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

• Published in: Biochemical pharmacology Vol. 72; no. 11; pp. 1396 - 1404
• Main Authors: Coquelle, Arnaud, Mouhamad, Shahul, Pequignot, Marie O., Braun, Thorsten, Carvalho, Gabrielle, Vivet, Sonia, Métivier, Didier, Castedo, Maria, Kroemer, Guido
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 30-11-2006; Elsevier Science
• Subjects: Apoptosis - drug effects; Biological and medical sciences; Bromodeoxyuridine - metabolism; Camptothecin - pharmacology; Cell death; Cell Line, Tumor; Cell sorting; Cell Survival - drug effects; Chemoresistance; Colonic Neoplasms - genetics; Colonic Neoplasms - metabolism; Colonic Neoplasms - pathology; Dose-Response Relationship, Drug; Enzyme Inhibitors - pharmacology; Flow Cytometry - methods; Fluorescent Antibody Technique; Humans; IKK; Interphase - drug effects; Medical sciences; Nitriles - pharmacology; Pharmacology. Drug treatments; Ploidies; Ploidy; Staurosporine - pharmacology; Sulfones - pharmacology; Ploidy; H3P; Chemoresistance; IKK; Cell sorting; FACS; Casp-3a; GFP; STS; Cell death; H2B; PI; BrdU; Enrichment; Treatment resistance; In vitro; Sorting; G1 Phase; S Phase; Cell cycle; G2 Phase; Apoptosis
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/j.bcp.2006.04.014

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.