Suicidal Death of Human Erythrocytes Following Exposure to Pentostatin

Abstract

Background/Aims: Pentostatin (2'-deoxycoformycin), a purine analog, is used for the treatment of diverse B and T-cell malignancies as well as for immunosuppression. Pentostatin is at least in part effective by triggering apoptosis. Pentostatin sensitive mechanisms leading to apoptosis include accumulation of DNA strand breaks, altered transcription and mitochondrial depolarization. Erythrocytes lack nuclei and mitochondria but nevertheless may enter eryptosis, an apoptosis-like suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), ceramide formation and energy depletion. The present study tested, whether and how pentostatin induces eryptosis. Methods: The phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, ceramide abundance utilizing specific antibodies, and cytosolic ATP concentration utilizing a luciferin–luciferase assay kit. Results: A 48 hours exposure of human erythrocytes to pentostatin (≥5 µg/ml) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Pentostatin significantly increased [Ca2+]i, and significantly decreased cytosolic ATP, but did not significantly modify ceramide abundance. The effect of pentostatin on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusion: Pentostatin triggers erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, effects paralleled by and at least partially due to entry of extracellular Ca2+ and cellular energy depletion.