Metabolism of pregnenolone by human breast cancer. Evidence for 17 alpha-hydroxylase and 17,20-lyase.

The metabolism of 7-(3)H-pregnenolone was studied in vitro using 16 human breast carcinomas. All mammary tumors transformed pregnenolone to progesterone. All estrogen receptor poor tumors and 4 out of 8 estrogen receptor rich tumors converted pregnenolone to 17-hydroxypregnenolone. Five estrogen receptor poor tumors showed the presence of 17,20-lyase as evidenced by formation of dehydroepiandrosterone and androstenedione. In two estrogen receptor poor tumors, conversions of pregnenolone to progesterone, 17-hydroxy pregnenolone, dehydroepiandrosterone, androstenedione and finally to estradiol was documented, providing a hypothetical pathway for steroid metabolism in human breast cancer. The conversion of pregnenolone to 17-hydroxypregnenolone was significantly less in receptor rich tumors and was totally absent in 4 receptor rich tumors with estrogen receptors of over 45 fmol/mg protein.     

Influence of neonatal diethylstilbestrol treatment on androgen and estrogen receptor levels in the mouse anterior prostate, ventral prostate and seminal vesicle

Perinatal exposure to the synthetic estrogen, diethylstilbestrol (DES), affects the structure of both male and female reproductive systems. Changes may also occur in the levels of steroid hormone receptors. Cytosolic and nuclear androgen and estrogen receptor levels (expressed per mg DNA) from the sex accessory glands of male BALB/c mice exposed neonatally to DES were analyzed by exchange assays. Neonatal DES exposure caused significant decreases in: (1) cytosolic androgen and cytosolic and nuclear estrogen receptor levels in the anterior prostate and (2) cytosolic estrogen receptor levels in the ventral prostate. A significant increase was seen in the cytosolic estrogen receptor levels in the seminal vesicle. Significant decreases in cytosolic protein levels occurred in all DES-exposed glands.     

Aromatase in the normal breast and breast cancer

Adipose tissue and muscle constitute the larger proportion of body mass, and therefore aromatization in these tissues is the major source of circulating estrogens in postmenopausal women. Although plasma estrogen concentrations are very low, levels in breast cancers from postmenopausal patients are reported to be 10-fold higher than in plasma and normal tissue. Whereas studies on aromatase activity in the tumor suggest that estrogen may be produced locally, the significance of this contribution has been questioned. Using immunocytochemistry (ICC) to an anti-aromatase antibody, a relatively strong immunoreaction was detected in tumor epithelial cells as well as in the terminal ductal lobular units (TDLUs) of the normal breast. Aromatase expression was detected in the cytoplasm of tumor epithelial cells and the surrounding stromal cells of over 50% of tumors in a series of 19 breast cancers. In situ hybridization (ISH) to aromatase mRNA confirmed the immunocytochemical result that the epithelial cells are the primary site of estrogen synthesis in the breast and breast cancers. In the 10 tumors which showed immunoreaction to aromatase, the average aromatase activity measured in cryosections was 286.5 ± 18.6 fmol estrogen/mg protein/h (SE), whereas in nine tumors with weak aromatase immunoreaction, the enzyme activity was 154.7 ± 19.3 fmol estrogen/mg protein/h (P < 0.05) (SE). The functional significance of tumor aromatase and locally produced estrogens on the growth of tumors was suggested by the correlation between aromatase activity and proliferating cell nuclear antigen (PCNA), a marker of cell proliferation (P < 0.005). Although intratumoral aromatase activity did not correlate with steroid receptors significantly, there was a trend for estrogen receptor (ER)-positive tumors to express aromatase. In addition, proliferation ([³H]-thymidine incorporation into DNA) during histoculture, was increased by both estradiol and testosterone in tumors with high aromatase activity. Our results suggest that some tumors synthesize sufficient estrogen to stimulate their proliferation. It may thus be important to inhibit tumor aromatase as well as to reduce circulating levels of estrogen for effective breast cancer treatment.     

An ultrahigh-throughput screening assay for estrogen receptor ligands.

Members of the steroid and thyroid hormone receptor superfamily (nuclear receptors) play diverse roles in mammalian physiology, in both normal and pathological states. For this reason, and because nuclear receptors are natural receptors for lipophilic small molecules, they are important therapeutic targets for the pharmaceutical industry. Here we describe a method for screening for ligands for one of these receptors, the estrogen receptor. The method is rapid, robust, and reliable, and has been used in an ultrahigh-throughput robotic screen. The receptor is crosslinked to a scintillant-containing solid support (FlashPlate) via a receptor-specific antibody. Test compounds are assayed for competition with a radiolabeled estrogen for binding to the immobilized receptor. Receptor-ligand complexes are allowed to form and receptor-bound radioactivity is detected in a scintillation counter. The assay has been designed for both isoforms of the estrogen receptor, alpha and beta, using separate antibodies for each. Given a radioactive tracer and an appropriate antibody, many of which are now commercially available, the assay could be established for any nuclear receptor.     

Spatial and molecular aspects of estrogen and progesterone receptor expression in human uteri and uterine carcinomas

The expression of steroid hormone receptors as molecules reflecting processes of development and differentiation in the human uterine tissue was analysed in a spatial distinct fashion: tissue samples were excised at the fundus and at different, spatially distinct positions of the uterus. They were analysed for concentrations of cytosolic estrogen and progesterone receptors in supernatants from frozen sections using an isoelectric focusing technique. The spatial and molecular distinct, qualitative and quantitative pattern of their expression in the human uterus and uterine adenocarcinomas were studied by sectioning tissue sample from the functionalis through the basalis of the endometrium until reaching deep myometrial parts of the tissue: (1) Specific spatial patterns of estrogen and progesterone receptor levels were detectable throughout the menstrual cycle. (2) For proliferative endometrium from the functionalis to the basalis of the endometrium, the content of both cytosolic receptor species increased up to 6-fold. (3) Differences detectable were less pronounced in the myometrial part of the tissue. (4) Differences of steroid receptor concentrations measured in the endometrium at different uterine positions were highest between fundus and corpus of the endometrium. (5) Maximal differences were detectable around ovulation. (6) After secretory transformation of the organ, specific patterns were still detectable, however quantitative differences were less pronounced. (7) Additionally, quantitative differences measurable were accompanied by variations of molecular properties of the progesterone receptor as demonstrated in an isoelectric focusing gel. (8) In endometrial adenocarcinomas, not only significant quantitative alterations in steroid receptor content were measured, but also a significantly changed spatial pattern of receptor concentrations, also a change of the molecular properties of the progesterone receptor was resolved if these tumor parameters were compared to those detected in the normal tissue of the same organ surrounding the tumor.     

Significance of lipoidal estradiol in human mammary tumors

Incubations of p-nitrophenyl fatty acyl esters and estradiol-17 beta fatty acyl 17-esters with porcine esterase, human mammary tumor cytosol and rat uterine cytosol leads to ester hydrolysis of compounds with short chain fatty acids. Esters with long chain fatty acids show no hydrolysis except in the presence of Tween 80. Short chain fatty acid esters have a higher binding potency to the estrogen receptor than long chain fatty acid esters. Extraction of the nuclear receptor peak sedimenting at 4.6S and identification of the steroid showed that about 90% of the radioactivity was associated with estradiol and only 10% with estradiol esters. These studies show that estradiol fatty acyl esters act as a storage form from which estradiol is released by enzymatic hydrolysis.     

Preferential nuclear binding of estrogen in the formalin-fixed rat uterus

Rat uterus fixed overnight in buffered formalin retains the ability to specifically bind estradiol. However, the estrogen binding property of fixed tissue appears preferentially localized in the nuclear fraction regardless of hormonal status. Furthermore, the quantity of the nuclear estrogen receptor in fresh or fixed uterus is virtually identical in the presence or absence of estrogenic hormone. Yet, while both tissue preparations exhibit equivalent increases in the total nuclear receptor occupancy after hormone exposure, only the fresh uterus contains a major cytosolic estrogen binder which decreases in availability upon the estrogen-induced elevation of the nuclear bound steroid. However, the cytosolic estrogen receptor exhibits a significant loss in its ligand binding property after formalin exposure. Thus, the preferential localization of estrogen binding in the nuclear fraction of fixed whole tissue may just reflect that only the tightly bound nuclear estrogen receptor's functional and/or structural integrity survives long-term formation fixation. Our observation of estrogen binding in preserved tissue may also be a clinically useful tool in therapy analysis.     

In vivo application of [111In-DTPA-D-Phe1]-octreotide for detection of somatostatin receptor-positive tumors in rats.

Radioiodinated somatostatin analogues are useful ligands for the in vitro and in vivo detection of somatostatin receptors. [111In-DTPA-D-Phe1]-octreotide, a somatostatin analogue labeled with a different radionuclide, also binds specifically to somatostatin receptors in vitro. In this study we investigated its in vivo application in the visualization of somatostatin receptor-positive tumors in rats. The distribution of the radiopharmaceutical was investigated after intravenous injection in normal rats and in rats bearing the somatostatin receptor-positive rat pancreatic carcinoma CA 20948. After injection the radiopharmaceutical was rapidly cleared (50% decrease in maximal blood radioactivity in 4 min), predominantly by the kidneys. Excreted radioactivity was mainly in the form of the intact radiopharmaceutical. Ex vivo autoradiographic studies showed that specific accumulation of radioactivity occurred in somatostatin receptor-containing tissue (anterior pituitary gland). However, in contrast to the adrenals and pituitary, the tracer accumulation in the kidneys was not mediated by somatostatin receptors. Increasing radioactivity over the somatostatin receptor-positive tumors was measured rapidly after injection and the tumors were clearly visualized by gamma camera scintigraphy. In rats pretreated with 1 mg octreotide accumulation of [111In-DTPA-D-Phe1]-octreotide in the tumors was prevented. Because of its relatively long effective half-life, [111In-DTPA-D-Phe1]-octreotide is a radionuclide-coupled somatostatin analogue which can be used to visualize somatostatin receptor-bearing tumors efficiently after 24 hr, when interfering background radioactivity is minimized by renal clearance. This is an advantage over the previously used [123I-Tyr3]-octreotide which has a shorter effective half-life and shows high abdominal interference due to its hepato-biliary clearance. Therefore, [111In-DTPA-D-Phe1]-octreotide seems a better alternative for scintigraphic imaging of somatostatin receptor-bearing tumors.     

In vivo assessment of 6-deoxy-6-[18F]fluoro-d-galactose as a PET tracer for studying galactose metabolism

The potential of 6-deoxy-6-[¹⁸F]fluoro-d-galactose (6-[¹⁸F]FdGal) as an in vivo tracer for studying galactose metabolism in tumors and liver was investigated. High uptake and rapid clearance of the radioactivity were observed in many organs of mice after i.v. injection of the tracer. d-Galactose loading did not affect liver uptake. Three experimental tumors showed a slightly higher uptake than other tissues, and rat brain tumor was clearly visualized by autoradiography. However, the radioactivity in tumors decreased rapidly. In the liver, a significant amount of the tracer was found in a galactonate form, while this oxidation was a minor metabolic pathway in the tumors. In both tumor and liver tissues, small amounts of the tracer were incorporated into macromolecular glycoconjugate via phosphate and uridylate forms as intermediate precursors. These results indicate that 6-[¹⁸F]FdGal is not suitable for studying galactose metabolism in vivo because of the low affinity of the tracer for the metabolism.     

Biological and biochemical characterization of estrogen-dependent mouse Leydig cell tumors

Leydig cell tumors formed in BALB/c mice were found to be able to be transplanted subcutaneously in the same strain. One of the sublines, called T 22137, showed growth inhibition in response to estrogenization of host mice. The other subline (T 124958-O), which was originally classified as an estrogen-independent line, was observed to contain the low-affinity estradiol binding component in the cytosol fraction. At a later stage of transplantation, however, the growth of this subline was modestly but significantly enhanced by estrogenic stimuli. This alteration was accompanied by appearance of an estrogen receptor-like molecule which was associated with chromatin even in the absence of estrogen stimuli. In the presence of estrogen selection pressure, a new tumor line, designated as T 124958-R, was established, which showed marked estrogen-dependent growth. T 124958-R was found to contain the cytosolic estrogen receptor. The additional difference was that the annulate lamellae were identified only in T 124958-R, but not in T 124958, by electron microscopic studies. The estrogen dependency of T 124958-R was further substantiated by demonstration of an estrogen secretory protein as well as the estrogen-enhanced formation of 5 alpha-steroids. T 22137 showed growth inhibition in tamoxifen-treated mice. The growth of T 124958-R was enhanced by the administration of tamoxifen to mice. This tamoxifen-induced tumor growth was further stimulated by the simultaneous administration of estrogen. These observations would suggest that mouse Leydig cell tumor systems provide us with a valuable model to investigate the influence of estrogen as well as antiestrogen on malignant cells.     

Physical separation of aortic corticoid receptors with type I and type II specificities

Previous gel filtration binding assay studies indicated that rat vascular smooth muscle cells contained corticoid receptor I and corticoid receptor II sites which could be distinguished on the basis of their relative affinities for aldosterone and dexamethasone. Ion-exchange chromatography experiments were designed to separate the two sites for further studies on their physical characteristics and role in vascular smooth muscle cell physiology. Cultured aortic cells were incubated with 5-10 nM 3H steroid alone or in the presence of 10-fold non-radioactive steroid competitor for 30 min at 37 degrees C. Following cell lysis, total cellular protein-bound steroid was isolated using Sephadex G-25 and applied to a DEAE-cellulose ion-exchange column. Three peaks of radioactivity were eluted using a 1-200 mM sodium phosphate gradient: peak I (30-38 mM), peak II (52-64 mM), and peak III (92-102 mM). Peaks I and II contained 60% of the eluted radioactivity and exhibited the same steroid specificity as corticoid receptor II sites (dexamethasone greater than aldosterone). Peak III contained 40% of the eluted radioactivity and exhibited the same steroid specificity as corticoid receptor I sites (aldosterone greater than dexamethasone). These studies support the binding assay data on steroid specificity and relative proportion of type I and II sites. They also document the existence of type I and II corticoid receptors with different physicochemical characteristics in rat aortic smooth muscle cells.     

Estrogen and progestin receptors in intracranial tumors

Tissue samples from 17 intracranial tumors were analysed for the cytosolic estrogen and progestin receptor concentration. Three of the four meningiomas were progestin receptor-positive, but all were estrogen receptor-negative. All the four meningioma patients were postmenopausal women. The intracranial metastasis of a mammary carcinoma contained both estrogen and progestin receptors. Low progestin receptor concentration was found in an astrocytoma of a postmenopausal women. In addition, the presence of estrogen and progestin receptors was studied in four prolactinomas, two glioblastomas, two oligodendrogliomas, two mixed-cell carcinomas and one astrocytoma. All these tumors were estrogen and progestin receptor-negative.     

Effects of temperature, storage and sodium molybdate on the analysis of estrogen and progesterone receptors in rabbit uterine tissue and gynecologic tumor

The cytoplasmic estrogen receptor (ERc) and progesterone receptor (PRc) in mammary tumors have been recognized as useful biochemical markers for predicting the objective response of patients with advanced breast cancers to endocrine therapy. These proteins are also useful in the prognosis of gynecologic carcinoma. This report presents data showing the effect of sodium molybdate in the stabilization of estrogen and progesterone receptors. In rabbit uterine tissue, molybdate (20 mM) increased the binding of progesterone and estrogen to the receptors in several ways: (a) the apparent loss of detectable receptors during lengthy sucrose gradient analysis and at elevated temperature (30 degrees C) was reduced; (b) the instability of receptors due to storage at -70 degrees C was lessened, and (c) the conversion of the 7S PRc to the 3.5S form was minimized. Similarly, molybdate caused a qualitative and/or a statistically significant quantitative difference in receptor values for some human gynecologic tumors presented herein; the molybdate-associated changes vary with tumor specimen. Of the 8 tumors for which receptor values in the presence of molybdate (M+) and its absence (M-) can be compared, detectable ERc of 6 and PRc of 7 tumors increased with molybdate, and ERc of 2 and PRc of 1 tumor showed no change. In addition to the increase in receptor values, a concomitant shift of the 3-4S molecules to the 7-8S moieties was noted for some tumors (1 of 6 for ERc and 3 of 7 for PRc). In 2 receptor-poor tumor samples, ERc was only detected in M+ cytosols. These results show that molybdate is effective in reducing receptor degradation and stabilizes the 7-8S molecules from converting to 4S moieties. The addition of molybdate may be helpful for better quantitation of steroid receptors in clinical specimens.     

Steroid hormone receptors in MCCLX, a transplantable lactogen-dependent mammary tumor of the rat

MCCLX is a transplantable rat mammary tumor which, for sustained growth, requires the elevated levels of circulating lactogen provided by pregnancy or the implantation of an estrogen pellet. High affinity receptors for estradiol, as well as for the glucocorticoids, dexamethasone and triamcinolone acetonide and the progestin R5020 were measured in the cytosols of these tumors. Estrogen binding capacities were significantly lower in the cytosols of tumors from estrogen pellet treated animals compared with tumors from pregnant animals. Ligand exchange assays demonstrated that nuclei of tumors from estrogen-treated rats contained 3-4 times the estrogen receptors but that there was a definite decrease in total estrogen binding capacity compared with tumors from pregnant rats. It was concluded that this lactogen-dependent tumor contains steroid receptors with molecular properties similar to those of normal target tissues, including estrogen receptors capable of nuclear translocation, the levels of which are modulated by the specific growth conditions.     

Effects of molybdate on steroid receptors in intact GH1 cells. Evidence for dissociation of an intracellular 10 S receptor oligomer prior to nuclear accumulation

Treatment of intact GH1 cells with sodium molybdate inhibits the subsequent rate of nuclear accumulation of hormone-occupied glucocorticoid and estrogen receptors. Cells were incubated at 23 degrees C for 1 h with 30 mM molybdate and then for up to 30 min with [3H]triamcinolone acetonide or [3H]estradiol in the continued presence of molybdate. Although molybdate did not affect the rate of receptor occupancy with either steroid, cells treated with molybdate had more occupied cytosolic and fewer occupied nuclear receptors than control cells. For the glucocorticoid receptor, cells treated with molybdate had more 10 S and fewer 4 S cytosolic receptors than control cells. In low salt cytosol molybdate inhibits the temperature-mediated subunit dissociation of occupied 10 S glucocorticoid receptor. These results suggest that a hormone-mediated dissociation of an intracellular 10 S oligomeric glucocorticoid receptor form to its 4 S subunits is required prior to accumulation of occupied receptors in the nuclear fraction. In cells incubated at 37 degrees C for 1 h or longer with [3H]triamcinolone acetonide, molybdate shifts the steady state intracellular distribution of receptor toward the 10 S cytosolic receptor form, consistent with the interpretation that molybdate affects the rapidly exchanging subunit equilibrium between the 10 S and 4 S cytosolic forms by slowing the rate of 10 S receptor dissociation. Molybdate prevents loss of glucocorticoid-occupied 10 S but not 4 S receptors in heated cytosol by stabilizing the relatively protease-resistant 10 S receptor. Since molybdate stabilizes 10 S oligomeric steroid receptors in vitro, the effects of molybdate on nuclear accumulation of occupied receptors in intact cells support the intracellular existence and physiological relevance of 10 S glucocorticoid and estrogen receptors. These results support a general model for steroid receptor activation in which binding of hormone promotes dissociation of intracellular 8-10 S oligomeric receptors to their DNA-binding subunits.     

Androgenic and estrogenic 17beta-hydroxysteroid dehydrogenase/17-ketosteroid reductase in human ovarian epithelial tumors: evidence for the type 1, 2 and 5 isoforms

17β-Hydroxysteroid dehydrogenase/17-ketosteroid reductases (17HSD/KSR) play a key role in regulating steroid receptor occupancy in normal tissues and tumors. Although 17HSD/KSR activity has been detected in ovarian epithelial tumors, our understanding of which isoforms are present and their potential for steroid metabolism is limited. In this investigation, 17HSD/KSR activity from a series of ovarian epithelial tumors was assayed in cytosol and microsomes under conditions which differentiate between isoforms. Inhibition studies were used to further characterize the steroid specificities of isoforms in the two subcellular fractions. Activity varied widely between tumors of the same histopathologic classification. The highest levels of activity were observed in mucinous tumors. Michaelis constants, maximum velocities, estradiol-17β/testosterone (E₂/T) activity ratios and inhibition patterns were consistent with a predominance of microsomal 17HSD/KSR2 and cytosolic 17HSD/KSR5, isoforms reactive with both E₂ and T, with evidence of estrogenic 17HSD/KSR1 in cytosol from some samples. In tumors where activity and mRNA expression were both characterized, Northern blots, PCR and sequence analysis indicated 17HSD/KSR5 was the predominant isoform. The presence of 17HSD/KSR5, which also has both 3-HSD/KSR and 20HSD/KSR activity, and 17HSD/KSR2 which also has 20-HSD activity, could influence not only estrogen and androgen binding but progesterone receptor occupancy, as well, in receptor-containing tumors.