Cyclooxygenase Inhibitors Decrease Apoptosis Initiated by Actinomycin D,Cycloheximide,and Staurosporine in Amnion-Derived WISH Cells

Abstract

Apoptosis is a process by which external or developmental factors induce a specific series of evetns leading to cell death. Recently, apoptotic cells have been described in rat amnion membrane at late getation, suggesting apoptosis may be involved in membrane rupture. Mechanisms controlling amnion cell apoptosis are unknwon. The objective of this study was to investigate whether cyclooxygenase and prostaglandins are integral to apoptosis in amnion, as reported in intestinal epithelial cells and renal mesangial cells. Amnion-derived WISH cells underwent apoptosis in a dose- and time-dependnet manner after incubation with actinomycin D, cycloheximide, or staurosporine, as determined by cell viability, DNA fragmentation analysis, and fluorescent in situ fragmentation analysis. Cells cultured with increasing does of these agents also demonstrated concomitant increases in prostaglandin E₂ outpuot. WISH cell coincubation with these agents and the cyclooxygenase inhibitors indomethacin or piroxican resulted in dose-dependent decreases in both prostaglandin E₂ and apoptosis. Cultures incubated with 0.5 μg/mL actinomycin D showed 80.7% cell apoptosis after 12 hours compared with 1.1% in untreated cultures. After 24 hours incubation with actinomycin D, 0.8% of the original cell number remained attached to the plate. In cultures coincubated with 0.5 μg/mL actinomycin D and 100 μmol/L indomethacin, only 19.2%, 24.7%, and 39.3% of the cells were found to be apoptotic after 12, 24, and 48 hours in culture, respectively. Similar trends were observed after the use of cycloheximide or staurosporine in combination with indomethacin or prioxicam. These data suggest that cyclooxygenase and/or prostaglandins play a role in programmed cell death of amnion-derived WISH cells in culture.