QUANTIFICATION OF APOPTOTIC CELLS WITH FLUORESCEIN ISOTHIOCYANATE-LABELED ANNEXIN-V IN CHINESE-HAMSTER OVARY CELL-CULTURES TREATED WITH CISPLATIN

Plasma membrane binding of annexin V was used to detect and quantitate apoptotic cells induced by cytotoxic drug treatment in epithelial cel l cultures. Chinese hamster ovary (CHO) cells were incubated for 2 h w ith the ID90 concentration of Cisplatin (20 mu M), and 24, 48, 72, and 96 h later the unfixed cells were stained with fluorescein isothiocya nate (FITC)-conjugated annexin V, The fluorescence signal was quantita ted by now cytometry (FCM), During the early phase of the apoptotic re sponse, the annexin V-binding frequency histograms showed two separate cell populations, a dimly and a brightly fluorescent one, Att = 96 h after drug incubation, when the process of apoptosis was completed, on ly the brightly fluorescent population was present, A dose-effect rela tionship could be established between the Cisplatin concentration used in the 2 h incubation and the binding of annexin V on the cell membra ne, as estimated by FITC fluorescence. The dimly and brightly fluoresc ent populations were sorted on the basis of annexin V binding, and ass ayed for 1) DNA breaks by in situ nick translation assay and DNA conte nt by DNA-propidium iodine fluorescence in a bivariate analysis, 2) me mbrane integrity by dye exclusion, and 3) morphological characteristic s of apoptosis, The dimly fluorescent cell population appeared to repr esent apoptotic cells in the early phase of the death process, as demo nstrated by intact cell membranes, normal DNA content, few DNA breaks, and chromatin condensation, The brightly fluorescent cells predominan tly had sub-G(1), DNA content, nuclear fragmentation, leaky cell membr anes, and probably represent late apoptotic cells, These results demon strate that cytotoxic drug-induced apoptosis can be quantitated try an nexin V binding and that by using this assay early and late apoptotic cells can be identified. (C) 1996 Wiley-Liss, Inc.