Erythrocytes are devoid of mitochondria and nuclei and were considered unable to undergo apoptosis. As shown recently, however, the Ca 2+ -ionophore ionomycin triggers breakdown of phosphatidylserine asymmetry (leading to annexin binding), membrane blebbing and shrinkage of erythrocytes, features typical for apoptosis in nucleated cells. In the present study, the effects of osmotic shrinkage and oxidative stress, well-known triggers of apoptosis in nucleated cells, were studied. Exposure to 850 mOsm for 24 h, to tert-butylhydroperoxide (1 mM) for 15 min, or to glucose-free medium for 48 h, all elicit erythrocyte shrinkage and annexin binding, both sequelae being blunted by removal of extracellular Ca 2+ and mimicked by ionomycin (1 lM). Osmotic shrinkage and oxidative stress activate Ca 2+ -permeable cation channels and increase cytosolic Ca 2+ concentration. The channels are inhibited by amiloride (1 mM), which further blunts annexin binding following osmotic shock, oxidative stress and glucose depletion. In conclusion, osmotic and oxidative stress open Ca 2+ -permeable cation channels in erythrocytes, thus increasing cytosolic Ca 2+ activity and triggering erythrocyte apoptosis.

Recent evidence suggests chronic myeloid leukemia (CML) stem cells are insensitive to kinase inhibitors and responsible for minimal residual disease in treated patients. We investigated whether CML stem cells, in a transgenic mouse model of CML-like disease or derived from patients, are dependent on Bcr-Abl. In the transgenic model, after retransplantation, donor-derived CML stem cells in which Bcr-Abl expression had been induced and subsequently shut off were able to persist in vivo and reinitiate leukemia in secondary recipients on Bcr-Abl reexpression. Bcr-Abl knockdown in human CD34(+) CML cells cultured for 12 days in physiologic growth factors achieved partial inhibition of Bcr-Abl and downstream targets p-CrkL and p-STAT5, inhibition of proliferation and colony forming cells, but no reduction of input cells. The addition of dasatinib further inhibited p-CrkL and p-STAT5, yet only reduced input cells by 50%. Complete growth factor withdrawal plus dasatinib further reduced input cells to 10%; however, the surviving fraction was enriched for primitive leukemic cells capable of growth in a long-term culture-initiating cell assay and expansion on removal of dasatinib and addition of growth factors. Together, these data suggest that CML stem cell survival is Bcr-Abl kinase independent and suggest curative approaches in CML must focus on kinase-independent mechanisms of resistance.

Erythrocytes lack nuclei and mitochondria, the organelles important for apoptosis of nucleated cells. However, following increase of cytosolic Ca 2 þ activity, erythrocytes undergo cell shrinkage, cell membrane blebbing and breakdown of phosphatidylserine asymmetry, all features typical for apoptosis in nucleated cells. The same events are observed following osmotic shock, an effect mediated in part by activation of Ca 2 þ -permeable cation channels. However, erythrocyte death following osmotic shock is blunted but not prevented in the absence of extracellular Ca 2 þ pointing to additional mechanisms. As shown in this study, osmotic shock (950 mOsm) triggers sphingomyelin breakdown and formation of ceramide. The stimulation of annexin binding following osmotic shock is mimicked by addition of ceramide or purified sphingomyelinase and significantly blunted by genetic (aSM-deficient mice) or pharmacologic (50 lM 3,4-dichloroisocoumarin) knockout of sphingomyelinase. The effect of ceramide is blunted but not abolished in the absence of Ca 2 þ . Conversely, osmotic shock-induced annexin binding is potentiated in the presence of sublethal concentrations of ceramide. In conclusion, ceramide and Ca 2 þ entry through cation channels concert to trigger erythrocyte death during osmotic shock.

Erythrocyte diseases such as sickle cell anemia, thalassemia and glucose-6-phosphate dehydrogenase deficiency decrease the erythrocyte life span, an effect contributing to anemia. Most recently, erythro-cytes have been shown to undergo apoptosis upon increase of cytosolic Ca²⁺ activity. The present study has been performed to explore whether sickle cell anemia, thalassemia and glucose-6-phosphate dehydrogenase deficiency enhance the sensitivity of erythrocytes to osmotic shock, oxidative stress or energy depletion, all maneuvers known to increase cytosolic Ca²⁺ activity. To this end, annexin binding as an indicator of apoptosis has been determined by FACS analysis. Erythrocytes from healthy individuals, from patients with sickle cell anemia, thalassemia or glucose-6-phosphate dehydrogenase deficiency all responded to osmotic shock (up to 950 mOsm by addition of sucrose for 24 hours), to oxidative stress (up to 1.0 mM tetra-butyl-hydroxyperoxide tBOOH) and to energy depletion (up to 48 hours glucose deprivation) with enhanced annexin binding. However, the sensitivity of sickle cells and of glucose-6-phosphate dehydrogenase deficient cells to osmotic shock and of sickle cells, thalassemic cells and glucose-6-phosphate dehydrogenase deficient cells to oxidative stress and to glucose depletion was significantly higher than that of control cells. Annexin binding was further stimulated by Ca²⁺ ionophore ionomycin with significantly higher sensitivity of sickle cells and glucose-6-phosphate dehydrogenase deficient cells as compared to intact cells. In conclusion, sickle cells, thalassemic cells and glucose-6-phosphate dehydrogenase deficient erythrocytes are more sensitive to osmotic shock, oxidative stress and/or energy depletion, thus leading to enhanced apoptosis of those cells. The accelerated apoptosis then contributes to the shortened life span of the defective erythrocytes.

Abstract. Recombinant human erythropoietin therapy is used to counteract anemia that is the result of renal insufficiency. It stimulates the formation of peripheral blood erythrocytes by inhibiting apoptosis of erythrocyte precursor cells. Mature erythrocytes have similarly been shown to undergo apoptosis. Hyperosmotic shock and Cl Ϫ removal activate a Ca 2ϩ -permeable, ethylisopropylamiloride-inhibitable cation channel. The subsequent increase of cytosolic Ca 2ϩ activates a scramblase that breaks down cell membrane phosphatidylserine asymmetry, leading to annexin binding. Studied was whether channel activity and erythrocyte cell death are regulated by erythropoietin. Scatchard plot analysis disclosed low-abundance, highaffinity binding of 125 I-erythropoietin to erythrocytes. Whole cell patch clamp experiments revealed significant inhibition of the ethylisopropylamiloride-sensitive current by 1 U/ml erythropoietin. Cl Ϫ removal triggered annexin binding, an effect abrogated by erythropoietin (1 U/ml) but not by GM-CSF (10 ng/ml). Osmotic shock (700 mOsm) stimulated annexin binding within 24 h in the majority of the erythrocytes, an effect blunted by erythropoietin (1 U/ml) but not by GM-CSF (10 ng/ml). In the nominal absence of Ca 2ϩ , the effect of osmotic shock was blunted and the effect of erythropoietin abolished. In hemodialysis patients, intravenous administration of erythropoietin (50 IU/kg) within 4 h decreased the number of annexin binding circulating erythrocytes. Erythropoietin binds to erythrocytes and inhibits volume-sensitive erythrocyte cation channels and thus the breakdown of phosphatidylserine asymmetry after activation of this channel. The effect could prolong the erythrocyte lifespan and may contribute to the enhancement of the erythrocyte number during erythropoietin therapy in dialysis patients.