A Dual-Color Fluorescence-Based Platform to Identify Selective Inhibitors of Akt Signaling
A Dual-Color Fluorescence-Based Platform to Identify Selective Inhibitors of Akt Signaling
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DOI：
10.1371/journal.pone.0001823
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发表时间：
2008-03-19
期刊：
影响因子：
3.7
通讯作者：
Link, Wolfgang
中科院分区：
文献类型：
--
作者：
Rosado, Aranzazu;Zanella, Fabian;Link, Wolfgang
背景：Akt 信号传导的抑制被认为是许多癌症最有前途的治疗策略之一。然而，基于细胞的抗癌药物筛选的合理靶向方法历来因缺乏合适的对照细胞而受到损害。方法/主要发现：为了解决这一根本问题，我们开发了 BaFiso，一个活细胞筛选平台，用于识别该途径的特定抑制剂。 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，可维持亲本 Ba/F3 细胞独立于白细胞介素 3 的存活，并且单独标记有不同的荧光蛋白。虽然在这两个系中的第一个中，在没有 IL-3 的情况下细胞的存活取决于激活的 Akt 的表达，但表达组成型激活的 Stat5 信号传导的细胞以不依赖于 Akt 的方式表现出不依赖于 IL-3 的生长和存活。然后可以在这些细胞系中筛选小分子，以鉴定可挽救 IL-3 依赖性的抑制剂。结论/意义：BaFiso 使用多参数活细胞成像测量差异细胞存活率，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 介导的生存信号传导的小分子抑制剂。
Background: 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.