Mitochondria from these mice are more numerous and smaller, indicative of mitochondrial dysfunction, and mitochondrial membrane potential is altered under conditions of oxidative stress. We also generated a PC12 cell line overexpressing RCAN1 (PC12RCAN1). Similar to RCAN1ox neurons, PC12RCAN1 cells have an increased susceptibility to oxidative stress and produce more mitochondrial ROS. This study demonstrates that increasing RCAN1 expression alters mitochondrial function and increases the susceptibility of neurons to oxidative stress in mammalian cells. These findings further contribute to our understanding of RCAN1 and its potential role in the pathogenesis of neurodegenerative disorders such as AD and DS.Mitochondria are the primary site of cellular energy generation and reactive oxygen species (ROS) accumulation. Elevated ROS levels are detrimental to normal cell function and have been linked to the pathogenesis of neurodegenerative disorders such as Down's syndrome (DS) and Alzheimer's disease (AD). RCAN1 is abundantly expressed in the brain and overexpressed in brain of DS and AD patients. Data from nonmammalian species indicates that increased RCAN1 expression results in altered mitochondrial function and that RCAN1 may itself regulate neuronal ROS production. In this study, we have utilized mice overexpressing RCAN1 (RCAN1 RCAN1 iecretion , and immecretion . Short- ecretion . RCAN1.4ecretion , and uprecretion . Overexp neurons , while R neurons  and prot neurons .β toxicity and tau hyperphosphorylation [β have failed to show any significant reduction in disease burden [β deposits in the brains of AD subjects without impact on either clinical disease progression or progression of tau aggregation pathology [RCAN1.1 is overexpressed in the brains of both DS and AD individuals , 4, 16. rylation . The pate burden . The facathology  indicateathology  and highathology . This meathology  and calcathology . Furtherathology .β-amyloid peptide-containing neuritic plaques, tau-containing neurofibrillary tangles, basal forebrain cholinergic neuron degeneration, and dementia [β and tau, with tau preferentially impairing complex I of the respiratory chain and amyloid-β blocking complex IV-dependent respiration [β and oxidative stress [It is worth noting that most DS individuals experience seizures and all are mentally impaired and develop Alzheimer's-like neuropathology by their mid-30's, characterized by dementia . Furtherdementia  and AD [dementia . Mitochodementia , 29 as idementia . Mitochodementia  and oxiddementia , 33. Mitpiration . A signipiration . Additioe stress . RCAN1 ae stress . Drosophila melanogaster suggests that nebula, the homolog of mammalian RCAN1, is involved in the regulation of mitochondrial function [ nebula in Drosophila neurons results in reduced mitochondrial size, increased mitochondrial number, and increased reactive oxygen species generation [Evidence fromfunction . Alteredneration . Howeverox) mice were generated using the human RCAN1 cDNA encoding the exon 1 splice variant as previously described [RCAN1 transgenic  D-glucose, 0.3% (w/v) BSA, and pH 7.4). Foetuses were rapidly killed by decapitation and the cerebral cortices were excised and pooled in 15 mL dissecting solution containing 0.25% (w/v) trypsin  at 37°C. After 20 minutes, digestion was terminated by the addition of 15 mL dissecting solution containing 0.01% DNase . The solution was mixed well and the cells pelleted and resuspended in 2 mL dissecting solution containing 0.02% (w/v) glycine max and 0.004% (w/v) DNase. The cells were dissociated by passing through a blunt-ended glass Pasteur pipette for 2 minutes. Finally, neuronal cells were collected after centrifugation at 500 g for 5 minutes and resuspended in 10 mL plating media , 10% FCS, 2% B27 supplement, 0.5 mM glutamycin , and 50 μg/mL gentamicin  before counting. Five hours after plating, the media were changed to plating media without FCS. Half the media was changed every two days during culture. All dishes and plates used for culture were treated with 0.005% poly-L-lysine for at least two hours before use . To obtain near pure neuronal cultures, 2.5 μg/mL cytosine D-arabinofuranoside  was applied to cultures on day four after plating for 24 hours. Experiments were performed on days 8-9 of cultures.For isolation of mouse cortical neurons,pregnant female mice were killed by cervical dislocation at 14 to 16 days after coitus and the foetuses were dissected from the uterine horns in dissecting solution -2,5-diphenyltetrazolium bromide) (Sigma) was added at a concentration of 500 μg/mL to each well and incubated at 37°C for 4 hours. Media were discarded and cells were resuspended in 200 μL DMSO by repeated pipetting to solubilize the formazan. Suspensions were transferred to Eppendorf tubes and centrifuged at 12,000 g in a MiniSpin bench top microcentrifuge (Eppendorf) for 4 minutes. 50 μL supernatant was transferred in triplicate to the wells of 96-well plate and absorbance was measured at 590 nm on a Fluostar Optima plate reader (BMG Lab Technologies).Primary cortical neurons were grown at a density of 8 × 10Adrenal medulla was dissected from mice and whole tissue was fixed for 1 hour in EM fixative  and postfixed in 2% osmium tetroxide in sodium cacodylate buffer. Fixed tissue was stained with 2% uranyl acetate and dehydrated through 70%, 90%, and 100% ethanol. Tissue was processed through 1,2-epoxypropane, a 1 : 1 mixture of 1,2-epoxypropane and Procure 812 resin , and two changes of 100% resin. Tissue and resin were transferred to Beem capsules and placed overnight at 90°C. Survey sections were stained with toluidine blue and 100 nm sections were cut on a Porter-Blum ultramicrotome  using a diamond knife . Thin sections were stained with Reynold's lead citrate and examined on a Hitachi H-600 transmission electron microscope . All analysis was done on chromaffin cells photographed at 4000x magnification unless otherwise mentioned. Electron micrographs were analysed by ImageJ image analysis software. Within the adrenal sections, chromaffin cells were distinguished by their characteristic granule morphology and are easily distinguished from cortical cells at the ultrastructural level . Mitocho3, 5.6 mM D-Glucose, 5.0 mM HEPES, and pH 7.4) and incubated with collagenase-A,  in Locke's buffer at a concentration of 3 mg/mL, in a shaking water bath at 37°C. The collagenase was diluted in cold Locke's buffer, and cells were pelleted and resuspended in DMEM medium supplemented with 1% penicillin/streptomycin  and 10% FCS  and filtered through nylon mesh. Cells were pelleted, resuspended in supplemented DMEM, and plated on 35 mm culture dishes and incubated at 37°C with 5% CO2. Cells were maintained in primary culture for 3-4 days prior to experiments. Chromaffin cells were washed twice and incubated in serum free culture media containing JC-1 (0.125 μM) for 30 min. The cultures were then rinsed and left in dye-free media for 20 minutes at 37°C before being subjected to real-time imaging in a Leica SP5 Spectral Confocal Microscope . High resolution digital images were analyzed using ImageJ.The ratiometric fluorescent dye JC-1 was used to provide relative measurements of mitochondrial membrane potential –41. At dgattccgagggggttaacggcgga-3′ and RCAN1-3′-FLAGR 5′-  tcacttgtcatcgtcgtccttgtagtcgctgaggtggatcggcgtgt-3′ (sequence encoding the FLAG tag is underlined whilst the stop codon is in bold). Template cDNA used in the PCR reaction was generated by RT-PCR of RNA extracted from a Ntera 2/D1 (NT2) human cell line. The resulting 901 bp PCR product had a FLAG tag sequence (DYKDDDDK) incorporated in frame directly upstream of theRCAN1 stop codon and was cloned into pGEM-T Easy (Promega). Sequence analysis carried out using T7 and SP6 primers (Promega) confirmed that the epitope-tag was in frame with the RCAN1 coding sequence. The 901 bp PCR product was excised from pGEM-T utilizing the EcoR1 restriction sites present on either side of the insert. The resulting DNA fragment was ligated into the EcoR1 restriction site of pcDNA3 (Invitrogen). As this ligation was not directional, clones were screened by PCR amplification using the T7 forward and the RCAN1-3′-FLAGR primers. Those clones with correct orientation produced a 958 bp PCR product, whilst clones with incorrect orientation produced a 57 bp product. For cell transfection experiments the pcDNA3/RCAN1-FLAG construct was prepared using an endo-free Qiagen Maxi prep. kit. The construct was linearized prior to transfection by digestion with the restriction endonuclease PvuI.Full length cDNA encoding RCAN1 fused to FLAG was generated by PCR using the following primers: RCAN1-5′-utrF 5′-μg of linearized plasmid DNA was transfected into 1.5 × 106 rat pheochromocytoma PC12 cells by electroporation. As a control, untransfected PC12 cells were grown on a separate plate. To select for transfectants, cells were treated with Geneticin (G418) containing media replaced every 48 hours for 10–15 days or until all the untransfected PC12 cells on the control plate had died. Transfectants were then grown in G418-free media and allowed to form colonies which were picked and subsequently expanded. PC12 cells were cultured in F-12 K containing 10% horse serum, 5% FCS, 1% Pen-Strep (penicillin-streptomycin), and 1% glutamine . Cells were grown at 37°C in a 5% CO2 humidified environment 95% air and were grown to 80–90% confluency before being passaged. For passaging, cells were treated with 0.125% trypsin  for 3 minutes, collected with fresh media, centrifuged at 400 g for 5 minutes, and seeded at a density of 2.5 × 105 cells/mL. Cells were plated out at a density of 2.5 × 103 cells/mL on 35 mm culture dishes and allowed at least 24 hours to adhere to plates before being used for experiments. In selected experiments, 10 mM of N-acetyl-cysteine  was added to culture media for 24 hours before experiments were performed.7 α-FLAG antibody conjugated to horseradish peroxidase (Sigma) was used to probe the membrane at a dilution of 1 : 1000.To test for expression of RCAN1-FLAG, RNA was extracted from cells grown to confluence on a 6 cm plate using TRIzol reagent (Invitrogen). cDNA was generated by RT-PCR using Superscript III enzyme (Invitrogen). PCR reactions were carried out using RCAN1-5′-utrF and RCAN1-3′-FLAGR primers. RCAN1-FLAG protein expression levels were assessed by Western blot analysis. Protein was extracted from PC12 clones and run on a 12% SDS-PAGE gel and transferred onto a PVDF membrane . An 2, 1 mM MgCl2, 5 mM D-Glucose, 10 mM HEPES, and pH 7.4). MitoSOX mitochondrial superoxide indicator  was added to cells at a final concentration of 5 μM. Cells were incubated for 10 minutes at 37°C, protected from light, and then gently washed three times with warm Krebs solution. Fluorescence was viewed using an Olympus IX71 fluorescence microscope . Images were captured at 20x magnification and at the same exposure time for each experimental group. Images were analysed using Image J analysis software . Background fluorescence of each image was measured in duplicate and removed from the mean fluorescence intensity of cells to obtain final values of mean cell fluorescence intensity.Media were removed from PC12 cells in culture dishes and replaced with warm (37°C) Krebs solution  decrease in cell viability at 50 μM H2O2 with 39.7 ± 0.4% of neurons viable versus 77.7 ± 6.1% of wild-type neurons. At 100 μM H2O2, there was a larger difference (P < 0.0001) in neuronal viability between the two populations; 31 ± 0.6% RCAN1ox neurons were viable versus the 53.9 ± 1.9% of wild-type neurons  and the average mitochondrial area reduced (P < 0.05) in RCAN1ox cells compared to WT. Analyzing the distribution of mitochondrial area in these cells  which rapidly reduced the JC-1 ratio to 0.48 ± 0.05 in WT cells and 0.37 ± 0.12 in RCAN1ox cells (2O2 (100 μM: 60 minutes) resulted in a significant difference in JC-1 fluorescence between WT and RCAN1ox cells. The change in fluorescence in JC-1 fluorescent ratio between the resting potential in these cells and that obtained after H2O2 indicated a more severe effect on mitochondrial membrane potential in RCAN1ox cells .To further ascertain whether mitochondrial function is altered by RCAN1 overexpression, we evaluated mitochondrial membrane potential under various conditions in WT and RCAN1.4 ± 0.4  in WT anox cells . Howeverox cells are not due to changes associated with developmental influences from outside the cells being studied, we developed a PC12 cell line overexpressing RCAN1 (PC12RCAN1 cells). Twenty-four independent PC12 clones stably transfected with RCAN1-FLAG . ROS production was decreased in PC12RCAN1cells treated with the antioxidant N-acetylcysteine (NAC) compared to untreated PC12RCAN1cells (P < 0.001). NAC treatment had no effect on mitochondrial ROS production in control PC12 cells.ROS production was measured in control and PC12 Figures . MitochoRCAN1 cells were subjected to the naturally occurring ROS H2O2 and viability measured similarly to our experiments in RCAN1ox neurons. PC12RCAN1 cells and control PC12 cells were treated with concentrations of H2O2 ranging from 50 to 250 μM for 18 hours. 41.5 ± 9.8% of PC12RCAN1cells were viable after treatment with 50 μM H2O2 whilst 78.1 ± 3.7% of the control cells remained viable (P < 0.01). This difference in cell viability was also observed when cells were treated with 100 μM H2O2, with 10.5 ± 2.8% of PC12RCAN1 cells remaining viable compared to 32.5 ± 9.0% of treated control cells. These results in PC12RCAN1 cells confirm that the effect of RCAN1 on cell susceptibility to oxidative stress is due to cell autonomous effects of RCAN1.PC122O2. Similarly, primary neurons overexpressing RCAN1 had a greater susceptibility to H2O2-induced oxidative stress. Altered mitochondrial morphology, characteristic of mitochondrial dysfunction, was observed via an ultrastructural analysis of RCAN1ox cells and these cells demonstrate altered mitochondrial membrane potential in response to increased ROS levels. Thus, we demonstrate that overexpressing RCAN1 in primary mammalian neuronal cells causes significant changes to mitochondrial function and oxidative stress responses.This study investigated the role of RCAN1 in regulating mitochondrial function and susceptibility to oxidative stress in a neuronal cell line and primary cells overexpressing RCAN1. Elevated RCAN1 expression in PC12 cells resulted in ROS accumulation, as well as decreased viability in response to HNebula, the Drosophila melanogaster ortholog of RCAN1, is localized in mitochondria where it interacts with the ADP/ATP translocator on the inner mitochondrial membrane. The elevated ROS accumulation we observe in PC12RCAN1 cells and which has been noted in neurons overexpressing nebula [ox chromaffin cells could be related to the regulation of calcineurin by RCAN1 and their various downstream targets. One important calcineurin target is dynamin-related protein 1 (Drp1), a protein which increases mitochondrial fission when dephosphorylated and is dephosphorylated by calcineurin [ox cells.g nebula  may be ag nebula , which ig nebula , 44. Theg nebula . The altcineurin . IncreasRCAN1 cells in response to H2O2 exposure. Calcineurin activity is significantly attenuated in response to H2O2 and the inhibition of calcineurin activity by exogenous pharmacological agents under these conditions increases ROS-induced damage [2+   overloading [2O2 induced cell death [Elevated RCAN1 expression resulted in decreased cell viability in primary neurons and PC12d damage . We postrloading , 48 whilll death . These cll death . It is tOur findings in mammalian cells that RCAN1 overexpression induces multiple facets of mitochondrial dysfunction may have relevance to both DS and AD. In these disorders, neuronal RCAN1 expression is significantly increased , 4, 16 aβ accumulation, can induce RCAN1 expression in neurons, which inhibits the activity of the protein-phosphatase calcineurin. Reduced calcineurin activity leads to the accumulation of hyperphosphorylated tau and subsequent neurofibrillary tangles, a hallmark of AD brains. Further to this, we now show that elevated RCAN1 expression leads to mitochondrial dysfunction including altered mitochondrial morphology and membrane potential and high mitochondrial ROS production. This accumulation of ROS and ensuing oxidative stress can have pathogenic consequences in mammalian neurons that are strongly implicated in AD pathogenesis and also induce a feed forward cycle by increasing RCAN1 expression further. Thus, our data provide a framework for the involvement of RCAN1, mitochondrial dysfunction, and oxidative stress that may be causative in AD progression.Based on our observations in this study and our prior knowledge of RCAN1 function and effectors of RCAN1 expression, we postulate that elevated RCAN1 expression may have a detrimental effect on multiple facets of neuronal function associated with mitochondrial function and AD pathology . Cellula


In this respect, rucaparib inhibits nine kinases with micromolar affinity, including PIM1, PIM2, PRKD2, DYRK1A, CDK1, CDK9, HIPK2, CK2, and ALK. In contrast, olaparib does not inhibit any of the sixteen kinases tested. In between, veliparib inhibits only two, namely, PIM1 and CDK9. The differential kinase pharmacology observed among PARP inhibitors provides a plausible explanation to their different cellular effects and offers unexplored opportunities for this drug class, but alerts also on the risk associated to transferring directly both preclinical and clinical outcomes from one PARP drug candidate to another.PARP inhibitors hold promise as a novel class of targeted anticancer drugs. However, their true mechanism of action is still not well understood following recent reports that show marked differences in cellular effects. Here, we demonstrate that three PARP drug candidates, namely, rucaparib, veliparib, and olaparib, have a clearly different  The evidence that a Poly(ADP-ribose)polymerase (PARP) inhibitor provided clinical benefit to patients carrying breast-cancer-associated BRCA1 or BRCA2 gene mutations was a breakthrough in cancer therapy  in spite50 = 3.7 µM) [i = 1.3 µM) from a large kinase profiling campaign [in vitro affinity for PIM1 and explore further their potential off-target kinase pharmacology as a means to better understand their mechanism of action.We previously reported that PJ34, an early chemical tool widely used to probe the biological function of PARP-1, is a micromolar inhibitor of PIM1 kinase (IC 3.7 µM) . The sig 3.7 µM) , one may 3.7 µM)  revealedcampaign . Howeverin vitro kinase profiling clearly demonstrate that PARP drug candidates have different affinity for PIM1 and related kinases, as summarized in Figure 50 values) for 9 of them, namely, PIM1 (1.2 µM), PIM2 (7.7 µM), PRKD2 (9.7 µM), DYRK1A (1.4 µM), CDK1 (1.4 µM), CDK9 (2.7 µM), HIPK2 (4.4 µM), CK2 (7.8 µM), and ALK (18 µM). In this respect, olaparib appears to be a markedly more selective PARP inhibitor than rucaparib. In between, veliparib shows low micromolar affinities for PIM1 (17 µM) and CDK9 (8.2 µM). Dose-response curves of the in vitro binding affinity of rucaparib and veliparib for PIM1 kinase are shown in Figure The results of the 50 = 0.56 µM) [Having confirmed that different PARP drug candidates are linked to essentially different kinase profiles, we wondered whether that could just be the tip of the iceberg. A recent HTS screening against S6K1 kinase surprisingly unraveled that a PARP inhibitor (Nu1085)  . The cry0.56 µM)  enabled 0.56 µM) . Moreove0.56 µM) . This waTo strengthen this hypothesis, we searched ChEMBL  for dual50 = 18 µM), which might partially explain the increased efficacy of rucaparib in cancer cells with alterations in ALK [The differential kinase polypharmacology among PARP drug candidates offers a reasonable explanation for some of the differences observed in their cellular effects -7. For eThe experimental confirmation that PARP drug candidates have a unique and differential off-target profile across multiple kinases known to be involved in cancer-relevant processes provides a completely new perspective of PARP inhibitors in clinical trials. Some of the new affinities identified may offer opportunities for expanding the current clinical scope of PARP inhibitors, just like other off-targets have led to new indications of cancer drugs . For insBut most importantly, due to known synergistic effects between PARPs and kinases , compoun50 values obtained for those kinases. For example, the highest peak concentration for a 50 mg single daily dose of veliparib is estimated to be around 1 µM [In spite of the biological relevance of the low-micromolar off-target affinities identified, one may argue that their true clinical significance is unclear due to the fact that the peak plasma concentration of PARP drugs is likely to be well below the ICund 1 µM . Under tund 1 µM . With veund 1 µM . Under tund 1 µM , an ongoWe may have just scratched the surface of the off-target pharmacology linked to PARP inhibitors but we have learned enough to realize that they have a clear and differential kinase pharmacology beyond their primary PARP targets. Recent clinical trials are resurrecting the interest on PARP inhibitors, despite ignoring the existence of a wealth of additional interactions outside the PARP target space . In the 50 > 5) known in publicly available repositories [www.cerep.fr). The selection included members of three different kinase groups distantly related by sequence , namely, CAMK , CMGC  and AGC . To this list, we added also two kinases belonging to the TK group that have been identified as biomarkers of PARP drug sensitivity [in vitro screening of PARP inhibitors.Taking PIM1 as a reference kinase based on previous findings , we usedsitories  were alssitivity  and sharsitivity ,13. In twww.cerep.fr) by measuring the phosphorylation of appropriate peptide substrates by human recombinant enzymes and using FRET as a detection method (a detailed description of each kinase assay is available in online Rucaparib, veliparib and olaparib were purchased from Selleckchem. Kinase in vitro assays were perfomed at Cerep (


Little is known about the effect of (−)-epigallocatechin-3-gallate (EGCG) on angiogenesis in adipocytes. We aimed to test the effect of EGCG on the expression of vascular endothelial growth factor (VEGF) in adipocytes. The levels of VEGF secretion, the expression of VEGF message ribonucleic acid (mRNA) and VEGF protein in 3T3-L1 cells were measured by enzyme linked immunosorbent assay (ELISA), real time polymerase chain reaction (PCR), and immunofluorescence staining, respectively. The xCELLigence real time cell analysis system was used to study the growth and differentiation of 3T3-L1 preadipocytes. A coculture system was used to test the effects of 3T3-L1 cells on proliferation of human umbilical vein endothelial cells (HUVECs). The conditioned media derived from 3T3-L1 cells treated with or without EGCG was used to culture the HUVECs for a tube formation assay. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα), two transcription factors related to both adipogenesis and angiogenesis, were examined to explore the potential mechanism. We found that all the three measurements of VEGF expression in adipocytes  were reduced after EGCG treatment. The growth of HUVECs co-cultured with 3T3-L1 cells was significantly increased and the conditioned media from EGCG treated 3T3-L1 adipocytes inhibited tube formation in HUVECs. Both PPARγ and C/EBPα expression in adipocytes were decreased with EGCG treatment. In conclusion, findings from this study suggest that EGCG may inhibit angiogenesis by regulating VEGF expression and secretion in adipocytes. Adipose tissue is characterized by its lifelong proliferation and relatively unlimited expansion. Development and maintenance of fat depots require angiogenesis, or the formation of new blood vessels, to enable delivery of oxygen and nutrients . PreviouIncreasing evidence shows that adipose tissue does not simply store energy. It is also an essential endocrine organ that secretes various angiogenic growth factors to promote angiogenesis during its expansion. Examples of the angiogenic growth factors are vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), placental growth factor (PlGF) and leptin ,5,6,7.VEGF is critical for maintaining the viability and metabolic/endocrine functions of adipocytes because of its role in regulating vascularization and blood perfusion . It has (−)-Epigallocatechin-3-gallate (EGCG) is the most abundant and active component of green tea. It has been reported to have chemopreventive and chemotherapeutic properties for obesity and obesity-related chronic diseases ,14. The 2. Cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium  that contained 10% FBS , 50 U/mL penicillin and 50 mg/mL streptomycin . Two days after confluent (day 0), the 3T3-L1 cells were incubated in a differentiation medium containing 0.5 mM 3-iso-butyl-1-methylxanthine , 1 µM dexamethasone  and 10 µg/mL insulin  in DMEM with 10% fetal bovine serum. After 48 h of stimulation, cells were cultured with DMEM containing 10 µg/mL insulin for 2 days and standard culture media for 4 days. The total duration of differentiation is 8 days. EGCG  was dissolved in phosphate buffered saline (PBS) and added to the cell culture medium at concentrations of 0–25 µg/mL on day 0 [3T3-L1 preadipocyte cells from the American Type Culture Collection  were cultured at 37 °C in a humidified atmosphere of 5% COon day 0 ,19. The on day 0 .time x) divided by the CI at the selected normalization time point (CInorm time) as follows: CI = CItime x/CInorm time  [We used the xCELLigence RTCA DP instrument  to perform real-time monitoring of cell survival according to the manufacturer’s instructions . The bac index”) . The nor4 cells/well) were cultured in 96-well plates. After confluent, 3T3-L1 cells were treated with differentiation medium and different concentrations of EGCG  for 8 days. Culture media was removed, and cells were washed twice with PBS. Cells were then fixed using 4% paraformaldehyde for 1 h at room temperature. After fixation, the cells were washed with PBS three times and stained with 0.5% Oil red O (in 60% isopropanol) for 30 min at room temperature. After washing three times with PBS, the cells were inspected using an optical microscope. In addition to this gross evaluation, the dye was eluted for 10 min with 100% isopropanol. The concentration of the eluted dye was then determined by measuring absorbance  510 nm).3T3-L1 cells (4 × 105 cells/well) were cultured in 12-well plates. After confluent, 3T3-L1 cells treated with differentiation medium and different concentrations of EGCG  for 8 days. Then, 3T3-L1 cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.2% Triton X-100 for 20 min at room temperature. Cells were washed 3× with 300 µL PBS and incubated with a primary antibody against VEGF  for 2 h in a humidified chamber. After three further washes with PBS, cells were incubated with Alexa Fluor®488 labeled anti-mouse antibody  for 1 h. Subsequently, cells were washed three times with PBS. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Then, cells were washed with PBS and viewed using a fluorescence microscope.3T3-L1 cells (5 × 105 cells/well) were cultured in 12-well plates. The VEGF concentration of the conditioned media derived from 3T3-L1 cells treated with or without EGCG was measured using a VEGF enzyme-linked immunosorbent kit . 50 µL of Standard, Control and samples were added to each well, and then incubate for 2 h at room temperature. The assay procedure followed the manufacturer’s protocols. The VEGF concentration of each well was determined by measuring absorbance (OD 450 nm).3T3-L1 cells (5 × 105 cells/well) were cultured in 12-well plates. After confluent, 3T3-L1 cells treated with differentiation medium and different concentrations of EGCG for 8 days. Then, cells were rinsed twice with ice-cold PBS, treated with Radio Immunoprecipitation Assay (RIPA) lysis buffer  and scraped. Next, cell lysates were centrifuged at 12,000× g for 10 min at 4 °C, and the supernatant was collected and stored at −80 °C. The protein concentrations of 3T3-L1 cells were measured using the bicinchoninic acid (BCA) method . An aliquot of 50 µg of supernatant protein was separated by 12% SDS-PAGE with 2× gel-loading buffer (100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.2% bromophenol blue, and 10% β-mercaptoethanol) and then blotted onto Immobilon-NC transfer membranes . Western blotting was performed using antibodies against PPARγ and C/EBPα  following the manufacturer’s protocols. The antibody for the internal control, α-tubulin, was purchased from Proteintech Group, Inc. .3T3-L1 cells (5 × 105 cells/well) were cultured in 12-well plates and were treated with or without EGCG. Total ribonucleic acid (RNA) was extracted with TRIzol reagent  according to the manufacturer’s instructions. The quality and quantity of total RNA were determined by spectrophotometry (absorbance 260/280 nm). The total RNA samples (2 µg) were converted into complementary deoxyribonucleic acid (cDNA) by reverse transcription using the GoScript™ Reverse Transcription System . Briefly, the reaction was performed in a final volume of 20 µL, which included reaction buffer, PCR Nucleotide mix, random primers, MgCl2, GoScript™ Reverse Transcriptase, RNase inhibitor and RNA. The reaction mixtures were heated at 25 °C for 5 min, 42 °C for 60 min and 70 °C for 15 min. Real-time PCR was performed using the 7000 Real-Time PCR System . Each well was brought to a final volume of 20 µL, which included GoTaq® qPCR Master Mix , an optimized concentration of each primer and 2 µL of cDNA. The reaction mixtures were heated at 95 °C for 15 min to activate the enzyme and then subjected to 40 cycles of melting at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. The mRNA levels of all genes were normalized using β-actin as an internal control. The following primers were used in the PCR reactions: VEGF-A forward, 5′-GAAAGGCTTCAGTGTGG-3′ and reverse, 5′-CAGGAATGGGTTTGTCG-3′; PPARγ forward, 5′-TCACAATGCCATCAGGT-3′ and reverse, 5′-GCGGGAAGGACTTTATGTA-3′; C/EBPα forward, 5′-GCCCCTCAGTCCCTGTCTTTA-3′ and reverse, 5′-AGCCCTCCACCTCCCTGTAG-3′; β-actin forward, 5′-CCTCTATGCCAACACAGT-3′ and reverse, 5′-AGCCACCAATCCACACAG 3′.3T3-L1 cells  were seeded (6000 cells/well) into E-plates. The cell growth curves were recorded at 15 min intervals on the xCELLigence System in real time. 3T3-L1 preadipocytes were adjusted to 1.25 × 103 cells/well) were incubated for 24 h at 37 °C with 50 µL of MCDB131 and 150 µL of the conditioned media derived from post-differentiated 3T3-L1 cells in the presence or absence of anti-mouse VEGF-neutralizing antibody. Three different phase-contrast microscopic low-power fields (×40) per well were photographed. The total length of capillary tubes in each photograph was measured using a scale ruler.For the tube formation assay, HUVECs were incubated with VEGF-free MCDB131 medium  for 24 h. Matrigel was thawed at 4 °C in an ice-water bath. Matrigel was then carefully added to a pre-chilled 96-well plate (50 µL per well) using a cold pipette and allowed to polymerize for 1 h at 37 °C. After polymerization, HUVECs  followed by Dunnett’s test  or Dunnett’s T3 test . All analyses were performed by using SPSS for windows (release 19.0). A value of To explore the effects of EGCG on preadipocyte growth, 3T3-L1 preadipocytes were treated with physiologically attainable concentrations  and higher concentrations of EGCG  for 2 days after seeding. The media with or without EGCG were not replaced during the incubation time. Compared with that in the control group, the normalized cell index in the groups using 0.5 µg/mL and 1 µg/mL EGCG, which reflects cell density, were significantly decreased A. As shoFollowing incubation of 3T3-L1 preadipocytes with differentiation medium (DMI) containing different concentrations of EGCG  , we examSince we found that the lipid accumulation was significantly decreased when 3T3-L1 cells were incubated with 25 μg/mL of EGCG compared to the control group, 10–25 μg/mL of EGCG were used to examine the effect of EGCG on regulating the relevant transcript factors. We measured the expression of PPARγ and C/EBPα to determine whether EGCG treatment altered adipocyte marker gene expression in 3T3-L1 adipocytes. As shown in According to its effect on the proliferation and differentiation of 3T3-L1 cells, we used 5–25 μg/mL of EGCG to explore the effect of EGCG on angiogenesis. We tested the effects of EGCG on VEGF expression in adipocytes with four sets of experiments. First, real time PCR results showed that EGCG decreased VEGF gene expression in both 3T3-L1 preadipocytes and adipocytes A,B. SecoIn order to test the role of adipocytes in angiogenesis, coculture of 3T3-L1 cells and HUVECs was performed. The results showed that cell density of HUVECs rose with the increase of 3T3-L1 preadipocytes A. Comparin vitro angiogenesis. We observed that the conditioned media, which contained the secretions from 3T3-L1 adipocytes , enhanced tube formation in HUVECs (Since 25μg/mL EGCG significantly decreased VEGF concentrations and suppressed VEGF mRNA expression in the culture of preadipocytes and adipocytes, the conditioned media from cultured 3T3-L1 adipocytes which was treated with 25 μg/mL EGCG was then used to perform the tube formation assay for n HUVECs . The conPrevious studies have shown that the growth and expansion of adipose tissue is accompanied by vascularization via angiogenesis . VEGF, oIn the HUVEC experiment, we did not measure the adipokine composition of the adipocyte conditioned media before addition to the HUVECs. However, there should be angiogenic factors, various pro-inflammatory cytokines and growth factors in the media as previously reported ,27. BecaOf note, EGCG is not stable in solution and can be autooxidized at neural pH and produces some metabolites, e.g., dimers . Indeed,There are few published data reported the concentration of EGCG in adipose tissue after its intake, which should be further studied. Previous studies have showed that the concentration of EGCG in human plasmais < 1 μM after consuming one cup of green tea ,31. Howein vivo [The mechanism through which EGCG inhibits adipocyte to produce VEGF has been unclear thus far. One possible mechanism is that, as mentioned above, EGCG can inhibit angiogenesis by ameliorating hypoxia that is induced by accumulation and expansion of the adipose tissue. The other possible mechanism is that EGCG has an inhibitory effect on PPARγ. PPARγ and C/EBPα are the main transcription factors in adipogenesis and lipogenesis. Down-regulation of PPARγ and C/EBPα expression by EGCG would be expected to be inhibitory to lipid deposition and adipocyte differentiation. In particular, PPARγ has a net pro-angiogenic role in the context of adipose tissue . Howeverin vivo . This isin vivo . In thisin vitro. However, the effect of EGCG on adipose tissue’s angiogenesis should be further investigated in vivo.The findings from this study suggested that EGCG could inhibit the growth and expansion of adipocytes via down regulation of VEGF expression and secretion


Mutations in genes that are part of the splicing machinery for myelodysplastic syndromes (MDS), including MDS without ring sideroblasts (RS), have been widely investigated. The effects of these mutations on clinical outcomes have been diverse and contrasting.de novo MDS patients, who did not harbor RS, for mutations affecting three spliceosomal genes .We examined a cohort of 129 SF3B1, U2AF1, and SRSF2 were 7.0 %, 7.8 %, and 10.1 %, respectively. Compared with previously reported results, these rates were relatively infrequent. The SRSF2 mutation strongly correlated with old age (P < 0.001), while the mutation status of SF3B1 did not affect overall survival (OS), progression-free survival (PFS), or acute myeloid leukemia (AML) transformation. In contrast, MDS patients with mutations in U2AF1 or SRSF2 exhibited inferior PFS. The U2AF1 mutation was associated with inferior OS in low-risk MDS patients (P = 0.035). The SRSF2 mutation was somewhat associated with AML transformation (P = 0.083).The mutation rates of SF3B1, U2AF1, and SRSF2 splicing gene mutations in MDS without RS were relatively low. We also demonstrated that the U2AF1 and SRSF2 mutations were associated with an unfavorable prognostic impact in MDS patients without RS.Our findings suggest that the frequencies of the The online version of this article (doi:10.1186/s12885-015-1493-5) contains supplementary material, which is available to authorized users. The myelodysplastic syndromes (MDS) represent myeloid clonal hemopathies, with a relatively heterogeneous spectrum of presentation. The major clinical problems of these disorders are morbidities caused by cytopenias and the potential for MDS to evolve into acute myeloid leukemia (AML) . AlthougRAS, TP53, and RUNX1. However, mutations in these genes do not fully explain the pathogenesis of MDS as these mutations are also commonly found in other myeloid malignancies. In addition, approximately 20 % of MDS cases are not associated with any genetic changes. The genetic alterations responsible for dysplastic phenotypes and ineffective hematopoiesis of myelodysplasia are poorly understood [The broad range of individual genes affected by mutations indicates that a variety of molecular mechanisms are involved in the pathogenesis of MDS . A numbeet al. [et al. [et al. [A previous report by Murati et al.  describe [et al.  and Maki [et al. , mutatioet al. [A recent study by Damm et al.  revealedSF3B1), U2 small nuclear RNA auxiliary factor 1 (U2AF1), and serine arginine-rich splicing factor 2 (SRSF2) among a cohort of MDS patients without RS.We investigated the prevalence and clinical impact of mutations in splicing factor 3 subunit b1  2008 criteria, at Chonnam National University Hwasun Hospital  were enrolled into this study. The patient cohort comprised 129 MDS patients without RS. A detailed summary of the enrolled patients is shown in Table 3/μl or platelets < 100 × 103/μl); and (3) intermediate-2 or high risk. Azacitidine was administered subcutaneously at a dose of 75 mg/m2 per day for seven consecutive days, every 28 days. Decitabine was administered intravenously at a dose of 20 mg/m2 per day for five consecutive days, every 28 days. When we retrospectively applied the IPSS-R for treated patients (n = 58), 3.5, 24.1, 29.3, 29.3, and 13.8 % of patients were considered to be at very low, low, intermediate, high, and very high risk, respectively. Clinical and laboratory data for MDS patients were analyzed and reviewed, based on their electronic medical records. All enrolled MDS patients gave their written, informed consent in accordance with the Declaration of Helsinki. This study was approved by the institutional review board of Chonnam National University Hwasun Hospital.From 2003–2011, 129 adult patients with SF3B1, U2AF1, and SRSF2 was conducted using polymerase chain reaction (PCR) followed by direct sequencing. For direct sequencing of the spliceosomal genes, six primer pairs were used  to obtain preliminary evidence regarding polymorphisms, mutations, and for translation of amino acids. Results obtained from MDS patients were confirmed on an online database  according to the manufacturer’s instructions. The detection of mutations in Chromosomal analysis (G-banding) was performed on preparations from 48-h bone marrow cell cultures where mitogens were not added, according to a protocol from the American Type Culture Collection. Aberrations in chromosomes were described according to the international system for cytogenetic nomenclature 2005 and 2009.χ2 test or Fisher’s exact test was performed to determine the significance of associations between SF3B1, U2AF1, and SRSF2 mutations and other parameters, including sex, WHO classification, karyotypes, and IPSS-R risk classification. Student’s t-test was used to compare continuous variables such as age and hemograms. Kaplan-Meier estimation was used to plot survival curves, and log-rank tests were used to calculate the difference between survival curves. Cox proportional hazard regression analysis was used to dissect the individual impact of prognostic factors for overall survival (OS), progression-free survival (PFS), and acute myeloid leukemia (AML) transformation. All tests were two-tailed, and a P-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using PASW version 18.0 .The SF3B1, U2AF1, and SRSF2) were observed in 24.8 % (32/129) of MDS patients . Among the 129 MDS patients, nine were identified as harboring a mutation in SF3B1 (7.0 %), 10 patients had mutations in U2AF1 (7.8 %), and 13 patients exhibited a mutation in SRSF2 (10.1 %). All 129 MDS patients in this study were without RS. The SF3B1, U2AF1, and SRSF2 mutations were mutually exclusive, with none of the patients having more than one of these genes affected . Positive cytogenetic findings, such as normal karyotype, −Y only, del(5q) alone, and del(20q) alone were more frequent in SF3B1mut patients than in SF3B1wt patients (88.9 vs. 75.8 %). Poor cytogenetic findings, such as complex karyotype, and abnormalities in chromosome 7 were more apparent in SF3B1wt patients than in SF3B1mut patients (10.0 vs. 0 %). There were no significant differences in age, sex, blood counts, bone marrow blasts, WHO subtype, and IPSS-R risk classification between SF3B1mut and SF3B1wt patients. Nevertheless, lower risk MDS patients, such as those with refractory cytopenia with unilineage dysplasia (RCUD) or refractory cytopenia with multilineage dysplasia (RCMD), were represented in higher proportions among SF3B1mut patients than SF3B1wt patients (66.7 vs. 57.5 %). For higher risk MDS patients, such as those with refractory anemia with excess blasts-1 (RAEB-1) or RAEB-2, there was a lower proportion of SF3B1mut patients than SF3B1wt patients (22.2 vs. 36.7 %).The clinical and hematological characteristics of patients with mutated (mut) versus wild-type (wt) U2AF1 were mainly male (8/10) and exhibited lower hemoglobin levels . Our cytogenetic results revealed meaningful differences between U2AF1mut and U2AF1wt patients (P = 0.022). Positive cytogenetic findings were more frequently observed for U2AF1wt than U2AF1mut patients (78.3 vs. 60.0 %), while poor cytogenetic findings were more common in U2AF1mut patients (10.0 vs. 9.2 %). In contrast, no significant differences were identified between U2AF1mut and U2AF1wt patients for age, sex, blood counts, bone marrow blasts, WHO subtype, and IPSS-R risk classification. The higher risk MDS patients (RAEB-1 or RAEB-2) were more likely to be U2AF1mut patients (60.0 vs. 33.6 %), while lower risk MDS patients (RCUD or RCMD) were less likely to be U2AF1mut individuals (40.0 vs. 59.7 %) (P = 0.629).Patients harboring mutations in SRSF2mut patients were older than SRSF2wt patients  and mostly male (9/13). Similar to the U2AF1mut patients, those with SRSF2 mutations displayed a significant difference in cytogenetic results (P = 0.048). Good cytogenetic findings were more frequently seen for SRSF2wt patients (79.4 vs. 53.8 % in SRSF2mut patients), while poor cytogenetic findings were more common for SRSF2mut patients (23.1 vs. 7.8 % in SRSF2wt patients). We observed no significant differences in sex, blood counts, bone marrow blasts, WHO subtype, and IPSS-R risk classification between SRSF2mut and SRSF2wt patients. The higher risk MDS patients (RAEB-1 or RAEB-2) were more likely to be SRSF2mut patients (46.2 vs. 34.5 %), while lower risk MDS patients (RCUD or RCMD) were less likely to be SRSF2mut patients (53.9 vs. 58.6 %) (P = 0.094).The U2AF1mut patients  and SRSF2mut patients .We investigated the effects of each spliceosomal mutation on clinical outcomes. Using univariate analyses, OS and AML transformation rates according to the mutation status of the three genes were not significant Table . An infeSF3B1/U2AF1/SRSF2 mutation status  and SRSF2  remained an independent predictor for PFS. AML transformation was not associated with the mutation status of SF3B1.The IPSS-R was used to derive clinical prognosis for MDS. To establish whether the mutation status of spliceosomal genes can add to the predictive power of IPSS-R, we performed multivariable Cox regression analyses, examining age, sex, IPSS-R total score, and us Table . The IPSSF3B1  defined by IPSS-R  defined by WHO 2008 criteria  defined by WHO 2008 criteria  MDS patients .MDS subgroup analysis revealed that the poor impact of a et al. [et al. [SF3B1, U2AF1, and SRSF2 mutations in MDS patients without RS from Korea.Recent reports regarding whole exome sequencing in MDS patients by Yoshida et al.  and Papa [et al.  suggest  [et al. . The exa [et al. . The mol [et al. . To dateSF3B1, U2AF1, and SRSF2 mutations were relatively infrequent in MDS patients without RS, contradicting the results from a previous study. In addition, our results demonstrate that the U2AF1 and SRSF2 mutations, unlike SF3B1, were associated with a negative prognostic impact for MDS patients without RS.Our findings indicate that the et al., mutations affecting spliceosomal genes that result in defective splicing belong to a new leukemogenic pathway, with these mutations possibly constituting diagnostic biomarkers that could be therapeutic targets [Spliceosomes in the nucleus are complexes composed of small nuclear RNAs (snRNA) and numerous protein subunits. These spliceosomes serve to remove introns from genes that encode proteins . Identif targets .SF3B1 gene is located on chromosome 2q33.1 and codes for the SF3B1 protein complex, which is involved in the early stages of spliceosome assembly. U2AF1 gene is located on chromosome 21q22, and encodes proteins that play a role in the early steps of 3′ splice site recognition. The SRSF2 gene is located on chromosome 17q25.2, with the coding protein known to play a role in preventing exon skipping and ensuring the accuracy of splicing [These spliceosomal gene mutations occur at varying frequencies for different disease subtypes, and contribute to differences in survival outcomes . The SF3splicing .SF3B1, U2AF1, and SRSF2 were 7.0, 7.8, and 10.1 %, respectively  of patients with AML evolving from MDS carried a somatic mutation in SF3B1 [SF3B1mut MDS patients progressed into AML, and these patients were more likely to present with advantageous cytogenetic findings. However, U2AF1mut and SRSF2mut patients were considered to belong to higher risk MDS groups or to have a poor cytogenetic findings , similar to the findings of Wu et al. [We also found that the in vitro , possiblu et al.   and that there was somewhat of an association between AML transformation and mutations in SRSF2.In summary, we observed that mutations in


Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the presence of a derivative chromosome 22 [der(22)] commonly called Philadelphia chromosome (Ph). The Ph chromosome is a product of the reciprocal translocation t. Additional genetic changes occur in less than 10 % of CML cases at the time of diagnosis and other genetic changes are seen in 60–80 % of the cases in advanced disease. Even though deletions in chromosome 9 are not rare findings in advanced phase-CML, del(9)(p23p11.1) as sole additional abnormality detected by fluorescence in situ hybridization (FISH) technique, to our knowledge has not been described in the literature.CDKN2A (cyclin-dependent kinase inhibitor 2A) before Imatinib mesylate (IM) treatment.A complete cytogenetic and molecular cytogenetic analysis, molecular biology method (reverse transcription polymerase chain reaction (RT-PCR)), and immunophenotype confirmed to be a CML case in blast crisis (BC). It revealed del(9)(p23p11.1) as sole abnormality detected by FISH technique besides Ph chromosome, which leads to monoallely of tumor suppressor gene The patient did not demonstrate a good response to IM treatment. The underlying mechanisms and prognostic implications of these cytogenetic abnormalities are discussed. BCR) gene 22q11.2 and the c-Abelson (ABL1) gene 9q34, resulting in the BCR-ABL1 fusion gene, encoding a constitutively active tyrosine kinase protein [Chronic myeloid leukemia (CML) is a myeloproliferative disorder. It is mainly characterized by the presence of a derivative chromosome 22 [der(22)], the so-called Philadelphia chromosome (Ph), which is due to a reciprocal translocation tq34.1;q11.2). This rearrangement involves the breakpoint cluster region  of CML (in more than 90 % of the CML cases), additional genetic changes occur in less than 10 % of cases at the time of diagnosis and other genetic changes are seen in 60–80 % of the cases in advanced disease  and for CDKN2A (cyclin-dependent kinase inhibitor 2A). This patient did not demonstrate a good response to IM treatment.Here we presented a de novo untreated Ph-positive CML case in BC phase with del(9)(p23p11.1) as sole additional abnormality detected by FISH technique, which leads to monoallely of tumor suppressor gene 9/l (41 % of cells were blasts), red blood cell (RBC) count was 3.62 × 106/mm3, hemoglobin level was 6.8 g/dl and the platelet count 26 × 109/l. Serum lactate dehydrogenase value (LDH) was 1,543 U/l . The patient was diagnosed as CML-BC according to WHO recommendations, in a high Sokal risk of 29.7 (0.8–1.2), high Hasford (Euro) risk of 2,856 , low Eutos risk of 40 (<87) and Etous probability of no complete cytogenetic response (CCgR) at 18 months was 15 %. In fact, the patient started on IM treatment (400 mg/day) for overall 1 month; then he increased IM daily dose to 800 mg for 1 month but the treatment was not successful and the patient had a toothache, anemia, and thrombocytopenia. Later he was given IM 400 mg daily dose for 2 months. Under the latter therapy the previous reported relevant symptoms disappeared. Unfortunately the patient died 7 months after diagnosis from the disease under the treatment for unknown reason.In October 2013 a 40-year-old male was diagnosed as suffering from CML. Physical examination revealed the indicative symptoms as hepatosplenomegaly, loss of weight, fever, anemia, and thrombocytopenia. Routine peripheral blood test showed elevated white blood cells (WBC) of 243.1 × 10CDKN2A confirmed that the deletion visible in the short arm encompassed subband 9p21 11]/ 46,XY,t,del(9)(p?)/ 1/ 46,XY,p21 Fig. . Multicop21 Fig. . RT-PCR 46,XY,t9;22)[11]/46,XY,der(9)(9pter- > 9p23::9p11.1- > 9q34::22q11.2- > 22qter),der(22)t[;22 showed the following immunophenotype, which was consistent with CML-BC (WHO recommendations): CD45CDKN2A as located at 9p21. Up to date, there are 7 CML cases listed in Mitelman Database showed the involvement of the short arm of chromosome 9, two of them revealed involvement of 9p21 [We described a de novo untreated CML case in BC with del(9)(p23p11.1) as sole additional acquired abnormality, which leads to monoallely of tumor suppressor gene  of 9p21 . Also, l of 9p21 , 6. To tAccording to the literature, approximately 10 % of CML patients present in advanced phases called accelerated phase (CML-AP) or CML-BC, without a clinically evident CP . Even wiThe progression to advanced phases of disease is caused by the development of genetic lesions in addition to BCR-ABL1 translocation; secondary cytogenetic changes are identified in more than 80 % of patients by the time of progression to CML-BC . Even inThe t or a variant Ph are frequently the sole chromosomal anomaly during the CP . The mosThe median time to progression from CML-CP to CML-BC is 3–5 years in untreated patients or in patients treated with hydroxyurea . HoweverThe molecular events associated with CML progression are complex and are likely related to genetic instability and impaired DNA repair mechanisms that are consequences of BCR-ABL1 activity –19. In aCDKN2A and CDKN2B are tumor suppressor genes located in 9p21. They belong to the family of inhibitors of cyclin-dependent kinases. The CDKN2A gene encodes two proteins, p14ARF and p16INK4a, and the CDKN2B gene p15 INK4b protein, which as key regulators of G1 phase cell-cycle arrest and senescence [nescence . These gnescence , 23,CDKN2A deletions in pediatric ALL appears controversial [Deletion at 9p21 is especially frequent in acute lymphocytic leukemia (ALL), occurring at more than of 20 % in B-cell precursor ALL and approximately 50 % T-ALL patients . The 9p2oversial –27.CDKN2A expression [Recently, FOXA1 is a key transcription (TF) factor for pression , 28. FOXpression , 30.CDKN2A as located at 9p21. The patient did not demonstrate a good response to Imatinib treatment.In conclusion, we described de novo untreated CML case in BC with del(9)(p23p11.1) as sole additional acquired abnormality detected by FISH technique, which leads to monoallely of tumor suppressor gene Chromosome analysis applying GTG-banding according to standard procedures  was perfFluorescence in situ hybridization (FISH) using the LSI BCR/ABL dual color dual fusion translocation probe , a locus specific probe for CDKN2A gene (LSI p16 in 9p21) with a probe for centromere nine, and a subtelomeric probe for 9p and 9q  were applied together with whole chromosome painting (WCP) probe for chromosome 9  according to manufacturer’s instructions . Also a Total RNA extracted from peripheral blood sample using the InviTrap RNA kit  according to the manufacturer’s recommendations. cDNA was prepared from 5 μg of total RNA with the Genequality BCR-ABL1 kit  and BCR-ABL1 fusion transcript was performed according to the manufacturer’s instructions .Flow cytometry of leukemic blasts was performed using a general panel of fluorescent antibodies against the following antigens typical for different cell lineages and cell types: CD1a, CD2, CD3, CD4, CD5, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD22, CD23, CD32, CD33, CD34, CD38, CD41a, CD45, CD56, CD57, CD64, CD103, CD117, CD123, CD209, CD235a and CD243; in addition antibodies against Kappa and Lambda light Chains, sIgD, sIgM, and HLADr were applied (BD Biosciences). Four-color immunophenotyping on peripheral blood specimen was performed. Samples were stained and analyzed on a BD FACSCalibur™ flow cytometer according to BD Biosciences manuals and products insert sheets. Autofluorescence, viability, and isotype controls were included. Flow cytometric data acquisition and analysis were conducted by BD Cellquest™ Pro software.Written informed consent was obtained from the patient for publication of this Case Report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Background: The sarcoplasmic reticulum Ca2+-ATPase (SERCA2) pump is an important component of the Ca2+-clock pacemaker mechanism that provides robustness and flexibility to sinus node pacemaking. We have developed transgenic mice with reduced cardiac SERCA2 abundance (Serca2 KO) as a model for investigating SERCA2's role in sinus node pacemaking.Methods and Results: In Serca2 KO mice, ventricular SERCA2a protein content measured by Western blotting was 75% (P < 0.05) lower than that in control mice (Serca2 FF) tissue. Immunofluorescent labeling of SERCA2a in ventricular, atrial, sinus node periphery and center tissue sections revealed 46, 45, 55, and 34%  lower labeling, respectively and a mosaic pattern of expression. With telemetric ECG surveillance, we observed no difference in basal heart rate, but the PR-interval was prolonged in Serca2 KO mice: 49 ± 1 vs. 40 ± 1 ms (P < 0.001) in Serca2 FF. During exercise, heart rate in Serca2 KO mice was elevated to 667 ± 22 bpm, considerably less than 780 ± 17 bpm (P < 0.01) in Serca2 FF. In isolated sinus node preparations, 2 mM Cs+ caused bradycardia that was equally pronounced in Serca2 KO and Serca2 FF (32 ± 4% vs. 29 ± 5%), indicating no change in the pacemaker current, If. Disabling the Ca2+-clock with 2 μM ryanodine induced bradycardia that was less pronounced in Serca2 KO preparations , suggesting a disrupted Ca2+-clock. Mathematical modeling was used to dissect the effects of membrane- and Ca2+-clock components on Serca2 KO mouse heart rate and sinus node action potential. Computer modeling predicted a slowing of heart rate with SERCA2 downregulation and the heart rate slowing was pronounced at >70% reduction in SERCA2 activity.Conclusions:Serca2 KO mice show a disrupted Ca2+-clock-dependent pacemaker mechanism contributing to impaired sinus node and atrioventricular node function. Together, they regulate the rate and rhythm of the spontaneous action potentials in the sinus node  is the main inward current, and thus a vital component of the voltage-clock and essential to pacemaking in the sinus node. Mice with a cardiac specific conditional knockout (KO) of the HCN4 channel have reduced If in sinus node cardiomyocytes and exhibit severe bradycardia  by exchanging one Ca2+ for three Na+  pump  mice were studied. Mice with Serca2 downregulation in the heart were generated using the Cre-lox system conditional gene KO strategy as described previously by Andersson et al. (Serca2 KO (Serca2flox∕flox Tg(αMHC-MerCreMer); n = 22) and control  mice were injected with 1 mg of tamoxifen intraperitoneally to induce Serca2 excision in cardiomyocytes. All experiments were carried out 7 weeks after tamoxifen administration in accordance with the Norwegian National Committee for Animal Welfare Act, consistent with the NIH guidelines . SERCA2a abundance and expression pattern was investigated by Western blotting and immunohistochemistry (n = 10). Telemetry transmitters were implanted in one cohort of mice (n = 16) as previously described . Tissue preparations of Wistar rat hearts (n = 13), with intact sinus and AV nodes were used in some investigations.Homozygous, conditional cardiac specific n et al. . At 12 w2+ dynamics (Ca2+-clock). The model code was implemented in C programming language, and was integrated using an implicit backward difference formula that provided a high accuracy of O(dt5), computational efficiency and unconditional numerical stability. The model code, including dependencies, is available from the authors on request. Standard library based code is available from the model repository, ModelsDB . All data are expressed as mean ± s.e.m. and n refers to the number of hearts. Either an unpaired t-test or one-way ANOVA followed by Tukey's multiple comparisons post-test was conducted to determine significant differences. The difference was considered to be significant when P < 0.05. Detailed methodology is available in the Supplementary Material.Mathematical modeling of the mouse heart rate was performed using our biophysically detailed single pacemaker cell model of the mouse sinus node  and 32 ± 3 g (n = 14), respectively at 7 weeks after tamoxifen administration  in Serca2 FF to 1.0 ± 0.3 a.u.  in Serca2 KO ventricular myocardium  in Serca2 FF to 62 ± 6 a.u.  in Serca2 KO sections  in Serca2 FF and 54 ± 2 a.u. (n = 6) in Serca2 KO sections. The Cx43 protein expression remained unchanged , a major gap junction connexin in the heart, was labeled in adjacent ventricular tissue sections and used as a marker of ventricular cardiomyocytes Figures . The quad Figure .Serca2 FF and Serca2 KO animals was 536 ± 27 and 536 ± 31 beats per minute (bpm), respectively . The maximum running speed was significantly higher in Serca2 FF . Subsequently, animals were injected intraperitoneally (i.p.) with adrenaline (0.5 mg/kg bw) and at 10 min after adrenaline administration, the heart rates in Serca2 FF and Serca2 KO animals was 681 ± 9 and 581 ± 23 bpm, respectively , 37 ± 2 (P < 0.05 vs. Serca2 FF) and 51 ± 4 ms (P < 0.05 vs. Serca2 FF), respectively  node function was assessed in vitro. Ryanodine (2 μM) prolonged the sinus node intrinsic cycle length from 241.1 ± 9.63 ms (n = 13) to 369.8 ± 25.4 ms . The tissue was overdrive paced and the atrio-His (AH) interval (a measure of conduction through the AV node) and the AV node Wenckebach cycle length were measured. Ryanodine (2 μM) prolonged the AH-interval from 47 ± 6.1 ms to 59 ± 7.7 ms (P ≤ 0.01) and the AV node Wenckebach cycle length from 161.5 ± 11.7 ms to 205.1 ± 22.7 ms (P ≤ 0.05).In rat right atrial preparations, the role of sarcoplasmic reticulum Can = 8) in Serca2 FF and 431 ± 30 bpm (n = 8) in Serca2 KO  and 2 μM ryanodine (for disabling Ca2+ release via the ryanodine receptor). In the presence of Cs+, the sinus rate reduced to 294 ± 59 bpm (n = 8) in Serca2 FF and 291 ± 33 bpm (n = 8) in Serca2 KO  in Serca2 FF mice whereas in Serca2 KO mice, the drop in sinus rate was to 377 ± 26 bpm . The histology images were used to locate the sinus node and identify suitable sections for labeling with immunofluorescent markers. HCN4 was used as the positive marker to delineate sinus node myocytes and Cx43 was used to label atrial muscle  in the sinus node center, sinus node periphery and atrial muscle, respectively . HCN4 and Cx43 expression patterns were similar in Serca2 FF and Serca2 KO sections  caused an overall increase of heart rate at all levels of SERCA2  resulted in lowering of heart rate: 272 bpm at 0% SERCA2 downregulation  and SERCA2 KO remodeled (black) at 0% SERCA2 downregulation is shown in Figure Serca2 downregulation on the sinus node of the adult mouse heart. Cardiomyocyte-specific excision of Serca2 in adult mice resulted in reduced SERCA2a protein abundance in the sinus node, atrial muscle and ventricular muscle. In vivo, baseline heart rate was not affected; however, during exercise and in the presence of adrenaline, heart rate was lower than Serca2 FF. In vitro, the intrinsic sinus node beating rate remained unaltered in Serca2 KO. Separate assessment of the voltage- and Ca2+-clock components of sinus node pacemaking revealed an unaltered voltage-clock and a diminished Ca2+-clock. Our Serca2 KO mouse model provides novel information on the importance of SERCA2a, and thus the sarcoplasmic reticulum Ca2+ clock, in the sinus node pacemaking mechanism.We have investigated the consequence of substantial Serca2 deletion. The reduction in SERCA2a protein content occurs in the sinus node, as well as atrial and ventricular tissues, suggesting Cre expression in all of these tissues. α-MHC is a contractile protein and mRNA corresponding to α-MHC has been reported in the ventricular myocardium, as well as in the atrium and sinus node  and sinus node beating rates measured in vitro (431 ± 30 vs. 415 ± 16 bpm in Serca2 FF tissues) were not different from those in Serca2 FF? In the present study, although SERCA2a is significantly downregulated (by 70–75%) in Serca2 KO hearts, some degree of residual SERCA2a activity will exist and is likely to contribute to the maintenance of baseline heart/sinus rates. Previously, we have observed baseline bradycardia in Serca2 KO with >95% SERCA2a downregulation and have demonstrated that very low SERCA levels are capable of partially refilling the sarcoplasmic reticulum  and activation of inward currents such as the L- and T-type Ca2+ currents , INaCa, tetrodotoxin-sensitive Na+ current (INa) and sustained inward current (Ist), amongst others  does not result in a straightforward overlay of the outcomes  because the cell model is complex and non-linear .Studies on isolated ventricular myocytes of In vivo, with the sympathetic innervation intact, the resting heart rates of both Serca2 KO and Serca2 FF mice are higher than that observed in vitro in isolated right atrial preparations. In our study, Serca2 KO mice with 75% reduction in left ventricular SERCA2a protein content show a smaller increase in heart rate vs. Serca2 FF in response to exercise and adrenaline injection. This is consistent with the smaller increase in heart rate in response to isoproterenol observed in Serca2 KO mice with >95% SERCA2a downregulation .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.