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.

Figures

Figure 2.

Comparison of the MiCK assay and TLVM of HL-60 cells exposed to etoposide. HL-60 cells were cultured with 10 μmol/L etoposide in complete medium for 24 hours. The cell cultures were analyzed by (A) MiCK assay for changes in OD and (B) the TLVM for percentages of blebbed cells (shown at 15-minute intervals). In A, time to the maximum apoptotic response (Tm), initiation time (Ti), development time (Td), and best-fit line (dotted line) of the increasing portion of the OD-versus-time curve are presented. Results of one representative experiment of three performed are shown. Phase contrast photomicrographs of cell culture were taken at the following times of exposure to etoposide: C, 2.5 hours; D, 4.2 hours; E, 7.25 hours; F, 12 hours; G, 16 hours; H, 24 hours. Original magnification, ×200.

Figure 3.

Comparison of the MiCK assay and TLVM of HL-60 cells exposed to cisplatin. HL-60 cells were cultured with 5 μmol/L cisplatin in complete medium for 24 hours. The cell cultures were analyzed by (A) MiCK assay for changes in OD and (B) the TLVM for percentages of blebbed cells. In A, time to the maximum apoptotic response (Tm), initiation time (Ti), development time (Td), and best fit line (dotted line) of the rapidly increasing portion of the OD-versus-time curve are shown. Results of one representative experiment of three performed are presented.

Figure 4.

Sequential morphological modifications during a single cell apoptosis recorded by TLVM. A single cell from the culture exposed to 10 μmol/L etoposide is shown at various time points during the process of apoptosis. Time points are: A, 0.0 hours; B, 3.5 hours; C, 4.12 hours; D, 4.32 hours; E, 6.75 hours; F, 6.8 hours; G, 7.0 hours; H, 8.75 hours; I 8.83 hours. Original magnification, ×400. See text for descriptions.

Figure 5.

Changes in the forward and side scattering of the light by HL-60 cells during apoptosis. Cells were cultured in complete medium for 24 hours with 10 μmol/L etoposide or for 36 hours with 5 μmol/L cisplatin. At indicated times of culture, aliquots of cells were removed and analyzed by flow cytometry for light scattering properties as described in Materials and Methods. The data presented are bivariate cytograms of 10,000 events. Quadrants R1, R2, R3, and R4 were set using control (0 hour) cultures in which more than 96% of events were located in quadrant R1. The percentages of events in each quadrant are shown.

Figure 6.

Comparisons between the results of Annexin V binding assay, DNA fragmentation and morphological tests in studying apoptosis in HL-60 cells. Cells were cultured in complete medium for 24 hours with 10 μmol/L etoposide or for 36 hours with 5 μmol/L cisplatin. At indicated times of culture, extent of apoptosis was determined using the three endpoint assays. In Annexin V binding assay, extent of apoptosis was determined as the percentage of early apoptotic cells with preserved plasma membrane integrity (An+PI−). The percentage of DNA fragmentation was determined by densitometric analysis of a digital image of the gel shown in Figure 7 ▶ . Results of one representative experiment of three performed are shown.

Figure 7.

Agarose gel analysis of DNA fragmentation in HL-60 cells exposed to 10 μmol/L etoposide and 5 μmol/L cisplatin. Cells were cultured in complete medium for 24 hours with 10 μmol/L etoposide or for 36 hours with 5 μmol/L cisplatin. At indicated times of culture, aliquots of cells were removed, DNA extracted, purified and separated on 1.5% agarose gel as described in Materials and Methods. Molecular weight markers (FMC Bioproducts, Rockland, ME) are shown in the first and last lanes. DNA with size of ≥21 kbp was considered intact; all DNA

Figure 1.

Dose-response effects of etoposide (column A) and cisplatin (column B) in the MiCK assay of apoptosis. HL-60 cells were plated in complete medium with various concentrations of either etoposide or cisplatin added to the cells at the initiation of the cultures. OD of the cultures at 600 nm were measured every 5 minutes for 24 hours. Graphs of OD plotted against time of culture in hours are shown. In each graph, the drug-treated culture is shown as a heavy line and an untreated control culture is shown as a thin line. Results of one representative experiment of three performed with each drug are shown.