Elevated Expression of LGR5 and WNT Signaling Factors in Neuroblastoma Cells With Acquired Drug Resistance.

Neuroblastoma (NB) is a pediatric solid cancer with high fatality, relapses, and acquired resistance to chemotherapy, that requires new therapeutic approaches to improve survival. LGR5 is a receptor that potentiates WNT/signaling pathway and has been reported to promote development and survival in several adult cancers. In this study we investigated LGR5 expression in a panel of NB cell lines with acquired resistance to vincristine or doxorubicin. We show LGR5-LRP6 cooperation with enhanced expression in drug resistant NB cell lines compared to parental cells, suggesting a role for LGR5 in the emergence of drug resistance, warranting further investigation.

Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival.

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.

Optimization of methods for the detection of BCR-ABL activity in Philadelphia-positive cells.

OBJECTIVE: The recent success in treating chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKI), such as imatinib mesylate (IM), has created a demand for reproducible methods to accurately assess inhibition of BCR-ABL activity within CML cells, including rare stem and progenitor cells, either in vitro or in vivo. The purpose of this study was to develop an enzyme-linked immunosorbent (ELISA) method to measure total tyrosine phosphorylation (P-Tyr) in small samples of cells that express BCR-ABL and to compare to more established methods. MATERIALS AND METHODS: The assay was first validated in BCR-ABL wild-type and mutant vs BCR-ABL-negative cell lines. P-Tyr levels were then measured by ELISA in primary CD34(+) CML cells treated with IM. RESULTS: In vitro exposure to TKI resulted in decreases in the level of P-Tyr, in both BCR-ABL-positive cell lines and primary CD34(+) CML samples, which were comparable to the reduction in P-Tyr by flow cytometry and phosphorylation of CrkL by either Western blot or flow cytometry. CONCLUSION: We have developed an accurate ELISA method to measure BCR-ABL activity within Ph(+) cells, which is comparable to other in vitro BCR-ABL assessment techniques in terms of sensitivity and could be adapted for high throughput.

Combined BCR-ABL inhibition with lentiviral-delivered shRNA and dasatinib augments induction of apoptosis in Philadelphia-positive cells.

OBJECTIVE: This study investigated two approaches, short hairpin RNA (shRNA) and the potent ABL inhibitor, dasatinib, alone and together, to achieve complete inhibition of BCR-ABL activity in Philadelphia-positive (Ph(+)) cells. MATERIALS AND METHODS: shRNA specific for BCR-ABL b3a2 were delivered, by lentiviral transduction or electroporation, to K562 cells, with or without dasatinib. mRNA and protein knockdown were measured by quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and Western blotting. BCR-ABL activity was assessed by intracellular flow cytometry for pCrkL. Cell death and apoptosis were assayed using trypan blue exclusion, Annexin-V, and active caspase-3 staining. RESULTS: Forty-eight hours after transduction or electroporation of shRNA, BCR-ABL mRNA, and protein were reduced by 75% and >90%, respectively, and sustained for 5 days. Lentiviral delivery and electroporation were equally effective. pCrkL was inhibited in association with cell death. By 5 days after transduction or electroporation, viable cells represented 50% of input, with a 12-fold reduction vs control, which expanded 6-fold. When shRNA, titrated by green fluorescent protein into low and high, was combined with dasatinib (concentration range, 0-10 nM), low shRNA was additive with low dasatinib (0.6 and 1 nM), leading to inhibition of pCrkL, induction of activated caspase-3, expression of Annexin-V, and marked reduction in viable cells. CONCLUSION: These results confirm that by lowering BCR-ABL levels with shRNA, complete inhibition of oncoprotein activity can be achieved with a lower concentration of dasatinib, thus providing a rationale for combining these approaches in the setting of high target expression, such as found in advanced phase disease and in the stem cell compartment.

Stimulation of erythrocyte ceramide formation by platelet-activating factor.

Osmotic erythrocyte shrinkage leads to activation of cation channels with subsequent Ca2+ entry and stimulates a sphingomyelinase with subsequent formation of ceramide. Ca2+ and ceramide then activate a scramblase leading to breakdown of phosphatidylserine asymmetry of the cell membrane. The mediators accounting for activation of erythrocyte sphingomyelinase and phosphatidylserine exposure remained elusive. The study demonstrates that platelet-activating factor (PAF) is released from erythrocytes upon hyperosmotic cell shrinkage. The experiments further disclose the presence of PAF receptors in erythrocytes and show that PAF stimulates the breakdown of sphingomyelin and the release of ceramide from erythrocytes at isotonic conditions. PAF further triggers cell shrinkage (decrease of forward scatter) and phosphatidylserine exposure (annexin binding) of erythrocytes. The stimulation of annexin-binding is blunted by a genetic knockout of PAF receptors, by the PAF receptor antagonist ABT491 or by inhibition of sphingomyelinase with urea. In conclusion, PAF activates an erythrocyte sphingomyelinase and the then formed ceramide leads to the activation of scramblase with subsequent phosphatidylserine exposure.

Regulation of cardiac shal-related potassium channel Kv 4.3 by serum- and glucocorticoid-inducible kinase isoforms in Xenopus oocytes.

The human cardiac transient outward potassium current I(to) is formed by co-assembly of voltage-dependent K(+) channel (Kv 4.3) pore-forming alpha-subunits with differently spliced K channel interacting protein (KChIP) accessory proteins. I(to) is of considerable importance for the normal course of the cardiac ventricular action potential. The present study was performed to determine whether isoforms of the serum- and glucocorticoid-inducible kinase (SGK) family influence Kv 4.3/KChIP2b channel activity in the Xenopus laevis heterologous expression system. Co-expression of SGK1, but not of SGK2 or SGK3, increased Kv 4.3/KChIP2b channel currents. The up-regulation of the current was not due to changes in the activation curve or changes of channel inactivation. The currents in oocytes expressing Kv 4.3 alone were smaller than those in Kv 4.3/KChIP2b expressing oocytes, but were still stimulated by SGK1. The effect of wild-type SGK1 was mimicked by constitutively active SGK1 (SGK1 S422D) but not by an inactive mutant (SGK1 K127N). The current amplitude increase mediated by SGK1 was not dependent on NEDD4.2 or RAB5, nor did it reflect increased cell surface expression. In conclusion, SGK1 stimulates Kv 4.3 potassium channels expressed in Xenopus oocytes by a novel mechanism distinct from the known NEDD4.2-dependent pathway.

Deranged Kv channel regulation in fibroblasts from mice lacking the serum and glucocorticoid inducible kinase SGK1.

Coexpression of the serum and glucocorticoid inducible kinase 1 (SGK1) up-regulates Kv channel activity in Xenopus oocytes and human embryonic kidney cells. To investigate the physiological impact of SGK1 dependent Kv channel regulation, we recorded whole-cell currents in lung fibroblasts from SGK1 knockout mice (sgk1-/-) and wild-type littermates (sgk1+/+). Serum-grown mouse lung fibroblasts (MLF) from both genotypes exhibited voltage-gated outwardly rectifying K(+)-currents with time-dependent activation (tau(act) approximately 3 msec), slow inactivation (tau(inact) approximately 700 msec), use-dependent inactivation, and (partial) inhibition by K(+) channel blockers TEA, 4-AP, and margatoxin. In serum grown MLF peak Kv current density at +100 mV was significantly lower in sgk1-/- (14 +/- 2 pA/pF, n = 13) than in sgk1+/+ (31 +/- 4 pA/pF, n = 16). PCR amplification of different Kv1 and Kv3 subunits from mouse fibroblasts demonstrated the expression of Kv1.1-1.7, Kv3.1, and Kv3.3 mRNA in both sgk1+/+ and sgk1-/- cells. Upon serum deprivation Kv currents almost disappeared in sgk1+/+ (4 +/- 1 pA/pF, n = 11) but not in sgk1-/- (10 +/- 1 pA/pF, n = 6) MLF. Accordingly, following serum deprivation Kv current density was significantly lower in sgk1+/+ than in sgk1-/-. Stimulation of serum-depleted cells with dexamethasone (dex) (1 microM, 1 day), IGF-1 (6.7 microM, 4-6 h) or both, significantly activated Kv currents in sgk1+/+ but not in sgk1-/- MLF. In the presence of both, dex and IGF-1, the Kv current density was significantly larger in sgk1+/+ (27 +/- 3 pA/pF, n = 12) than in sgk1-/- (13 +/- 3 pA/pF, n = 10) cells. Similar to MLF, Kv currents were significantly higher in sgk1+/+ mouse tail fibroblasts (MTF). In sgk1+/+ but not sgk1-/- MTF the Kv currents were inhibited upon serum deprivation and reincreased after stimulation of serum deprived MTF with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h). According to Fura-2-fluorescence capacitative Ca(2+) entry was lower in sgk1-/- MTF compared to sgk1+/+ MTF. Upon serum deprivation capacitative Ca(2+) entry decreased significantly in sgk1+/+ but not in sgk1-/- MTF. Stimulation of depleted cells with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h) reincreased capacitative Ca(2+) entry in sgk1+/+ MTF, whereas in sgk1-/- cells it remained unchanged. In conclusion, lack of SGK1 does not abrogate Kv channel activity but abolishes regulation of those channels by serum, glucocorticoids and IGF-1, an effect influencing capacitative Ca(2+) entry.

Cl- channel blockers NPPB and niflumic acid blunt Ca(2+)-induced erythrocyte 'apoptosis'.

Exposure to Ca2+ ionophore ionomycin, osmotic shock, oxidative stress and glucose depletion trigger cell shrinkage and scramblase-mediated phosphatidylserine exposure at the outer leaflet of the erythrocyte cell membrane. The effects are partially due to activation of GARDOS channels and subsequent cellular K+ loss leading not only to cell shrinkage but also participating in the triggering of erythrocyte scramblase. As conductive loss of K+ would depend on the parallel loss of anions we hypothesised that activation of scramblase is similarly dependent on the activity of Cl- channels. To test this hypothesis, we used Cl- channel blockers NPPB and niflumic acid. It is shown here that treatment of erythrocytes with 1 microM ionomycin leads to cellular K+ loss, decrease of hematocrit and decrease of forward scatter in FACS analysis reflecting cell shrinkage as well as increase of annexin positive cells reflecting phosphatidylserine exposure. Those events were significantly blunted in the presence of 100 microM NPPB by 34% (K+ loss), 45% (hematocrit), 32% (forward scatter) and 69% (annexin binding), or in the presence of 100 microM niflumic acid by 15% (forward scatter) and 45% (annexin binding), respectively. Moreover, oxidative stress triggered annexin binding which was again significantly inhibited (by 51%) in the presence of 100 microM NPPB. In conclusion, Cl- channels presumably participate in the regulation of erythrocyte 'apoptosis'.

Enhanced susceptibility to erythrocyte "apoptosis" following phosphate depletion.

Among the sequelae of phosphate depletion is anaemia, due in part to a decreased life span of mature erythrocytes. Recent studies have disclosed that cellular stress leads to an increase of cytosolic Ca(2+) activity in erythrocytes thereby triggering cell shrinkage and breakdown of phosphatidylserine asymmetry of the cell membrane, both typical features of apoptosis. In the present experiments, phosphatidylserine exposure and cell size were measured by fluorescence-activated cell sorting (FACS) analysis of annexin binding and forward scatter, respectively. Erythrocytes from intact mice were compared with erythrocytes from mice exposed to a low-phosphate diet for 4 days. Annexin binding of freshly drawn erythrocytes was slightly but significantly enhanced by the low-phosphate diet. Furthermore, intracellular phosphate and ATP concentrations were significantly decreased in those erythrocytes whereas intracellular Ca(2+) activity was unaltered. Osmotic shock (exposure to 700 mOsm by addition of sucrose for 12 h), removal of Cl(-) (replaced by gluconate for 15 h) or removal of glucose (12 h) decreased cell volume and increased the number of annexin-binding erythrocytes. Interestingly, these effects were significantly larger in erythrocytes from phosphate-depleted animals. The experiments reveal a novel mechanism triggered by phosphate depletion that presumably contributes to the enhanced vulnerability and accelerated sequestration of erythrocytes and, thus, to anaemia.

Erythrocyte ion channels in regulation of apoptosis.

Erythrocytes lack mitochondria and nuclei, key organelles in the regulation of apoptosis. Until recently, erythrocytes were thus not considered subject to this type of cell death. However, exposure of erythrocytes to the Ca2+ ionophore ionomycin was shown to induce cell shrinkage, cell membrane blebbing and breakdown of phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the cell surface, all typical features of apoptosis. Further studies revealed the participation of ion channels in the regulation of erythrocyte "apoptosis." Osmotic shock, oxidative stress and energy depletion all activate a Ca2(+)-permeable non-selective cation channel in the erythrocyte cell membrane. The subsequent increase of Ca2+ concentration stimulates a scramblase leading to breakdown of cell membrane phosphatidylserine asymmetry and activates Ca2+ sensitive K+ (Gardos) channels leading to KCl loss and (further) cell shrinkage. Phosphatidylserine exposure and cell shrinkage are blunted in the nominal absence of extracellular Ca2+, in the presence of the cation channel inhibitors amiloride or ethylisopropylamiloride, at increased extracellular K+ or in the presence of the Gardos channel inhibitors clotrimazole or charybdotoxin. Thus, increase of cytosolic Ca2+ and cellular loss of K+ participate in the triggering of erythrocyte scramblase. Nevertheless, phosphatidylserine exposure is not completely abrogated in the nominal absence of Ca2+, pointing to additional Ca2(+)-independent pathways. One of those is activation of sphingomyelinase with subsequent formation of ceramide which in turn leads to stimulation of erythrocyte scramblase. The exposure of phosphatidylserine at the extracellular face of the cell membrane stimulates phagocytes to engulf the apoptotic erythrocytes. Thus, sustained activation of the cation channels eventually leads to clearance of affected erythrocytes from peripheral blood. Erythropoietin inhibits the non-selective cation channel and thus interferes with erythrocyte "apoptosis." Susceptibility to scramblase activation is enhanced in thalassemia, sickle cell disease and glucose-6-phosphate dehydrogenase deficiency. Infection with Plasmodium falciparum leads to activation of the cation channel eventually triggering erythrocyte "apoptosis."

Blunted apoptosis of erythrocytes from taurine transporter deficient mice.

In nucleated cells cellular taurine is released prior to DNA fragmentation and the breakdown of phosphatidylserine asymmetry within the plasma membrane. Similar to what is seen in nucleated cells, phosphatidylserine asymmetry is also abolished in erythrocytes exposed to osmotic shock or oxidative stress. The present study has been performed to explore the sensitivity of erythrocytes from a taurine transporter knockout mouse (taut-/-) against osmotic shock and oxidative stress. Erythrocyte cell volume was estimated from forward scatter and breakdown of phosphatidylserine asymmetry was identified by determination of annexin binding using FACS analysis. Erythrocytes from taut-/- mice were compared to erythrocytes from wild type littermates (taut+/+). Plasma concentration and erythrocyte content of taurine was significantly lower in taut-/- than in taut+/+ mice, but the intraerythrocyte taurine concentration did not exceed the plasma concentration. Hyperosmotic shock (exposure to 700 mOsm) and oxidative stress (exposure to 0.1 mM tert-butyl-hydroperoxide) significantly decreased the cell volume and increased the number of annexin binding sites of erythrocytes from both, taut-/- and taut+/+ mice. However, decrease of cell volume and increase of annexin binding was significantly blunted in erythrocytes from taut-/- mice as compared to their taut+/+ littermates. Stimulation of erythropoiesis by prior hemorrhage did not abrogate the difference between taut+/+ and taut-/- erythrocytes. The present observations point to a decreased sensitivity of mature erythrocytes from taut-/- mice to osmotic shock and oxidative stress, rendering them more resistant to apoptosis.

Inhibition of erythrocyte cation channels by erythropoietin.

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(-) removal activate a Ca(2+)-permeable, ethylisopropylamiloride-inhibitable cation channel. The subsequent increase of cytosolic Ca(2+) 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, high-affinity 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(-) 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(2+), 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.

Cation channels, cell volume and the death of an erythrocyte.

Similar to a variety of nucleated cells, human erythrocytes activate a non-selective cation channel upon osmotic cell shrinkage. Further stimuli of channel activation include oxidative stress, energy depletion and extracellular removal of Cl-. The channel is permeable to Ca2+ and opening of the channel increases cytosolic [Ca2+]. Intriguing evidence points to a role of this channel in the elimination of erythrocytes by apoptosis. Ca2+ entering through the cation channel stimulates a scramblase, leading to breakdown of cell membrane phosphatidylserine asymmetry, and stimulates Ca(2+)-sensitive K+ channels, thus leading to KCl loss and (further) cell shrinkage. The breakdown of phosphatidylserine asymmetry is evidenced by annexin binding, a typical feature of apoptotic cells. The effects of osmotic shock, oxidative stress and energy depletion on annexin binding are mimicked by the Ca2+ ionophore ionomycin (1 microM) and blunted in the nominal absence of extracellular Ca2+. Nevertheless, the residual annexin binding points to additional mechanisms involved in the triggering of the scramblase. The exposure of phosphatidylserine at the extracellular face of the cell membrane stimulates phagocytes to engulf the apoptotic erythrocytes. Thus, sustained activation of the cation channels eventually leads to clearance of affected erythrocytes from peripheral blood. Susceptibility to annexin binding is enhanced in several genetic disorders affecting erythrocyte function, such as thalassaemia, sickle-cell disease and glucose-6-phosphate dehydrogenase deficiency. The enhanced vulnerability presumably contributes to the shortened life span of the affected erythrocytes. Beyond their role in the limitation of erythrocyte survival, cation channels may contribute to the triggering of apoptosis in nucleated cells exposed to osmotic shock and/or oxidative stress.

Enhanced erythrocyte apoptosis in sickle cell anemia, thalassemia and glucose-6-phosphate dehydrogenase deficiency.

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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.