New Method for the Analysis of Cell Cycle-Specific Apoptosis

Abstract

Background: In this study, a new method for the analysis of cell cycle specificity of apoptosis was designed by using a modified annexin V and propidium iodide (API) method. Methods: Cells of the human promyelocytic HL-60 line treated with camptothecin (CPT) or ultraviolet light (UV) were labeled with fluorescein isothiocyanate-conjugated annexin V and prefixed with 1% methanol-free formaldehyde on ice, and their DNA was stained stoichiometrically with propidium iodide in the presence of digitonin. Cellular green and red fluorescences were measured by flow cytometry. Results: Cell cycle specificity of apoptosis obtained by the API method and those analyzed for the presence of DNA strand breaks by using terminal deoxynucleotidyl transferase (TdT) assay were similar: CPT- or UV-induced apoptosis preferentially in S- or G 1-phase cells, respectively. When the internucleosomal DNA degradation was prevented by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone, apoptotic cells could not be detected by the TdT assay but were identified by the API method. Conclusions: The API method, similar to the TdT assay, accurately detects the cell cycle phase specificity of apoptosis. Also, the API method appears to detect earlier stages of apoptosis than the TdT assay. © 2004 Wiley-Liss, Inc.