title: Comparative analysis of different methodological approaches to the in vitro study of drug-induced apoptosis.
abstract: Apoptosis is a dynamic process in which a characteristic morphological or biochemical event used in an assay as a specific marker of apoptosis may be observed over a limited period of time. Asynchronous involvement of cells in apoptosis results in different proportions of apoptotic cells with blebbed membrane, broken nuclei, modified mitochondrial units or fragmented DNA coexisting in the culture at any single moment. Thus, depending on the method 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 micromol/L etoposide and cisplatin. Both the extent and timing of apoptotic responses were dependent on the drug and drug concentration. Time-lapse video microscopy (TLVM), flow cytometry analysis of the light scattering properties of cells, morphological studies of Giemsa-stained cells, annexin V binding, and DNA fragmentation assays were performed at multiple times of cell exposure to 10 micromol/L etoposide and 5 micromol/L cisplatin. Steep linear increases in optical density, indicating apoptosis in the MiCK assay, correlated with both linear increases in the proportion of cells with plasma membrane blebbing in TLVM and with increased side scattering properties of apoptotic cells in flow cytometry. During a 24-hour culture period, the MiCK assay and TLVM provided multiple consecutive appraisals of nondisturbed cell microcultures at intervals of 5 and 2.5 minutes, respectively, and thus could be considered as real time kinetic assays. With the three endpoint assays, each of which was applied 12 times at 2-hour intervals, maximum apoptotic responses 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 apoptosis 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, being the lowest with annexin V and the greatest with DNA fragmentation assays. The best correlations of both extent and timing of apoptosis were observed between the MiCK, TLVM, and morphological assays. In conclusion, both a maximum apoptotic response and the time at which it was achieved are the obligatory requirements for determining the apoptosis-inducing potency of an agent and for comparing results of studies performed in different laboratories.
authors.affiliation: Division of Hematology, Department of Medicine, Vanderbilt University Medical Center Nashville, Tennessee, USA. vladimir.d.kravtsov@vanderbilt.edu
authors.forename: V D
authors.initials: VD
authors.lastname: Kravtsov
authors.forename: T O
authors.initials: TO
authors.lastname: Daniel
authors.forename: M J
authors.initials: MJ
authors.lastname: Koury
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D048429:Cell Size / Q000187:drug effects; D002945:Cisplatin / Q000493:pharmacokinetics; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D005047:Etoposide / Q000493:pharmacokinetics; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D018922:HL-60 Cells; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism / Q000473:pathology*; D008027:Light; D018715:Microscopy, Video; D012542:Scattering, Radiation; D013997:Time Factors
chemical_list: D017304:Annexin A5; D005047:Etoposide; D016650:Fluorescein-5-isothiocyanate; D002945:Cisplatin