Enrichment of non-synchronized cells in the G1, S and G2 phases of the cell cycle for the study of apoptosis

Arnaud Coquelle, Shahul Mouhamad, Marie O. Pequignot, Thorsten Braun, Gabrielle Carvalho, Sonia Vivet, Didier Métivier, Maria Castedo, Guido Kroemer

Résultats de recherche: Contribution à un journalArticleRevue par des pairs

21 Citations (Scopus)

Résumé

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-κB 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.

langue originaleAnglais
Pages (de - à)1396-1404
Nombre de pages9
journalBiochemical Pharmacology
Volume72
Numéro de publication11
Les DOIs
étatPublié - 30 nov. 2006
Modification externeOui

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