Françoise Capony

Formation of estrogen nuclear receptor in uterus: Effect of androgens, estrone and nafoxidine

Abstract Different steroids and nafoxidine were tested in vitro on rat uteri for their ability to transfer the estrogen cytosol receptor (Rc) to the nucleus. This transfer was evaluated by testing the simultaneous decrease of the number of specific binding sites for estradiol in cytosol and increase of the same binding sites in nuclear extracts. Three classes of compounds could be distinguished according to their affinity for Rc and activity to transfer Rc to the nucleus ; those which bind and transfer Rc (estradiol, estrone and nafoxidine), those which neither bind nor transfer Rc (progesterone, cortisol) and finally those which do not bind Rc, but transfer it to the nucleus (androgens). It is suggested that androgens modify membrane permeability for the estrogen cytosol receptor.

The 52-kDa estrogen-induced protein secreted by MCF7 cells is a lysosomal acidic protease

An estrogen-induced 52-kDa glycoprotein secreted by human breast cancer cells and able to autostimulate the growth of MCF7 cells has been purified, using monoclonal antibodies, and characterized. The protein contains mannose 6-phosphate signals on its N-linked high-mannose chains, suggesting that it is a lysosomal enzyme. Both the secreted 52-kDa protein and its processed cellular forms (52-, 48- and 34-kDa) were identified as carboxyl proteinases having an optimal activity at pH 3.5 and being specifically inhibited by pepstatin. This protease is characterized by its inducibility by estrogens and its high concentration in proliferative benign and malignant mammary tissue, when detected by immunohistochemistry. The estrogen-induced secretion of this protease may help to understand how estrogens stimulate mammary tumor growth and/or invasion.

High-affinity binding of the antiestrogen [3H]tamoxifen to the 8S estradiol receptor

The interaction of tamoxifen (ICI 46,474), a synthetic antiestrogen, with uterine cytosol proteins of immature calf and rat has been studied directly using the tritiated compound labeled with a high specific activity. The binding complexes were measured by the dextrancoated charcoal, protamine sulfate and hydroxyapatite assays. Scatchard plots revealed a single class of high-affinity (KD congruent to 1.7 nM) binding sites, with a binding capacity similar to that of estradiol. Competitive experiments showed the same binding specificity for estrogens and antiestrogens. Sucrose gradient analysis revealed an 8S binding protein which could be partially proteolysed by trypsin into a 4S binding protein. Kinetic studies showed that the association rate of tamoxifen was 5 times lower than that of estradiol and reacted according to a second order kinetics. The first-order kinetics of dissociation was considerably higher than that of estradiol, giving a half-dissociation time of 20--40 min at 0--2 degrees C. In some cases tamoxifen displayed two slopes of dissociation, but the proportion of the slow-dissociating complex was always inferior to that found with estradiol. In contrast to estradiol, the kinetic constants ratio (k-/k+) gave a calculated dissociation constant, similar to that determined in equilibrium conditions (KD), agreeing with a simple reactional scheme. We conclude that the antiestrogen tamoxifen binds directly to the 8S cytosol receptor for estrogens and not to another receptor for the antagonists. In contrast to estradiol, the antagonist is rapidly dissociated from the receptor sites and is unable to protect them against thermal inactivation. The affinity of tamoxifen for its receptor sites as determined directly is surprisingly high when compared to its affinity evaluated indirectly by competitive experiments. It is then suggested that the two ligands either bind on two different sites of the same protein or induce a different conformational change of the same binding site.

Estradiol dependent decrease of binding inhibition by anti-estrogens (a possible test of receptor activation)

Abstract The interaction of the uterine estrogen receptor (R) with three anti-estrogens, Dimethylstilbestrol 1 , Tamoxifen and Nafoxidine, have been studied either indirectly by competitive experiments or directly using radioactive compounds. The affinity of these anti-estrogens for R was found much higher when determined directly without estradiol (E2) than when evaluated by competitive experiments. In the presence of E2, but not in its absence, the inhibitory activity of the anti-estrogens decreased slowly with time. The present report strongly suggests that R is transformed by E2 into a form less sensitive to anti-estrogen. This “desensitization” of R to the estrogen antagonists is proposed as another in vitro test for the E2 induced “activation” of its receptor.

Effect of Antiestrogens on Uterine Estradiol Receptors

Synthetic and natural antiuterotrophic and uterotrophic compounds were tested invitro in their ability to bind to the uterine cytosol receptor for estradiol (Rc) and to favor the formation of the nuclear receptor. These compounds were compared to estradiol in their capacity to decrease the number of specifically bound estradiol in cytosol and increase those in nuclear extracts when incubated with uteri invitro. For each compound, a ‘nuclear transfer activity’ was defined and compared on the one hand to its binding affinity for Rc and on the other hand to its invivo uterotrophic activity. For estrogens devoided of antiestrogenic activity, estradiol and estrone, the nuclear transfer activity was parallel to the affinity for Re and the biological activity. The antiestrogen nafoxidine (U11,100) when added to the cytosol was found to be a reversible and competitive inhibitor of estradiol for Re with an affinity about 30-fold less than estradiol (Ki 7 nM). A complete inhibition could be obtained both before and after the dissociation of Rc by KCl. However, these binding properties of nafoxidine were not in agreement with its partial nuclear transfer activity and its partial uterotrophic activity. The androgens do not bind to Rc; however, they were shown to activate the transfer of the estrogen Rc to the nucleus whereas progesterone did not. These results are discussed in relation to the mechanism of action of uterotrophic and antiuterotrophic compounds.

Estradiol increases the production of α1-antichymotrypsin in MCF7 and T47D human breast cancer cell lines

alpha 1-Antichymotrypsin (Achy) is an antiprotease of the acute inflammation phase, which is also released by MCF7 human breast cancer cells in culture. Using a fluorimetric assay with the synthetic substrate L-Seryl-L-Tyrosyl-2-N-naphthylamide, we have shown that a medium conditioned by MCF7 cells treated by estradiol inhibits the activity of alpha-chymotrypsin. This inhibition increased when physiological concentrations of estradiol were added to the cells for 2 days. It was due to an increased production of Achy and not to a direct effect of estradiol on alpha-chymotrypsin activity as shown by double immunoprecipitation with an antiserum against human alpha 1-antichymotrypsin. An increased accumulation by estradiol of an antigen located in the cytoplasm of MCF7 cells, which was revealed by immunoperoxidase staining with antibodies to Achy, also indicated that estradiol increased the production of Achy in these cells. Similar immunostaining was observed in a breast cancer tissue. Most of the estrogen regulated 60-68 kDa protein secreted by T47D cells (Chalbos et al. (1982) J. Clin. Endocrinol. Metab. 55, 276-283) was also specifically immunoprecipitated by the antibodies to Achy. Thus, alpha 1-antichymotrypsin is the first protein to be identified which is induced by estradiol and secreted by breast cancer cells.

In vitro and in vivo interactions of [3H]dimethylstilbestrol with the estrogen receptor ☆

The binding of an antiestrogen with the uterine estrogen receptor (R) has been studied directly in vitro using tritiated dimethylstilbestrol (DMS). The affinity of DMS for R as determined at equilibrium was similar to that of estradiol-17beta (E2) (K(D) approximately .3 nN) when taking into account the higher nonspecific binding of DMS. The number of DMS binding sites was constantly found to be inferior to that of E2. The fact that the DMS binding entity specifically bound estrogen and antiestrogen, was destroyed by pronase, displayed an 8S sedimentation constant, and interacted in vitro with DNA, strongly suggested that DMS interacted directly with R. The association of DMS to R was a simple 2nd-order reaction while its dissociation was a 1st-order reaction with 2 slopes. The association and dissociation rate constants of the R-DMS complex were, respectively, slower and higher than those of the R-E2 complex. The rapid dissociation rate of DMS could be responsible for its inability to protect the receptor binding sites against thermo-inactivation. Tritiated DMS was able in vivo to induce the nuclear translocation of the receptor. However, as with other short-acting antiestrogens and contrary to Nafoxidine, the time of nuclear retention of R was short. These results are in agreement with the assumption that the length of the nuclear retention of R is determinant in explaining the weak agonist activity of this compound.

Estradiol increases the secretion by MCF7 cells of several lysosomal pro-enzymes.

The synthesis and secretion of pro-cathepsin D is increased by estrogens in MCF7 cells. We quantified the effect of estradiol on other lysosomal enzymes in order to investigate the mechanism of this hypersecretion. Precursors of beta-hexosaminidase, cathepsin B and beta-galactosidase, which are routed to lysosomes via the mannose-6-phosphate (Man-6-P) receptor, were secreted in much lower amounts than pro-cathepsin D, but their secretion was also increased by estradiol. The activity of acid phosphatase, which is routed to lysosomes via a different transmembrane mechanism, was not altered by estradiol. While estradiol stimulated gene expression of pro-cathepsin D, it had no effect on that of pro-cathepsin B. We conclude that estradiol stimulates the secretion of several lysosomal pro-enzymes in MCF7 cells, suggesting that a general mechanism is responsible for this derouting rather than a specific alteration of cathepsin D structure.

Antibodies to the estrogen induced 52 K protein released by human breast cancer cells

Abstract Polyclonal antibodies against an estrogen induced 52 K protein released by human breast cancer cells have been developed by injecting rabbits with a crude cellular pellet of MCF7 human breast cancer cells. The rabbit antisera have been tested against [35S]Methionine labelled proteins released by the MCF7 cells followed by separation of the immune complexes with Protein A Sepharose. In spite of their low specificity and titer, these antisera allowed us to investigate the release of the 52 K protein in vitro by other mammary cancer or normal cells.

Effect of tunicamycin and endoglycosidase H and F on the estrogen regulated 52000-Mr protein secreted by breast cancer cells

The glycosylation and immunoreactivity of an estrogen regulated glycoprotein secreted by breast cancer cells in culture and defined by its molecular mass (52 000-Mr protein) have been studied indirectly using an inhibitor of glycosylation and specific endoglycosidases. The protein and its deglycosylated forms were immunoprecipitated with specific monoclonal antibodies to the 52 000-Mr protein and analyzed by SDS polyacrylamide gel electrophoresis. The 52 000-Mr protein was intensely labelled by [3H] mannose or [35S] methionine. Tunicamycin treatment of the cells, endoglycosidase H or endoglycosidase F digestion of conditioned media, gave two identical deglycosylated forms of 50 000-Mr and 48 000-Mr which remained immunoreactive. The 48 000-Mr protein, in contrast to the 52 000 and 50 000-Mr proteins, was unable to bind concanavalin A. The 52 000-Mr protein was resolved into five spots of decreasing pI on two-dimensional gels following immunoprecipitation. Endoglycosidase H treatment decreased the molecular weight and reduced the intensity of spots of lower pI, suggesting that the N-glycosylated chains contain acidic molecules. We conclude that: The 52 000-Mr secreted protein contains at least two high mannose or hybrid N-glycosylated chains of approximately 2,000 molecular weight corresponding to 8% of the mass of the 52 000-Mr protein. The two types of monoclonal antibodies (site 1 and 2) raised against the 52 000-Mr glycoprotein are still able to recognize the 48 000-Mr N-deglycosylated form indicating that they do not interact with the N-glycosylated moiety of the molecule.