C. Gelly

Importance of estrogen sulfates in breast cancer

Estrogen sulfates are quantitatively the most important form of circulating estrogens during the menstrual cycle and in the post-menopausal period. Huge quantities of estrone sulfate and estradiol sulfate are found in the breast tissues of patients with mammary carcinoma. It has been demonstrated that different estrogen-3-sulfates (estrone-3-sulfate, estradiol-3-sulfate, estriol-3-sulfate) can provoke important biological responses in different mammary cancer cell lines: there is a significant increase in progesterone receptor. On the other hand, no significant effect was observed with estrogen-17-sulfates. The reason for the biological response of estrogen-3-sulfates is that these sulfates are hydrolyzed, and no sulfatase activity for C17-sulfates is present in these cell lines. [3H]Estrone sulfate is converted in a very high percentage to estradiol (E2) in different hormone-dependent mammary cancer cell lines (MCF-7, R-27, T-47D), but very little or no conversion was found in the hormone-independent mammary cancer cell lines (MDA-MB-231, MDA-MB-436). Different anti-estrogens (tamoxifen and derivatives) and another potent anti-estrogen: ICI 164,384, decrease the concentration of estradiol very significantly after incubation of estrone sulfate with the different hormone-dependent mammary cancer cell lines. No significant effect was observed for the uptake and conversion of estrone sulfate in the hormone-independent mammary cancer cell lines. Progesterone provokes an important decrease in the uptake and in estradiol levels after incubation of [3H]estrone sulfate with the MCF-7 cells. It is concluded that in breast cancer: (1) Estrogen sulfates can play an important role in the biological response of estrogens; (2) Anti-estrogens and progesterone significantly decrease the uptake and estradiol levels in hormone-dependent mammary cancer cell lines; (3) The control of the sulfatase and 17 beta-hydroxysteroid dehydrogenase activities, which are key steps in the formation of estradiol in the breast, can open new possibilities in the treatment of hormone-dependent mammary cancer.

Transcriptional Activation of the Human ucp1 Gene in a Rodent Cell Line SYNERGISM OF RETINOIDS, ISOPROTERENOL, AND THIAZOLIDINEDIONE IS MEDIATED BY A MULTIPARTITE RESPONSE ELEMENT

Uncoupling protein 1 (UCP1) is uniquely expressed in brown adipocytes and generates heat production by uncoupling respiration from ATP synthesis. The activatory effects of norepinephrine and retinoic acid (RA) on rodent ucp1gene transcription have been well characterized. These effects are mediated by a 211-base pair (bp) enhancer which is also sufficient to restrict expression to brown adipose tissue. The molecular mechanisms controlling the transcription of the human ucp1 gene are unknown. In order to study the transcriptional regulation of the human gene, we set up chloramphenicol acetyltransferase constructs containing the entire or deleted 5′ regions upstream of the transcriptional start site of the gene. These constructs were transiently transfected in a mouse cell line. A 350-bp hormone response region showing a significant homology with the rat ucp1 enhancer and located between theBclI polymorphic site and an AatII site (bp −3820/−3470) was detected. This region was sufficient to mediate the stimulation by RA and by combined treatments (RA + isoproterenol (ISO), RA + thiazolidinedione (TZD), or RA + ISO + TZD). The highest stimulation, a 26-fold increase in basal activity, was obtained by RA + ISO + TZD treatment. In contrast to the rodent gene, under our conditions, the effect of ISO and/or TZD is dependent on RA stimulation. Analysis of 105 bp inside the 350-bp element by site-directed mutagenesis and gel retardation experiments demonstrated that a multipartite response element mediates the drug stimulation. This region binds RARs and RXRs nuclear factors, CREB/ATF factors, and also PPARγ despite the absence of a consensus peroxisome-proliferator response element. The activation of the human ucp1 gene transcription by certain hormones or drugs, and the identification of the cis-elements involved, will help to identify new compounds activating fat oxidation and energy expenditure in humans.

Specific [3H]-estradiol binding in the fetal uterus and testis of guinea pig. Quantitative evolution of [3H]-estradiol receptors in the different fetal tissues (kidney, lung, uterus and testis) during fetal development

Abstract Specific estradiol receptors have been found in fetal uterus in the cytosol fraction and in the nuclear extracts obtained by successive extractions with (A) 0.1 M Tris-HCl-0.0015 M EDTA, (B) 0.3 M NaCl-0.01 M Tris-HCl, and (C) 1 M NaCl-0.01 M Tris-HCl. In uterine cytosol, an estradiol-specific component with a sedimentation coefficient of 8.5-9 S in sucrose density gradient was detected. The K d of the binding of estradiol in fetal uterine cytosol is 4 × 10-10 M. Similar [3H] -estradiol receptors are present in fetal testis. A systematic study of the evolution of specific [3H]-estradiol binding during fetal development has shown the following results. The specific binding of [3H]-estradiol per mg protein in fetal kidney cytosol increased from no detectable binding at 34–35 days of gestation to 35 fmol/mg protein at 59 days with a subsequent decrease to 22 at 24 h after birth. Specific binding in the three nuclear extracts studied (0.1 M Tris, 0.3 M NaCI and 1 M NaCl) also increased from 11 fmol/mg protein at 34–35 days of gestation to 147 fmol/mg protein at 59 days and decreased to 50 at 24 h after birth. In fetal lung cytosol, specific [3H] -estradiol binding increased dramatically from 24 fmol/mg protein at 34–35 days to 434 at 59 days, decreasing to 345 fmol/mg protein at 24 h after birth. Nuclear binding also increased from 27 fmol/mg protein at 34–35 days to 329 at 59 days and decreased to 114 at 24 h after birth. In the fetal uterus, specific binding in the cytosol fraction is 900 fmol/mg protein at 34–35 days of gestation and increases to 500 fmol/mg protein at the end of gestation (55–60 days). In contrast, fetal heart showed very little or no specific binding of [3H]-estradiol.

Biological effects and morphological responses to estriol, estriol-3-sulfate, estriol-17-sulfate and tamoxifen in a tamoxifen-resistant cell line (R-27) derived from MCF-7 human breast cancer cells

The R-27 cell line is a variant clone derived from the MCF-7 human breast cancer cell line which has lost its inhibitory response to anti-estrogens. In the present study, we have compared the biological responses to estriol (E3), estriol-3-sulfate (E3-3-S), and estriol-17-sulfate (E3-17-S) in these cells and in the parent MCF-7 cells. In the R-27 cell line after 7 days of culture, the progesterone receptor (PR) concentrations were greatly increased by E3 and E3-3-sulfate; however, tamoxifen did not block this effect. The effect in PR provoked by E3-17-S was significantly less intense. The concentrations of PR (pmol/mg DNA +/- S.D.) in the R-27 cells were as follows: control: 1.1 +/- 0.8; +E3: 10.5 +/- 2.4; +E3-3-S: 5.4 +/- 2.3; +E3-17-S: 2.6 +/- 0.8. E3 and E3-3-S also stimulated PR in the MCF-7 cells but to a lesser extent. No stimulation was observed in the E3-17-S treatment. A fraction (0.5-1%) of the E3-3-S was found to be hydrolysed in the medium during the incubation in both cell lines, but no hydrolysis occurred after incubation with E3-17-S. Ultrastructural observations showed that in the E3 and E3-3-S treated cells, there was an important development of the ergastoplasm, bundles of filaments and an accumulation of ribosomes. No significant morphological alteration was observed in cells exposed to E3-17-S. In conclusion, E3 is biologically very active in both the R-27 and the MCF-7 cell lines and E3-3-S could play a role in the control of the estrogenic activity of E3.

Effect of tamoxifen and tamoxifen derivatives on the conversion of estrone sulfate to estradiol in the MCF-7 mammary cancer cell line

The human mammary cancer cell line MCF-7 in culture was used to study the effect of tamoxifen and its derivatives: 4-hydroxytamoxifen (4-OH-Tam), N-desmethyltamoxifen (Dem-Tam) and cis-tamoxifen (cis-Tam) on the uptake and conversion of [3H]estrone sulfate (3H-E1S) to estradiol (E2). When [3H]-E1S (4 X 10(-9) M) was incubated by itself (control) a great proportion of the radioactivity was found as [3H]E2, predominantly in the nuclear fraction. All of the anti-estrogens (10(-6) M - 10(-5) M) studied decreased the total uptake of radioactivity by the cells by 50-60% and the quantity of E2 formed. The calculated concentrations (in pg/mg DNA +/- S.E.M.) of E2 (cytosol + 0.6 M KCl nuclear extract) with the anti-estrogens at 10(-5) M were as follows: control 56 +/- 3; Tam treated cells 4 +/- 1; + 4-OH-Tam 2 +/- 1; + Dem-Tam 5 +/- 2; + cis-Tam 8 +/- 4. A significant decrease in the concentrations of E2 was also observed in the mitochondria-microsomal fractions after the different treatments. It is suggested that the MCF-7 cells can use estrone-3-sulfate as a source of E2 and that the inhibitory effect of tamoxifen and its derivatives on the conversion of this sulfate to E2 could be involved in the anti-estrogenic process of these triphenylethylene derivatives.

Mineralocorticosteroid receptors in the foetal compartment

Abstract Mineralocorticosteroid receptors have been found in the foetal kidney of the guinea pig (at 25–40 days of gestation) in experiments carried out both in vivo and in vitro. More than 50% of the total d-aldosterone receptors were found in the nucleus. 20% of the total nuclear radio-activity was extracted by the 0.1 M Tris-HCl solution and 50% by the 1 M NaCl -0.01 M Tris solution, suggesting that at this period of the foetal evolution the aldosterone receptors are localized principally in the chromatin fraction. The formation of these [ 3 H]-aldosteronc-macro-molecule complexes is very rapid, maximum values being found at 4 min of incubation at 37°C. The nuclear receptors, but not those of the cytosol fraction, are temperature-dependent, d-Aldosterone and deoxycorticosterone compete with the [ 3 H]-aldosterone complex. On the other hand, d-aldosterone has no effect on the [ 3 H]-aldosterone-macromolecule complex if reincubation is carried out with the nuclear extracts (at 4°C) after extraction. Also, the 0.1 M Tris and 0.01 M Tris-1 M NaCl nuclear extracts do not form [ 3 H]-aldosterone complexes after incubation (separately or combined) at 4°C or 37°C, suggesting that the configuration necessary for the formation of the [ 3 H]-aldosterone complexes is altered by the extraction procedure. Incubation of the purified nucleus produces more [ 3 H]-aldosterone-macromolecule complexes (per mg of protein) than incubation with the total cell or with the crude nuclear fraction. These data suggest that unbound aldosterone can cross the nuclear membrane and form the aldosterone complexes without the participation of a cytosol intermediate.

Steroid hormone receptors in fetal guinea-pig kidney

Cytosol and nuclear subcellular fractions isolated from fetal kidney of guinea pigs (35–55 days of gestation) form steroid macromolecule complexes both “in vivo” and “in vitro” with [3H]-aldosterone or [3H]-estradiol but not with [3H]-progesterone. d-Aldosterone competes in the formation of the cytosol and nuclear [3H]-aldosterone complexes but estradiol has no effect. Estradiol competes in the formation of the [3H]-estradiol complexes of both the cytosol and the nuclear extracts but d-aldosterone has no effect, suggesting the presence of two different receptors for these hormones in the fetal kidney during this period of development. In the [3H]-estradiol experiments 30 min after administration of this hormone to the fetus or 15 min after incubation at 37°C with the cell suspensions of the fetal kidney, the bulk of the [3H]-complexes (60–70%) is located in the nucleus; of this, 60% is found in the chromatin fraction. 62–95 % of the radioactivity in the complexes consists of non-metabolized estradiol. In addition to estradiol, estrone and estriol were found to compete for binding sites albeit less intensively, whereas testosterone and cortisol had no effect. Ultracentrifugation in a sucrose density gradient (in 0.5 M NaCl) of the 0.3 M NaCl and 1 M NaCl nuclear extracts yielded a component with a sedimentation coefficient of 3.7 S in which estradiol had a significant competitive effect. Experiments carried out with the [3H]-estradiol cytosol-complex obtained at 4°C and then reincubated with the purified nuclei at 4° or 37°C show that the quantity of [3H]-estradiol found in the different nuclear extracts was 2.5–3 times greater at 37°C than at 4°C. By the application of the Scatchard method, the presence of two binding sites was established, the first with a dissociation constant of 2.5 × 10−10 M and the second with a Kd of 7.7 × 10−9 M. When [3H]-estrone was administered subcutaneously “in situ” to the fetus, it was noted that most of the radioactive material in the 3H-macromolecule complexes of the cytosol and nuclear extracts of the kidney is [3H]-estradiol. It is concluded that during this period of fetal development (35–55 days of gestation): estradiol receptors are present in the fetal kidney; in the interconversion of estrone ai estradiol in the same fetal tissue the reaction proceeds predominantly in the formation of estradiol.

Estrogen concentrations and effect of estradiol on progesterone receptors in the fetal and new-born guinea-pigs

Abstract Estradiol concentration in plasma of fetal guinea-pigs, determined by radioimmunoassay, varies from 9 to 40 pg/ml and the concentration of estrone between 80 to 105 pg/ml. The concentrations of both estrogens do not change significantly during fetal evolution. The total number of specific estradiol binding sites in fetal guinea-pig uterus reaches 18 to 20pmol/mg DNA at the end of gestation, and the concentrations of estradiol plus estrone is ∼1500 pg per g of this tissue which correlates well with the number of specific binding sites occupied by the endogenous estrogens (2–4 pmol/mg DNA). In the fetal uterus, estradiol receptors appear at about mid-gestation (from 34 days) but progesterone receptors are detectable only after 50–52 days of gestation. After estradiol treatment of the pregnant guinea pig (1 mg/kg/day for 3 days), the following changes take place in the fetal uterus: (1) Induction of progesterone receptor protein at an earlier period (37–42 days) when this receptor is not detectable in control animals. (2) Five to ten-fold increase in progesterone receptors at the end of gestation. (3) An increase of [3H]-progesterone uptake by fetal uterus (localized by autoradiography). (4) The stimulation of progesterone receptors is of long duration. Five days after treatment the values are similar to those obtained 24 h after the administration of the last dose of estradiol. (5) Similar effects were noted also in fetal ovary, but not in lungs, kidney, brain and heart or placenta. (6) The induction of progesterone receptors by estradiol is significantly lower in the uterus of new-born guinea pigs (2–7 days old). It is concluded that estradiol expresses its biological effect in the fetal compartment, increasing the levels of progesterone receptors.

Contributions of studies on uncoupling proteins to research on metabolic diseases.

Abstract. Ricquier D, Fleury C, Larose M, Sanchis D, Pecqueur C, Raimbault S, Gelly C, Vacher D, Cassard‐Doulcier A‐M, Lévi‐Meyrueis C, Champigny O, Miroux B, Bouillaud F (Centre National de la Recherche Scientifique, Centre de Recherche sur l’Endocrinologie Moléculaire et le Développement, Meudon, France). Contributions of studies on uncoupling proteins to research on metabolic diseases. (Minisymposium: Genes & Obesity). J Intern Med 1999; 245: 637–642.

Biological responses of tamoxifen in the fetal and newborn vagina and uterus of the guinea-pig and in the R-27 mammary cancer cell line

The biological and morphological responses of tamoxifen were studied in two models: the uterus and vagina of fetal and newborn guinea-pigs: R-27 cells--a mammary cancer cell line (tamoxifen resistant) derived from the MCF-7 cancer cell line. Tamoxifen (TAM) alone or in combination with estradiol (E2) was administered to pregnant (50-52 days of gestation) or to newborn (2-day-old) guinea-pigs for a long period (12 days). TAM alone produced a great trophic effect on the uterus and vagina which was markedly enhanced when TAM was administered together with E2. Histological studies showed that TAM provokes morphological changes in both the endometria and the myometria and this effect was also greater when TAM was administered together with E2. In the fetal uterus and vagina, the ultrastructural studies showed that TAM induces morphological alterations in different cytoplasmic organelles. This effect was much more intense in newborns where TAM provoked a significant vacuolization of the epithelial cells. Concerning progesterone receptor (PR) in the fetal or newborn tissues (uterus or vagina) TAM provoked a less intense effect than those provoked by E2, but TAM did not block the effect provoked by E2. It was observed that [3H]TAM binds specifically to the estrogen receptor (ER) of fetal guinea pig uterus and this complex is partially recognized by a monoclonal antibody which recognizes the activated form of this receptor, supporting the suggestion that the biological action of TAM is mediated by the ER. The biological and ultrastructural effects provoked by TAM (1 X 10(-6) M), estriol (E3)(5 X 10(-8) M) and the combination of TAM + E3 were studied in the R-27 mammary cancer cell line in culture. E3 stimulated the PR content by 7-10 times. However, TAM did not provoke a significant decrease in the concentration of PR, and in the mixture of TAM + E3 the concentration of PR was of the same order as that in E3 treatment. Ultrastructural observations indicate an intense concentration of ribosomes in the pericytoplasmic area after exposure to E3 and with exposure to TAM an increase in vacuoles and a significant enlargement of the size of the mitochondria were observed. It is concluded that TAM in the target tissues of fetal and newborn guinea pigs acts as a real estrogen and in the R-27 mammary cancer cell line TAM does not block the effect provoked by E3, however it does provoke intense ultrastructural modifications.