ESTROGENIC AND ANTIESTROGENIC REGULATION OF THE HALF-LIFE OF COVALENTLY LABELED ESTROGEN-RECEPTOR IN MCF-7 BREAST-CANCER CELLS

Effect of estrogens and antiestrogens (AEs) on estrogen receptor (ER) half-life was analyzed in MCF-7 cells by assessing its progressive dis appearance after covalent labeling in situ with [H-3]tamoxifen aziridi ne ([H-3]TAZ). Cells were incubated for 1 h with 20 nM [H-3]TAZ either in the absence or presence of a 500-fold excess of unlabeled estradio l (E(2)) (non-specific binding). The entire ER population was labeled by this method as established by subsequent incubation of the cells wi th [I-125]E(2). [H-3]TAZ labeled cells were maintained in culture for additional 5 h in the absence (control) or presence of increasing amou nts (0.1 nM-1 mu M) of either a given estrogen (E(2), estrone, diethyl stilbestrol, bisphenol), a pure AE (RU 58 668, ICI 164 384) or an AE w ith residual estrogenic activity (RU 39 411, 4-hydroxytamoxifen keoxif ene). The progressive disappearance of nuclear and cytosolic [H-3]TAZ- ER complex during 5 h incubation were assessed by their immunoprecipit ation with anti-ER monoclonal antibody (H 222) followed by scintillati on counting or SDS-PAGE and fluorography. Fading of labeled receptors was extremely slow (approximate to 10% loss after 6 h) in absence of a ny hormone/antihormone indicating a long half-life of the [H-3]TAZ-ER complex. Addition of estrogens as well as pure AEs led to a dramatic r eduction of the half-life while AEs with residual estrogenic activity were extremely less efficient in this regard providing an explanation for the ability of latter compounds to up-regulate the receptor since they do not affect ER mRNA synthesis and stability. Receptor disappear ance induced by estrogens was closely related to their binding affinit y for ER. Newly synthesized ER emerged during the treatment with hormo nes or antihormones seems to be implicated in the phenomenon since [H- 3]TAZ was covalently bound and could, therefore, not be displaced by t hese compounds. Induction of synthesis of a short half-life peptide(s) with degradative activity was demonstrated by addition of cycloheximi de or puromycine (both at 50 mu M) which completely blocked ER disappe arance. The fact that no cleavage products of ER were detected by SDS- PAGE suggested a lysosomial hydrolysis. Hence, hormonal modulation of only a part of ERs may down-regulate their total population until it r eaches the steady-state level. Copyright (C) 1996 Elsevier Science Ltd