However, how Ca2+ coordinates structural components and signaling molecules for proper cell motility had remained elusive. With the advance of fluorescent live-cell Ca2+ imaging in recent years, we gradually understand how Ca2+ is regulated spatially and temporally in migrating cells, driving polarization, protrusion, retraction, and adhesion at the right place and right time. Here we give an overview about how cells create local Ca2+ pulses near the leading edge, maintain cytosolic Ca2+ gradient from back to front, and restore Ca2+ depletion for persistent cell motility. Differential roles of Ca2+ in regulating various effectors and the interaction of roles of Ca2+ signaling with other pathways during migration are also discussed. Such information might suggest a new direction to control cancer metastasis by manipulating Ca2+ and its associating signaling molecules in a judicious manner.Proper control of Ca At the organismic level, calcium together with other materials composes bone to support our bodies . At the neuronal  activitiigration . Aberrantabolism , neuron tabolism , immunittabolism , and maltabolism . However2+ signaling in cell migration. As an essential cellular process, cell migration is critical for proper physiological activities, such as embryonic development [2+ signaling is meticulously controlled temporally and spatially in both local and global manners, it serves as a perfect candidate to regulate cell migration modules. However, although the significant contribution of Ca2+ to cell motility has been well recognized [2+ was linked to the machinery of cell migration. The advances of live-cell fluorescent imaging for Ca2+ and cell migration in recent years gradually unravel the mystery, but there is still a long way to go.Such a puzzling situation also exists when we consider Caelopment , angiogeelopment , and immelopment , and patelopment , wound helopment , and canelopment . In eithelopment . Since Ccognized , it had 2+ signaling is polarized and regulated in migrating cells, its local actions on the cytoskeleton, and its global effect on cell migration and cancer metastasis. The strategies employing Ca2+ signaling to control cell migration and cancer metastasis will also be discussed.In the present paper, we will give a brief overview about how Ca2+ in cell migration can be traced back to the late 20th century, when fluorescent probes were invented [2+ in live cells [2+ sensor Fura-2, Brundage et al. revealed that the cytosolic Ca2+ level was lower in the front than the back of the migrating cells. Furthermore, the decrease of regional Ca2+ levels could be used as a marker to predict the cell front before the eosinophil moved [2+ gradient in migrating cells was also confirmed by other research groups [The attempt to unravel the roles of Cainvented  to monitve cells . Using mil moved . Such a h groups , though 2+ signals in migrating cells was also noticed. The use of small molecule inhibitors and Ca2+ channel activators suggested that local Ca2+ in the back of migrating cells regulated retraction and adhesion [2+ influx in the cell front as the polarity determinant of migrating macrophages [2+ signals was not available in those reports due to the limited capabilities of imaging and Ca2+ indicators in early days.In the meantime, the importance of local Caadhesion . Similarrophages . Unfortu2+ pulses of nanomolar scales [2+ indicators (GECIs) [2+-binding statuses, revolutionized Ca2+ imaging. Compared to small molecule Ca2+ indicators, GECIs' high molecular weights make them less diffusible, enabling the capture of transient local signals. Furthermore, signal peptides could be attached to GECIs so the recombinant proteins could be located to different compartments, facilitating Ca2+ measurements in different organelles. Such tools dramatically improved our knowledge regarding the dynamic and compartmentalized characteristics of Ca2+ signaling.The above problems were gradually resolved in recent years with the advance of technology. First, the utilization of high-sensitive camera for live-cell imaging  reduced r scales . In addi (GECIs) , 23, whi2+ flickers” were observed in the front of migrating cells [2+, including the Ca2+ gradient in migrating cells, was also gradually clarified [2+ signaling in migrating cells is briefly summarized as follows.With the above techniques, “Cang cells , and theng cells . Moreovelarified . Our preFor a polarized cell to move efficiently, its front has to coordinate activities of protrusion, retraction, and adhesion . The for2+ signals are also pulsatile in the junction of lamellipodia and lamella [2+-calmodulin complexes and MLCK [2+ pulses in nanomolar scales are sufficient to trigger significant myosin activities.For the above actions to proceed and persist, the structural components, actin and myosin, are regulated in a cyclic manner. For actin regulation, activities of small GTPases, Rac, RhoA, and Cdc42 , and pro lamella . Those p lamella , 32. Impand MLCK , small l2+ pulses in migrating cells raise the question where those Ca2+ signals come from. In a classical signaling model, most intracellular Ca2+ signals originate from endoplasmic reticulum (ER) through inositol triphosphate (IP3) receptors [3 generated via receptor-tyrosine kinase- (RTK-) phospholipase C (PLC) signaling cascades. It is therefore reasonable to assume that local Ca2+ pulses are also generated from internal Ca2+ storage, that is, the ER. In an in vitro experiment, when Ca2+ chelator EGTA was added to the extracellular space, local Ca2+ pulses were not immediately eliminated from the migrating cells [3 signaling cascades reduced local Ca2+ pulses efficiently in moving cells [2+ pulses in the cell front [3 signaling enhances the ER in the cell front to release local Ca2+ pulses, which are responsible for cyclic moving activities in the cell front.The critical roles of local Caeceptors , 35, whing cells , supportng cells . The obsll front . Therefo2+ pulses during cell migration. As the major pathway to activate PLC, GPCRs coupled to the Gαq/11 subunit [2) by PLC to generate diacylglycerol (DAG) and IP3, which subsequently releases Ca2+ from the ER as Ca2+ pulses and spikes [2+ oscillations induced by GPCR pathways have been observed in various cell types [αq/11 and PLC include serotonergic, adrenergic, muscarinic, glutamatergic, and histamine receptors [2+ pulses in migrating cells. Nonetheless, more studies are required to clarify this important question.In addition to RTK, the readers may wonder about the potential roles of G protein-coupled receptors (GPCRs) on local Ca subunit  trigger d spikes . Indeed,ll types , 39. Howeceptors , most ofeceptors , epidermeceptors , and vaseceptors , activat2+ pulses. In fact, IP3-induced Ca2+ oscillation has been reported repeatedly in various physiological circumstances [3 receptors in the front ER but does not directly open those Ca2+ channels. Alternatively, the cyclic Ca2+ channel opening could be triggered by Ca2+-induced Ca2+ release (CICR), which is the activation of IP3 receptors by small changes of local Ca2+ levels [2+ influx [2+ flickers [2+ pulses in the migrating cell front are probably the integrated results of polarized RTK signaling interacting with pulsatile membrane stretch and TRP channel opening, to release Ca2+ periodically from the front ER.The readers may also be curious how nonpulsatile RTK-PLC signaling generates oscillatory local Camstances , 43, 44.+ levels , 46. In + levels , offerin+ levels . Specifi+ levels , 50, by + influx , alterinflickers , 52, andflickers . Therefo2+ signals induce significant changes of cell motility implies that the basal cytosolic Ca2+ level, especially that at the front of migrating cells, has to be extremely low, so the cell migration machinery, specifically myosin and focal adhesion complexes, can promptly respond to small Ca2+ changes. To achieve the above goal, the migrating cells meticulously utilize two types of Ca2+-ATPase pumps, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) and plasma membrane Ca2+-ATPase (PMCA).The fact that tiny cyclic Ca2+ into the ER lumen with fast speed and high affinity [2+ storage with high fidelity. Once SERCA is inactivated, the ER luminal Ca2+ leaks out to the cytoplasm in no time [2+ will saturate MLCK and induce persistent contraction of myosin [2+, disabling further Ca2+ signaling through IP3 receptors. Hence, SERCA is essential for physiological and pathological cell migration. It is therefore not surprising to see aberrant SERCA expressions in cancer progression, invasion, and metastasis [SERCA is transmembranously located at the ER, continuously pumping cytosolic Caaffinity . Althougaffinity , these p no time . The resf myosin , renderitastasis , 57.2+ out of the cytoplasm into the ER, it is convenient to hypothesize that cytosolic Ca2+ gradient in migrating cells results from differential SERCA activities. If the SERCA activity was higher in the front than in the back, more Ca2+ in the front cytosol would be pumped into the front ER, resulting in the low-in-front, high-in-back Ca2+ gradient in the cytoplasm and a reverse  gradient in the ER. However, blocking SERCA activities with small molecule inhibitors caused a paradoxical increase of Ca2+ gradient, in addition to the global increase of cytosolic Ca2+ [2+ with the T1ER FRET probe [2+ gradient when the cell moved [2+ free at the basal status, SERCA does not contribute to the Ca2+ gradient in migrating cells.Since SERCA continuously and efficiently removes Calic Ca2+ . MonitorET probe  also revll moved . Therefo2+ pumps at the plasma membrane might be better candidates for the Ca2+ gradient during cell migration. Similar to SERCA, PMCA also continuously removes cytosolic Ca2+, by pumping it to the extracellular space [2+ gradient and cell motility during cell migration [2+ efflux through plasma membrane also demonstrated an enhancement of PMCA activity by 30–50% in the front of migrating cells [2+ gradient during cell migration.Based on the above data, Caar space , 59. Unligration . Direct ng cells . Hence, 2+ gradient during cell migration. Still, how PMCA is accumulated in the cell front requires further investigation.It is still not totally understood how cells adjust local PMCA activities to make them high in the front and low in the back. Several modulators have been demonstrated to regulate PMCA, including calmodulin , PKA 6161, and c2+ storage [3 receptor-based Ca2+ signaling, during which the luminal ER Ca2+ is evacuated. After IP3-induced Ca2+ release, although Ca2+ can be recycled back to the ER through SERCA, a significant amount of cytosolic Ca2+ will be pumped out of the cell through PMCA, resulting in the depletion of internal Ca2+ storage. To rescue this, low luminal Ca2+ activates STIM1 [2+ influx channel ORAI1 [2+ homeostasis could therefore be maintained during active signaling processes including cell migration.SOC influx is an essential process to maintain internal Ca storage  for IP3 es STIM1 , 64, whies STIM1 , 66. Actes STIM1 , openingel ORAI1 , 69. Ca22+-regulatory molecules to affect cell migration, the molecular mechanism is still not totally clear. Recent experimental evidence implied that STIM1 helped the turnover of cell-matrix adhesion complexes [2+ pulses in the front of migrating cells. In a moving cell, local Ca2+ pulses near its leading edge result in the depletion of Ca2+ in its front ER. Such depletion subsequently activates STIM1 at the cell front. Compatible with the above assumption, more STIM1 was translocated to the ER-plasma membrane junction in the cell front compared to its back during cell migration [2+ channels is meticulously regulated in a spatial manner maintaining cell polarity and motility.Since the identification of STIM1 and ORAI1 as the major players of SOC influx, numerous reports have emerged confirming their significant roles in cell migration and cancer metastasis Tables . Althougomplexes , 25, so igration , 66, the2+ is regulated locally and globally for effective cytoskeletal remodeling, cell migration, and cancer metastasis. Ca2+ pulses and spikes occur at the right place and right time, activating numerous downstream structural and signaling targets, which have been investigated separately over the past decades. The clarification of Ca2+ signaling in recent years has dramatically improved our understanding about how those components are regulated temporally and spatially in migrating cells. However, such advancement has revealed more questions than answers. More efforts are required to resolve those problems in the future.As described above, intracellular Ca2+ in receptor tyrosine kinase signaling pathways, during which the growth factor binds to the receptor and activates its tyrosine kinase through dimerization and autophosphorylation [3, which subsequently induces Ca2+ release from the ER. DAG and Ca2+ then bind separately to the C1 and C2 domains of classical PKC  [PKC is a typical downstream target of Carylation . The res, and γ) . Dependi, and γ) . The actα is enriched in the front of migrating cells [β phosphorylates the heavy chains of myosin II, inhibiting myosin contraction and facilitating the process of directional determination in migrating cells [2+ signaling and coordinate for effective moving activities requires further investigation.The classical PKC family has direct and significant impact on cell migration. PKCng cells . It direng cells  for a mong cells –77. How 2+ for activation [2+ dynamics during cell migration also awaits more research in the future.Besides classical PKCs, atypical PKCs  also regtivation . Togethetivation , 79, thetivation , 80. How2+ [Rho GTPases, including Rac1, RhoA, and Cdc42, have been known as the key components for the regulation of actin dynamics . It is t2+ , exert a2+ and Rho GTPases, it is reasonable to expect their mutual interactions considering their perfect coordination during cell migration [2+ influx at the leading edges of polarized macrophages resulted in the disassembly of actin filaments and lamellipodia activities [2+ on Rho GTPases. Moreover, the transamidation of Rac1 was shown to be dependent on intracellular Ca2+ and calmodulin in rat cortical cells, suggesting the biochemical link between Rho GTPases and Ca2+ signaling [2+ interacts with Rho GTPases.Although the present data reveals no evidence of direct binding between Caigration , 29, 30.tivities . The factivities  also indignaling . Hopeful2+ pulses at the junction of lamellipodia and lamella activate MLCK [Kd) of about 1 nM [2+ concentration is sufficient to induce significant activation of MLCK and subsequent contraction of myosin II. Moreover, the high sensitivity of MLCK to Ca2+ implies that the front cytoplasm has to be free of Ca2+ at the basal status, so MLCK can be inactive at baseline but respond to small rises of Ca2+ promptly. Such design justifies the physiological importance of the front-low, back-high Ca2+ gradient in migrating cells.As mentioned above, local Caate MLCK , which sout 1 nM . Therefo2+ pulses are tightly linked to the oscillatory dynamics of cell protrusion, retraction, and adhesion.In cell migration, the immediate effect of myosin contraction is the retraction of actin bundles, which not only facilitates the disassembly of F-actin at lamella but also allows the protruding front to attach to the extracellular matrix , 31. In 2+ also affects the dynamics of actin, the major component of cytoskeleton [2+ does not directly bind to actin, it affects the activities of multiple actin regulators. First of all, Ca2+ activates protein kinase C and calmodulin-dependent kinases, both of which interact with actin affecting its dynamics [2+ signaling regulates the Rho GTPases [2+ for its proper activity. Moreover, myosin, as one the major actin regulators, is totally dependent on Ca2+ for its proper activity [2+ modulates actin dynamics through multiple signaling pathways and structural molecules.Besides myosin, Caskeleton , 86. Altdynamics –89. Seco GTPases , which a GTPases , 91 alsoactivity . Therefo2+ also affects cell migration through protein cleavage and degradation. Calpain, as a Ca2+-dependent intracellular protease [2+ influx at the back of migrating cells facilitated retraction and detachment at their rear ends [2+ and PMCA in migrating cells [2+ level and higher calpain activity in the back than in the front. However, such speculation requires more experimental data to be validated.In addition to kinase activities and physical force, Caprotease , 93, is protease , paxilliprotease , and focprotease , compatiear ends . Beside ear ends . Since tng cells , decreas2+, including Pyk2, plectin, and matrix metallopeptidases.In addition to calpain, several adhesion-related proteins are also regulated by Ca2+ and protein kinase C [As a cytoplasmic protein tyrosine kinase, Pyk2 is activated by intracellular Cakinase C . It regukinase C  and the kinase C . In humakinase C .2+ also regulates the conveyance of integrin-based signaling into the cytoskeleton, with its interaction with plectin, the bridge between integrin complexes and actin filaments. Recent biochemical and biophysical evidence indicated that the binding of plectin 1a with Ca2+ effectively decreased its interactions with integrin β and with F-actin, decoupling cell-matrix adhesion with cytoskeletal structures [2+ regulation, cells could determine how many environmental signals would be conducted into the cells for cytoskeleton modification. More studies are required to clarify the above hypothesis.Caructures , 101. We2+. In prostate cancer, increased expression of TRPV2 elevated cytosolic Ca2+ levels, which enhanced MMP9 expression and cancer cell aggressiveness [2+ induced the binding of Ca2+-modulating cyclophilin ligand to basigin, stimulating the production of MMP [2+ not only modulates the outside-in (integrin to actin) signaling but also regulates the inside-out (Ca2+ to MMP) signaling for cell migration and cancer metastasis.Furthermore, matrix metallopeptidases (MMP), as facilitating factors for cancer metastasis, are also regulated by intracellular Casiveness . Furthern of MMP . Therefo2+ signaling in migrating cells, we would expect extensive interactions between Ca2+ and other signaling modules during cell migration. Indeed, though still preliminary, recent work has revealed potential cross talk between Ca2+ and other pathways controlling cell motility. These findings will shed new light on our pilgrimage toward a panoramic view of cell migration machinery.Regarding the complicated temporal and spatial regulation of Ca2+ storage in the ER, which releases local Ca2+ pulses to enhance the formation of nascent focal adhesion complexes [2+ sensor for the activation of the SOC influx, had been reported as an oncogene [In the present model, SOC influx maintains Caomplexes . Therefooncogene  or a tumoncogene  by diffe2+ and basal cell-matrix adhesion. Primary cells are usually well attached to the matrix, so further enhancing their adhesion capability might trap them in the matrix and deter them from moving forward. In contrast, metastatic cancer cells often have weak cell-matrix adhesion, so strengthening their attachment to the matrix facilitates the completion of cell migration cycles. Indeed, recent evidence suggested that, in an in vitro cell migration assay [One possible explanation of the confusing results uses the interaction between Caon assay , SOC inf2+ pulses synchronize with the retraction phases of lamellipodial cycles [2+ signaling and Rho GTPases. Clarifying how these molecules are regulated to coordinate with each other will dramatically improve our understanding of lamellipodia and help developing better strategies to control physiological and pathological cell migration.Previous reports have revealed the oscillatory activities of Rho GTPases in the front of migrating cells, including Rac1, RhoA, and Cdc42 , 30. Thel cycles , there p2+ signaling in migrating cells, together with the enrichment of RTK, phosphatidylinositol -triphosphate (PIP3), and DAG in the cell front [2+ pulses will improve our knowledge about the spatial and temporal regulation of signal transduction inside the cells. Such information will further enhance our capability to develop novel strategies targeting pathological processes and manipulating diseases.The meticulous spatial control of Call front , reveals


Mesenteric arterial EC were isolated from PAR2 wild-type (WT) and null mice, after saline (controls) or angiotensin II (AngII) infusion, for imaging intracellular calcium and characterizing the calcium-release system by immunofluorescence. EC Ca2+ signals comprised two forms of Ca2+-release events that had distinct spatial-temporal properties and occurred near either the plasmalemma  or center of EC. In healthy EC, PAR2-dependent increases in the densities and firing rates of both forms of Ca2+-release were abolished by inositol 1,4,5- trisphosphate receptor (IP3R) inhibitor, but partially reduced by transient potential vanilloid channels inhibitor ruthenium red (RR). Acetylcholine (ACh)-induced less overall Ca2+-release than PAR2 activation, but enhanced selectively the incidence of central events. PAR2-dependent Ca2+-activity, inhibitors sensitivities, IP3R, small- and intermediate-conductance Ca2+-activated potassium channels expressions were unchanged in EC from AngII WT. However, the same cells exhibited decreases in ACh-induced Ca2+-release, RR sensitivity, and endothelial nitric oxide synthase expression, indicating AngII-induced dysfunction was differentiated by receptor, Ca2+-release, and downstream targets of EC activation. We conclude that PAR2 and muscarinic receptors selectively elicit two elementary Ca2+ signals in single EC. PAR2-selective IP3R-dependent peripheral Ca2+-release mechanisms are identical between healthy and diseased states. Further study of PAR2-selective Ca2+-release for eliciting pathological and/or normal EC functions is warranted.Endothelial cell (EC)-dependent vasodilation by proteinase-activated receptor 2 (PAR2) is preserved in small caliber arteries in disease states where vasodilation by muscarinic receptors is decreased. In this study, we identified and characterized the PAR2-mediated intracellular calcium (Ca Protease activities were stopped by rapidly cooling samples on ice, and diluting the reaction mixture with ECIB containing 2 mmol/L CaCl2 (ECIB-Ca2+). Suspensions of EC and VSMC were generated by titurating samples, washed twice by centrifuging, and resuspending cell pellets in ECIB-Ca2+. Suspensions were kept on ice until used for Ca2+ imaging or immunofluorescence experiments. This method yielded 150–200 EC per mouse. EC were distinguished from VSMC by their distinct appearances (cell shapes and sizes) when viewed in different z-planes using bright field and confocal fluorescence microscopy and by the lack of platelet endothelial cell adhesion molecule-1 (PECAM-1) detection in immunofluorescence studies.Exposure to trypsin-like serine protease activity, which could desensitize PAR2, was avoided by selection of appropriate grades of reagents. Briefly, mesenteric arterial cascades were removed from heparinized (10 units) mice and arterial branches excluding the superior-mesenteric artery were dissected free of adherent tissues in ice-chilled EC isolation buffer , and a concentration range of agonists , in the absence of inhibitors of Ca2+-release channels. To control for variation between animals (within genotype and pump treatment) for agonist-mediated responses, three of the five to six runs collected internal reference data in every animal: baseline, 30 nmol/L 2fly, and 300 nmol/L ACh; the other runs collected data for one of a range of concentrations of 2fly, and ACh. In the second series, experiments were performed in the absence, and presence of inhibitors without agonists (baseline) and with single mid-range effective concentrations of agonists .The first series of Ca2+ were superfused for 1 min, buffer flow stopped and Ca2+-imaging data acquired. In the second series, inhibitors in ECIB-Ca2+ were superfused for 1 min, buffer flow stopped for 10 min, test compounds added directly to cells, and Ca2+-imaging data acquired.In the first series of experiments, test compounds in ECIB-Caμg mL−1) at room temperature for 10 min were transferred to 35 mm uncoated Fluorodish , allowed to settle from suspension (adhere to the bottom) and washed with ECIB-Ca2+ (gassed with 95% O2/5% CO2) at 37°C for 10 min. These washed preparations were placed in the experimental chamber of an inverted motorized microscope  equipped with a confocal Nipkow spinning disk unit  and superfused with O2-bubbled ECIB-Ca2+ at 37°C.Cells incubated with Fluo4-AM (10 2+ dynamics were recorded by 2-dimension (2D) CM  emitted fluorescence filtered at 512 nm and collected by a low light sensitive CCD camera . 2D full field snapshots of instantaneous fluorescence were taken at video rate (30 frames sec−1) and converted into 512 × 512 pixels resolution images. Stacks of 300 frames (∼10 sec) were converted into ratio (F/F0) images by dividing pixel-to-pixel each (F) image by the reference (F0) image selected before the Ca2+ variation of interest. Offline image processing was performed using the NIH  software ImageJ.Intracellular Ca2+ dynamics were analyzed from F/F0-image stacks. The stacks were inspected visually, Ca2+-release sites identified, and counted; the number of Ca2+ events was estimated per site over 10 sec periods. Spatial properties of Ca2+ events were determined from F/F0-images by measuring the pixel (voxel) profiles along virtual line-scans in the x/y directions; time-course of Ca2+ events estimated by measuring variations of the pixel (voxel) profiles through the stacks.Briefly, Ca2HPO4, 10; KH2PO4, 2.0), and permeabilized with 0.1% saponin in PBS containing 3% (w/v) bovine serum albumin, for blocking non-specific protein binding by antibodies. Fixed permeabilized cells were centrifuged (120g) for 5 min, resuspended in PBS, and incubated overnight with PECAM-1 polyclonal goat antibody (1:500 dilution) and a selected target antibody at 4°C. Cells were washed with PBS and incubated for 90 min at 4°C with Texas Red® dye-conjugated AffinPure Bovine anti-goat IgG  and fluorescein isothiocyanate (FITC)-conjugated AffinPure Goat anti-rabbit IgG ; both purchased from Jackson ImmunoResearch Laboratories .We modified a method  and intermediate-conductance Ca2+-activated potassium channel KCNN4 (KCa3.1); all are known to label these proteins in the mouse. Rabbit antibody for PAR2, B5 , and KCa3.1  were purchased from Abcam . Mouse antibody for human inositol 1,4,5-trisphosphate receptor (IP3R) type 1  was purchased from EMD Millipore ; the targeted amino acids sequence (22–230) is nearly identical with mouse IP3R1 and very highly conserved with mouse IP3R2 and IP3R3.Rabbit polyclonal antibodies were used to detect PAR2, endothelial or nitric oxide synthase 3 (eNOS), small-conductance Caz-steps: 0.25 μm) assessed cell volume distributed fluorescent antibodies.Standard (laser scanning) fluorescence confocal microscopy (CM) (Olympus Fluoview 1000) detected antibodies. 2D line scan images were sampled at 1024 × 1024 pixels resolution. Co-localization experiments used dual excitation . Vertical optical slicing  as percentage of Maximal Fluorescence Intensity. In space, the fluorescence distribution was characterized across the full cell width and normalized to % Max Cell Width. Signal intensity for immunoreactive protein expression data were expressed as area-under-the-curve for statistical comparisons between groups.Unless otherwise indicated general reagents and chemicals were purchased from Sigma-Aldrich .t-tests, and ANOVA with Bonferroni post hoc test were used to compare variables as appropriate. P < 0.05 was considered significant.Data are presented as mean ± SEM. Student unpaired To validate the protocol for isolated cell preparations, we measured PAR2 and EC-specific marker PECAM-1 immunofluorescence in fixed permeabilized EC. Saline WT (control) EC expressed PAR2 and PECAM-1. PAR2 immunofluorescence detection by B5-antibody co-localized with PECAM-1 near the plasmalemma, and heterogeneously spread within the cytoplasm in saline WT EC  events occurring near the plasmalemma  analyses of events at peripheral  peripheral versus central: 1.77 ± 0.01 versus 3.14 ± 0.02; majority of PAR2 Ca2+-release  occurred at peripheral sites.To identify PAR2 CaT EC Fig.B left exbserved:  events observed:  events observed:  events observed:  events observed:  events observed:  events o2+-release and was 70% larger (P < 0.05) for central versus peripheral events. The rising phase of the transient (time-to-rise [trise] from baseline to maximum amplitude) was 18% faster (P < 0.05) for peripheral than central events. The transient decline  was 1.8-times slower (P < 0.05) for peripheral than central events. Central events occurred at constant frequency (0.64 ± 0.12 Hz) in the presence of 2fly, and originated from the exact same loci in the cells.From Table2+ events which remained same as saline WT EC at basal (untreated) conditions. In saline WT and KO EC, central and peripheral events characteristics  were compared to those pretreated with xestospongin C (XeC) (IP3R inhibition), ruthenium red (RR) , and XeC + RR (IP3R and TRPV inhibition). In saline WT EC, 2fly (at EC50) increased the peripheral events . This increase was nearly abolished by XeC, partly reduced by RR, and blocked by XeC + RR (right y-axis). Similarly in saline WT EC, 2fly increased the central events  and this increase was nearly abolished by XeC, partly reduced by RR, and blocked by XeC + RR (right y-axis). This inhibition by XeC, RR, and XeC + RR of peripheral and central events densities was identical in WT AngII and saline WT EC.To identify the molecular nature of the Ca3R in the isolated EC of small caliber arteries in the healthy and diseased states, we measured the IP3R-antibody fluorescence intensity across the mid-plane of EC. IP3R colocalized with PECAM-1 at the periphery and spread heterogeneously throughout the cytoplasm in saline WT and AngII WT EC . However, the majority of Ca2+-release still occurred along the cell periphery. ACh concentration-dependent increases in the densities of total, peripheral, and central Ca2+-release events reached plateaus of 50–60% of the maximum effects by PAR2  were compared in EC exposed to XeC, RR, and XeC + RR. In saline WT EC, ACh at EC50 increased the peripheral events  and was inhibited by 80% with XeC, 30% with RR, and 90% with XeC + RR (right y-axis). Similarly in saline WT EC, ACh at EC50 increased the central events  and was nearly abolished by XeC, partly reduced by RR, and blocked by XeC + RR (right y-axis).To identify the molecular nature of the Caents Fig.C left de2+-release in WT AngII than in saline EC. In AngII WT EC, ACh increased the peripheral events density by four times, and increased the central events density by eight times  and Ca2+-uptake (t1/2) functions are different for each event type. IP3- and Ca2+-sensitivities differ between the three types of IP3R . The 2D confocal video frame rate was sufficiently fast to capture Ca2+-events reported in EC. We show EC receptor-specific dysfunction at the level of whole arteries extends to the level of single EC. On one hand, the single-cell level provides a level of cellular specificity for investigation and facilitates translating experiments with human cells. On the other hand, the single-cell level restricted the scope to EC, and thus, future studies may consider PAR2 Ca2+ signaling in the context of EC to EC and EC to VSMC.PAR2 agonists cause global cytosolic Ca2+-release in EC of small caliber arteries in the healthy state comprised two distinct specific Ca2+ events. In EC of small caliber arteries in a diseased state, PAR2 mechanisms of IP3R-mediated Ca2+-release were preserved. Further studies of PAR2-selective Ca2+-release for eliciting pathological and/or normal EC functions in tissues and in vivo are warranted.PAR2 Ca


The activation of cannabinoid receptor subtype 2 (CB2R) prevents acinar cell pathogenesis in animal models of acute pancreatitis. However, whether CB2Rs modulate intracellular Ca2+ signals in pancreatic acinar cells is largely unknown. We evaluated the roles of CB2R agonist, GW405833 (GW) in agonist-induced Ca2+ oscillations in pancreatic acinar cells using multiple experimental approaches with acute dissociated pancreatic acinar cells prepared from wild type, CB1R-knockout (KO), and CB2R-KO mice. Immunohistochemical labeling revealed that CB2R protein was expressed in mouse pancreatic acinar cells. Electrophysiological experiments showed that activation of CB2Rs by GW reduced acetylcholine (ACh)-, but not cholecystokinin (CCK)-induced Ca2+ oscillations in a concentration-dependent manner; this inhibition was prevented by a selective CB2R antagonist, AM630, or was absent in CB2R-KO but not CB1R-KO mice. In addition, GW eliminated L-arginine-induced enhancement of Ca2+ oscillations, pancreatic amylase, and pulmonary myeloperoxidase. Collectively, we provide novel evidence that activation of CB2Rs eliminates ACh-induced Ca2+ oscillations and L-arginine-induced enhancement of Ca2+ signaling in mouse pancreatic acinar cells, which suggests a potential cellular mechanism of CB2R-mediated protection in acute pancreatitis.Emerging evidence demonstrates that the blockade of intracellular Ca For example, activation of CB2Rs inhibits adenylyl cyclase via Gi/Goα subunits and causes a reduction in the intracellular level of cyclic adenosine monophosphate (cAMP)52R agonists may be useful candidates for treating inflammatory diseases and pain2R expression has been observed in spinal cord, dorsal root ganglion, and activated microglia in a rodent model of neuropathic pain, as well as in human hepatocellular carcinoma tumor samples2R mRNA and protein are expressed in pancreatic acinar cells, and activation of these CB2Rs prevents acinar cell pathogenesis in an animal model of pancreatitis2R modulates intracellular Ca2+ signals in pancreatic acinar cells is largely unknown. Specifically, it is unknown whether an agent that induces pancreatitis  enhances Ca2+ oscillations and whether application of a CB2R agonist eliminates L-arginine-induced enhancement of Ca2+ oscillations in pancreatic acinar cells.The cannabinoid receptor type 2 (CB2+ imaging approaches combined with immunohistochemistry using wild-type (WT), CB1R-knockout (KO), and CB2R-KO mice.In this study, we address these important questions using patch-clamp and confocal CaAll experimental protocols were approved by and performed in accordance with guidelines set by the animal care and use and ethical committees at the Barrow Neurological Institute, Xiangya Hospital , and Shantou University Medical College .1RKO2RKO miceMice used for this study were adult (4–6 month old), male, CD1 mice . In addition, WT, CB2+. After collagenase digestion, the cell suspension was gently pipetted to obtain further separation of the cells, and then washed with physiological saline. A 100-μL volume of cell suspension was then poured into extracellular solution in a 2-mL experimental bath. The isolated cells usually adhered to the bottom within 15–20 min and were used for recording within 3 h after preparation. All experiments were performed at room temperature (22 ± 1 °C).Acute isolated pancreatic cells were prepared as previously described13142+-activated Cl− currents for monitoring intracellular Ca2+ signal oscillations, as reported previously13H) of −30 mV. For perforated-patch recording, amphotericin B (200 μg/mL) was dissolved into the pipette solution. In these studies, we did not compensate for series resistance.Conventional whole-cell patch-clamp recording was used to record the CaA stream of standard extracellular solution was continuously perfused over the cell during recording. A computer-controlled U-tube system was used for drug applicationSerum amylase activity was measured using the AMS assay kit  and a microplate reader, following the manufacturer’s recommendations.To measure myeloperoxidase (MPO) activity, lung tissues were immediately homogenized on ice in 10 volumes of normal saline. MPO activity was measured using the MPO assay kit  and a microplate reader, following the manufacturer’s recommendations.1R-KO, and CB2R- KO mice (3 mice for each group) were anesthetized and quickly perfused with saline to flash all blood cells. Both whole striatum and spleen tissue were dissected out, snap frozen, and kept on dry ice. All the tissues were homogenized in cell lysis buffer  using a sonicator and centrifuge at 15,000 rpm for 15 min at 4 °C to get supernatant. The protein concentration for each sample was quantified with a Bio-Rad Protein Assay . A total of 20-μg protein (spleen) or 40-μg protein (striatum) were loaded and separated by SDS-PAGE in a 4–15% gradient gel for the detection of endogenous calnexin  and CB2R (NIDA-5633) by using Invitrogen blotting and transferring modules . Membranes were blocked for 2 h at room temperature with Licor Odyssey blocking buffer  after washing 3 times with phosphate-buffered saline containing 0.1% Tween-20. Membranes were first incubated with either anti-CB2 (1:500 NIDA-5633 Ab) or anti-calnexin  antibody overnight at 4 °C. After washing 3 times, the membranes were incubated with goat anti-rabbit IgG (IRDye 680CW)  for 1.5 h at room temperature. Then the membranes were washed 3 times and then scanned in a Licor Odyssey Sa Imaging System (LI-COR Biosciences).WT, CB2R antibody  at 4 °C overnight. After washing 3 times with 0.1 M PB, sections were incubated with Alexa Fluor 488 goat anti-rabbit secondary antibody  in 5% BSA and 0.5% Triton X-100 PB for 2 h at room temperature. Sections were then washed, mounted, and cover slipped. Images were taken with a fluorescence microscope (Nikon Eclipse 80i) equipped with a digital camera .Sections were first blocked in 5% bovine serum albumin (BSA) and 0.5% Triton X-100 in phosphate buffer (PB) for 2 h at room temperature. Then, sections were incubated with 1:500 NIDA-5633 mCBhttp://imagej.nih.gov/ij/).Dissociated pancreatic acinar cells were first incubated with fluo-4-AM (15 μM)  for 15 min, followed by a 10-min rest allowing for de-esterification of the indicator. Confocal imaging was performed using an Olympus FluoView FV1000 microscope  equipped with an argon laser (488 nm) and a UPLSAPO 40×, 0.95 NA objective. X-Y imaging was performed at a rate of 1.644 s per frame, 400 frames total, with a resolution of 512 × 512. Fluorescent fluo-4 signal was measured using ImageJ v.1.47  140 KCl, 1.13 MgCl2, 5 Na2ATP, 0.24 EGTA, 10 glucose and 10 HEPES, pH 7.2. Drugs used in this study were GW405833 (Standard extracellular solution contained (in mM): 140 NaCl, 1.0 CaClGW405833 , JWH133,2R antibodies, NIDA-5633 mCB2-Ab  that recognize the C-terminal (326-340 aa) of mCB2Rs, were produced by Genemed Synthesis Inc. .CB2+-activated Cl− current responses were presented as the current charge (current area/Cm/min), and then the drug-induced changes were compared to the baseline level of charge (induced by ACh). When data were obtained from the same recorded cell and the changes of ACh response were compared before, during, and after testing drug exposure, a paired Student t test was used. To compare the effect of the tested drug between 2 groups of animals , the unpaired Student t test was used. To analyze multiple effects, one-way analysis of variance (ANOVA) with Tukey’s post hoc tests were used.For patch-clamp experiments, the Ca2-positive band was detected (at ~40 kD) in both the spleen and striatal tissues of WT and CB1-KO (CB1−/−), while the densities of this band in CB2-KO mice (CB2−/−) were substantially reduced in CB2-rich spleen tissues and almost undetectable in striatal tissues. 2R immunostaining with the CB2R antibody (NIDA-5633), illustrating that the high densities of CB2R immunostaining were detected in the majority of spleen cells of WT mice. In contrast, a very low density of CB2-like staining was detected in a minority of spleen cells in CB2R KO mice, suggesting that the NIDA-5633 antibody used is highly mouse CB2R-specific. We then used this antibody to detect CB2R expression in single isolated acinar cells. 2R antibody , DAPI (Cc), and merged mCB2-ir and DAPI (Cd). We found high densities of CB2R immunolabeling in pancreatic acinar cells , on ACh-induced Ca2+ oscillations. 2+ oscillation response (as a baseline). Then, the GW is added to the bath perfusion in the presence of ACh. Finally, the GW is washed out with the same concentration of ACh. With this protocol, the ACh is continuously perfused throughout the recording period, and we can compare the change of ACh-induced Ca2+ oscillations before GW perfusion (baseline), during GW perfusion, and after GW washout in the same recorded cell. For statistical analysis of the effects of GW on ACh-induced Ca2+ oscillations, we measured baseline oscillations as the charge (current area/Cm/min)2+ oscillations during GW perfusion and after washout of GW to the baseline. Our data showed that in the continuous presence of 10 nM ACh, 10 μM GW reduced Ca2+ oscillations, and this inhibitory effect was reversed after washout . In 10 μm GW group, Ca2+ oscillation levels were reduced from baseline −4.69 ± 0.32 to −1.68 ± 0.32 nC/min . In 100 μm GW group, Ca2+ oscillation levels were reduced from baseline −5.77 ± 1.75 to −0.50 ± 0.15 nC/min . Further comparisons determined that Ca2+ oscillation levels differed significantly between the following groups: GW 1 μM vs. 10 μM (p < 0.05), GW 1 μM vs. 100 μM (p < 0.05), and GW 10 μM vs. 100 μM (p < 0.01), which confirms that GW inhibition occurs in a concentration-dependent manner.To profile the pharmacological effect of GW on ACh-induced Ca2+ oscillations is mediated through CB2Rs, we designed three sets of experiments. 1) We tested the effect of a selective CB2R antagonist (AM630) on GW inhibition of Ca2+ oscillations. 2) We examined GW inhibitory effects on pancreatic acinar cells prepared from CB1-KO and CB2-KO mice. 3) We evaluated the effects of a selective CB1R agonist (ACEA) on ACh-induced Ca2+ oscillations. The results of these experiments demonstrated that GW inhibition of ACh-induced Ca2+ oscillations was presented in WT , CB1R-KO , and CB2R-KO  mice. Furthermore, co-application of AM630 (0.1 μM) and GW (10 μM) abolished the inhibitory effect of GW on 10 nM ACh-induced Ca2+ oscillations , while AM630 alone had no affect  also reduced ACh-induced Ca2+ oscillations but this effect was likely mediated through ethanol that was used to dissolve ACEA  receptors2R agonist (GW) or antagonist (AM630) was applied internally or in which, IP3 was applied internally. When GW (100 μM) was added into the recording electrode and a perforated whole-cell recording (amphotericin B) was performed, bath-application of 10 nM ACh induced Ca2+ oscillations. When the recording mode was switched from perforated to conventional whole-cell recording by a brief suction, GW was infused into the recorded cell, and no detectable inhibitory effect on ACh-induced Ca2+ oscillations was present (2+ oscillations (3 (30 μM), which causes IP3-induced Ca2+ oscillations, GW produced little inhibitory effect on the IP3-induced Ca2+ oscillations , but GW inhibited ACh-induced Ca2+ oscillations in the same recorded cell .Data presented thus far demonstrate that GW inhibited ACh-induced Ca 100 μM, . In the llations . Figure 2+ oscillations from baseline level of 4.93 ± 0.39 to 10.34 ± 1.83 nC/min  in an irreversible manner  and pulmonary peritoneal macrophages (MPO) levels, which are two major effects present in early-stage of acute pancreatitis. We injected L-arginine  to establish an acute pancreatitis model232+ oscillations between saline- and L-arginine-treated groups using Ca2+ imaging. Systemic L-arginine injection enhanced ACh-induced Ca2+ oscillations compared to systemic saline injection, but GW and L-arginine co-injected showed similar level of ACh-induced Ca2+ oscillations , while co-injection of GW and L-arginine reduced L-arginine’s effect . These results suggest that the activation of pancreatic acinar cell CB2Rs may prevent early pathogenesis of acute pancreatitis through the inhibition of enhanced intracellular Ca2+ signals. In addition, co-injection of GW  and L-arginine  also significantly reduced pancreatic L-arginine-induced enhancement of AMS  or is absent in CB2-KO mice, but not in CB1-KO mice. The pancreatitis inducer, L-arginine, significantly enhances ACh-induced intracellular Ca2+ oscillations, and the CB2R agonist, GW, abolishes this L-arginine effect. In addition, this CB2R agonist also improved L-arginine-induced pathological changes. Collectively, our data demonstrate that CB2R agonist GW reduces ACh-enhanced intracellular Ca2+ signals in mouse pancreatic acinar cells, and this may underlie an important cellular mechanism for a CB2R agonist to serve as a new candidate for treating acute pancreatitis.The novel findings of this study are that the activation of membrane CB2R protein expression was found using immunohistochemical staining and Western blot101R and CB2R mRNA were identified using real-time RT-PCR and immunohistochemical staining2R proteins were expressed in freshly isolated mouse pancreatic acinar cells, which is consistent with previous report2Rs are expressed in mouse pancreatic acinar cells and they may play an important role in modulating acinar cells function.Previously, in rodent pancreatic acinar cells, CB2+ oscillations for studying pancreatitis2R agonist, GW, affected ACh-induced Ca2+ oscillations in the isolated pancreatic acinar cells through CB2Rs. Using both patch-clamp recording and confocal Ca2+ imaging techniques, we found that GW significantly reduced ACh-induced Ca2+ oscillations, and this inhibition is GW-concentration dependent. We also tested another selective CB2R agonist, JWH-133, on the ACh-induced Ca2+ oscillations, and found a similar inhibition  intracellular Ca2+ levels in endothelia cells1R2R2+ oscillations and insulin release. It has been reported that anandamide reduced intracellular Ca2+ concentration through the suppression of a Na+/Ca2+ exchanger current in rat cardiac myocytes2R agonist reduces intracellular Ca2+ signals in mouse pancreatic acinar cells. Considering that Ca2+ plays an important role in cellular function, especially enzyme secretion in pancreatic acinar cells, our data suggest that CB2R modulates an important aspect of pancreatic acinar cell physiology and pathophysiology.Accumulating evidence demonstrates a complex relationship between the cannabinoid ligand (and receptors) and intracellular Ca1R or CB2R, but in some cases they also can act on non-cannabinoid targets2+ signals through a cell membrane or cytosolic CB2Rs. First, we examined the effects of pharmacological manipulations of CB1R and CB2R and found that the CB2R selective antagonist AM630 abolished GW-induced reduction of Ca2+ oscillations, suggesting that GW modulates ACh-induced Ca2+ oscillations through the CB2Rs. Then, we genetically manipulated cannabinoid receptors and compared the effects of GW on Ca2+ oscillations between WT and CB2R-KO mice, and also WT and CB1R-KO mice. We found that in CB2R-KO but not CB1R-KO mice, GW lost its inhibitory effect, further confirming that CB2R is the key target for mediating GW-induced reduction in Ca2+ oscillations.Cannabinoid ligands exert their pharmacological effects through CB1R agonist, ACEA  reduced ACh-induced Ca2+ oscillations , and found that 1 μM DMSO itself did not affect ACh-induced Ca2+ oscillations . To address this question, we designed three experiments. We first examined the effect of bath-applied GW on the Ca2+ oscillations induced by intracellular application of IP3, and found that GW did not affect IP3-induced Ca2+ oscillations, suggesting that the target that mediated GW-induced inhibition in Ca2+ oscillations is located in the signal pathway before IP3 receptors, and not on the IP3 receptor itself. We then intracellularly applied GW through a recording electrode to examine the effect of intracellular administration of GW on bath ACh-induced Ca2+ oscillations, and found that intracellular infusion of GW (even at 100 μM) did not alter ACh-induced Ca2+ oscillations. Finally, we intracellularly applied AM630 through a recording electrode to examine the effect of bath-applied GW on ACh-induced Ca2+ oscillations. Our data showed that intracellular infusion of AM630 did not prevent bath-applied GW-induced reduction of Ca2+ oscillations. Collectively, our data support the conclusion that GW modulates intracellular Ca2+ signaling through the membrane CB2Rs in pancreatic acinar cells.Finally, we asked where the CB2+ signals is unclear. Our data show that membrane CB2Rs are necessary for mediating GW’s effect. GW’s action in ACh-induced Ca2+ oscillations should occur at the G-protein-mediated signal pathway between muscarinic receptor (M3) activation and IP3 production because GW did not affect IP3-induced Ca2+ oscillations. We also demonstrated that GW failed to affect ACh-induced Ca2+ oscillations in pancreatic acinar cells prepared from CB2R-KO mice, suggesting that GW likely did not affect muscarinic receptor function. In addition, we found that bath-applied GW failed to inhibit CCK-induced Ca2+ oscillations even at 100 μM, suggesting that GW selectively modulates muscarinic receptor-mediated G-protein signaling2+ oscillations may involve cross talk between muscarinic receptor- and CB2R-mediated G-protein signal pathways, such as homologous and/or heterologous desensitization of G-protein coupled receptors (GPCRs)39383 and Ca2+ responses to ACh in rat submandibular acini. Investigators found that intracellular cAMP increased IP3 formation in response to ACh, while blocking PKA by H89 reduced IP3 formation2Rs significantly reduces intracellular cAMP levels, we thus postulated that GW may activate CB2Rs, reduce cAMP, and in turn reduce intracellular IP3 production, and lead to a reduction of ACh-induced Ca2+ oscillations. Our findings warrant further testing of this hypothesis.The precise mechanism by which GW modulates intracellular Ca2+ persist, intracellular signaling is disrupted, cell damage occurs, and acute pancreatitis forms. Emerging evidence suggests that the earliest abnormalities of acute pancreatitis arise by aberrant elevation of intracellular Ca2+ within acinar cells because the sustained intracellular Ca2+ elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation, and pharmacological blockade of store-operated or Ca2+ release-activated Ca2+ channels would prevent sustained elevation of intracellular Ca2+, and consequence protease activation and necrosis2R agonist, GW, reduces ACh-induced Ca2+ oscillations, abolishes L-arginine–induced enhancement of Ca2+ oscillations and prevents L-arginine–induced elevation of both pancreatic AMS and pulmonary MPO levels. These results suggest that a CB2R agonist may serve as a novel therapeutic strategy to prevent and/or treat acute pancreatitis. This conclusion is consistent with previous report that a CB2R agonist exhibits a protective effect on pathogenesis in an acute pancreatitis animal model2+ signaling by GW also provide a new target to interpret the role of CB2R agonists in treating acute pancreatitis in addition to CB2R-mediated anti-inflammation.Pancreatic acinar cells are functional units of the exocrine pancreas. They synthesize, store, and secrete inactive preforms of digestive enzymes into the lumen of the acinus. The activity of pancreatic acinar cells is crucially modulated by the secretagogues ACh and CCK; both can act on their specific membrane receptors  and then induce an elevation in cytoplasmic calcium. If high concentrations of intracellular CaHow to cite this article: Huang, Z. et al. Cannabinoid receptor subtype 2 (CB2R) agonist, GW405833 reduces agonist-induced Ca2+ oscillations in mouse pancreatic acinar cells. Sci. Rep.6, 29757; doi: 10.1038/srep29757 (2016).


SOCE is important for a multitude of physiological processes, including muscle development, T-cell activation, and fertilization. Therefore, understanding the molecular regulation of SOCE is imperative. SOCE activation requires conformational and spatial changes in proteins located in both the endoplasmic reticulum and plasma membrane. This leads to the generation of an ionic current of very small amplitude. Both biochemical and electrophysiological parameters of SOCE can be difficult to record in small mammalian cells. In this protocol we present the different methodologies that enable the study of SOCE in a unique model system, the frog oocyte, which provides several advantages and have contributed significantly to our understanding of SOCE regulation.Store-operated Ca Cytoplasmic Ca2+ levels are maintained at low levels (on the order of 100 nM) providing a low baseline for Ca2+ transients, and as such enhancing signaling efficiency. In contrast to the low cytoplasmic Ca2+ levels, Ca2+ concentrations in the extracellular space are high, 1–2 mM. The other source for Ca2+ is intracellular stores, primarily in the lumen of the endoplasmic reticulum (ER) with Ca2+ concentrations in the hundreds of micromolar range  and its required effectors have been identified in some protozoa as well as vertebrates and invertebrates but have not yet been identified in plants   that localize to distinct cellular compartments. Imaging the spatial organization of SOCE regulators in small mammalian cells is a tedious task given the complexity of the spatial distribution of organelles such as the ER. The large size of the Xenopus laevis oocyte, which allows for good spatial separation between the ER and the plasma membrane makes it an excellent model to study the distribution of the membrane proteins involved in SOCE. Another limiting factor in mammalian cells is the SOCE current density that can be as small as 1 pA/pF. Although the density of the endogenous SOCE current in the Xenopus oocyte is similar to what is observed in mammalian cells (≈0.5 nA/nF), the large oocyte surface area (200–250 nF) results in a significant SOCE current (~100 nA)  that can be used as an amplifying system to report Ca2+ influx through SOCE , the animals are quarantined after shipment for 4 weeks in static aquaria before being transferred to a recirculating water system . They live on a 12/12 dark/light photoperiod and are fed twice a week.Animals: hCG (human chorionic gonadotropin) injected wild 3 (2 g/L).Anesthetics: Tricaine  is dissolved at a concentration of 5 g/L in deionized water containing NaHCO2+-free solution Ringer contains 96 mM NaCl, 2 mM KCl, 5 mM MgCl2-6H2O, 5 mM Hepes. The pH is adjusted to 7.6 with NaOH. The Ca2+ containing solution is made by adding 0.6 mM Ca2+ to the Ca2+ free media, both solutions are filtered sterilized.Solutions: nominally Ca2+-free solution (2 mg/ml) and placed in 50 ml conical tubes (10 ml per tube).Enzyme: Collagenase IA (Affymetrix), is dissolved on the day of the surgery in CaThe oocytes are stored in 12 cm Petri dishes at 18°C.Oocyte media: The oocytes are kept in 0.5x L15 media  supplemented with 10 mM Hepes, penicillin (10 units/mL), and streptomycin (10 μg/mL) and gentamycin (100 μg/mL), pH is adjusted to 7.6 with NaOH and the solution is filter sterilized. The L15 solutions are stored at 4°C and used within a week.RNA synthesis is performed using the “mMessage mMachine T7 Kit” according to the manufacturer instructions (Life Technologies).Injection needle: Injection needles are pulled from glass capillaries  using a P2000 puller (Sutter Instruments), the tip is then broken with tweezers to generate a tip diameter of ~20 μmMicro-injection setup: A plastic grid (1.5 mm mesh opening) is glued  at the bottom of a 50 mm plastic dish to hold the oocytes in place while injecting. The injection needle is mounted on a micro-injector  attached to a three axis manipulator. The injection is performed under a stereomicroscope . Some oocytes will die after injection and some will not express consequently, for imaging and electrophysiology, 25–50 oocytes are injected allowing to record 5–10 individual cells.Sharp microelectrodes: Intracellular microelectrodes are pulled from glass capillaries  using a P97 puller (Sutter instruments). They have a resistance of 1~2 MΩ when filled with 3M KCl.Recording setup: The two electrodes are connected to a GeneClamp 500B amplifier controlled by pClamp 10.5 (Axon Instruments). The signals are digitized using a Digidata 1322 (Axon Instruments). The reference electrode consists of a chlorinated silver wire.Perfusion: The recording chamber is a custom made half bowl (~1 cm in diameter) milled in a block of plexiglas. The cells are continuously superfused using a peristaltic pump . A gravity perfusion device is added to the system to allow fast application of drugs contained in 50 ml syringes. Both perfusion systems are connected to the recording chamber using a 4-way manifold to minimize the dead volume .2, 2 mM MgCl2, and 10 mM Hepes, pH adjusted to 7.4 with NaOH.Recording solutions: The composition of the standard Ringer solution is: 96 mM NaCl, 2.5 mM KCl, 1.8 mM CaClExpression screening: The cells expressing fluorescent proteins are tested for expression using a stereomicroscope .The oocytes are dropped in a holder made of a metal plate (0.8 mm thick) drilled with a hole of 1.3 mm in diameter. The grid is placed in a recording chamber whose bottom is a glass coverslip (Warner Instruments Series 20).Confocal imaging: We use two different inverted confocal microscopes for the imaging of live cells: a LSM710 (Zeiss) fitted with a Plan Apo 63x/1.4 oil immersion objective or a TCS SP5 (Leica) fitted with a 63x/1.4–0.6 oil immersion objective. The images are recorded with Zen Black 2012 (Zeiss) or LAS AF 2.4.1 (Leica).Analysis software: All imaging analysis are performed using the MBF version of ImageJ or FIJIThe frog is anesthetized in a 2 L beaker containing 50 ml of Tricaine and put in a closed box for 20 min. The nostrils of the animal are left intentionally outside the liquid to prevent drowning during sedation. The leg of the animal is pinched to ascertain complete sedation before dissection.A double pithing is performed. First, hard scissors are placed through the mouth of the frog and the superior part of the skull is cut out. Second, a sharp dissection needle is inserted in the spinal canal to destroy the spinal cord.2+-free media to limit cell toxicity during the enzymatic processTwo vertical paramedian incisions (2 cm) are made on the left and right side of the abdomen through the skin and the muscles. The ovaries are gently pulled out through the incisions and placed in a 12 cm petri dish containing CaThe ovarian lobes are then carefully cut open to flatten the sacs and allow access to the enzyme.The tissue is then placed in two 50 ml tubes filled each with 10 ml of the collagenase solution and left on a rocking platform for 1 h. The collagenase solution is then renewed and the cells incubated for another hour.The digestion of the oocytes is carefully monitored by removing a few cells and checking for the efficiency of the digestion under a dissecting microscope. Different batches of collagenase will have different activity, which will affect the timing of the digestion. Over-digestion will decrease oocyte quality. Efficient defolicullation of the oocytes can be assessed by simply staining the cells with the nuclear dye Hoechst 33342 to visualize the nuclei of follicle cells around the oocyte.2+-free solution and 4x with Ca2+-containing Ringer to remove all the collagenase before transferring them to L15 media. Undigested clusters of cells are discarded using a plastic Pasteur pipette and stage VI oocytes can be selected according to their size (above 1 mm). The quality of the oocytes often correlates with the sharpness of the ring separating the animal (dark) and vegetal (yellowish) poles. The oocytes are stored at 18°C and used within a week of their isolation.The cells are then washed 4x with CaXenopus β-Globin gene. This helps decrease cRNA degradation once injected in the oocyte.Coding sequences of interest are inserted into the multiple cloning site of the pSGEM vector  of the RNA solution placed onto a piece of parafilm under a stereomicroscope.Oocytes are injected at least 24 h after isolation. The oocytes are held in a plastic grid and bathed into L15 during the injection procedure. The cells are injected in either pole with volumes typically ranging between 9.2 and 46 nlXenopus oocytes variations in intracellular Ca2+ concentrations near the plasma membrane can be monitored using the activation of large CaCC that amplifies the Ca2+ signals . In 2+ imaging has been used to monitor the increase in Ca2+ concentration induced by the hyperpolarizing voltage pulse to −140 mV, as well as the clearance kinetics after depolarization , termed ICl1. The second pulse to -140 mV increases the driving force for Ca2+ entry through any open Ca2+-permeant channel and activates an inward Cl− current, termed ICl2 with smaller amplitude than ICl1 due to the fast inactivation and outward rectifying properties of the CaCC. The third pulse is identical to the first one , the Cl− current obtained is therefore a reading of [Ca2+]i after the second pulse. The difference in amplitude between the Cl− currents obtained in the +40 mV depolarizing pulses 1 and 3 reflects Ca2+ influx triggered during the second hyperpolarizing pulse to −140 mV and is termed IClT (for transient). IClT decays with similar kinetics as the clearance of intracellular sub-membrane Ca2+  and Orai1 (1 ng).2+ release can be measured as a large transient increase in ICl1  to empty the ER stores. The intracellular Ca Figures .2+ influx induced by SOCE can be monitored as the development of the IClT component and is larger in STIM1/Orai1 injected oocytes  is tip-filled with a stock solution of 250 mM BAPTA and inserted into the oocyte. The required amount of BAPTA to be injected will depend on the depth of the pipette tip in the oocyte and on the size of the Cat Figure , or at t2+ can also be raised from 1.8 to 5 mM.To increase the amplitude of the recorded current, the extracellular CaThe SOCE current is studied using a voltage ramp from −120 mV to +40 mV.3+ (100 μM) is bath applied to fully inhibit the SOCE current  and GFP-Orai1 (2 ng) are injected into the oocyte and allowed 2–3 days to express  or by depleting the stores with a short term (20 s) application of ionomycin (10 μM)  to mimic store depletion Figures .The same z-stacks reveal large bright clusters at the plasma membrane level in both channels indicating the translocation of STIM1 to the cortical ER, as well as the co-clustering of the two proteins. The cortical ER following store depletion localizes to the same focal plane as the plasma membrane.Xenopus oocyte .Eggs can be spotted by the white spot in the animal pole indicating Germinal Vesicle Breakdown (GVBD). This can be further confirmed by fixing the oocytes and cutting them in half to visualize the breakdown of the nuclear envelope.Confocal imaging is performed on oocytes and eggs according to the procedure described above in Section Visualizing STIM1 and Orai1 clustering.On the egg, Orai1-GFP fluorescence is reduced at the plasma membrane and is enriched in intracellular vesicles Figure .T current development is visible in cells overexpressing STIM/Orai as well as in naïve oocytes. As stated before, the size of the IClT is measured using the peak difference between the current induced by the voltage pulse to +40 mV before and after the −140 mV hyperpolarizing pulse. The increase in the current amplitude when overexpressing SOCE proteins is very important  a small number of cells (5–10) will give an adequate reading. When expressing STIM or Orai mutants, a larger number of cells (and frogs) will be required to detect any effect on the ionic currents. Statistical analysis is usually performed using an unpaired Student “t” test and if more than two conditions are used an ANOVA test is used. There is usually no need to normalize the current amplitude unless some great variability appears in the current obtained with the wild type proteins. The currents in the naïve cells being very small when compared to overexpressing ones it is not advisable to use them as a normalization reference.After store depletion, the IClt Figure . If too Several parameters can be extracted from the confocal images to define the behavior of STIM1 and Orai1 during store depletion  reflects the distance between the Ch-STIM1 and GFP-Orai1 protein populations.The fluorescence intensity of each channel of each optical slice is normalized to the maximum value of the stack and is plotted as a function of its position on the z axis Figure 9. The did.After store depletion with TPEN, the translocation of Ch-STIM1 to the cortical ER can be measured as a dramatic reduction in An alternative method is to define the peak of the Orai1 curve as a reference for plasma membrane plane and to measure the area under the curve of the STIM1 signal at the left and right of the Orai1 maximum Figure . This giThe enhanced co-localization of Ch-STIM1 and GFP-Orai1 in the clusters can be visualized in the co-localization plots Figure , and meaCluster numbers, area and distribution can be measured using the ImageJ “Analyze particles” plugin  and the cluster density. The density is evaluated by measuring the distance between the centers of each cluster using the “Nearest Neighbor Distances” plugin of image JTo measure Orai internalization in the egg, since Orai is moving toward the cytoplasm, it cannot be used anymore as a membrane reference. Then either the STIM1 maximum is used as an intracellular reference  or another reference has to be chosen at the plasma membrane Figure . The CaC2+ signaling the oocyte expresses a very small number of Ca2+-permeable channels at the plasma membrane (probably resticted to Orai1 and TRPV6) and the Ca2+ influx at rest is reduced. It therefore provides a very good background for recording overexpressed channels. Similarly, the only source for intracellular Ca2+ release from the ER is the IP3 receptors creating as simple mechanism of store depletion. The size of the oocyte allows the simultaneous recording of the formation of the SOCE clusters in 3 days  as well as the development of the Ca2+ current using CaCC as amplifiers.In terms of Ca2+ signaling (Courjaret and Machaca, Using the different experimental approaches described in this chapter, in the frog oocyte one can study structure-function properties of STIM1 and Orai1 (Yu et al., RC performed experiments and analyzed data. KM analyzed data. RC and KM developed methods and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It has been considered as an important signaling molecule that mediates various physiological and biochemical processes in plants. Normal metabolism in plant cells results in H2O2 generation, from a variety of sources. Also, it is now clear that nitric oxide (NO) and calcium (Ca2+) function as signaling molecules in plants. Both H2O2 and NO are involved in plant development and abiotic responses. A wide range of evidences suggest that NO could be generated under similar stress conditions and with similar kinetics as H2O2. The interplay between H2O2 and NO has important functional implications to modulate transduction processes in plants. Moreover, close interaction also exists between H2O2 and Ca2+ in response to development and abiotic stresses in plants. Cellular responses to H2O2 and Ca2+ signaling systems are complex. There is quite a bit of interaction between H2O2 and Ca2+ signaling in responses to several stimuli. This review aims to introduce these evidences in our understanding of the crosstalk among H2O2, NO, and Ca2+ signaling which regulates plant growth and development, and other cellular and physiological responses to abiotic stresses.Hydrogen peroxide (H H2O2 is continually synthesized through various sources including enzyme and non-enzyme pathways in plants. To date, it has become accepted that H2O2 plays important roles in plant developmental and physiological processes including seed germination  signaling is also a core regulator of plant physiological process and stress adaption such as cell polarity regulation , as a small signaling molecule, appears to be involved in plant developmental and physiological processes such as seed germination , and redox reaction.HMittler, . Figure 2O2 production in plants through several enzymes includingcell wall peroxidases  and glutathione (GSH), as non-enzymatic compounds, are constantly participated in regulating ROS level  seedlings  to induce thermotolerance in maize seedlings. Moreover, the interaction among H2O2, NO and Ca2+ could relieve copper stress in Ulva compressa , which improved tolerance to cold stress  and H2O2 also played a significant role in antioxidant defense in ABA signaling in maize leaves  which could regulate catalase3 (CAT3) activity mediating stomatal movement  could enhance heat stress tolerance by modulating H2O2 production. Over production of H2O2 induced by heat stress increased OsANN1 expression and up-regulated the level of SOD and CAT expression, which constructed a signaling mechanism for stress defense in plants  and NO in Ca2+-dependent guard cell signaling. It was shown that CAS may transduce Ca2+ signaling through activating its downstream target NO and H2O2 signaling pathway  through activating plasma membrane- and intracellular membrane-associated Ca2+ channels. Besides, the interaction among H2O2, NO, and Ca2+ signaling may regulate ABA-induced antioxidant defense in maize (Ma et al., 2O2, NO and Ca2+ may increase antioxidant system and induce disease defense in plants.It has been suggested that there is a connection among Hs Figure . For inse et al.  has repo2O2, NO, and Ca2+ also have an effect on abiotic stress response in plants. For example, Lang et al. (2+ and H2O2 in Aegiceras corniculatum to up-regulate Na+/H+ antiport system of plasma membrane under salt stress. There were species-specific interactions between H2O2, Ca2+, NO, and ATP in salt-induced reduction of K+ efflux (Lang et al., 2O2, NO, and Ca2+ when Ulva compressa exposed to copper excess and the interaction had a significant effect on transcriptional activation of target genes (Alberto et al., 2O2-induced NO generation could be inhibited by Ca2+ channel blockers, implicating that Ca2+ may mediate the effect of H2O2 on NO production. Furthermore, Ca2+ release through different type of Ca2+ channels was also shown to be activated by NO and H2O2 (Alberto et al., 2O2, NO and Ca2+ may provide additional layers of responses to abiotic stresses through controlling ion transport, increasing antioxidant enzyme activities and affecting expression of resistance genes, indicating a feedback mechanism between H2O2, NO and Ca2+ under abiotic stresses. In a word, the combination of these findings strongly supports the view that there has an interaction among H2O2, NO, and Ca2+ signaling pathway in plant growth, development and abiotic stress responses. During signaling transduction, Ca2+ signaling could be activated by H2O2 and NO; it could also regulate H2O2 and NO signaling. Ca2+ may act as a point of signaling convergence between H2O2 and NO signaling pathways in plants. However, the network of H2O2, NO, and Ca2+ seems to be intricate and multidimensional. Therefore, considerably more work will need to be done to determine the interaction among H2O2, NO and Ca2+ signaling in plants.Furthermore, the interplay among Hg et al.  reported2O2 was once considered as a poisonous molecule in plants. Based on current studies, H2O2 may be a vital signaling molecule which controls plant growth and development. Interestingly, NO and Ca2+ which also act as the key component of signaling transduction in plants seem to be as upstream or downstream signaling molecules of H2O2. Meanwhile, H2O2 modulates NO and Ca2+ signaling pathways. There is a complex interactive network among H2O2, NO, and Ca2+ in plants. Moreover, the interplay among them has functional implications for regulating developmental and physiological processes which may increase the possibility of signal reception and transduction in plants. Future work will need to focus on the molecular mechanism of the interplay among H2O2, NO, and Ca2+ during signaling transduction in plants.HLN wrote the paper. WL provided the idea and revised the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antibody-based immunotherapy represents a promising strategy in cancer. BiTE® antibodies have demonstrated efficacy in hematologic malignancies, both preclinically and clinically.BiTE® antibody, Blinatumomab, directs cytotoxic T cells to CD19-expressing target cells. Blinatumomab has shown anti-leukemia activity in adult relapsed/refractory (r/r) B-precursor ALL. Its efficacy and toxicity was evaluated in a large confirmatory Phase II study. Patients with Ph-negative r/r ALL (N = 189; refractory; 1st relapseTwo investigational BiTE antibodies are under development targeting leukemia. The most advanced BiTE® antibody targeting CD33, AMG 330, was developed for its suitability as immunotherapy in AML. To simulate the natural setting of target and T cells in AML patients, a long-term culture system was developed that supports the growth of primary AML cells ex-vivo for up to 5 weeks. AMG 330 activated and expanded residual autologous T cells within primary AML patient samples and eliminated CD33+ blasts even at very low effector to target ratios. The functional relevance of CD33 expression levels was shown by faster lysis kinetics of CD33BRIGHT versus CD33DIM AML cell lines and primary AML cells in ex-vivo cytotoxicity assays. However, by extending the exposure time to AMG 330, potent anti-leukemic activity was observed in both CD33BRIGHT and CD33DIM cells. AMG 330 treated T cells were shown to up-regulate the activation markers CD25, PD-1, TIM3 and LAG3, which was partially reversible after complete target cell eliminationGiven the anti-leukemia activity of single-agent Blinatumomab in a difficult-to-treat population with r/r ALL, another BiTE® antibodies for targeted T cell-mediated immunotherapy of patients with malignancies.Clinical experience with Blinatumomab in ALL and ex-vivo activity of AMG 330 in primary AML samples supports further development of BiTE


Chromosomal translocations are a major cause of cancer. At the same time, the mechanisms that lead to specific chromosomal translocations that associate different gene regions remain largely unknown. Translocations are induced by double strand breaks (DSBs) in DNA. Here we review recent data on the mechanisms of generation, mobility and repair of DSBs and stress the importance of the nuclear organization in this process. Many cancers are characterized by chromosomal translocations. First evidence of translocations and their possible role in cancer was put forward by German cytologist Theodor Boveri 100 years ago, in 1914 ). A combc-myc and immunoglobulin heavy chain (IgH) loci when c-myc dynamically colocalizes with IgH upon activation of B-lymphocytes [Currently, two hypotheses have been formulated to explain the proximity of damaged loci resulting in translocation events. The “contact first” hypothesis proposes that the translocation partner loci are proximal within the nucleus during the event leading to DSBs. The key prediction of this model is close spatial proximity of translocation partners . This prphocytes . This cophocytes ).Positions of the chromosomes in the nuclear space also affect the localization of potential translocation partners. Gene-rich and small chromosomes tend to be clustered in the center of the nucleus ; thus a An alternative “breakage first” hypothesis postulates that chromosome ends with DSBs can freely move within the nuclear space and that collision of damaged loci on different chromosomes may lead to translocations. In this case, the probability of translocation may increase with the scale of DSB movement. This movement can be either directed or stochastic, resembling the Brownian motion of particles. The amplitude of stochastic movements might increase after the DSBs thus increasing the volume of the nuclear space scanned by the damaged locus. The probability of collision of distant loci followed by a translocation might also increase in this case. A combination of both directed and stochastic movements is also possible: the stochastic movement might bring the locus in contacts with a structure, e.g. a nuclear membrane, interchromosomal space or a perinucleolar region , and in DSBs arise in cells under the influence of external factors, such as oxidative stress and ionizing radiation, but they are also associated with physiological processes such as DNA replication . RAG1/2 In normal conditions, DSBs are repaired either by homologous recombination (HR) which requires the presence of the sister chromatid or by non-homologous end joining (NHEJ), a direct ligation of damaged DNA ends . HR is aThe movement of DSBs in the nuclear space could be dependent on the break stability: both ends of a DSB may stay associated before the translocation. In case of the encounter of two neighboring loci with DSBs, a reciprocal translocation may occur. Alternately, a DSB may disturb the integrity of the chromosome. The ends of the DSB separate and the translocations events for each end become independent. In this case, the translocations will not be reciprocal. Repair kinetics and the time of persistence of DSBs also play a role in the mechanisms of translocation. Indeed, persistent DSBs are more likely to produce translocations .Currently it is not yet clear whether DSBs lead to an increased mobility of the damaged locus. The data both confirming this hypothesis , 17–21 aTranslocations may be initiated by induction of a limited number of DSBs by ultrasoft X-rays, laser micro-irradiation, γ-irradiation, α-particles. DSBs may also be randomly induced by chemical agents, including topoisomerase poisons used in cancer therapy. Non-random DSBs can now be induced using meganucleases and TALEN nucleases. A direct comparison between results obtained using different types of DSBs is possible only in the case when the amount of generated DSBs and local DSB densities are comparable . Even inDSBs can be easily detected in fixed cell preparations using antibodies against DSB-specific proteins, DNA repair proteins or by BrdU incorporation at the DSB site. Of course, it is currently impossible to follow the movements of a single damaged locus in a particular fixed cell. The dynamics of DSBs and/or repair loci can only be studied in this case by comparing the position of the introduced break(s) to some control points at different time points followed by statistical analysis. The mobility of the damaged loci may also be followed by induction of regularly spaced DSBs. This can be done by using an irradiation-masking microgrid that allows to obtain a regular pattern of DSBs and evaluate the mobility of DSBs vs. this regular pattern . The dynA progress in time lapse microscopy allows now to study the dynamics of DSBs in living cells in real time , 29. Sevin vivo a unique site in the genome with a fluorescent probe, to introduce a DSB and then follow the DSB movement. This can be done by integrating into the genome a construct containing the I-SceI recognition site surrounded by lacO and/or tetO sites [lacO and/or tetO sites induces formation of heterochromatin structures and affects gene expression [The most sophisticated approach allows labeling tO sites , 30. In pression , 32. The2(t), where x reflects the path of the locus after DSB induction and t is time after DSB induction. MSD also depends on the viscosity of the medium via a diffusion coefficient. MSD analysis allows to determine the volume of nucleus scanned by the locus with the DSB. If the mobility of the locus is restricted, the MSD value rapidly reaches a plateau.DSB mobility is estimated using mean square displacement (MSD); MSD = ΔxRelative mobility of two loci can be characterized by mean square change in distance (MSCD). This method implies that the mobility of both loci has a similar pattern, which is not always the case , 26.There is a significant difference between the behavior of DSBs in yeast and in higher eukaryotes: in yeast, DNA is repaired in specific nuclear compartments, repair foci. The damaged loci may be relocated into these repair foci as observed in . Higher A relative consensus exists on the mobility of damaged loci in yeast: several DSBs assemble at Rad52-containing repair foci , suggestLarge-scale movement of damaged loci is specific for persistent DSBs. These DSBs are particularly dangerous for the cells. In yeast, persistent DSBs move towards the nuclear periphery through association with Mps3, telomerase and proteins of the nuclear pore complex . DSBs inImportantly, the damaged locus moves as a whole and the damaged ends remain associated within the locus , 39, 40.In contrast to yeast, the published data on mobility of DSBs in higher eukaryotes are quite controversial. Mobility of DSBs induced by X-rays or γ-irradiation did not differ from that of intact loci –21, reviThe majority of the I-SceI-induced single DSBs also did not change their position in the nuclear space . It shouThe opposite results on relocalization and clusterization of loci with DSBs were obtained by Aten et al., . Cells wChromatin context in the damaged locus may significantly affect DSB mobility. Histone variant incorporation, histone post-translational modifications, and ATP-dependent chromatin remodelling - three major strategies for chromatin manipulation - may occur at DSBs . HiHow chromatin remodelling at DSB can affect the locus mobility? This subject has been extensively studied during the past few years (for review see –49). ChrAnother factor that can potentially affect DSB mobility is chromatin decompaction in the damaged locus Figure . ChromatPhosphorylation of the H2AX histone is one of the early responses of the cell to DNA damage. Phosphorylated H2AX (γH2AX) is spread over several megabases on both sides of a DSB, and attracts the MRN complex. This is followed by accumulation of 53BP1, RAD51, Nbs1, BRCA1 and chromatin remodeling complexes (INO80 и SWR1) at the DSB site (reviewed in ). BesideUnfolding of a damaged locus is also due to DSB-induced post-translational modifications of all core histones (reviewed in ). HistonIt is likely that a specific chromatin organization at and around the DSB site regulates repair by creating a specific compartment that coordinates DNA repair. The increase in DSB mobility may be due to gradual restructuring of chromatin in this repair-competent compartment. The probability of translocation may depend not on the collision of broken chromosome ends, but rather on a collision and fusion of repair-competent chromatin domains. The latter seems more likely. If these chromatin domains are structured, their fusion in a single compartment makes translocation a highly probable event.Multiple DSBs are particularly dangerous since they may lead to translocations, but even isolated DSBs may produce massive damage to cell if they are located in regions that ensure structural and functional integrity of the genome. When a limited number of DSBs are introduced in DNA, the mobility of the damaged locus may be limited by a chromatin domain or a subcompartment, and thus depend on chromatin organization and the genome architecture. Currently there are several models of nuclear organization, most of these models predict the existence of chromatin domains separated by specific border elements . A When multiple and clustered DSBs are introduced into DNA, DSBs in key regions responsible for chromatin folding may destabilize the nuclear organization, thus greatly increasing the DSB mobility Figure . These kDNA topoisomerase II is an essential enzyme regulating DNA and chromatin topology. DNA topoisomerase II functions by forming a covalent complex with DNA, introducing a DSB and resealing the cleaved molecule , 70. DNAbcr) in topoisomerase II poison-induced translocations [Chromosomal breakpoints are clustered in so-called breakpoints cluster regions  [AML1 and its most frequent translocation partner ETO1 demonstrated that their colocalization frequency increased from 4.8% to 9.9% after treatment with topoisomerase poison etoposide [MLL and its partners in the nuclear space was shown to be inversely proportional to the MLL/AF4 and MLL/ENL translocation frequency [MLL colocalization with its translocation partners AF9 or AF4 in one transcription factory did not exceed 2-3%, being almost a random event [MLL locus revealed several interesting observations: at least in 17% of etoposide-treated cells, topoisomerase II-induced DSB was converted into a chromosomal break with two DSB ends being distant in the nuclear space; the frequency of this situation increased when etoposide was removed and the cell could repair the DSB [Approximately 20-30% of therapy-related hematologic diseases contain balanced chromosomal translocations . Treatmegy.org/) . Currenttoposide , 88. Therequency . It has om event . 3D FISHbcr-targeted DSB in the MLL gene introduced by a Zn-finger nuclease was also converted into a chromosomal break [bcr did not induce translocations [MLL bcr may be clustered and heterogeneous, some are introduced by topoisomerase II, leaving DNA end bound via the topoisomerase II subunits; these can be converted into DSBs after ubiquitin-26S proteasome proteolysis of topoisomerase II [The bcr region is also a target of apoptotic nucleases, and apurinic and apyrimidinic sites may arise in the region from oxidative stress induced by etoposide [Interestingly, a single al break . Inhibitocations , suggesterase II , 93. Thetoposide . Such coMLL are reciprocal [Importantly, most translocations concerning ciprocal , and onlAn interesting insight on mobility of DSBs might be obtained from the analysis of primary translocations in cancer. Indeed, reciprocal translocations occur when both partner loci have DSBs that are not converted into a chromosomal break. Such translocations are characteristic for Non-Hodgkin lymphomas and leukaemias.DSBs followed by chromosomal breakage mostly produce non-reciprocal translocations. Primary non-reciprocal translocations are characteristic of epithelial tumours  and soliTherefore, a caution must be taken when applying results on DSB mobility obtained on one cell line to other cell types and tissues. Moreover, some results that we have reviewed above have been obtained on cancer cell lines that might be prone to specific types of translocation.Other factors might induce translocations, e.g. telomeric dysfunction provokes appearance of non-reciprocal translocations in epithelial cells . TelomerResults obtained with different DSB models in higher eukaryotes are often inconsistent. Indeed, single DSBs are rarely converted into a chromosomal break. In this situation, an increase in the locus mobility is mostly linked to chromatin decompaction  that incStudies of DSB mobility, its mechanisms and factors are important for better understanding of the mechanisms of translocations. A directed reduction of DSB mobility upon X-ray irradiation or DNA-damaging drug treatment may reduce a risk of secondary translocations in cancer.


The diagnostic classification of pediatric AML includes a combination of morphology, cytochemistry, immunophenotyping and molecular genetics. Outcome is mainly dependent on the initial response to treatment and molecular and cytogenetic aberrations. Treatment consists of a combination of intensive anthracycline- and cytarabine-containing chemotherapy and stem cell transplantation in selected genetic high-risk cases or slow responders. In general, ~30% of all pediatric AML patients will suffer from relapse, whereas 5%–10% of the patients will die due to disease complications or the side-effects of the treatment. Targeted therapy may enhance anti-leukemic efficacy and minimize treatment-related morbidity and mortality, but requires detailed knowledge of the genetic abnormalities and aberrant pathways involved in leukemogenesis. These efforts towards future personalized therapy in a rare disease, such as pediatric AML, require intensive international collaboration in order to enhance the survival rates of pediatric AML, while aiming to reduce long-term toxicity.Pediatric acute myeloid leukemia (AML) represents 15%–20% of all pediatric acute leukemias. Survival rates have increased over the past few decades to ~70%, due to improved supportive care, optimized risk stratification and intensified chemotherapy. In most children, AML presents as a  In adult and elderly patients, AML is often preceded by myelodysplastic syndrome (MDS), but in children, the occurrence of AML preceded by clonal evolution of preleukemic myeloproliferative diseases, such as MDS or juvenile myelomonocytic leukemia (JMML), is rare. Germline affected individuals, such as those with Fanconi anemia or Bloom syndrome, have an increased risk for developing AML as a secondary malignancy [In children, the most frequently occurring hematological malignancies include acute leukemias, of which 80% are classified as acute lymphoblastic leukemia (ALL) and 15%–20% as acute myeloid leukemia (AML). The incidence of AML in infants is 1.5 per 100,000 individuals per year, the incidence decreases to 0.9 per 100,000 individuals aged 1–4 and 0.4 per 100,000 individuals aged 5–9 years, after which it gradually increases into adulthood, up to an incidence of 16.2 per 100,000 individuals aged over 65 years [lignancy . Recentllignancy ,8.GATA1 gene. The leukemic cells of patients with Down syndrome are usually highly sensitive to chemotherapy with an exceptional high survival rate, and therefore it is possible to treat these patients with adjusted treatment protocols [Children with Down syndrome classically present with a unique megakaryoblastic subtype of AML, classically following a transient myeloproliferative disorder in the neonatal period, which is characterized by somatic mutations in the rotocols . In addirotocols ,11.HLXB9-MNX1, t/NUP98-KDM5A and t/RBM15-MKL1 [AML is a heterogeneous disease with respect to morphology, immunophenotyping, cooperating underlying germline and somatic genetic abnormalities, as well as clinical behavior. The standard diagnostic process of AML is based on a combination of morphology, cytochemistry, immunophenotyping, cytogenetic and molecular characterization of the leukemic blasts derived from the bone marrow or peripheral blood . Each AMNX1, t11;M15-MKL1 ,18,19.RUNX1/RUNX1T1 translocated cases and caused less treatment-related mortality [The clinical outcome of pediatric AML has improved significantly over the past few decades, with current long-term survival rates of ~70%  20,21,2,28,29,30ortality .The added value of hematopoietic stem cell transplantation (SCT) in newly-diagnosed pediatric AML is under discussion, as in general, the occurrence of procedure-related deaths needs to be counterbalanced by the reduction in relapse risk. The procedure-related deaths are dependent on the intensity of the prior induction chemotherapy. SCT in first CR is therefore currently only recommended for a selected subset of high risk cases in most European protocols. SCT plays a more prominent role in most North-American treatment protocols ,36. ReceDespite intensive treatment, ~30% of the pediatric patients relapse, and outcome is poor, reflected by the ~30%–40% of patients surviving in the largest and most recent series reported to date ,42. NeveFLT3, KIT, N-RAS, K-RAS and PTPN11. Type II abnormalities impair differentiation and mainly result from genetic aberrations in hematopoietic transcription factors, due to, for instance, the AML-characteristic translocations t/AML1-ETO and 11q23/MLL rearrangements or from mutations in genes, such as NPM1 and CEBPA [MLL-rearranged abnormalities  is associated with a very favorable outcome in pediatric AML. In contrast, poor survival rates were reported in pediatric AML with translocations t and t [EVI1 caused by 3q26 abnormalities predicts an adverse outcome in adult AML, but EVI1 overexpression is not an independent prognostic factor in pediatric AML [WT1 and FLT3-itd predict a poor outcome, the latter dependent on the allele ratio, and these mutations are described as events in clonal evolution towards relapse [Most important prognostic factors for the survival of pediatric AML are the initial response to treatment and the underlying genetic and molecular aberrations ,77,78. Cp12;q23) ,81. The p12;q23) ,17. Monop12;q23) ,82. Delep12;q23) ,82. The p12;q23) ,83,84,85tric AML ,87. The  relapse .NPM1 and bi-allelic CEBPA mutations, conferring a favorable prognosis, while the cryptic translocation NUP98/NSD1 confers a poor prognosis, due to a poor response to treatment and a high risk for relapse, independent of the poor prognostic Type I FLT3-itd abnormality [A special subtype of pediatric AML is the cytogenetically normal (CN) AML group, where clinical outcome is highly dependent on the presence of single-gene mutations or cryptic translocations. Of special interest are ormality ,57,89.WT1, NF1 and TET2, the latter being more common in adults than in children [In order to provide more insight into the heterogeneity and biology of AML, genome-wide approaches have been recently employed, although the success rate is variable. Array-based comparative genomic hybridization (array-CGH) and single-nucleotide polymorphism (SNP) arrays identified several regions with loss of heterozygosity and recurrent copy number variations (CNVs), albeit with low frequency in AML . These Cchildren ,65,67,91BRE and IGSF4 [Gene expression profiling could predict the cytogenetic subtypes of AML with high accuracy, although its value for diagnostic purposes remains limited, since most aberrations can be identified with conventional karyotyping ,93,94,95nd IGSF4 ,97.In addition to discovering novel gene mutations, next generation sequencing has also proven to be a powerful tool in the study of the clonal evolution of both adult and pediatric AML ,99. By cEpigenetic profiling was able to distinguish cytogenetic subtypes of adult AML . DiffereDifferences in microRNA expression levels can classify several types of cancer . ProfiliThe translation from molecular aberrations towards targeted therapy might be the solution to improve outcome in the next few decades. Since further intensification of current chemotherapy treatment seems not feasible in pediatric AML, due to high morbidity and mortality rates, new therapeutic approaches that are more tumor-specific and cause less severe side effects are urgently needed. Some new compounds directed at specific molecular targets have already been investigated in early clinical trials in pediatric AML.FLT3 gene are among the best studied drugs in this respect in pediatric AML and include trials using PKC412, CEP701, AC220 and sorafenib [FLT3 gene mutation are treated with sorafenib in addition to standard intensive chemotherapy [FLT3 mutated patients with such therapeutic regimens.Tyrosine kinase inhibitors directed at inhibiting the constitutive activation of the orafenib ,110,111.orafenib ,113. In otherapy . HoweverKIT and RAS gene mutations. Patients with KIT mutations include the imatinib-resistant patients with the D816V/Y mutation, who are sensitive to dasatinib [Other potential targets in AML include asatinib ,115. A pasatinib . There iasatinib . No triaasatinib . To inhiasatinib ,120.MLL-rearranged AML, efforts are directed at developing targeted therapy, for instance by inhibiting DOT1L, which is part of the MLL-complex, with current clinical trials ongoing [In  ongoing . Interes ongoing .Gemtuzumab ozogamicin is a conjugated monoclonal antibody against CD33 and linked to a cytostatic agent, calicheamicin, an anti-tumor antibiotic. AML cells often express CD33 and are therefore targeted by this drug. The first phase III studies did not show an improvement in disease-free and overall survival in pediatric AML patients, but it was favorable in patients with refractory or relapsed disease and was effective at reducing MRD levels before SCT ,124,125.Clofarabine is a purine nucleoside antimetabolite, registered for relapsed or refractory pediatric ALL. Early trials in pediatric AML did not show convincing efficacy, probably due to the intensive pre-treatment in these cases . HoweverIn xenograft models, treatment of AML with a combination of decitabine, a hypomethylating agent, and cytarabine was more effective at reducing tumor burden in comparison to cytarabine alone . Low-dosInternational collaboration, which has been pursued over the last few decades on the levels of the International Berlin-Frankfurt-Munster Study Group (IBFM-SG), Innovative Therapies for Children with Cancer (ITCC), European Network for Cancer Research in Children and Adolescents (ENCCA), Therapeutic Advances in Childhood Leukemia (TACL) and Childhood Oncology Group (COG), has been proven successful in clinical and biological studies and will speed up efforts to enhance therapeutic options and the availability of novel agents for individual pediatric AML patients ,80,81,82Current survival of pediatric AML is ~70%, and a therapeutic plateau has been reached with current chemotherapy. Further intensification of treatment is not feasible because of toxicity. The heterogeneity of AML is illustrated by the various prognostically relevant non-randomly associated molecular and cytogenetic aberrations that were discovered in recent years. However, many cooperating events in leukemogenesis still remain unknown. The application of new techniques, especially next generation sequencing, will contribute to our understanding of the genetic landscape of AML and enable the development of more targeted and personalized therapy in the near future. To achieve such goals for such a rare disease as pediatric AML, international collaboration is crucial.