The anti-aromatase effect of progesterone and of its natural metabolites 20alpha- and 5alpha-dihydroprogesterone in the MCF-7aro breast cancer cell line.

BACKGROUND: Progesterone is metabolized in the normal breast mainly into 4-ene-pregnenes (e.g. 20alpha-dihydroprogesterone, 20alphaDHP) but, in contrast, in breast cancer tissue the 5alpha-dihydropregnanes (e.g. 5alpha-dihydroprogesterone, 5alphaDHP) are prevalent. In the present study the effect of progesterone and its main metabolites 20alphaDHP and 5alphaDHP on the aromatase activity in a stable aromatase-expressing estrogen receptor-positive human breast cancer cell line, MCF-7aro, was explored. MATERIALS AND METHODS: The MCF-7aro cells were stripped of endogenous steroids and incubated with physiological concentrations of [3H]-testosterone ([3H]-testos: 5 x 10(-9)M) alone or in the presence of progesterone, 20alphaDHP or 5alphaDHP (5 x 10(-6) or 5 x 10(-8)M) for 24 h at 37 degrees C. The cellular radioactivity uptake was determined in the ethanolic supernatant and the DNA content in the remaining pellet. [3H]-Estradiol (E2), [3H]-estrone (E1) and [3H]-testos were characterized by thin layer chromatography and quantified using the corresponding standard. RESULTS: Aromatase activity was present at a high level in the MCF-7aro cells after incubation with [3H]-testos when the concentration of [3H]-E2 was 3.70 pmol/mg DNA; 20alphaDHP at concentrations of 5 x 10(-6)M or 5 x 10(-8)M significantly inhibited this conversion by 50.3% and 36.5%, respectively. No significant effect was found with the metabolite 5alphaDHP or the parent hormone, progesterone. CONCLUSION: The MCF-7aro cell line shows high detectable aromatase activity. The present data indicate that the progesterone metabolite 20alphaDHP, found mainly in normal breast tissue, can act as an anti-aromatase agent.

Estradiol inhibits the estrone sulfatase activity in normal and cancerous human breast tissues.

It is well accepted that estradiol (E2) plays an important role in the genesis and evolution of breast cancer. Quantitative evaluation indicates that in human breast tumor, estrone sulfate (E1S) 'via sulfatase' is a much more likely precursor for E2 than is androstenedione 'via aromatase'. In previous studies, it was demonstrated that in isolated MCF-7 and T-47D breast cancer cell lines, estradiol can block estrone sulfatase activity. In the present study, the effect of E2 was explored using total normal and cancerous breast tissues. This study was carried out with post-menopausal patients with breast cancer. None of the patients had a history of endocrine, metabolic or hepatic diseases or had received treatment in the previous 2 months. Each patient received local anaesthetic (lidocaine 1%) and two regions of the mammary tissue were selected: (A) the tumoral tissue and (B) the distant zone (glandular tissue) which was considered as normal. Samples were placed in liquid nitrogen and stored at -80 degrees C until enzyme activity analysis. Breast cancer histotypes were ductal and post-menopausal stages were T2. Homogenates of tumoral or normal breast tissues (45-75 mg) were incubated in 20 mM Tris-HCl, pH 7.2 with physiological concentrations of [3H]-E1S (5 x 10(-9)M) alone or in the presence of E2 (5 x 10(-5) to 5 x 10(-7) M) during 30 min or 3 h. E1S, E1 and E2 were characterized by thin layer chromatography and quantified using the corresponding standard. The sulfatase activity is significantly more intense with the breast cancer tissue than normal tissue, since the concentration of E1 was 3.20 +/- 0.15 and 0.42 +/- 0.07 pmol/mg protein, respectively after 30 min incubation. The values were 27.8 +/- 1.8 and 3.5 +/- 0.21 pmol/mg protein, respectively after 3 h incubation. Estradiol at the concentration of 5 x 10(-7) M inhibits this conversion by 33% and 31% in cancerous and normal breast tissues, respectively and by 53% and 88% at the concentration of 5 x 10(-5) M after 30 min incubation. The values were 24% and 18% for 5 x 10(-7) M and 49% and 42% for 5 x 10(-5) M, respectively after 3h incubation. It was observed that [3H]-E1S is only converted to [3H]-E1 and not to [3H]-E2 in normal or cancerous breast tissues, which suggests a low or no 17beta-hydroxysteroid dehydrogenase (17beta-HSD) Type 1 reductive activity in these experimental conditions. In conclusion, estradiol is a strong anti-sulfatase agent in cancerous and normal breast tissues. This data can open attractive perspectives in clinical trials using this hormone.

Estradiol as an anti-aromatase agent in human breast cancer cells.

Estradiol (E(2)) is an important risk factor in the development and progression of breast cancer. However, a "direct effect" of E(2) in breast cancerization has not yet been demonstrated. The estrogen receptor complex can mediate the activation of oncogens, proto-oncogens, nuclear proteins and other target genes that can be involved in the transformation of normal to cancerous cells. Breast cancer cells possess all the enzymes (sulfatase, aromatase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD)) necessary for the local bioformation of E(2). In the last years, many studies have shown that treatment of breast cancer patients using anti-aromatase agents has beneficial therapeutic effects. The aromatase activity is very low in most breast cancer cells but was significantly increased in a hormone-dependent breast cancer cell line: the MCF-7aro, using the aromatase cDNA transfection and G-418 (neomycin) selection. In the present study, we explore the effect of E(2) on the aromatase activity of this cell line. The MCF-7aro cell line was a gift from Dr. S. Chen (Beckman Research Institute, Duarte, U.S.A.). For experiments the cells were stripped of endogenous steroids and incubated with physiological concentrations of [(3)H]-testosterone (5 x 10(-9)mol/l) alone or in the presence of E(2) (5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol/l) for 24h at 37 degrees C. The cellular radioactivity uptake was determined in the ethanolic supernatant and the DNA content in the remaining pellet. [(3)H]-E(2), [(3)H]-estrone ([(3)H]-E(1)) and [(3)H]-testosterone were characterized by thin layer chromatography and quantified using the corresponding standard. It was observed that [(3)H]-testosterone is converted mainly into [(3)H]-E(2) and not to E(1), which suggests very low or absence of oxidative 17beta-HSD (type 2) activity in these experimental conditions. The aromatase activity, corresponding to the conversion of [(3)H]-testosterone to [(3)H]-E(2) after 24h, is relatively high, since the concentration of E(2) was 2.74+/-0.11pmol/mg DNA in the non-treated cells. E(2) inhibits this conversion by 77, 57 and 21%, respectively, at the concentrations of 5 x 10(-5), 5 x 10(-7) and 5 x 10(-9)mol. In previous studies, it was demonstrated that E(2) exerts a potent anti-sulfatase activity in the MCF-7 and T-47D breast cancer cells. The present data show that E(2) can also block the aromatase activity. The dual inhibition of the aromatase and sulfatase activities, two crucial enzymes for the biosynthesis of E(2) by E(2) itself in breast cancer add interesting and attractive information for the use of estrogen therapeutic treatments.

Heterogeneity of binding sites for tamoxifen and tamoxifen derivatives in estrogen target and nontarget fetal organs of guinea pig.

The present paper shows that the antiestrogen tamoxifen and different tamoxifen derivatives bind to two distinct cytoplasmic binding sites in the fetal uterus of guinea pig. The first one (Site A) corresponds to the estrogen receptor and binds tamoxifen with a dissociation constant (Kd) of 1.8 +/- 0.4 nM. The dissociation rate constant (k-1) of th Site A:tamoxifen complex at 4 degrees is 8.3 +/- 2 x 10(-4) sec-1 and at 26 degrees is 123 +/- 26 x 10(-4) sec-1. The binding ability of Site A appears to be thermolabile, being destroyed by heating at 37 degrees. The hydroxylated derivatives of tamoxifen (Metabolites B and D) have a higher affinity for Site A as compared to tamoxifen. The second binding site for tamoxifen (Site B) appears to be specific for the triphenylethylene class of antiestrogens (nafoxidine and several tamoxifen metabolites), the hydroxylation of tamoxifen decreasing the affinity for Site B. In contrast, natural and synthetic estrogens as well as cortisol, testosterone, and progesterone do not compete for Site B. Site B shows a higher affinity for tamoxifen (Kd 0.39 +/- 0.01 nM) as compared to Site A and a higher stability of the complex at both 4 and 26 degrees (k-1 0.81 +/- 0.14 and 3.0 +/- 0.4 x 10(-4) sec-1, respectively). The binding ability of Site B appears to be resistant to heating at 37 degrees. Both Sites A and B are destroyed by proteolytic treatment and are precipitated by 36% saturated ammonium sulfate. The tamoxifen:Site A complex translocates into the uterine nuclei in a "cell free" system by a temperature-dependent process, but the tamoxifen:Site B complex does not. Nevertheless, a site with a Kd similar to cytoplasmic Site B (0.47 +/- 0.1 nM) is spontaneously present in untreated fetal uterine nuclei. In other fetal organs which contain no estrogen receptor (heart) or very low levels (lung), the concentration of Site B is found to be significantly lower than in the fetal uterus (5 to 6 times). Furthermore, progressively lower levels of Site B are found in neonatal, immature, and mature uteri (as compared to fetal uterus) which contain also decreasing amounts of estrogen receptors. It is concluded that, besides binding to the estrogen receptor, the triphenylethylene antiestrogens bind to a specific site distinct from the estrogen receptor, that appears to be localized mainly in estrogen target cells.

Specific binding and biological response of antiestrogens in the fetal uterus of the guinea pig.

The present data show that tamoxifen and nafoxidine compete specifically with estradiol for the estrogen receptor in the fetal uterus of the guinea pig. Furthermore, [3H]tamoxifen binds to the cytosol macromolecules of this fetal tissue at two classes of binding sites, one with a dissociation constant (Kd) of 1.8 +/- 0.4 x 10(-9) M and a concentration of specific binding sites (n) of 1800 +/- 100 fmol/mg protein, which corresponds to the same specific binding site as does estradiol, and a second class of sites with a Kd of 3.9 +/- 0.1 x 10(-10) M (617 +/- 77 fmol/mg protein), which does not bind estrogens. In another series of experiments, it was demonstrated that tamoxifen or nafoxidine injected into the mother (1 mg/kg/day for 3 consecutive days) stimulates fetal uterine weight with an intensity similar to that of estradiol but, in contrast to estradiol, the stimulatory effect of the progesterone receptor is limited. Also, it is observed that estradiol decreases by 80% the total number of specific estradiol binding sites (cytoplasmic plus nuclear). On the other hand, both tamoxifen and nafoxidine can translocate the estrogen receptors into the nucleus, but they have only a limited effect on the total number of specific binding sites of estradiol. It is concluded that the fetal uterus of the guinea pig responds to the effect of estrogens and antiestrogens.

Specific binding of estrogens in different fetal tissues of guinea pig during fetal development.

Cytosol and nuclear specific estradiol binding was evaluated in the fetal uterus, kidney, lung, and brain of guinea pig during fetal development and after birth. The quantity of specific binding sites increases during fetal development and decreases after birth, particularly that of the nuclei. An exception is the binding in the cytosol fraction of lung in which the number of available binding sites continues to increase in newborn animals. In the fetal uterus the amount of specific binding of estradiol and estrone is similar. The selective uptake of radioactivity and its localization by autoradiography in cell nuclei are in accordance with the high levels of estrogen receptors, particularly at the end of gestation.

Activation and immunorecognition by a monoclonal antibody of the tamoxifen-estrogen receptor complex.

The interaction of tamoxifen with the estrogen receptor of fetal guinea pig uterus, the activation of the tamoxifen-estrogen receptor complex and its immunorecognition by a monoclonal antibody raised against the human estrogen receptor is described in the present paper. The results show that: the tamoxifen-receptor complex sediments at 8 S in low-salt and at 4.5 S in high-salt sucrose gradients, this complex is partially recognized by the monoclonal antibody allowing the differentiation of two forms: the alpha form, which binds to the monoclonal antibody, and the beta form, which does not react with it; several factors such as time, temperature and high salt concentrations were capable of activating the tamoxifen-receptor complex, as determined by the increase of its binding to DNA-cellulose; these factors also induced a partial transformation of the beta form to the alpha form; sodium molybdate inhibited both activation and transformation of the beta into the alpha form. The correlation between activation and induction of the alpha form suggests that the monoclonal antibody recognizes selectively the activated form of the tamoxifen-receptor complex. These results indicate similar properties of the estrogen receptor when bound to either tamoxifen or estradiol; however, the differences observed in the behavior of the tamoxifen-receptor complex as compared with the estradiol-receptor complex, though quantitative rather than qualitative, suggest that the estrogen receptor is affected differently by tamoxifen and estradiol.

Immunological difference between ribonuclease and temperature, time and salt-induced forms of the estrogen receptor detected by a monoclonal antibody.

The effect of RNAase A on the activation of the estrogen receptor from fetal guinea pig uterus was studied by DNA-cellulose binding assay and immunorecognition of the estradiol-receptor complex by the monoclonal antibody D547 raised against the human estrogen receptor. After RNAase treatment at 4 degrees C or 25 degrees C the binding of the receptor to DNA-cellulose doubled. This stimulation was partially prevented by sodium molybdate. RNAase treatment did not modify the interaction of the receptor with the monoclonal antibody D547; this antibody, as was demonstrated previously, selectively recognizes the activated form of the receptor when activation has been induced by temperature, time or high salt concentrations. In addition, RNAase had little or no effect on the transformation of the 8-9 S receptor to more slowly sedimenting forms under low salt concentrations. These observations suggest that even if RNAase induces receptor activation, which can be inferred from the increase in its binding to DNA-cellulose, the conformational modifications of the receptor molecule involved in this process are apparently different from those induced by factors such as temperature, time or high-salt concentrations.

Rapid modulation by progesterone and tamoxifen of estradiol effects on nuclear histone acetylation in the uterus of the fetal guinea pig.

The effect of estradiol, progesterone, tamoxifen, estradiol + progesterone or estradiol + tamoxifen on the [3H]acetylation of histones in the fetal uterus of guinea pig was studied. The fetuses were injected subcutaneously 'in situ' with the hormones or tamoxifen + [3H]acetate alone. In 10 min, estradiol stimulated the acetylation of histone 10-12-times with respect to the control animals. Progesterone and tamoxifen blocked this effect. It is suggested that histone acetylation is an early step induced by estrogen action during intrauterine life and that progesterone and tamoxifen suppress this mechanism very effectively.

Enzymes involved in the formation and transformation of steroid hormones in the fetal and placental compartments.

Human fetal and placental compartments have all the enzymatic systems necessary to produce steroid hormones. However, their activities are different and complementary: the fetus is very active in converting acetate into cholesterol, in transforming pregnanes to androstanes, various hydroxylases, sulfotransferases, whilst all these transformations are absent or very limited in the placenta. This compartment can transform cholesterol to C21-steroids, convert 5-ene to 4-ene steroids, and has a high capacity to aromatize C19 precursors and to hydrolyse sulfates. Steroid hormone receptors are present at an early stage of gestation and are functional for important physiological activities. The production rate of some steroids increases drastically with fetal evolution (e.g. estriol increases 500-1000 times in relation to non-pregnant women). We can hypothesize that the control of active steroid hormones could be carried out by fetal and placental factors, which act by stimulating or inhibiting the enzymes involved in their formation and transformation during pregnancy evolution and, consequently, limiting the high levels of the biologically active hormone.

Effect of nomegestrol acetate on estrogen biosynthesis and transformation in MCF-7 and T47-D breast cancer cells.

Although ovaries serve as the primary source of estrogen for pre-menopausal women, after menopause estrogen biosynthesis from circulating precursors occurs in peripheral tissues by the action of several enzymes, 17beta-hydroxysteroid dehydrogenase 1 (17beta-HSD1), aromatase and estrogen sulfatase. In the breast, both normal and tumoral tissues have been shown to be capable of synthesizing estrogens, and this local estrogen production can be implicated in the development of breast tumors. In these tissues, estradiol (E(2)) can be synthesized by three pathways: (1) estrone sulfatase transforms estrogen sulfates into bioactive estrogens, (2) 17beta-HSD1 converts estrone (E(1)) into E(2), (3) aromatase which converts androgens into estrogens is also present and contributes to the in situ synthesis of active estrogens but to a far lesser extent than estrone sulfatase. Quantitative assessment of E(2) formation in human breast tumors indicates that metabolism of estrone sulfate (E(1)S) via the sulfatase pathway produces 100-500 times more E(2) than androgen aromatization. Breast tissue also possesses the estrogen sulfotransferase involved in the conversion of estrogens into their sulfates that are biologically inactive. In the present review, we summarized the action of the 19-nor-progestin nomegestrol acetate (NOMAC) on the sulfatase, 17beta-HSD1 and sulfotransferase activities in the hormone-dependent MCF-7 and T47-D human breast cancer cell lines. Using physiological doses of substrates NOMAC blocks very significantly the conversion of E(1)S to E(2). It inhibits the transformation of E(1) to E(2). NOMAC has a stimulatory effect on sulfotransferase activity in both cell lines, with a strong stimulating effect at low doses but only a weak effect at high concentrations. The effects on the three enzymes are always stronger in the progesterone-receptor rich T47-D cell line as compared with the MCF-7 cell line. Besides, no effect is found for NOMAC on the transformation of androstenedione to E(1) in the aromatase-rich choriocarcinoma cell line JEG-3. In conclusion, the inhibitory effect provoked by NOMAC on the enzymes involved in the biosynthesis of E(2) (sulfatase and 17HSD pathways) in estrogen-dependent breast cancer, as well as the stimulatory effect on the formation of the inactive E(1)S, can open attractive perspectives for future clinical trials.

Modulation of tamoxifen-specific binding sites and estrogen receptors by estradiol and progesterone in the neonatal uterus of guinea pig.

Estradiol administration to newborn guinea pigs (30 ng/g body wt) provokes an 84 +/- 10% and 145 +/- 10% (means +/- SE) increase of tamoxifen-specific binding site (site B) in the uterine cytosol and in the 0.6 M KCl nuclear extract, respectively, 48 h after hormone administration. The simultaneous injection of progesterone (15 micrograms/g body wt) with estradiol antagonizes this estrogen-induced increase of site B. Parallel modifications of estrogen receptor are observed following the same estradiol treatment: after translocation to the nuclear fraction, cytoplasmic estrogen receptor is replenished 24 h after hormone administration with a net increase of 60 +/- 10% (mean +/- SE) after 48 h. The increase of estrogen receptor is also antagonized by the simultaneous administration of progesterone. It is concluded that the levels of both tamoxifen-specific binding site and estrogen receptor are modulated by estradiol and progesterone in the neonatal uterus of guinea pig.

Immunorecognition of cytosol and nuclear estradiol receptor of fetal guinea pig uterus using monoclonal antibody.

Using a monoclonal antibody to estrogen receptor obtained from a preparation of extranuclear receptor of MCF-7 human breast cancer, it is observed that this antibody recognizes the estrogen receptor in both the cytosol and nuclear fractions of the fetal guinea pig uterus. In sucrose gradient ultracentrifugation (10-30% w/v, 0.4 M KCl), the cytosol estrogen receptor complex, which migrates with a coefficient of sedimentation of 4.5 S, is shifted to 7.4 S when bound to the estrogen receptor monoclonal antibody. For the nuclear fraction, the respective values are 5 S and 7.8 S.

Interaction of three monoclonal antibodies with the nonactivated and activated forms of the estrogen receptor.

The 9S nonactivated oligomeric estrogen receptor from fetal guinea pig uterus interacts with the H222 monoclonal antibody (whose epitope is located in the hormone-binding domain) to yield an 11S complex and with the H226 monoclonal antibody (whose epitope is located in the A/B region, just N-terminal to the DNA-binding domain) to yield a 9.4S complex. No reaction was detected with the D547 monoclonal antibody (whose epitope is located between the hormone-binding and DNA-binding domains). In high salt gradients, the 11S oligomeric receptor-H222 complex dissociates to a 8S monomer-H222 complex and the 9.4S oligomeric receptor-H226 complex to a 7S monomer-H226 complex plus the 4.5S monomer receptor not bound to the antibody. These observations suggest that the nonactivated oligomeric receptor contains more than one estradiol-binding subunit with a structure such that the H222 epitopes are fully accessible, the H226 epitopes are partially accessible, and the D547 epitopes are masked. The temperature-activated receptor reacts with the H226 antibody to yield two complexes that sediment at 7S and 9S in high salt gradients. The 9S complex corresponds to a receptor form complexed with more than one antibody molecule; therefore, this suggests the formation of a receptor homodimer where the two H226 epitopes are exposed. However, a single 8S peak is observed when the H222 antibody reacts with the activated receptor, suggesting that only one H222 epitope is accessible in the dimeric receptor. In addition, binding to the H222 antibody before activation prevents dimerization. Thus, the H222 appears to be close to the dimerization domain. The activated receptor reacts with the D547 antibody to yield a 8S complex that apparently contains only one antibody molecule. On the other hand, the receptor extracted from nuclei was found to be a single 5.5S form with the same immunological characteristics as the receptor activated in cytosol. In conclusion, interaction of these monoclonal antibodies with the different forms of the estrogen receptor reveals a structural transformation during activation, with a concomitant change in the exposure of the receptor functional domains.

Antiestrogens antagonize the stimulatory effect of epidermal growth factor on the induction of progesterone receptor in fetal uterine cells in culture.

In fetal uterine cells in culture, epidermal growth factor (EGF) increased progesterone receptor concentrations more than 2-fold. Two other growth factors, transforming growth factor-alpha and fibroblast growth factor, were not able to cause the same increase. This response to EGF was dose dependent; a half-maximal effect was obtained at 10(-10) M. The antiestrogens tamoxifen and 4-hydroxytamoxifen were able to antagonize the stimulatory effect of EGF on progesterone receptor concentrations, but they did not affect its mitogenic effect. The inhibitory effect of 4-hydroxytamoxifen depended on concentration; half-maximal inhibition was observed between 0.5-1 X 10(-9) M. 4-Hydroxytamoxifen could completely inhibit the progesterone receptor increase due to EGF even when added to cells already exposed to the growth factor for 6 days. EGF seems to be acting as an estrogen in increasing progesterone receptors in fetal uterine cells, and antiestrogens are potent antagonists of this response, indicating that growth factors may also be involved in some protein-inducing effects of estrogens. Since estrogen receptor levels were at the limits of detectability under all of the experimental conditions studied, nonestrogen receptor-mediated pathways may be involved. These observations show the potential importance of other factors acting in combination with estrogens in the modulation of progesterone receptor levels.

Control of progesterone receptors in fetal uterine cells in culture: effects of estradiol, progestins, antiestrogens, and growth factors.

Cells from the fetal uterus of the guinea pig have been grown as monolayer cell cultures both as primary cultures and through several passages. The cells have a fibroblast-like morphology and ultrastructure, and the subcultures are estrogen responsive. Estradiol induced a 2- to 3-fold increase in specific binding of [3H]R5020 by 9 days in culture, with no effect on proliferation. This binding has the characteristics of the progesterone receptor from the fetal guinea pig uterus (saturable, high affinity, specific for progestins). The increase in progesterone receptor depended on the dose of estradiol, with a half-maximal response at about 5 X 10(-11) M. Progesterone receptor concentrations were inhibited to below basal levels by progesterone and R5020 and the nonsteroidal antiestrogens, tamoxifen, and 4-hydroxytamoxifen. Both progestins and antiestrogens antagonized the stimulatory effect of estradiol. None of these compounds had any effect on cell growth. On the other hand, insulin and epidermal growth factor caused a great increase in cell proliferation. Insulin alone had no effect on progesterone receptor concentrations, but epidermal growth factor stimulated the progesterone receptor about as much as estradiol. Furthermore, coincubation of insulin with estradiol produced a synergistic effect. Estrogen receptor levels were low or undetectable at any time in either the primary culture or the subcultures. It is concluded that fetal uterine cells in culture can serve as a good in vitro model for study of the control of the progesterone receptor in a fetal target tissue.

Estrogen and antiestrogen effects on different lymphoid cell populations in the developing fetal thymus of guinea pig.

Estrogen responsiveness in the developing fetal thymus of guinea pig has been investigated. One-day estradiol (E2) treatment (1 mg/kg BW) of pregnant animals selectively decreases the number of larger lymphoid cells of the outer cortex of the fetal thymus, without affecting the global number of cortical lymphocytes. E2 treatments for longer periods (from 2 until 6 days) provoke a further impairment in the pattern of outer cortical lymphocytes, selectively reducing larger lymphoid cells, and also decrease the global number of cortical lymphocytes without affecting medullary lymphocytes. Treatment with the antiestrogen tamoxifen (2 mg/kg BW . day for 6 days) results in a transfer of cytoplasmic estrogen receptor to the nuclei of fetal thymus and also affects the histology of the fetal organ, although to a lesser extent than E2. However, when tamoxifen is administered with E2 (1 mg/kg BW . day during the last 3 days of tamoxifen treatment), it antagonizes all of the effects induced by E2 alone on the fetal thymus. The 6-day E2 treatment decreases the weight of the fetal thymus in an age-dependent manner; this effect is progressively more intense from 42 days of gestation at the time of treatment (mean +/- SE, 25 +/- 5% decrease) up to the end of gestation (63 +/- 7% decrease). The effect of E2 on large lymphoid cells also increases in an age-dependent manner. The extent of the estrogen responses observed is correlated with the levels of cytoplasmic estrogen receptors, which increase from 38 days to the end of gestation. The intracellular compartmentalization of estrogen receptor shows that cytoplasmic estrogen receptors are transferred to the nuclei by E2 at both younger and older ages. It is concluded that E2 has differential actions on the lymphoid cell population of the fetal thymus of guinea pig by events antagonized by the antiestrogen tamoxifen and related to the availability and development of cytoplasmic estrogen receptors.

The fetal thymus of guinea pig as an estrogen target organ.

[3H]Estradiol ([3H]E2) shows a saturable, high affinity binding (Kd = 0.18 nM) in the cytosol of the guinea pig fetal thymus. The association rate constant at 4 C of [3H]E2 to cytoplasmic receptor is 1.0 X 10(5) M-1 sec-1. The dissociation rate constant of this complex is 4.4 X 10(-6) sec-1 at 4 C and 3.3 X 10(-4) sec-1 at 26 C. Other physicochemical characteristics, such as binding specificity, which is limited to natural and synthetic estrogens, and sedimentation coefficient, which is 8S under low salt conditions and 4S under high salt conditions, are typical for estrogen receptor. The [3H]E2-cytoplasmic receptor complex chromatographed in DEAE-Trisacryl columns is eluted in a continuous 0- to 0.5-M KCl gradient by 0.15- to 0.2-M KCl concentrations. The cytoplasmic [3H]E2-receptor complex can be transferred to the nucleus in vitro by a temperature-dependent process; it can be extracted from the nucleus by 0.4 M KCl as a 5S sedimenting macromolecule. The levels of available cytoplasmic receptors increase during fetal development from 9.7 fmol/mg protein at 36 days of gestation to 31 fmol/mg protein at the end of gestation. Three or six daily injections of E2 or estrone (1 mg/kg BW) to pregnant animals (58-64 days gestation) significantly decreased the weight of the fetal thymus by 50% with respect to values in vehicle-injected animals. Histologically, a reduction in the size of thymic lobules with a decrease in the width of the cortical lymphoid area and an increase in the medulla-cortex ratio was observed. The in vitro incorporation of [3H]thymidine into thymic DNA was reduced by 50% after E2 treatment. The same estrogen treatment also induced a decrease in cytoplasmic estrogen receptors by 63-72% and a concomitant 5- to 6-fold increase in nuclear estrogen receptors, with respect to values in vehicle-injected animals. It is concluded that estrogen receptors are present in the fetal thymus of the guinea pig, and that this fetal organ is estrogen responsive.

Estrogen and progesterone receptors in the fetal and newborn vagina of guinea pig: biological, morphological, and ultrastructural responses to tamoxifen and estradiol.

Estrogen and progesterone receptors were characterized in the fetal and newborn vagina of guinea pig. The concentrations of estrogen receptor (total sites, cytosol plus nuclei) which were very high [7090 +/- 1700 (+/- SD) fmol/mg DNA] in the fetal vagina (62-64 days gestation) decreased significantly after birth and increased slightly at 2 weeks of age. Tamoxifen (TAM) and estradiol (E2) or the combined treatment (TAM + E2) induced a great increase in weight and DNA content in both the fetal and newborn vagina. After a 12-day treatment to pregnant or newborn guinea pigs, the fetal vagina wet weights (milligrams +/- SD) were as follows: control animals, 87 +/- 14; +TAM, 254 +/- 38; +E2, 177 +/- 19; +(TAM+E2), 218 +/- 41. The values in newborn vaginas were: 155 +/- 40, 434 +/- 75, 477 +/- 49, and 512 +/- 76, respectively. The DNA contents per vagina (in micrograms per +/- SD) in the fetal tissues were as follows: control, 187 +/- 64; +TAM, 563 +/- 74; +E2, 650 +/- 100; and +(TAM+E2), 776 +/- 113; in the newborn vagina these values were: 592 +/- 75, 880 +/- 113; 781 +/- 75, and 901 +/- 15, respectively. Histological studies showed drastic morphological alterations after each of the treatments, particularly in the epithelial cells. Similarly, the ultrastructural examination with transmission electron microscopy showed the alteration of mitochondria, the development of the rough endoplasmic reticulum, and the formation of numerous vacuoles and secretory granules. TAM stimulated the number of specific sites for progesterone, but less intensely than did E2. However, in the combined experiment (TAM+E2), TAM did not block the action of E2. It is concluded that TAM acts in the fetal and newborn vagina of guinea pigs as a real estrogen agonist.

Concentrations of estrone, estradiol, and estrone sulfate and evaluation of sulfatase and aromatase activities in pre- and postmenopausal breast cancer patients.

This report concerns the evaluation of various estrogens, estrone (El), estradiol (E2), and estrone sulfate (E1S), as well as E1S-sulfatase and aromatase activities in pre- and postmenopausal women with breast cancer. The levels (in picomoles per g; mean +/- SEM) of the various estrogens in the breast tissue from premenopausal patients (n = 11) are: El, 1.4 +/- 0.5; E2, 1.2 +/- 0.6; and E1S, 1.2 +/- 0.3. In postmenopausal patients (n = 23), the values are, respectively, 1.0 +/- 0.4, 1.4 +/- 0.7, and 3.3 +/- 1.9. These concentrations of estrogens in the tumors of postmenopausal patients are significantly higher than those found in plasma. The activity of E1S-sulfatase in both pre- and postmenopausal patients was 50-200 times higher than that of aromatase. E1S-sulfatase and aromatase activities are significantly higher in post-menopausal than in cycling patients. It is concluded that despite the low levels of circulating estrogens in postmenopausal patients, the tissue concentrations of these steroids are several-fold higher than those in plasma, suggesting tumor accumulation of these estrogens. The physiopathology and clinical significance of these high levels of the various estrogens (E1, E2, and E1S) as well as sulfatase and aromatase activities in postmenopausal patients with breast cancer is yet to be explored.