BCR-ABL activity and its response to drugs can be determined in CD34+ CML stem cells by CrkL phosphorylation status using flow cytometry.

In chronic myeloid leukaemia, CD34(+) stem/progenitor cells appear resistant to imatinib mesylate (IM) in vitro and in vivo. To investigate the underlying mechanism(s) of IM resistance, it is essential to quantify Bcr-Abl kinase status at the stem cell level. We developed a flow cytometry method to measure CrkL phosphorylation (P-CrkL) in samples with <10(4) cells. The method was first validated in wild-type (K562) and mutant (BAF3) BCR-ABL(+) as well as BCR-ABL(-) (HL60) cell lines. In response to increasing IM concentration, there was a linear reduction in P-CrkL, which was Bcr-Abl specific and correlated with known resistance. The results were comparable to those from Western blotting. The method also proved to be reproducible with small samples of normal and Ph(+) CD34(+) cells and was able to discriminate between Ph(-), sensitive and resistant Ph(+) cells. This assay should now enable investigators to unravel the mechanism(s) of IM resistance in stem cells.

PGE(2) in the regulation of programmed erythrocyte death.

Hyperosmotic shock, energy depletion, or removal of extracellular Cl(-) activates Ca(2+)-permeable cation channels in erythrocyte membranes. Subsequent Ca(2+) entry induces erythrocyte shrinkage and exposure of phosphatidylserine (PS) at the erythrocyte surface. PS-exposing cells are engulfed by macrophages. The present study explored the signalling involved. Hyperosmotic shock and Cl(-) removal triggered the release of prostaglandin E(2) (PGE(2)). In whole-cell recording, activation of the cation channels by Cl(-) removal was abolished by the cyclooxygenase inhibitor diclophenac. In FACS analysis, phospholipase-A(2) inhibitors quinacrine and palmitoyltrifluoromethyl-ketone, and cyclooxygenase inhibitors acetylsalicylic acid and diclophenac, blunted the increase of PS exposure following Cl(-) removal. PGE(2) (but not thromboxane) induced cation channel activation, increase in cytosolic Ca(2+) concentration, cell shrinkage, PS exposure, calpain activation, and ankyrin-R degradation. The latter was attenuated by calpain inhibitors-I/II, while PGE(2)-induced PS exposure was not. In conclusion, hyperosmotic shock or Cl(-) removal stimulates erythrocyte PS exposure through PGE(2) formation and subsequent activation of Ca(2+)-permeable cation channels.

Involvement of ceramide in hyperosmotic shock-induced death of erythrocytes.

Erythrocytes lack nuclei and mitochondria, the organelles important for apoptosis of nucleated cells. However, following increase of cytosolic Ca(2+) 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+)-permeable cation channels. However, erythrocyte death following osmotic shock is blunted but not prevented in the absence of extracellular Ca(2+) 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 microM 3,4-dichloroisocoumarin) knockout of sphingomyelinase. The effect of ceramide is blunted but not abolished in the absence of Ca(2+). Conversely, osmotic shock-induced annexin binding is potentiated in the presence of sublethal concentrations of ceramide. In conclusion, ceramide and Ca(2+) entry through cation channels concert to trigger erythrocyte death during osmotic shock.

Inhibition of erythrocyte cation channels and apoptosis by ethylisopropylamiloride.

Even though lacking mitochondria and nuclei erythrocytes do undergo apoptotic cell death which is characterized by breakdown of phosphatidylserine asymmetry (leading to annexin binding), membrane blebbing and cell shrinkage. Previously, we have shown that erythrocyte apoptosis is triggered by osmotic shrinkage at least in part through activation of cell volume-sensitive cation channels and subsequent Ca2+ entry. The channels could not only be activated by cell shrinkage but as well by replacement of Cl- with gluconate. Both, channel activity and annexin binding were sensitive to high concentrations of amiloride (1 mM). The present study has been performed to search for more effective blockers. To this end channel activity has been evaluated utilizing whole-cell patch-clamp and annexin binding determined by FACS analysis as an indicator of erythrocyte apoptosis. It is shown that either, increase of osmolarity or replacement of Cl- by gluconate triggers the activation of the cation channel which is inhibited by amiloride at 1 mM but not at 100 microM. Surprisingly, the cation channel was significantly more sensitive to the amiloride analogue ethylisopropylamiloride (EIPA, IC(50)=0.6+/-0.1 microM, n=5). Exposure of the cells to osmotic shock by addition of sucrose (850 mOsm) led to stimulation of annexin binding which was inhibited similarly by EIPA (IC(50)=0.2+/-0.2 microM, n=4). Moreover, annexin binding was inhibited by higher concentrations of HOE 642 (IC(50)=10+/-5 microM, n=5) and HOE 694 (IC(50)=12+/-6 microM, n=4). It is concluded that osmotic shock stimulates a cation channel which participates in the triggering of erythrocyte apoptosis. EIPA is an effective inhibitor of this cation channel and of channel mediated triggering of erythrocyte apoptosis.

Cation channels trigger apoptotic death of erythrocytes.

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-butyl-hydroperoxide (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 microM). 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.