Journal of Steroid Biochemistry & Molecular Biology 87 (2003) 207–221 Mechanisms governing the accumulation of estrogen receptor alpha in MCF-7 breast cancer cells treated with hydroxytamoxifen and related antiestrogens Ioanna La¨ ıos a , Fabrice Journe b , Guy Laurent c , Denis Nonclercq c , Robert-Alain Toillon a , Hye-Sook Seo a , Guy Leclercq a,∗ a Laboratoire J.-C. Heuson de Cancérologie Mammaire, Service de Médecine Interne, Institut Jules Bordet, Université Libre de Bruxelles, 1 rue Héger-Bordet, B-1000 Brussels, Belgium b Laboratoire d’Endocrinologie et de Métabolisme Osseux, Service de Médecine Interne, Institut Jules Bordet, Université Libre de Bruxelles, 1 rue Héger-Bordet, B-1000 Brussels, Belgium c Service d’Histologie et de Cytologie Expérimentale, Faculté de Médecine et de Pharmacie, Université de Mons-Hainaut, 6 Avenue du Champ de Mars, B-7000 Mons, Belgium Received 10 February 2003; accepted 9 September 2003 Abstract This study aimed at a better understanding of estrogen receptor  (ER) up regulation induced by partial estrogen antagonists. Effect of treatment with hydroxytamoxifen (OH-Tam) on ER level in MCF-7 cells was investigated by an approach combining ER measurement (enzyme immunoassay) and morphological demonstration (immunofluorescence). Furthermore, the influence of drug exposure on the rates of ER synthesis and degradation was assessed by determining [ 35 S]methionine incorporated into the receptor in different experimental conditions (measurement of synthesis or pulse-chase experiments). ER up regulation was already induced by a 1-h pulse treatment with OH-Tam, thus a continuous exposure was not required. This process appeared reversible (i.e. ER accumulation due to OH-Tam rapidly vanished upon subsequent exposure to 17-estradiol (E 2 ) or the pure antiestrogen RU 58668). While OH-Tam did not affect the rate of [ 35 S]methionine incorporation into ER, it clearly caused an impairment of ER degradation (pulse-chase experiments) indicating that up regulation results from a stabilization of the receptor associated with the maintenance of its synthesis. Various tamoxifen derivatives, as well as a few related partial antiestrogens, were compared on the basis of binding ability and propensity to induce ER up regulation. A close relationship was found between both properties. Structure-activity analysis revealed that the capacity of these compounds to induce ER up regulation is associated with characteristics of their aminoalkyle side-chain, similar to those required for antiestrogenicity. © 2003 Elsevier Ltd. All rights reserved. Keywords: Breast cancer cell; MCF-7; Partial antiestrogen; Receptor turnover; Receptor up regulation 1. Introduction Considerable attention has been paid to the mechanism of action of triphenylethylenic antiestrogens after they were demonstrated to antagonize the development of breast can- cers, especially those expressing the estrogen receptor  (ER). Among these drugs, the partial antiestrogen tamox- ifen (Tam) has become a reference compound in view of its high clinical efficacy and lack of major side effects [1–4]. As a matter of fact, Tam still remains the most widely used antiestrogen for the treatment of breast cancer at different stages. ∗ Corresponding author. Tel.: +32-2-5413744; fax: +32-2-5413498. E-mail address: lcanmamm@ulb.ac.be (G. Leclercq). Experimental studies conducted with the MCF-7 breast cancer cell line have clearly shown that short-term ex- posure to Tam, as well as to its active metabolite 4-hydroxytamoxifen (OH-Tam), leads to a significant in- crease of ER content (up regulation) [5–7]. Actually, addi- tional investigations with other partial antiestrogens reveal that ER up regulation could be a hallmark feature of this particular class of pharmacological compounds [8,9]. This behavior contrasts with that observed with other ligands (i.e. estrogens, pure antiestrogens), which down regulate the receptor [10,11]. ER up regulation upon Tam treatment is associated with its strong anchorage to the nuclear matrix [12], which results in a progressive loss of 17-estradiol (E 2 ) binding ability [13]. The “partial antiestrogenicity” of Tam suggests that this 0960-0760/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2003.09.011