Badia E, Duchesne M J, Semlali A, Fuentes M, Giamarchi C, Richard-Foy H, Nicolas J C, Pons M
Unité 439, Institut National de la Santé et de la Recherche Médicale, Montpellier, France.
Cancer Res. 2000 Aug 1;60(15):4130-8.
Antiestrogen resistance is frequently observed in patients after longterm treatment with tamoxifen, a nonsteroidal antiestrogen widely used for endocrine therapy of breast cancer. In vitro studies in resistant cells showed that the expression of natural estrogen-responsive genes is frequently altered. Using MVLN cells, an MCF-7-derived cell model, we previously demonstrated that 4-hydroxytamoxifen (OHT) treatment irreversibly inactivated an estrogen-regulated chimeric luciferase response by a direct effect of the drug and not through a cell selection process (E. Badia et al., Cancer Res., 54: 5860-5866, 1994). In the present study, we present tamoxifen-resistant but still estrogen-dependent clones isolated after long-term treatment of MVLN cells with OHT and show that progesterone receptor (PR) expression was irreversibly decreased in some of these clones, whereas the PRA:PRB ratio of residual PR remained unchanged. The irreversible inactivation of both chimeric luciferase gene and PR gene expression was associated with the disappearance of DNase 1-hypersensitive sites. In the case of the chimeric gene, at least one of these sites was close to the estrogen responsive element. Genomic sequencing analysis of a clone with very low PR content did not reveal any methylation on CpG dinucleotides or any mutation in the PR gene promoter region. In all of the resistant clones tested and independently of their PR content, estrogen receptor expression was only lowered by half and remained functional, whereas pS2 expression was not modified. We also observed that the residual luciferase activity level (1-2%) of the MVLN clones, the luciferase expression of which had been irreversibly inactivated, was raised 4-fold by trichostatin A treatment. We conclude that long-term OHT treatment may modify the chromatin structure and thus could contribute to differentially silencing natural target genes.
长期使用他莫昔芬治疗的乳腺癌患者中经常出现抗雌激素耐药性，他莫昔芬是一种广泛用于乳腺癌内分泌治疗的非甾体类抗雌激素药物。对耐药细胞的体外研究表明，天然雌激素反应基因的表达经常发生改变。我们之前使用源自MCF-7的细胞模型MVLN细胞证明，4-羟基他莫昔芬（OHT）处理通过药物的直接作用而非细胞选择过程不可逆地使雌激素调节的嵌合荧光素酶反应失活（E. Badia等人，《癌症研究》，54: 5860-5866，1994）。在本研究中，我们展示了用OHT长期处理MVLN细胞后分离得到的他莫昔芬耐药但仍依赖雌激素的克隆，并表明其中一些克隆中孕激素受体（PR）的表达不可逆地降低，而残留PR的PRA:PRB比值保持不变。嵌合荧光素酶基因和PR基因表达的不可逆失活与DNase 1超敏位点消失有关。就嵌合基因而言，这些位点中至少有一个靠近雌激素反应元件。对一个PR含量极低的克隆进行基因组测序分析，未发现CpG二核苷酸甲基化或PR基因启动子区域的任何突变。在所有测试的耐药克隆中，无论其PR含量如何，雌激素受体表达仅降低一半且仍保持功能，而pS2表达未改变。我们还观察到，其荧光素酶表达已不可逆失活的MVLN克隆的残留荧光素酶活性水平（1-2%）通过曲古抑菌素A处理提高了4倍。我们得出结论，长期OHT处理可能会改变染色质结构，从而可能导致天然靶基因的差异沉默。
