Centre for Biological Systems Analysis (ZBSA), Albert-Ludwigs-University Freiburg, Habsburgerstraße 49, 79104 Freiburg, Germany.
Cell Commun Signal. 2011 Jul 13;9:17. doi: 10.1186/1478-811X-9-17.
Non-transformed mammary epithelial cell lines such as MCF-10A recapitulate epithelial morphogenesis in three-dimensional (3D) tissue culture by forming acinar structures. They represent an important tool to characterize the biological properties of oncogenes and to model early carcinogenic events. So far, however, these approaches were restricted to cells with constitutive oncogene expression prior to the set-up of 3D cultures. Although very informative, this experimental setting has precluded the analysis of effects caused by sudden oncoprotein expression or withdrawal in established epithelial cultures. Here, we report the establishment and use of a stable MCF-10A cell line (MCF-10Atet) fitted with a novel and improved doxycycline (dox)-regulated expression system allowing the conditional expression of any transgene.
MCF-10Atet cells were generated by stable transfection with pWHE644, a vector expressing a second generation tetracycline-regulated transactivator and a novel transcriptional silencer. In order to test the properties of this new repressor/activator switch, MCF-10Atet cells were transfected with a second plasmid, pTET-HABRAF-IRES-GFP, which responds to dox treatment with the production of a bi-cistronic transcript encoding hemagglutinin-tagged B-Raf and green fluorescent protein (GFP). This improved conditional expression system was then characterized in detail in terms of its response to various dox concentrations and exposure times. The plasticity of the phenotype provoked by oncogenic B-RafV600E in MCF-10Atet cells was analyzed in 3D cultures by dox exposure and subsequent wash-out.
MCF-10Atet cells represent a tightly controlled, conditional gene expression system. Using B-RafV600E as a model oncoprotein, we show that its sudden expression in established 3D cultures results in the loss of acinar organization, the induction of an invasive phenotype and hallmarks of epithelial-to-mesenchymal transition (EMT). Importantly, we show for the first time that this severe transformed phenotype can be reversed by dox wash-out and concomitant termination of oncogene expression.
Taken together, we have generated a stable MCF-10A subline allowing tight dox-controlled and reversible expression of any transgene without the need to modify its product by introducing artificial dimerization or ligand-binding domains. This system will be very valuable to address phenomena such as EMT, oncogene addiction, oncogene-induced senescence and drug resistance.
非转化的乳腺上皮细胞系，如 MCF-10A，通过形成腺泡结构在三维（3D）组织培养中再现上皮形态发生。它们是表征致癌基因生物学特性和模拟早期致癌事件的重要工具。然而，到目前为止，这些方法仅限于在建立 3D 培养物之前具有组成型致癌基因表达的细胞。尽管非常有信息，但这种实验设置排除了分析在已建立的上皮培养物中突然表达或撤回癌蛋白表达引起的影响。在这里，我们报告了一种稳定的 MCF-10A 细胞系（MCF-10Atet）的建立和使用，该细胞系配备了一种新的改良的强力霉素（dox）调控表达系统，允许任何转基因的条件表达。
通过稳定转染 pWHE644 生成 MCF-10Atet 细胞，该载体表达第二代四环素调控的转录激活子和新型转录沉默子。为了测试这种新的阻遏物/激活物开关的特性，将 MCF-10Atet 细胞用第二质粒 pTET-HABRAF-IRES-GFP 转染，该质粒对 dox 处理的反应是产生编码带有血凝素标签的 B-Raf 和绿色荧光蛋白（GFP）的双顺反子转录本。然后，详细研究了该改进的条件表达系统对不同 dox 浓度和暴露时间的反应。在 3D 培养物中，通过 dox 暴露和随后的冲洗来分析致癌 B-RafV600E 引起的 MCF-10Atet 细胞表型的可塑性。
MCF-10Atet 细胞代表一种严格控制的、条件性基因表达系统。使用 B-RafV600E 作为模型致癌蛋白，我们表明其在已建立的 3D 培养物中的突然表达导致腺泡组织的丧失、侵袭表型的诱导和上皮-间充质转化（EMT）的特征。重要的是，我们首次表明，这种严重的转化表型可以通过 dox 冲洗和同时终止致癌基因表达来逆转。
总之，我们生成了一种稳定的 MCF-10A 亚系，允许任何转基因的紧密 dox 控制和可逆表达，而无需通过引入人工二聚化或配体结合结构域来修饰其产物。该系统对于解决 EMT、致癌基因成瘾、致癌基因诱导的衰老和耐药性等现象将非常有价值。
