Rosado Aranzazú, Zanella Fabian, Garcia Beatriz, Carnero Amancio, Link Wolfgang
Experimental Therapeutics Program, Centro Nacional de Investigaciones Oncologicas, Madrid, Spain.
PLoS One. 2008 Mar 19;3(3):e1823. doi: 10.1371/journal.pone.0001823.
Inhibition of Akt signaling is considered one of the most promising therapeutic strategies for many cancers. However, rational target-orientated approaches to cell based drug screens for anti-cancer agents have historically been compromised by the notorious absence of suitable control cells.
METHODOLOGY/PRINCIPAL FINDINGS: In order to address this fundamental problem, we have developed BaFiso, a live-cell screening platform to identify specific inhibitors of this pathway. BaFiso relies on the co-culture of isogenic cell lines that have been engineered to sustain interleukin-3 independent survival of the parental Ba/F3 cells, and that are individually tagged with different fluorescent proteins. Whilst in the first of these two lines cell survival in the absence of IL-3 is dependent on the expression of activated Akt, the cells expressing constitutively-activated Stat5 signaling display IL-3 independent growth and survival in an Akt-independent manner. Small molecules can then be screened in these lines to identify inhibitors that rescue IL-3 dependence.
CONCLUSIONS/SIGNIFICANCE: BaFiso measures differential cell survival using multiparametric live cell imaging and permits selective inhibitors of Akt signaling to be identified. BaFiso is a platform technology suitable for the identification of small molecule inhibitors of IL-3 mediated survival signaling.
抑制Akt信号传导被认为是针对多种癌症最有前景的治疗策略之一。然而，基于细胞的抗癌药物筛选中，合理的靶向方法历来因缺乏合适的对照细胞而受到影响。
方法/主要发现：为了解决这一根本问题，我们开发了BaFiso，这是一种用于识别该信号通路特异性抑制剂的活细胞筛选平台。BaFiso依赖于同基因细胞系的共培养，这些细胞系经过工程改造，能够维持亲代Ba/F3细胞在无白细胞介素-3（IL-3）情况下的存活，并且分别用不同的荧光蛋白进行标记。在这两个细胞系中的第一个中，无IL-3时细胞存活依赖于活化Akt的表达，而组成性激活Stat5信号的细胞以不依赖Akt的方式显示出IL-3非依赖性生长和存活。然后可以在这些细胞系中筛选小分子，以鉴定能够恢复IL-3依赖性的抑制剂。
结论/意义：BaFiso使用多参数活细胞成像来测量细胞存活差异，并允许鉴定Akt信号传导的选择性抑制剂。BaFiso是一种平台技术，适用于鉴定IL-3介导的存活信号的小分子抑制剂。
