Demirpence Ediz, Semlali Abdelhabib, Oliva Joan, Balaguer Patrick, Badia Eric, Duchesne Marie-Josèphe, Nicolas Jean-Claude, Pons Michel
INSERM U439, 34090 Montpellier, France.
Cancer Res. 2002 Nov 15;62(22):6519-28.
We showed previously that prolonged treatment of a MCF-7-derived cell line with hydroxytamoxifen (OHT) induces the irreversible silencing of some estrogen-responsive genes, whereas OHT-resistant cell growth appears simultaneously (E. Badia et al., Cancer Res., 60: 4130-4138, 2000). Based on the hypothesis that particular gene silencings could be involved in triggering the resistance phenomenon, we focused our study on the mechanism of OHT-induced silencing. More precisely, we wished to determine to what extent the recruited histone deacetylase (HDAC) activity, which is known to be involved in the repressive effect induced by antagonist ligands of nuclear receptors, could participate in various aspects of OHT effects, particularly in gene silencing. A fusion protein (HDAC-EG) of human HDAC1 fused with the estrogen receptor DNA-binding domain and the glucocorticoid receptor ligand-binding domain allowed targeting of chimeric HDAC1 activity on estrogen-responsive elements (EREs) in the presence of glucocorticoid ligands. When HDAC-EG was transiently expressed in HeLa cells together with estrogen receptor, an antiestrogen-like effect was obtained on an ERE-controlled luciferase reporter gene in the presence of agonist or antagonist glucocorticoids. In MCF-7-derived cells stably expressing HDAC-EG and an estrogen-regulated luciferase, liganded HDAC-EG again produced an antiestrogenic effect on expression of natural estrogen-regulated genes such as pS2, progesterone receptor, and cathepsin D and cell growth together with chimeric luciferase gene expression. However, a prolonged HDAC-EG-mediated antiestrogen effect did not lead to irreversible luciferase gene silencing, as OHT does. It nevertheless accelerated the OHT-driven phenomenon. The antiestrogen effect of OHT thus differs from that of an ERE-targeted HDAC1 activity that might participate in irreversible silencing but is not sufficient to trigger it.
我们之前的研究表明，用羟基他莫昔芬（OHT）对源自MCF-7的细胞系进行长时间处理会导致一些雌激素反应性基因的不可逆沉默，而同时出现对OHT耐药的细胞生长现象（E. Badia等人，《癌症研究》，60: 4130 - 4138，2000）。基于特定基因沉默可能参与引发耐药现象这一假说，我们将研究重点放在OHT诱导沉默的机制上。更确切地说，我们希望确定已知参与核受体拮抗剂配体诱导的抑制作用的募集组蛋白脱乙酰酶（HDAC）活性在OHT效应的各个方面，特别是在基因沉默中能发挥多大作用。一种将人HDAC1与雌激素受体DNA结合结构域和糖皮质激素受体配体结合结构域融合的融合蛋白（HDAC-EG），使得在存在糖皮质激素配体的情况下，嵌合HDAC1活性能够靶向雌激素反应元件（ERE）。当HDAC-EG与雌激素受体在HeLa细胞中瞬时共表达时，在存在激动剂或拮抗剂糖皮质激素的情况下，对ERE控制的荧光素酶报告基因产生了抗雌激素样效应。在稳定表达HDAC-EG和雌激素调节荧光素酶的源自MCF-7的细胞中，结合配体的HDAC-EG再次对天然雌激素调节基因如pS2、孕激素受体和组织蛋白酶D的表达以及细胞生长产生抗雌激素作用，并伴有嵌合荧光素酶基因表达。然而，与OHT不同，HDAC-EG介导的长时间抗雌激素作用并未导致荧光素酶基因的不可逆沉默。不过，它加速了OHT驱动的现象。因此，OHT的抗雌激素作用不同于靶向ERE的HDAC1活性的抗雌激素作用，后者可能参与不可逆沉默，但不足以引发这种沉默。
