Estrogen mitogenic action. I. Demonstration of estrogen-dependent MTW9/PL2 carcinogen-induced rat mammary tumor cell growth in serum-supplemented culture and technical implications

Summary The MTW9/PL cell line was established by our laboratory in culture from the carcinogen-induced hormone-responsive MT-W9A rat mammary tumor of a Wistar-Furth (W/Fu) rat. This tumor formed estrogen, androgen, and progesterone responsive tumors in W/Fu rats (Sirbasku, D. A., Cancer Res. 38:1154–1165; 1978). It was later used to derive the MTW9/PL2 cell population which was also estrogen-responsive in vivo (Danielpour, D., et al., In Vitro Cell. Dev. Biol. 24∶42–52; 1988). In the study presented here, we describe serum-supplemented culture conditions in which the MTW9/PL2 cells demonstrate≥80-fold steroid hormone growth responses. All sera used were steroid hormone-depleted by charcoal-dextran treatment at 34°C. The studies were done with horse serum as well as serum from other mammalian species. The growth of the MTW9/PL2 cells was biphasic in response to hormone-depleted serum. Concentrations of ≤5% (v/v) promoted optimum growth. Above this concentration, serum was inhibitory. Concentrations ≥40% (v/v) inhibited growth altogether. Addition of 1.0×10⁻¹³−1.0×10⁻⁸ M 17β-estradiol (E₂) reversed the inhibition completely. At 1.0×10⁻⁸ M, estrone, estriol and diethylstilbestrol promoted growth as well as E₂. Testosterone and dihydrotestosterone promoted growth only at ≥10⁻⁷ M. Progesterone was effective only at≥10⁻⁶ M. Cortisol was ineffective. Labeled-hormone-binding analysis and Western immunoblotting documented that MTW9/PL2 cells had estrogen and progesterone receptors but not androgen or cortisol receptors. Estrogen treatment of MTW9/PL2 cells induced a concentration and time dependent increase in progesterone receptors. We conclude (1) the MTW9/PL2 population is the first highly steroid hormone-responsive rat mammary tumor cell line to be established in culture from a carcinogen-induced tumor, and (2) sera from a number of species including horse, rat and human contain an inhibitor which mediates estrogen sensitive MTW9/PL2 cell growth in culture.     

Estrogen mitogenic action. II. Negative regulation of the steroid hormone-responsive growth of cell lines derived from human and rodent target tissue tumors and conceptual implications

Summary In an accompanying report (Moreno-Cuevas, J. E.; Sirbasku, D. A., In Vitro Cell. Dev. Biol.; 2000), we demonstrated 80-fold estrogen mitogenic effects with MTW9/PL2 rat mammary tumor cells in cultures supplemented with charcoaldextran-treated serum. All sera tested contained an estrogen reversible inhibitor(s). The purpose of this report is to extend those observations to additional sex steroid-responsive human and rodent cell lines. Every line tested showed a biphasic response to hormone-depleted serum. Concentrations of ≤10% (v/v) promoted substantive growth. At higher concentrations, serum was progressively inhibitory. With estrogen receptor-positive (ER⁺) human breast cancer cells, rat pituitary tumor cells, and Syrian hamster kidney tumor cells, 50% (v/v) serum caused significant inhibition, which was reversed by very low physiologic concentrations of estrogens. This same pattern was observed with the steroid hormone-responsive LNCaP human prostatic carcinoma cells. Because steroid hormone mitogenic effects are now easily demonstrable using our new methods, the identification of positive results has nullified our original endocrine estromedin hypothesis. We also evaluated autocrine/paracrine growth factor models of estrogen-responsive growth. We asked if insulin-like growth factors I and II, insulin, transforming growth factor alpha, or epidermal growth factor substituted for the positive effects of estrogens. Growth factors did not reverse the serum-caused inhibition. We asked also if transforming growth factor beta (TGFβ) substituted for the serum-borne inhibitor. TGFβ did not substitute. Altogether, our results are most consistent with the concept of a unique serum-borne inhibitor as has been proposed in the estrocolyone model. However, the aspect of the estrocolyone model related to steroid hormone mechanism of action requires more evaluation. The effects of sex steroids at picomolar concentrations may reflect mediation via inhibitor “activated” intracellular signaling pathways.     

Platelet derived growth factor(s) for a hormone-responsive rat mammary tumor cell line.

Mammary tumor cell growth factor(s) has been identified in extracts of platelets from both male and female rats, as well as in extracts prepared from pooled outdated human platelets. When assayed by the growth promotion of MTW9/PL rat mammary tumor cells in culture, platelet extracts alone were able to support growth 50--75% as well as whole serum. The mitogenic activity from crude human platelet lysates was shown to be trypsin sensitive, relatively stable to extremes of pH, labile to heat treatment at 70 degrees, non-dialysable, ammonium sulfate precipitable, not removed by 56 degrees charcoal treatment, and of apparent molecular weight of 30,000 to 50,000 daltons as estimated by G-100 Sephadex chromatography. The platelet derived mammary growth factor activity was not replaced or potentiated by thrombin or known hormones and growth factors such as prolactin, insulin, 17-beta-estradiol, progesterone, hydrocortisone, L-thyroxine, and mouse epidermal growth factor. The experimental report demonstrates that platelets are a rich source growth factor activity for rat epithelial mammary tumor cells, and that the activity appears to be a polypeptide(s) different from other mitogenic activities known to influence growth of mammary tissue.     

Characterization of a sheep pituitary-derived growth factor for rat and human mammary tumor cells

A growth factor for rat and human mammary tumor cells (MTGF-Pit) was isolated from lyophilized powders of whole sheep pituitaries by a rapid four-step procedure utilizing acetic acid extraction, heating at 93 degrees C, and sequential chromatography in 0.10 M acetic acid on sulphopropyl Sephadex and Sephadex G-50. From 10 g of pituitary powder, 8-10-mg amounts of MTGF-Pit were isolated. By 8 M urea, 0.1% SDS-12.5% polyacrylamide gel electrophoresis analysis followed by Coomassie blue staining, this preparation was shown to be one major stained band. When assayed for growth effects on cells maintained in serum-free medium, 5.1-19.2 nM MTGF-Pit half replaced the growth of MTW9/PL rat and MCF-7 and T-47D human mammary tumor cells in response to 2% to 10% serum. MTGF-Pit shows mitogenic activity toward normal human diploid fibroblasts only at concentrations in excess of 2.5 X 10(-4) M, while rat fibroblasts are unresponsive even at this high concentration. From data available, we conclude that a mitogenic activity for epithelial-type mammary cells has been isolated, and this growth factor appears to be a previously undetected acid- and heat-stable activity that is highly abundant (estimated at 0.16% or more of the total dry weight of the pituitary powder). The isolated ovine MTGF-Pit (3,900 +/- 200 daltons) does not share the molecular weight of native prolactin (24,000 daltons), "cleaved" prolactin (16,000 daltons), or growth hormone (22,000 daltons), and by all tests applied cannot be replaced with other known hormones and purified growth factors. We conclude a potent new mammary tumor cell mitogenic activity has been identified from sheep pituitaries.     

Growth of MTW9/PL2 estrogen-responsive rat mammary tumor cells in hormonally defined serum-free media

Summary Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4 insulin 10 μg/ml, transferrin 10 μg/ml, sodium selenite 10 ng/ml, triiodo-l-thyronine 0.3 nM, phosphoethanolamine 5 μM, epidermal growth factor (20 ng/ml), 17 β-estradiol 2 nM, and bovine serum albumin 20 μg/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When ethanolamine (50 μM), glutathione (20 μg/ml), and linoleic acid/bovine serum albumin (150 μg/ml) were added (formulation DDM-2), the growth rate was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone. This work was supported by National Cancer Institute grants CA-38024 and CA-26617 and American Cancer Society grant BC255.     

Isolation and characterization of a serially cultivated,neoplastic, epithelial cell line from theN-nitrosomethylurea induced rat mammary adenocarcinoma

Summary A new in vitro model for human breast cancer is described. Derived from anN-nitrosomethylurea (NMU) induced rat mammary adenocarcinoma, this serially cultivated cell line has been demonstrated, by a variety of criteria, to be an authentic neoplastic, rat mammary epithelial cell line. The criteria used include morphological and growth characteristics; the presence of specific cell surface antigens; steroid hormone receptors; hormone responsiveness; casein production; karyotype and isoenzyme profile analysis; anchorage independent growth and oncogenicity. Inasmuch as the NMU cell line possesses high concentrations of glucocorticoid and androgen receptors, it may provide a useful model for study of the action of these hormones in human breast cancer. In addition, the NMU line may serve as a valuable in vitro model in which to assess the effects of a variety of endogenous and exogenous agents known to influence mammary tumor growth in vivo, including drugs, nutrients, and growth factors. This work was supported by Grants CA29602 and RR05775-05 from the National Cancer Institute, Bethesda, Maryland.     

Purification and properties of a mammary-uterine-pituitary tumor cell growth factor from pregnant sheep uterus

A mammary-uterine-pituitary tumor cell growth factor has been purified from lyophilized powders of pregnant sheep uteri by a five-step procedure. Uterine-derived growth factor (UDGF) was extracted from the powders with 0.1 M acetic acid, heated at 95 degrees C, and further purified by sulfopropyl-Sephadex C-25, Sephadex G-50, and carboxymethyl-Sephadex C-25 chromatography. From 500 g of uterine powder, 40 to 50 mg of UDGF can be isolated at an overall yield of 33%. The degree of homogeneity of the final preparations was estimated by 8 M urea, 0.1% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), and by PAGE under nondissociating conditions at either pH 8.5 or 4.5. In all PAGE experiments, the purified UDGF preparation showed a single Coomassie blue-stained band that directly corresponded to the only area of elution of UDGF activity from duplicate unstained gels. Molecular sieve high performance liquid chromatography HPLC, reverse phase HPLC on an octylsilyl (C8) column, and hydrophobic chromatography on octyl-Sepharose CL-4B all confirm a similar degree (i.e. greater than 90%) of homogeneity. The Mr of UDGF estimated by urea/sodium dodecyl sulfate-PAGE was 4200 +/- 500 and, by molecular sieve HPLC, 6200 +/- 1000. The isoelectric point of UDGF was estimated as pI = 7.3. The UDGF isolated showed marked cell-type specificity for established cell lines that were derived from estrogen-responsive tumors; purified sheep UDGF was mitogenic for MTW9/PL rat mammary tumor cells (at 10(-10) to 10(-9) M concentrations) while showing no mitogenic activity toward normal rat diploid fibroblasts. UDGF also promoted growth of uterine-derived tumor cells and the GH3/C14 rat pituitary line. Measuring growth as an increase in cell number, UDGF supported the logarithmic growth of the MTW9/PL rat mammary tumor cells over 6 days; other known hormones and growth factors were not able to substitute for the UDGF mitogenic action on MTW9/PL cells. It is concluded that a rapid, high-yield method of purification of a new uterine-derived growth factor activity has been developed.     

Glucocorticoid-receptor interaction and induction of murine mammary tumor virus.

The relationship between the cellular uptake of glucocorticoid hormones, the binding of these hormones to specific in vitro receptors, and the induction of mouse mammary tumor viruses in an established mouse mammary tumor cell line was highly correlated. These results suggest that the induction of mouse mammary tumor virus by glucocorticoid hormones is a physiological process acting through a mechanism of high affinity, saturable steroid-receptors. A temperature-sensitive or salt-dependent step following glucocorticoid-receptor interaction was required for nuclear uptake of the steroid. Induction studies with different adrenocorticoids indicate that the synthetic glucocorticoid, dexamethasone (1,4-pregnadiene-9-fluor-16alpha-methyl-11beta,17alpha,21-triol-3,20-dione), is the most potent inducer of mouse mammary tumor viruses and all steroids which caused significant induction were glucocorticoids. Other glucocorticoids appear to stimulate murine mammary tumor virus production by a mechanism similar to that of dexamethasone; for example, corticosterone competes with dexamethasone for binding to the glucocorticoid receptor and blocks the uptake of dexamethasone into cells. Progesterone also blocks the cellular uptake of dexamethasone and can bind to the glucocorticoid receptor at low concentrations (10-7 to 10-8 M) but progesterone does not consistently induce virus at hormone concentrations even as high as 10-4 M. Thus, in this system, binding to a cytoplasmic receptor is necessary but not sufficient for induction by glucocorticoids. Estrogens and androgens interfere with receptor binding and cellular uptake of dexamethasone but only at much higher concentration (10-4 M) than progesterone, and do not induce mammary tumor virus production. Although there was a positive correlation between steroid structure, binding, and biologic induction, other factors clearly affect the physiological manifestations of steroid actions. Mouse cells with comparable cytoplasmic receptor levels and comparable nuclear uptake differed absolutely in their degree of murine mammary tumor virus induction following hormone treatment. Although all mouse cells examined contain comparable levels of murine mammary tumor virus DNA, only cells producing constitutive levels of murine mammary tumor virus RNA could be induced to higher levels by a variety of glucocorticoids.     

Body zinc distribution profile during N-methyl-N-nitrosourea-induced mammary tumorigenesis in rats at various levels of dietary zinc intake

Zinc distribution is apparently altered in breast cancer patients. It is unclear if this apparent zinc redistribution is a consequence of altered zinc nutrition or tissue-specific response to breast cancer. Our objectives were to assess effects of N-methyl-N-nitrosourea-treatment and N-methyl-N-nitrosourea-induced mammary tumorigenesis on body zinc-distribution profile in rats and to assess effects of dietary zinc intake on the body zinc-distribution profile during N-methyl-N-nitrosourea treatment and N-methyl-N-nitrosourea-induced mammary tumorigenesis in rats. Female Sprague-Dawley rats were assigned to zinc-deficient (3 mg/kg diet) or zinc-adequate (31 mg/kg diet) ad libitum or pair-fed group. Rats were sham treated or N-methyl-N-nitrosourea treated (50 mg/kg body weight; Experiment 1 or 40 mg/kg body weight; Experiment 2) (n=6). In both experiments, the zinc concentration was significantly higher (6–19 times) in mammary tumor than in mammary gland. Tissue zinc concentration was essentially unaffected by N-methyl-N-nitrosourea treatment and tumor bearing, but was reduced by zinc deficiency in the bone, kidney, and liver. Overall, higher mammary tumor zinc concentration and absence of zinc redistribution during N-methyl-N-nitrosourea treatment and N-methyl-N-nitrosourea-induced mammary tumorigenesis, regardless of zinc intakes, indicates zinc accumulation in mammary tumors. Because zinc is essential for growth and cancer is characterized by uncontrolled growth, this zinc accumulation suggests an involvement of zinc in N-methyl-N-nitrosourea-induced rat mammary tumorigenesis.     

Metabolic inhibitors increase prolactin binding to cultured mammary tumor cells

We determined the effects of metabolic inhibitors on ¹²⁵I-labeled prolactin binding in monolayers of cultured rat mammary tumors. Chemical agents that blocked energy production increased binding by 8–20 fold, as did lowering the temperature from 37°C to 4°C. This difference was not due to blocking degradation of the hormone and inhibitors of degradation (lysosomotropic amines, bacitracin) did not increase binding. In the presence of a metabolic inhibitor at 37°C, binding reached a steady state within 3 h and had an apparent dissociation constant of ～6 × 10^?10 M. Studies with fresh tumor slices produced comparable results. The findings indicate that the level of metabolic energy in mammary tumor cells can regulate prolactin binding.     

Stromal expression of tenascin is inversely correlated to epithelial differentiation of hormone dependent tissues

We were previously investigating the expression of the extracellular matrix glycoprotein tenascin in normal and malignant endometrial tissues of humans and rodents. These studies suggested that the expression of tenascin was induced by proliferating epithelia (normal and particularly malignant) and was downregulated with their differentiation. The aim of this study was to investigate the hormone dependency of tenascin expression in (a) the transplantable EnDA endometrial tumor model with or without estrogen deprivation (overiectomy) of the animals, (b) DMBA-induced rat mammary tumors with or without a hormonal treatment of the animals [ovariectomy, antiestrogen (tamoxifen) or antiprogestin (ZK 98299) treatment] and (c) in the rat prostate of untreated or androgen deprived animals (orchiectomy, flutamide-, casodex- or cyproterone acetate (CPA)- treatment). 1. Estrogen withdrawal by ovariectomy did not affect tenascin expression in transplantable EnDA endometrial adenocarcinoma, meaning the entire extracellular space of the stromal mesenchyme was decorated by tenascin immunoreactivity. 2. In untreated DMBA-induced rat mammary tumors almost the entire extracellular space of the stroma was stained by tenascin immunoreactivity. Ovariectomy and antiestrogen treatment did not affect tenascin expression. In contrast, antiprogestin treatment induced terminal differentiation of mammary tumor cells and in parallel downregulated tenascin expression. 3. In the normal rat prostate no tenascin was detectable by immunocytochemistry. However, following androgen deprivation we found tenascin expression in the stroma of the prostate. The most prominent expression was observable after CPA-treatment, possibly due to its progestagenic potency. In conclusion, the hormones and antihormones tested show no direct effect on the stromal expression of tenascin. However, proliferative activity and a low degree of differentiation of the epithelium induces tenascin expression, whereas epithelial differentiation apparently shuts down tenascin expression. Preliminary in vitro studies suggest that paracrine acting growth factors trigger the hormonal regulation of tenascin expression.     

Bombesin-dependent pro-MMP-9 activation in prostatic cancer cells requires beta1 integrin engagement

In Con8 rat mammary epithelial tumor cells, the synthetic glucocorticoid dexamethasone stimulates transepithelial electrical resistance (TER), promotes the remodeling of apical junctions, and down-regulates the level of fascin, an actin-bundling protein that can bind to beta-catenin. We have previously shown that ectopic expression of fascin prevented the glucocorticoid-mediated recruitment of tight junction and adherens junction proteins to the site of cell-cell contact. Here we demonstrate that exogenous treatment or constitutive production of transforming growth factor-alpha (TGF-alpha) ablated the dexamethasone down-regulation of the fascin protein level and disrupted the dexamethasone-induced remodeling of the apical junction and stimulation of the monolayer TER. The response to TGF-alpha was polarized in that basolateral, but not apical, exposure to this growth factor coordinately reversed the steroid control of fascin production and tight junction formation. Expression of dominant negative RasN17 or treatment with the PD098059 MEK inhibitor abolished or attenuated the TGF-alpha disruptive effects on TER, junction remodeling, and fascin protein levels. Our results implicate the regulation of fascin protein levels as a target of cross-talk between the Ras-dependent growth factor signaling and glucocorticoid signaling pathways that controls tight junction dynamics in mammary epithelial tumor cells. We propose that reversing the down-regulation of fascin is critical for the ability of TGF-alpha to disrupt the glucocorticoid-induced remodeling of the apical junction that leads to tight junction formation.     

Sex steroids and growth factors in the regulation of mammary gland proliferation, differentiation, and involution

The mammary gland is subjected to major morphological and biochemical changes during the lactation cycle. It is therefore not surprising that this dynamic process is strictly controlled. The importance of the sex steroid hormones 17beta-estradiol and progesterone for normal development of the mammary gland was recognized several decades ago and has been unequivocally confirmed since. Furthermore, it is now also established that the influence of sex steroids is not restricted to mammogenesis, but that these hormones also control involution. Another important regulatory role is played by growth factors that have been shown to modulate survival (epidermal growth factor, amphiregulin, transforming growth factor alpha, insulin like growth factor, and tumor necrosis factor alpha) or apoptosis (tumor necrosis factor alpha, transforming growth factor beta) of mammary cells. However, the molecular mechanism underlying the influence of sex steroid hormones and/or growth factors on the development and function of the mammary gland remains largely unknown to date. Also scarce is information on the interaction between both groups of modulators. Nevertheless, based on the current indications compiled in this review, an important functional role for sex steroid hormones in the lactation cycle in co-operation with growth factors can be suggested.     

Mitogenic activity of pituitary hormones on cell cultures of normal and carcinogen-induced tumor epithelium from rat mammary glands

Cell suspension containing normal or tumor epithelium were readily obtained by enzymatically digesting rat mammary glands from perphenazine-treated (prolactin-hypersecreting) cycling, female virgin animals or hormone- responsive mammary tumors from animal treated with dimethylbenzanthracene. Cell suspensions were fractioned into predominantly epithelial and predominantly stromal cells by their differential rates of attachment to culture dishes. Both normal mammary and tumor epithelial cells were characterized by the presence of specific cell-junctional complexes, desmosome-like structures, surface microvilli, and their ability to synthesize casein. Serum-dependent protease activity was greater in cultures derived from tumors, and cells from such cultures grew in agarose whereas those from the non-neoplastic gland did not. The addition of prolactin to the culture medium stimulated DNA synthesis in primary or secondary epithelial cultures from tumors, whereas additional insulin and hydrocortisone with prolactin were required for similar levels of DNA synthesis in cultures from non-neoplastic glands. The fraction of cells synthesizing DNA was, however, smaller than that with 10 percent serum measured in the same time period. Both growth hormone and epidermal growth factor stimulated DNA synthesis but to a lesser extent than did prolactin. Prolactin with hydrocortisone and insulin were relatively inactive in promoting DNA synthesis of the nonepithelial cells whereas pituitary fibroblast growth factor was more active. These mitogenic effects were obtained when the hormones were added to the medium at near physiological concentrations, and paralleled the known activities of the hormones in control of mammary gland growth and development in the rat.     

Increased yield of mouse mammary tumor virus (MMTV) by cultivation of monolayer-derived mammary tumor cells in suspension

Summary The MJY-alpha epithelial-like mammary tumor cell line was adapted for cultivation in suspension using a shaker culture technique. Replication of suspension (MJY-beta) cells was more sensitive than monolayer cells to decreases in the concentration of serum in the medium. Comparison of amino acid incoerporation and lactate production rates revealed additional differences between monolayer and suspension cultures. In addition, growth in susfpension resulted in 10- to 400-fold increases in mouse mammary tumor virus (MMTV) production by the mammary tumor cells. Incrases in MMTV yield were detected within 48 h of culture initiation and MMTV production remained elevated throughout 20 cell passages in suspension. Exposure of MJY-beta cells to 14 μM hydrocorticone further increased MMTV yield two-to five-fold. The MJY-beta suspension cultures demonstrated that these epithelial-like cells do not require attachment to a solid substrate for replication or for MMTV production. Loss of structural polarization associated with growth as a monolayer resulted in stimulation of MMTV production greater than and independent of steroid exposure. This work was supported by the T. J. Martell Foundation for Cancer and Leukemia Research and by USPHS grant 5P-30CA23102. F. M. is a trainee on MSTP grant GM07280 from the National Institute of Health. This work was submitted in partial fullfillment of the requirements for the Ph. D. degree (F. M.).     

Glucocorticoid modulation of prolactin receptors on mammary cells of lactating mice

Prolactin receptors were monitored by measuring ¹²⁵I-labeled prolactin binding to collagenase-dissociated mammary epithelial cells of lactating BALB/c mice. Specific receptors for iodine-labeled prolactin with an apparent dissociation constant (K_d) of 0.99 · 10^?9 M were present on the dissociated mammary cells. The binding was inhibited by ovine prolactin, human growth hormone and human placental lactogen but not by follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, bovine growth hormone or insulin. Adrenal ablation of nursing mothers caused a reduction of the number of prolactin receptors and this effect was preventable by hydrocortisone therapy. Hydocortisone injections to mothers 3 days after adrenalectomy also induced a replenishment of the prolactin receptors on the mammary cells. Injections of progesterone failed to sustain the high level of mammary cell prolactin receptors in adrenalectomized animals. Stimultaneous injections of hydrocortisone and progesterone to animals 3 days after adrenalectomy caused a partial suppression of the stimulatory action of hydrocortisone alone. The results suggest that hydrocortisone can exert a modulatory influence on mammary cell prolactin receptors in non-hypophysectomized post-partum mice without altering the dissociation constant (K_d) of the receptors.     

Purification of an equine apotransferrin variant (thyromedin) essential for thyroid hormone dependent growth of GH1 rat pituitary tumor cells in chemically defined culture

Pituitary tumor cells require thyroid hormones for growth in vivo [Sorrentino, J. M., Kirkland, W. L., & Sirbasku, D. A. (1976) J. Natl. Cancer Inst. 56, 1155-1158]. In vitro, GH1 rat pituitary tumor cells were studied in a serum-free defined medium (PCM-10) formulated with Ham's F12 and Dulbecco's modified Eagle's media (1:1, v/v) supplemented with 2.2 g/L sodium bicarbonate, 15 mM 4-(2-hydroxy-ethyl)-1-piperazineethanesulfonic acid (pH 7.2), 10 micrograms/mL human transferrin, 50 microM ethanolamine, 10 micrograms/mL insulin, 10 ng/mL selenous acid, 0.1 nM 3,5,3'-triiodothyronine (T3) and 500 micrograms/mL bovine serum albumin and in the same medium without T3 (PCM-0). The cells only grew in PCM-10 when low concentrations of horse serum were added. Attempts to replace the serum factor requirement with known growth factors and adhesion proteins were unsuccessful. The Mr 65,000-72,000 serum factor regulating T3-induced growth (thyromedin) was purified to homogeneity and identified as equine transferrin R and/or D by amino acid sequencing. The ED50 in PCM-10 was 17-40 micrograms/mL (260-620 nM) while in PCM-0 half-maximum growth was not achieved at 200 micrograms/mL. Concentrations of 75 micrograms/mL in PCM-10 caused 80% of serum-stimulated growth rate. Removal of iron from thyromedin, and assay in iron salts reduced PCM-10, increased the specific activity 110-270-fold to ED50 150 ng/mL (2.3 nM); at 1.0 micrograms/mL, growth in PCM-10 was 16-fold greater than in PCM-0. Iron saturation of thyromedin caused total loss of biological activity. We conclude that the horse transferrin variant isolated in this report is active as apotransferrin.     

Steroid hormone responsive cells in serum-free culture--analyses of hormone-mediated gene expression and cell growth

Steroid hormone-responsive cell lines were clones from mouse mammary cancer (Shionogi Carcinoma 115) and Leydig cell tumor. SC-3 and SC-4 cells from Shionogi Carcinoma were androgen-responsive and -unresponsive in a serum-free medium, respectively. SC-3 cells secreted FGF-like growth factor as well as 24 K glycoprotein in response to androgen stimuli. B-1 and B-1F cells from mouse Leydig cell tumor were growth-stimulated in a serum-free medium by estrogen, androgen or retinoic acid. Transfection of ERE-TK-CAT gene into B-1F cells revealed that both estrogen and retinoic acid activated the CAT activity. In addition, the presence of corresponding receptors for steroid hormones or retinoic acid was demonstrated by hormone binding assays and/or Northern blot analysis. Thus, these serum-free culture systems seem to be very useful for analysing hormone action mechanisms in vitro.     

Pharmacology of vorozole

Vorozole (R83842) is a potent and selective, non-steroidal aromatase inhibitor. It is the dextro-enantiomer of the triazole derivative R 76 713. In FSH-stimulated rat granulosa cells, vorozole inhibited aromatase activity with an IC₅₀-value of 1.4±0.5 nM. In pregnant mare serum gonadotropin (PMSG)-primed female rats, plasma estradiol levels measured 2 h after single oral administration of vorozole were significantly reduced by drug doses of 0.001 mg/kg and higher, with an ED₅₀-value of 0.0034 mg/kg. In ovariectomized nude mice, bearing an estrogen-producing JEG-3 choriocarcinoma, 5 days treatment with vorozole, dose-dependently reduced uterus weight and completely inhibited tumor aromatase, measured ex vivo. Vorozole showed IC₅₀-values higher than 10 μM for inhibition of progesterone synthesis in rat granulosa cells, for inhibition of steroid biosynthesis in isolated rat testicular and adrenal cells and for inhibition of steroid binding to estrogen-, progestin-, androgen- and gluco- and mineralocorticoid-receptors. In LHRH/ACTH-injected male rats and in rats fed a sodium-deprived diet, single oral administration of up to 10 mg/kg vorozole did not affect plasma levels of testicular and adrenal steroids. The compound also had no in vivo estrogen or androgen (ant)agonistic properties. In the DMBA-induced rat mammary carcinoma model, vorozole at an oral dose of 2.5 mg/kg b.i.d. inhibited tumor growth similarly to ovariectomy.