Comparative analysis of different methodological approaches to the in vitro study of drug-induced apoptosis

Apoptosis is a dynamic process in which a characteristic morphological or b iochemical event used in an assay as a specific marker of apoptosis may be observed over a Limited period of time. Asynchronous involvement of cells i n apoptosis results in different proportions of apoptotic cells with blebbe d membrane, broken nuclei, modified mitochondrial units or fragmented DNA c oexisting in the culture at any single moment. Thus, depending on the metho d used, the extent of apoptosis determined in the same cell population may vary. In the present study, a microculture kinetic (MiCK) assay was used to monitor apoptosis in HL-60 cells exposed to 1, 2.5, 5, 10, and 20 mu mol/L etoposide and cisplatin, Both the extent and timing of apoptotic responses were dependent on the drug and drug concentration. Time-lapse video micros copy (TLVM), flow cytometry analysis of the light scattering properties of cells, morphological studies of Giemsa-stained cells, annexin V binding, an d DNA fragmentation assays were performed at multiple times of cell exposur e to 10 pmol/L etoposide and 5 mu mol/L cisplatin, Steep linear increases i n optical density, indicating apoptosis In the MiCK assay, correlated with both linear increases in the proportion of cells with plasma membrane blebb ing in TLVM and with increased side scattering properties of apoptotic cell s in flow cytometry, During a 24-hour culture period, the MiCK assay and TL VM provided multiple consecutive appraisals of nondisturbed cell microcultu res at intervals of 5 and 2.5 minutes, respectively, and thus could be cons idered as real time kinetic assays. With the three endpoint assays, each of which was applied 12 times at 2-hour intervals, maximum apoptotic response s varied from 22.5 to 72% in etoposide-treated cells and from 30 to 57% in cisplatin-treated cells. With the annexin V binding assay, maximum apoptosi s could always be detected 4 to 5 hours earlier than it was seen in Giemsa- stained preparations and 8 hours earlier than it was detected by measuring of DNA fragmentation. Values of the maximum extent of apoptosis varied, bei ng the lowest with annexin V and the greatest with DNA fragmentation assays . The best correlations of both extent and timing of apoptosis were observe d between the MiCK, TLVM, and morphological assays. In conclusion, both a m aximum apoptotic response and the time at which it was achieved are the obl igatory requirements for determining the apoptosis-inducing potency of an a gent and for comparing results of studies performed in different laboratori es.