Reischmann Patricia, Fiebeck Johanna, von der Weiden Nadine, Müller Oliver
University of Applied Sciences Kaiserslautern, Molecular Biology, Amerikastraße 1, 66482 Zweibrücken, Germany.
PHAST GmbH, Entenmühlstraße 48, 66424 Homburg, Germany.
Int J Cell Biol. 2015;2015:928502. doi: 10.1155/2015/928502. Epub 2015 Dec 22.
The Wnt signaling pathway has been associated with many essential cell processes. This study aims to examine the effects of Wnt signaling on proliferation of cultured HEK293T cells. Cells were incubated with Wnt3a, and the activation of the Wnt pathway was followed by analysis of the level of the β-catenin protein and of the expression levels of the target genes MYC and CCND1. The level of β-catenin protein increased up to fourfold. While the mRNA levels of c-Myc and cyclin D1 increased slightly, the protein levels increased up to a factor of 1.5. Remarkably, MTT and BrdU assays showed different results when measuring the proliferation rate of Wnt3a stimulated HEK293T cells. In the BrdU assays an increase of the proliferation rate could be detected, which correlated to the applied Wnt3a concentration. Oppositely, this correlation could not be shown in the MTT assays. The MTT results, which are based on the mitochondrial activity, were confirmed by analysis of the succinate dehydrogenase complex by immunofluorescence and by western blotting. Taken together, our study shows that Wnt3a activates proliferation of HEK293 cells. These effects can be detected by measuring DNA synthesis rather than by measuring changes of mitochondrial activity.
Wnt信号通路与许多重要的细胞过程相关。本研究旨在探讨Wnt信号对培养的HEK293T细胞增殖的影响。将细胞与Wnt3a一起孵育，通过分析β-连环蛋白的水平以及靶基因MYC和CCND1的表达水平来追踪Wnt信号通路的激活情况。β-连环蛋白的水平增加了四倍。虽然c-Myc和细胞周期蛋白D1的mRNA水平略有增加，但蛋白质水平增加了1.5倍。值得注意的是，在测量Wnt3a刺激的HEK293T细胞的增殖率时，MTT和BrdU检测显示出不同的结果。在BrdU检测中，可以检测到增殖率的增加，这与所应用的Wnt3a浓度相关。相反，在MTT检测中未显示出这种相关性。基于线粒体活性的MTT结果通过免疫荧光和蛋白质印迹法对琥珀酸脱氢酶复合物的分析得到了证实。综上所述，我们的研究表明Wnt3a激活了HEK293细胞的增殖。这些效应可以通过测量DNA合成来检测，而不是通过测量线粒体活性的变化来检测。
