Stimulation of Suicidal Erythrocyte Death by Phosphatase Inhibitor Calyculin A

Abstract

Background/Aims: The serine/threonine protein phosphatase 1 and 2a inhibitor Calyculin A may trigger suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺] _i). Eryptosis is fostered by activation of staurosporine sensitive protein kinase C, SB203580 sensitive p38 kinase, and D4476 sensitive casein kinase. Eryptosis may further involve zVAD sensitive caspases. The present study explored, whether Calyculin A induces eryptosis and, if so, whether its effect requires Ca²⁺ entry, kinases and/or caspases Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, and [Ca²⁺] _i from Fluo-3 fluorescence, as determined by flow cytometry. Results: A 48 hours exposure of human erythrocytes to Calyculin A (≥ 2.5 nM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly increased Fluo-3 fluorescence. The effect of Calyculin A on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺, by staurosorine (1 µM), SB203580 (2 µM), D4476 (10 µM), and zVAD (10 µM). Conclusions: Calyculin A triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part requiring Ca²⁺ entry, kinase activity and caspase activation.