Coquelle Arnaud, Mouhamad Shahul, Pequignot Marie O, Braun Thorsten, Carvalho Gabrielle, Vivet Sonia, Métivier Didier, Castedo Maria, Kroemer Guido
Centre National de la Recherche Scientifique, UMR8125, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France.
Biochem Pharmacol. 2006 Nov 30;72(11):1396-404. doi: 10.1016/j.bcp.2006.04.014. Epub 2006 Apr 29.
The susceptibility of cells to apoptosis induction is deeply influenced by their position in the cell cycle. Unfortunately, however, current methods for the enrichment of cells in defined phases of the cell cycle are mostly based on the synchronization of cells by agents or conditions that are intrinsically toxic and induce apoptosis on their own. We developed a novel procedure for the purification of cells in distinct phases of the cell cycle. This method is based on the stable transfection of cells with a chimeric protein made up by histone H2B and green fluorescent protein (GFP). Cytofluorometric purification of cells defined by their size and their H2B-GFP-dependent fluorescence (which reflects chromatin and hence DNA content) allowed for the efficient separation of diploid and tetraploid cells in the fluorescence-activated cell sorter (FACS). Moreover, when applied to diploid cells, this method allowed for the enrichment of live, functional cells in the G1, S and G2 phases of the cell cycle. FACS-purified cells were viable and readily resumed the cell cycle upon reculture. While staurosporine was equally toxic for cells in any phase of the cell cycle, camptothecin was particularly toxic for cells in the S phase. Moreover, BAY11-7082, a specific inhibitor of the IKK complex required for NF-kappaB activation, exhibited a particular cell cycle-specific profile of toxicity (G2>S>G1). These results delineate a novel procedure for studying the intersection between cell cycle regulation and cell death mechanisms.
细胞对凋亡诱导的敏感性深受其在细胞周期中所处位置的影响。然而，遗憾的是，目前用于富集处于特定细胞周期阶段细胞的方法大多基于用本质上具有毒性且自身会诱导凋亡的试剂或条件使细胞同步化。我们开发了一种用于纯化处于不同细胞周期阶段细胞的新方法。该方法基于用由组蛋白H2B和绿色荧光蛋白（GFP）组成的嵌合蛋白对细胞进行稳定转染。通过细胞荧光分选法根据细胞大小及其依赖H2B - GFP的荧光（反映染色质进而反映DNA含量）对细胞进行纯化，能够在荧光激活细胞分选仪（FACS）中高效分离二倍体细胞和四倍体细胞。此外，当应用于二倍体细胞时，该方法能够富集处于细胞周期G1、S和G2期的活的、有功能的细胞。经FACS纯化的细胞具有活力，重新培养后能迅速恢复细胞周期。虽然星形孢菌素对处于细胞周期任何阶段的细胞毒性相同，但喜树碱对处于S期的细胞毒性尤其大。此外，BAY11 - 7082是NF - κB激活所需的IKK复合物的特异性抑制剂，它表现出特定的细胞周期特异性毒性特征（G2>S>G1）。这些结果描绘了一种用于研究细胞周期调控与细胞死亡机制交叉点的新方法。
