Formation of glucuronides of estradiol-17 beta by human mammary cancer cells

The formation of glucuronides of estradiol-17 beta by human mammary cancer cell lines is reported for the first time. When incubated with [3H]estradiol-17 beta (1 nM) for 16 h, ZR-75-1 and T47-D cells formed estradiol-3-glucuronide and estradiol-17 beta-glucuronide in approximately equal proportions, whereas MCF-7 cells formed E2-3-glucuronide only. Yields of monoglucuronides from MCF-7 and ZR-75-1 cells were 0.35 pmol/mg DNA, which represented 20-26% of the yield of estradiol-monosulphates. A HPLC system capable of separating most estradiol monosulphates, monoglucuronides and mixed conjugates, is described.     

Long-chain fatty acid esters of 5-androstene-3 beta, 17 beta-diol: composition and turnover in human mammary cancer cells in culture

Long-chain fatty acid esters of the adrenal-derived estrogen 5-androstene-3 beta, 17 beta-diol (ADIOL) were found to accumulate in four human mammary cancer cell lines (MCF-7, ZR-75-1, MDA-MB-231 and MDA-MB-330) when explosed to 10-30 nM ADIOL for variable time periods. At each time point examined, the monoester fraction, which represented the major component of the total lipoidal fraction, contained fatty acids linked to either the 3 beta- or 17 beta-positions. However, there was considerable variation in the ratio of 3 beta- to 17 beta-monoesters in the four cell lines. By means of reverse phase HPLC and referral to authentic synthesized compounds, each monoester fraction was found to contain a number of long-chain fatty acid components whose composition resembled that previously determined for the fatty acid esters formed from 17 beta-estradiol. A specific and measurable turnover of a subfraction of ADIOL-17 beta-monoesters composed of essential fatty acids (22:6, 20:4, 18:3) occurred in MCF-7 cells, and to a lesser extent in ZR-75-1 cells. No changes were observed with time in any of the components of the 3 beta- or 17 beta-monoester fractions in MDA-MB-231 and MDA-MB-330 cells. These results, coupled with other studies, now suggest that a very rapid turnover of some components of these lipoidal derivatives may be occurring. If so, it is possible that the system of acylation-deacylation may be involved in a transport mechanism for estrogens and perhaps other steroid hormones.     

Formation and turnover of long-chain fatty acid esters of 5-androstene-3 beta, 17 beta -diol in estrogen receptor positive and negative human mammary cancer cell lines in culture

Microsomal preparations derived from bovine placenta cotyledons, previously investigated as a convenient source of fatty acyl coenzyme A: estradiol-17 beta-acyl transferase, have been shown to acylate other steroids bearing 3 beta- or 17 beta-hydroxyl groups. In the presence of 0.1 mM oleoyl CoA, the apparent Km values for dehydroepiandrosterone, testosterone, and 5-androstene-3 beta,17 beta-diol (delta 5-DIOL) were 45, 67, and 20 microM, respectively. Acylation of delta 5-DIOL occurred at either the 3 beta- or 17 beta-positions to give monoesters. Testosterone, estradiol-17 beta, and delta 5-DIOL acted as competitive inhibitors for the acylation of the 3 beta-hydroxyl group of dehydroepiandrosterone (Ki values 71, 75, and 41 microM, respectively). Such data indicate that a single enzyme of wide substrate specificity may be involved in these acylation reactions. When estrogen receptor (ER) positive and negative human mammary cancer cell lines were incubated with 10 nM [3H]delta 5-DIOL, intracellular accumulation of delta 5-DIOL long-chain fatty acid esters occurred; rates being higher (p less than 0.001) in ER negative cells (MDA-MB-231 and MDA-MB-330) compared to MCF-7 cells (ER positive), and higher (P less than 0.005) in MDA-MB-231 cells compared to ZR-75-1 cells (ER positive). After exposure to 10 nM [3H]delta 5-DIOL for 16 h, the total labeled steroid fatty acid fraction was composed predominantly of delta 5-DIOL-3 beta- and 17 beta-monoesters (approximately 85%), the remainder containing approximately equal amounts of delta 5-DIOL-diesters and dehydroepiandrosterone-3 beta-esters. Subsequent transfer to medium lacking delta 5-DIOL was accompanied by a breakdown of the labeled esters, which was more rapid in the ER positive cell lines. During this period, intracellular free delta 5-DIOL levels rapidly declined in MDA-MB-330 cells but were maintained in MCF-7 cells, presumably by binding to ER. This behavior parallels that of estradiol-17 beta previously observed in these cell lines and further emphasizes the potential importance of the adrenal-derived estrogen delta 5-DIOL in consideration of a hormone-based etiology of human breast cancer.     

Secretion of estradiol-17beta by porcine mammary gland of ovariectomized steroid-treated sows

Although the mammary gland of many species secretes estradiol (E(2)), nothing is known of E(2) secretion in the porcine gland. The present study was designed to investigate whether porcine mammary gland was a source of E(2), and to test the influence of individual and combined effects of exogenous progesterone and estradiol benzoate (EB) on the secretion of E(2). Immature crossbred gilts were ovariectomized at 7 months of age followed by 4 weeks later by steroid hormone replacement therapy to produce estradiol and progesterone (P(4)) blood concentrations similar to those observed during a normal estrous cycle. Arterial and venous blood plasma (from carotid artery and anterior mammary vein, respectively) were sampled for 2h at 10 min intervals. Plasma concentrations of progesterone, androstenedione (A(4)), testosterone (T), estrone (E(1)) and estradiol were determined by RIA. In all gilts treated with progesterone alone or in combination with EB, concentrations of P(4), A(4) and E(1) in blood collected from venous outflow were lower compared to concentrations in arterial blood, whereas concentrations of E(2) were higher in blood plasma from the anterior mammary vein compared to plasma from the carotid artery. The results indicated that the porcine mammary gland secreted E(2). Increased concentrations of plasma E(2) collected only from P(4)-treated animals suggested that progesterone activated enzymes involved in steroidogenesis in porcine mammary gland, or those utilized in its metabolism.     

Esterification-deesterification of estradiol by human mammary cancer cells in culture

Formation of lipoidal derivatives of estradiol-17 beta (E2) esterified to long-chained fatty acids has been reported to occur in estrogen target tissues. Employing human breast cancer cells in culture, we have detected the rapid synthesis of such compounds upon exposure of the cells to concentrations of [3H]E2 as low as 1 nM. When exposed to 10 nM [3H]E2 in the culture medium, synthesis of E2-lipoidal derivative (E2-L) reached 270 fmol/mg DNA in 2 h in the estrogen receptor positive MCF-7 human mammary cancer cell line. Higher rates (approximately 900 fmol/mg DNA in 2 h) were reached in 2 estrogen receptor negative human mammary cancer cell lines; MDA-MB-231 and MDA-MB-330. E2-L was the major form of estrogen in the latter cells at this time interval (E2-L/E2 approximately 3.0). Far higher concentrations of E2 were found in MCF-7 cells compared to 231 and 330 cells, and, in contrast to the latter, this was mostly specifically bound. Upon subsequent withdrawal of E2 from the medium, intracellular concentrations of E2-L decreased very rapidly in the first 5 h period, then declined more slowly to approximately 50 fmol/mg DNA at 24 h. Intracellular concentrations of E2 were maintained over this time period. E2-L was not present in the medium. Thus, accumulation of E2-L in cells upon continuous exposure to E2 represents the net result of esterification and deesterification reactions. These hydrophobic E2-derivatives may then be involved in the "capture" of E2 for transport through membranes and subsequent regeneration of E2 to maintain occupancy of the nuclear receptor.     

Comparison of the methylation of estradiol-17 beta and of estradiol-17 alpha by dimethyl sulfate

E₂-17β and E₂-17α give substantially similar yields of 3-methyl ether and dimethyl ether when methylated by Brown's procedure.     

Purification of estradiol-17 beta dehydrogenase from human placenta by affinity chromatography

Estriol 16-hemisuccinate has been synthesized and covalently attached to Sepharose through 1,5-diaminopentane. A crude preparation of estradiol-17β dehydrogenase from human placenta was adsorbed on the gel. After extensive washing, the enzyme was eluted by $\text{M}$ hydroxylamine in 0.1 $\text{M}$ potassium phosphate buffer (20–50% glycerol), pH 7, at room temperature. An apparently homogeneous enzyme with a specific activity of 7.2 U/mg (82% recovery) was obtained. It is stable for weeks in the eluting buffer. The hydroxylamine can be removed by passing the enzyme solution over a Sephadex G-100 column or by dialyzing it against 0.1 $\text{M}$ potassium phosphate buffer containing 20% glycerol. This one-step process makes purification of the enzyme simple and easy.     

Biosynthesis of estradiol-17 beta fatty acyl esters by microsomes derived from bovine liver and adrenals

A fatty acyl coenzyme A:estradiol-17 beta acyl transferase activity has been detected in bovine hepatic and adrenocortical microsomes. It is thoroughly increased when adenosine triphosphate (5 mM) and coenzyme A (1 mM) are added to incubation buffer. Using a substrate concentration of 185 microM, the hepatic and adrenocortical microsomal activities have been found to be to 2.4 +/- 0.1 and 5.5 +/- 0.2 nmol/h/mg prot., respectively. Five major estradiol-17-esters have been isolated by reverse phase high performance liquid chromatography from both microsomal incubations, the fatty acid moieties being: arachidonate, linoleate, oleate, palmitate and stearate. However, the distribution of hepatic metabolites is quite different from that obtained with adrenocortical membranes, this is well explained by the corresponding differences between the endogenous contents of free fatty acids. With any of the two types of microsomal membranes used, the results show that estradiol is more susceptible to be esterified to polyunsaturated fatty acids than saturated ones. The possible physiological implications of such an activity in liver and adrenals are discussed.     

Regulation of protein synthesis by estradiol-17beta in cultured hepatocytes

Estradiol-17beta added to cultured chick embryo hepatocytes induced the appearance in the medium of a phosphoprotein, identified as phosvitin on the basis of: (i) its behaviour on ionic exchange columns; (ii) its SDS-acrylamide gel electrophoretic mobility; (iii) its amino acid composition. The hormone treatment was also followed by a decreased synthesis of other proteins secreted by the hepatocytes.     

Markers of atresia in ovarian follicular components from rhesus monkeys treated with estradiol-17 beta

A new model for the investigation of atresia in rhesus monkeys is presented. This model is based upon the reliable induction of an atresia-like process in the dominant preovulatory follicle (DF) by estradiol-17 beta administered subcutaneously via Silastic capsules for 24 h. Data obtained from follicular contents aspirated from treated animals demonstrated alterations in the putative markers of atresia similar to those described in other models of atresia. Although follicle size and appearance and volume of follicular fluid (FF) were unaltered in treated animals, FF was much more viscous than that aspirated from follicles in untreated animals; this was apparently due to a greater quantity of intercellular matrix that was sensitive to digestion by hyaluronidase. In treated animals, FF concentrations of estrogen (E) and progesterone (P) were depressed 3- and 6.6-fold, respectively. Viability of granulosa cells (GC) from these animals was reduced by 40%, as was their ability to release basal amounts of E and P in vitro. Accumulation of P by GC from treated animals approximated unstimulated control levels when human follicle-stimulating hormone (hFSH) was included in the culture. Therefore, FSH may have a limited capability to "rescue" GC from atresia induced by estradiol. The percentage of cells that bound 125I-hFSH maximally, as measured by autoradiography following 72 h in culture, was not altered by treatment. Oocytes from animals treated with estradiol showed signs of degeneration at aspiration, and deteriorated further in culture. This model is unique in that atresia can be induced in the single DF of a primate species, and thus avoids the disadvantages inherent to studying atresia of heterogeneous follicles in polytocous species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytokine-induced production of IFN-beta 2/IL-6 by freshly explanted human endometrial stromal cells. Modulation by estradiol-17 beta

The cytokine IFN-beta 2/IL-6 has emerged as an important means of communication between cells--both within the immune system as well as outside it. In exploring the link between the endocrine and the immune systems, we have studied the secretion of IFN-beta 2/IL-6 by freshly explanted human endometrial stromal cells and its modulation by estrogens. Endometrial stromal cells produced IFN-beta 2/IL-6 in response to other inflammation-associated cytokines such as IL-1 alpha or beta, TNF, and IFN-gamma. This secretion was strongly inhibited by estradiol-17 beta at concentrations as low as 10(-9) M. Multiple species of stromal cell IFN-beta 2/IL-6 in the size range 23 to 30 kDa were detected using immunoprecipitation or immunoblotting procedures. The endometrial stromal cell IFN-beta 2/IL-6 species were phosphorylated and differentially glycosylated in a manner comparable to IFN-beta 2/IL-6 secreted by induced human peripheral blood monocytes or foreskin fibroblasts. However, in contrast to peripheral blood monocytes and fibroblasts, bacterial LPS did not induce IFN-beta 2/IL-6 production in endometrial stromal cells. Additionally, the IFN-beta 2/IL-6 identified in medium from IL-1 alpha-induced stromal cells is biologically active on hepatocytes. These observations, taken together with the observation that IFN-beta 2/IL-6 strongly inhibits the proliferation of human epithelial cells, suggest the possibility that stromal cell secreted IFN-beta 2/IL-6 may affect the physiology of the overlying epithelium in an hormonally modulated manner. Estrogen-regulated production of endometrial IFN-beta 2/IL-6 may participate in gender-specific systemic immunomodulation.     

Stimulation of estradiol-17 beta secretion by 7,12-dimethylbenz (a) anthracene during mammary tumor induction in Sprague-Dawley rats

Testosterone, androstenedione, progesterone, 17-hydroxyprogesterone, estrone and estradiol-17 beta serum levels were measured at given times after dimethylbenz (a) anthracene (DMBA) treatment of a sensitive rat strain Sprague-Dawley (S-D) and a resistant strain Wistar (W). Tumors appeared with a 100% incidence around the 14th to 15th estrous cycle after DMBA treatment in in Sprague-Dawley rats. Hormonal determinations were made, during the 5th or 6th estrous cycle after DMBA treatment, in groups of 4-day cycling rats of both strains which were given DMBA or the carrier solution (sesame oil) when they were about 55-days old. In Sprague-Dawley female rats, DMBA treatment significantly stimulated estradiol-17 beta and estrone preovulatory surge on proestrous days. No such stimulation was found for any other steroid at any time of the estrous cycle. On the other hand, the resistant Wistar rats did not show any disturbed preovulatory or basal steroid hormone release after the carcinogen treatment. These results complete and explain previous findings concerning the hypothalamo-pituitary activity after DMBA treatment of S-D rats: an early and persistent alteration in the centers involved in the hormonal cyclicity of the hypothalamo-pituitary-ovarian axis must be a result of the DMBA treatment. This deregulation could probably account for the distant and selective production of tumors in the mammary gland induced by a single gastric administration of DMBA.     

Action of estradiol-17 beta on the expression of c-Fos gene in endometrial cells in cultured

The c-fos expression was investigated in primary culture of guinea-pig endometrial cells. Cells were made quiescent by serum depletion. Stimulation of these cells by estradiol (E2, 10(-8)M) alone or in combination with epidermal growth factor (EGF, 100 ng/ml) or insulin (10 micrograms/ml) failed to induce c-fos gene. The c-fos expression was early and transiently increased by fetal calf serum (15%) or estradiol plus EGF plus insulin. Protein synthesis inhibitors (cycloheximide or anisomycin) in association with E2 induced a superinduction of c-fos gene. In the same conditions puromycin had no effect. It appears that E2 acts in a multiple step process including an initial c-fos gene derepression by either EGF plus insulin or some protein synthesis inhibitors.