Insights into the influences of carboxymethyl-β-cyclodextrin on DNA formulations characteristics and gene transfection efficiency

BACKGROUND Gene therapy is an expanding field and it can treat genetic and acquired diseases. OBJECTIVE It was found that formulations with DNA: CM-β-CD (Carboxymethyl-beta-cyclodextrin): Pluronic-F127 1:3:3 and 1:3 DNA: CM-β-CD are the most stable formulations indicating high incorporation of DNA within CM-β -CD. METHOD Gel electrophoresis revealed DNA with low CM-β -CD concentration has formed a more stable complex. Samples 1:3 DNA: CM-β-CD and 1:3:3 DNA: CM-β-CD: Pluronic-127 show no DNA fragment, suggesting good condensation of DNA inside cyclodextrin cavity. RESULTS This was confirmed by fluorescence data where fluorescence intensity was reduced for samples DNA: CM-β-CD 1:3. Overall, the findings showed that Carboxymethyl-beta-cyclodextrin (as a novel non-viral gene vector) was able to provide condensation and protection to the DNA, with and without Pluronic-F127, at low concentration. CONCLUSION pDNA/CM-β-CD complex has not only shown to be able to transfect COS 7 and SHSY5Y cell lines, but it gives a higher transfection efficiency than that produced by the TransIT-LT1 commercial transfection reagent

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

Role of Ca2+-activated K+ channels in human erythrocyte apoptosis.

Exposure of erythrocytes to the Ca2+ ionophore ionomycin has recently been shown to induce cell shrinkage, cell membrane blebbing, and breakdown of phosphatidylserine asymmetry, all features typical of apoptosis of nucleated cells. Although breakdown of phosphatidylserine asymmetry is thought to result from activation of a Ca2+-sensitive scramblase, the mechanism and role of cell shrinkage have not been explored. The present study was performed to test whether ionomycin-induced activation of Ca2+-sensitive Gardos K+ channels and subsequent cell shrinkage participate in ionomycin-induced breakdown of phosphatidylserine asymmetry of human erythrocytes. According to on-cell patch-clamp experiments, ionomycin (1 microM) induces activation of inwardly rectifying K+-selective channels in the erythrocyte membrane. Fluorescence-activated cell sorter analysis reveals that ionomycin leads to a significant decrease of forward scatter, reflecting cell volume, an effect blunted by an increase of extracellular K+ concentration to 25 mM and exposure to the Gardos K+ channel blockers charybdotoxin (230 nM) and clotrimazole (5 microM). As reflected by annexin binding, breakdown of phosphatidylserine asymmetry is triggered by ionomycin, an effect again blunted, but not abolished, by an increase of extracellular K+ concentration and exposure to charybdotoxin (230 nM) and clotrimazole (5 microM). Similar to ionomycin, glucose depletion leads (within 55 h) to annexin binding of erythrocytes, an effect again partially reversed by an increase of extracellular K+ concentration and exposure to charybdotoxin. K-562 human erythroleukemia cells similarly respond to ionomycin with cell shrinkage and annexin binding, effects blunted by antisense, but not sense, oligonucleotides against the small-conductance Ca2+-activated K+ channel isoform hSK4 (KCNN4). The experiments disclose a novel functional role of Ca2+-sensitive K+ channels in erythrocytes, i.e., their participation in regulation of erythrocyte apoptosis

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

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'

Investigating the role of cancer stem cell related genes in acquired drug resistance in neuroblastoma

ntroduction: Neuroblastoma is a paediatric tumour that develops from embryonic neural crest cells that give rise to the sympathetic nervous system. Aggressive high risk disease remains a clinical challenge and despite multimodal therapy survival rates are poor. A major obstacle in the successful treatment of the disease is the development of acquired resistance to chemotherapeutic agents. We hypothesize that aggressive neuroblastomas acquire a more immature phenotype and an increase in expression of cancer stem cell related genes that contributes to drug resistance. Aim: To investigate the role of cancer stem cell genes in neuroblastoma cell line models of acquired drug resistance. Methods: To study the drivers of acquired drug resistance we performed RT2 profiler - Human Cancer Stem Cell Array (Qiagen, UK) on three paired parental and vincristine resistant cell lines (IMR5, IMR32 and NGP). The drug-adapted cancer cell lines IMR5, IMR32, NGP were derived from the Resistant Cancer Cell Line (RCCL) collection (www.kent.ac.uk/stms/cmp/RCCL/RCCLabout.html). Raw expression data were log transformed and processed using quantile normalization method. Linear models were applied for the selection of significantly differentially expressed genes using criteria P.Val.Adj 1 & < -1. Functional enrichment analysis was performed on 11 genes appearing among three comparisons for their potential involvement in functional processes and pathways of drug resistance. Results and Summary: Several genes were identified that were deregulated in 3/3 drug resistant cell lines to compared to parental cell lines including Sox-2, Lin28A, Lin28B, MYC and Snai1. Many of these genes are associated with drug resistance mechanisms in other solid cancers and have been shown to be involved in pathways related to stem cell signalling, differentiation and development, as well as the Notch signalling pathway. Our data suggests a potential enrichment of cancer stem cell related genes that contribute to the development of acquired drug resistance in neuroblastoma and warrants further study on the functional effects of these genes/ pathways

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

Effect of Vibrio parahaemolyticus haemolysin on human erythrocytes.

Haemolysin Kanagawa, a toxin from Vibrio parahaemolyticus, is known to trigger haemolysis. Flux studies indicated that haemolysin forms a cation channel. In the present study, channel properties were elucidated by patch clamp and functional significance of ion fluxes by fluorescence-activated cell sorting (FACS) analysis. Treatment of human erythrocytes with 1 U ml-1 haemolysin within minutes induces a non-selective cation permeability. Moreover, haemolysin activates clotrimazole-sensitive K+ channels, pointing to stimulation of Ca2+-sensitive Gardos channels. Haemolysin (1 U ml-1) leads within 5 min to slight cell shrinkage, which is reversed in Ca2+-free saline. Erythrocytes treated with haemolysin (0.1 U ml-1) do not undergo significant haemolysis within the first 60 min. Replacement of extracellular Na+ with NMDG+ leads to slight cell shrinkage, which is potentiated by 0.1 U ml-1 haemolysin. According to annexin binding, treatment of erythrocytes with 0.1 U ml-1 haemolysin leads within 30 min to breakdown of phosphatidylserine asymmetry of the cell membrane, a typical feature of erythrocyte apoptosis. The annexin binding is significantly blunted at increased extracellular K+ concentrations and by K+ channel blocker clotrimazole. In conclusion, haemolysin Kanagawa induces cation permeability and activates endogenous Gardos K+ channels. Consequences include breakdown of phosphatidylserine asymmetry, which depends at least partially on cellular loss of K+

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

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