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BACKGROUND: Preeclampsia, a maternal hypertensive disease, is characterized by shallow invasion of the maternal spiral arterioles resulting in hypoxia/reperfusion type insult; however, the molecular mechanism is unknown. The aim of this study was to determine the mechanism of altered oxygen tension or inhibition of phosphatidyl-inositol-3-kinase (PI3K) on trophoblast survival and to investigate the effect of epidermal growth factor (EGF) on maintaining cellular integrity. MATERIALS AND METHOD: We have used flow cytometry, immunoblotting, and fluoroimmunocytochemistry to study apoptosis in a characterized, spontaneously transformed first trimester extravillous-like trophoblast cell line that exhibits many characteristics of in vivo trophoblast. RESULTS: Time-dependent exposure of first trimester extravillous-like trophoblast to all oxygen tensions tested promoted dissipation of the mitochondrial membrane potential (psi(m)) and resulted in a significant increase in celldeath by 48 hr as determined by dual staining flow cytometry. Western blot analysis revealed expression ofcleaved caspase-3 and caspase-9 increased with time with hypoxia and hyperoxia promoting the greatest elevation indicating that longer duration of exposure to a change inoxygen tension causes increased apoptosis via a mitochondrial-mediated pathway. Disruption of the anti-apoptotic PI3K pathway by LY294002 (40 microM), its specific inhibitor, caused further significant dissipation of the psi(m) (p< 0.01) and cleavage of caspase-3. EGF was able to maintain the psi(m) and to prevent cleavage of caspase-3 even in the presence of LY294002, indicating that its survival effects were independent of the PI3K pathway. CONCLUSIONS: These results suggest that inhibition of the PI3K/Akt pathway can sensitize first-trimester trophoblast-like cells into oxygen-induced cell death and that EGF exerts its anti-apoptotic effect independently of PI3K/Akt.