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.

Real-time imaging of single nerve cell apoptosis in retinal neurodegeneration

“…3 F-J). This finding is consistent with the morphological analysis of apoptosis in vitro, which suggests a 2-to 4-h period between the appearance of plasma membrane protrusions to the shedding of blebs of apoptotic cells (27)(28)(29).…”

“…Despite the fact that our measurement of RGC loss over time is similar to that of previous authors (22)(23)(24)(25)(26), we have recorded much higher rates of detection of RGC apoptosis (15,23,25). Compared with other methods such as DNA fragmentation, morphometry, time-lapse video microscopy, and microculture kinetic assays, annexin 5 has been shown to reveal apoptosis significantly earlier, with positive labeling of cells over a wide range of different stages of the apoptotic pathway (27).…”

“…The retina as a window on the brain may also enable this procedure to highlight possible links to other neural degenerative diseases that may include a parallel damage to RGCs. Annexin 5 detects apoptosis significantly earlier than other methods (27). Its binding to the apoptotic cell membrane (phosphotidyl serine) is linked to key steps in the apoptotic process, particularly activation of caspase 3.…”

“…This has great implications for our technique being used to assess potential therapies preventing early apoptosis, e.g., caspase inhibitors (31). In addition, because binding of annexin 5 continues for Ͼ24 h after initiation of apoptosis (27), our ability to observe changes in individual cells over time would facilitate comparisons of strategies targeting different components of the apoptotic pathway, right up to the final end point of cell death.…”

Endometrial carcinoma in vitro chemosensitivity testing of single and combination chemotherapy regimens using the novel microculture kinetic apoptosis assay: implications for endometrial cancer treatment

“…The more recently developed, automated microculture kinetic (MiCK) assay measures sensitivity of tumor cells to chemotherapy using clinical doses of drug by measuring the fraction of cells that have undergone apoptosis. [1][2][3][4] The MiCK assay has been previously tested on cells from patients with acute myeloid leukemia and has been used to direct chemotherapy choice in leukemia patients. 5 Studies comparing clinical criteria to the MiCK assay showed the assay was better able to predict complete remission and survival in acute myeloid leukemia patients.…”