Monoclonal Antibody D547 Research Articles

A one minute pulse of estradiol to MCF-7 breast cancer cells changes estrogen receptor binding properties and commits cells to induce estrogenic responses.

Changes in estradiol (E2)-binding parameters can be detected within minutes, while the estrogenic responses are manifested after several hours or days of continuous exposure to the steroid. The goal of this study was to determine the time of commitment for the induction of transcription-dependent responses in the human breast cancer cell line MCF-7. In cultures grown in steroid-deprived serum, a pulse of 1 nM E2 as short as 1 min was sufficient to maximally increase the level of the progesterone receptor, as determined by binding of the progestin [3H]ORG.2058 after 2 days, and to partially stimulate cell proliferation for 5 days. From uptake experiments it was calculated that after 1 min about 7000 E2 molecules were bound per cell, enough to occupy 5% of the approx. 150,000 estrogen receptors per cell. Preincubating cells with unlabelled E2 for 1 min lead to a loss of [3H]E2-binding capacity. As analysed by Scatchard plot, this loss was due to a decrease in the number of exchangeable binding sites and, to a lesser extent, to an increase in the dissociation constant. For up to 30 min of E2-incubation the level of receptor protein remained constant as determined by immunoassay with the anti-ER monoclonal antibodies D547 and H222. The dissociation kinetics of [3H]E2 bound by MCF-7 cells after a 5 min pulse were biphasic, with the slower phase having a rate of 2.3 x 10(-3) min. This rate is characteristic of the activated ER. The estrogenic response is thus committed by E2 within less than 1 min and evoked by the activation of a small fraction of estrogen receptors.

RNA-induced transformation of the estrogen receptor detected by a monoclonal antibody which recognizes the activated receptor

The effect of RNA and polyribonucleotides on the estrogen receptor from fetal guinea pig uterus was studied through the analysis of the sedimentation properties of this receptor and its interaction with the monoclonal antibody D547. Different exogenous RNAs (calf thymus RNA, yeast RNA and rabbit liver transfer RNA) were able to induce a transformation of the 9S native receptor to 4.5–7S sedimenting forms in low salt sucrose density gradients, as activating factors such as temperature and time do. This transformation was prevented by 20mM sodium molybdate. Moreover, the RNA treated receptor was partially recognized by the monoclonal antibody D547. This antibody, as was demonstrated previously, selectively reacts with the activated form of this receptor. When different homo-polyribonucleotides were tested, the effect depended on their composition. In contrast, DNA did not affect either the sedimentation properties of the receptor or its reaction with the antibody. These observations suggest that RNA induces a dissociation of the 9S receptor and that at least one of the resulting forms is the activated receptor. However, RNA and polyribonucleotides inhibited the receptor binding to DNA-cellulose apparently by competing with DNA. The data suggest a role of RNA in estrogen receptor activation.

Binding of antiestrogens exposes an occult antigenic determinant in the human estrogen receptor.

Treatment of human breast cancer cytosol with tamoxifen (Tam) or 4-monohydroxytamoxifen (MHT) enhances the immunoreactivity of the estrogen receptor toward monoclonal antibody H222 but not monoclonal antibodies D547 or D75. This effect is evident from an increase in the apparent receptor content measured by the Abbott enzyme immunoassay, which uses peroxidase-labeled H222 as the chromogenic marker, and in the rate and size of the sedimentation peak of the immune complex of the receptor with radiolabeled H222. In contrast, MHT shows no effect in reversed immunoassay systems that use peroxidase-labeled D547 or D75 as chromogenic markers, nor does it affect the sedimentation peak of the complex of D547 with the receptor. MHT can exert its action on receptor bound to immobilized antibody. These results indicate that reaction with antiestrogens causes a change, probably conformational, in the receptor protein that exposes an occult antigenic determinant recognized uniquely by H222. Since this can occur in cytosol previously treated with excess estradiol in the cold, it appears to result from an interaction of antiestrogens with a region of the receptor distinct from the estrogen-binding site, suggesting that agonist and antagonist actions may involve different parts of the receptor molecule.

Immunological differences between the estradiol-, tamoxifen- and 4-hydroxy-tamoxifen-estrogen receptor complexes detected by two monoclonal antibodies

Two monoclonal antibodies (D547 and H222), obtained against the estrogen receptor from MCF-7 breast cancer cells, were used to study the estrogen receptor from fetal guinea-pig uterus bound to estradiol or to the antiestrogens tamoxifen and 4-hydroxytamoxifen. The estradiol-receptor complex binds partially to the monoclonal antibody D547, shifting its sedimentation coefficient in high salt sucrose density gradients from 4.5S to 7.5S. Recently, we demonstrated that the form selectively recognized by this monoclonal antibody is the activated form of the receptor. The estrogen receptor complexed with tamoxifen or 4-hydroxytamoxifen is also partially recognized by this monoclonal antibody but the fraction of total receptor bound to the antibody is significantly less than for the receptor complexed with estradiol. Another series of experiments showed that the monoclonal antibody H222, which recognizes a different antigenic site on the receptor molecule, binds all the estradiol-receptor complex (independently of the degree of activation), shifting its sedimentation coefficient to 7.5S. However, even if all the 4-hydroxytamoxifen-receptor complex is bound by this antibody, only a fraction of the receptor is recognized when it is complexed with tamoxifen. These data show different interactions between the estradiol-, tamoxifen- and 4-hydroxytamoxifen-receptor complexes and the two monoclonal antibodies tested and suggest that these compounds induce different conformational modifications of the estrogen receptor molecule.

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°C or 25°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.

Characterization of estrogen receptors and associated protein kinase activity by high-performance hydrophobic-interaction chromatography

We have determined that high-performance hydrophobic-interaction chromatography (HPHIC) with weakly hydrophobic columns permit the rapid separation of the labile isoforms of estrogen receptor proteins. Previously we reported the use of the SynChrom propyl 500 column for HPHIC of steroid receptors. However, due to the strongly hydrophobic characteristics of the ligand, [ 125Iiodoestradiol-17β, and the receptor protein, organic solvent was required in the mobile phase for greater recovery of receptor proteins. Here, we report separation of steroid receptors from human breast tumors and rat uteri, using the Beckman CAA-HIC, a non-ionic polyether-bonded column, without the need for organic solvents and with virtually 100% recoveries. Receptors were extracted in 10 m M phosphate buffer (pH 7.4). Maximum resolution and separation were acheived when a descending salt gradient of ammonium sulfate in phosphate buffer (pH 7.4) was used (2−0 M in 30 min.). Estrogen receptor (ER) was resolved into two isoforms with R = 22 ± 1 min ( n = 16, designated as peak I) and 27.5 ± 0.5 min ( n = 14, designated as peak II) and a purification of five- to twenty-fold in a single pass. Free steroid was eluted at t R = 35 ± 1 min ( n = 4). Separation was dependent on adjusting the ionic strength of cytosol to 1.5 M ammonium sulfate. ER, purified by HPHIC, retained ligand binding capacity and exhibited protein kinase activity, which was dominant in the less hydrophobic peak I ( t R = 22 min) when immunoprecipitated with the monoclonal antibody D547. This method of rapidly purifying ER with high retention of biological activity may now be applied to the study of the molecular interrelationships of steroid receptor isoforms.

The inhibition of prolactin synthesis in GH 3 rat pituitary tumor cells by monohydroxytamoxifen is associated with changes in the properties of the estrogen receptor

E 2 (1 nM) stimulated the synthesis of PRL in GH 3 cells. OH TAM (100 nM) did not affect basal PRL synthesis, but completely inhibited the increase produced by 1 nM E 2. [ 3H]E 2 and [ 3H]OH TAM both bound to the cytosolic 8S ER and these were split into 4S subunits on sucrose gradients containing 0.4 M KCl. By comparison, ER complexes extracted from nuclei of GH 3 cells cultured in media containing [ 3H]E 2 or [ 3H]OH TAM both sedimented at 5S on sucrose gradients containing 0.4 M KCl. Both 4S and 5S ER complexes were recognized by the monoclonal antibody D547 which increased their sedimentation coefficients to 8–9S. In contrast, a polyclonal antibody raised to calf uterine ER in the goat, interacted with the cytosolic ER so that the binding of [ 3H]E 2 was inhibited but the binding of [ 3]OH TAM was only slightly reduced. A molecular model is proposed to describe the binding of E 2 and OH TAM to the ER that might contribute to an understanding of estrogen and antiestrogen action.

Interaction of [3H] estradiol - and [3H] monohydroxytamoxifen-estrogen receptor complexes with a monoclonal antibody.

The aim of this study was to compare and contrast the interaction of estrogen [( 3H]17 beta-estradiol)- or antiestrogen [( 3H]monohydroxytamoxifen)-receptor complexes from human breast tumor cytosols with monoclonal antibodies raised to the human breast tumor estrogen receptor. Breast tumor cytosols containing estrogen receptor which sedimented as radiolabeled peaks in either the 8S, 8S and 4S, or 4S regions of sucrose density gradients, interacted with the monoclonal antibody D547 to produce a broad 9-10S peak, a broad 8S-10S peak, or a more discrete 8S peak, respectively. On high salt (0.4M KC1) sucrose density gradients the 4S ligand-receptor complex plus antibody produced a binding peak at approximately the 8S region of the gradient. These sedimentation studies with the monoclonal antibody D547, and similar studies with the monoclonal antibody D58, could detect no differences in the cytosolic estrogen receptor whether complexed with [3H]estradiol or with [3H]monohydroxytamoxifen. These observations were confirmed by Scatchard equilibrium saturation analysis and sucrose density gradient analysis of cytosols from the MCF-7 human breast cancer cell line. The antibody D547 interacted with 8S ER from these cytosols to produce a broad 8S-10S peak, but the antibody produced no change in the affinity or number of binding sites present in these cytosols. It seems, therefore, that the antigenic determinants recognized by these particular antibodies on the breast tumor cytosolic receptor are not significantly altered by the binding of either an estrogen or an antiestrogen to the receptor.