Borrás M, Laios I, el Khissiin A, Seo H S, Lempereur F, Legros N, Leclercq G
Laboratoire J-C. Heuson de Cancérologie Mammaire, Service de Médecine Interne, Institut Jules Bordet, Brussels, Belgium.
J Steroid Biochem Mol Biol. 1996 Feb;57(3-4):203-13. doi: 10.1016/0960-0760(95)00272-3.
Effect of estrogens and antiestrogens (AEs) on estrogen receptor (ER) half-life was analyzed in MCF-7 cells by assessing its progressive disappearance after covalent labeling in situ with [3H]tamoxifen aziridine ([3H]TAZ). Cells were incubated for 1 h with 20 nM [3H]TAZ either in the absence or presence of a 500-fold excess of unlabeled estradiol (E2) (non-specific binding). The entire ER population was labeled by this method as established by subsequent incubation of the cells with [125I]E2. [3H]TAZ labeled cells were maintained in culture for additional 5 h in the absence (control) or presence of increasing amounts (0.1 nM - 1 microM) of either a given estrogen (E2, estrone, diethylstilbestrol, bisphenol), a pure AE (RU 58 668, ICI 164 384) or an AE with residual estrogenic activity (RU 39 411, 4-hydroxytamoxifen, keoxifene). The progressive disappearance of nuclear and cytosolic [3H]TAZ-ER complex during 5 h incubation were assessed by their immunoprecipitation with anti-ER monoclonal antibody (H 222) followed by scintillation counting or SDS-PAGE and fluorography. Fading of labeled receptors was extremely slow (approximately 10% loss after 6 h) in absence of any hormone/antihormone indicating a long half-life of the [3H]TAZ-ER complex. Addition of estrogens as well as pure AEs led to a dramatic reduction 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 disappearance induced by estrogens was closely related to their binding affinity for ER. Newly synthesized ER emerged during the treatment with hormones or antihormones seems to be implicated in the phenomenon since [3H]TAZ was covalently bound and could, therefore, not be displaced by these compounds. Induction of synthesis of a short half-life peptide(s) with degradative activity was demonstrated by addition of cycloheximide or puromycine (both at 50 microM) which completely blocked ER disappearance. 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 reaches the steady-state level.
通过评估用[3H]他莫昔芬氮丙啶（[3H]TAZ）原位共价标记后雌激素受体（ER）的逐渐消失情况，分析了雌激素和抗雌激素（AEs）对MCF-7细胞中ER半衰期的影响。细胞在不存在或存在500倍过量未标记雌二醇（E2）（非特异性结合）的情况下，用20 nM [3H]TAZ孵育1小时。如随后用[125I]E2孵育细胞所证实的，通过这种方法标记了整个ER群体。[3H]TAZ标记的细胞在不存在（对照）或存在递增剂量（0.1 nM - 1 microM）的给定雌激素（E2、雌酮、己烯雌酚、双酚）、纯抗雌激素（RU 58 668、ICI 164 384）或具有残留雌激素活性的抗雌激素（RU 39 411、4-羟基他莫昔芬、凯昔芬）的情况下，在培养中再维持5小时。通过用抗ER单克隆抗体（H 222）进行免疫沉淀，随后进行闪烁计数或SDS-PAGE及荧光自显影，评估在5小时孵育期间核和胞质[3H]TAZ-ER复合物的逐渐消失情况。在不存在任何激素/抗激素的情况下，标记受体的消退极其缓慢（6小时后约损失10%），表明[3H]TAZ-ER复合物的半衰期很长。添加雌激素以及纯抗雌激素导致半衰期显著缩短，而具有残留雌激素活性的抗雌激素在这方面的效率极低，这为后一类化合物上调受体的能力提供了解释，因为它们不影响ER mRNA的合成和稳定性。雌激素诱导的受体消失与其对ER的结合亲和力密切相关。在用激素或抗激素处理期间新合成的ER似乎与该现象有关，因为[3H]TAZ是共价结合的，因此不能被这些化合物取代。添加放线菌酮或嘌呤霉素（均为50 microM）证明诱导了具有降解活性的短半衰期肽的合成，这完全阻断了ER的消失。SDS-PAGE未检测到ER的裂解产物这一事实表明是溶酶体水解。因此，仅对部分ER的激素调节可能会下调其总数，直至达到稳态水平。
