Elwood V. Jensen

A role for estrogen receptor β in the regulation of growth of the ventral prostate

In normal rats and mice, immunostaining with specific antibodies revealed that nuclei of most prostatic epithelial cells harbor estrogen receptor β (ERβ). In rat ventral prostate, 530- and 549-aa isoforms of the receptor were identified. These sediment in the 4S region of low-salt sucrose gradients, indicating that prostatic ERβ does not contain the same protein chaperones that are associated with ERα. Estradiol (E2) binding and ERβ immunoreactivity coincide on the gradient, with no indication of ERα. In prostates from mice in which the ERβ gene has been inactivated (BERKO), androgen receptor (AR) levels are elevated, and the tissue contains multiple hyperplastic foci. Most epithelial cells express the proliferation antigen Ki-67. In contrast, prostatic epithelium from wild-type littermates is single layered with no hyperplasia, and very few cells express Ki-67. Rat ventral prostate contains an estrogenic component, which comigrates on HPLC with the testosterone metabolite 5α-androstane-3β,17β-diol (3βAdiol). This compound, which competes with E2 for binding to ERβ and elicits an estrogenic response in the aorta but not in the pituitary, decreases the AR content in prostates of wild-type mice but does not affect the elevated levels seen in ERβ knockout (BERKO) mice. Thus ERβ, probably as a complex with 3βAdiol, is involved in regulating the AR content of the rodent prostate and in restraining epithelial growth. These findings suggest that ligands specific for ERβ may be useful in the prevention and/or clinical management of prostatic hyperplasia and neoplasia.

Immunochemical studies of estrogen receptors

Fusion of splenic lymphocytes from Lewis rats, immunized with affinity-purified estrogen receptor from the cytosol of MCF-7 human breast cancer cells, with two different mouse myeloma lines, has provided 13 monoclonal hybridoma lines secreting antiestrophilin antibodies, each of which (with one possible exception) recognizes a different antigenic determinant in the human receptor molecule. Of this library of monoclonal antibodies, some react with estrophilin from all sources tested, some react with mammalian but not avian receptors, whereas one preparation appears specific for estrophilin from primate sources. By proteolytic digestion under controlled conditions with mercury-deactivated papain, chymotrypsin, and trypsin, respectively, it is possible to remove sequentially the determinants recognized by one, two or three of the monoclonal antibodies, leaving the epitopes for the six remaining antibodies investigated on the steroid-binding portion of the receptor. The proteolytic fragment containing the epitope most readily removed (by mercuripapain) also contains the DNA-binding domain of the activated receptor molecule. Immunocytochemical staining, using the peroxidase procedure with various monoclonal antibody preparations, of frozen sections of human breast cancer tissue, fixed in ethanol or in picric acid-formaldehyde reagent, shows clearly that the majority of the native receptor, which appears in the cytosol after tissue homogenization, is actually localized within the nuclear compartment in the intact cell. The immunocytochemical technique also permits the identification of mixed populations of receptor-containing and non-containing cells in human breast cancers.

Cloning of the human oestrogen receptor cDNA

Poly A+ RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and those fractions enriched in oestrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the lambda gt11 vectors. Clones corresponding to the ER were isolated from both libraries after screening with either ER monoclonal antibodies (lambda gt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (lambda gt10). Five cDNA clones were isolated by antibody screening and five after screening with synthetic oligonucleotides. The two largest ER cDNA clones, lambda OR3 (1.3 kbase) and lambda OR8 (2.1 kbase), isolated using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, lambda OR8 contains DNA sequences which cross-hybridize with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Using lambda OR8 as a hybridization probe revealed a single poly A+ RNA band of approx. 6.2 kbase in the ER containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER-cell line HeLa. The same probe hybridizes to a chicken gene which is expressed in oviduct tissue as a 7.5 kbase poly A+ RNA.

Estrogen-binding substances of target tissues.

Abstract A variety of evidence supports the concept that the interaction of estradiol with uterine tissue takes place by a two stage mechanism. The hormone associates spontaneously with an extranuclear protein to form an 8S complex, made up of 4S subunits, which possesses a strong affinity for nuclei. In the nucleus a new estradiol-protein complex, extractable as a 5S unit, is formed from the 8S complex by a temperature-dependent process which consumes 8S receptor. The concept of “induced derepression” is proposed to explain how the nuclear transformation of 4S to a 5S estradiol-protein complex might initiate uterine growth.

Receptors Reconsidered: A 20-Year Perspective

Publisher Summary The original discovery of steroid hormone receptors and essentially all information concerning their interaction and function in target cells have depended on experiments in which a radioactive steroid serves as a marker for the receptor protein to which it binds. The female reproductive tissues, such as uterus, vagina, and anterior pituitary, contain a characteristic estrogen-binding component which was first indicated by their striking ability to take up and retain tritiated hexestrol and estradiol after the administration of physiological doses of these substances to immature animals. This chapter discusses the biochemical mechanism by which the reaction of estradiol and other estrogenic hormones with receptor substances elicits hormonal response has been the subject of extensive investigation. The activated steroid-receptor complex is translocated to the nucleus where it binds to chromatin and in some way modulates RNA synthesis which appears to be characteristically restricted in hormone-dependent tissues.

Monoclonal antibodies as probes for estrogen receptor detection and characterization

Splenic lymphocytes from a Lewis rat, immunized with purified estradiol-receptor complex of calf uterine nuclei, were fused with cells of three mouse myeloma lines to yield several monoclonal lines of hybridoma cells that secrete antiestrophilin antibodies, which, like the antiserum of the immunized rat, react specifically with estrophilin of calf tissues. In contrast, a Lewis rat, immunized with cytosol estradiol-receptor complex from MCF-7 human breast cancer cells after purification by a novel affinity chromatography technique, gave antiserum that crossreacts with receptor from mammalian as well as avian tissues. Monoclonal antibodies secreted by three hybridoma cell lines derived from this immunized rat showed interesting differences in cross reactivity. All recognized receptor from primate sources, two of the three monoclonal preparations recognized receptor from calf and rat uterus as well, but none reacted with estrophilin from hen oviduct. Thus, in addition to similarities, there appear to be immunocytochemical differences between estrogen receptors from mammalian and avian sources and between receptors from primate and non-primate tissues. These monoclonal antibody preparations, recognizing different determinants on the estrophilin molecule, provide a novel approach to the study of receptor structure and function as well as the basis for a simple immunoradiometric determination of estrogen receptors in human breast cancers. Preliminary studies indicate that they also may prove useful for the immunocytochemical detection of estrophilin in tissue sections.

Hormone dependency of breast cancer

Quantitative determination of the estrogen receptor (estrophilin) content of an excised tumor specimen provides information useful in selecting the type of systemic therapy best suited to the individual patient with advanced breast cancer. Women whose tumors contain low or negligible amounts of estrophilin rarely respond to endocrine ablation or other hormone therapy, whereas most but not all patients with receptor‐rich cancers receive benefit from endocrine treatment. Properly interpreted, estrophilin assay predicts hormone dependency correctly in 85 to 90 percent of the cases. Analysis of the primary tumor at the time of mastectomy not only serves as a guide to subsequent therapy if metastases should appear but it also provides a clue to the probability and rapidity of recurrence. Because present methods for receptor determination have certain limitations, we are developing an immunoassay, based on monoclonal antibodies to human estrophilin, that promises to provide a simple, inexpensive procedure for the routine analysis of breast cancer specimens.

Tissue-specific stimulation of RNA synthesis by transformed estradiol-receptor complex.

Summary Exposure to the estradiol-receptor complex of uterine cytosol increases the RNA synthetic capacity of purified uterine nuclei but not of liver or kidney nuclei. This stimulation is effected only by complex in which the binding unit of the receptor has undergone estrogen-induced conversion from the native (4S) to the transformed (5S) form. It is suggested that an important function of estrogenic hormone is to promote transformation of the receptor protein to an active form which can enter the nucleus and stimulate RNA synthesis.

Estrophile to nucleophile in two easy steps

Abstract A variety of self-consistent experimental evidence supports the concept that estrogcnic hormones interact with their target tissues by a multi-stage mechanism in which the hormone first associates with the 4S binding unit of an 8S extranuclear receptor protein, activating it to undergo conversion to a 5S form. This temperature-dependent transformation takes place when the receptor is bound to estradiol, but not when it is uncomplexed or bound to estrone. The transformed hormone-receptor complex migrates to the nucleus where it associates with an acceptor site in the chromatin. Analytical amounts of the 5S nuclear complex and a calciumstabilized 4S cytosol complex have been isolated from calf uterus in apparently pure state, opening the way for the large scale purification of these different forms of the receptor protein. The estrogen-receptor interaction sequence appears to be involved in the acceleration of biosynthetic reactions in hormone-dependent tissues. Exposure of isolated uterine nuclei either to transformed complex or to native complex under conditions where transformation can take place causes a significant increase in their ability to synthesize RNA. Similar treatment does not enhance the already high RNA synthetic capacity of liver or kidney nuclei. It is suggested that an important function of the hormone is to promote the conversion of the receptor protein to an active form which can enter the nucleus and alleviate a defficiency in RNA synthesis, characteristic of estrogen-responsive tissues.

Hormone dependency in breast cancer.

Hormone deprivation by surgical ablation of endocrine glands affords the most effective treatment presently available for advanced breast cancer in those patients whose tumors are of the hormone-dependent type. Less than half of premenopausal patients and a smaller fraction of postmenopausal patients respond to endocrine ablation. The estrogen receptor (estrophilin) content of an excised specimen of the tumor can provide information useful for selecting optimal therapy. Of 123 women with advanced breast cancer whose without ovarian function who responded to hormone therapy had 750 or more fmol/gm of tumor tissue. Of premenopausal patients only those with 300 fmol or more per gm of tumor tissue responded. Therefore 750 fmol/gm of tumor tissue for postmenopausal and 250 for premenopausal patients have been resported as positive or receptor-rich. Those with less have been clarified as receptor-poor. Nearly 66% of patients whose cancers are reported as receptor-rich for estrophilin can expect to benefit from endocrine therapy. Those with lesser amounts of estrophilin have very little chance of benefiting from hormone therapy and are better treated by chemotherapy. Of more than 1200 women whose breast cancers have been assayed about 30% were classified as receptor-rich and 70% as intermediate or receptor-poor. The simultaneous presence of a progesterone receptor has also been shown to be of importance in some cases. Routinely characterizing primary breast cancers at the time of mastectomy is recommended. Subsequent recurrences or metastases have been shown to have estrophilin content similar to the primary tumor. In a discussion the receptor-rich and receptor-poor classification was thought by some to be misleading. Wider use of antiestrogens was recommended instead of ablative surgery.