Correlating cell cycle with apoptosis in a cell line expressing a tandem green fluorescent protein substrate specific for group II caspases

Background: We describe a rapid flow cytometric assay that correlates cell cycle with apoptotic cell death in a cell line expressing a tandem green fl uorescent protein (GFP). Methods: A jurkat cell line was transfected with a gene construct coding fo r constitutive expression of a tandem GFP molecule carrying a consensus cle avage site (DEVD) for group II caspases (C-2-Y). Cells were treated with CD 95 antibody (Ab), then incubated with annexin V-phycoerythrin (PE), propidi um iodide (PI), and Hoechst 33342. Results: After CD95 treatment, the C-2-Y cell line had twice the number of nonapoptotic cells compared with both control cell lines. This proportion o f viable, nonapoptotic cells after treatment was unaffected by the level of GFP (DEVD) expression in the cells, as confirmed by sorted populations. Th e early apoptotic cells in the C-2-Y cell line had an increased G0-G1 phase population compared with the control cell lines. Conclusions: Apoptosis is delayed in the C-2-Y cell line and the early apop totic cells have a higher G0-G1 cell cycle frequency. The artificial substr ate competes with the natural substrate(s), thereby slowing the apoptotic p rocess. The expression level of DEVD-GFP does not alter the delayed inducti on of apoptosis. Caspase activation occurs prior to phosphatidylserine tran slocation. Cytometry 45:225-234, 2001. (C) 2001 Wiley-Liss, Inc.