Endocrine Abstracts

Apoptosis is an important process influencing tissues growth, however the assessment of this process in monolayer culture is difficult because apoptotic cells disappear quickly due to secondary necrosis. Most of the methods used with success in vivo is useless in vitro. Our approach to evaluating apoptosis in vitro is based on the possibility to perform multiple assays on the same sample well. The aim of the study was to evaluate the effect of mifepristone (RU-486), a strong competitive antagonist of progesterone and glucocorticoid receptors, used at the previously selected concentration of 2×10⁻⁵M on viability of cells of the HECa10 high endothelial line assessed in real time and on apoptotic intensity assessed in cultures carried out with different numbers of sown cells: 1000, 2000, 4000 and 8000 cells/well in a 96-well plate. Two compatible assays were used: luminescence method for assessment of cell viability in real time and fluorescence method for apoptosis detection based on activities of caspase-3 and 7 (Promega Corporation). It was shown that the longer the exposure time to mifepristone and the higher the cell density in the wells, the inhibitory effect of mifepristone on viability of cells is earlier and stronger (observed already after 2 hours for 8000 cells/well, after 4 hours for 4000 cells, and only after 24 hours for 1000 and 2000 cells/well). The inhibitory effect of mifepristone grew with the duration of the culture what allowed to choose the right time point for the assessment of the apoptosis intensity. After 26 hours mifepristone caused an increase in apoptosis e.g. by 19% for 8000 cells and by 15% for 1000 cells/well. At that time point mifepristone inhibited cell viability e.g. by 40% for 8000 cells and by 20% for 1000 cells/well. Tracing the viability of cells in controls found out that the luminescence signal remains linear for 1000 and 2000 cells/well over the 3 day period, whereas the signal from higher cell number (4000 and 8000 cells/well) lose linearity after 48 hours. Mifepristone, a drug commonly used as medical abortion, evokes strong antiangiogenic effect on HECa10 line via inhibition of cells viability and induction of apoptosis.Tracing the viability of cells in cultures in real time allows to choose the optimal time to complete the culture and to determine the apoptosis intensity under the influence of mifepristone.