depletion of erythrocytes leads to activation of Ca 2ϩ -permeable cation channels, Ca 2ϩ entry, activation of a Ca 2ϩ -sensitive erythrocyte scramblase, and subsequent exposure of phosphatidylserine at the erythrocyte surface. Ca 2ϩ entry into erythrocytes was previously shown to be stimulated by phorbol esters and to be inhibited by staurosporine and chelerythrine and is thus thought to be regulated by protein phosphorylation/dephosphorylation, presumably via protein kinase C (PKC) and the corresponding phosphoserine/threonine phosphatases. The present experiments explored whether PKC could contribute to effects of energy depletion on erythrocyte phosphatidylserine exposure and cell volume. Phosphatidylserine exposure was estimated from annexin binding and cell volume from forward scatter in fluorescence-activated cell sorter analysis. Removal of extracellular glucose led to depletion of cellular ATP, stimulated PKC activity, led to translocation of PKC␣, enhanced serine phosphorylation of membrane proteins, decreased cell volume, and increased annexin binding, the latter effect being blunted but not abolished in the presence of 1 M staurosporine or 50 nM calphostin C. The PKC stimulator phorbol-12-myristate-13-acetate (3 M) and the phosphatase inhibitor okadaic acid (1-10 M) mimicked the effect of glucose depletion and similarly led to translocation of PKC␣ and enhanced serine phosphorylation, increased annexin binding, and decreased forward scatter, the latter effects being abrogated by PKC inhibitor staurosporine (1 M). Fluo-3 fluorescence measurements revealed that okadaic acid also enhanced erythrocyte Ca 2ϩ activity. The present observations suggest that protein phosphorylation and dephosphorylation via PKC and the corresponding protein phosphatases contribute to phosphatidylserine exposure and cell shrinkage after energy depletion. cell volume; eryptosis; calcium; okadaic acid; staurosporine AS SHOWN RECENTLY, erythrocytes exposed to oxidative stress, osmotic shock, or glucose depletion activate a Ca 2ϩ -permeable cation channel (22,29,30,32)

Diabetes increases the percentage of circulating erythrocytes exposing phosphatidylserine (PS) at the cell surface. PS-exposing erythrocytes are recognized, bound, engulfed and degraded by macrophages. Thus, PS exposure, a feature of suicidal erythrocyte death or eryptosis, accelerates clearance of affected erythrocytes from circulating blood. Moreover, PS-exposing erythrocytes bind to the vascular wall thus interfering with microcirculation. The present study explored mechanisms involved in the triggering of PS exposure by methylgloxal, an extra- and intracellular metabolite which is enhanced in diabetes. PS exposure, cell size and cytosolic Ca²⁺-activity after methylglyoxal treatment were measured by FACS analysis of annexin V binding, forward scatter and Fluo-3-fluorescence, respectively, and it was shown that the treatment significantly enhanced the percentage of PS-exposing erythrocytes at concentrations (0.3 µM) encountered in diabetic patients. Surprisingly, methylglyoxal did not significantly increase cytosolic Ca²⁺ concentration, and at concentrations up to 3 µM, did not decrease the forward scatter. Instead, exposure to methylglyoxal inhibited glycolysis thus decreasing ATP and GSH concentrations. In conclusion, methylglyoxal impairs energy production and anti-oxidative defense, effects contributing to the enhanced PS exposure of circulating erythrocytes and eventually resulting in anemia and deranged microcirculation.

IntroductionMicroRNAs are regulators of central cellular processes and are implicated in the pathogenesis and prognosis of human cancers. MicroRNAs also modulate responses to anti-cancer therapy. In the context of radiation oncology microRNAs were found to modulate cell death and proliferation after irradiation. However, changes in microRNA expression profiles in response to irradiation have not been comprehensively analyzed so far. The present study's intend is to present a broad screen of changes in microRNA expression following irradiation of different malignant cell lines.Materials and methods1100 microRNAs (Sanger miRBase release version 14.0) were analyzed in six malignant cell lines following irradiation with clinically relevant doses of 2.0 Gy. MicroRNA levels 6 hours after irradiation were compared to microRNA levels in non-irradiated cells using the "Geniom Biochip MPEA homo sapiens".ResultsHierarchical clustering analysis revealed a pattern, which significantly (p = 0.014) discerned irradiated from non-irradiated cells. The expression levels of a number of microRNAs known to be involved in the regulation of cellular processes like apoptosis, proliferation, invasion, local immune response and radioresistance (e. g. miR-1285, miR-24-1, miR-151-5p, let-7i) displayed 2 - 3-fold changes after irradiation. Moreover, several microRNAs previously not known to be radiation-responsive were discovered.ConclusionIonizing radiation induced significant changes in microRNA expression profiles in 3 glioma and 3 squamous cell carcinoma cell lines. The functional relevance of these changes is not addressed but should by analyzed by future work especially focusing on clinically relevant endpoints like radiation induced cell death, proliferation, migration and metastasis.

Nitric oxide (NO) is known to counteract apoptosis by S-nitrosylation of protein thiol groups. NO is generated and stored in erythrocytes, which may undergo eryptosis, a suicidal cell death similar to apoptosis of nucleated cells. Eryptosis is triggered by increased cytosolic Ca2+ activity and/or ceramide and characterized by cell shrinkage and phosphatidylserine exposure at the cell surface. The present study explored whether nitric oxide could interfere with the machinery underlying eryptosis. To this end, erythrocyte phosphatidylserine exposure (annexin V-binding) and cell volume (forward scatter) were determined by flow cytometry. The Ca2+ ionophore ionomycin (0.1 microM) increased cytosolic Ca2+ activity, triggered annexin binding, and decreased forward scatter. The annexin binding and decrease of forward scatter but not the increase of cytosolic Ca2+ activity were reversed by the NO-donor nitroprusside (1 microM) and papanonoate (100 microM). Higher concentrations of nitroprusside (0.1 and 1 mM) stimulated eryptosis. Glucose depletion, exposure to C6-ceramide (3 microM), hypertonic (addition of 550 mM sucrose), and isotonic (replacement of Cl- with gluconate) cell shrinkage all triggered annexin V binding, effects all reversed by nitroprusside (1 microM). Dibutyryl-cGMP (1 mM) blunted the ionomycin- but not the ceramide-induced annexin V binding. Ionomycin decreased protein nitrosylation and thioredoxin activity, effects reversed by the NO-donor papanonoate. Clearance of erythrocytes from circulating blood was significantly faster in eNOS knockout mice than in their wild-type littermates. In conclusion, nitric oxide participates in the regulation of erythrocyte survival, an effect partially mimicked by cGMP and paralleled by alterations of protein nitrosylation and thioredoxin activity.

Accelerated suicidal death or eryptosis of infected erythrocytes may delay development of parasitemia in malaria. Eryptosis is inhibited by nitric oxide (NO). The present study has been performed to explore, whether inhibition of NO synthase by L-NAME modifies the course of malaria. We show here that L-NAME (>10 µM) increased phosphatidylserine exposure of Plasmodium falciparum infected human erythrocytes, an effect significantly more marked than in noninfected human erythrocytes. We further show that parasitemia in Plasmodium berghei infected mice was significantly decreased (from 50% to 18% of circulating erythrocytes 20 days after infection) by addition of 1 mg/ml L-NAME to the drinking water. According to CFSE labelling L-NAME treatment accelerated the clearance of both, noninfected and infected, erythrocytes from circulating blood, but did not significantly extend the life span of infected animals. In conclusion, treatment with L-NAME shortens the life span of circulating erythrocytes and thus delays development of parasitemia during malaria.