iodine found in the tumors was shown to be free iodide. This finding suggests that these two parameters may be independent of each other, but both are higher in HR tumors.

Isolation and purification of rat mammary tumor peroxidase.

7,12-Dimethylbenz(a)anthracene-induced rat mammary tumors often contain high levels of the enzyme perioxidase, a putative marker of estrogen dependence. This enzyme can be effectively extracted with 0.5 M CaCl2, giving rise to a soluble peroxidase with a molecular weight of about 50,000 as determined by gel filtration. This is the same size as the estrogen-induced peroxidase of rat uterus but smaller than other mammalian peroxidases. Further purification of the rat mammary tumor peroxidase by concanavalin A-Sepharose chromatography and hydrophobic interaction chromatography on phenyl Sepharose provides a 640-fold purification of the enzyme.

Regression of rat mammary tumors effected by a gonadoliberin analog.

A synthetic analog of gonadoliberin (gonadtropin-releasing factor of luteinizing hormone/follicle-stimulating hormone-releasing hormone), designated A-43818, was evaluated for its ability to effect regression of carcinogen-induced mammary tumors in the Sprague-Dawley rat. This analog, specifically (D-leuyl6, desglycyl-NH210, prolyl ethylamide9), gonadoliberin, is a potent synthetic luteinizing hormone/follicle-stimulating hormone-releasing hormone at low dose levels but, at the higher dose levels used in these studies, the effect appears to be that of a potent gonadoliberin antagonist. Administration of 5 or 20 mug of A-43818 per day to tumor-bearing rats was essentially as effective as ovariectomy in causing mammary tumor regression. At least 80% of the tumors in the A-43818-treated animals underwent regression; about one-half of the regressing tumors disappeared in the 6-week period of continuous treatment, and, unlike the 0.9% NaCl solution control group, no new tumors appeared during the treatment period. A subsequent 4-week period of drug withdrawal resulted in the regrowth of palpable tumors and the appearance of new tumors, most of which again regressed on further A-43818 administration.

Identification, subcellular localization, and estrogen regulation of peroxidase in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors.

An estrogen-induced, intensely staining peroxidase 3,3-diaminobenzidine-positive reaction product is found to be characteristic of hormone-dependent, 7,12-dimenthylbenz(a)anthracene-induced mammary tumors of the rat. This product is demonstrated in thick sections of such tumors from intact or estrogen-treated castrate rats but is not seen in tumors that are in regression due to castration or estrogen deprivation. It is, furthermore, absent from tumors whose growth is unaffected by castration. The subcellular localization of this enzyme activity is restricted mainly to the nuclear envelope and cisternae of the granular endoplasmic reticulum in addition to secretory granules. This provides the first evidence for a criterion that would allow differentiation of hormone-dependent and hormone-independent mammary cancer on histological sections and, as such, may have considerable potential as an aid in the classification of human breast cancer.

Estrogen and prolactin receptor concentrations in rat mammary tumors and response to endocrine ablation.

Estrogen and prolactin receptor concentrations were measured in 24 carcinogen-induced rat mammary tumors and correlated with the tumor response to host ovariectomy or hypophysectomy. It was found that essentially all of the tumors contained some specific estrogen receptor, and all but three contained prolactin receptor. The values for each receptor comprised a continuum from very low to relatively high concentrations, suggesting that previous considerations of hormone dependence on the basis of presence or absence of hormone receptors may be oversimplified. The concentration of each receptor tended to be lower in the hormone-independent than in the hormone-dependent tumors, but there were a number of hormone-independent tumors with higher receptor levels than some of the hormone-dependent tumors had. A better correlation of tumor response to endocrine ablation resulted from a combination of the 2 receptor levels than from either receptor concentration alone. These results suggest that there is a complex relationship between mammary tumor response to endocrine ablatin and levels of estrogen and prolactin receptors and that some tumors may be dependent upon 1 or both of these hormones for growth.

Comparison of immunocytochemical and steroid-binding assays for estrogen receptor in human breast tumors.

An estrogen receptor immunocytochemical assay which uses monoclonal antibodies to the estrogen receptor protein [Nature (Lond.), 307: 745-747, 1984] was applied to several human tissues, including human breast tumors, and the results were compared to those of steroid-binding assays performed on cytosol extracts of the same tissues. Specific immunoperoxidase staining in fixed, frozen sections was confined to the nucleus of selected cell populations within each tissue examined. In 117 human breast cancers, the presence or absence of nuclear staining was significantly associated with the concentration of cytosolic estrogen receptor. Thirty-eight estrogen receptor immunocytochemical assay-positive tumors were further assessed for several quantifiable features of the staining, including intensity, cellularity, and the proportion of tumor cells stained. Of these, epithelial cellularity showed the highest degree of correlation with the results of steroid-binding assays.

Bromine-80m-labeled estrogens: Auger electron-emitting, estrogen receptor-directed ligands with potential for therapy of estrogen receptor-positive cancers.

To assess their possible use for estrogen receptor (ER)-directed radiotherapy of estrogen receptor-containing cancers, two estrogens were synthesized with the Auger electron-emitting nuclide bromine-80m and administered to immature female rats. Both the triphenylethylene-based estrogen, [80mBr]-2-bromo-1,1-bis(4-hydroxyphenyl)phenylethylene (Br-BHPE) and the steroidal estrogen [80mBr]17 alpha-bromovinylestradiol, showed substantial diethylstilbestrol-inhibitable localization only in the estrogen target tissues, the uterus, pituitary, ovaries, and vagina and, except for the liver and intestines, generally lower concentrations in all other tissues at both 0.5 and 2 h. The [80mBr]Br-BHPE (specific activity, 8700 Ci/mmol), was shown to bind specifically to the low salt extractable ER of the rat uterus. Comparing i.p., i.v., and s.c. administration of [80mBr]BHPE the i.p. route was found to be particularly advantageous to effect maximum, DES-inhibitable concentrations of radiobromine in the ER-rich target organs in the peritoneal cavity. When the tissue distribution of the [80mBr]Br-BHPE was compared with that of sodium bromide-80m, it was apparent that no substantial amounts of radiobromine were released from the bromoestrogen prior to its target tissue localization. The substantial concentration of these bromine-80m-labeled estrogens in ER-rich tissues, combined with previously reported evidence for the effective radiotoxicity of Auger electron-emitting nuclides within cell nuclei suggest a good potential for such ligands for therapy of ER positive cancers.

Regulation of estrogen and progestin receptor concentrations in an experimental rat prostatic carcinoma by estrogen, antiestrogen, and progesterone.

In order to assess prostatic tissue as a target for receptor-mediated estrogen action, we have examined the regulation of estrogen (ER) and progestin receptors (PgR) by estrogen, antiestrogen, and progesterone in cytosolic and nuclear fractions of the R3327H (Dunning) prostatic adenocarcinoma of the rat. Twenty micrograms diethylstilbestrol (DES) with or without 800 micrograms tamoxifen (Tam) were injected s.c. in oil 5 times weekly for 2 weeks. Controls were given oil only. Estrogen receptor assays were carried out using [3H]estradiol and a hydroxylapatite exchange method. Progestin receptors were assayed using [3H]R5020 and dextran-coated charcoal to separate free and bound steroid. All binding data were evaluated by using Scatchard analysis. Treatment with DES depleted cytosolic ER, promoted association of ER with the nuclear fraction, and concomitantly increased PgR concentrations in amounts proportional to nuclear ER. Treatment with Tam alone resulted in higher nuclear ER concentrations than treatment with DES, but induced only one-fifth the amount of PgR. Treatment with DES plus Tam resulted in similar nuclear ER concentrations as with Tam alone, but PgR concentrations were intermediate between those observed with DES alone and Tam alone. Thus Tam exhibited both estrogenic and antiestrogenic properties. In this experiment, the same cytosolic and nuclear extracts were also assayed for ER by using monoclonal antibodies to the receptor in an enzyme immunoassay. No significant differences were observed between the results obtained by the radioligand and enzyme immunoassay methods in the cytosol and nuclear fractions from the control and DES-treated tumors. However in both Tam-treated groups, the ER values obtained by the enzyme immunoassay method were significantly higher than those obtained by the radioligand method in both cytosolic and nuclear fractions. This confirms the observations made by others in female target organs, that monoclonal antibody to ER reacts differently with the Tam-bound ER complex than with the estradiol-bound ER complex. In a separate experiment, administration of progesterone with DES decreased the concentration of nuclear ER to less than one-half that observed after administration of DES alone, with proportional decreases in both cytosolic and nuclear PgR. All these observations indicate that the control of ER and PgR concentrations in this prostatic tumor is identical to that observed in female rat target organs. Use of an immunohistochemical method for the detection of ER in frozen sections indicated that the receptor was localized in the glandular epithelium in both control and DES-treated tumors.

Estrogen receptor-directed radiotoxicity with Auger electrons: specificity and mean lethal dose.

To assess the feasibility of using estrogen receptor-directed therapy with Auger electron-emitting ligands for therapy of estrogen receptor (ER)-containing cancers, we synthesized and evaluated the radiotoxicity of several 123I-labeled estrogens to specifically kill ER+ cells in culture. Auger electrons have been previously shown to be of short range, generally less than the dimensions of a cell, so that to use them therapeutically a mechanism is needed to deliver the Auger electron-emitting nuclide to the vicinity of the DNA. Since it is now well established that the estrogen receptor, when bound to estrogen, forms a high affinity association with distinct estrogen response elements in the DNA, we wished to test the hypothesis that a short exposure of cells to a 123I-labeled estrogen would be specifically radiotoxic to ER+ cells, and that the decays per cell needed for cell killing would be compatible with reasonable levels of receptor occupancy. Using the halodestannylation reaction with tributyl tin precursors of several estrogens and commercially available iodine-123, we prepared the iodoestrogens, E-17 alpha(-)[123I]-iodo-11 beta-methoxyestradiol and 2(-)[123I]iodo-1,1-bis(4-hydroxyphenyl)--2-phenylethylene, at high specific activities, in several cases at essentially the specific activity of 123I itself, 240,000 Ci/mmol. When various concentrations of either of the 123I-labeled estrogens were incubated for 1 h with a subline of ER+ Chinese hamster ovary cells and the washed cells plated for survival assays, a dose-dependent, unlabeled estradiol-inhibitable reduction in survival was observed. In contrast, Chinese hamster ovary cells not expressing estrogen receptor showed little sensitivity to the radiotoxicity of the 123I-labeled estrogens. Calculations based on the assayed residence time of the iodoestrogens in the cells indicate that several hundred decays per cell are sufficient to kill cells.

Differences between estrogen- and antiestrogen-estrogen receptor complexes from human breast tumors identified with an antibody raised against the estrogen receptor.

Radiolabeled estrogens 17 beta-[3H]estradiol and diethylstilbestrol ( [3H]DES) and the antiestrogen [3H]monohydroxytamoxifen ( [3H]MHT) all bind with high affinity to the extranuclear estrogen receptor (ER) from the MCF-7 human breast tumor cell line (Kd = 3 X 10(-10), 2 X 10(-10), and 0.63 X 10(-10) M, respectively). A polyclonal antibody raised in a goat to the calf nuclear ER selectively decreased the binding affinity and number of binding sites for 17 beta-[3H]estradiol, but did not appear to affect these binding parameters for [3H]MHT. In the presence of goat antibody, the binding of the nonsteroidal estrogen DES was so perturbed that it was not possible to quantitate the decreased number of binding sites or affinity of this compound as assessed by Scatchard saturation analysis. These results were confirmed in human breast tumor cytosols by sucrose density gradient analysis. The binding of 17 beta-[3H]-estradiol and [3H]DES to the ER was significantly reduced by preincubation with the polyclonal antibody, whereas the binding of [3H]MHT was reduced only when the tumor cytosol was preincubated with a very high concentration of antibody. At these concentrations of antibody, the binding of 17 beta-[3H]estradiol and [3H]DES to the receptor was prevented completely. In contrast, when the antibody was added to the tumor cytosol after the 3H-ligand had bound to the receptor, the binding properties of all 3H-ligands were unaffected. The [3H]MHT-ER antibody complex consistently sedimented as a higher-molecular-weight complex on sucrose density gradients than did the corresponding estrogenic complexes. The decrease in the affinity of estrogenic ligands can be explained in part by an increase in the dissociation rate at 4 degrees of these compounds from the ER. The dissociation rate of MHT was unaffected by the goat antibody. These results imply that there are important differences in the binding of antiestrogen and estrogens to the tumor cytosol ER. A ligand-binding model is proposed that may aid in the understanding of antiestrogen action.

Bromine-80m radiotoxicity and the potential for estrogen receptor-directed therapy with auger electrons.

While theoretically feasible, estrogen receptor (ER)-directed radiotherapy of hormone-dependent cancers has not been realized because no ER-seeking ligand with an appropriate radiotoxic potential has been identified. Since an appropriate nuclide is a key component we studied the 4.4-h half-life, Auger electron-emitting nuclide bromine-80m. When incorporated into DNA this nuclide was radiotoxic to cells in culture and caused substantial chromosomal damage, while similar concentrations of bromine-80m as bromide or bromoantipyrine were without effect. The mean lethal dose for bromine-80m was 45 atoms per nucleus which is consistent with use in receptor-positive cancers with limited numbers of ER.

Prognostic usefulness of estrogen receptor immunocytochemical assays for human breast cancer.

Breast cancers of postmenopausal patients at high risk for recurrence participating in an adjuvant therapy protocol were independently assayed for estrogen receptor by conventional dextran-coated charcoal steroid binding assays and by immunocytochemistry (ER-ICA) to compare the two assays and to assess the prognostic usefulness of ER-ICA. The ER-ICA was based on a monoclonal antibody to the estrogen receptor and was applied to lightly fixed, frozen sections of the cancers. Excellent agreement was found between the two estrogen receptor methods. It was found that a combination of the distribution of ER-ICA stained cells and the overall staining intensity gave a statistically significant correlation with the quantitative estrogen receptor dextran-coated charcoal steroid binding assay value. In addition, the overall appraisal of the lesion as ER-ICA positive or negative as well as the ER-ICA staining intensity and proportion of ER-ICA stained cancer cells related to patient disease-free interval and survival, independent of patient lymph node involvement. This relationship of ER-ICA status to prognosis appeared not to relate only to responses to adjuvant tamoxifen treatment since it also was observed with patients who did not receive the antiestrogen.

Epithelial peroxidase and endometrial granulocytes in the normal cyclic human endometrium.

Studies in animal models have clearly shown a relationship between the administration of estrogens and the appearance of peroxidase activity in growth-responsive estrogen target tissues (endometrium, cervix, vagina, breast, and DMBA rat mammary tumor). We have studied the ultrastructural localization of endogenous peroxidase activity in the normal cyclic human endometrium. Endogenous peroxidase activity was not identified in proliferative phase endometria, with the exception of one very late proliferative phase endometrium. Most secretory phase endometria showed at least some ultrastructurally identified peroxidase activity in glandular epithelial cells. The number of epithelial cells showing peroxidase activity varied from less than 10% to 85%. The peroxidase activity was present throughout the endoplasmic reticulum of these epithelial cells, extending from the perinuclear cistern to the most peripheral portions of the endoplasmic reticulum adjacent to the apical lumen. Biochemical assays of peroxidase activity in these endometria were compared with the ultrastructurally identified epithelial peroxidase and the endometrial granulocyte count. Uterine granulocyte peroxidase appeared to make a substantial contribution to the total peroxidase activity assayed by biochemical methods. Standard biochemical techniques alone, therefore were not considered to be adequate to evaluate epithelial peroxidase activity.