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Sample records for bcr-abl positive cells

  1. Imatinib-dependent tyrosine phosphorylation profiling of Bcr-Abl-positive chronic myeloid leukemia cells

    OpenAIRE

    Preisinger, C.; Schwarz, J. P.; Bleijerveld, O.B.; et al

    2012-01-01

    Bcr-Abl is the major cause and pathogenetic principle of chronic myeloid leukemia (CML). Bcr-Abl results from a chromosomal translocation that fuses the bcr and abl genes, thereby generating a constitutively active tyrosine kinase, which stimulates several signaling networks required for proliferation and survival. peer-reviewed

  2. Unsupervised explorative data analysis of normal human leukocytes and BCR/ABL positive leukemic cells mid-infrared spectra

    NARCIS (Netherlands)

    Bellisola, G.; Bolomini-Vittori, M.; Cinque, G.; Dumas, P.; Fiorini, Z.; Laudanna, C.; Mirenda, M.; Sandt, C.; Silvestri, G.; Tomasello, L.; Vezzalini, M.; Wehbe, K.; Sorio, C.

    2015-01-01

    We proved the ability of Fourier Transform Infrared microspectroscopy (microFTIR) complemented by Principal Component Analysis (PCA) to detect protein phosphorylation/de-phosphorylation in mammalian cells. We analyzed by microFTIR human polymorphonuclear neutrophil (PMNs) leukocytes, mouse-derived p

  3. UV Differentially Induces Oxidative Stress, DNA Damage and Apoptosis in BCR-ABL1-Positive Cells Sensitive and Resistant to Imatinib

    Directory of Open Access Journals (Sweden)

    Ewelina Synowiec

    2015-08-01

    Full Text Available Chronic myeloid leukemia (CML cells express the active BCR-ABL1 protein, which has been targeted by imatinib in CML therapy, but resistance to this drug is an emerging problem. BCR-ABL1 induces endogenous oxidative stress promoting genomic instability and imatinib resistance. In the present work, we investigated the extent of oxidative stress, DNA damage, apoptosis and expression of apoptosis-related genes in BCR-ABL1 cells sensitive and resistant to imatinib. The resistance resulted either from the Y253H mutation in the BCR-ABL1 gene or incubation in increasing concentrations of imatinib (AR. UV irradiation at a dose rate of 0.12 J/(m2·s induced more DNA damage detected by the T4 pyrimidine dimers glycosylase and hOGG1, recognizing oxidative modifications to DNA bases in imatinib-resistant than -sensitive cells. The resistant cells displayed also higher susceptibility to UV-induced apoptosis. These cells had lower native mitochondrial membrane potential than imatinib-sensitive cells, but UV-irradiation reversed that relationship. We observed a significant lowering of the expression of the succinate dehydrogenase (SDHB gene, encoding a component of the complex II of the mitochondrial respiratory chain, which is involved in apoptosis sensing. Although detailed mechanism of imatinib resistance in AR cells in unknown, we detected the presence of the Y253H mutation in a fraction of these cells. In conclusion, imatinib-resistant cells may display a different extent of genome instability than their imatinib-sensitive counterparts, which may follow their different reactions to both endogenous and exogenous DNA-damaging factors, including DNA repair and apoptosis.

  4. The L-amino acid oxidase from Calloselasma rhodostoma snake venom modulates apoptomiRs expression in Bcr-Abl-positive cell lines.

    Science.gov (United States)

    Burin, Sandra Mara; Berzoti-Coelho, Maria Gabriela; Cominal, Juçara Gastaldi; Ambrosio, Luciana; Torqueti, Maria Regina; Sampaio, Suely Vilela; de Castro, Fabíola Attié

    2016-09-15

    Anti-apoptotic genes and apoptomiRs deregulated expression contribute to apoptosis resistance in chronic myeloid leukemia (CML) Bcr-Abl(+) cells. Here, the L-amino acid oxidase from Calloselasma rhodostoma (CR-LAAO) venom altered the apoptotic machinery regulation by modulating the expression of the miR-145, miR-26a, miR-142-3p, miR-21, miR-130a, and miR-146a, and of the apoptosis-related proteins Bid, Bim, Bcl-2, Ciap-2, c-Flip, and Mcl-1 in Bcr-Abl(+) cells. CR-LAAO is a potential tool to instigate apoptomiRs regulation that contributes to drive CML therapy. PMID:27421670

  5. Induction of apoptosis by shikonin through a ROS/JNKmediated process in Bcr/Abl-positive chronic myelogenous leukemia (CML) cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/ Abl-positive chronic myelogenous leukemia (CML) cells (e.g., K562, LAMA84). Treatment of K562 cells with shikonin (e.g., 0.5 μM) resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species (ROS), striking activation of c-Jun-N-terminai kinase (JNK) and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events (i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis) following shikonin treatment. Inhibition of JNK and knock-down of JNKI significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS.

  6. INHIBITION OF APOPTOSIS BY bcr-abl FUSION GENE IN K562 CELLS

    Institute of Scientific and Technical Information of China (English)

    WANG Chun-hong; SUN Bing-zhong; YUAN Yue-chuan

    1999-01-01

    Objective: To investigate the effect of bcr-abl fusion gene on CML cell apoptosis. Methods: Apoptosis of exvivo cultured K562 cells were observed after exposure to synthetic 18 mer antisense oligodeoxynucleotide complementary to the bcr-abl junction (b3a2). Results: Apoptosis of K562 cells was significantly increased associated with inhibition of bcr-abl expression. Conclusion: bcr-abl fusion gene formation due to chromosome translocation may be the major mechanism of CML via inhibition of apoptosis.

  7. Environment-mediated drug resistance in Bcr/Abl-positive acute lymphoblastic leukemia

    OpenAIRE

    Feldhahn, Niklas; Arutyunyan, Anna; Stoddart, Sonia; ZHANG Bin; Schmidhuber, Sabine; Yi, Sun-ju; Kim, Yong-Mi; Groffen, John; Heisterkamp, Nora

    2012-01-01

    Although cure rates for acute lymphoblastic leukemia (ALL) have increased, development of resistance to drugs and patient relapse are common. The environment in which the leukemia cells are present during the drug treatment is known to provide significant survival benefit. Here, we have modeled this process by culturing murine Bcr/Abl-positive acute lymphoblastic leukemia cells in the presence of stroma while treating them with a moderate dose of two unrelated drugs, the farnesyltransferase i...

  8. Establishment and characterization of A novel Philadelphia-chromosome positive chronic myeloid leukemia cell line, TCC-S, expressing P210 and P190 BCR/ABL transcripts but missing normal ABL gene.

    Science.gov (United States)

    Van, Phan Nguyen Thanh; Xinh, Phan Thi; Kano, Yasuhiko; Tokunaga, Katsushi; Sato, Yuko

    2005-03-01

    A novel Philadelphia-chromosome positive (Ph+) cell line, TCC-S, has been established from a patient with Ph+ chronic myeloid leukemia (CML) in the blastic crisis. TCC-S cells were shown to express both P210 and P190 BCR/ABL transcripts by reverse transcriptase-polymerase chain reaction (PCR), although quantitative-PCR revealed that TCC-S cells mainly expressed P210 BCR/ABL transcript. Karyotype analysis revealed several triploid clones which constantly harbored two der(9)del(9) (p12)t(9;22) (q34;qll)s and two del(9) (q21)s. The der(9)del(9) (p12)t(9;22) (q34;q11) is rarely found in other CML cell lines. Moreover, to the best of our knowledge, del(9) (q21) resulting in missing of a restrict region including normal ABL gene has not been found among CML cell lines previously described. Thus, TCC-S cells with only BCR/ABL gene and no normal ABL gene may be a useful tool for functional study of ABL in Ph+ CML.

  9. Allogeneic stem cell transplantation for patients harboring T315I BCR-ABL mutated leukemias

    DEFF Research Database (Denmark)

    Nicolini, Franck Emmanuel; Basak, Grzegorz W; Soverini, Simona;

    2011-01-01

    T315I(+) Philadelphia chromosome-positive leukemias are inherently resistant to all licensed tyrosine kinase inhibitors, and therapeutic options remain limited. We report the outcome of allogeneic stem cell transplantation in 64 patients with documented BCR-ABL(T315I) mutations. Median follow...... myeloid leukemia. The occurrence of chronic GVHD had a positive impact on overall survival (P = .047). Transplant-related mortality rates were low. Multivariate analysis identified only blast phase at transplantation (hazard ratio 3.68, P = .0011) and unrelated stem cell donor (hazard ratio 2.98, P = .011......) as unfavorable factors. We conclude that allogeneic stem cell transplantation represents a valuable therapeutic tool for eligible patients with BCR-ABL(T315I) mutation, a tool that may or may not be replaced by third-generation tyrosine kinase inhibitors....

  10. Comparative study of DNA damage, cell cycle and apoptosis in human K562 and CCRF-CEM leukemia cells: role of BCR/ABL in therapeutic resistance.

    Science.gov (United States)

    Pytel, Dariusz; Wysocki, Tomasz; Majsterek, Ireneusz

    2006-09-01

    The Philadelphia translocation t(9;22) resulting in the bcr/abl fusion gene is the pathogenic principle of almost 95% of human chronic myelogenous leukemia (CML). Imatinib mesylate (STI571) is a specific inhibitor of the BCR/ABL fusion tyrosine kinase that exhibits potent antileukemic effects in CML. BCR/ABL-positive K562 and -negative CCRF-CEM human leukemia cells were investigated. MTT survival assay and clonogenic test of the cell proliferation ability were used to estimate resistance against idarubicin. DNA damage after cell treatment with the drug at the concentrations from 0.001 to 3 microM with or without STI571 pre-treatment were examined by the alkaline comet assay. We found that the level of DNA damages was lower in K562 cells after STI571 pre-treatment. It is suggested that BCR/ABL activity may promote genomic instability, moreover K562 cells were found to be resistant to the drug treatment. Further, we provided evidence of apoptosis inhibition in BCR/ABL-positive cells using caspase-3 activity colorimetric assay and DAPI nuclear staining for chromatin condensation. We suggest that these processes associated with cell cycle arrest in G2/M checkpoint detected in K562 BCR/ABL-positive compared to CCRF-CEM cells without BCR/ABL expression might promote clone selection resistance to drug treatment.

  11. Evaluation of Morpholino Antisense Oligos’ Role on BCR-ABL Gene Silencing in the K562 Cell Line

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    Bahman Delalat

    2010-01-01

    Full Text Available Objective: Chronic myeloid leukemia (CML develops when a hematopoietic stem cellacquires the BCR/ABL fusion gene. This causes these transformed hematopoietic cellsto have a greater than normal proliferation rate. Scientists attempt to improve the CMLtreatment process by silencing the BCR/ABL oncogene. In this work, we used morpholinoantisense oligos to silence the BCR/ABL oncogene.Materials and Methods: In this study, the K562 was used as a BCR/ABL fusion-genepositive cell line and the Jurkat cell line as a control. We explored the inhibiting capacityof morpholino antisense oligos in the the expression of the BCR/ABL oncogene andstudied their p210 BCR/ABL suppression, inhibition of cell proliferation and stimulation ofapoptosis in the K562 cells after 24 and 48 hours. Endo-Porter was used for delivery ofmorpholino antisense oligos into cell cytosols. Meanwhile, flow cytometric analysis wasperformed in order to determine the appropriate concentration of morpholino antisenseoligos.Results: Prolonged exposure of the K562 cell line to the morpholino antisense oligostargeted against the BCR-ABL gene showed proliferation inhibition as its main feature.After western blotting, we found that complete silencing of BCR/ABL was achieved, butflow cytometric analysis showed no broad apoptosis.Conclusion: The results indicate that the Morpholino antisense oligo is able to inhibitp210 BCR/ABL; however, it cannot induce broad apoptosis due to co-silencing of BCR.

  12. Curcumin synergistically augments bcr/abl phosphorethieate antisense oligonucleotides to inhibit growth of chronic myelogenous leukemia cells

    Institute of Scientific and Technical Information of China (English)

    Kun-zhong ZHANG; Jian-hua XU; Xiu-wang HUANG; Li-xian WU; Yu SU; Yuan-zhong CHEN

    2007-01-01

    Aim: To investigate the growth inhibition effect of the combination of bcr/abl phosphorothioate antisense oligonucleotides (PS-ASODN) and curcumin (cur), and the possible mechanisms of cur on the chronic myelogenous leukemia cell line K562. Methods: The K562 cell line was used as a P210bcr/abl-positive cell model in vitro and was exposed to different concentrations of PS-ASODN (0-20 μmol/L), cur (0-20 μmol/L), or a combination of both. Growth inhibition and apoptosis of K562 cells were assessed by MTT assay and AO/EB fluorescent staining, respec-tively. The expression levels of P210bct/abl, NF-κB and heat shock protein 90 (Hsp90) were assessed by Western blot. Results: Exposure to cur (5-20 μmol/L) and PS-ASODN (5-20 μmol/L) resulted in a synergistic inhibitory effect on cell growth.Growth inhibition was associated with the inhibition of the proliferation and in-duction of apoptosis. Western blot analysis showed that the drugs synergisti-cally downregulated the level of P210bcr/abl and NF-κB. Cur downregulated Hsp90,whereas no synergism was observed when cur was combined with PS-ASODN.Conclusion: PS-ASODN and cur exhibited a synergistic inhibitory effect on the cell growth of K562. The synergistic growth inhibition was mediated through different mechanisms that involved the inhibition of P210bcr/abl.

  13. Susceptibility of Ph-positive all to TKI therapy associated with Bcr-Abl rearrangement patterns: a retrospective analysis.

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    Yu Jing

    Full Text Available BACKGROUND: Tyrosine kinase inhibitors (TKIs have demonstrated success in the treatment of acute lymphoblastic leukemia (ALL in patients that express BCR-ABL rearrangements (Philadelphia chromosome [Ph]. The current study aimed to assess the efficacy of TKIs and prognostic factors in the treatment of adults with Ph+-ALL. METHODS: In this multicenter retrospective study, the relationship between Ph+-ALL and treatment outcomes among Chinese patients receiving TKI-containing induction/consolidation chemotherapy was examined. A total of 86 Ph+-ALL patients were included and followed for 3.85 (0.43-9.30 years. Overall survival (OS and event-free survival (EFS were analyzed. RESULTS: A total of 86 Ph+-ALL patients (40 females and 46 males; median age: 34.0 years were enrolled, including those with BCR/ABL transcripts 190 (n = 52, 210 (n = 25, and 230 (n = 2; BCR/ABL isoform determination was not available for 7 patients. Mortality was influenced by variable BCR/ABL transcripts and TKI administration, and BCR/ABL transcripts, hematopoietic stem cell transplantation (HSCT, and TKI administration were associated with the occurrence of events. The OS rate in the TKI administration group during steady state was significantly higher compared with those patients who did not receive TKI administration (P = 0.008, the EFS rate in the TKI administration group during steady state was significantly higher compared with those patients who did not receive TKIs (P = 0.012, and also higher than those with TKI salvage administration (P = 0.004. BCR/ABL transcripts 210 showed preferable OS and EFS compared with BCR/ABL transcripts 190 and 230 (P<0.05 for each. CONCLUSIONS: The susceptibility of Ph+-ALL to TKI associated with the patterns of BCR-ABL rearrangement is demonstrated for the first time, thus adding another risk-stratifying molecular prognostic tool for the management of patients with Ph+-ALL.

  14. Targeting Hedgehog signaling pathway and autophagy overcomes drug resistance of BCR-ABL-positive chronic myeloid leukemia.

    Science.gov (United States)

    Zeng, Xian; Zhao, Hui; Li, Yubin; Fan, Jiajun; Sun, Yun; Wang, Shaofei; Wang, Ziyu; Song, Ping; Ju, Dianwen

    2015-01-01

    The frontline tyrosine kinase inhibitor (TKI) imatinib has revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, drug resistance is the major clinical challenge in the treatment of CML. The Hedgehog (Hh) signaling pathway and autophagy are both related to tumorigenesis, cancer therapy, and drug resistance. This study was conducted to explore whether the Hh pathway could regulate autophagy in CML cells and whether simultaneously regulating the Hh pathway and autophagy could induce cell death of drug-sensitive or -resistant BCR-ABL(+) CML cells. Our results indicated that pharmacological or genetic inhibition of Hh pathway could markedly induce autophagy in BCR-ABL(+) CML cells. Autophagic inhibitors or ATG5 and ATG7 silencing could significantly enhance CML cell death induced by Hh pathway suppression. Based on the above findings, our study demonstrated that simultaneously inhibiting the Hh pathway and autophagy could markedly reduce cell viability and induce apoptosis of imatinib-sensitive or -resistant BCR-ABL(+) cells. Moreover, this combination had little cytotoxicity in human peripheral blood mononuclear cells (PBMCs). Furthermore, this combined strategy was related to PARP cleavage, CASP3 and CASP9 cleavage, and inhibition of the BCR-ABL oncoprotein. In conclusion, this study indicated that simultaneously inhibiting the Hh pathway and autophagy could potently kill imatinib-sensitive or -resistant BCR-ABL(+) cells, providing a novel concept that simultaneously inhibiting the Hh pathway and autophagy might be a potent new strategy to overcome CML drug resistance.

  15. Autophagy induction by Bcr-Abl-expressing cells facilitates their recovery from a targeted or nontargeted treatment.

    LENUS (Irish Health Repository)

    Crowley, Lisa C

    2012-01-31

    Although Imatinib has transformed the treatment of chronic myeloid leukemia (CML), it is not curative due to the persistence of resistant cells that can regenerate the disease. We have examined how Bcr-Abl-expressing cells respond to two mechanistically different therapeutic agents, etoposide and Imatinib. We also examined Bcr-Abl expression at low and high levels as elevated expression has been associated with treatment failure. Cells expressing low levels of Bcr-Abl undergo apoptosis in response to the DNA-targeting agent (etoposide), whereas high-Bcr-Abl-expressing cells primarily induce autophagy. Autophagic populations engage a delayed nonapoptotic death; however, sufficient cells evade this and repopulate following the withdrawal of the drug. Non-Bcr-Abl-expressing 32D or Ba\\/F3 cells induce both apoptosis and autophagy in response to etoposide and can recover. Imatinib treatment induces both apoptosis and autophagy in all Bcr-Abl-expressing cells and populations rapidly recover. Inhibition of autophagy with ATG7 and Beclin1 siRNA significantly reduced the recovery of Imatinib-treated K562 cells, indicating the importance of autophagy for the recovery of treated cells. Combination regimes incorporating agents that disrupt Imatinib-induced autophagy would remain primarily targeted and may improve response to the treatment in CML.

  16. Cytoprotective effect of imatinib mesylate in non-BCR-ABL-expressing cells along with autophagosome formation

    Energy Technology Data Exchange (ETDEWEB)

    Ohtomo, Tadashi [Department of Biochemistry, Tokyo Medical University, Tokyo (Japan); Miyazawa, Keisuke, E-mail: miyazawa@tokyo-med.ac.jp [Department of Biochemistry, Tokyo Medical University, Tokyo (Japan); Naito, Munekazu [Department of Anatomy, Tokyo Medical University, Tokyo (Japan); Moriya, Shota [Department of Biochemistry, Tokyo Medical University, Tokyo (Japan); Kuroda, Masahiko [Department of Molecular Pathology, Tokyo Medical University, Tokyo (Japan); Itoh, Masahiro [Department of Anatomy, Tokyo Medical University, Tokyo (Japan); Tomoda, Akio [Department of Biochemistry, Tokyo Medical University, Tokyo (Japan)

    2010-01-01

    Treatment with imatinib mesylate (IM) results in an increased viable cell number of non-BCR-ABL-expressing cell lines by inhibiting spontaneous apoptosis. Electron microscopy revealed an increase of autophagosomes in response to IM. IM attenuated the cytotoxic effect of cytosine arabinoside, as well as inhibiting cell death with serum-deprived culture. Cytoprotection with autophagosome formation by IM was observed in various leukemia and cancer cell lines as well as normal murine embryonic fibroblasts (MEFs). Complete inhibition of autophagy by knockdown of atg5 in the Tet-off atg5{sup -/-} MEF system attenuated the cytoprotective effect of IM, indicating that the effect is partially dependent on autophagy. However, cytoprotection by IM was not mediated through suppression of ROS production via mitophagy, ER stress via ribophagy, or proapoptotic function of ABL kinase. Although the target tyrosine kinase(s) of IM remains unclear, our data provide novel therapeutic possibilities of using IM for cytoprotection.

  17. The Role of MEK Kinase 1 in Bcr-Abl-Induced Self-Renewal Activity of Embryonic Stem Cells

    OpenAIRE

    NAKAMURA, Yukinori; Yujiri, Toshiaki; Tanizawa, Yukio

    2006-01-01

    BCR-ABL oncogene, the moecular hallmark of chronic mylogenous leukemia, arises in a promitive hematopoietic stem cell that has the capacity for both differentiation and self-renewal. Its product, Bcr-Abl protein, has been shown to sctivate signal transducers and activators of transcription 3 (STAT3) and to promote self-renewal in embryonic stem (ES)cells, even in the absencs of leukemia in hibitory factor (LIF). MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase ...

  18. Inhibitory Effects of Omacetaxine on Leukemic Stem Cells and BCR-ABL-Induced Chronic Myeloid Leukemia and Acute Lymphoblastic Leukemia in Mice

    OpenAIRE

    Chen, Yaoyu; Hu, Yiguo; Michaels, Shawnya; Segal, David; Brown, Dennis; Li, Shaoguang

    2009-01-01

    Omacetaxine mepesuccinate (formerly homoharringtonine) is a molecule with a mechanism of action that is different from tyrosine kinase inhibitors and its activity in chronic myeloid leukemia (CML) seems to be independent of BCR-ABL mutation status. Using BCR-ABL-expressing myelogenous and lymphoid cell lines and mouse models of CML and B cell acute lymphoblastic leukemia (B-ALL) induced by wild type BCR-ABL or T315I mutant-BCR-ABL, we evaluated the inhibitory effects of omacetaxine on CML and...

  19. MEK kinase 1 is essential for Bcr-Abl-induced STAT3 and self-renewal activity in embryonic stem cells.

    Science.gov (United States)

    Nakamura, Yukinori; Yujiri, Toshiaki; Nawata, Ryouhei; Tagami, Kozo; Tanizawa, Yukio

    2005-11-17

    BCR-ABL oncogene, the molecular hallmark of chronic myelogenous leukemia, arises in a primitive hematopoietic stem cell that has the capacity for both differentiation and self-renewal. Its product, Bcr-Abl protein, has been shown to activate signal transducers and activators of transcription 3 (STAT3) and to promote self-renewal in embryonic stem (ES) cells, even in the absence of leukemia inhibitory factor (LIF). MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase kinase involved in Bcr-Abl signal transduction. To investigate the role of MEKK1 in Bcr-Abl-induced transformation of stem cells, p210 Bcr-Abl was stably transfected into wild-type (WT(p210)) and MEKK1-/- (MEKK1-/-(p210)) ES cells. Bcr-Abl enhanced MEKK1 expression in ES transfectants, as it does in other Bcr-Abl-transformed cells. In the absence of LIF, WT(p210) cells showed constitutive STAT3 activation and formed rounded, compact colonies having strong alkaline phosphatase activity, a characteristic phenotype of undifferentiated ES cells. MEKK1-/-(p210) cells, by contrast, showed less STAT3 activity than WT(p210) cells and formed large, flattened colonies having weak alkaline phosphatase activity, a phenotype of differentiated ES cells. These results indicate that MEKK1 plays a key role in Bcr-Abl-induced STAT3 activation and in ES cells' capacity for LIF-independent self-renewal, and may thus be involved in Bcr-Abl-mediated leukemogenesis in stem cells. PMID:16044153

  20. Reactive Oxygen Species and Mitochondrial DNA Damage and Repair in BCR-ABL1 Cells Resistant to Imatinib.

    Science.gov (United States)

    Blasiak, Janusz; Hoser, Grazyna; Bialkowska-Warzecha, Jolanta; Pawlowska, Elzbieta; Skorski, Tomasz

    2015-01-01

    Imatinib revolutionized the therapy of chronic myeloid leukemia (CML), but the resistance to it became an emerging problem. We reported previously that CML cells expressing the BCR/ABL1 fusion gene, accumulated a high level of reactive oxygen species (ROS) due to deregulated mitochondrial electron transport chain, which in turn led to genomic instability, resulting in imatinib resistance. In the present work, we hypothesize that imatinib-resistant cells may show higher instability of mitochondrial DNA (mtDNA) than their sensitive counterparts. To verify this hypothesis, we checked the ROS level and mtDNA damage and repair in model CML cells sensitive and resistant to imatinib and exposed to doxorubicin (DOX), a DNA-damaging agent. The extent of endogenous ROS in imatinib-resistant cells was higher than in their sensitive counterparts and DOX potentiated this relationship. ROS level in cells with primary resistance, which resulted from the T315I mutation in BCR/ABL1, was higher than in cells with acquired resistance. DOX-induced mtDNA damage in T315I imatinib-resistant cells was more pronounced than in imatinib-sensitive cells. All kinds of cells were repairing mtDNA damage with similar kinetics. In conclusion, imatinib-resistant cells can show increased instability of mtDNA, which can result from increased ROS production. PMID:26309809

  1. Additive antileukemia effects by GFI1B- and BCR-ABL-specific siRNA in advanced phase chronic myeloid leukemic cells.

    Science.gov (United States)

    Koldehoff, M; Zakrzewski, J L; Beelen, D W; Elmaagacli, A H

    2013-07-01

    Previous studies demonstrated selective inhibition of the BCR-ABL (breakpoint cluster region-Abelson murine leukemia oncogene) tyrosine kinase by RNA interference in leukemic cells. In this study, we evaluated the effect of BCR-ABL small interfering RNA (siRNA) and GFI1B siRNA silencing on chronic myeloid leukemia (CML) cells in myeloid blast crises. The GFI1B gene was mapped to chromosome 9 and is, therefore, located downstream of the BCR-ABL translocation in CML cells. Co-transfection of BCR-ABL siRNA and GFI1B siRNA dramatically decreased cell viability and significantly induced apoptosis and inhibited proliferation in K562 cells (P<0.0001) and primary advanced phase CML cells (P<0.0001) versus controls. Furthermore, combining of BCR-ABL siRNA and GFI1B siRNA significantly modified the expression of several relevant genes including Myc, MDR1, MRP1 and tyrosyl-phosphoproteins in primary CML cells. Our data suggest that silencing of both BCR-ABL siRNA and GFI1B siRNA is associated with an additive antileukemic effect against K562 cells and primary advanced CML cells, further validating these genes as attractive therapeutic targets. PMID:23788109

  2. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl+ K562 and Jak2(V617F)+ HEL Leukemia Cells

    International Nuclear Information System (INIS)

    Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl+ K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells, Stat5 is

  3. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl+ K562 and Jak2(V617F)+ HEL Leukemia Cells.

    Science.gov (United States)

    Weber, Axel; Borghouts, Corina; Brendel, Christian; Moriggl, Richard; Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd

    2015-01-01

    Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl+ K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells, Stat5 is

  4. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl+ K562 and Jak2(V617F+ HEL Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Axel Weber

    2015-03-01

    Full Text Available Signal transducers and activators of transcription (Stats play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML and Jak2(V617F in other myeloproliferative diseases (MPD. We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl+ K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL

  5. Structure-Activity Relationship Studies of Mitogen Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 and BCR-ABL1 Inhibitors Targeting Chronic Myeloid Leukemic Cells.

    Science.gov (United States)

    Cherian, Joseph; Nacro, Kassoum; Poh, Zhi Ying; Guo, Samantha; Jeyaraj, Duraiswamy A; Wong, Yun Xuan; Ho, Melvyn; Yang, Hai Yan; Joy, Joma Kanikadu; Kwek, Zekui Perlyn; Liu, Boping; Wee, John Liang Kuan; Ong, Esther H Q; Choong, Meng Ling; Poulsen, Anders; Lee, May Ann; Pendharkar, Vishal; Ding, Li Jun; Manoharan, Vithya; Chew, Yun Shan; Sangthongpitag, Kanda; Lim, Sharon; Ong, S Tiong; Hill, Jeffrey; Keller, Thomas H

    2016-04-14

    Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented. PMID:27011159

  6. A report case of mixed phenotype acute leukemia with BCR - ABL positive and literature review%BCR - ABL 融合基因阳性的急性混合细胞白血病1例并文献复习

    Institute of Scientific and Technical Information of China (English)

    符爽; 张男; 陈芳; 刘璇; 胡延平; 张旻昱; 张继红

    2014-01-01

    Objective:To investigate the clinical characteristics,biological features,clinical curative effect and out-come of mixed PhenotyPe acute leukemia(MPAL). Methods:Results of clinical data,cell morPhology,immunoPheno-tyPe,cytogenetics and molecular genetics of the MPAL Patient with BCR - ABL Positive were analyzed with literature reviews. Results:The clinical and hematological findings were comPatible with the diagnosis of MPAL. LymPhoid and myeloid markers were Positive on the leukemia cells. KaryotyPe analysis showed that the Patient had t(9;22)(q34;q11)translocation. Quantitative reverse transcriPtion PCR results showed that BCR - ABL(P210)exPressed 100% . And no deletion,Point or insert mutation was found among C - Kit(8,17 exon),FLT3 - ITD/ TKD,NPM1 and CEBPα. Conclusion:Primary MPAL with BCR - ABL Positive is rarely rePorted which have a Poor Prognosis. Some of them are cases of chronic myeloid leukemia blast crisis. BCR - ABL gene exPression is closely related to Patients' Prognosis.%目的:探讨 BCR - ABL 融合基因阳性表达的急性混合细胞白血病(MPAL)的临床特点、生物学特性、临床疗效及预后。方法:对1例 BCR - ABL 融合基因阳性的急性混合细胞白血病患者的临床资料、细胞形态学、免疫分型、细胞遗传学及分子生物学的检测结果进行综合分析。结果:患者的临床表现和实验室检测结果均符合急性混合细胞白血病诊断标准,免疫表型分析白血病细胞同时表达髓系和淋系抗原,染色体核型为46,XX,t(9;22)(q34;q11),荧光定量 PCR 检测结果示 BCR - ABL(P210)融合基因表达100%,白血病预后基因检测结果示患者 C - Kit(8、17 exon)、FLT3- ITD/ TKD、NPM1和 CEBPα基因均为野生型。结论:初发的 BCR - ABL 融合基因阳性的急性混合细胞白血病病例罕见,部分病例由慢性粒细胞白血病急变而来,预后差。BCR - ABL 基因阳性与患者预后密切相关。

  7. Allogeneic stem cell transplantation for patients harboring T315I BCR-ABL mutated leukemias

    DEFF Research Database (Denmark)

    Nicolini, Franck Emmanuel; Basak, Grzegorz W; Soverini, Simona;

    2011-01-01

    , still alive]) for those with chronic myeloid leukemia in the blast phase and 7.4 months (range 1.4 months to not reached [ie, still alive]) for those with Philadelphia chromosome-positive acute lymphoblastic leukemia but has not yet been reached for those in the chronic and accelerated phases of chronic...

  8. Involvement of primary mesenchymal precursors and hematopoietic bone marrow cells from chronic myeloid leukemia patients by BCR-ABL1 fusion gene.

    Science.gov (United States)

    Chandia, Mauricio; Sayagués, José-María; Gutiérrez, María-Laura; Chillón, María-Laura; Aristizábal, José-Alejandro; Corrales, Alejandro; Castellanos, Marta; Melón, Alberto; Sánchez, María-Luz; Bárcena, Paloma; Matarraz, Sergio; González-González, María; Barrena, Susana; López, Antonio; Cañizo, María-Consuelo; Sánchez-Guijo, Fermín; Orfao, Alberto

    2014-03-01

    For decades now, it is well established that chronic myeloid leukemia (CML) is a hematopoietic stem cell(HPC) disorder. However, it remains to be determined whether BCR-ABL1 gene rearrangement occurs in a HPC or at an earlier stem cell and whether the degree of involvement of hematopoiesis by the BCR-ABL1 fusion gene relates to the response to therapy. Here, we have investigated by interphase fluorescence in situ hybridization (iFISH) the distribution of BCR-ABL1 fusion gene in FACS-sorted bone marrow (BM) populations of mesenchymal precursor cells (MPC) and other hematopoietic cell populations from 18 newly diagnosed CML patients. Overall, our results showed systematic involvement at relatively high percentages of BM maturing neutrophils (97%615%), basophils (95%612%), eosinophils (90%68%), CD341 precursors cells (90%67%),monocytes (84%630%), nucleated red blood cells (87%624%), and mast cells (77%633%). By contrast, MPC(30%634%), B-cells (15%627%), T-lymphocytes (50%626%), and NK-cells (35%634%) were involved at lower percentages. In 8/18 CML patients, 2 tumor BCR-ABL11 subclones were detected by iFISH. Of note, all tumor cell subclones were systematically detected in CD341 cells, whereas MPC were only involved by the ancestral tumor cell subclone. In summary, here we confirm the presence at diagnosis of the BCR-ABL1 fusion gene inMPC, CD341 precursors, and other different BM hematopoietic myeloid cell lineages from CML patients,including also in a significant fraction of cases, a smaller percentage of T, B, and NK lymphocytes.Interestingly, involvement of MPC was restricted to the ancestral BCR-ABL11 subclone. PMID:24779036

  9. Gads (Grb2-related adaptor downstream of Shc) is required for BCR-ABL-mediated lymphoid leukemia

    Science.gov (United States)

    Gillis, LC; Berry, DM; Minden, MD; McGlade, CJ; Barber, DL

    2016-01-01

    Philadelphia chromosome-positive leukemias, including chronic myeloid leukemia and B-cell acute lymphoblastic leukemia (B-ALL), are driven by the oncogenic BCR-ABL fusion protein. Animal modeling experiments utilizing retroviral transduction and subsequent bone marrow transplantation have demonstrated that BCR-ABL generates both myeloid and lymphoid disease in mice receiving whole bone marrow transduced with BCR-ABL. Y177 of BCR-ABL is critical to the development of myeloid disease, and phosphorylation of Y177 has been shown to induce GRB2 binding to BCR-ABL, followed by activation of the Ras and phosphoinositide 3 kinase signaling pathways. We show that the GRB2-related adapter protein, GADS, also associates with BCR-ABL, specifically through Y177 and demonstrate that BCR-ABL-driven lymphoid disease requires Gads. BCR-ABL transduction of Gads(−/−) bone marrow results in short latency myeloid disease within 3–4 weeks of transplant, while wild-type mice succumb to both a longer latency lymphoid and myeloid diseases. We report that GADS mediates a unique BCR-ABL complex with SLP-76 in BCR-ABL-positive cell lines and B-ALL patient samples. These data suggest that GADS mediates lymphoid disease downstream of BCR-ABL through the recruitment of specific signaling intermediates. PMID:23399893

  10. miR-29b suppresses CML cell proliferation and induces apoptosis via regulation of BCR/ABL1 protein

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yajuan; Wang, Haixia; Tao, Kun [Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated of Ministry of Education, Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 400016 (China); Xiao, Qing [Department of Hematology, The First Affiliated Hospital, Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 400016 (China); Huang, Zhenglan; Zhong, Liang; Cao, Weixi [Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated of Ministry of Education, Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 400016 (China); Wen, Jianping [Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5 (Canada); Feng, Wenli, E-mail: fengwlcqmu@sina.com [Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated of Ministry of Education, Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 400016 (China)

    2013-05-01

    MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally and are critical for many cellular pathways. Recent evidence has shown that aberrant miRNA expression profiles and unique miRNA signaling pathways are present in many cancers. Here, we demonstrate that miR-29b is markedly lower expressed in CML patient samples. Bioinformatics analysis reveals a conserved target site for miR-29b in the 3′-untranslated region (UTR) of ABL1. miR-29b significantly suppresses the activity of a luciferase reporter containing ABL1-3′UTR and this activity is not observed in cells transfected with mutated ABL1-3′UTR. Enforced expression of miR-29b in K562 cells inhibits cell growth and colony formation ability thereby inducing apoptosis through cleavage of procaspase 3 and PARP. Furthermore, K562 cells transfected with a siRNA targeting ABL1 show similar growth and apoptosis phenotypes as cells overexpression of miR-29b. Collectively, our results suggest that miR-29b may function as a tumor suppressor by targeting ABL1 and BCR/ABL1. - Highlights: ► miR-29b expression was downregulated in CML patients. ► ABL1 was identified as a direct target gene of miR-29b. ► Enforced expression of miR-29b inhibits cell proliferation and induces apoptosis. ► miR-29b might be a therapeutic target to CML.

  11. Expression of P190 and P210 BCR/ABL1 in normal human CD34(+) cells induces similar gene expression profiles and results in a STAT5-dependent expansion of the erythroid lineage

    DEFF Research Database (Denmark)

    Järås, Marcus; Johnels, Petra; Agerstam, Helena;

    2009-01-01

    and systematically compared the effects of retroviral P190 BCR/ABL1 and P210 BCR/ABL1 expression on cell proliferation, differentiation, and global gene expression in human CD34(+) cells from cord blood. RESULTS: Expression of either P190 BCR/ABL1 or P210 BCR/ABL1 resulted in expansion of erythroid cells...... and stimulated erythropoietin-independent burst-forming unit-erythroid colony formation. By using a lentiviral anti-signal transducer and activator of transcription 5 (STAT5) short-hairpin RNA, we found that both P190 BCR/ABL1- and P210 BCR/ABL1-induced erythroid cell expansion were STAT5-dependent. Under...... that the early cellular and transcriptional effects of P190 BCR/ABL1 and P210 BCR/ABL1 expression are very similar when they are expressed in the same human progenitor cell population, and that STAT5 is an important regulator of BCR/ABL1-induced erythroid cell expansion....

  12. Expression of BCR/ABL p210 from a Knockin Allele Enhances Bone Marrow Engraftment without Inducing Neoplasia

    Directory of Open Access Journals (Sweden)

    Samantha B. Foley

    2013-10-01

    Full Text Available Chronic myeloid leukemia (CML and some acute lymphoblastic leukemias are characterized by the t(9;22 chromosome, which encodes the BCR/ABL oncogene. Multiple mouse models of CML express BCR/ABL at high levels from non-Bcr promoters, resulting in the development of leukemias. In contrast, a significant fraction of healthy humans have been found to have BCR/ABL-positive hematopoietic cells. To bridge the gap between the information derived from current mouse models and nonleukemic humans with the BCR/ABL oncogene, we generated a knockin model with BCR/ABL p210 expressed from the Bcr locus. Unlike previous models, expression of BCR/ABL from the knockin allele did not induce leukemia. BCR/ABL mutant cells did exhibit favorable bone marrow engraftment compared to control cells. These data suggest that BCR/ABL expression alone is insufficient to induce disease. This model allows for inducible spatial and temporal control of BCR/ABL expression for analysis of early steps in the pathogenesis of BCR/ABL-expressing leukemias.

  13. MicroRNA-1301-Mediated RanGAP1 Downregulation Induces BCR-ABL Nuclear Entrapment to Enhance Imatinib Efficacy in Chronic Myeloid Leukemia Cells.

    Directory of Open Access Journals (Sweden)

    Tsung-Yao Lin

    Full Text Available Chronic myeloid leukemia (CML is a myeloproliferative disease. Imatinib (IM, the first line treatment for CML, is excessively expensive and induces various side effects in CML patients. Therefore, it is essential to investigate a new strategy for improving CML therapy. Our immunoblot data revealed that RanGTPase activating protein 1 (RanGAP1 protein levels increased by approximately 30-fold in K562 cells compared with those in normal cells. RanGAP1 is one of the important components of RanGTPase system, which regulates the export of nuclear protein. However, whether RanGAP1 level variation influences BCR-ABL nuclear export is still unknown. In this report, using shRNA to downregulate RanGAP1 expression level augmented K562 cell apoptosis by approximately 40% after treatment with 250 nM IM. Immunofluorescence assay also indicated that three-fold of nuclear BCR-ABL was detected. These data suggest that BCR-ABL nuclear entrapment induced by RanGAP1 downregulation can be used to improve IM efficacy. Moreover, our qRT-PCR data indicated a trend of inverse correlation between the RanGAP1 and microRNA (miR-1301 levels in CML patients. MiR-1301, targeting the RanGAP1 3' untranslated region, decreased by approximately 100-fold in K562 cells compared with that in normal cells. RanGAP1 downregulation by miR-1301 transfection impairs BCR-ABL nuclear export to increase approximately 60% of cell death after treatment of 250 nM IM. This result was almost the same as treatment with 1000 nM IM alone. Furthermore, immunofluorescence assay demonstrated that Tyr-99 of nuclear P73 was phosphorylated accompanied with nuclear entrapment of BCR-ABL after transfection with RanGAP1 shRNA or miR-1301 in IM-treated K562 cells. Altogether, we demonstrated that RanGAP1 downregulation can mediate BCR-ABL nuclear entrapment to activate P73-dependent apoptosis pathway which is a novel strategy for improving current IM treatment for CML.

  14. Cell sorting enables interphase fluorescence in situ hybridization detection of low BCR-ABL1 producing stem cells in chronic myeloid leukaemia patients beyond deep molecular remission

    DEFF Research Database (Denmark)

    van Kooten Niekerk, Peter Buur; Petersen, Charlotte Christie; Nyvold, Charlotte Guldborg;

    2014-01-01

    The exact disease state of chronic myeloid leukaemia (CML) patients in deep molecular remission is unknown, because even the most sensitive quantitative reverse transcription polymerase chain reaction (qPCR) methods cannot identify patients prone to relapse after treatment withdrawal. To elucidate......) ), n = 11) using both sensitive qPCR and interphase fluorescence in situ hybridization (iFISH). Despite evaluating fewer cells, iFISH proved superior to mRNA-based qPCR in detecting residual Ph(+) stem cells (P = 0·005), and detected Ph(+) stem- and progenitor cells in 9/10 patients at frequencies of 2......-14%. Moreover, while all qPCR(+) samples also were iFISH(+) , 9/33 samples were qPCR-/iFISH(+) , including all positive samples from MR(4) patients. Our findings show that residual Ph(+) cells are low BCR-ABL1 producers, and that DNA-based methods are required to assess the content of persisting Ph(+) stem...

  15. Allosteric inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated BCR-ABL and BCR-ABL-T315I

    Directory of Open Access Journals (Sweden)

    Mian Afsar

    2012-09-01

    Full Text Available Abstract Background Chronic myelogenous leukemia (CML and Philadelphia chromosome-positive (Ph+ acute lymphatic leukemia (Ph + ALL are caused by the t(9;22, which fuses BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase “escapes” the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the ‘gatekeeper’ mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia. Methods The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC from Ph + ALL-patients. Results Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner. Conclusions Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors.

  16. Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line

    Science.gov (United States)

    Yang, Chengbin; Panwar, Nishtha; Wang, Yucheng; Zhang, Butian; Liu, Maixian; Toh, Huiting; Yoon, Ho Sup; Tjin, Swee Chuan; Chong, Peter Han Joo; Law, Wing-Cheung; Chen, Chih-Kuang; Yong, Ken-Tye

    2016-04-01

    First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro

  17. BCR/ABL stimulates WRN to promote survival and genomic instability

    Science.gov (United States)

    Slupianek, Artur; Poplawski, Tomasz; Jozwiakowski, Stanislaw K.; Cramer, Kimberly; Pytel, Dariusz; Stoczynska, Ewelina; Nowicki, Michal O.; Blasiak, Janusz; Skorski, Tomasz

    2010-01-01

    BCR/ABL-transformed chronic myeloid leukemia (CML) cells accumulate numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) and genotoxic agents. To repair these lesions BCR/ABL stimulate unfaithful DSB repair pathways, homologous recombination repair (HRR), non-homologous end-joining (NHEJ) and single-strand annealing (SSA). Here we show that BCR/ABL enhances the expression and increase nuclear localization of WRN (mutated in Werner syndrome), which is required for processing DSB ends during the repair. Other fusion tyrosine kinases (FTKs) such as TEL/ABL, TEL/JAK2, TEL/PDGFβR, and NPM/ALK also elevate WRN. BCR/ABL induces WRN mRNA and protein expression in part by c-MYC -mediated activation of transcription and Bcl-xL –dependent inhibition of caspase-dependent cleavage, respectively. WRN is in complex with BCR/ABL resulting in WRN tyrosine phosphorylation and stimulation of its helicase and exonuclease activities. Activated WRN protects BCR/ABL-positive cells from the lethal effect of oxidative and genotoxic stresses, which causes DSBs. In addition, WRN promotes unfaithful recombination-dependent repair mechanisms HRR and SSA, and enhances the loss of DNA bases during NHEJ in leukemia cells. In summary, we postulate that BCR/ABL-mediated stimulation of WRN modulates the efficiency and fidelity of major DSB repair mechanisms to protect leukemia cells from apoptosis and to facilitate genomic instability. PMID:21123451

  18. BCR/ABL stimulates WRN to promote survival and genomic instability.

    Science.gov (United States)

    Slupianek, Artur; Poplawski, Tomasz; Jozwiakowski, Stanislaw K; Cramer, Kimberly; Pytel, Dariusz; Stoczynska, Ewelina; Nowicki, Michal O; Blasiak, Janusz; Skorski, Tomasz

    2011-02-01

    BCR/ABL-transformed chronic myeloid leukemia (CML) cells accumulate numerous DNA double-strand breaks (DSB) induced by reactive oxygen species (ROS) and genotoxic agents. To repair these lesions BCR/ABL stimulate unfaithful DSB repair pathways, homologous recombination repair (HRR), nonhomologous end-joining (NHEJ), and single-strand annealing (SSA). Here, we show that BCR/ABL enhances the expression and increase nuclear localization of WRN (mutated in Werner syndrome), which is required for processing DSB ends during the repair. Other fusion tyrosine kinases (FTK), such as TEL/ABL, TEL/JAK2, TEL/PDGFβR, and NPM/ALK also elevate WRN. BCR/ABL induces WRN mRNA and protein expression in part by c-MYC-mediated activation of transcription and Bcl-xL-dependent inhibition of caspase-dependent cleavage, respectively. WRN is in complex with BCR/ABL resulting in WRN tyrosine phosphorylation and stimulation of its helicase and exonuclease activities. Activated WRN protects BCR/ABL-positive cells from the lethal effect of oxidative and genotoxic stresses, which causes DSBs. In addition, WRN promotes unfaithful recombination-dependent repair mechanisms HRR and SSA, and enhances the loss of DNA bases during NHEJ in leukemia cells. In summary, we postulate that BCR/ABL-mediated stimulation of WRN modulates the efficiency and fidelity of major DSB repair mechanisms to protect leukemia cells from apoptosis and to facilitate genomic instability.

  19. Down-regulation of Thanatos-associated protein 11 by BCR-ABL promotes CML cell proliferation through c-Myc expression.

    Science.gov (United States)

    Nakamura, Satoki; Yokota, Daisuke; Tan, Lin; Nagata, Yasuyuki; Takemura, Tomonari; Hirano, Isao; Shigeno, Kazuyuki; Shibata, Kiyoshi; Fujisawa, Shinya; Ohnishi, Kazunori

    2012-03-01

    Bcr-Abl activates various signaling pathways in chronic myelogenous leukemia (CML) cells. The proliferation of Bcr-Abl transformed cells is promoted by c-Myc through the activation of Akt, JAK2 and NF-κB. However, the mechanism by which c-Myc regulates CML cell proliferation is unclear. In our study, we investigated the role of Thanatos-associated protein 11 (THAP11), which inhibits c-Myc transcription, in CML cell lines and in hematopoietic progenitor cells derived from CML patients. The induction of THAP11 expression by Abl kinase inhibitors in CML cell lines and in CML-derived hematopoietic progenitor cells resulted in the suppression of c-Myc. In addition, over-expression of THAP11 inhibited CML cell proliferation. In colony forming cells derived from CML-aldehyde dehydrogenase (ALDH)(hi) /CD34(+) cells, treatment with Abl kinase inhibitors and siRNA depletion of Bcr-Abl induced THAP11 expression and reduced c-Myc expression, resulting in inhibited colony formation. Moreover, overexpression of THAP11 significantly decreased the colony numbers, and also inhibited the expression of c-myc target genes such as Cyclin D1, ODC and induced the expression of p21(Cip1) . The depletion of THAP11 inhibited JAK2 or STAT5 inactivation-mediated c-Myc reduction in ALDH(hi) /CD34(+) CML cells. Thus, the induced THAP11 might be one of transcriptional regulators of c-Myc expression in CML cell. Therefore, the induction of THAP11 has a potential possibility as a target for the inhibition of CML cell proliferation. PMID:21400515

  20. Sensitive detection of pre-existing BCR-ABL kinase domain mutations in CD34+ cells of newly diagnosed chronic-phase chronic myeloid leukemia patients is associated with imatinib resistance: implications in the post-imatinib era.

    Directory of Open Access Journals (Sweden)

    Zafar Iqbal

    Full Text Available BACKGROUND: BCR-ABL kinase domain mutations are infrequently detected in newly diagnosed chronic-phase chronic myeloid leukemia (CML patients. Recent studies indicate the presence of pre-existing BCR-ABL mutations in a higher percentage of CML patients when CD34+ stem/progenitor cells are investigated using sensitive techniques, and these mutations are associated with imatinib resistance and disease progression. However, such studies were limited to smaller number of patients. METHODS: We investigated BCR-ABL kinase domain mutations in CD34+ cells from 100 chronic-phase CML patients by multiplex allele-specific PCR and sequencing at diagnosis. Mutations were re-investigated upon manifestation of imatinib resistance using allele-specific PCR and direct sequencing of BCR-ABL kinase domain. RESULTS: Pre-existing BCR-ABL mutations were detected in 32/100 patients and included F311L, M351T, and T315I. After a median follow-up of 30 months (range 8-48, all patients with pre-existing BCR-ABL mutations exhibited imatinib resistance. Of the 68 patients without pre-existing BCR-ABL mutations, 24 developed imatinib resistance; allele-specific PCR and BCR-ABL kinase domain sequencing detected mutations in 22 of these patients. All 32 patients with pre-existing BCR-ABL mutations had the same mutations after manifestation of imatinib-resistance. In imatinib-resistant patients without pre-existing BCR-ABL mutations, we detected F311L, M351T, Y253F, and T315I mutations. All imatinib-resistant patients except T315I and Y253F mutations responded to imatinib dose escalation. CONCLUSION: Pre-existing BCR-ABL mutations can be detected in a substantial number of chronic-phase CML patients by sensitive allele-specific PCR technique using CD34+ cells. These mutations are associated with imatinib resistance if affecting drug binding directly or indirectly. After the recent approval of nilotinib, dasatinib, bosutinib and ponatinib for treatment of chronic myeloid

  1. Modeling the low-LET dose-response of BCR-ABL formation: predicting stem cell numbers from A-bomb data.

    Science.gov (United States)

    Radivoyevitch, T; Hoel, D G

    1999-01-01

    Formation of the BCR-ABL chromosomal translocation t(9;22)(q34;q11) is essential to the genesis of chronic myeloid leukemia (CML). An interest in the dose-response of radiation induced CML therefore leads naturally to an interest in the dose-response of BCR-ABL formation. To predict the BCR-ABL dose-response to low-linear energy transfer (LET) ionizing radiation, three models valid over three different dose ranges are examined: the first for doses greater than 80 Gy, the second for doses less than 5 Gy and the third for doses greater than 2 Gy. The first of the models, due to Holley and Chatterjee, ignores the accidental binary eurejoining of DNA double-strand break (DSB) free ends ('eurejoining' refers to the accidental restitution of DSB free ends with their own proper mates). As a result, the model is valid only in the limit of high doses. The second model is derived directly from cytogenetic data. This model has the attractive feature that it implicitly accounts for single-track effects at low doses. The third model, based on the Sax-Markov binary eurejoining/misrejoining (SMBE) algorithm, does not account for single-track effects and is therefore limited to moderate doses greater than approximately 2 Gy. Comparing the second model to lifetime excess CML risks expected after 1 Gy, estimates of the number of hematopoietic stem cells capable of causing CML were obtained for male and female atomic bomb survivors in Hiroshima and Nagasaki. The stem cell number estimates lie in the range of 5 x 10(7)-3 x 10(8) cells. PMID:10616282

  2. GENE THERAPY USING RETROVIRAL VECTOR OF bcr-abl SPECIFIC MULTI-UNIT RIBOZYMES COULD INHIBIT CML CELL GROWTH AND INDUCE APOPTOSIS

    Institute of Scientific and Technical Information of China (English)

    冯琦; 孙凯; 赵永同; 张涛; 尚振川; 王莎; 王玮; 赵宁; 颜真; 韩苇; 张英起; 孙秉中

    2003-01-01

    Objective: To investigate the in vitro cleavage ability and effects on apoptosis and cell growth of the bcr-abl fusion gene specific multi-unit ribozymes. Methods: Three fusion point specific ribozymes were designed and the multi-unit ribozymes' in vitro transcription vector and retroviral vector were constructed. The in vitro cleavage ability was tested. The retroviral vector was transfected into K562 cell and the effects on proliferation, apoptosis, cell cycle and cell structure were observed. Results: Multi-unit ribozymes had in vitro cleavage efficiency of 70.8%, which was more efficient than single-unit and double-unit ribozymes. Transfection of the retroviral vector of the ribozyme into K562 cells, induced inhibition of cell growth and apoptosis. The incorporation rate of DNA in ribozymes transfected K562 cells was greatly decreased along with time passed, with an inhibition rate of more than 50% after 96 h of transfection. Under FCM, 18.4% of the cells underwent apoptosis 72 h after transfection and more cells were blocked in G phase, with the ratio in S phase greatly decreased (41.9%). Under electron microscope, compaction of nuclear chromatin and apoptosis bodies were observed.Conclusion: Multi-unit ribozymes specific to bcr-abl fusion gene can be used to treat CML and to purge bone marrow for self-grafting.

  3. Induction of autophagy by Imatinib sequesters Bcr-Abl in autophagosomes and down-regulates Bcr-Abl protein.

    LENUS (Irish Health Repository)

    Elzinga, Baukje M

    2013-06-01

    Chronic Myeloid Leukemia (CML) is a disease of hematopoietic stem cells which harbor the chimeric gene Bcr-Abl. Expression levels of this constitutively active tyrosine kinase are critical for response to tyrosine kinase inhibitor treatment and also disease progression, yet the regulation of protein stability is poorly understood. We have previously demonstrated that imatinib can induce autophagy in Bcr-Abl expressing cells. Autophagy has been associated with the clearance of large macromolecular signaling complexes and abnormal proteins, however, the contribution of autophagy to the turnover of Bcr-Abl protein in imatinib treated cells is unknown. In this study, we show that following imatinib treatment, Bcr-Abl is sequestered into vesicular structures that co-localize with the autophagy marker LC3 or GABARAP. This association is inhibited by siRNA mediated knockdown of autophagy regulators (Beclin 1\\/ATG7). Pharmacological inhibition of autophagy also reduced Bcr-Abl\\/LC3 co-localization in both K562 and CML patient cells. Bcr-Abl protein expression was reduced with imatinib treatment. Inhibition of both autophagy and proteasome activity in imatinib treated cells was required to restore Bcr-Abl protein levels to those of untreated cells. This ability to down-regulate Bcr-Abl protein levels through the induction of autophagy may be an additional and important feature of the activity of imatinib.

  4. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl{sup +} K562 and Jak2(V617F){sup +} HEL Leukemia Cells

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Axel [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany); Borghouts, Corina [Ganymed Pharmaceuticals AG, Mainz 55131 (Germany); Brendel, Christian [Boston Children’s Hospital, Division of Hematology/Oncology, Boston, MA 02115 (United States); Moriggl, Richard [Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna 1090 (Austria); Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd, E-mail: Groner@em.uni-frankfurt.de [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany)

    2015-03-19

    Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl{sup +} K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells

  5. SGX393 inhibits the CML mutant Bcr-Abl[superscript T315I] and preempts in vitro resistance when combined with nilotinib or dasatinib

    Energy Technology Data Exchange (ETDEWEB)

    O' Hare, Thomas; Eide, Christopher A.; Tyner, Jeffrey W.; Corbin, Amie S.; Wong, Matthew J.; Buchanan, Sean; Holme, Kevin; Jessen, Katayoun A.; Tang, Crystal; Lewis, Hal A.; Romero, Richard D.; Burley, Stephen K.; Deininger, Michael W. (OHSU- Cancer Instit.); (SGX)

    2010-01-12

    Imatinib inhibits Bcr-Abl, the oncogenic tyrosine kinase that causes chronic myeloid leukemia. The second-line inhibitors nilotinib and dasatinib are effective in patients with imatinib resistance resulting from Bcr-Abl kinase domain mutations. Bcr-Abl{sup T315I}, however, is resistant to all Abl kinase inhibitors in clinical use and is emerging as the most frequent cause of salvage therapy failure. SGX393 is a potent inhibitor of native and T315I-mutant Bcr-Abl kinase that blocks the growth of leukemia cell lines and primary hematopoietic cells expressing Bcr-Abl{sup T315I}, with minimal toxicity against Bcr-Abl-negative cell lines or normal bone marrow. A screen for Bcr-Abl mutants emerging in the presence of SGX393 revealed concentration-dependent reduction in the number and range of mutations. Combining SGX393 with nilotinib or dasatinib preempted emergence of resistant subclones, including Bcr-Abl{sup T315I}. These findings suggest that combination of a T315I inhibitor with the current clinically used inhibitors may be useful for reduction of Bcr-Abl mutants in Philadelphia chromosome-positive leukemia.

  6. Doxorubicin Differentially Induces Apoptosis, Expression of Mitochondrial Apoptosis-Related Genes, and Mitochondrial Potential in BCR-ABL1-Expressing Cells Sensitive and Resistant to Imatinib

    Directory of Open Access Journals (Sweden)

    Ewelina Synowiec

    2015-01-01

    Full Text Available Imatinib resistance is an emerging problem in the therapy of chronic myeloid leukemia (CML. Because imatinib induces apoptosis, which may be coupled with mitochondria and DNA damage is a prototype apoptosis-inducing factor, we hypothesized that imatinib-sensitive and -resistant CML cells might differentially express apoptosis-related mitochondrially encoded genes in response to genotoxic stress. We investigated the effect of doxorubicin (DOX, a DNA-damaging anticancer drug, on apoptosis and the expression of the mitochondrial NADH dehydrogenase 3 (MT-ND3 and cytochrome b (MT-CYB in model CML cells showing imatinib resistance caused by Y253H mutation in the BCR-ABL1 gene (253 or culturing imatinib-sensitive (S cells in increasing concentrations of imatinib (AR. The imatinib-resistant 253 cells displayed higher sensitivity to apoptosis induced by 1 μM DOX and this was confirmed by an increased activity of executioner caspases 3 and 7 in those cells. Native mitochondrial potential was lower in imatinib-resistant cells than in their sensitive counterparts and DOX lowered it. MT-CYB mRNA expression in 253 cells was lower than that in S cells and 0.1 μM DOX kept this relationship. In conclusion, imatinib resistance may be associated with altered mitochondrial response to genotoxic stress, which may be further exploited in CML therapy in patients with imatinib resistance.

  7. Association of HLA Class I and Class II genes with bcr-abl transcripts in leukemia patients with t(9;22 (q34;q11

    Directory of Open Access Journals (Sweden)

    Cano Pedro

    2004-06-01

    Full Text Available Abstract Background Based on the site of breakpoint in t(9;22 (q34;q11, bcr-abl fusion in leukemia patients is associated with different types of transcript proteins. In this study we have seen the association of HLA genes with different types of bcr-abl transcripts. The association could predict the bcr-abl peptide presentation by particular HLA molecules. Methods The study included a total of 189 patients of mixed ethnicity with chronic myelogenous leukemia and acute lymphocytic leukemia who were being considered for bone marrow transplantation. Typing of bcr-abl transcripts was done by reverse transcriptase PCR method. HLA typing was performed by molecular methods. The bcr-abl and HLA association was studied by calculating the relative risks and chi-square test. Results Significant negative associations (p Conclusions The negative associations of a particular bcr-abl transcript with specific HLA alleles suggests that these alleles play a critical role in presenting peptides derived from the chimeric proteins and eliciting a successful T-cell cytotoxic response. Knowledge of differential associations between HLA phenotypes and bcr-abl fusion transcript types would help in developing better strategies for immunization with the bcr-abl peptides against t(9;22 (q34;q11-positive leukemia.

  8. Nilotinib treatment in mouse models of P190 Bcr/Abl lymphoblastic leukemia

    Directory of Open Access Journals (Sweden)

    Groffen John

    2007-10-01

    Full Text Available Abstract Background Ph-positive leukemias are caused by the aberrant fusion of the BCR and ABL genes. Nilotinib is a selective Bcr/Abl tyrosine kinase inhibitor related to imatinib, which is widely used to treat chronic myelogenous leukemia. Because Ph-positive acute lymphoblastic leukemia only responds transiently to imatinib therapy, we have used mouse models to test the efficacy of nilotinib against lymphoblastic leukemia caused by the P190 form of Bcr/Abl. Results After transplant of 10,000 highly malignant leukemic cells into compatible recipients, untreated mice succumbed to leukemia within 21 days, whereas mice treated with 75 mg/kg nilotinib survived significantly longer. We examined cells from mice that developed leukemia while under treatment for Bcr/Abl kinase domain point mutations but these were not detected. In addition, culture of such cells ex vivo showed that they were as sensitive as the parental cell line to nilotinib but that the presence of stromal support allowed resistant cells to grow out. Nilotinib also exhibited impressive anti-leukemia activity in P190 Bcr/Abl transgenic mice that had developed overt leukemia/lymphoma masses and that otherwise would have been expected to die within 7 days. Visible lymphoma masses disappeared within six days of treatment and leukemic cell numbers in peripheral blood were significantly reduced. Treated mice survived more than 30 days. Conclusion These results show that nilotinib has very impressive anti-leukemia activity but that lymphoblastic leukemia cells can become unresponsive to it both in vitro and in vivo through mechanisms that appear to be Bcr/Abl independent.

  9. MPT0B169, a New Antitubulin Agent, Inhibits Bcr-Abl Expression and Induces Mitochondrion-Mediated Apoptosis in Nonresistant and Imatinib-Resistant Chronic Myeloid Leukemia Cells.

    Science.gov (United States)

    Wong, Shuit-Mun; Liu, Fu-Hwa; Lee, Yueh-Lun; Huang, Huei-Mei

    2016-01-01

    Chronic myeloid leukemia (CML) is a clonal disorder of hematopoietic stem/progenitor cells that is caused by the Bcr-Abl oncoprotein. Clinical resistance to the Bcr-Abl inhibitor imatinib is a critical problem in treating CML. This study investigated the antitumor effect and mechanism of MPT0B169, a new antitubulin agent, in K562 CML cells and their derived imatinib-resistant cells, IMR2 and IMR3. IMR2 and IMR3 cells showed complete resistance to imatinib-induced growth inhibition and apoptosis. Resistance involved ERK1/2 overactivation and MDR1 overexpression. MPT0B169 inhibited the growth of K562, IMR2, and IMR3 cells in a dose- and time-dependent manner. MPT0B169 substantially inhibited the mRNA and protein levels of Bcr-Abl, followed by its downstream pathways including Akt, ERK1/2, and STAT3 in these cells. MPT0B169 treatment resulted in a decrease in the polymer form of tubulin according to Western blot analysis. It triggered cell cycle arrest at the G2/M phase before apoptosis, which was related to the upregulation of the mitotic marker MPM2 and the cyclin B1 level, and a change in the phosphorylation of Cdk1. MPT0B169 induced apoptosis in nonresistant and imatinib-resistant cells via a mitochondrion-mediated caspase pathway. Further study showed that the agent led to a decrease in the antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 and an increase in the apoptotic protein Bax. Taken together, our results suggest that MPT0B169 might be a promising agent for overcoming imatinib resistance in CML cells. PMID:26815740

  10. MPT0B169, a New Antitubulin Agent, Inhibits Bcr-Abl Expression and Induces Mitochondrion-Mediated Apoptosis in Nonresistant and Imatinib-Resistant Chronic Myeloid Leukemia Cells.

    Directory of Open Access Journals (Sweden)

    Shuit-Mun Wong

    Full Text Available Chronic myeloid leukemia (CML is a clonal disorder of hematopoietic stem/progenitor cells that is caused by the Bcr-Abl oncoprotein. Clinical resistance to the Bcr-Abl inhibitor imatinib is a critical problem in treating CML. This study investigated the antitumor effect and mechanism of MPT0B169, a new antitubulin agent, in K562 CML cells and their derived imatinib-resistant cells, IMR2 and IMR3. IMR2 and IMR3 cells showed complete resistance to imatinib-induced growth inhibition and apoptosis. Resistance involved ERK1/2 overactivation and MDR1 overexpression. MPT0B169 inhibited the growth of K562, IMR2, and IMR3 cells in a dose- and time-dependent manner. MPT0B169 substantially inhibited the mRNA and protein levels of Bcr-Abl, followed by its downstream pathways including Akt, ERK1/2, and STAT3 in these cells. MPT0B169 treatment resulted in a decrease in the polymer form of tubulin according to Western blot analysis. It triggered cell cycle arrest at the G2/M phase before apoptosis, which was related to the upregulation of the mitotic marker MPM2 and the cyclin B1 level, and a change in the phosphorylation of Cdk1. MPT0B169 induced apoptosis in nonresistant and imatinib-resistant cells via a mitochondrion-mediated caspase pathway. Further study showed that the agent led to a decrease in the antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 and an increase in the apoptotic protein Bax. Taken together, our results suggest that MPT0B169 might be a promising agent for overcoming imatinib resistance in CML cells.

  11. Association of HLA antigens and BCR-ABL transcripts in leukemia patients with the Philadelphia chromosome

    Directory of Open Access Journals (Sweden)

    Daiana Landenberger de Carvalho

    2012-01-01

    Full Text Available OBJECTIVE: This study aimed to verify the association between human leukocyte antigens and the bcr-abl fusion protein resulting from t(9;22(q34;q11 in chronic leukemia myeloid and acute lymphoblastic leukemia patients. METHODS: Forty-seven bcr-abl positive individuals were evaluated. Typing was performed bymicrolymphocytotoxicity and molecular biological methods (human leukocyte antigens Class I and Class II. A control group was obtained from the data of potential bone marrow donors registered in the Brazilian Bone Marrow Donor Registry (REDOME. RESULTS: Positive associations with HLA-A25 and HLA-B18 were found for the b2a2 transcript, as well as a tendency towards a positive association with HLA-B40 and a negative association with HLA-A68. The b3a2 transcript showed positive associations with HLA-B40 and HLA-DRB1*3. CONCLUSION: The negative association between human leukocyte antigens and the BCR-ABL transcript suggests that binding and presentation of peptides derived from the chimeric protein are effective to increase a cytotoxic T lymphocyte response appropriate for the destruction of leukemic cells.

  12. Association of HLA Class I and Class II genes with bcr-abl transcripts in leukemia patients with t(9;22) (q34;q11)

    International Nuclear Information System (INIS)

    Based on the site of breakpoint in t(9;22) (q34;q11), bcr-abl fusion in leukemia patients is associated with different types of transcript proteins. In this study we have seen the association of HLA genes with different types of bcr-abl transcripts. The association could predict the bcr-abl peptide presentation by particular HLA molecules. The study included a total of 189 patients of mixed ethnicity with chronic myelogenous leukemia and acute lymphocytic leukemia who were being considered for bone marrow transplantation. Typing of bcr-abl transcripts was done by reverse transcriptase PCR method. HLA typing was performed by molecular methods. The bcr-abl and HLA association was studied by calculating the relative risks and chi-square test. Significant negative associations (p < 0.05) were observed with HLA-A*02 (b2a2, e1a2), -A*68 (b2a2, b3a2, e1a2), -B*14 (b2a2, b3a2, e1a2), -B*15 (b2a2, b3a2), -B*40 (b2a2), -DQB1*0303 (b2a2, b3a2), -DQB1*0603 (b2a2), -DRB1*0401 (e1a2), -DRB1*0701 (b3a2), and -DRB1*1101 (b2a2). The negative associations of a particular bcr-abl transcript with specific HLA alleles suggests that these alleles play a critical role in presenting peptides derived from the chimeric proteins and eliciting a successful T-cell cytotoxic response. Knowledge of differential associations between HLA phenotypes and bcr-abl fusion transcript types would help in developing better strategies for immunization with the bcr-abl peptides against t(9;22) (q34;q11)-positive leukemia

  13. WT1-mediated repression of the proapoptotic transcription factor ZNF224 is triggered by the BCR-ABL oncogene.

    Science.gov (United States)

    Montano, Giorgia; Vidovic, Karina; Palladino, Chiara; Cesaro, Elena; Sodaro, Gaetano; Quintarelli, Concetta; De Angelis, Biagio; Errichiello, Santa; Pane, Fabrizio; Izzo, Paola; Grosso, Michela; Gullberg, Urban; Costanzo, Paola

    2015-09-29

    The Kruppel-like protein ZNF224 is a co-factor of the Wilms' tumor 1 protein, WT1. We have previously shown that ZNF224 exerts a specific proapoptotic role in chronic myelogenous leukemia (CML) K562 cells and contributes to cytosine arabinoside-induced apoptosis, by modulating WT1-dependent transcription of apoptotic genes. Here we demonstrate that ZNF224 gene expression is down-regulated both in BCR-ABL positive cell lines and in primary CML samples and is restored after imatinib and second generation tyrosine kinase inhibitors treatment. We also show that WT1, whose expression is positively regulated by BCR-ABL, represses transcription of the ZNF224 gene. Finally, we report that ZNF224 is significantly down-regulated in patients with BCR-ABL positive chronic phase-CML showing poor response or resistance to imatinib treatment as compared to high-responder patients. Taken as a whole, our data disclose a novel pathway activated by BCR-ABL that leads to inhibition of apoptosis through the ZNF224 repression. ZNF224 could thus represent a novel promising therapeutic target in CML. PMID:26320177

  14. Essential role for telomerase in chronic myeloid leukemia induced by BCR-ABL in mice

    OpenAIRE

    Vicente-Dueñas, Carolina; Barajas-Diego, Marcos; Romero-Camarero, Isabel; González-Herrero, Inés; Flores, Teresa; Sánchez García, Isidro

    2012-01-01

    The telomerase protein is constitutively activated in malignant cells from many patients with cancer, including the chronic myeloid leukemia (CML), but whether telomerase is essential for the pathogenesis of this disease is not known. Here, we used telomerase deficient mice to determine the requirement for telomerase in CML induced by BCR-ABL in mouse models of CML. Loss of one telomerase allele or complete deletion of telomerase prevented the development of leukemia induced by BCR-ABL. Howev...

  15. A Requirement for SOCS-1 and SOCS-3 Phosphorylation in Bcr-Abl-Induced Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Xiaoxue Qiu

    2012-06-01

    Full Text Available Suppressors of cytokine signaling 1 and 3 (SOCS-1 and SOCS-3 are inhibitors of the Janus tyrosine kinase (JAK/signal transducers and activators of transcription (STAT pathway and function in a negative feedback loop during cytokine signaling. Abl transformation is associated with constitutive activation of JAK/STAT-dependent signaling. However, the mechanism by which Abl oncoproteins bypass SOCS inhibitory regulation remains poorly defined. Here, we demonstrate that coexpression of Bcr-Abl with SOCS-1 or SOCS-3 results in tyrosine phosphorylation of these SOCS proteins. Interestingly, SOCS-1 is highly tyrosine phosphorylated in one of five primary chronic myelogenous leukemia samples. Bcr-Abl-dependent tyrosine phosphorylation of SOCS-1 and SOCS-3 occurs mainly on Tyr 155 and Tyr 204 residues of SOCS-1 and on Tyr 221 residue of SOCS-3. We observed that phosphorylation of these SOCS proteins was associated with their binding to Bcr-Abl. Bcr-Abl-dependent phosphorylation of SOCS-1 and SOCS-3 diminished their inhibitory effects on the activation of JAK and STAT5 and thereby enhanced JAK/STAT5 signaling. Strikingly, disrupting the tyrosine phosphorylation of SOCS-1 or SOCS-3 impaired the expression of Bcl-XL protein and sensitized K562 leukemic cells to undergo apoptosis. Moreover, selective mutation of tyrosine phosphorylation sites of SOCS-1 or SOCS-3 significantly blocked Bcr-Abl-mediated tumorigenesis in nude mice and inhibited Bcr-Abl-mediated murine bone marrow transformation. Together, these results reveal a mechanism of how Bcr-Abl may overcome SOCS-1 and SOCS-3 inhibition to constitutively activate the JAK/STAT-dependent signaling, and suggest that Bcr-Abl may critically requires tyrosine phosphorylation of SOCS-1 and SOCS-3 to mediate tumorigenesis when these SOCS proteins are present in cells.

  16. Molecular Detection of BCR-ABL in Chronic Myeloid Leukemia.

    Science.gov (United States)

    Qin, Ya-Zhen; Huang, Xiao-Jun

    2016-01-01

    All chronic myeloid leukemia (CML) patients have the BCR-ABL fusion gene. The constitutively activated BCR-ABL tyrosine kinase is a critical pathogenetic event in CML. Tyrosine kinase inhibitors (TKIs), such as imatinib, are synthesized small molecules that primarily target BCR-ABL tyrosine kinases and have become a first-line treatment for CML. Detection of BCR-ABL transcript level by real-time quantitative polymerase chain reaction (RQ-PCR) is a clinical routine for evaluating TKI treatment efficacy and predicting long-term response. Furthermore, because they are a main TKI resistance mechanism, the BCR-ABL tyrosine kinase domain (TKD) point mutations that are detected by Sanger sequencing can help clinicians make decisions on subsequent treatment selections. Here, we present protocols for the two abovementioned molecular methods for CML analysis. PMID:27581134

  17. Outcomes of Children With BCR-ABL1–Like Acute Lymphoblastic Leukemia Treated With Risk-Directed Therapy Based on the Levels of Minimal Residual Disease

    Science.gov (United States)

    Roberts, Kathryn G.; Pei, Deqing; Campana, Dario; Payne-Turner, Debbie; Li, Yongjin; Cheng, Cheng; Sandlund, John T.; Jeha, Sima; Easton, John; Becksfort, Jared; Zhang, Jinghui; Coustan-Smith, Elaine; Raimondi, Susana C.; Leung, Wing H.; Relling, Mary V.; Evans, William E.; Downing, James R.; Mullighan, Charles G.; Pui, Ching-Hon

    2014-01-01

    Purpose BCR-ABL1–like acute lymphoblastic leukemia (ALL) is a recently identified B-cell ALL (B-ALL) subtype with poor outcome that exhibits a gene expression profile similar to BCR-ABL1-positive ALL but lacks the BCR-ABL1 fusion protein. We examined the outcome of children with BCR-ABL1–like ALL treated with risk-directed therapy based on minimal residual disease (MRD) levels during remission induction. Patients and Methods Among 422 patients with B-ALL enrolled onto the Total Therapy XV study between 2000 and 2007, 344 had adequate samples for gene expression profiling. Next-generation sequencing and/or analysis of genes known to be altered in B-ALL were performed in patients with BCR-ABL1–like ALL who had available material. Outcome was compared between patients with and those without BCR-ABL1–like ALL. Results Forty (11.6%) of the 344 patients had BCR-ABL1–like ALL. They were significantly more likely to be male, have Down syndrome, and have higher MRD levels on day 19 and at the end of induction than did other patients with B-ALL. Among 25 patients comprehensively studied for genetic abnormalities, 11 harbored a genomic rearrangement of CRLF2, six had fusion transcripts responsive to ABL tyrosine kinase inhibitors or JAK inhibitors, and seven had mutations involving the Ras signaling pathway. There were no significant differences in event-free survival (90.0% ± 4.7% [SE] v 88.4% ± 1.9% at 5 years; P = .41) or in overall survival (92.5% ± 4.2% v 95.1% ± 1.3% at 5 years; P = .41) between patients with and without BCR-ABL1–like ALL. Conclusion Patients who have BCR-ABL1–like ALL with poor initial treatment response can be salvaged with MRD-based risk-directed therapy and may benefit from identification of kinase-activating lesions for targeted therapies. PMID:25049327

  18. The Study on BCR/ABL Fusion Gene in Adult Acute Lymphoblastic Leukemia

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to assess the significance of BCR/ABC fusion gene in adult acute lymphoblastic Leukemia (ALL), 28 patients who were diagnosed as ALL were enrolled to detect BCR/ABC gene using nested-RT PCR. The results showed that 9 cases (31.25%) were BCR/ABL positive ,and expressed P210 subtype. A mong them 7 cases were B-ALL, and one was T-ALL. The diagnosis was proved by monoclonal antibodies recognition by indirect immunofluorescence. Adult patients with BCR/ABL positive ALL were significantly older (p<0. 01) and had higher WBC count (p<0. 01) as compared with BCR/ABL-negative patients. There was no significant difference in sex, hemoglobin and splenomegaly between two group (p>0. 05). The induc tion failure rate was high in BCR/ABL positive patients and those who achieved complete remission usually relapsed earlier. In conclusion, adult ALL patients with BCR/ABL-positive have poorer prognosis.

  19. Expression of p89c-Mybex9b, an alternatively spliced form of c-Myb, is required for proliferation and survival of p210BCR/ABL-expressing cells

    International Nuclear Information System (INIS)

    The c-Myb gene encodes the p75c-Myb isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is pc-Mybex9b, which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein–protein interactions and negative regulation. In hematopoietic cells, expression of pc-Mybex9b accounts for 10–15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of pc-Mybex9b and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75c-Myb, pc-Mybex9b is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of pc-Mybex9b enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of pc-Mybex9b reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34+ cells, without affecting the levels of p75c-Myb. Together, these studies indicate that expression of the low-abundance pc-Mybex9b isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells

  20. BCR-ABL Promotes PTEN Downregulation in Chronic Myeloid Leukemia

    OpenAIRE

    Cristina Panuzzo; Sabrina Crivellaro; Giovanna Carrà; Angelo Guerrasio; Giuseppe Saglio; Alessandro Morotti

    2014-01-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the t(9;22) translocation coding for the chimeric protein p210 BCR-ABL. The tumor suppressor PTEN plays a critical role in the pathogenesis of CML chronic phase, through non genomic loss of function mechanisms, such as protein down-regulation and impaired nuclear/cytoplasmic shuttling. Here we demonstrate that BCR-ABL promotes PTEN downregulation through a MEK dependent pathway. Furthermore, we describe a novel n...

  1. Targeting the SH2-Kinase Interface in Bcr-Abl Inhibits Leukemogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Grebien, Florian; Hantschel, Oliver; Wojcik, John; Kaupe, Ines; Kovacic, Boris; Wyrzucki, Arkadiusz M.; Gish, Gerald D.; Cerny-Reiterer, Sabine; Koide, Akiko; Beug, Hartmut; Pawson, Tony; Valent, Peter; Koide, Shohei; Superti-Furga, Giulio (AAS); (Mount Sinai Hospital); (Med U. Vienna); (UC); (IMP-CNRS)

    2012-10-25

    Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention.

  2. BCR-ABL Translocation in Pediatric Acute Lymphoblastic Leukemia in Southern India.

    Science.gov (United States)

    Sugapriya, D; Preethi, S; Shanthi, P; Chandra, N; Jeyaraman, G; Sachdanandam, P; Thilagavathy, S; Venkatadesilalu, S

    2012-03-01

    Cytogenetics and polymerase chain reaction (PCR) based assays provide important information regarding biologically defined and prognostically relevant subgroups in acute leukemias. We utilized karyotyping and molecular analysis by reverse transcriptase PCR for the BCR-ABL translocation, in addition to morphological study, cytochemistry and immunophenotyping, to study 24 cases of pediatric acute lymphoblastic leukemia (ALL). Our objective was to determine the frequency of the BCRABL translocation in childhood ALL from southern India. Karyotyping showed one case of hyperdiploidy, one case of t (12; 21) translocation and one case of 46, XY-21+mar. The BCR-ABL translocation was found in 8.3% of these cases. One of these was a cryptic translocation, the karyotype being normal. BCR-ABL positivity in ALL is associated with aggressive disease and has been shown to be a poor prognostic factor, especially in children. PMID:23449388

  3. MicroRNA-320a acts as a tumor suppressor by targeting BCR/ABL oncogene in chronic myeloid leukemia.

    Science.gov (United States)

    Xishan, Zhu; Ziying, Lin; Jing, Du; Gang, Liu

    2015-01-01

    Accumulating evidences demonstrated that the induction of epithelial-mesenchymal transition (EMT) and aberrant expression of microRNAs (miRNAs) are associated with tumorigenesis, tumor progression, metastasis and relapse in cancers, including chronic myeloid leukemia (CML). We found that miR-320a expression was reduced in K562 and in CML cancer stem cells. Moreover, we found that miR-320a inhibited K562 cell migration, invasion, proliferation and promoted apoptosis by targeting BCR/ABL oncogene. As an upstream regulator of BCR/ABL, miR-320a directly targets BCR/ABL. The enhanced expression of miR-320a inhibited the phosphorylation of PI3K, AKT and NF-κB; however, the expression of phosphorylated PI3K, AKT and NF-κB were restored by the overexpression of BCR/ABL. In K562, infected with miR-320a or transfected with SiBCR/ABL, the protein levels of fibronectin, vimentin, and N-cadherin were decreased, but the expression of E-cadherin was increased. The expression of mesenchymal markers in miR-320a-expressing cells was restored to normal levels by the restoration of BCR/ABL expression. Generally speaking, miR-320a acts as a novel tumor suppressor gene in CML and miR-320a can decrease migratory, invasive, proliferative and apoptotic behaviors, as well as CML EMT, by attenuating the expression of BCR/ABL oncogene.

  4. A BCR/ABL-hIL-2 DNA Vaccine Enhances the Immune Responses in BALB/c Mice

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    Yanan Qin

    2013-01-01

    Full Text Available The use of a DNA vaccine encoding the BCR/ABL fusion gene is thought to be a promising approach for patients with chronic myeloid leukemia (CML to eradicate minimal residual disease after treatment with chemotherapy or targeted therapy. In this study, our strategy employs genetic technology to create a DNA vaccine encoding the BCR/ABL fusion and human interleukin-2 (hIL-2 genes. The successfully constructed plasmids BCR/ABL-pIRES-hIL-2, BCR/ABL-pIRES, and pIRES-hIL-2 were delivered intramuscularly to BALB/c mice at 14-day intervals for three cycles. The transcription and expression of the BCR/ABL and hIL-2 genes were found in the injected muscle tissues. The interferon-γ (IFN-γ serum levels were increased, and the splenic CD4+/CD8+ T cell ratio was significantly decreased in the BCR/ABL-pIRES-hIL-2-injected mice. Furthermore, specific antibodies against K562 cells could be detected by indirect immunofluorescence. These results indicate that a DNA vaccine containing BCR/ABL and hIL-2 together may elicit increased in vivo humoral and cellular immune responses in BALB/c mice.

  5. BCR-ABL DERIVED PEPTIDE VACCINES FOR CHRONIC MYELOID LEUKAEMIA

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    M. Bocchia

    2012-01-01

    Full Text Available Chronic Myeloid Leukemia (CML is a myeloproliferative pluripotent stem cell disorder characterized by the presence of a cytogenetic hallmark, the Philadelphia (Ph chromosome, and accounts for 15% of adult leukemias. The disease progresses from a chronic phase through an accelerated phase to a blast phase and its natural course accounts for a median 4 years survival1. The Ph chromosome is derived by a reciprocal translocation termed t(9;22 in which the c-abl oncogene has moved from chromosome 9 into the breakpoint cluster region (bcr, within the bcr gene on chromosome 22, resulting in a chimeric bcr-abl fusion gene that encodes a 210 KD protein (p210 with constitutive tyrosine kinase activity. Two major alternative chimeric p210 can result from this fusion gene: p210-b2a2 where the junction occurs between bcr exon 2 (b2 and abl exon 2 (a2 and p210-b3a2 where the the junction occurs between bcr exon 3 (b3 and abl exon 2 (a2. About 40% of CML patients harbor the p210-b2a2 and about 60% of them show the p210-b3a2.

  6. AP24534, a Pan-BCR-ABL Inhibitor for Chronic Myeloid Leukemia, Potently Inhibits the T315I Mutant and Overcomes Mutation-Based Resistance

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    O’Hare, Thomas; Shakespeare, William C.; Zhu, Xiaotian; Eide, Christopher A.; Rivera, Victor M.; Wang, Frank; Adrian, Lauren T.; Zhou, Tianjun; Huang, Wei-Sheng; Xu, Qihong; Metcalf, III, Chester A.; Tyner, Jeffrey W.; Loriaux, Marc M.; Corbin, Amie S.; Wardwell, Scott; Ning, Yaoyu; Keats, Jeffrey A.; Wang, Yihan; Sundaramoorthi, Raji; Thomas, Mathew; Zhou, Dong; Snodgrass, Joseph; Commodore, Lois; Sawyer, Tomi K.; Dalgarno, David C.; Deininger, Michael W.N.; Druker, Brian J.; Clackson, Tim; (OHSU- Cancer Instit.); (ARIAD)

    2010-09-07

    Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL{sup T315I} mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL{sup T315I}-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

  7. A critical role of CDKN3 in Bcr-Abl-mediated tumorigenesis.

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    Qinghuang Chen

    Full Text Available CDKN3 (cyclin-dependent kinase inhibitor 3, a dual specificity protein phosphatase, dephosphorylates cyclin-dependent kinases (CDKs and thus functions as a key negative regulator of cell cycle progression. Deregulation or mutations of CDNK3 have been implicated in various cancers. However, the role of CDKN3 in Bcr-Abl-mediated chronic myelogenous leukemia (CML remains unknown. Here we found that CDKN3 acts as a tumor suppressor in Bcr-Abl-mediated leukemogenesis. Overexpression of CDKN3 sensitized the K562 leukemic cells to imanitib-induced apoptosis and dramatically inhibited K562 xenografted tumor growth in nude mouse model. Ectopic expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence. In contrast, depletion of CDKN3 expression conferred resistance to imatinib-induced apoptosis in the leukemic cells and accelerated the growth of xenograph leukemia in mice. In addition, we found that CDKN3 mutant (CDKN3-C140S devoid of the phosphatase activity failed to affect the K562 leukemic cell survival and xenografted tumor growth, suggesting that the phosphatase of CDKN3 was required for its tumor suppressor function. Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression. Moreover, overexpression of CDKN3 delayed G1/S transition in K562 leukemic cells. Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.

  8. Identification of drug combinations containing imatinib for treatment of BCR-ABL+ leukemias.

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    Yunyi Kang

    Full Text Available The BCR-ABL translocation is found in chronic myeloid leukemia (CML and in Ph+ acute lymphoblastic leukemia (ALL patients. Although imatinib and its analogues have been used as front-line therapy to target this mutation and control the disease for over a decade, resistance to the therapy is still observed and most patients are not cured but need to continue the therapy indefinitely. It is therefore of great importance to find new therapies, possibly as drug combinations, which can overcome drug resistance. In this study, we identified eleven candidate anti-leukemic drugs that might be combined with imatinib, using three approaches: a kinase inhibitor library screen, a gene expression correlation analysis, and literature analysis. We then used an experimental search algorithm to efficiently explore the large space of possible drug and dose combinations and identified drug combinations that selectively kill a BCR-ABL+ leukemic cell line (K562 over a normal fibroblast cell line (IMR-90. Only six iterations of the algorithm were needed to identify very selective drug combinations. The efficacy of the top forty-nine combinations was further confirmed using Ph+ and Ph- ALL patient cells, including imatinib-resistant cells. Collectively, the drug combinations and methods we describe might be a first step towards more effective interventions for leukemia patients, especially those with the BCR-ABL translocation.

  9. BCR-ABL1 kinase inhibits uracil DNA glycosylase UNG2 to enhance oxidative DNA damage and stimulate genomic instability

    Science.gov (United States)

    Slupianek, Artur; Falinski, Rafal; Znojek, Pawel; Stoklosa, Tomasz; Flis, Sylwia; Doneddu, Valentina; Pytel, Dariusz; Synowiec, Ewelina; Blasiak, Janusz; Bellacosa, Alfonso; Skorski, Tomasz

    2013-01-01

    Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of CML-CP. Unfortunately, 25% of TKI-naive patients and 50–90% of TKI-responding patients carry CML clones expressing TKI resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen species (ROS), which may result in accumulation of uracil derivatives in genomic DNA. Unfaithful and/or inefficient repair of these lesions generates TKI resistant point mutations in BCR-ABL1 kinase. Using an array of specific substrates and inhibitors/blocking antibodies we found that uracil-DNA glycosylase UNG2 were inhibited in BCR-ABL1 –transformed cell lines and CD34+ CML cells. The inhibitory effect was not accompanied by downregulation of nuclear expression and/or chromatin association of UNG2. The effect was BCR-ABL1 kinase-specific because several other fusion tyrosine kinases did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI we found that reduction of UNG2 activity increased the number of uracil derivatives in genomic DNA detected by modified comet assay and facilitated accumulation of ouabain-resistant point mutations in reporter gene Na+/K+ATPase. In conclusion, we postulate that BCR-ABL1 kinase-mediated inhibition of UNG2 contributes to accumulation of point mutations responsible for TKI-resistance causing the disease relapse, and perhaps also other point mutations facilitating malignant progression of CML. PMID:23047475

  10. A target-disease network model of second-generation BCR-ABL inhibitor action in Ph+ ALL.

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    Uwe Rix

    Full Text Available Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL is in part driven by the tyrosine kinase bcr-abl, but imatinib does not produce long-term remission. Therefore, second-generation ABL inhibitors are currently in clinical investigation. Considering different target specificities and the pronounced genetic heterogeneity of Ph+ ALL, which contributes to the aggressiveness of the disease, drug candidates should be evaluated with regard to their effects on the entire Ph+ ALL-specific signaling network. Here, we applied an integrated experimental and computational approach that allowed us to estimate the differential impact of the bcr-abl inhibitors nilotinib, dasatinib, Bosutinib and Bafetinib. First, we determined drug-protein interactions in Ph+ ALL cell lines by chemical proteomics. We then mapped those interactions along with known genetic lesions onto public protein-protein interactions. Computation of global scores through correlation of target affinity, network topology, and distance to disease-relevant nodes assigned the highest impact to dasatinib, which was subsequently confirmed by proliferation assays. In future, combination of patient-specific genomic information with detailed drug target knowledge and network-based computational analysis should allow for an accurate and individualized prediction of therapy.

  11. Distinct GAB2 signaling pathways are essential for myeloid and lymphoid transformation and leukemogenesis by BCR-ABL1.

    Science.gov (United States)

    Gu, Shengqing; Chan, Wayne W; Mohi, Golam; Rosenbaum, Joel; Sayad, Azin; Lu, Zhibin; Virtanen, Carl; Li, Shaoguang; Neel, Benjamin G; Van Etten, Richard A

    2016-04-01

    Tyrosine kinase inhibitors (TKIs) directed against BCR-ABL1, the product of the Philadelphia (Ph) chromosome, have revolutionized treatment of patients with chronic myeloid leukemia (CML). However, acquired resistance to TKIs is a significant clinical problem in CML, and TKI therapy is much less effective against Ph(+)B-cell acute lymphoblastic leukemia (B-ALL). BCR-ABL1, via phosphorylated Tyr177, recruits the adapter GRB2-associated binding protein 2 (GAB2) as part of a GRB2/GAB2 complex. We showed previously that GAB2 is essential for BCR-ABL1-evoked myeloid transformation in vitro. Using a genetic strategy and mouse models of CML and B-ALL, we show here that GAB2 is essential for myeloid and lymphoid leukemogenesis by BCR-ABL1. In the mouse model, recipients of BCR-ABL1-transducedGab2(-/-)bone marrow failed to develop CML-like myeloproliferative neoplasia. Leukemogenesis was restored by expression of GAB2 but not by GAB2 mutants lacking binding sites for its effectors phosphatidylinositol 3-kinase (PI3K) or SRC homology 2-containing phosphotyrosine phosphatase 2 (SHP2). GAB2 deficiency also attenuated BCR-ABL1-induced B-ALL, but only the SHP2 binding site was required. The SHP2 and PI3K binding sites were differentially required for signaling downstream of GAB2. Hence, GAB2 transmits critical transforming signals from Tyr177 to PI3K and SHP2 for CML pathogenesis, whereas only the GAB2-SHP2 pathway is essential for lymphoid leukemogenesis. Given that GAB2 is dispensable for normal hematopoiesis, GAB2 and its effectors PI3K and SHP2 represent promising targets for therapy in Ph(+)hematologic neoplasms. PMID:26773044

  12. Expression patterns of WT-1 and Bcr-Abl measured by TaqMan quantitative real-time RT-PCR during follow-up of leukemia patients with the Ph chromosome

    Institute of Scientific and Technical Information of China (English)

    CHEN Zi-xing陈子兴; Jaspal Kaeda; Sue Saunders; John M Goldman

    2004-01-01

    Background This study was designed to quantitatively measure WT-1 expression levels in patients with chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) during follow-up and to clarify the value of WT-1 as a molecular marker in minimal residual disease monitoring.Methods The TaqMan quantitative real-time RT-PCR method was established by using cloned WT-1 cDNA or synthesized oligonucleotides resembling WT-1 cDNA fragments in limit dilution as template until a stable and reliable standard curve was obtained. In a 25-month follow-up, the transcriptional levels of WT-1, Bcr-Abl, and Abl gene, were quantitatively measured in bone marrow cells from 25 CML or acute lymphoblastic leukemia (ALL) patients with the Ph chromosome. In addition, the expression of these genes in 40 samples of normal peripheral blood was also examined using the same method. The ratios of WT-1/Abl and Bcr-Abl/Abl were both plotted, and the two expression patterns were compared as well as their clinical significance.Results The levels of WT-1 expression in normal peripheral blood were detectable. In CML and Ph positive ALL patients, WT-1 expression levels changed in parallel with the Bcr-Abl expression pattern as the disease progressed or responded to effective treatment.Conclusion WT-1 expression provides a novel molecular marker in addition to Bcr-Abl for monitoring minimal residual disease (MRD) and targeting therapy in Ph chromosome-positive leukemia patients.

  13. A multiple reaction monitoring (MRM method to detect Bcr-Abl kinase activity in CML using a peptide biosensor.

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    Tzu-Yi Yang

    Full Text Available The protein kinase Bcr-Abl plays a major role in the pathogenesis of chronic myelogenous leukemia (CML, and is the target of the breakthrough drug imatinib (Gleevec™. While most patients respond well to imatinib, approximately 30% never achieve remission or develop resistance within 1-5 years of starting imatinib treatment. Evidence from clinical studies suggests that achieving at least 50% inhibition of a patient's Bcr-Abl kinase activity (relative to their level at diagnosis is associated with improved patient outcomes, including reduced occurrence of resistance and longer maintenance of remission. Accordingly, sensitive assays for detecting Bcr-Abl kinase activity compatible with small amounts of patient material are desirable as potential companion diagnostics for imatinib. Here we report the detection of Bcr-Abl activity and inhibition by imatinib in the human CML cell line K562 using a cell-penetrating peptide biosensor and multiple reaction monitoring (MRM on a triple quadrupole mass spectrometer. MRM enabled reproducible, selective detection of the peptide biosensor at fmol levels from aliquots of cell lysate equivalent to ~15,000 cells. This degree of sensitivity will facilitate the miniaturization of the entire assay procedure down to cell numbers approaching 15,000, making it practical for translational applications in patient cells in which the limited amount of available patient material often presents a major challenge.

  14. Prospective molecular monitoring of BCR/ABL transcript in children with Ph+ acute lymphoblastic leukaemia unravels differences in treatment response.

    Science.gov (United States)

    Cazzaniga, Giovanni; Lanciotti, Marina; Rossi, Vincenzo; Di Martino, Daniela; Aricò, Maurizio; Valsecchi, Maria Grazia; Basso, Giuseppe; Masera, Giuseppe; Micalizzi, Concetta; Biondi, Andrea

    2002-11-01

    Children with Philadelphia-chromosome-positive (Ph+) acute lymphoblastic leukaemia (ALL) represent a subgroup at very high risk for treatment failure, despite intensive chemotherapy. However, recent retrospective studies showed that Ph+ childhood ALL is a heterogeneous disease with regard to treatment response. We have prospectively monitored, by reverse transcription polymerase chain reaction (RT-PCR) during follow-up, the presence of the BCR/ABL fusion transcript in Ph+ ALL children diagnosed in the Italian multicentre Associazione Italiana Ematologia Oncologia Pediatrica ALL-AIEOP-95 therapy protocol. To our knowledge, this is the first report on the evaluation of minimal residual disease (MRD) in childhood Ph+ ALL prospectively enrolled in an intensive, Berlin-Frankfurt-Munster (BFM)-type treatment protocol. Twenty-seven of 36 (75.0%) Ph+ patients consecutively enrolled into the high-risk group of the AIEOP-ALL protocol between May 1995 and October 1999 were successfully analysed. Twenty were good responders to the pre-phase of prednisone/intrathecal methotrexate treatment (PGR) and seven were poor responders (PPR). Within the PPR group, the RT-PCR monitoring constantly showed positivity for the BCR/ABL fusion transcript and all the patients died of disease progression. In contrast, highly sensitive qualitative RT-PCR monitoring revealed heterogeneity within the PGR group of Ph+ childhood ALL patients. Three different subgroups could be defined, according to the clearance of Ph+ cells within the first 5 months of treatment. This provides useful information on the capability of chemotherapy to reduce the leukaemic clone, with prognostic implications.

  15. BCR-ABL1 kinase inhibits uracil DNA glycosylase UNG2 to enhance oxidative DNA damage and stimulate genomic instability.

    Science.gov (United States)

    Slupianek, A; Falinski, R; Znojek, P; Stoklosa, T; Flis, S; Doneddu, V; Pytel, D; Synowiec, E; Blasiak, J; Bellacosa, A; Skorski, T

    2013-03-01

    Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in chronic phase (CML-CP). Unfortunately, 25% of TKI-naive patients and 50-90% of patients developing TKI-resistance carry CML clones expressing TKI-resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen species, which may result in accumulation of uracil derivatives in genomic DNA. Unfaithful and/or inefficient repair of these lesions generates TKI-resistant point mutations in BCR-ABL1 kinase. Using an array of specific substrates and inhibitors/blocking antibodies we found that uracil DNA glycosylase UNG2 were inhibited in BCR-ABL1-transformed cell lines and CD34(+) CML cells. The inhibitory effect was not accompanied by downregulation of nuclear expression and/or chromatin association of UNG2. The effect was BCR-ABL1 kinase-specific because several other fusion tyrosine kinases did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI, we found that reduction of UNG2 activity increased the number of uracil derivatives in genomic DNA detected by modified comet assay and facilitated accumulation of ouabain-resistant point mutations in reporter gene Na(+)/K(+)ATPase. In conclusion, we postulate that BCR-ABL1 kinase-mediated inhibition of UNG2 contributes to accumulation of point mutations responsible for TKI resistance causing the disease relapse, and perhaps also other point mutations facilitating malignant progression of CML.

  16. Inverse regulation of bridging integrator 1 and BCR-ABL1 in chronic myeloid leukemia.

    Science.gov (United States)

    Trino, Stefania; De Luca, Luciana; Simeon, Vittorio; Laurenzana, Ilaria; Morano, Annalisa; Caivano, Antonella; La Rocca, Francesco; Pietrantuono, Giuseppe; Bianchino, Gabriella; Grieco, Vitina; Signorino, Elisabetta; Fragasso, Alberto; Bochicchio, Maria Teresa; Venturi, Claudia; Rosti, Gianantonio; Martinelli, Giovanni; Del Vecchio, Luigi; Cilloni, Daniela; Musto, Pellegrino

    2016-01-01

    Endocytosis is the major regulator process of tyrosine kinase receptor (RTK) functional activities. Bridging integrator 1 (BIN1) is a key protein involved in RTK intracellular trafficking. Here, we report, by studying 34 patients with chronic myeloid leukemia (CML) at diagnosis, that BIN1 gene is downregulated in CML as compared to healthy controls, suggesting an altered endocytosis of RTKs. Rab interactor 1 (RIN1), an activator of BIN1, displayed a similar behavior. Treatment of 57 patients by tyrosine kinase inhibitors caused, along with BCR-ABL1 inactivation, an increase of BIN1 and RIN1 expression, potentially restoring endocytosis. There was a significant inverse correlation between BIN1-RIN1 and BCR-ABL1 expression. In vitro experiments on both CML and nontumorigenic cell lines treated with Imatinib confirmed these results. In order to provide another proof in favor of BIN1 and RIN1 endocytosis function in CML, we demonstrated that Imatinib induced, in K562 cell line, BIN1-RIN1 upregulation accompanied by a parallel AXL receptor internalization into cytoplasmic compartment. This study shows a novel deregulated mechanism in CML patients, indicating BIN1 and RIN1 as players in the maintenance of the abnormal RTK signaling in this hematological disease.

  17. Quantification of BCR-ABL mRNA in Plasma/Serum of Patients with Chronic Myelogenous Leukemia

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    Miwako Narita, Anri Saito, Aya Kojima, Minami Iwabuchi, Naoya Satoh, Takayoshi Uchiyama, Akie Yamahira, Tatsuo Furukawa, Hirohito Sone, Masuhiro Takahashi

    2012-01-01

    Full Text Available Quantification of tumor-associated mRNA extracted from blood cells/tissues containing tumor cells is used for evaluation of treatment efficacy or residual tumor cell burden in tumors including leukemia. However, this method using tumor cell-containing blood/tissue is difficult to evaluate the whole tumor cell burden in the body. In order to establish an efficient method to evaluate the whole tumor cell burden in the body, we tried to quantify tumor-associated mRNA existing in plasma/serum instead of leukemia cell-containing blood cells in patients with chronic myelogenous leukemia (CML and compared the levels of BCR-ABL mRNA between plasma/serum and peripheral blood cells. mRNA of BCR-ABL, WT1 or GAPDH (control molecule was detected by real-time RT-PCR using RNA extracted from plasma/serum of almost all the patients with CML. Copy numbers of BCR-ABL mRNA were significantly correlated between plasma/serum and peripheral blood cells. However, levels of BCR-ABL mRNA extracted from serum were low compared with those extracted with peripheral blood cells. The present findings suggest that although real-time RT-PCR of mRNA existing in plasma/serum could be used for evaluating the whole tumor cell burden in the body, it's required to establish an efficient method to quantify plasma/serum mRNA by nature without degrading during the procedure.

  18. A clinical and laboratory study of chronic myeloid leukemia with atypical BCR-ABL fusion gene subtypes

    Institute of Scientific and Technical Information of China (English)

    桂晓敏

    2014-01-01

    Objective To explore the clinical and laboratory features of chronic myeloid leukemia(CML)with atypical e14a3 and e19a2 BCR-ABL fusion gene subtypes.Methods We retrospectively analyzed a cohort of CML patients with Ph chromosome positive confirmed by cytogenetic and FISH but classical e13a3(b2a2),e14a2(b3a2)and e1a2 fusion transcripts negative identified by

  19. Frequency of p190 and p210 BCR-ABL rearrangements and survival in Brazilian adult patients with acute lymphoblastic leukemia

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    Ilana de França Azevedo

    2014-10-01

    Full Text Available Objective: This study investigated the occurrence of the p190 and p210 break point clusterregion-Abelson (BCR-ABL rearrangements in adults with acute lymphoblastic leukemia and possible associations with clinical and laboratory characteristics and survival. Methods: Forty-one over 18-year-old patients with acute lymphoblastic leukemia of both genders followed-up between January 2008 and May 2012 were included in this study. Clinical and laboratory data were obtained from the medical charts of the patients. Reverse transcription polymerase chain reaction (RT-PCR using specific primers was employed to identify molecular rearrangements. Results: At diagnosis, the median age was 33 years, and there was a predominance of males (61%. The most common immunophenotype was B lineage (76%. BCR-ABL rearrangements was detected in 14 (34% patients with the following distribution: p190 (28%, p210 (50% and double positive (22%. Overall survival of patients with a mean/median of 331/246 days of follow up was 39%, respectively, negative BCR-ABL (44% and positive BCR-ABL (28%. Conclusion: These results confirm the high frequency of BCR-ABL rearrangements and the low survival rate of adult Brazilian patients with acute lymphoblastic leukemia.

  20. The diagnosis of BCR/ABL-negative chronic myeloproliferative diseases (CMPD): a comprehensive approach based on morphology, cytogenetics, and molecular markers.

    Science.gov (United States)

    Haferlach, Torsten; Bacher, Ulrike; Kern, Wolfgang; Schnittger, Susanne; Haferlach, Claudia

    2008-01-01

    Recent years showed significant progress in the molecular characterization of the chronic myeloproliferative disorders (CMPD) which are classified according to the WHO classification of 2001 as polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), essential thrombocythemia (ET), CMPD/unclassifiable (CMPD-U), chronic neutrophilic leukemia, and chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome, all to be delineated from BCR/ABL-positive chronic myeloid leukemia (CML). After 2001, the detection of the high frequency of the JAK2V617F mutation in PV, CIMF, and ET, and of the FIP1L1-PDGFRA fusion gene in CEL further added important information in the diagnosis of CMPD. These findings also enhanced the importance of tyrosine kinase mutations in CMPD and paved the way to a more detailed classification and to an improved definition of prognosis using also novel minimal residual disease (MRD) markers. Simultaneously, the broadening of therapeutic strategies in the CMPD, e.g., due to reduced intensity conditioning in allogeneic hematopoietic stem cell transplantation and the introduction of tyrosine kinase inhibitors in CML, in CEL, and in other ABL and PDGRFB rearrangements, increased the demands to diagnostics. Therefore, today, a multimodal diagnostic approach combining cytomorphology, cytogenetics, and individual molecular methods is needed in BCR/ABL-negative CMPD. A stringent diagnostic algorithm for characterization, choice of treatment, and monitoring of MRD will be proposed in this review. PMID:17938925

  1. The chimeric ubiquitin ligase SH2-U-box inhibits the growth of imatinib-sensitive and resistant CML by targeting the native and T315I-mutant BCR-ABL.

    Science.gov (United States)

    Ru, Yi; Wang, Qinhao; Liu, Xiping; Zhang, Mei; Zhong, Daixing; Ye, Mingxiang; Li, Yuanchun; Han, Hua; Yao, Libo; Li, Xia

    2016-01-01

    Chronic myeloid leukemia (CML) is characterized by constitutively active fusion protein tyrosine kinase BCR-ABL. Although the tyrosine kinase inhibitor (TKI) against BCR-ABL, imatinib, is the first-line therapy for CML, acquired resistance almost inevitably emerges. The underlying mechanism are point mutations within the BCR-ABL gene, among which T315I is notorious because it resists to almost all currently available inhibitors. Here we took use of a previously generated chimeric ubiquitin ligase, SH2-U-box, in which SH2 from the adaptor protein Grb2 acts as a binding domain for activated BCR-ABL, while U-box from CHIP functions as an E3 ubiquitin ligase domain, so as to target the ubiquitination and degradation of both native and T315I-mutant BCR-ABL. As such, SH2-U-box significantly inhibited proliferation and induced apoptosis in CML cells harboring either the wild-type or T315I-mutant BCR-ABL (K562 or K562R), with BCR-ABL-dependent signaling pathways being repressed. Moreover, SH2-U-box worked in concert with imatinib in K562 cells. Importantly, SH2-U-box-carrying lentivirus could markedly suppress the growth of K562-xenografts in nude mice or K562R-xenografts in SCID mice, as well as that of primary CML cells. Collectively, by degrading the native and T315I-mutant BCR-ABL, the chimeric ubiquitin ligase SH2-U-box may serve as a potential therapy for both imatinib-sensitive and resistant CML. PMID:27329306

  2. Mutations in the BCR-ABL1 Kinase Domain and Elsewhere in Chronic Myeloid Leukemia.

    Science.gov (United States)

    Soverini, Simona; de Benedittis, Caterina; Mancini, Manuela; Martinelli, Giovanni

    2015-06-01

    Chronic myeloid leukemia (CML) has been the first human malignancy to be associated, more than 50 years ago, with a consistent chromosomal abnormality--the t(9;22)(q34;q11) chromosomal translocation. The resulting BCR-ABL1 fusion gene, encoding a tyrosine kinase with deregulated activity, has a central role in the pathogenesis of CML. Ancestral or additional genetic events necessary for CML to develop have long been hypothesized but never really demonstrated. CML can successfully be treated with tyrosine kinase inhibitors (TKIs). Mutations in the BCR-ABL1 kinase domain might arise, however, that confer resistance to 1 or more of the currently available TKIs. Hence, the critical role of BCR-ABL1 mutation screening for optimal therapeutic management, with the current gold standard technique, conventional sequencing, likely to be replaced soon by ultra-deep sequencing. Mutations in genes other than BCR-ABL1 include ASXL1, TET2, RUNX1, DNMT3A, EZH2, and TP53 in chronic phase patients and RUNX1, ASXL1, IKZF1, WT1, TET2, NPM1, IDH1, IDH2, NRAS, KRAS, CBL, TP53, CDKN2A, RB1, and GATA-2 mutations in advanced phase patients. The latter also display additional cytogenetic abnormalities, including submicroscopic regions of gain or loss that only single nucleotide polymorphism arrays or array comparative genomic hybridization can detect. Whether whole genome/exome sequencing studies will uncover novel mutations relevant for pathogenesis, progression, and risk-adapted therapy is still unclear. PMID:26297264

  3. Modeling the influence of stromal microenvironment in the selection of ENU-induced BCR-ABL1 mutants by tyrosine kinase inhibitors.

    Science.gov (United States)

    Aggoune, Djamel; Tosca, Lucie; Sorel, Nathalie; Bonnet, Marie-Laure; Dkhissi, Fatima; Tachdjian, Gérard; Bennaceur-Griscelli, Annelise; Chomel, Jean-Claude; Turhan, Ali G

    2014-01-01

    Tyrosine kinase inhibitors (TKIs) have profoundly changed the natural history of chronic myeloid leukemia (CML). However, acquired resistance to imatinib, dasatinib or nilotinib (1(st) and 2(nd) generation TKIs), due in part to BCR-ABL1 kinase mutations, has been largely described. These drugs are ineffective on the T315I gatekeeper substitution, which remains sensitive to 3(rd) generation TKI ponatinib. It has recently been suggested that the hematopoietic niche could protect leukemic cells from targeted therapy. In order to investigate the role of a stromal niche in mutation-related resistance, we developed a niche-based cell mutagenesis assay. For this purpose, ENU (N-ethyl-N-nitrosourea)-exposed UT-7 cells expressing non-mutated or T315I-mutated BCR-ABL1 were cultured with or without murine MS-5 stromal cells and in the presence of imatinib, dasatinib, nilotinib, or ponatinib. In the assays relative to 1(st) and 2(nd) generation TKIs, which were performed on non-mutated BCR-ABL1 cells, our data highlighted the increasing efficacy of the latter, but did not reveal any substantial effect of the niche. In ponatinib assays performed on both non-mutated and T315I-mutated BCR-ABL1 cells, an increased number of resistant clones were observed in the presence of MS-5. Present data suggested that T315I mutants need either compound mutations (e.g. E255K/T315I) or a stromal niche to escape from ponatinib. Using array-comparative genomic hybridization experiments, we found an increased number of variations (involving some recurrent chromosome regions) in clones cultured on MS-5 feeder. Overall, our study suggests that the hematopoietic niche could play a crucial role in conferring resistance to ponatinib, by providing survival signals and favoring genetic instability.

  4. Low expression of miR-196b enhances the expression of BCR-ABL1 and HOXA9 oncogenes in chronic myeloid leukemogenesis.

    Directory of Open Access Journals (Sweden)

    Yue Liu

    Full Text Available MicroRNAs (miRNAs can function as tumor suppressors or oncogene promoters during tumor development. In this study, low levels of expression of miR-196b were detected in patients with chronic myeloid leukemia. Bisulfite genomic sequencing PCR and methylation-specific PCR were used to examine the methylation status of the CpG islands in the miR-196b promoter in K562 cells, patients with leukemia and healthy individuals. The CpG islands showed more methylation in patients with chronic myeloid leukemia compared with healthy individuals (P<0.05, which indicated that low expression of miR-196b may be associated with an increase in the methylation of CpG islands. The dual-luciferase reporter assay system demonstrated that BCR-ABL1 and HOXA9 are the target genes of miR-196b, which was consistent with predictions from bioinformatics software analyses. Further examination of cell function indicated that miR-196b acts to reduce BCR-ABL1 and HOXA9 protein levels, decrease cell proliferation rate and retard the cell cycle. A low level of expression of miR-196b can cause up-regulation of BCR-ABL1 and HOXA9 expression, which leads to the development of chronic myeloid leukemia. MiR-196b may represent an effective target for chronic myeloid leukemia therapy.

  5. The impact of multiple low-level BCR-ABL1 mutations on response to ponatinib

    Science.gov (United States)

    Yeung, David T. O.; Yeoman, Alexandra L.; Altamura, Haley K.; Jamison, Bronte A.; Field, Chani R.; Hodgson, J. Graeme; Lustgarten, Stephanie; Rivera, Victor M.; Hughes, Timothy P.; Branford, Susan

    2016-01-01

    The third-generation tyrosine kinase inhibitor (TKI) ponatinib shows activity against all common BCR-ABL1 single mutants, including the highly resistant BCR-ABL1-T315I mutant, improving outcome for patients with refractory chronic myeloid leukemia (CML). However, responses are variable, and causal baseline factors have not been well-studied. The type and number of low-level BCR-ABL1 mutations present after imatinib resistance has prognostic significance for subsequent treatment with nilotinib or dasatinib as second-line therapy. We therefore investigated the impact of low-level mutations detected by sensitive mass-spectrometry before ponatinib initiation (baseline) on treatment response in 363 TKI-resistant patients enrolled in the PONATINIB for Chronic Myeloid Leukemia Evaluation and Ph+ Acute Lymphoblastic Leukemia trial, including 231 patients in chronic phase (CP-CML). Low-level mutations were detected in 53 patients (15%, including low-level T315I in 14 patients); most, however, did not undergo clonal expansion during ponatinib treatment and, moreover, no specific individual mutations were associated with inferior outcome. We demonstrate however, that the number of mutations detectable by mass spectrometry after TKI resistance is associated with response to ponatinib treatment and could be used to refine the therapeutic approach. Although CP-CML patients with T315I (63/231, 27%) had superior responses overall, those with multiple mutations detectable by mass spectrometry (20, 32%) had substantially inferior responses compared with those with T315I as the sole mutation detected (43, 68%). In contrast, for CP-CML patients without T315I, the inferior responses previously observed with nilotinib/dasatinib therapy for imatinib-resistant patients with multiple mutations were not seen with ponatinib treatment, suggesting that ponatinib may prove to be particularly advantageous for patients with multiple mutations detectable by mass spectrometry after TKI resistance

  6. Molecular Imaging of Bcr-Abl Phosphokinase in a Xenograft Model*

    OpenAIRE

    Wu, Ji Yuan; David J. Yang; Angelo, Laura S.; Kohanim, Saady; Kurzrock, Razelle

    2009-01-01

    The purpose of this study was to determine whether the Bcr-Abl tyrosine kinase can be assessed by gamma imaging using an 111Indium-labeled anti-phosphotyrosine antibody, and if the response to treatment with imatinib could be detected using this imaging technique. Anti-phosphotyrosine antibody (APT) was labeled with indium (111In) using ethylenedicysteine (EC) as a chelator. To determine if 111In-EC-APT could assess a non-receptor tyrosine kinase, xenografts of the human chronic myelogenous l...

  7. BCR-ABL融合基因T315I突变的白血病行异基因HSCT的疗效及复发后的治疗分析%Curative effectiveness of allogeneic stem cell transplantation and treatment of recurrence in patients harboring T315I BCR-ABL mutated leukemias

    Institute of Scientific and Technical Information of China (English)

    桂晓敏; 仇惠英; 潘金兰; 周敏; 鲍协炳; 陈苏宁; 孙爱宁; 吴德沛; 岑建农

    2014-01-01

    Objective To retrospectively analyze the curative effectiveness of allogeneic stem cell transplantation (allo-SCT) for the leukemia patients with the T315I mutation,and to discuss the treatments for the relapsing patients after allo-SCT.Method Through observing 10 leukemia patients harboring a T315I BCR-ABL mutation who underwent allo-SCT,we analyzed the remission rate,recurrence rate and long-term survival situation of these patients and explored the treatments for relapsing patients after allo-SCT.Result Of 10 patients,4 patients were female and 6 were male.The median age was 29 years (range,18-49).Before HSCT,5 were given imatinib,3 nilotinib and 2 dasatinib,respectively.Ten patients (100%) obtained complete remission (CR) and the median remission time was 23 days (range,14-56).During the follow-up period,1 case of CML and 3 cases of Ph+-ALL (40%) obtained CR.The survival time was 75 to 741 days.In the 6 dead patients,1 died of upper gastrointestinal hemorrhage on the day 137 after allo-SCT; 2 died of graft versus host disease on the day 53 and 617 respectively after allo-SCT; 1 case of CML and 2 cases of Ph+ ALL relapsed on the day 27,50 and 130 respectively after allo-SCT,and 2 of them died on the day 5 and 40 respectively after relapse,and 1 recurrent case did not obtain remission after chemotherapy and died on the day 97 after relapse.Conclusion The allo-SCT can be used to treat the leukemia patients with T315I mutation,and high remission rate can be obtained,tut there is the high risk of recurrence.The patients with recurrent leukemia can take further relevant treatment to prolong survival time.%目的 分析BCR-ABL融合基因T315I突变的白血病患者接受异基因造血干细胞移植(HSCT)的资料,探讨移植疗效及复发后的治疗.方法 观察10例伴T315I突变的费城染色体阳性(Ph+)的急性淋巴细胞白血病(ALL)和慢性粒细胞白血病(CML)接受异基因HSCT后缓解率、复发率以及长期存活情况,分析这

  8. Frequency of the ETV6-RUNX1, BCR-ABL1, TCF3-PBX1, and MLL-AFF1 fusion genes in Guatemalan pediatric acute lymphoblastic leukemia patients and their ethnic associations.

    Science.gov (United States)

    Carranza, Claudia; Granados, Lilian; Morales, Oneida; Jo, Wendy; Villagran, Swuanny; Tinti, Damaris; Villegas, Mauricio; Antillón, Federico; Torselli, Silvana; Silva, Gabriel

    2013-06-01

    Fusion genes involved in acute lymphoblastic leukemia (ALL) occur mostly due to genetic and environmental factors, and only a limited number of studies have reported any ethnic influence. This study assesses whether an ethnic influence has an effect on the frequency of any of the four fusion genes: BCR-ABL1, ETV6-RUNX1, TCF3-PBX1, and MLL-AFF1 found in ALL. To study this ethnic influence, mononuclear cells were obtained from bone marrow samples from 143 patients with ALL. We performed RNA extraction and reverse transcription, then assessed the quality of the cDNA by amplifying the ABL1 control gene, and finally evaluated the presence of the four transcripts by multiplex polymerase chain reaction. We found 10 patients who had the BCR-ABL1 fusion gene (7%); 3 patients (2%) were TCF3-PBX1 positive; and 6 patients (4.5%) were ETV6-RUNX1 positive. The incidence of this last fusion gene is quite low when compared to the values reported in most countries. The low incidence of the ETV6-RUNX1 fusion gene found in Guatemala matches the incidence rates that have been reported in Spain and Indian Romani. Since it is known that an ethnic resemblance exists among these three populations, as shown by ancestral marker studies, the ALL data suggests an ethnic influence on the occurrence and frequency of this particular fusion gene.

  9. Transferred BCR/ABL DNA from K562 extracellular vesicles causes chronic myeloid leukemia in immunodeficient mice.

    Directory of Open Access Journals (Sweden)

    Jin Cai

    Full Text Available Our previous study showed that besides mRNAs and microRNAs, there are DNA fragments within extracellular vesicles (EVs. The BCR/ABL hybrid gene, involved in the pathogenesis of chronic myeloid leukemia (CML, could be transferred from K562 EVs to neutrophils and decrease their phagocytic activity in vitro. Our present study provides evidence that BCR/ABL DNAs transferred from EVs have pathophysiological significance in vivo. Two months after injection of K562 EVs into the tail vein of Sprague-Dawley (SD rats, they showed some characteristics of CML, e.g., feeble, febrile, and thin, with splenomegaly and neutrophilia but with reduced neutrophil phagocytic activity. These findings were also observed in immunodeficient NOD/SCID mice treated with K562 EVs; BCR/ABL mRNA and protein were found in their neutrophils. The administration of actinomycin D, an inhibitor of de novo mRNA synthesis, prevented the abnormalities caused by K562 EVs in NOD/SCID mice related to CML, including neutrophilia and bone marrow hyperplasia. As a specific inhibitor of tyrosine kinases, imatinib blocked the activity of tyrosine kinases and the expression of phospho-Crkl, induced by the de novo BCR/ABL protein caused by K562 EVs bearing BCR/ABL DNA. Our current study shows the pathophysiological significance of transferred tumor gene from EVs in vivo, which may represent an important mechanism for tumorigenesis, tumor progression, and metastasis.

  10. Expression of p190 BCR-ABL fusion gene in a patient with chronic myeloid leukemia Expressão do rearranjo gênico BCR-ABL com ponto de quebra na região menor do gene BCR em um paciente com leucemia mielóide crônica

    Directory of Open Access Journals (Sweden)

    P. V. B. Carvalho

    2003-01-01

    Full Text Available A minority of chronic myeloid leukemia cases have breakpoints in the minor cluster region (m-bcr of the BCR-ABL gene. We report on a patient with Ph-positive and m-bcr breakpoint at diagnosis. She was treated with hydroxyurea and interferon-alpha. Two years later, she developed a lymphoid blast crisis and died shortly after. We discuss herein the different forms of the BCR-ABL oncogene, its products, and the possible influence of them on the clinical outcome of patients with the disease.A leucemia mielóide crônica (LMC é uma doença mieloproliferativa clonal e caracteriza-se pela presença da translocação cromossômica entre os braços longos dos cromossomos 9 e 22, o denominado cromossomo Ph. Esta translocação determina a fusão dos genes BCR e ABL. Os diferentes pontos de quebra no gene BCR determinam a síntese de proteínas com diferentes pesos moleculares pelo gene BCR-ABL. Nós relatamos o caso de uma paciente portadora de LMC com ponto de quebra cromossômico na região menor do gene BCR. Foi tratada com hidroxiuréia e interferon alfa. Dois anos após o diagnóstico desenvolveu crise blástica linfóide e evoluiu rapidamente para o óbito. Nós discutimos nesta apresentação as diferentes formas do gene BCR-ABL e seus produtos e a possível influência dos mesmos na evolução clínica dos pacientes com a doença.

  11. Epidemiologic study on survival of chronic myeloid leukemia and Ph(+) acute lymphoblastic leukemia patients with BCR-ABL T315I mutation

    DEFF Research Database (Denmark)

    Nicolini, Franck E; Mauro, Michael J; Martinelli, Giovanni;

    2009-01-01

    ), or blastic-phase (BP) and Philadelphia chromosome-positive (Ph)(+) acute lymphoblastic leukemia (ALL) patients with T315I mutation. Medical records of 222 patients from 9 countries were reviewed; data were analyzed using log-rank tests and Cox proportional hazard models. Median age at T315I mutation......The BCR-ABL T315I mutation represents a major mechanism of resistance to tyrosine kinase inhibitors (TKIs). The objectives of this retrospective observational study were to estimate overall and progression-free survival for chronic myeloid leukemia in chronic-phase (CP), accelerated-phase (AP...

  12. In-silico identification of inhibitors against mutated BCR-ABL protein of chronic myeloid leukemia: a virtual screening and molecular dynamics simulation study.

    Science.gov (United States)

    Kumar, Himansu; Raj, Utkarsh; Gupta, Saurabh; Varadwaj, Pritish Kumar

    2016-10-01

    Aberrant and proliferative expression of the oncogene BCR-ABL in the bone marrow cells had been proven as the prime cause of chronic myeloid leukemia (CML). It has been established that tyrosine kinase domain of BCR-ABL protein is a potential therapeutic target for the treatment of CML. Imatinib is considered as a first-generation drug that can inhibit the enzymatic action by inhibiting the ATP binding with BCR-ABL protein. Later on, insensitivity of CML cells towards Imatinib has been observed may be due to mutation in tyrosine kinase domain of the ABL receptor. Subsequently, some other second-generation drugs have also been reported viz. Baustinib, Nilotinib, Dasatinib, Ponatinib, Bafetinib, etc., which can able to combat against mutated domain of ABL tyrosine kinase protein. By taking into account of bioavailability and resistance developed, there is an utmost need to find some more inhibitors for the mutated ABL tyrosine kinase protein. For virtual screening, a data-set has been generated by collecting the all available drug like natural compounds from ZINC and Drug Bank databases. Comparative docking analysis was also carried out on the active site of ABL tyrosine kinase receptor with reported reference inhibitors. Molecular dynamics simulation of the best screened interacting complex was done for 50 ns to validate the stability of the system. These selected inhibitors were further validated and analyzed through pharmacokinetics properties and series of ADMET parameters by in silico methods. Considering the above said parameters proposed molecules are concluded as potential leads for drug designing pipeline against CML. PMID:26479578

  13. Increased acetylation of lysine 317/320 of p53 caused by BCR-ABL protects from cytoplasmic translocation of p53 and mitochondria-dependent apoptosis in response to DNA damage

    NARCIS (Netherlands)

    Kusio-Kobialka, Monika; Wolanin, Kamila; Podszywalow-Bartnicka, Paulina; Sikora, Ewa; Skowronek, Krzysztof; McKenna, Sharon L.; Ghizzoni, Massimo; Dekker, Frank J.; Piwocka, Katarzyna

    2012-01-01

    Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cells caused by the expression of BCR-ABL. Loss of p53 has not been implicated as important for the development of CML. Mutations in p53 protein are infrequent, however they correlate with the disease progression. The absence of p53

  14. Comparative quantitative analysis of BCR-ABL transcripts with the T315I mutant clone by polymerase chain reaction (PCR)-Invader method.

    Science.gov (United States)

    Tadokoro, Kenichi; Ishikawa, Maho; Suzuki, Makoto; Saito, Tomoyoshi; Suzuki, Yoshie; Yamaguchi, Toshikazu; Yagasaki, Fumiharu

    2011-09-01

    Drug resistance is a serious complication in the treatment of chronic myeloid leukemia (CML). The most common and best-characterized mechanism of secondary imatinib resistance in CML is the development of kinase domain mutations in the BCR-ABL gene. Second-generation tyrosine kinase inhibitors, such as dasatinib or nilotinib, overcome most of these mutations, but they are not effective against the T315I mutant. To determine whether these mutations contribute to clinical resistance, it is necessary to monitor the ratio of the mutant and wild-type forms. Here, we developed a polymerase chain reaction (PCR)-Invader assay for comparative quantitative analysis (qPI assay) of BCR-ABL transcripts with the T315I mutant clone. T315I ratios were calculated for the wild-type and mutant fold-over-zero (FOZ) values. In examination with 2 kinds of plasmids containing wild-type or T315I mutant PCR amplicons, mutant FOZ values were detected down to 1% of the total. The results of 12 serial samples from 2 patients (case A: Philadelphia-positive acute lymphoblastic leukemia and case B: CML) with the T315I mutant clone were compared with those of direct sequencing or 2 kinds of allele-specific oligonucleotide (ASO)-PCR. All samples showed the T315I mutation by qPI assay and ASO-PCR, and 10 samples showed it by direct sequencing. Significant correlation (correlation coefficient; r2 = 0.951) was noted between the qPI assay and quantitative ASO-PCR to analyze T315I mutant ratios. Thus, the qPI assay is a useful method for evaluating the T315I mutant clone in BCR-ABL transcripts.

  15. Functionally deregulated AML1/RUNX1 cooperates with BCR-ABL to induce a blastic phase-like phenotype of chronic myelogenous leukemia in mice.

    Directory of Open Access Journals (Sweden)

    Kiyoko Yamamoto

    Full Text Available Patients in the chronic phase (CP of chronic myelogenous leukemia (CML have been treated successfully following the advent of ABL kinase inhibitors, but once they progress to the blast crisis (BC phase the prognosis becomes dismal. Although mechanisms underlying the progression are largely unknown, recent studies revealed the presence of alterations of key molecules for hematopoiesis, such as AML1/RUNX1. Our analysis of 13 BC cases revealed that three cases had AML1 mutations and the transcript levels of wild-type (wt. AML1 were elevated in BC compared with CP. Functional analysis of representative AML1 mutants using mouse hematopoietic cells revealed the possible contribution of some, but not all, mutants for the BC-phenotype. Specifically, K83Q and R139G, but neither R80C nor D171N mutants, conferred upon BCR-ABL-expressing cells a growth advantage over BCR-ABL-alone control cells in cytokine-free culture, and the cells thus grown killed mice upon intravenous transfer. Unexpectedly, wt.AML1 behaved similarly to K83Q and R139G mutants. In a bone marrow transplantation assay, K83Q and wt.AML1s induced the emergence of blast-like cells. The overall findings suggest the roles of altered functions of AML1 imposed by some, but not all, mutants, and the elevated expression of wt.AML1 for the disease progression of CML.

  16. Detection of BCR-ABL Fusion mRNA Using Reverse Transcriptase Loop-mediated Isothermal Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Dugan, L C; Hall, S; Kohlgruber, A; Urbin, S; Torres, C; Wilson, P

    2011-12-08

    RT-PCR is commonly used for the detection of Bcr-Abl fusion transcripts in patients diagnosed with chronic myelogenous leukemia, CML. Two fusion transcripts predominate in CML, Br-Abl e13a2 and e14a2. They have developed reverse transcriptase isothermal loop-mediated amplification (RT-LAMP) assays to detect these two fusion transcripts along with the normal Bcr transcript.

  17. Structural Mechanism of the Pan-BCR-ABL Inhibitor Ponatinib (AP24534): Lessons for Overcoming Kinase Inhibitor Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Tianjun; Commodore, Lois; Huang, Wei-Sheng; Wang, Yihan; Thomas, Mathew; Keats, Jeff; Xu, Qihong; Rivera, Victor M.; Shakespeare, William C.; Clackson, Tim; Dalgarno, David C.; Zhu, Xiaotian (ARIAD)

    2012-01-20

    The BCR-ABL inhibitor imatinib has revolutionized the treatment of chronic myeloid leukemia. However, drug resistance caused by kinase domain mutations has necessitated the development of new mutation-resistant inhibitors, most recently against the T315I gatekeeper residue mutation. Ponatinib (AP24534) inhibits both native and mutant BCR-ABL, including T315I, acting as a pan-BCR-ABL inhibitor. Here, we undertook a combined crystallographic and structure-activity relationship analysis on ponatinib to understand this unique profile. While the ethynyl linker is a key inhibitor functionality that interacts with the gatekeeper, virtually all other components of ponatinib play an essential role in its T315I inhibitory activity. The extensive network of optimized molecular contacts found in the DFG-out binding mode leads to high potency and renders binding less susceptible to disruption by single point mutations. The inhibitory mechanism exemplified by ponatinib may have broad relevance to designing inhibitors against other kinases with mutated gatekeeper residues.

  18. Nilotinib and Combination Chemotherapy in Treating Patients With Newly Diagnosed Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia or Blastic Phase Chronic Myelogenous Leukemia

    Science.gov (United States)

    2015-10-29

    B-cell Adult Acute Lymphoblastic Leukemia; Blastic Phase Chronic Myelogenous Leukemia; Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Philadelphia Chromosome Positive Adult Precursor Acute Lymphoblastic Leukemia; Untreated Adult Acute Lymphoblastic Leukemia

  19. Determination of lactate dehydrogenase (LDH and Bcr-Abl transcript in the follow-up of patients with chronic myeloid leukemia = Determinação da lactate desidrogenase (LDH e do transcrito Bcr-Abl em pacientes com leucemia mielóide crônica

    Directory of Open Access Journals (Sweden)

    Roberto Iemitsu Tatakihara

    2010-07-01

    Full Text Available Chronic myeloid leukemia (CML is a malignant myeloproliferative disorder that originates from a pluripotent stem cell characterized by abnormal release of the expanded, malignant stem cell clone from the bone marrow into the bloodstream. The vast majority of patients with CML present Bcr-Abl transcripts. Lactate dehydrogenase (LDH is considered a biochemical marker common for tumor growth, anaerobic glycolysis and has been considered a poor prognostic factor for acute myeloid leukemia. Therefore, this study aimed to evaluate the concentration of LDH in plasma and the detection of the Bcr-Abl transcripts in patients with CML and healthy donors. We analyzed 22 patients demonstrably diagnosed with CML and 56 healthy donors. LDH concentration in plasma was higher in patients with CML. All patients with CML in this study were under treatment, but even so four patients had the Bcr-Abl (b3a2 transcript in peripheral blood. Two out of the four patients with b3a2 showed higher LDH (486 U L-1 and 589 U L-1. Thus, although the study was conducted with small numbers of samples, it is possible to suggest therapy alteration for two patients who presented transcript b3a2 in the peripheral blood samples and whose LDH concentration was high, in order to improve the disease. Leucemia mieloide crônica (LMC é uma desordem mieloproliferativa maligna que é originada de célula-tronco pluripotente caracterizada por expansão anormal, maligna de clones de células tronco da medula óssea na circulação. A grande maioria dos pacientes com LMC apresentam transcritos Bcr-Abl. Lactato desidrogenase (LDH,considerado um marcador bioquímico para crescimento tumoral, glicólise anaeróbica, e tem sido considerado um fator de pior prognóstico da LMC. Portanto, este estudo visa avaliar a concentraçãode LDH no plasma e a detecção do transcrito Bcr-Abl em 22 pacientes com LMC e 56 indivíduos saudáveis. Foram avaliados 22 pacientes com LMC e 56 doadores saudáveis. A

  20. Conventional and fluorescence in situ hybridization analysis of three-way complex BCR-ABL rearrangement in a chronic myeloid leukemia patient

    Directory of Open Access Journals (Sweden)

    Ganguly Bani

    2007-01-01

    Full Text Available Chromosomal analysis was carried out in bone marrow sample of an 11-year-old girl suspected of myeloproliferative disorder. Conventional G-banding study detected a complex three-way translocation involving 7, 9 and 22, which has resulted in the formation of a variant Philadelphia chromosome causing rearrangement of abl and bcr genes in 87% cells. Fluorescence in situ hybridization (FISH confirmed the fusion of bcr-abl oncogene. Thus the bone marrow karyotype was observed as 46,XX (13% / 46,XX,t(7;9;22(q11;q34;q11 (87%. Hyperdiploidy was present in two cells. In this study, both conventional cytogenetic and FISH diagnosis proved to be significant to identify the variant nature of the Philadelphia chromosome and hyperdiploid condition for introduction of a suitable treatment regimen and estimation of life expectancy of the young girl.

  1. Synthesis and characterization of a BODIPY conjugate of the BCR-ABL kinase inhibitor Tasigna® (Nilotinib): Evidence for transport of Tasigna® and its fluorescent derivative by ABC drug transporters

    OpenAIRE

    Shukla, Suneet; Skoumbourdis, Amanda P.; Walsh, Martin J.; Hartz, Anika M. S.; Fung, King Leung; Wu, Chung-pu; Gottesman, Michael M.; Bauer, Björn; Thomas, Craig J.; Suresh V Ambudkar

    2011-01-01

    Tasigna® (Nilotinib) is a recently approved BCR-ABL kinase inhibitor by the Food and Drug Administration, which is indicated for the treatment of drug-resistant chronic myelogenous leukemia (CML). The efflux of tyrosine kinase inhibitors by ATP-binding cassette (ABC) drug transporters, which actively pump these drugs out of cells utilizing ATP as an energy source, has been linked to the development of drug resistance in CML patients. We report here synthesis and characterization of a fluoresc...

  2. Next-generation sequencing for sensitive detection of BCR-ABL1 mutations relevant to tyrosine kinase inhibitor choice in imatinib-resistant patients

    Science.gov (United States)

    Soverini, Simona; De Benedittis, Caterina; Polakova, Katerina Machova; Linhartova, Jana; Castagnetti, Fausto; Gugliotta, Gabriele; Papayannidis, Cristina; Mancini, Manuela; Klamova, Hana; Salvucci, Marzia; Crugnola, Monica; Iurlo, Alessandra; Albano, Francesco; Russo, Domenico; Rosti, Gianantonio; Cavo, Michele; Baccarani, Michele; Martinelli, Giovanni

    2016-01-01

    In chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients who fail imatinib treatment, BCR-ABL1 mutation profiling by Sanger sequencing (SS) is recommended before changing therapy since detection of specific mutations influences second-generation tyrosine kinase inhibitor (2GTKI) choice. We aimed to assess i) in how many patients who relapse on second-line 2GTKI therapy next generation sequencing (NGS) may track resistant mutations back to the sample collected at the time of imatinib resistance, before 2GTKI start (switchover sample) and ii) whether low level mutations identified by NGS always undergo clonal expansion. To this purpose, we used NGS to retrospectively analyze 60 imatinib-resistant patients (CML, n = 45; Ph+ ALL, n = 15) who had failed second-line 2GTKI therapy and had acquired BCR-ABL1 mutations (Group 1) and 25 imatinib-resistant patients (CML, n = 21; Ph+ ALL, n = 4) who had responded to second-line 2GTKI therapy, for comparison (Group 2). NGS uncovered that in 26 (43%) patients in Group 1, the 2GTKI-resistant mutations that triggered relapse were already detectable at low levels in the switchover sample (median mutation burden, 5%; range 1.1%–18.4%). Importantly, none of the low level mutations detected by NGS in switchover samples failed to expand whenever the patient received the 2GTKI to whom they were insensitive. In contrast, no low level mutation that was resistant to the 2GTKI the patients subsequently received was detected in the switchover samples from Group 2. NGS at the time of imatinib failure reliably identifies clinically relevant mutations, thus enabling a more effective therapeutic tailoring. PMID:26980736

  3. Molecular monitoring of BCR-ABL transcripts in patients with chronic myelogenous leukemia: is high sensitivity of clinical value?

    Science.gov (United States)

    Norkin, Maxim; Schiffer, Charles A

    2010-04-01

    Monitoring of disease response during treatment with tyrosine kinase inhibitors of patients with chronic myelogenous leukemia dramatically changed after the introduction of real-time PCR, which allows quantification of BCR-ABL transcript levels with high sensitivity and precision. However, its role in patients who have achieved complete cytogenetic response is not entirely clear; incorrect interpretation of results could lead to unnecessary changes from an effective treatment. This review discusses the current evidence regarding the benefits, uncertainties, and potential drawbacks of molecular monitoring in patients with chronic myelogenous leukemia in chronic phase.

  4. 用COLD-PCR方法提高BCR-ABL1激酶区耐药突变检测灵敏度%Application of COLD-PCR for improved detection of BCR-ABL1 kinase domain drug-resistant mutation

    Institute of Scientific and Technical Information of China (English)

    李庆; 刘红星; 孙慧; 王倩; 张之芬; 武焕玲; 李元堂; 陈永金; 田文君

    2013-01-01

    Objective To evaluate the feasibility of the co-amplification using a low denaturation temperature PCR (COLD-PCR) method in improving the detection of BCR-ABL1 fusion gene kinase domain drug-resistant mutation.Methods Conventional nested PCR (CN-PCR) and COLD-PCR protocol were designed to amplify full-length of BCR-ABL1 kinase domain.PCR products were purified and then Sanger sequenced to detect BCR-ABL1 kinase domain mutation (KDM).20 newly diagnosed CML patients and 32 Imatinib (IM) resistant patients were chosen and peripheral blood or bone marrow samples were collected.PCR products of the clinical specimens were purified and then cloned to evaluate the proportion of the KDM allele.Standard samples with different proportion of KDMs were prepared using T-A vectors with KDM and wild type BCR-ABL1 kinase domain sequence,and then CN-PCR,COLD-PCR and Sanger sequencing were performed to compare the detection sensitivity.Results In the 32 IM-resistant patients,23 were identified KDM-positive using CN-PCR and 28 KDM-positive using COLD-PCR.In the 20 newly diagnosed CML patients,none was identified KDM-positive using CN-PCR and 1 was identified carrying a lower proportion of KDM using COLD-PCR.For 16 different types of KDMs,COLD-PCR method could improve the detection sensitivity 4-12 times.Conclusion Detection sensitivity of BCR-ABL1 drug-resistant kinase domain mutation can be effectively improved by using COLD-PCR method and may contribute to early detection of resistance KDMs.%目的 探讨用低变性温度共扩增PCR(COLD-PCR)方法提高BCR-ABL1融合基因激酶区耐药突变检测灵敏度的可行性.方法 本研究为实验诊断研究.设计常规巢式PCR(CN-PCR)和COLD-PCR方案扩增BCR-ABL1激酶区全长,扩增产物纯化后用Sanger测序法检测激酶区突变(KDM).采集32例经伊马替尼(IM)治疗并出现耐药的慢性粒细胞性白血病(CML)患者和20例初诊CML患者外周血或骨髓标本,用上述2种方案检测标本中的KDM.

  5. Selective Depletion of CD45RA+ T Cells From Allogeneic Peripheral Blood Stem Cell Grafts From HLA-Matched Related and Unrelated Donors in Preventing GVHD

    Science.gov (United States)

    2016-07-08

    Accelerated Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Acute Biphenotypic Leukemia; Acute Leukemia of Ambiguous Lineage; Acute Undifferentiated Leukemia; Adult Acute Lymphoblastic Leukemia in Remission; Adult Acute Myeloid Leukemia in Remission; Blast Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Blastic Plasmacytoid Dendritic Cell Neoplasm; Childhood Acute Lymphoblastic Leukemia in Remission; Childhood Acute Myeloid Leukemia in Remission; Graft Versus Host Disease; Lymphoblastic Lymphoma; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Recurrent Childhood Acute Lymphoblastic Leukemia; Recurrent Childhood Acute Myeloid Leukemia; Recurrent Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Refractory Anemia With Excess Blasts

  6. Overexpression or knock-down of runt-related transcription factor 1 affects BCR-ABL-induced proliferation and migration in vitro and leukemogenesis in vivo in mice

    Institute of Scientific and Technical Information of China (English)

    YANG Li-jun; YU Wei-dong; DU Jun-bao; CHAO Shuang; CHEN Min-xia; ZHAO He-hua; GUO Jing-zhu

    2009-01-01

    Background Runt-related transcription factor 1 (Runx1) plays a crucial role in hematogenesis and its dysfunction may contribute to leukemogenesis. However, it is not clear whether or not abnormal expression of Runx1 will induce leukemia and how the change of Runx1 expression level could affect BCR-ABL-induced leukemogenesis. In the present study, we aimed to analyze if abnormal expression of Runx1 in BaF3 cells alone would induce leukemogenesis. And we also wanted to know if abnormal expression of Runx1 in leukemic cells would affect leukemogenesis. Furthermore, we investigated whether overexpression or knock-down of Runx1 in BaF3 cells would induce leukemogenesis.Methods Plasmids containing full-length Runx1 cDNA were transduced into BaF3 cells and BaF3-P185wt cells (BCR-ABL transformed BaF3 cells) by electroporation. Plasmids containing a short hairpin RNA of Runx1 were transduced into BaF3 cells and BaF3-P185wt cells by electroporation. Runx1 expression level was quantified by Western blotting and quantitative real-time PCR. The effects of overexpression or knock-down of Runx1 on proliferation, apoptosis and migration of cells were detected in vitro. Then, using MSCV-P185wt-FGFP as a control, we transplanted MSCV-P185wt-Runx1 cells or MSCV-P185wt-shRNA cells into Balb/c mice through tail vein and observed tumorgenesis of the different phenotypes.Results In vitro analysis revealed that overexpression of Runx1 in P185wt cells could inhibit cell proliferation and slow down cell migration; while knock-down of Runx1 could promote cell proliferation and speed up cell migration. In vivo analysis indicated that mice transplanted with MSCV-P185wt-Runx1 survived longer than controls. In contrast, mice transplanted with MSCV-P185wt-shRNA survived shorter than the control group. Gross pathological analysis revealed that the MSCV-P185wt-Runx1 group had less severe splenomegaly and hepatomegaly compared to the control group, and the MSCV-P185wt-shRNA group had more severe

  7. Allelic expression imbalance of JAK2 V617F mutation in BCR-ABL negative myeloproliferative neoplasms.

    Directory of Open Access Journals (Sweden)

    Hye-Ran Kim

    Full Text Available The discovery of a single point mutation in the JAK2 gene in patients with BCR/ABL-negative myeloproliferative neoplasms (MPNs has not only brought new insights and pathogenesis, but also has made the diagnosis of MPNs much easier. Although, to date, several mechanisms for the contribution of single JAK2V617F point mutation to phenotypic diversity of MPNs have been suggested in multiple studies, but it is not clear how a unique mutation can cause the phenotypic diversity of MPNs. In this study, our results show that allelic expression imbalance of JAK2 V617F mutant frequently occurs and contributes to phenotypic diversity of BCR-ABL-negative MPNs. The proportion of JAK2 V617F mutant allele was significantly augmented in RNA levels as compared with genomic DNA differently by distinct MPNs subtypes. In detail, preferential expression of JAK2 mutant allele showed threefold increase from the cDNA compared with the genomic DNA from patients with essential thrombocythemia and twofold increase in polycythemia vera. In conclusion, allelic expression imbalance of JAK2 V617F mutant proposes another plausible mechanism for the contribution of single JAK2 point mutation to phenotypic diversity of MPNs.

  8. Biosensing of BCR/ABL fusion gene using an intensity-interrogation surface plasmon resonance imaging system

    Science.gov (United States)

    Wu, Jiangling; Huang, Yu; Bian, Xintong; Li, DanDan; Cheng, Quan; Ding, Shijia

    2016-10-01

    In this work, a custom-made intensity-interrogation surface plasmon resonance imaging (SPRi) system has been developed to directly detect a specific sequence of BCR/ABL fusion gene in chronic myelogenous leukemia (CML). The variation in the reflected light intensity detected from the sensor chip composed of gold islands array is proportional to the change of refractive index due to the selective hybridization of surface-bound DNA probes with target ssDNA. SPRi measurements were performed with different concentrations of synthetic target DNA sequence. The calibration curve of synthetic target sequence shows a good relationship between the concentration of synthetic target and the change of reflected light intensity. The detection limit of this SPRi measurement could approach 10.29 nM. By comparing SPRi images, the target ssDNA and non-complementary DNA sequence are able to be distinguished. This SPRi system has been applied for assay of BCR/ABL fusion gene extracted from real samples. This nucleic acid-based SPRi biosensor therefore offers an alternative high-effective, high-throughput label-free tool for DNA detection in biomedical research and molecular diagnosis.

  9. Sphingomyelin synthase 1 activity is regulated by the BCR-ABL oncogene[S

    OpenAIRE

    Burns, Tara Ann; Subathra, Marimuthu; signorelli, Paola; Choi, Young; Yang, Xiaofeng; Wang, Yong; Villani, Maristella; Bhalla, Kapil; Zhou, Daohong; Luberto, Chiara

    2013-01-01

    Sphingomyelin synthase (SMS) produces sphingomyelin while consuming ceramide (a negative regulator of cell proliferation) and forming diacylglycerol (DAG) (a mitogenic factor). Therefore, enhanced SMS activity could favor cell proliferation. To examine if dysregulated SMS contributes to leukemogenesis, we measured SMS activity in several leukemic cell lines and found that it is highly elevated in K562 chronic myelogenous leukemia (CML) cells. The increased SMS in K562 cells was caused by the ...

  10. Donor Lymphocyte Infusion in Treating Patients With Persistent, Relapsed, or Progressing Cancer After Donor Hematopoietic Cell Transplant

    Science.gov (United States)

    2016-07-25

    Blast Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Recurrent Hodgkin Lymphoma; Recurrent Non-Hodgkin Lymphoma; Recurrent Plasma Cell Myeloma

  11. Pentostatin and Lymphocyte Infusion in Preventing Graft Rejection in Patients Who Have Undergone Donor Stem Cell Transplant

    Science.gov (United States)

    2016-02-29

    Acute Lymphoblastic Leukemia; Acute Myeloid Leukemia; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Graft Versus Host Disease; Hodgkin Lymphoma; Myelodysplastic/Myeloproliferative Neoplasm; Non-Hodgkin Lymphoma; Plasma Cell Myeloma; Waldenstrom Macroglobulinemia

  12. Molecular approach to diagnose BCR/ABL negative chronic myeloproliferative neoplasms

    Directory of Open Access Journals (Sweden)

    Michelle Maccarini Barcelos

    2011-01-01

    Full Text Available Chronic myeloproliferative neoplasms arise from clonal proliferation of hematopoietic stem cells. According to the World Health Organization myeloproliferative neoplasms are classified as: chronic myelogenous leukemia, polycythemia vera, essential thrombocythemia, primary myelofibrosis, chronic neutrophilic leukemia, chronic eosinophilic leukemia, hypereosinophilic syndrome, mast cell disease, and unclassifiable myeloproliferative neoplasms. In the revised 2008 WHO diagnostic criteria for myeloproliferative neoplasms, mutation screening for JAK2V617F is considered a major criterion for polycythemia vera diagnosis and also for essential thrombocythemia and primary myelofibrosis, the presence of this mutation represents a clonal marker. There are currently two hypotheses explaining the role of the JAK2V617F mutation in chronic myeloproliferative neoplasms. According to these theories, the mutation plays either a primary or secondary role in disease development. The discovery of the JAK2V617F mutation has been essential in understanding the genetic basis of chronic myeloproliferative neoplasms, providing some idea on how a single mutation can result in three different chronic myeloproliferative neoplasm phenotypes. But there are still some issues to be clarified. Thus, studies are still needed to determine specific molecular markers for each subtype of chronic myeloproliferative neoplasm.

  13. BCR-ABL negative myeloproliferative neoplasia: a review of involved molecular mechanisms.

    Science.gov (United States)

    Koopmans, Suzanne M; Schouten, Harry C; van Marion, Ariënne M W

    2015-02-01

    The clonal bone marrow stem cell disorders essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) belong to the group of Philadelphia chromosome negative myeloproliferative neoplasia (Ph- MPN). In 2005 the JAK2(V617F) mutation was discovered which has generated more insight in the pathogenetic mechanism of the MPNs. More mutations have been detected in MPN patients since. However, the underlying cause of MPN has not been discovered so far. The mechanism of increased angiogenesis in MPNs and the development of fibrosis in the bone marrow in PMF patients and in some ET and PV patients is still not known. This review will focus on the most important molecular pathogenetic mechanisms in MPN patients. PMID:25196073

  14. Recent advances in the bcr-abl negative chronic myeloproliferative diseases

    Directory of Open Access Journals (Sweden)

    Stroncek David F

    2006-10-01

    Full Text Available Abstract The chronic myeloproliferative disorders are clonal hematopoietic stem cell disorders of unknown etiology. In one of these (chronic myeloid leukemia, there is an associated pathognomonic chromosomal abnormality known as the Philadelphia chromosome. This leads to constitutive tyrosine kinase activity which is responsible for the disease and is used as a target for effective therapy. This review concentrates on the search in the other conditions (polycythemia vera, essential thrombocythemia and idiopathic mylofibrosis for a similar biological marker with therapeutic potential. There is no obvious chromosomal marker in these conditions and yet evidence of clonality can be obtained in females by the use of X-inactivation patterns. PRV-1mRNA over expression, raised vitamin B12 levels and raised neutrophil alkaline phosphatase scores are evidence that cells in these conditions have received excessive signals for proliferation, maturation and reduced apoptosis. The ability of erythroid colonies to grow spontaneously without added external erythropoietin in some cases, provided a useful marker and a clue to this abnormal signaling. In the past year several important discoveries have been made which go a long way in elucidating the involved pathways. The recently discovered JAK2 V617F mutation which occurs in the majority of cases of polycythemia vera and in about half of the cases with the two other conditions, enables constitutive tyrosine kinase activity without the need for ligand binding to hematopoietic receptors. This mutation has become the biological marker for these conditions and has spurred the development of a specific therapy to neutralize its effects. The realization that inherited mutations in the thrombopoietin receptor (c-Mpl can cause a phenotype of thrombocytosis such as in Mpl Baltimore (K39N and in a Japanese family with S505A, has prompted the search for acquired mutations in this receptor in chronic myeloproliferative

  15. BCR/ABL-negative primitive progenitors suitable for transplantation can be selected from the marrow of most early-chronic phase but not accelerated-phase chronic myelogenous leukemia patients

    OpenAIRE

    Verfaillie, Catherine; R Bhatia; Miller, W.; F. Mortari; Van Roy, V.; Burger, S.; Mccullough, J; Stieglbauer, K; Dewald, G; Heimfeld, S; Miller, J. S.; McGlave, P B

    1996-01-01

    We have previously reported that selection of marrow cells on the basis of the CD34+HLA-DR- phenotype (34+DR-) may result in the recovery of Philadelphia chromosome (Ph)- and BCR/ABL-negative long-term culture-initiating cells (LTC-IC) in selected patients with chronic myelogenous leukemia (CML). We now present data on 27 early chronic-phase ([ECP] studied within 1 year after diagnosis) and 23 advanced-phase ([AP] late chronic phase, ie, studied >1 year from diagnosis, or accelerated phase) C...

  16. Expression of PTD-bcr/abl fusion oncoprotein fragment carrying protein transduction domain and its transmembrane transportation%PTD-bcr/abl融合癌蛋白片段的表达及其跨膜转运

    Institute of Scientific and Technical Information of China (English)

    梁英民; 孙强; 蒋姗姗; 陈萍; 王冀姝; 刘利; 韩骅

    2001-01-01

    Aim To explore the method of transfering oncoprotein bcr/abl, mediated by protein transduction domain (PTD), in order to provide the experimental basis for immune therapy. Methods DNA fragment encoding PTD was synthesized and fused with PCR-amplified bcr/abl gene fragment. The fusion protein was expressed in E.coli as His-tagged protein. The purified fusion protein was loaded to cultured HL-60 cells. Whether the fusion protein entered into HL-60 cells was determined by Western blot. Results It is shown that PTD could mediate transportation of bcr/abl protein to enter into cells. Conclusion These results may provide a new approach for loading exogenous proteins to antigen presenting cells.%目的探讨蛋白转导结构域 (PTD)介导的转导 bcr/abl癌蛋白片段的方法,为进行免疫治疗提供实验依据。方法合成编码 PTD的基因片段,并与 PCR扩增的慢性粒细胞白血病癌蛋白 bcr/abl基因片段融合 ,在大肠杆菌中表达。将纯化的融合蛋白 PTD-bcr/abl与 HL-60 细胞共孵育,用 Western blot分析融合蛋白的跨膜转运。结果 PTD基序可介导 bcr/abl蛋白由细胞外跨膜转导进入细胞内。结论这一结果可能为应用外源蛋白负载 (loading)抗原提呈细胞 (APC)提供新的途径。

  17. Myeloproliferative neoplasms (BCR-ABL1 negative) and myelodysplastic/myeloproliferative neoplasms: current diagnostic principles and upcoming updates.

    Science.gov (United States)

    Geyer, J T; Orazi, A

    2016-05-01

    Since the publication of the latest World Health Organization (WHO) classification in 2008, there has been a significant effort for clarification of unresolved questions, especially with the help of the rapidly developing field of molecular genetic studies, next-generation sequencing in particular. Numerous entities within the WHO categories of myeloproliferative neoplasms (MPNs) and myelodysplastic (MDS)/MPNs have been extensively studied, with large published series attempting to characterize and better define their morphologic and molecular genetic features. This emerging genetic landscape maintains a robust correlation with the various disease entities recognized by the WHO classification scheme based on a careful integration of detailed clinical information, bone marrow and peripheral blood morphology, immunohistology, and genomics. This brief review summarizes the current guidelines as they apply to diagnosing both the classical BCR-ABL1 negative MPN (polycythemia vera, essential thrombocythemia, and primary myelofibrosis) and the more common subtypes of MDS/MPN overlap syndromes. The more important recent molecular updates as well as the upcoming changes to the current WHO classification, expected to be published in late 2016, will also be briefly reviewed. PMID:27161873

  18. 荧光原位杂交在慢性粒细胞白血病细胞BCR/ABL融合基因检测中的应用及其意义%The detection of BCR/ABL fusion gene by interphase fluorescence in situ hybridization in patients with chronic myeloid leukemia and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    张济; 李君君; 颜家运; 邹礼衡; 刘静

    2012-01-01

    目的:探讨荧光原位杂交(FISH)技术检测慢性粒细胞白血病(CML)骨髓和外周血细胞的BCR /ABL融合基因的临床价值.方法:应用FISH 对正常对照组及CML患者骨髓和外周血细胞BCR/ ABL融合基因进行检测和分析.结果:慢性期CML 患者骨髓和外周血细胞该融合基因阳性细胞率分别为(61.9 ± 22.3)%和(68.4 ± 19.8)%,加速期为(77.2 ± 16.7)% 和(86.8 ± 12.1)%,急变期为(80.6 ± 17.5)%和(81.4 ± 18.0)%,两者细胞中BCR/ABL 基因的阳性率未见统计学差异(P>0.05),且骨髓和外周血细胞之间融合基因阳性细胞比率呈直线正相关.同时发现在完全临床缓解患者中,经伊马替尼治疗的CML 患者(71.4%)较经干扰素和羟基脲联合治疗者(10.0%)有更高的分子生物学缓解(P 0.05). A significant correlation was found between the BCR/ABL positive cells in peripheral blood and in bone marrow samples. Moreover, the higher rate of complete molecular remission was achieved by treating with imatinib than with interferon and hydroxyurea during complete remission phase (P < 0.05). Conclusions Detecting BCR/ABL fusion gene in peripheral blood and bone marrow samples by FISH are beneficial to diagnosis, treatment and minimal residual disease monitor in CML.

  19. Rapid Evolution to Blast Crisis Associated with a Q252H ABL1 Kinase Domain Mutation in e19a2 BCR-ABL1 Chronic Myeloid Leukaemia

    Directory of Open Access Journals (Sweden)

    Sarah L. McCarron

    2013-01-01

    Full Text Available A minority of chronic myeloid leukaemia (CML patients express variant transcripts of which the e19a2 BCR-ABL1 fusion is the most common. Instances of tyrosine kinase inhibitor (TKI resistance in e19a2 BCR-ABL1 CML patients have rarely been reported. A case of e19a2 BCR-ABL1 CML is described in whom imatinib resistance, associated with a Q252H ABL1 kinase domain mutation, became apparent soon after initiation of TKI therapy. The patient rapidly transformed to myeloid blast crisis (BC with considerable bone marrow fibrosis and no significant molecular response to a second generation TKI. The clinical course was complicated by comorbidities with the patient rapidly succumbing to advanced disease. This scenario of Q252H-associated TKI resistance with rapid BC transformation has not been previously documented in e19a2 BCR-ABL1 CML. This case highlights the considerable challenges remaining in the management of TKI-resistant BC CML, particularly in the elderly patient.

  20. SPR Detection and Discrimination of the Oligonucleotides Related to the Normal and the Hybrid bcr-abl Genes by Two Stringency Control Strategies

    Science.gov (United States)

    Matsishin, M. J.; Ushenin, Iu. V.; Rachkov, A. E.; Solatkin, A. P.

    2016-01-01

    In this study, we applied two stringency control strategies for surface plasmon resonance (SPR) detection of DNA hybridization and discrimination of completely and partially complementary 24-mer sequences. These sequences are specific to the human normal bcr and the hybrid bcr-abl genes, protein products of which are responsible for some leukemia. SPR sensors based on resonance phenomena in nanoscale gold films are well suited for label-free, real-time investigations of the macromolecule interactions. Thermodynamic parameters obtained using the web server DINAMelt allowed supposing the possibility for realization (a) stringency control based on the ionic strength of the hybridization buffer and (b) stringency control based on the temperature elevation. The first one resulted in that the discrimination index of completely complementary and partially complementary oligonucleotides depending on the target concentration varied from 1.3 to 1.8 in 2 × SSC and from 2.0 to 2.9 in 0.5 × SSC. For implementation of the second stringency control strategy, SPR spectrometer measuring flow cell with built-in high-precision temperature control and regulation as well as corresponding software was created. It is shown that the duplexes formed by the immobilized probes mod-Ph and completely complementary oligonucleotides P1 remained without significant changes until ~50 °C, while the duplexes formed with partially complementary oligonucleotide Bcrex14 almost entirely disrupted at 40 °C. Thus, the absolutely effective thermodiscrimination of this pair of oligonucleotides was achieved in this temperature range (40-50 °C).

  1. SPR Detection and Discrimination of the Oligonucleotides Related to the Normal and the Hybrid bcr-abl Genes by Two Stringency Control Strategies.

    Science.gov (United States)

    Matsishin, M J; Ushenin, Iu V; Rachkov, A E; Solatkin, A P

    2016-12-01

    In this study, we applied two stringency control strategies for surface plasmon resonance (SPR) detection of DNA hybridization and discrimination of completely and partially complementary 24-mer sequences. These sequences are specific to the human normal bcr and the hybrid bcr-abl genes, protein products of which are responsible for some leukemia. SPR sensors based on resonance phenomena in nanoscale gold films are well suited for label-free, real-time investigations of the macromolecule interactions. Thermodynamic parameters obtained using the web server DINAMelt allowed supposing the possibility for realization (a) stringency control based on the ionic strength of the hybridization buffer and (b) stringency control based on the temperature elevation. The first one resulted in that the discrimination index of completely complementary and partially complementary oligonucleotides depending on the target concentration varied from 1.3 to 1.8 in 2 × SSC and from 2.0 to 2.9 in 0.5 × SSC. For implementation of the second stringency control strategy, SPR spectrometer measuring flow cell with built-in high-precision temperature control and regulation as well as corresponding software was created. It is shown that the duplexes formed by the immobilized probes mod-Ph and completely complementary oligonucleotides P1 remained without significant changes until ~50 °C, while the duplexes formed with partially complementary oligonucleotide Bcrex14 almost entirely disrupted at 40 °C. Thus, the absolutely effective thermodiscrimination of this pair of oligonucleotides was achieved in this temperature range (40-50 °C). PMID:26759355

  2. BCR-ABL transcript variations in chronic phase chronic myelogenous leukemia patients on imatinib first-line: Possible role of the autologous immune system.

    Science.gov (United States)

    Clapp, Geoffrey D; Lepoutre, Thomas; Nicolini, Franck E; Levy, Doron

    2016-05-01

    Many chronic myelogenous leukemia (CML) patients in chronic phase who respond well to imatinib therapy show fluctuations in their leukemic loads in the long-term. We developed a mathematical model of CML that incorporates the intervention of an autologous immune response. Our results suggest that the patient's immune system plays a crucial role in imatinib therapy in maintaining disease control over time. The observed BCR-ABL/ABL oscillations in such patients provide a signature of the autologous immune response. PMID:27467931

  3. Coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites for the detection of BCR/ABL fusion gene

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xueping [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Wang, Li [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Department of Medical Laboratory, Chongqing Emergency Medical Center (Chongqing The Fourth Hospital), Chongqing, 400016 (China); Sheng, Shangchun [The No.2 Peoples' Hospital of Yibin, Sichuan, 644000 (China); Wang, Teng; Yang, Juan [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Xie, Guoming, E-mail: guomingxie@cqmu.edu.cn [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China); Feng, Wenli, E-mail: fengwlcqmu@sina.com [Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016 (China)

    2015-08-19

    This article described a novel method by coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites (GS/PANI/AuNPs) for highly sensitive and specific detection of BCR/ABL fusion gene (bcr/abl) in chronic myeloid leukemia (CML). DNA circuit known as catalyzed hairpin assembly (CHA) is enzyme-free and can be simply operated to achieve exponential amplification, which has been widely employed in biosensing. However, application of CHA has been hindered by the need of specially redesigned sequences for each single-stranded DNA input. Herein, a transducer hairpin (HP) was designed to obtain a universal DNA circuit with favorable signal-to-background ratio. To further improve signal amplification, GS/PANI/AuNPs with excellent conductivity and enlarged effective area were introduced into this DNA circuit. Consequently, by combining the advantages of CHA and GS/PANI/AuNPs, bcr/abl could be detected in a linear range from 10 pM to 20 nM with a detection limit of 1.05 pM. Moreover, this protocol showed excellent specificity, good stability and was successfully applied for the detection of real sample, which demonstrated its great potential in clinical application. - Highlights: • A transducer hairpin was designed to improve the versatility of DNA circuit. • GS/PANI/AuNPs were introduced to the DNA circuit for further signal amplification. • The established biosensor displayed high sensitivity and good specificity.

  4. Coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites for the detection of BCR/ABL fusion gene

    International Nuclear Information System (INIS)

    This article described a novel method by coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites (GS/PANI/AuNPs) for highly sensitive and specific detection of BCR/ABL fusion gene (bcr/abl) in chronic myeloid leukemia (CML). DNA circuit known as catalyzed hairpin assembly (CHA) is enzyme-free and can be simply operated to achieve exponential amplification, which has been widely employed in biosensing. However, application of CHA has been hindered by the need of specially redesigned sequences for each single-stranded DNA input. Herein, a transducer hairpin (HP) was designed to obtain a universal DNA circuit with favorable signal-to-background ratio. To further improve signal amplification, GS/PANI/AuNPs with excellent conductivity and enlarged effective area were introduced into this DNA circuit. Consequently, by combining the advantages of CHA and GS/PANI/AuNPs, bcr/abl could be detected in a linear range from 10 pM to 20 nM with a detection limit of 1.05 pM. Moreover, this protocol showed excellent specificity, good stability and was successfully applied for the detection of real sample, which demonstrated its great potential in clinical application. - Highlights: • A transducer hairpin was designed to improve the versatility of DNA circuit. • GS/PANI/AuNPs were introduced to the DNA circuit for further signal amplification. • The established biosensor displayed high sensitivity and good specificity

  5. RT-PCR ANALYSIS OF E2A-PBX1, TEL-AML1, BCR-ABL AND MLL-AF4 FUSION GENE TRANSCRIPTS IN B-LINEAGE ACUTE LYMPHOBLASTIC LEUKEMIA

    Directory of Open Access Journals (Sweden)

    Iuliu-Cristian Ivanov

    2013-11-01

    Full Text Available Acute lymphoblastic leukemia represents a heterogeneous group of hematological malignancies, defined by clonal proliferation of lymphoid cells. Immunophenotyping by flow cytometry and molecular analysis for the detection of genetic anomalies are clinical standard procedures for diagnosis, sub-classification and post-therapeutic evaluation. Samples from 105 patients diagnosed with acute lymphoblastic leukemia were immunophenotyped at diagnosis and were investigated by molecular analysis in order to identify the occurrence of four fusion genes: MLL-AF4, TEL-AML-1, BCR-ABL-p190, E2A-PBX-1. There were no associations found between the immunophenotype and the presence of any fusion genes evaluated. Both methods in combination remain a prerequisite for an improved subclassification of hematological malignancies, therapeutic decision, and evaluation of treatment response.

  6. Leucine replaced by methionine at 273 position in chronic myeloid leukemia: Knowns and unknowns…

    Directory of Open Access Journals (Sweden)

    Aditi Harsh Thanky

    2016-01-01

    Full Text Available Chronic myeloid leukemia is a clonal bone marrow stem cell disorder characterized by the presence of Philadelphia chromosome t(9;22(q34;q11 leading to fusion oncogene BCR-ABL. Tyrosine kinase inhibitors (TKIs act by competitively inhibiting BCR-ABL oncoprotein with significant response rates. However, up to 30% of patients fail to achieve complete cytogenetic remission on 1st line TKI imatinib, one of the reasons being mutations in BCR-ABL kinase domain leading to imatinib resistance. Over 80 such mutations have been documented in the literature; however, some of the rare mutations still remain to be studied for their impact in development of resistance and their responsiveness to currently available therapeutic options. Here, we report one such case of a rare mutation leucine replaced by methionine at 273 position and its clinical implications.

  7. Hypoxia-Like Signatures Induced by BCR-ABL Potentially Alter the Glutamine Uptake for Maintaining Oxidative Phosphorylation

    NARCIS (Netherlands)

    Sontakke, Pallavi; Koczula, Katarzyna M; Jaques, Jennifer; Wierenga, Albertus T J; Brouwers-Vos, Annet Z; Pruis, Maurien; Günther, Ulrich L; Vellenga, Edo; Schuringa, Jan Jacob

    2016-01-01

    The Warburg effect is probably the most prominent metabolic feature of cancer cells, although little is known about the underlying mechanisms and consequences. Here, we set out to study these features in detail in a number of leukemia backgrounds. The transcriptomes of human CB CD34+ cells transduce

  8. Optimization of novel combi-molecules: identification of balanced and mixed bcr-abl/DNA targeting properties.

    Science.gov (United States)

    Rachid, Zakaria; Katsoulas, Athanasia; Williams, Christopher; Larroque, Anne-Laure; McNamee, James; Jean-Claude, Bertrand J

    2007-08-01

    Steps toward the identification of combi-molecules with strong abl tyrosine kinase (TK) inhibitory property and significant DNA damaging potential are described. The optimized combi-molecule 13a was shown to induce approximately twofold stronger abl TK inhibitory activity than Gleevec and high levels of DNA damage in chronic myelogenous leukemic cells. PMID:17572088

  9. 伴不典型BCR-ABL融合基因的慢性髓性白血病的临床和实验研究%A clinical and laboratory study of chronic myeloid leukemia with atypical BCR-ABL fusion gene subtypes

    Institute of Scientific and Technical Information of China (English)

    桂晓敏; 潘金兰; 仇惠英; 岑建农; 薛永权; 陈苏宁; 沈宏杰; 姚利; 张俊

    2014-01-01

    目的 探讨伴不典型BCR-ABL融合基因亚型el4a3和e19a2型慢性髓性白血病(CML)的临床和实验室特点.方法 对2004至2012年染色体核型分析有t(9;22) (q34;q11),荧光原位杂交(FISH)证实为BCR-ABL融合基因阳性,而常规实时定量PCR(RQ-PCR)检测常见BCR-ABL融合基因(b3a2、b2a2和e1a2)阴性的6例CML患者,重新设计引物进行PCR扩增,并将扩增产物测序,以明确不典型BCR-ABL融合基因类型.对BCR-ABL融合基因扩增产物进行突变检测.对患者的临床资料进行回顾性分析.结果 6例患者PCR扩增产物经测序分析,其中5例为e14a3型,1例为e19a2型.5例e14a3型CML患者中,男4例,女1例,中位年龄48岁,慢性期4例,加速期1例;1例e19a2型患者为女性,40岁,CML慢性期,PLT>1 000× 109/L.5例e14a3型CML患者中4例在羟基脲或IFN治疗无效后予以伊马替尼(IM)治疗,1例行造血干细胞移植(HSCT).前4例患者中1例因有E255K突变而对IM耐药,改用达沙替尼后获完全细胞遗传学反应(CCyR);1例在IM治疗获CCyR后3个月复发并急变,最终死亡;2例IM治疗后获CCyR,目前状态稳定,仍处于CCyR,尽管其中1例伴有I293T突变.行HSCT治疗患者目前处于CCyR.1例e19a2型CML患者羟基脲治疗后获得完全血液学反应,后改用IM治疗,很快获得CCyR.结论 伴不典型BCR-ABL融合基因的CML发病率极低,酪氨酸激酶抑制剂或HSCT都可以取得疗效,常规RQ-PCR可能漏检少见的不典型BCR-ABL融合基因亚型.%Objective To explore the clinical and laboratory features of chronic myeloid leukemia (CML) with atypical e14a3 and e19a2 BCR-ABL fusion gene subtypes.Methods We retrospectively analyzed a cohort of CML patients with Ph chromosome positive confirmed by cytogenetic and FISH but classical el3a3 (b2a2),e14a2 (b3a2)and ela2 fusion transcripts negative identified by conventional realtime quantification RT-PCR (RQ-PCR).Further RQ-PCR was done with the forward primer and reverse primer designed to

  10. A combination of STI571 and BCR-ABL1 siRNA with overexpressed p15INK4B induced enhanced proliferation inhibition and apoptosis in chronic myeloid leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Xia, D.Y.; Liu, L.; Hao, M.W.; Liu, Q.; Chen, R.A.; Liang, Y.M. [Department of Hematology, Tangdu Hospital, Fourth Military Medical University, Xi' an (China)

    2014-10-14

    p15INK4B, a cyclin-dependent kinase inhibitor, has been recognized as a tumor suppressor. Loss of or methylation of the p15INK4B gene in chronic myeloid leukemia (CML) cells enhances myeloid progenitor formation from common myeloid progenitors. Therefore, we examined the effects of overexpressed p15INK4B on proliferation and apoptosis of CML cells. Overexpression of p15INK4B inhibited the growth of K562 cells by downregulation of cyclin-dependent kinase 4 (CDK4) and cyclin D1 expression. Overexpression of p15INK4B also induced apoptosis of K562 cells by upregulating Bax expression and downregulating Bcl-2 expression. Overexpression of p15INK4B together with STI571 (imatinib) or BCR-ABL1 small interfering RNA (siRNA) also enhanced growth inhibition and apoptosis induction of K562 cells. The enhanced effect was also mediated by reduction of cyclin D1 and CDK4 and regulation of Bax and Bcl-2. In conclusion, our study may provide new insights into the role of p15INK4B in CML and a potential therapeutic target for overcoming tyrosine kinase inhibitor resistance in CML.

  11. BCR-ABL1: Test

    Science.gov (United States)

    ... Complete Blood Count ; Bone Marrow Aspiration and Biopsy ; Blood Smear ; WBC Differential At a Glance Test Sample The ... are often evaluated as part of the initial diagnosis, but the majority of follow-up monitoring is performed on blood samples. There is significant test variability among laboratories ...

  12. First Case of Biphenotypic/bilineal (B/myeloid, B/monocytic) Mixed Phenotype Acute Leukemia with t(9;22)(q34;q11.2);BCR-ABL1.

    Science.gov (United States)

    Kim, Hyeong Nyeon; Hur, Mina; Kim, Hanah; Ji, Misuk; Moon, Hee-Won; Yun, Yeo-Min; Lee, Mark Hong

    2016-07-01

    Mixed phenotype acute leukemia (MPAL) includes biphenotypic leukemia, bilineal leukemia, or its combination by the 2008 WHO classification. A few cases of combined biphenotypic/bilineal MPAL have been reported so far; they all had biphenotypic expressions in only one of the two distinct leukemic populations. A 43-year-old female presented with leukocytosis and bicytopenia. Her complete blood counts were: hemoglobin, 6.9 g/dL; white blood cells, 62.8×10(9)/L; and platelets, 83×10(9)/L. Neither lymphadenopathy nor organomegaly was observed. Blasts and promonocytes/monoblasts were increased in her peripheral blood (42%) and bone marrow (60.1%). Flow cytometric analysis revealed two distinct populations of leukemic cells, which expressed CD11c, CD19, and cytoplasmic CD79a in common. Additionally, the first population expressed CD10 and CD117 (B/myeloid), and the second one expressed CD14 and CD20 (B/monocytic). She had a karyotype of 46,XX,inv(9)(p12q13),t(9;22)(q34;q11.2)[20] and BCR/ABL1 rearrangement. To the best of our knowledge, this is the first reported case of biphenotypic/bilineal MPAL with B/myeloid and B/monocytic expressions.

  13. Standardization of real-time quantitative polymerase chain reaction for detection of the JAK2V617F mutation in BCR-ABL1 negative myeloproliferative neoplasms: A tertiary care centre experience

    Directory of Open Access Journals (Sweden)

    Tathagat Chatterjee

    2015-01-01

    Full Text Available Background: Identification of JAK2V617F mutations has led to a significant development in our understanding of the pathogenesis and therapy of BCR-ABL1 negative myeloproliferative neoplasms (MPNs. However, not all cases of BCR-ABL1 negative MPNs carry JAK2V617F mutations. The present study was undertaken with an aim to standardize the real-time quantitative polymerase chain reaction (PCR for the detection of JAK2V617F mutations and to find the prevalence of Janus kinase 2 (JAK2 mutations in MPNs in the Indian scenario. Materials and Methods: Real-time quantitative PCR was used to detect the JAK2V617F mutation. Standardization of the detection procedure was carried out using recommended guidelines to ensure the accuracy and reproducibility of results. Forty-nine patients of BCR-ABL1 negative MPNs were included in the study. Results: The JAK2V617F mutation was detected in 63.3% patients of BCR-ABL1 negative MPNs. On classification of these BCR-ABL1 negative MPNs, JAK2V617F mutation was detected in 78.3% patients with polycythemia vera (PV, 62.5% patients with essential thrombocythemia (ET, and 44.4% patients with Primary myelofibrosis (PMF. Conclusion: Role of detection of JAK2 mutations in BCR-ABL1 negative MPN has begun to be described and can be used in the diagnosis of PV, ET, and PMF along with other criterias. In future, it may be suitable for treatment monitoring and prognostication.

  14. Safranal, a Crocus sativus L constituent suppresses the growth of K-562 cells of chronic myelogenous leukemia. In silico and in vitro study.

    Science.gov (United States)

    Geromichalos, George D; Papadopoulos, Theophanis; Sahpazidou, Despina; Sinakos, Zacharias

    2014-12-01

    Crocin, a main constituent of Crocus sativus L (saffron), has been found to inhibit the growth of K-562 human chronic myelogenous leukemia (CML) cells expressing Bcr-Abl protein tyrosine kinase activity. The aim of our study is to investigate the ability of the bioactive saffron's constituents, crocin (CRC) and safranal (SFR), to inhibit the Bcr-Abl protein activity employing an in silico approach, as well as the in vitro effect of these compounds on K-562 growth and gene expression of Bcr-Abl. In silico molecular docking studies revealed that mostly SFR can be attached to Bcr-Abl protein, positioned inside the protein's binding cavity at the same place with the drug used in the treatment of CML, imatinib mesylate (IM). The predicted polar interactions and hydrophobic contacts constructing a hydrophobic cavity inside the active site, explain the observed inhibitory activity. Cytotoxicity experiments showed that SFR and CRC mediate cytotoxic response to K562 cells. In vitro studies on the expression of Bcr-Abl gene revealed that SFR and in a lesser degree IM inhibited the expression of the gene, while in contrast CRC induced an increase. The ultimate goal was to evaluate the existence of a potential antitumor activity of saffron's constituents SFR and CRC.

  15. Total-Body Irradiation With or Without Fludarabine Phosphate Followed By Donor Stem Cell Transplant in Treating Patients With Hematologic Cancer

    Science.gov (United States)

    2016-02-02

    Acute Lymphoblastic Leukemia in Remission; Acute Myeloid Leukemia in Remission; Aggressive Non-Hodgkin Lymphoma; Chronic Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Diffuse Large B-Cell Lymphoma; Hematopoietic and Lymphoid Cell Neoplasm; Indolent Non-Hodgkin Lymphoma; Mantle Cell Lymphoma; Myelodysplastic/Myeloproliferative Neoplasm; Plasma Cell Myeloma; Refractory Chronic Lymphocytic Leukemia; Refractory Hodgkin Lymphoma; Waldenstrom Macroglobulinemia

  16. Which method better evaluates the molecular response in newly diagnosed chronic phase chronic myeloid leukemia patients with imatinib treatment, BCR-ABL(IS) or log reduction from the baseline level?

    Science.gov (United States)

    Qin, Ya-Zhen; Jiang, Qian; Jiang, Hao; Li, Jin-Lan; Li, Ling-Di; Zhu, Hong-Hu; Lai, Yue-Yun; Lu, Xi-Jing; Liu, Yan-Rong; Jiang, Bin; Huang, Xiao-Jun

    2013-09-01

    The molecular response of chronic myeloid leukemia (CML) patients to tyrosine kinase inhibitor treatment can be evaluated either by BCR-ABL mRNA levels on international scale (IS) or by log reduction from the baseline level of the laboratory. Both methods were compared in 248 newly diagnosed chronic phase CML patients treated with imatinib. The major molecular responses (MMR) obtained by both methods predict progression-free survival (PFS, all Plog reduction method, had the same PFS as MMR patients identified by both methods. The molecular responses of patients at 3 and 6 months, as evaluated by the two methods, have similar predictive values on their cytogenetic responses at 12 months and on their molecular responses at 18 months. Both ≤ 10%(IS) and ≥ 1 log reduction at 3 months and ≤ 1%(IS) at 6 months were significantly associated with PFS (P=0.0011, 0.0090, and 0.0064). The percentages of patients with BCR-ABL(IS) of ≤ 1%, >1-10%, and of >10% at 3 months and 6 months in the German CML Study IV were similar with those with corresponding BCR-ABL(IS) in our center, but was significantly different with those evaluated by the log reduction method. Therefore, the molecular response evaluated by BCR-ABL(IS) has similar trends in PFS and in response prediction, but can better differentiate patients than that by the log reduction method. Furthermore, the IS method allows comparison among molecular response results from different laboratories.

  17. 自身淬灭荧光定量PCR检测慢性粒细胞白血病bcr/abl mRNA%Quantification of bcr/abl mRNA in patients with chronic myeloid leukemia by using real-time quantitative fluorescence PCR with self-quenched primer

    Institute of Scientific and Technical Information of China (English)

    彭辉; 冯文莉; 王小中; 曾建明; 肖青; 潘健; 曹唯希; 罗云萍; 黄宗干

    2007-01-01

    目的 利用自身淬灭探针技术建立一种能检测慢性粒细胞白血病(CML)bcr/abl融合基因mRNA的实时荧光定量PCR方法,为CML的诊断、疗效观察以及微量残留白血病(MRD)的监测提供有效手段.方法 用逆转录PCR方法(RT-PCR)扩增K562细胞的bcr/abl融合基因,A-T克隆法构建定量标准模板;设计自身淬灭荧光引物(探针),建立自身淬灭荧光定量逆转录PCR方法(FQRT-PCR),并对该方法的线性检测范围、灵敏度、重复性、稳定性进行检测;然后检测临床白血病患者骨髓标本bcr/abl mRNA.结果 建立的FQ-RT-PCR方法可检测10 copies/μl的bcr/abl重组质粒,并能从105个正常细胞中检出1个白血病细胞,该方法的批内、批间变异系数(CV)分别为2.1%、6.1%,线性检测范围为102~109 copies/μl.25例CML患者bcr/abl融合基因mRNA表达量中位数为4.50×104[(0.45~89.00)×104]copies/μg RNA,其中11例慢性期初诊患者bcr/abl mRNA表达量中位数为5.45×104[(2.95~19.30)×104]copies/μg RNA,6例急变期患者bcr/abl mRNA表达量中位数为13.00×104[(4.10~89.00)×104]copies/μg RNA,8例慢性期治疗后复查患者bcr/ablmRNA表达量中位数为2.35×104[(0.45~5.12)×104]copies/μg RNA,CML急变期患者bcr/abl融合基因表达水平与慢性期患者之间差异有统计学意义(q=3.41,P<0.05).PCR产物经电泳分析,其中21例CML患者为b3a2型,4例CML患者为b2a2型;3例急性淋巴细胞白血病(ALL)患者中,1例有bcr/abl mRNA表达,为b2a2型.结论 所建立的基于自身淬灭探针技术的实时荧光定量PCR检测方法灵敏、特异、重复性好,结果用拷贝数表示,准确可靠,利于标准统一.可广泛用于CML的诊断、疗效观察以及MRD的监测.

  18. Relationship between JAK2 gene V617F mutation and clinical characteristics in patients with BCR-ABL-negative myeloproliferative neoplasms%BCR-ABL阴性的骨髓增殖性肿瘤JAK2基因V617F突变分析

    Institute of Scientific and Technical Information of China (English)

    张燕香; 魏蓉

    2013-01-01

    ,42 subjects harbored V617F mutation (64.62%).The positive rates of V617F mutation in PV and ET patients were 75.00% (21 subjects) and 56.67% (17 subjects),respectively.Four of the 7 patients with IMF were V617F mutation positive.For PV patients,the levels of white blood cell count and platelet count in V617F mutation carriers were significantly higher than that in non-carriers (P<0.05).For ET patients,the levels of white blood cell count and hemoglobin in V617F mutation carriers were significantly higher than that in non-carriers (P<0.05).For IMF patients,however,no significant differences were found between mutation carriers and non-carriers.The incidence of thrombosis in V617F mutation carriers was higher than that in non-carriers (P<0.001).Conclusions:V617F mutation in JAK2 is a major molecular genetic marker for Chinese patients with BCR-ABL-negative myeloproliferative neoplasms.This mutation has impacts on clinical characteristics and can be used as a criterion for the diagnosis of BCR-ABL-negative myeloproliferative neoplasm.

  19. A phase 2 study of MK-0457 in patients with BCR-ABL T315I mutant chronic myelogenous leukemia and philadelphia chromosome-positive acute lymphoblastic leukemia

    DEFF Research Database (Denmark)

    Seymour, J F; Kim, D W; Rubin, E;

    2014-01-01

    achieved major hematologic response. The most common adverse event (AE) was neutropenia (50%). The most common grade 3/4 AEs were neutropenia (46%) and febrile neutropenia (35%). MK-0457 demonstrated minimal efficacy and only at higher, intolerable doses; lower doses were tolerated and no unexpected...

  20. Pharmacophore Modeling of Nilotinib as an Inhibitor of ATP-Binding Cassette Drug Transporters and BCR-ABL Kinase Using a Three-Dimensional Quantitative Structure–Activity Relationship Approach

    OpenAIRE

    Shukla, Suneet; Kouanda, Abdul; Silverton, Latoya; Talele, Tanaji T.; Suresh V Ambudkar

    2014-01-01

    Nilotinib (Tasigna) is a tyrosine kinase inhibitor approved by the FDA to treat chronic phase chronic myeloid leukemia patients. It is also a transport substrate of the ATP-binding cassette (ABC) drug efflux transporters ABCB1 (P-glycoprotein, P-gp) and ABCG2 (BCRP), which may have an effect on the pharmacokinetics and toxicity of this drug. The goal of this study was to identify pharmacophoric features of nilotinib in order to potentially develop specific inhibitors of BCR-ABL kinase with mi...

  1. Graft-Versus-Host Disease Prophylaxis in Treating Patients With Hematologic Malignancies Undergoing Unrelated Donor Peripheral Blood Stem Cell Transplant

    Science.gov (United States)

    2016-08-18

    Acute Lymphoblastic Leukemia; Acute Myeloid Leukemia; Aggressive Non-Hodgkin Lymphoma; Chronic Lymphocytic Leukemia; Diffuse Large B-Cell Lymphoma; Hematopoietic and Lymphoid Cell Neoplasm; Indolent Non-Hodgkin Lymphoma; Mantle Cell Lymphoma; Myelodysplastic Syndrome; Myeloproliferative Neoplasm; Prolymphocytic Leukemia; Recurrent Chronic Lymphocytic Leukemia; Recurrent Plasma Cell Myeloma; Refractory Chronic Lymphocytic Leukemia; Refractory Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Refractory Hodgkin Lymphoma; Small Lymphocytic Lymphoma; T-Cell Chronic Lymphocytic Leukemia; Waldenstrom Macroglobulinemia

  2. Fludarabine Phosphate, Cyclophosphamide, Total-Body Irradiation, and Donor Bone Marrow Transplant Followed by Donor Natural Killer Cell Therapy, Mycophenolate Mofetil, and Tacrolimus in Treating Patients With Hematologic Cancer

    Science.gov (United States)

    2016-06-07

    Acute Lymphoblastic Leukemia; Acute Myeloid Leukemia; Aggressive Non-Hodgkin Lymphoma; Diffuse Large B-Cell Lymphoma; Previously Treated Myelodysplastic Syndrome; Recurrent Chronic Lymphocytic Leukemia; Recurrent Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Recurrent Indolent Adult Non-Hodgkin Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Plasma Cell Myeloma; Recurrent Small Lymphocytic Lymphoma; Refractory Chronic Lymphocytic Leukemia; Refractory Hodgkin Lymphoma; Refractory Plasma Cell Myeloma; Refractory Small Lymphocytic Lymphoma; Waldenstrom Macroglobulinemia

  3. Interferon-α Revisited: Individualized Treatment Management Eased the Selective Pressure of Tyrosine Kinase Inhibitors on BCR-ABL1 Mutations Resulting in a Molecular Response in High-Risk CML Patients.

    Science.gov (United States)

    Polivkova, Vaclava; Rohon, Peter; Klamova, Hana; Cerna, Olga; Divoka, Martina; Curik, Nikola; Zach, Jan; Novak, Martin; Marinov, Iuri; Soverini, Simona; Faber, Edgar; Machova Polakova, Katerina

    2016-01-01

    Bone marrow transplantation or ponatinib treatment are currently recommended strategies for management of patients with chronic myeloid leukemia (CML) harboring the T315I mutation and compound or polyclonal mutations. However, in some individual cases, these treatment scenarios cannot be applied. We used an alternative treatment strategy with interferon-α (IFN-α) given solo, sequentially or together with TKI in a group of 6 cases of high risk CML patients, assuming that the TKI-independent mechanism of action may lead to mutant clone repression. IFN-α based individualized therapy decreases of T315I or compound mutations to undetectable levels as assessed by next-generation deep sequencing, which was associated with a molecular response in 4/6 patients. Based on the observed results from immune profiling, we assumed that the principal mechanism leading to the success of the treatment was the immune activation induced with dasatinib pre-treatment followed by restoration of immunological surveillance after application of IFN-α therapy. Moreover, we showed that sensitive measurement of mutated BCR-ABL1 transcript levels augments the safety of this individualized treatment strategy. PMID:27214026

  4. Interferon-α Revisited: Individualized Treatment Management Eased the Selective Pressure of Tyrosine Kinase Inhibitors on BCR-ABL1 Mutations Resulting in a Molecular Response in High-Risk CML Patients.

    Directory of Open Access Journals (Sweden)

    Vaclava Polivkova

    Full Text Available Bone marrow transplantation or ponatinib treatment are currently recommended strategies for management of patients with chronic myeloid leukemia (CML harboring the T315I mutation and compound or polyclonal mutations. However, in some individual cases, these treatment scenarios cannot be applied. We used an alternative treatment strategy with interferon-α (IFN-α given solo, sequentially or together with TKI in a group of 6 cases of high risk CML patients, assuming that the TKI-independent mechanism of action may lead to mutant clone repression. IFN-α based individualized therapy decreases of T315I or compound mutations to undetectable levels as assessed by next-generation deep sequencing, which was associated with a molecular response in 4/6 patients. Based on the observed results from immune profiling, we assumed that the principal mechanism leading to the success of the treatment was the immune activation induced with dasatinib pre-treatment followed by restoration of immunological surveillance after application of IFN-α therapy. Moreover, we showed that sensitive measurement of mutated BCR-ABL1 transcript levels augments the safety of this individualized treatment strategy.

  5. Switching to second-generation tyrosine kinase inhibitor improves the response and outcome of frontline imatinib-treated patients with chronic myeloid leukemia with more than 10% of BCR-ABL/ABL ratio at 3 months

    Science.gov (United States)

    Casado, Luis-Felipe; García-Gutiérrez, José-Valentín; Massagué, Isabel; Giraldo, Pilar; Pérez-Encinas, Manuel; de Paz, Raquel; Martínez-López, Joaquín; Bautista, Guiomar; Osorio, Santiago; Requena, María-José; Palomera, Luis; Peñarrubia, María-Jesús; Calle, Carmen; Hernández-Rivas, José-Ángel; Burgaleta, Carmen; Maestro, Begoña; García-Ormeña, Nuria; Steegmann, Juan-Luis

    2015-01-01

    Chronic myeloid leukemia patients display heterogeneous responses to imatinib. Survival depends on baseline clinical characteristics (including prognostic scoring systems) and on early response (such as >10% BCR-ABL/ABL ratio at 3 months of therapy). The results of switching to second-generation tyrosine kinase inhibitors (2GTKIs) may contain a bias since, in the majority of these studies, patients who switch treatment due to intolerance or failure are censored or excluded. We analyzed the Spanish Registry data on switching in an intention-to-treat analysis of patients in standard clinical practice. Switching to 2GTKIs improves responses from 45% to 75% of complete cytogenetic response (CCyR) and from 15% to 45% of major molecular response (MMR) in the group without molecular response 1 (MR1) at 3 months and from 70% to 87% in CCyR and from 52% to 87% in MMR in the group with MR1. The final response rate is poorer in the group with no MR1 at 3 months. Nevertheless, the differences in the rates of response were not translated into differences in major events (transformations or deaths), and the final progression-free survival and overall survival were similar. PMID:25756742

  6. Estandarización de la TR-PCR para la detección de las fusiones génicas BCR-ABL en pacientes con leucemia Mieloide Crónica (LMC y Linfoide Aguda (LLA provenientes de HUSVP y Clíncia León XIII

    Directory of Open Access Journals (Sweden)

    Gonzálo Vásquez Palacio

    2006-04-01

    Full Text Available La translocación recíproca t(9:22(q34;q11 involucra el proto-oncogen ABL y el gen BCR, originando un gen de fusión BCR-ABL, que codifica una proteína con elevada actividad tirosina quinasa, implicada en la leucemogénesis.

  7. Rapid identification of compound mutations in patients with Ph-positive leukemias by long-range next generation sequencing

    Science.gov (United States)

    Kastner, R.; Zopf, A.; Preuner, S.; Pröll, J.; Niklas, N.; Foskett, P.; Valent, P.; Lion, T.; Gabriel, C.

    2016-01-01

    An emerging problem in patients with Ph-positive leukemias is the occurrence of cells with multiple mutations in the BCR-ABL1 tyrosine kinase domain (TKD) associated with high resistance to different tyrosine kinase inhibitors. Rapid and sensitive detection of leukemic subclones carrying such changes, referred to as compound mutations, is therefore of increasing clinical relevance. However, current diagnostic methods including next generation sequencing (NGS) of short fragments do not optimally meet these requirements. We have therefore established a long-range (LR) NGS approach permitting massively parallel sequencing of the entire TKD length of 933bp in a single read using 454 sequencing with the GS FLX+ instrument (454 Life Sciences). By testing a series of individual and consecutive specimens derived from six patients with chronic myeloid leukemia, we demonstrate that long-range NGS analysis permits sensitive identification of mutations and their assignment to the same or to separate subclones. This approach also facilitates readily interpretable documentation of insertions and deletions in the entire BCR-ABL1 TKD. The long-range NGS findings were reevaluated by an independent technical approach in select cases. PCR amplicons of the BCR-ABL1 TKD derived from individual specimens were subcloned into pGEM®-T plasmids, and >100 individual clones were subjected to analysis by Sanger sequencing. The NGS results were confirmed, thus documenting the reliability of the new technology. Long-range NGS analysis therefore provides an economic approach to the identification of compound mutations and other genetic alterations in the entire BCR-ABL1 TKD, and represents an important advancement of the diagnostic armamentarium for rapid assessment of impending resistant disease. PMID:24365090

  8. Potent, transient inhibition of BCR-ABL with dasatinib 100 mg daily achieves rapid and durable cytogenetic responses and high transformation-free survival rates in chronic phase chronic myeloid leukemia patients with resistance, suboptimal response or intolerance to imatinib

    Science.gov (United States)

    Shah, Neil P.; Kim, Dong-Wook; Kantarjian, Hagop; Rousselot, Philippe; Llacer, Pedro Enrique Dorlhiac; Enrico, Alicia; Vela-Ojeda, Jorge; Silver, Richard T.; Khoury, Hanna Jean; Müller, Martin C.; Lambert, Alexandre; Matloub, Yousif; Hochhaus, Andreas

    2010-01-01

    Background Dasatinib 100 mg once daily achieves intermittent BCR-ABL kinase inhibition and is approved for chronic-phase chronic myeloid leukemia patients resistant or intolerant to imatinib. To better assess durability of response to and tolerability of dasatinib, data from a 2-year minimum follow-up for a dose-optimization study in chronic-phase chronic myeloid leukemia are reported here. Design and Methods In a phase 3 study, 670 chronic-phase chronic myeloid leukemia patients with resistance, intolerance, or suboptimal response to imatinib were randomized to dasatinib 100 mg once-daily, 50 mg twice-daily, 140 mg once-daily, or 70 mg twice-daily. Results Data from a 2-year minimum follow-up demonstrate that dasatinib 100 mg once daily achieves major cytogenetic response and complete cytogenetic response rates comparable to those in the other treatment arms, and reduces the frequency of key side effects. Comparable 2-year progression-free survival and overall survival rates were observed (80% and 91%, respectively, for 100 mg once daily, and 75%–76% and 88%–94%, respectively, in other arms). Complete cytogenetic responses were achieved rapidly, typically by 6 months. In patients treated with dasatinib 100 mg once daily for 6 months without complete cytogenetic response, the likelihood of achieving such a response by 2 years was 50% for patients who had achieved a partial cytogenetic response, and only 8% or less for patients with minor, minimal, or no cytogenetic response. Less than 3% of patients suffered disease transformation to accelerated or blast phase. Conclusions Intermittent kinase inhibition can achieve rapid and durable responses, indistinguishable from those achieved with more continuous inhibition. PMID:20139391

  9. Selective Depletion of CD45RA+ T Cells From Allogeneic Peripheral Blood Stem Cell Grafts in Preventing GVHD in Children

    Science.gov (United States)

    2016-10-06

    Accelerated Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Acute Biphenotypic Leukemia; Acute Leukemia of Ambiguous Lineage; Acute Myeloid Leukemia in Remission; Acute Undifferentiated Leukemia; Adult Acute Lymphoblastic Leukemia in Remission; Blast Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Childhood Acute Lymphoblastic Leukemia in Remission; Chronic Myelogenous Leukemia, BCR-ABL1 Positive; RAEB-1; RAEB-2; Recurrent Adult Acute Myeloid Leukemia; Recurrent Childhood Acute Lymphoblastic Leukemia; Recurrent Childhood Acute Myeloid Leukemia; Refractory Adult Acute Lymphoblastic Leukemia; Refractory Childhood Acute Lymphoblastic Leukemia

  10. Open Label, Phase II Study to Evaluate Efficacy and Safety of Oral Nilotinib in Philadelphia Positive (Ph+) Chronic Myelogenous Leukemia (CML) Pediatric Patients.

    Science.gov (United States)

    2016-10-07

    Leukemia; Leukemia,Pediatric; Leukemia, Myleiod; Leukemia, Mylegenous, Chronic; Leukemia, Mylegenous, Accelerated; BCR-ABL Positive; Myeloproliferative Disorder; Bone Marrow Disease; Hematologic Diseases; Neoplastic Processes; Imatinib; Dasatinib; Enzyme Inhibitor; Protein Kinase Inhibitor

  11. Sirolimus, Cyclosporine, and Mycophenolate Mofetil in Preventing Graft-versus-Host Disease in Treating Patients With Hematologic Malignancies Undergoing Donor Peripheral Blood Stem Cell Transplant

    Science.gov (United States)

    2016-09-06

    Adult Acute Lymphoblastic Leukemia; Adult Acute Myeloid Leukemia; Adult Diffuse Large B-Cell Lymphoma; Adult Myelodysplastic Syndrome; Adult Non-Hodgkin Lymphoma; Aggressive Non-Hodgkin Lymphoma; Childhood Acute Lymphoblastic Leukemia; Childhood Acute Myeloid Leukemia; Childhood Diffuse Large B -Cell Lymphoma; Childhood Myelodysplastic Syndrome; Childhood Non-Hodgkin Lymphoma; Chronic Lymphocytic Leukemia; Chronic Lymphocytic Leukemia in Remission; Chronic Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Hematopoietic and Lymphoid Cell Neoplasm; Mantle Cell Lymphoma; Plasma Cell Myeloma; Prolymphocytic Leukemia; Recurrent Chronic Lymphocytic Leukemia; Recurrent Diffuse Large B-Cell Lymphoma; Recurrent Hodgkin Lymphoma; Refractory Chronic Lymphocytic Leukemia; T-Cell Chronic Lymphocytic Leukemia; T-Cell Prolymphocytic Leukemia; Waldenstrom Macroglobulinemia

  12. A rare case of a three way complex variant positive Philadelphia translocation involving chromosome (9;11;22)(q34;p15;q11) in chronic myeloid leukemia: A case report

    Science.gov (United States)

    Asif, Muhammad; Hussain, Abrar; Rasool, Mahmood

    2016-01-01

    The t(9;22)(q34;q11) translocation is present in 90–95% of patients with chronic myeloid leukemia (CML). Variant complex translocations have been observed in 5–8% of CML patients, in which a third chromosome other than (9;22) is involved. Imatinib mesylate is the first line breakpoint cluster region-Abelson gene (BCR/ABL)-targeted oral therapy for CML, and may produce a complete response in 70–80% of CML patients in the chronic phase. In the present study, a bone marrow sample was used for conventional cytogenetic analysis, and the fluorescence in situ hybridization (FISH) test was used for BCR/ABL gene detection. A hematological analysis was also performed to determine the white blood cell (WBC) count, red blood cell count, hemoglobin levels, packed and mean cell volumes, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and platelet values of the patient. The hematological analysis of the patient indicated the increased WBC of 186.5×103 cells/µl, and decreased hemoglobin levels of 11.1 g/dl. The FISH test revealed that 67% cells demonstrated BCR/ABL gene translocation. The patient was treated with 400 mg imatinib mesylate daily, and was monitored at various intervals over a 6-month period. The present study reports the rare case of a patient that demonstrates a three-way Philadelphia chromosome-positive translocation involving 46XY,t(9;11;22)(q34;p15;q11)[10], alongside CML in the chronic phase. The translocation was analyzed using cytogenetic and FISH tests. PMID:27602125

  13. Dasatinib inhibits proliferation and activation of CD8+ T-lymphocytes by down-regulation of the T-cell receptor signaling

    OpenAIRE

    Fei, Fei

    2007-01-01

    The novel Src/Abl inhibitor dasatinib (BMS-354825) is a promising therapeutic agent with imatinib-resistant BCR-ABL mutations in patients with chronic myelogenous leukemia (CML) or Ph-positive acute lymphoblastic leukemia (ALL). However, little is known about its effects on T-cell function, especially for patients undergoing allogeneic transplantation for leukemia. Here, we demonstrate that dasatinib at a concentration of 5 nM to 250 nM inhibits the proliferation and activation of CD8+ T-lymp...

  14. Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Okabe, Seiichi, E-mail: okabe@tokyo-med.ac.jp; Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

    2013-06-07

    Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance.

  15. Sustaining integrating imatinib and interferon-α into maintenance therapy improves survival of patients with Philadelphia positive acute lymphoblastic leukemia ineligible for allogeneic stem cell transplantation.

    Science.gov (United States)

    Kuang, Pu; Liu, Ting; Pan, Ling; Zhu, Huanling; Wu, Yu; Ye, Yuanxin; Xiang, Bing; Ma, Hongbing; Chang, Hong; Niu, Ting; Cui, Xu; He, Chuan; Li, Jianjun; Ji, Jie; Huang, Jie; Dong, Tian; Dai, Yang; Lu, Xiaojun; Qing, Shenglan; Wu, Huaxin; Liang, Xiaogong; Wang, Xiaoyu; Wu, Chunnong

    2016-10-01

    We report the clinical results of sustainedly integrating imatinib and interferon-α into maintenance therapy in the patients ineligible for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Maintenance therapy lasted for 5 years with imatinib 400 mg daily, interferon-α 3 million units, 2∼3 doses per week, and chemotherapy including vindesine and dexamethasone scheduled monthly in first year, once every 2 months in second year, and once every 3 months in third year. The chemotherapy was discontinued after 3 years and the imatinib and interferon-α continued for another 2 years. For 41 patients without allo-HSCT with a median follow-up of 32 months, the 3-year DFS and OS were 42.7  ± 8.6% and 57.9  ± 8.4%, respectively. Our study suggests that sustaining maintenance with low-dose chemotherapy, imatinib and interferon-α improved survival of adult Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) patients ineligible for allo-HSCT, and even provided an opportunity for cure. BCR/ABL persistent negativity at 6 and 9 months may have benefit to choose suitable patients for the imatinib/interferon-α maintenance strategy. PMID:26879808

  16. Quantitative assessment of the CD26+ leukemic stem cell compartment in chronic myeloid leukemia: patient-subgroups, prognostic impact, and technical aspects.

    Science.gov (United States)

    Culen, Martin; Borsky, Marek; Nemethova, Veronika; Razga, Filip; Smejkal, Jiri; Jurcek, Tomas; Dvorakova, Dana; Zackova, Daniela; Weinbergerova, Barbora; Semerad, Lukas; Sadovnik, Irina; Eisenwort, Gregor; Herrmann, Harald; Valent, Peter; Mayer, Jiri; Racil, Zdenek

    2016-05-31

    Little is known about the function and phenotype of leukemic stem cells (LSCs) in chronic myeloid leukemia (CML) or about specific markers that discriminate LSCs from normal hematopoietic stem cells (HSCs). CD26 has recently been described as a specific marker of CML LSCs. In the current study, we investigated this marker in a cohort of 31 unselected CML patients. BCR/ABL1 positivity was analyzed in highly enriched stem cell fractions using fluorescence in situ hybridization (FISH) and reverse transcription PCR (RT-PCR). The proportion of CD26+ LSCs and CD26- HSCs varied considerably among the patients analyzed, and the percentage of CD26+ cells correlated with leukocyte count. The CD26 expression robustly discriminated LSCs from HSCs. This required a strict gating of the stem cell compartment. Thus, in patients with very low LSC or HSC numbers, only the highly sensitive RT-PCR method discriminated between clonal and non-clonal cells, while a robust FISH analysis required larger numbers of cells in both compartments. Finally, our data show that the numbers of CD26+ CML LSCs correlate with responses to treatment with BCR-ABL1 inhibitors. PMID:27145281

  17. Quantitative Proteomics Analysis of Leukemia Cells.

    Science.gov (United States)

    Halbach, Sebastian; Dengjel, Jörn; Brummer, Tilman

    2016-01-01

    Chronic myeloid leukemia (CML) is driven by the oncogenic fusion kinase Bcr-Abl, which organizes its own signaling network with various proteins. These proteins, their interactions, and their role in relevant signaling pathways can be analyzed by quantitative mass spectrometry (MS) approaches in various models systems, e.g., in cell culture models. In this chapter, we describe in detail immunoprecipitations and quantitative proteomics analysis using stable isotope labeling by amino acids in cell culture (SILAC) of components of the Bcr-Abl signaling pathway in the human CML cell line K562. PMID:27581145

  18. An Adult Male Presenting with Concurrent Plasma Cell Myeloma Involving a CCND1-IGH Translocation and Chronic Myelogenous Leukemia with a Variant (9;22) Translocation.

    Science.gov (United States)

    Lee, Peter M; Siangchin, Ken; Song, Sophie; Shabsovich, David; Naeini, Yalda; Tirado, Carlos A

    2016-01-01

    The t(11;14)(q13;q32) involving IGH and CCND1 a nd t(9;22) (q34;q11.2) involving BCR and ABL1 are common abnormalities in plasma cell myeloma (PCM) and chronic myelogenous leukemia (CML), respectively. However, the concurrence of the two malignancies is extremely rare. Herein, we present a case of an 87-year-old male who presented with anemia and monocytosis. FISH studies on a bone marrow sample enriched for plasma cells detected a t(11;14) positive for IGH and CCND1 fusion in 92% of nuclei. However, cytogenetic analysis of the bone marrow revealed a t(9;22)(q34;q11.2) in 40% of the metaphases. Interphase and metaphase FISH studies on the sample confirmed the presence of the BCR-ABL1 fusion in 88% of nuclei but did not show any signals corresponding to the derivative 9, suggesting a variant t(9;22) with a deletion or additional material of unknown origin at the 9q34 band of the derivative 9 and a derivative 22 bearing the BCR-ABL1 fusion gene. The concurrence of plasma cell myeloma and chronic myelogenous leukemia is extremely rare with less than 20 cases reported. The molecular pathway in which the multiple malignancies arise is still poorly understood, and this case provides insight into the concurrence of PCM and CML. PMID:27584682

  19. The NF-κB Pathway and Cancer Stem Cells.

    Science.gov (United States)

    Rinkenbaugh, Amanda L; Baldwin, Albert S

    2016-04-06

    The NF-κB transcription factor pathway is a crucial regulator of inflammation and immune responses. Additionally, aberrant NF-κB signaling has been identified in many types of cancer. Downstream of key oncogenic pathways, such as RAS, BCR-ABL, and Her2, NF-κB regulates transcription of target genes that promote cell survival and proliferation, inhibit apoptosis, and mediate invasion and metastasis. The cancer stem cell model posits that a subset of tumor cells (cancer stem cells) drive tumor initiation, exhibit resistance to treatment, and promote recurrence and metastasis. This review examines the evidence for a role for NF-κB signaling in cancer stem cell biology.

  20. Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein

    DEFF Research Database (Denmark)

    Järås, Marcus; Johnels, Petra; Hansen, Nils Gunder;

    2010-01-01

    will require full eradication of Ph chromosome-positive (Ph(+)) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34(+) cells and also in cord blood CD34(+) cells as a consequence of retroviral BCR/ABL1 expression. To test...... their Ph-chromosome status. Interestingly, we found that the CML CD34(+)CD38(-)IL1RAP(+) cells were Ph(+), whereas CML CD34(+)CD38(-)IL1RAP(-) cells were almost exclusively Ph(-). By performing long-term culture-initiating cell assays on the two cell populations, we found that Ph(+) and Ph(-) candidate CML...

  1. [Prolonged molecular response induced by imatinib in Philadelphia positive acute lymphoblastic leukemia A case report and brief review].

    Science.gov (United States)

    Raissi, Abderrahim; Bouaouad, Majdouline; Drideb, Noufissa Alami; Jennane, Selim; Mahtat, El Mahdi; Doghmi, Kamal; Mikdame, Mohammed

    2015-01-01

    Philadelphia or BCR-ABL positive acute lymphoblastic leukemia (PH+ ALL) is the most common and severe of adult ALL. The only potentially curator treatment remains allogeneic hematopoietic stem cells transplantation (SCT) in first complete remission. The use of imatinib has revolutionized the treatment of chronic myeloid leukemia. Its incorporation into PH + ALL protocols also improved the prognosis of this disease giving better complete remission rates compared to chemotherapy alone. The treatment of patients not eligible for SCT remains controversial. Prolonged use of high dose tyrosine kinase inhibitors (TKI) (ie: imatinib at 600 or 800 mg/j) as maintenance therapy seems to be a reasonable approach. We present a case of prolonged molecular remission of PH+ ALL under TKI alone as maintenance therapy.

  2. Signal transduction factors on the modulation of radiosusceptibility in K562 cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Kwang Mo; Jeong, Soo Jin [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Youn, Seon Min [College of Medicine, Eulji Univ., Daejeon (Korea, Republic of)

    2003-09-01

    The human chronic myelogenous leukemia cell line, K562, expresses the chimeric bcr-abl oncoprotein, whose deregulated protein tyrosine kinase activity antagonizes the induction of apoptosis via DNA damaging agents. Previous experiments have shown that nanomolar concentrations of herbimycin A [HMA] coupled with X-irradiation have a synergistic effect in inducing apoptosis in the Ph-positive K562 leukemia cell line, but genistein, a PTK inhibitor, is non selective for the radiation-induced apoptosis of p210{sup bcr}/{sup abl} protected K562 cells. In these experiments, the cytoplasmic signal transduction pathways, the induction of a number of transcription factors and the differential gene expression in this model were investigated. K562 cells in the exponential growth phase were used in this study. The cells were irradiated with 0.5-12 Gy, using a 6 MeV Linac (Clinac 1800, Varian, USA). Immediately after irradiation, the cells were treated with 0.25{mu}M of HMA and 25{mu}M of genistein, and the expressions and the activities of ablkinase, MAPK family, NF-KB, c-fos, c-myc, and thymidine kinase1 (TK1) were examined. The differential gene expressions induced by PTK inhibitors were also investigated. The modulating effects of herbimycin A and genistein on the radiosensitivity of K562 cells were not related to the bcr-abl kinase activity. The signaling responses through the MAPK family of proteins, were not involved either. In association with the radiation-induced apoptosis, which is accelerated by HMA, the expression of c-myc was increased. The combined treatment of genistein, with irradiation, enhanced NF-KB activity and the TK 1 expression and activity. The effects of HMA and genistein on the radiosensitivity of the K562 cells were not related to the bcr-abl kinase activity. In this study, another signaling pathway, besides the MAPK family responses to radiation to K562 cells, was found. Further evaluation using this model will provide valuable information for the

  3. Ipilimumab After Allogeneic Stem Cell Transplant in Treating Patients With Persistent or Progressive Cancer

    Science.gov (United States)

    2013-03-26

    Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Atypical Chronic Myeloid Leukemia, BCR-ABL1 Negative; Childhood Myelodysplastic Syndromes; Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Disseminated Neuroblastoma; Malignant Neoplasm; Ovarian Choriocarcinoma; Ovarian Embryonal Carcinoma; Ovarian Immature Teratoma; Ovarian Mature Teratoma; Ovarian Mixed Germ Cell Tumor; Ovarian Monodermal and Highly Specialized Teratoma; Ovarian Polyembryoma; Ovarian Yolk Sac Tumor; Previously Treated Myelodysplastic Syndromes; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Recurrent Adult Burkitt Lymphoma; Recurrent Adult Diffuse Large Cell Lymphoma; Recurrent Adult Diffuse Mixed Cell Lymphoma; Recurrent Adult Hodgkin Lymphoma; Recurrent Adult Immunoblastic Large Cell Lymphoma; Recurrent Adult Lymphoblastic Lymphoma; Recurrent Grade 3 Follicular Lymphoma; Recurrent Malignant Testicular Germ Cell Tumor; Recurrent Mantle Cell Lymphoma; Recurrent Neuroblastoma; Recurrent Ovarian Epithelial Cancer; Recurrent Ovarian Germ Cell Tumor; Refractory Chronic Lymphocytic Leukemia; Refractory Multiple Myeloma; Relapsing Chronic Myelogenous Leukemia; Stage I Multiple Myeloma; Stage II Multiple Myeloma; Stage II Ovarian Epithelial Cancer; Stage III Malignant Testicular Germ Cell Tumor; Stage III Multiple Myeloma; Stage III Ovarian Epithelial Cancer; Stage IIIA Breast Cancer; Stage IIIB Breast Cancer; Stage IIIC Breast Cancer; Stage IV Breast Cancer; Stage IV Ovarian Epithelial Cancer; Testicular Choriocarcinoma; Testicular Choriocarcinoma and Embryonal Carcinoma; Testicular Choriocarcinoma and Seminoma; Testicular Choriocarcinoma and Teratoma; Testicular Choriocarcinoma and Yolk Sac Tumor; Testicular Embryonal Carcinoma; Testicular

  4. Imatinib and nilotinib inhibit hematopoietic progenitor cell growth, but do not prevent adhesion, migration and engraftment of human cord blood CD34+ cells.

    Directory of Open Access Journals (Sweden)

    Ludovic Belle

    Full Text Available BACKGROUND: The availability of tyrosine kinase inhibitors (TKIs has considerably changed the management of Philadelphia chromosome positive leukemia. The BCR-ABL inhibitor imatinib is also known to inhibit the tyrosine kinase of the stem cell factor receptor, c-Kit. Nilotinib is 30 times more potent than imatinib towards BCR-ABL in vitro. Studies in healthy volunteers and patients with chronic myelogenous leukemia or gastrointestinal stromal tumors have shown that therapeutic doses of nilotinib deliver drug levels similar to those of imatinib. The aim of this study was to compare the inhibitory effects of imatinib and nilotinib on proliferation, differentiation, adhesion, migration and engraftment capacities of human cord blood CD34(+ cells. DESIGN AND METHODS: After a 48-hour cell culture with or without TKIs, CFC, LTC-IC, migration, adhesion and cell cycle analysis were performed. In a second time, the impact of these TKIs on engraftment was assessed in a xenotransplantation model using NOD/SCID/IL-2Rγ (null mice. RESULTS: TKIs did not affect LTC-IC frequencies despite in vitro inhibition of CFC formation due to inhibition of CD34(+ cell cycle entry. Adhesion of CD34(+ cells to retronectin was reduced in the presence of either imatinib or nilotinib but only at high concentrations. Migration through a SDF-1α gradient was not changed by cell culture in the presence of TKIs. Finally, bone marrow cellularity and human chimerism were not affected by daily doses of imatinib and nilotinib in a xenogenic transplantation model. No significant difference was seen between TKIs given the equivalent affinity of imatinib and nilotinib for KIT. CONCLUSIONS: These data suggest that combining non-myeloablative conditioning regimen with TKIs starting the day of the transplantation could be safe.

  5. Imatinib induces H2AX phosphorylation and apoptosis in chronic myelogenous leukemia cells in vitro via caspase-3/Mst1 pathway

    Institute of Scientific and Technical Information of China (English)

    Yan-jun ZHANG; Lian-ning DUAN; Cheng-rong LU; Yan CAO; Yuan LUO; Rong-feng BAO; Shu YAN; Mei XUE; Feng ZHU; Zhe WANG

    2012-01-01

    Aim:Histone H2AX is a novel tumor suppressor and its phosphorylation at the C terminus (Ser139 and Tyr142)is required for tumor cell apoptosis.The aim of the present study was to elucidate the mechanisms underlying imatinib-induced C-terminal phosphorylation of H2AX in chronic myelogenous leukemia cells in vitro.Methods:BCR-ABL-positive K562 cells were used.Microscopy,Western blotting and flow cytometry were used to study the signaling pathways that regulate imatinib-induced H2AX phosphorylation and the apoptotic mechanisms.Results:Treatment of K562 cells with imatinib (1-8 μmol/L)induced phosphorylation of H2AX at Ser139 and Tyr142 in time-and dose-dependent manners.In contrast,imatinib at the same concentrations did not affect H2AX acetylation at Lys 5,and the acetylated H2AX maintained a higher level in the cells.Meanwhile,imatinib (1-8 μmol/L)activated caspase-3 and its downstream mammalian STE20-like kinase 1 (Mst1),and induced apoptosis of K562 cells.The caspase-3 inhibitor Z-VAD (40 μmol/L)reduced imatinibinduced H2AX phosphorylation at Ser139 and Tyr142 and blocked imatinib-induced apoptosis of K562 cells.Imatinib (4 μmol/L)induced expression of Williams-Beuren syndrome transcription factor (WSTF),but not wild-type p53-induced phosphatase 1 (Wip1)in K562 cells.Conclusion:The caspase-3/Mst1 pathway is required for H2AX C-terminal phosphorylation at Ser139 and Tyr142 and subsequent apoptosis in Bcr-Abl-positive K562 cells induced by imatinib.

  6. Novel Acylguanidine Derivatives Targeting Smoothened Induce Antiproliferative and Pro-Apoptotic Effects in Chronic Myeloid Leukemia Cells.

    Directory of Open Access Journals (Sweden)

    Alessandra Chiarenza

    Full Text Available The most relevant therapeutic approaches to treat CML rely on the administration of tyrosine kinase inhibitors (TKIs like Imatinib, which are able to counteract the activity of Bcr-Abl protein increasing patient's life expectancy and survival. Unfortunately, there are some issues TKIs are not able to address; first of all TKIs are not so effective in increasing survival of patients in blast crisis, second they are not able to eradicate leukemic stem cells (LSC which represent the major cause of disease relapse, and third patients often develop resistance to TKIs due to mutations in the drug binding site. For all these reasons it's of primary interest to find alternative strategies to treat CML. Literature shows that Hedgehog signaling pathway is involved in LSC maintenance, and pharmacological inhibition of Smoothened (SMO, one of the key molecules of the pathway, has been demonstrated to reduce Bcr-Abl positive bone marrow cells and LSC. Consequently, targeting SMO could be a promising way to develop a new treatment strategy for CML overcoming the limitations of current therapies. In our work we have tested some compounds able to inhibit SMO, and among them MRT92 appears to be a very potent SMO antagonist. We found that almost all our compounds were able to reduce Gli1 protein levels in K-562 and in KU-812 CML cell lines. Furthermore, they were also able to increase Gli1 and SMO RNA levels, and to reduce cell proliferation and induce apoptosis/autophagy in both the tested cell lines. Finally, we demonstrated that our compounds were able to modulate the expression of some miRNAs related to Hedgehog pathway such as miR-324-5p and miR-326. Being Hedgehog pathway deeply implicated in the mechanisms of CML we may conclude that it could be a good therapeutic target for CML and our compounds seem to be promising antagonists of such pathway.

  7. Novel Acylguanidine Derivatives Targeting Smoothened Induce Antiproliferative and Pro-Apoptotic Effects in Chronic Myeloid Leukemia Cells

    Science.gov (United States)

    Chiarenza, Alessandra; Manetti, Fabrizio; Petricci, Elena; Ruat, Martial; Naldini, Antonella; Taddei, Maurizio; Carraro, Fabio

    2016-01-01

    The most relevant therapeutic approaches to treat CML rely on the administration of tyrosine kinase inhibitors (TKIs) like Imatinib, which are able to counteract the activity of Bcr-Abl protein increasing patient’s life expectancy and survival. Unfortunately, there are some issues TKIs are not able to address; first of all TKIs are not so effective in increasing survival of patients in blast crisis, second they are not able to eradicate leukemic stem cells (LSC) which represent the major cause of disease relapse, and third patients often develop resistance to TKIs due to mutations in the drug binding site. For all these reasons it’s of primary interest to find alternative strategies to treat CML. Literature shows that Hedgehog signaling pathway is involved in LSC maintenance, and pharmacological inhibition of Smoothened (SMO), one of the key molecules of the pathway, has been demonstrated to reduce Bcr-Abl positive bone marrow cells and LSC. Consequently, targeting SMO could be a promising way to develop a new treatment strategy for CML overcoming the limitations of current therapies. In our work we have tested some compounds able to inhibit SMO, and among them MRT92 appears to be a very potent SMO antagonist. We found that almost all our compounds were able to reduce Gli1 protein levels in K-562 and in KU-812 CML cell lines. Furthermore, they were also able to increase Gli1 and SMO RNA levels, and to reduce cell proliferation and induce apoptosis/autophagy in both the tested cell lines. Finally, we demonstrated that our compounds were able to modulate the expression of some miRNAs related to Hedgehog pathway such as miR-324-5p and miR-326. Being Hedgehog pathway deeply implicated in the mechanisms of CML we may conclude that it could be a good therapeutic target for CML and our compounds seem to be promising antagonists of such pathway. PMID:26934052

  8. Tyrosine kinase inhibitors influence ABCG2 expression in EGFR-positive MDCK BCRP cells via the PI3K/Akt signaling pathway.

    Science.gov (United States)

    Pick, Anne; Wiese, Michael

    2012-04-01

    Multidrug resistance observed in cancer chemotherapy is commonly attributed to overexpression of efflux transporter proteins. These proteins act as ATP-dependent drug efflux pumps, actively extruding chemotherapeutic agents from cells and causing a decrease in intracellular drug accumulation. Besides the well-recognized role of P-glycoprotein (P-gp, ABCB1), the breast cancer resistance protein (BCRP, ABCG2) is becoming increasingly accepted as playing an important role in multidrug resistance. In contrast to P-glycoprotein, only a few inhibitors of ABCG2 are known. According to the literature, tyrosine kinase inhibitors (TKIs) can be considered to be broad-spectrum inhibitors, interacting with ABCB1, ABCC1 and ABCG2. Here, we investigated seven different TKIs, gefitinib, erlotinib, AG1478, PD158780, PD153035, nilotinib and imatinib, for their potential to restore ABCG2 sensitivity to cells. Furthermore, we analyzed the alteration of ABCG2 expression caused by TKIs and demonstrated that EGFR inhibitors such as gefitinib and PD158780 reduced both total and surface expression of ABCG2 in EGRF-positive MDCK BCRP cells by interaction with the PI3K/Akt signaling pathway. The reduced ABCG2 content led to an increased effect of XR9577, a well-known ABCG2 modulator, lowering the concentration required for half maximal inhibition. On the other hand, BCR-ABL inhibitors had no influence on ABCG2 expression and modulator activity. Interestingly, a combination of an EGFR inhibitor with the PI3K/Akt inhibitor LY294002 led to a significant reduction of ABCG2 expression at low concentrations of the drugs. Based on our results, we assume that EGFR exerts a post-transcriptional enhancing effect on ABCG2 expression via the PI3K/Akt signaling pathway, which can be attenuated by EGFR inhibitors. Blocking the key signaling pathway regulating ABCG2 expression with EGFR inhibitors, combined with the inhibition of ABCG2 with potent modulators might be a promising approach to circumvent MDR

  9. Donor Umbilical Cord Blood Transplant With or Without Ex-vivo Expanded Cord Blood Progenitor Cells in Treating Patients With Acute Myeloid Leukemia, Acute Lymphoblastic Leukemia, Chronic Myelogenous Leukemia, or Myelodysplastic Syndromes

    Science.gov (United States)

    2016-09-09

    Acute Biphenotypic Leukemia; Acute Lymphoblastic Leukemia in Remission; Acute Myeloid Leukemia Arising From Previous Myelodysplastic Syndrome; Acute Myeloid Leukemia in Remission; Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Mixed Phenotype Acute Leukemia; Myelodysplastic Syndrome; Pancytopenia; Refractory Anemia; Refractory Anemia With Excess Blasts; Refractory Anemia With Excess Blasts in Transformation; Secondary Acute Myeloid Leukemia

  10. CML Mouse Model Generated from Leukemia Stem Cells.

    Science.gov (United States)

    Hu, Yiguo

    2016-01-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disorder with a high number of well-differentiated neutrophils in peripheral blood and myeloid cells in bone marrow (BM). CML is derived from the hematopoietic stem cells (HSCs) with the Philadelphia chromosome (Ph(+), t(9;22)-(q34;q11)), resulting in generating a fusion oncogene, BCR/ABL1. HSCs with Ph(+) are defined as leukemia stem cells (LSCs), a subpopulation cell at the apex of hierarchies in leukemia cells and responsible for the disease continuous propagation. Several kinds of CML models have been developed to reveal the mechanism of CML pathogenesis and evaluate therapeutic drugs in the past three decades. Here, we describe the procedures to generate a CML mouse model by introducing BCR/ABL1 into Lin(-)Sca1(+) cKit(+) population cells purified from mouse bone marrow. In CML retroviral transduction/transplantation mouse models, this modified model can mimic CML pathogenesis on high fidelity. PMID:27581136

  11. AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR HIGH-RISK ACUTE LYMPHOBLASTIC LEUKEMIA: NON-RANDOMIZED STUDY WITH A MAXIMUM FOLLOW-UP OF MORE THAN 22 YEARS

    Directory of Open Access Journals (Sweden)

    Grzegorz Helbig

    2014-06-01

    Full Text Available Objective. To evaluate the efficacy and toxicity of autologous hematopoietic stem cell transplantation (AHSCT for high-risk acute lymphoblastic leukemia (ALL. Material and methods. Overall, 128 high-risk ALL patients at a median age of 26 years (range 18-56 years at diagnosis received AHSCT between 1991-2008. Induction treatment was anthracycline-based in all patients. Conditioning regimen consisted of CAV (cyclophosphamide, cytarabine, etoposide in 125 patients whereas 3 subjects received cyclophosphamide and TBI (total body irridation. Bone marrow was stored for 72 hours in 4oC and re-infused 24 hours after conditioning completion. Bone marrow was a source of stem cells in 119 patients, peripheral blood in 2 and 7 subjects received both bone marrow and peripheral blood. Results. With a median follow-up after AHSCT of 1.6 years (range 0.1-22.3 years, the probability of leukemia-free survival (LFS for the whole group at 10 years was 27% and 23% at 20 years. Transplant-related mortality at 100 days after AHSCT was 3.2%.. There was a strong tendency for better LFS for MRD-negative patients if compared with patients who had positive or unknown MRD status at AHSCT (32% vs 23% and 25%, respectively; p=0.06. There was no difference in LFS between B- and T-lineage ALL as well as between patients transplanted in first complete remission (CR1 and CR2. LFS at 10 years for patients with detectable BCR-ABL at transplant was 20% and this was comparable with subjects with negative and missing BCR-ABL status (26% and 28%; p=0.97. Conclusions. The results of AHSCT for high-risk ALL remains unsatisfactory with low probability of long-term LFS.

  12. Automated detection of residual cells after sex-mismatched stem-cell transplantation – evidence for presence of disease-marker negative residual cells

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    Johannes Tilman

    2009-05-01

    Full Text Available Abstract Background A new chimerism analysis based on automated interphase fluorescence in situ hybridization (FISH evaluation was established to detect residual cells after allogene sex-mismatched bone marrow or blood stem-cell transplantation. Cells of 58 patients were characterized as disease-associated due to presence of a bcr/abl-gene-fusion or a trisomy 8 and/or a simultaneous hybridization of gonosome-specific centromeric probes. The automatic slide scanning platform Metafer with its module MetaCyte was used to analyse 3,000 cells per sample. Results Overall 454 assays of 58 patients were analyzed. 13 of 58 patients showed residual recipient cells at one stage of more than 4% and 12 of 58 showed residual recipient cells less than 4%, respectively. As to be expected, patients of the latter group were associated with a higher survival rate (48 vs. 34 month. In only two of seven patients with disease-marker positive residual cells between 0.1–1.3% a relapse was observed. Besides, disease-marker negative residual cells were found in two patients without relapse at a rate of 2.8% and 3.3%, respectively. Conclusion The definite origin and meaning of disease-marker negative residual cells is still unclear. Overall, with the presented automatic chimerism analysis of interphase FISH slides, a sensitive method for detection of disease-marker positive residual cells is on hand.

  13. Tacrolimus and Mycophenolate Mofetil With or Without Sirolimus in Preventing Acute Graft-Versus-Host Disease in Patients Who Are Undergoing Donor Stem Cell Transplant for Hematologic Cancer

    Science.gov (United States)

    2015-10-14

    Myelodysplastic/Myeloproliferative Neoplasm, Unclassifiable; Previously Treated Myelodysplastic Syndrome; Refractory Chronic Lymphocytic Leukemia; Refractory Plasma Cell Myeloma; Waldenstrom Macroglobulinemia; Accelerated Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Adult Acute Lymphoblastic Leukemia in Remission; Adult Acute Myeloid Leukemia in Remission; Adult Acute Myeloid Leukemia With t(9;11)(p22;q23); MLLT3-; Adult Acute Myeloid Leukemia With Inv(16)(p13.1q22); CBFB-MYH11; Adult Acute Promyelocytic Leukemia With t(15;17)(q22;q12); PML-RARA; Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); RUNX1-RUNX1T1; Atypical Chronic Myeloid Leukemia, BCR-ABL1 Negative; Blast Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Childhood Acute Lymphoblastic Leukemia in Remission; Childhood Acute Myeloid Leukemia in Remission; Childhood Burkitt Lymphoma; Childhood Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Childhood Diffuse Large Cell Lymphoma; Childhood Immunoblastic Lymphoma; Childhood Myelodysplastic Syndrome; Stage II Contiguous Adult Burkitt Lymphoma; Stage II Contiguous Adult Diffuse Large Cell Lymphoma; Stage II Contiguous Adult Diffuse Mixed Cell Lymphoma; Stage II Contiguous Adult Diffuse Small Cleaved Cell Lymphoma; Stage II Adult Contiguous Immunoblastic Lymphoma; Stage II Contiguous Adult Lymphoblastic Lymphoma; Stage II Grade 1 Contiguous Follicular Lymphoma; Stage II Grade 2 Contiguous Follicular Lymphoma; Stage II Grade 3 Contiguous Follicular Lymphoma; Stage II Contiguous Mantle Cell Lymphoma; Stage II Non-Contiguous Adult Burkitt Lymphoma; Stage II Non-Contiguous Adult Diffuse Large Cell Lymphoma; Stage II Non-Contiguous Adult Diffuse Mixed Cell Lymphoma; Stage II Non-Contiguous Adult Diffuse Small Cleaved Cell Lymphoma; Stage II Adult Non-Contiguous Immunoblastic Lymphoma; Stage II Non-Contiguous Adult Lymphoblastic Lymphoma; Stage II Grade 1 Non-Contiguous Follicular Lymphoma; Stage II Grade 2 Non-Contiguous Follicular Lymphoma; Stage

  14. Secondary chromosomal changes in 34 Philadelphia-chromosome-positive chronic myelocytic leukemia patients from the Mexican West.

    Science.gov (United States)

    Meza Espinoza, Juan Pablo; Judith Picos Cárdenas, Verónica; Gutiérrez-Angulo, Melva; González García, Juan Ramón

    2004-01-15

    The clonal evolution in t(9;22)-positive chronic myelocytic leukemia (CML) is well established. Four major changes occur in more than 70% of patients: +8, i(17q), +19, and an extra Philadelphia chromosome. The frequencies of secondary chromosomal changes in 34 patients from the states of Jalisco, Nayarit, Michoacán, and Colima (the Mexican West) with Philadelphia-chromosome-positive CML were assessed. The most frequent abnormalities were tetraploidy (12 cases); +8, inv(3)(q21q26), and octoploidy (3 cases each); and +der(22)(2 cases). Some translocations not previously associated with CML were observed, such as t(2;7)(p12;q36), t(3;6)(q26;p25), t(3;17)(q26;p13), and t(6;17)(q21;q23 approximately q25). Significant differences were found for +8 with respect to population results from Japan and from southern, eastern, and western Europe; for i(17)(q10) from eastern Europe; for +19 from Japan and western Europe; and for +der(22) from Japan, southern Europe, and western Europe. Although polyploidy could result from endomitosis, there is no direct evidence that the BCR/ABL protein influences such a process; however, protein kinases such as MAPK, which are involved in endomitosis, are activated by the BCR/ABL protein, and so the BCR/ABL protein could promote endomitosis through the MAPK pathway.

  15. Management of side effects of BCR/ABL-negative chronic myeloproliferative neoplasm therapies. Focus on anagrelide.

    Science.gov (United States)

    Antelo, María Luisa; de Las Heras, Natalia; Gonzalez Porras, Jose Ramón; Kerguelen, Ana; Raya, Jose María

    2015-12-01

    Although hydroxyurea is considered the first-line cytoreductive therapy in high-risk patients with polycythemia vera or essential thrombocythemia, approximately 20-25% of patients develop resistance or intolerance and they need an alternative therapy. Anagrelide is the treatment of choice in patients with essential thrombocythemia intolerant or with resistance to hydroxyurea. Anagrelide is usually well tolerated. Although there is concern about the increased risk of cardiac side effects, in most cases these are mild, and easily manageable. In this paper, the available evidence about the management of patients with myeloproliferative neoplasms, with a special focus on the side effects of drug therapies is reviewed.

  16. Coexistance of JAK2V617F mutation and BCR/ABL translocation in one patient

    Directory of Open Access Journals (Sweden)

    Murat Albayrak

    2010-09-01

    Full Text Available Dear Editor,The myeloproliferative disorders (MPDs constitute a subcategory of chronic myeloid disorders and include chronic myeloid leukemia (CML, essential thrombocytemia (ET, polycythemia vera (PV and myelofibrosis (MF. In 1960, the discovery of the Philadelphia chromosome (Ph became a cornerstone in CML treatment and led to the development of moleculary targeted therapy. Recently, an acquired mutation in the Janus kinase 2 (JAK2 gene has been discovered in nearly all patents with PV and approximately half of the patients with primary MF and ET. Subsequently, the mutation has been demonstrated in atypical MPDs (chronic neutrophilic leukemia, unclassified, de novo myelodysplastic syndrome or acute myeloid leukemia.1 It has been hoped that targeted inhibition of JAK2V617F should achieve similar disease control as thyrosine kinases has produced in CML.

  17. Treatment of human pre-B acute lymphoblastic leukemia with the Aurora kinase inhibitor PHA-739358 (Danusertib

    Directory of Open Access Journals (Sweden)

    Fei Fei

    2012-06-01

    Full Text Available Abstract Background Treatment of Philadelphia chromosome-positive acute lymphoblastic leukemias (Ph-positive ALL with clinically approved inhibitors of the Bcr/Abl tyrosine kinase frequently results in the emergence of a leukemic clone carrying the T315I mutation in Bcr/Abl, which confers resistance to these drugs. PHA-739358, an Aurora kinase inhibitor, was reported to inhibit the Bcr/Abl T315I mutant in CML cells but no preclinical studies have examined this in detail in human ALL. Results We compared the sensitivity of human Bcr/Abl T315I, Bcr/Abl wild type and non-Bcr/Abl ALL cells to this drug. PHA-739358 inhibited proliferation and induced apoptosis independently of Bcr/Abl, the T315I mutation, or presence of the tumor suppressor p53, but the degree of effectiveness varied between different ALL samples. Since short-term treatment with a single dose of drug only transiently inhibited proliferation, we tested combination treatments of PHA-739358 with the farnesyltransferase inhibitor Lonafarnib, with vincristine and with dasatinib. All combinations reduced viability and cell numbers compared to treatment with a single drug. Clonogenic assays showed that 25 nM PHA-739358 significantly reduced the colony growth potential of Ph-positive ALL cells, and combined treatment with a second drug abrogated colony growth in this assay. PHA-739358 further effectively blocked Bcr/Abl tyrosine kinase activity and Aurora kinase B in vivo, and mice transplanted with human Bcr/Abl T315I ALL cells treated with a 3x 7-day cycle of PHA-739358 as mono-treatment had significantly longer survival. Conclusions PHA-739358 represents an alternative drug for the treatment of both Ph-positive and negative ALL, although combined treatment with a second drug may be needed to eradicate the leukemic cells.

  18. Pharmacokinetics of dasatinib for Philadelphia-positive acute lymphocytic leukemia with acquired T315I mutation

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    Takahashi Naoto

    2012-05-01

    Full Text Available Abstract Background The BCR-ABL T315I kinase domain mutation is insensitive to dasatinib therapy for Philadelphia-positive acute lymphoid leukemia (Ph + ALL patients. Resistant T315I clone may be present prior to initiating dasatinib, which could expand under selective pressures during treatment. However, it is also possible that Ph + ALL patients newly acquire the T315I mutation during dasatinib therapy. Despite the potent inhibition of BCR-ABL kinase by dasatinib, little is known about the relationship between dasatinib pharmacokinetics and the emergence of kinase domain mutations in vivo. Methods To determine whether plasma dasatinib pharmacokinetics influences the emergence of BCR-ABL mutations, we measured plasma dasatinib levels in 11 Ph + ALL patients undergoing dasatinib monotherapy. Results Bone marrow relapse occurred in 5 of the 11 Ph + ALL patients (45%. Importantly, a T315I mutation was detected in 4 of the 5 relapsed patients, despite the absence of BCR-ABL mutations in any patient at baseline. The median plasma concentration at 2 hours (C2h, the median plasma maximum concentration (Cmax, and the median area under the observed plasma concentration-time curve from 0 to 4 hours (AUC0-4 were all significantly lower in patients with T315I than those without the mutation (C2h, 22.3 ng/mL vs. 111.6 ng/mL, P = 0.0242; Cmax, 43.8 ng/mL vs. 112.4 ng/mL, P = 0.0242; AUC0-4, 108.3 ng·h/mL vs. 268.3 ng·h/mL, P = 0.0061, respectively. Conclusions These data indicate that the emergence of the T315I mutation among Ph + ALL patients treated with dasatinib is, in part, dependent on plasma dasatinib pharmacokinetics. Notably, these data also suggest that newly acquired BCR-ABL mutations may be inhibited by an increased exposure of dasatinib.

  19. Aging-associated inflammation promotes selection for adaptive oncogenic events in B cell progenitors

    OpenAIRE

    Henry, C J; Casas-Selves, M.; Kim, J; Zaberezhnyy, V.; Aghili, L.; Daniel, A.E.; Jimenez, L; Azam, T.; McNamee, E.N.; Clambey, E.T.; Klawitter, J; Serkova, N.J.; Tan, A.C.; Dinarello, C A; DeGregori, J.

    2015-01-01

    The incidence of cancer is higher in the elderly; however, many of the underlying mechanisms for this association remain unexplored. Here, we have shown that B cell progenitors in old mice exhibit marked signaling, gene expression, and metabolic defects. Moreover, B cell progenitors that developed from hematopoietic stem cells (HSCs) transferred from young mice into aged animals exhibited similar fitness defects. We further demonstrated that ectopic expression of the oncogenes BCR-ABL, NRASV1...

  20. Hypoxia selects bortezomib-resistant stem cells of chronic myeloid leukemia.

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    Michele Tanturli

    Full Text Available We previously demonstrated that severe hypoxia inhibits growth of Chronic Myeloid Leukemia (CML cells and selects stem cells where BCR/Abl(protein is suppressed, although mRNA is not, so that hypoxia-selected stem cells, while remaining leukemic, are independent of BCR/Abl signaling and thereby refractory to Imatinib-mesylate. The main target of this study was to address the effects of the proteasome inhibitor Bortezomib (BZ on the maintenance of stem or progenitor cells in hypoxic primary cultures (LC1, by determining the capacity of LC1 cells to repopulate normoxic secondary cultures (LC2 and the kinetics of this repopulation. Unselected K562 cells from day-2 hypoxic LC1 repopulated LC2 with rapid, progenitor-type kinetics; this repopulation was suppressed by BZ addition to LC1 at time 0, but completely resistant to day-1 BZ, indicating that progenitors require some time to adapt to stand hypoxia. K562 cells selected in hypoxic day-7 LC1 repopulated LC2 with stem-type kinetics, which was largely resistant to BZ added at either time 0 or day 1, indicating that hypoxia-selectable stem cells are BZ-resistant per se, i.e. before their selection. Furthermore, these cells were completely resistant to day-6 BZ, i.e. after selection. On the other hand, hypoxia-selected stem cells from CD34-positive cells of blast-crisis CML patients appeared completely resistant to either time-0 or day-1 BZ. To exploit in vitro the capacity of CML cells to adapt to hypoxia enabled to detect a subset of BZ-resistant leukemia stem cells, a finding of particular relevance in light of the fact that our experimental system mimics the physiologically hypoxic environment of bone marrow niches where leukemia stem cells most likely home and sustain minimal residual disease in vivo. This suggests the use of BZ as an enhanced strategy to control CML. in particular to prevent relapse of disease, to be considered with caution and to need further deepening.

  1. Update on the management of Philadelphia chromosome positive chronic myelogenous leukemia: role of nilotinib

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    Emole J

    2016-02-01

    Full Text Available Josephine Emole,1 Taiwo Talabi,2 Javier Pinilla-Ibarz1 1Department of Malignant Hematology, 2Moffitt Program for Outreach Wellness Education and Resources, H Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA Abstract: Chronic myelogenous leukemia (CML is a pluripotent stem cell disease characterized by the presence of the Philadelphia chromosome and the bcr-abl gene. The discovery of tyrosine kinase inhibitors (TKIs revolutionized therapy for CML, such that durable response, increased overall survival, and increased progression-free survival of patients in chronic phase CML is now possible. Due to resistance and intolerance to imatinib, there was need for development of second- and third-generation TKIs for the treatment of CML. This review examines the role of nilotinib, an oral second-generation TKI, in the treatment of Philadelphia positive CML. The pharmacology, efficacy, and safety of nilotinib are critically evaluated. Patient-related issues, including tolerance, drug interactions, and quality of life issues are also examined. Keywords: chronic myelogenous leukemia, nilotinib, tyrosine kinase inhibitor

  2. Gut microbiota-derived propionate reduces cancer cell proliferation in the liver

    OpenAIRE

    Bindels, Laure B.; Porporato, Paolo; Dewulf, Evelyne; Verrax, Julien; Audrey M Neyrinck; Martin, J C; Scott, K P; Buc Calderon, Pedro; Feron, Olivier; Muccioli, Giulio; Sonveaux, Pierre; Cani, Patrice D.; Delzenne, Nathalie M

    2012-01-01

    BACKGROUND: Metabolites released by the gut microbiota may influence host metabolism and immunity. We have tested the hypothesis that inulin-type fructans (ITF), by promoting microbial production of short-chain fatty acids (SCFA), influence cancer cell proliferation outside the gut. METHODS: Mice transplanted with Bcr-Abl-transfected BaF3 cells, received ITF in their drinking water. Gut microbiota was analysed by 16S rDNA polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis...

  3. Immature leukemic CD34+CXCR4+ cells from CML patients have lower integrin-dependent migration and adhesion in response to the chemokine SDF-1.

    Science.gov (United States)

    Peled, Amnon; Hardan, Izhar; Trakhtenbrot, Luba; Gur, Eyal; Magid, Michal; Darash-Yahana, Merav; Cohen, Ninette; Grabovsky, Valentin; Franitza, Suzana; Kollet, Orit; Lider, Ofer; Alon, Ronen; Rechavi, Gideon; Lapidot, Tsvee

    2002-01-01

    Chronic myelogenous leukemia (CML), a malignant myeloproliferative disorder originating from a pluripotent stem cell expressing the bcr-abl oncogene, is characterized by abnormal release of the expanded, malignant stem cell clone from the bone marrow (BM) into the circulation. Moreover, immature CD34+ CML cells have lower adhesion to stromal cells and fibronectin as well as lower engraftment potential in severe combined immunedeficient (SCID) and nonobese diabetic (NOD)/SCID mice than normal CD34+ cells. We report in this study that leukemic Philadelphia chromosome-positive (Ph+)CD34+ cells from newly diagnosed CML patients that express the chemokine receptor CXCR4 migrate in response to stromal-derived factor-1 (SDF-1). However, normal Ph-CD34+CXCR4+ cells derived from the same patient have significantly higher migration levels toward SDF-1. In contrast to their transwell migration potential, the SDF-1-mediated integrin-dependent polarization and migration of the Ph+CD34+CXCR4+ cells through extracellular matrix-like gels were significantly lower than for normal cells. Concomitantly, binding of these cells to vascular cell adhesion molecule-1 or fibronectin, in the presence of SDF-1, was also substantially lower. These findings suggest a major role for SDF-1-mediated, integrin-dependent BM retention of Ph+CD34+ cells. PMID:12004084

  4. The research on the immuno-modulatory defect of Mesenchymal Stem Cell from Chronic Myeloid Leukemia patients

    Directory of Open Access Journals (Sweden)

    Yuguang Song

    2011-05-01

    Full Text Available Abstract Overwhelming evidence from leukemia research has shown that the clonal population of neoplastic cells exhibits marked heterogeneity with respect to proliferation and differentiation. There are rare stem cells within the leukemic population that possess extensive proliferation and self-renewal capacity not found in the majority of the leukemic cells. These leukemic stem cells are necessary and sufficient to maintain the leukemia. While the hematopoietic stem cell (HSC origin of CML was first suggested over 30 years ago, recently CML-initiating cells beyond HSCs are also being investigated. We have previously isolated fetal liver kinase-1-positive (Flk1+ cells carrying the BCR/ABL fusion gene from the bone marrow of Philadelphia chromosome-positive (Ph+ patients with hemangioblast property. Here, we showed that CML patient-derived Flk1+CD31-CD34-MSCs had normal morphology, phenotype and karyotype but appeared impaired in immuno-modulatory function. The capacity of patient Flk1+CD31-CD34- MSCs to inhibit T lymphocyte activation and proliferation was impaired in vitro. CML patient-derived MSCs have impaired immuno-modulatory functions, suggesting that the dysregulation of hematopoiesis and immune response may originate from MSCs rather than HSCs. MSCs might be a potential target for developing efficacious cures for CML.

  5. Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

    OpenAIRE

    Gavrilescu, L Cristina; Richard A Van Etten

    2007-01-01

    Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFRα, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transf...

  6. Heterogeneity of leukemia-initiating capacity of chronic myelogenous leukemia stem cells.

    Science.gov (United States)

    Zhang, Bin; Li, Ling; Ho, Yinwei; Li, Min; Marcucci, Guido; Tong, Wei; Bhatia, Ravi

    2016-03-01

    Chronic myelogenous leukemia (CML) results from transformation of a long-term hematopoietic stem cell (LTHSC) by expression of the BCR-ABL fusion gene. However, BCR-ABL-expressing LTHSCs are heterogeneous in their capacity as leukemic stem cells (LSCs). Although discrepancies in proliferative, self-renewal, and differentiation properties of normal LTHSCs are being increasingly recognized, the mechanisms underlying heterogeneity of leukemic LTHSCs are poorly understood. Using a CML mouse model, we identified gene expression differences between leukemic and nonleukemic LTHSCs. Expression of the thrombopoietin (THPO) receptor MPL was elevated in leukemic LTHSC populations. Compared with LTHSCs with low MPL expression, LTHSCs with high MPL expression showed enhanced JAK/STAT signaling and proliferation in response to THPO in vitro and increased leukemogenic capacity in vivo. Although both G0 and S phase subpopulations were increased in LTHSCs with high MPL expression, LSC capacity was restricted to quiescent cells. Inhibition of MPL expression in CML LTHSCs reduced THPO-induced JAK/STAT signaling and leukemogenic potential. These same phenotypes were also present in LTHSCs from patients with CML, and patient LTHSCs with high MPL expression had reduced sensitivity to BCR-ABL tyrosine kinase inhibitor treatment but increased sensitivity to JAK inhibitors. Together, our studies identify MPL expression levels as a key determinant of heterogeneous leukemia-initiating capacity and drug sensitivity of CML LTHSCs and suggest that high MPL-expressing CML stem cells are potential targets for therapy. PMID:26878174

  7. Chronic Myelogenous Leukemia Cells Contribute to the Stromal Myofibroblasts in Leukemic NOD/SCID Mouse In Vivo

    Directory of Open Access Journals (Sweden)

    Ryosuke Shirasaki

    2012-01-01

    Full Text Available We recently reported that chronic myelogenous leukemia (CML cells converted into myofibroblasts to create a microenvironment for proliferation of CML cells in vitro. To analyze a biological contribution of CML-derived myofibroblasts in vivo, we observed the characters of leukemic nonobese diabetes/severe combined immunodeficiency (NOD/SCID mouse. Bone marrow nonadherent mononuclear cells as well as human CD45-positive cells obtained from CML patients were injected to the irradiated NOD/SCID mice. When the chimeric BCR-ABL transcript was demonstrated in blood, human CML cells were detected in NOD/SCID murine bone marrow. And CML-derived myofibroblasts composed with the bone marrow-stroma, which produced significant amounts of human vascular endothelial growth factor A. When the parental CML cells were cultured with myofibroblasts separated from CML cell-engrafted NOD/SCID murine bone marrow, CML cells proliferated significantly. These observations indicate that CML cells make an adequate microenvironment for their own proliferation in vivo.

  8. Centrosome positioning in non-dividing cells.

    Science.gov (United States)

    Barker, Amy R; McIntosh, Kate V; Dawe, Helen R

    2016-07-01

    Centrioles and centrosomes are found in almost all eukaryotic cells, where they are important for organising the microtubule cytoskeleton in both dividing and non-dividing cells. The spatial location of centrioles and centrosomes is tightly controlled and, in non-dividing cells, plays an important part in cell migration, ciliogenesis and immune cell functions. Here, we examine some of the ways that centrosomes are connected to other organelles and how this impacts on cilium formation, cell migration and immune cell function in metazoan cells. PMID:26319517

  9. Centrosome positioning in non-dividing cells.

    Science.gov (United States)

    Barker, Amy R; McIntosh, Kate V; Dawe, Helen R

    2016-07-01

    Centrioles and centrosomes are found in almost all eukaryotic cells, where they are important for organising the microtubule cytoskeleton in both dividing and non-dividing cells. The spatial location of centrioles and centrosomes is tightly controlled and, in non-dividing cells, plays an important part in cell migration, ciliogenesis and immune cell functions. Here, we examine some of the ways that centrosomes are connected to other organelles and how this impacts on cilium formation, cell migration and immune cell function in metazoan cells.

  10. Influence of different chromosomal abnormalities in Ph-positive bone marrow cells on the chronic myeloid leukemia course during tyrosine kinase inhibitors therapy

    Directory of Open Access Journals (Sweden)

    O. Yu. Vinogradova

    2014-07-01

    Full Text Available The additional molecular and chromosomal abnormalities (ACA in Phositive cells usually considered as a genetic marker of chronic myeloid leukemia (CML progression. 457 patients in different CML phases received tyrosine kinase inhibitors (1st and 2nd generation were studied. During therapy 50 cases with additional chromosomal abnormalities in Ph+ clone (22 of them in chronic CML phase were revealed (median follow-up from CML diagnosis – 117 months, median imatinib therapy – 62 months. 86 % of patients in chronic phase with Ph+- cell abnormalities were cytogenetic resistance, and their 5-years overall survival was 80 % which was significantly lower than in patients without ACA (p < 0.005. The treatment results depend on chromosomal abnormalities detected. In patients with additional chromosome 8 imatinib therapy is effective, although complete cytogenetic response (CCR is achieved only in the later therapy stages. In patients with additional translocations CCR also achieved with imatinib or 2nd generation TKI. Only a third of patients with additional Ph-chromosome or BCR/ABL amplification achieved complete suppression of Ph+ clone using 2nd generation TKI. The presence of additional chromosome 7 abnormalities and complex karyotype disorders involving isochromosome i(17(q10 are poor prognostic factors of TKI treatment failures.

  11. Influence of different chromosomal abnormalities in Ph-positive bone marrow cells on the chronic myeloid leukemia course during tyrosine kinase inhibitors therapy

    Directory of Open Access Journals (Sweden)

    O. Yu. Vinogradova

    2012-01-01

    Full Text Available The additional molecular and chromosomal abnormalities (ACA in Phositive cells usually considered as a genetic marker of chronic myeloid leukemia (CML progression. 457 patients in different CML phases received tyrosine kinase inhibitors (1st and 2nd generation were studied. During therapy 50 cases with additional chromosomal abnormalities in Ph+ clone (22 of them in chronic CML phase were revealed (median follow-up from CML diagnosis – 117 months, median imatinib therapy – 62 months. 86 % of patients in chronic phase with Ph+- cell abnormalities were cytogenetic resistance, and their 5-years overall survival was 80 % which was significantly lower than in patients without ACA (p < 0.005. The treatment results depend on chromosomal abnormalities detected. In patients with additional chromosome 8 imatinib therapy is effective, although complete cytogenetic response (CCR is achieved only in the later therapy stages. In patients with additional translocations CCR also achieved with imatinib or 2nd generation TKI. Only a third of patients with additional Ph-chromosome or BCR/ABL amplification achieved complete suppression of Ph+ clone using 2nd generation TKI. The presence of additional chromosome 7 abnormalities and complex karyotype disorders involving isochromosome i(17(q10 are poor prognostic factors of TKI treatment failures.

  12. CD34-positive interstitial cells of the human detrusor

    DEFF Research Database (Denmark)

    Rasmussen, Helle; Hansen, Alastair; Smedts, Frank;

    2007-01-01

    Interstitial cells of Cajal (ICC) are well described in the bowel wall. They are c-kit positive and play a role as pacemaker cells. Similar c-kit-positive cells have recently been described in the human bladder. The aim of this study was to characterize interstitial cells of the bladder detrusor ...

  13. BM microenvironmental protection of CML cells from imatinib through Stat5/NF-κB signaling and reversal by Wogonin.

    Science.gov (United States)

    Xu, Xuefen; Zhang, Xiaobo; Liu, Yicheng; Yang, Lin; Huang, Shaoliang; Lu, Lu; Wang, Shuhao; Guo, Qinglong; Zhao, Li

    2016-04-26

    Constitutive Stat5 activation enhanced cell survival and resistance to imatinib (IM) in chronic myelogenous leukemia (CML) cells. However, the mechanism of Stat5 activation in mediating resistance to IM in bone marrow (BM) microenvironment has not been evaluated precisely. In this study, we reported HS-5-derived conditioned medium (CM) significantly enhanced IM resistance in K562 and KU812. Interestingly, upregulation of the proportion of CD34+ subpopulation was found in CML cells. Subsequently, the BCR/ABL-independent activation of Stat5 increased P-glycoprotein (P-gp) activity in CM-mediated protection of CML stem cells (LSCs) from IM. Further research revealed Stat5 activation increased the DNA binding activity of NF-κB though binding of p-Stat5 and p-RelA in nucleus. Moreover, highly acetylated RelA was required for Stat5-mediated RelA nuclear binding. The study further confirmed that Wogonin potentiated the inhibitory effects of IM on leukemia development by suppressing Stat5 pathway both in CM model and the K562 xenograft model. In summary, results clearly demonstrated BCR/ABL-independent Stat5 survival pathway could contribute to resistance of CML LSCs to IM in BM microenvironment and suggested that natural durgs effectively inhibiting Stat5 may be an attractive approach to overcome resistance to BCR/ABL kinase inhibitors. PMID:27027438

  14. Reciprocal t(9;22 ABL/BCR fusion proteins: leukemogenic potential and effects on B cell commitment.

    Directory of Open Access Journals (Sweden)

    Xiaomin Zheng

    Full Text Available BACKGROUND: t(9;22 is a balanced translocation, and the chromosome 22 breakpoints (Philadelphia chromosome--Ph+ determine formation of different fusion genes that are associated with either Ph+ acute lymphatic leukemia (Ph+ ALL or chronic myeloid leukemia (CML. The "minor" breakpoint in Ph+ ALL encodes p185(BCR/ABL from der22 and p96(ABL/BCR from der9. The "major" breakpoint in CML encodes p210(BCR/ABL and p40(ABL/BCR. Herein, we investigated the leukemogenic potential of the der9-associated p96(ABL/BCR and p40(ABL/BCR fusion proteins and their roles in the lineage commitment of hematopoietic stem cells in comparison to BCR/ABL. METHODOLOGY: All t(9;22 derived proteins were retrovirally expressed in murine hematopoietic stem cells (SL cells and human umbilical cord blood cells (UCBC. Stem cell potential was determined by replating efficiency, colony forming--spleen and competitive repopulating assays. The leukemic potential of the ABL/BCR fusion proteins was assessed by in a transduction/transplantation model. Effects on the lineage commitment and differentiation were investigated by culturing the cells under conditions driving either myeloid or lymphoid commitment. Expression of key factors of the B-cell differentiation and components of the preB-cell receptor were determined by qRT-PCR. PRINCIPAL FINDINGS: Both p96(ABL/BCR and p40(ABL/BCR increased proliferation of early progenitors and the short term stem cell capacity of SL-cells and exhibited own leukemogenic potential. Interestingly, BCR/ABL gave origin exclusively to a myeloid phenotype independently from the culture conditions whereas p96(ABL/BCR and to a minor extent p40(ABL/BCR forced the B-cell commitment of SL-cells and UCBC. CONCLUSIONS/SIGNIFICANCE: Our here presented data establish the reciprocal ABL/BCR fusion proteins as second oncogenes encoded by the t(9;22 in addition to BCR/ABL and suggest that ABL/BCR contribute to the determination of the leukemic phenotype through their

  15. Matrix metalloproteinase-9 was involved in the immuno-modulatory defect of mesenchymal stem cell from chronic myeloid leukemia patients

    Institute of Scientific and Technical Information of China (English)

    ZHU Xi-shan; SHI Wei; AN Guang-yu; ZHANG Hong-mei; SONG Yu-guang; LI You-bin

    2011-01-01

    Background Overwhelming evidences on chronic myeloid leukemia (CML) indicate that patients harbor quiescent CML stem cells that are responsible for blast crisis. While the hematopoietic stem cell (HSC) origin of CML was first suggested over 30 years ago, recently CML-initiating cells beyond HSCs are also being investigated.Methods We have previously isolated fetal liver kinase-1-positive (Flk1+) cells carrying the BCR/ABL fusion gene from the bone marrow of Ph+ patients with hemangioblast property. In this study, we isolated CML patient-derived regulation using fluorescence in situ hybridization (FISH) analysis, fluorescence activated cell sorting (FACS),enzyme-linked immunoadsorbent assay, mixed lymphocyte reaction assays; then we compared these characters with those of the healthy donors.lymphocyte activation and proliferation was impaired in vitro.Conclusions CML patient-derived MSCs have impaired immuno-modulatory functions, suggesting that the dysregulation of hematopoiesis and immune response may originate from MSCs rather than hematopoietic stem cells (HSCs). MSCs might be a potential target for developing efficacious treatment for CML.

  16. Fangchinoline induces G0/G1 arrest by modulating the expression of CDKN1A and CCND2 in K562 human chronic myelogenous leukemia cells

    Science.gov (United States)

    WANG, YUPING; CHEN, JIE; WANG, LIN; HUANG, YUJI; LENG, YE; WANG, GUIYING

    2013-01-01

    Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease caused by the oncoprotein BCR-ABL, which exhibits a constitutive tyrosine kinase activity. Imatinib mesylate (IM), an inhibitor of the tyrosine kinase activity of BCR-ABL, has been used as a first-line therapy for CML. However, IM is less effective in the accelerated phase and blastic phases of CML and certain patients develop IM resistance due to the mutation and amplification of the BCR-ABL gene. Fangchinoline, an important chemical constituent from the dried roots of Stephaniae tetrandrae S. Moore, exhibits significant antitumor activity in various types of cancers, including breast, prostate and hepatocellular carcinoma. However, the effects and the underlying mechanisms of fangchinoline in CML remain unclear. In the present study, we identified that fangchinoline inhibits cell proliferation in a dose- and time-dependent manner in K562 cells derived from the blast crisis of CML. Additional experiments revealed that fangchinoline induces cell cycle arrest at the G0/G1 phase and has no effect on apoptosis, which is mediated through the upregulation of cyclin-dependent kinase (CDK)-N1A and MCL-1 mRNA levels, as well as the downregulation of cyclin D2 (CCND2) mRNA levels. These findings suggest the potential of fangchinoline as an effective antitumor agent in CML. PMID:23596478

  17. Clinical impact of ABL1 kinase domain mutations and IKZF1 deletion in adults under age 60 with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL): molecular analysis of CALGB (Alliance) 10001 and 9665.

    Science.gov (United States)

    DeBoer, Rebecca; Koval, Gregory; Mulkey, Flora; Wetzler, Meir; Devine, Steven; Marcucci, Guido; Stone, Richard M; Larson, Richard A; Bloomfield, Clara D; Geyer, Susan; Mullighan, Charles G; Stock, Wendy

    2016-10-01

    Recent studies have identified oncogenic lesions in Philadelphia chromosome-positive (Ph+)  acute lymphoblastic leukemia (ALL) and ABL1 kinase mutations that confer resistance to tyrosine kinase inhibitors. We sought to determine the prevalence and clinical impact of these lesions in patients on CALGB 10001, a previously reported Phase II study of imatinib, chemotherapy, and hematopoietic cell transplant in adult Ph + ALL. Of the 58 enrolled, 22 relapsed. By direct sequencing, an ABL1 kinase mutation known to induce imatinib resistance was present at relapse in 13 of 20. Using quantitative PCR assays, the mutations were detectable at diagnosis or early during treatment in most (62%) relapsed patients. Aberrations in IKZF1, CDKN2A/B, and PAX5 were assessed in 28 samples using SNP arrays and genomic DNA sequencing. Of these, 22 (79%) had IKZF1 deletion. The combination of IKZF1 deletion and p210 BCR-ABL1 (p < 0.0001), high white blood cell count (p = 0.021), and minimal residual disease (p = 0.013) were associated with worse disease-free survival. PMID:26892479

  18. Activity of ladanein on leukemia cell lines and its occurrence in Marrubium vulgare.

    Science.gov (United States)

    Alkhatib, Racha; Joha, Sami; Cheok, Meyling; Roumy, Vincent; Idziorek, Thierry; Preudhomme, Claude; Quesnel, Bruno; Sahpaz, Sevser; Bailleul, François; Hennebelle, Thierry

    2010-01-01

    Three methoxylated flavones isolated from Marrubium peregrinum - ladanein, scutellarein-5,7,4'-trimethyl ether, and scutellarein-5,6,7,4'-tetramethyl ether - were assayed for their cytotoxicity towards a recently developed dasatinib-resistant murine leukemia cell line (DA1-3b/M2 (BCR-ABL)), together with the structurally related non-methylated flavone scutellarein. The most active compound, ladanein, was looked for in 20 common Lamiaceae species by a quick HPLC screening. Among the possible positive results, the most interesting source was found to be Marrubium vulgare, which led to the isolation and identification of ladanein for the first time in this species. Ladanein also displayed moderate (20-40 microM) activities against K562, K562R (imatinib-resistant), and 697 human leukemia cell lines but was toxic neither to MOLM13 nor to human peripheral blood mononuclear cells. This work provides a common natural source for the hemi-synthesis of future ladanein-derived flavones and the study of their antileukemic activity. PMID:19644796

  19. Activity of ladanein on leukemia cell lines and its occurrence in Marrubium vulgare.

    Science.gov (United States)

    Alkhatib, Racha; Joha, Sami; Cheok, Meyling; Roumy, Vincent; Idziorek, Thierry; Preudhomme, Claude; Quesnel, Bruno; Sahpaz, Sevser; Bailleul, François; Hennebelle, Thierry

    2010-01-01

    Three methoxylated flavones isolated from Marrubium peregrinum - ladanein, scutellarein-5,7,4'-trimethyl ether, and scutellarein-5,6,7,4'-tetramethyl ether - were assayed for their cytotoxicity towards a recently developed dasatinib-resistant murine leukemia cell line (DA1-3b/M2 (BCR-ABL)), together with the structurally related non-methylated flavone scutellarein. The most active compound, ladanein, was looked for in 20 common Lamiaceae species by a quick HPLC screening. Among the possible positive results, the most interesting source was found to be Marrubium vulgare, which led to the isolation and identification of ladanein for the first time in this species. Ladanein also displayed moderate (20-40 microM) activities against K562, K562R (imatinib-resistant), and 697 human leukemia cell lines but was toxic neither to MOLM13 nor to human peripheral blood mononuclear cells. This work provides a common natural source for the hemi-synthesis of future ladanein-derived flavones and the study of their antileukemic activity.

  20. T cell depleted haploidentical transplantation: positive selection

    Directory of Open Access Journals (Sweden)

    Franco Aversa

    2011-06-01

    Full Text Available Interest in mismatched transplantation arises from the fact that a suitable one-haplotype mismatched donor is immediately available for virtually all patients, particularly for those who urgently need an allogenic transplant. Work on one haplotype-mismatched transplants has been proceeding for over 20 years all over the world and novel transplant techniques have been developed. Some centres have focused on the conditioning regimens and post transplant immune suppression; others have concentrated on manipulating the graft which may be a megadose of extensively T celldepleted or unmanipulated progenitor cells. Excellent engraftment rates are associated with a very low incidence of acute and chronic GVHD and regimen-related mortality even in patients who are over 50 years old. Overall, event-free survival and transplant-related mortality compare favourably with reports on transplants from sources of stem cells other than the matched sibling.

  1. In vitro effects of imatinib on CD34(+) cells of patients with chronic myeloid leukemia in the megakaryocytic crisis phase.

    Science.gov (United States)

    Meng, Fankai; Zeng, Wen; Huang, Lifang; Qin, Shuang; Miao, Ningning; Sun, Hanying; Li, Chunrui

    2014-03-01

    Imatinib is a tailored drug for the treatment of chronic myeloid leukemia (CML), and has substantial activity and a favorable safety profile when used as a single agent in patients with CML in myeloid blast crisis. The megakaryocytic blast crisis in CML occurs rarely and carries a poor prognosis. The aim of the present study was to investigate the effects of imatinib on cluster of differentiation (CD)34(+) cells from patients with CML in the megakaryocytic crisis phase. Bone marrow mononuclear cells (BMNCs) were isolated from patients with CML in the megakaryocytic crisis phase. CD34(+) cells were selected from BMNCs by positive immunomagnetic column separation. Imatinib significantly induced G1 arrest, reduced the phosphorylation of cyclin-dependent kinase 1 and retinoblastoma proteins and inhibited the proliferation of CD34(+) cells from patients with CML in the megakaryocytic crisis phase. Annexin V/propidium iodide and caspase-3 activity showed that imatinib induced apoptosis. Western blot analysis and protein tyrosine kinase activity assays showed that imatinib inhibited BCR-ABL protein tyrosine kinase activity. The in vitro data thus markedly indicate a potential clinical application of imatinib for patients with CML in the megakaryocytic crisis phase.

  2. Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

    OpenAIRE

    Christian Stemberger; Stefan Dreher; Claudia Tschulik; Christine Piossek; Jeannette Bet; Yamamoto, Tori N.; Matthias Schiemann; Michael Neuenhahn; Klaus Martin; Martin Schlapschy; Arne Skerra; Thomas Schmidt; Matthias Edinger; Riddell, Stanley R.; Lothar Germeroth

    2012-01-01

    A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve--especially for clinical cell product...

  3. Identification of galectin-1 as a novel mediator for chemoresistance in chronic myeloid leukemia cells.

    Science.gov (United States)

    Luo, Wu; Song, Li; Chen, Xi-Lei; Zeng, Xiang-Feng; Wu, Jian-Zhang; Zhu, Cai-Rong; Huang, Tao; Tan, Xiang-Peng; Lin, Xiao-Mian; Yang, Qi; Wang, Ji-Zhong; Li, Xiao-Kun; Wu, Xiao-Ping

    2016-05-01

    Multidrug resistance protein-1 (MDR1) has been proven to be associated with the development of chemoresistance to imatinib (Glivec, STI571) which displays high efficacy in treatment of BCR-ABL-positive chronic myelogenous leukemia (CML). However, the possible mechanisms of MDR1 modulation in the process of the resistance development remain to be defined. Herein, galectin-1 was identified as a candidate modulator of MDR1 by proteomic analysis of a model system of leukemia cell lines with a gradual increase of MDR1 expression and drug resistance. Coincidently, alteration of galectin-1 expression triggers the change of MDR1 expression as well as the resistance to the cytotoxic drugs, suggesting that augment of MDR1 expression engages in galectin-1-mediated chemoresistance. Moreover, we provided the first data showing that NF-κB translocation induced by P38 MAPK activation was responsible for the modulation effect of galectin-1 on MDR1 in the chronic myelogenous leukemia cells. Galectin-1 might be considered as a novel target for combined modality therapy for enhancing the efficacy of CML treatment with imatinib. PMID:27050374

  4. Hurdles overcome in technology transfer for AIET and Positive outcome in Indian patients

    Directory of Open Access Journals (Sweden)

    Dedeepiya V

    2012-01-01

    perigastric peritoneal deposits with a marked decrease in the size of the inguinal lymph nodes from and no recurrence in the pelvic region. There was also improvement in appetite and quality of life of the patient. There were no adverse reactions following the AIET infusions. [8] The latest follow-up in May 2012 revealed static non progressive disease with all the parameters including tumor markers within normal range. Case 2: A 15 year old girl presented with complaints of diffuse bone pain for 3 months, intermittent fever for 1 month and increasing pallor in October 2004. On examination, she was found to have pallor with hepatosplenomegaly. A low haemoglobin level (9.3 mg/dl, low platelets (21000 and a WBC count of 13600 with 83% lymphoblasts was revealed in her blood picture. Bone marrow examination and flow cytometry were suggestive of Acute Lymphoblastic Leukemia – CD 10+ CD 19+. Cytogenetics revealed Philadelphia chromosome and BCR-ABL positivity. Qualitative BCR-ABL was done serially to assess disease status. She was started on chemotherapy as per UK MRC protocol – Regimen B along with Imatinib Mesylate (in view of her BCR-ABL positivity and prophylactic cranial radiotherapy of 12 Gy at the end of delayed intensification. She had persistent disease with low positive BCR-ABL at 0.04% at the end of delayed intensification. As there were no matched HLA donors as she had high risk disease she was suggested for AIET using autologous natural killer (NK cells (CD3-CD56+ in January 2007. 562 x 106 in vitro expanded NK cells were infused to the patient intravenously and four weeks later, the BCR-ABL in the PB turned negative. She completed her treatment in October 2007. She has been on regular follow up for the last five years and she continues to be in remission. Case 3: A female patient aged 64 years with advanced serous papillary Adenocarcinoma of both ovaries with liver metastasis underwent omentectomy and resection of the liver nodule in the month of January 2011 and

  5. Influence of cell geometry on division-plane positioning.

    Science.gov (United States)

    Minc, Nicolas; Burgess, David; Chang, Fred

    2011-02-01

    The spatial organization of cells depends on their ability to sense their own shape and size. Here, we investigate how cell shape affects the positioning of the nucleus, spindle and subsequent cell division plane. To manipulate geometrical parameters in a systematic manner, we place individual sea urchin eggs into microfabricated chambers of defined geometry (e.g., triangles, rectangles, and ellipses). In each shape, the nucleus is positioned at the center of mass and is stretched by microtubules along an axis maintained through mitosis and predictive of the future division plane. We develop a simple computational model that posits that microtubules sense cell geometry by probing cellular space and orient the nucleus by exerting pulling forces that scale to microtubule length. This model quantitatively predicts division-axis orientation probability for a wide variety of cell shapes, even in multicellular contexts, and estimates scaling exponents for length-dependent microtubule forces. PMID:21295701

  6. Novel serial positive enrichment technology enables clinical multiparameter cell sorting.

    Directory of Open Access Journals (Sweden)

    Christian Stemberger

    Full Text Available A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve--especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4(high/CD25(high/CD45RA(high 'regulatory T cells' and CD8(high/CD62L(high/CD45RA(neg 'central memory T cells', have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research.

  7. Donor Umbilical Cord Blood Transplant in Treating Patients With Hematologic Cancer

    Science.gov (United States)

    2016-08-05

    Acute Biphenotypic Leukemia; Acute Lymphoblastic Leukemia; Acute Myeloid Leukemia; Aggressive Non-Hodgkin Lymphoma; Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Chronic Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Indolent Non-Hodgkin Lymphoma; Malignant Lymphoma, Large Cell Type; Mixed Phenotype Acute Leukemia; Myelodysplastic Syndrome; Myeloproliferative Neoplasm; Recurrent Chronic Lymphocytic Leukemia; Recurrent Follicular Lymphoma; Recurrent Lymphoplasmacytic Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Marginal Zone Lymphoma; Recurrent Plasma Cell Myeloma; Recurrent Small Lymphocytic Lymphoma; Recurrent T-Cell Non-Hodgkin Lymphoma; Refractory Chronic Lymphocytic Leukemia; Refractory Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Refractory Follicular Lymphoma; Refractory Hodgkin Lymphoma; Refractory Lymphoplasmacytic Lymphoma; Refractory Mantle Cell Lymphoma; Refractory Small Lymphocytic Lymphoma; T-Cell Non-Hodgkin Lymphoma

  8. Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

    Science.gov (United States)

    Tschulik, Claudia; Piossek, Christine; Bet, Jeannette; Yamamoto, Tori N.; Schiemann, Matthias; Neuenhahn, Michael; Martin, Klaus; Schlapschy, Martin; Skerra, Arne; Schmidt, Thomas; Edinger, Matthias; Riddell, Stanley R.; Germeroth, Lothar; Busch, Dirk H.

    2012-01-01

    A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve – especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4high/CD25high/CD45RAhigh ‘regulatory T cells’ and CD8high/CD62Lhigh/CD45RAneg ‘central memory T cells’, have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research. PMID:22545138

  9. SPARC expression in CML is associated to imatinib treatment and to inhibition of leukemia cell proliferation

    Directory of Open Access Journals (Sweden)

    Giallongo Cesarina

    2013-02-01

    Full Text Available Abstract Background SPARC is a matricellular glycoprotein with growth-inhibitory and antiangiogenic activity in some cell types. The study of this protein in hematopoietic malignancies led to conflicting reports about its role as a tumor suppressor or promoter, depending on its different functions in the tumor microenvironment. In this study we investigated the variations in SPARC production by peripheral blood cells from chronic myeloid leukemia (CML patients at diagnosis and after treatment and we identified the subpopulation of cells that are the prevalent source of SPARC. Methods We evaluated SPARC expression using real-time PCR and western blotting. SPARC serum levels were detected by ELISA assay. Finally we analyzed the interaction between exogenous SPARC and imatinib (IM, in vitro, using ATP-lite and cell cycle analysis. Results Our study shows that the CML cells of patients at diagnosis have a low mRNA and protein expression of SPARC. Low serum levels of this protein are also recorded in CML patients at diagnosis. However, after IM treatment we observed an increase of SPARC mRNA, protein, and serum level in the peripheral blood of these patients that had already started at 3 months and was maintained for at least the 18 months of observation. This SPARC increase was predominantly due to monocyte production. In addition, exogenous SPARC protein reduced the growth of K562 cell line and synergized in vitro with IM by inhibiting cell cycle progression from G1 to S phase. Conclusion Our results suggest that low endogenous SPARC expression is a constant feature of BCR/ABL positive cells and that IM treatment induces SPARC overproduction by normal cells. This exogenous SPARC may inhibit CML cell proliferation and may synergize with IM activity against CML.

  10. Treosulfan, Fludarabine Phosphate, and Total-Body Irradiation in Treating Patients With Hematological Cancer Who Are Undergoing Umbilical Cord Blood Transplant

    Science.gov (United States)

    2016-10-21

    Acute Biphenotypic Leukemia; Adult Acute Lymphoblastic Leukemia in Remission; Adult Acute Myeloid Leukemia in Remission; Adult Myelodysplastic Syndrome; Childhood Acute Lymphoblastic Leukemia in Remission; Childhood Acute Myeloid Leukemia in Remission; Childhood Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Childhood Myelodysplastic Syndrome; Chronic Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; RAEB-2; Recurrent Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Refractory Chronic Myelogenous Leukemia, BCR-ABL1 Positive

  11. The conformational control inhibitor of tyrosine kinases DCC-2036 is effective for imatinib-resistant cells expressing T674I FIP1L1-PDGFRα.

    Directory of Open Access Journals (Sweden)

    Yingying Shen

    Full Text Available The cells expressing the T674I point mutant of FIP1-like-1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRα in hypereosinophilics syndrome (HES are resistant to imatinib and some second-generation tyrosine kinase inhibitors (TKIs. There is a desperate need to develop therapy to combat this acquired drug resistance. DCC-2036 has been synthesized as a third-generation TKI to combat especially the Bcr-Abl T315I mutant in chronic myeloid leukemia. This study evaluated the effect of DCC-2036 on FIP1L1-PDGFRα-positive cells, including the wild type (WT and the T674I mutant. The in vitro effects of DCC-2036 on the PDGFRα signal pathways, proliferation, cell cycling and apoptosis of FIP1L1-PDGFRα-positive cells were investigated, and a nude mouse xenograft model was employed to assess the in vivo antitumor activity. We found that DCC-2036 decreased the phosphorylated levels of PDGFRα and its downstream targets without apparent effects on total protein levels. DCC-2036 inhibited proliferation, and induced apoptosis with MEK-dependent up-regulation of the pro-apoptotic protein Bim in FIP1L1-PDGFRα-positive cells. DCC-2036 also exhibited in vivo antineoplastic activity against cells with T674I FIP1L1-PDGFRα. In summary, FIP1L1-PDGFRα-positive cells are sensitive to DCC-2036 regardless of their sensitivity to imatinib. DCC-2036 may be a potential compound to treat imatinib-resistant HES.

  12. Vimentin positive acantholytic penile squamous cell carcinoma with rhabdoid features

    Directory of Open Access Journals (Sweden)

    Ramesh Y Chavan

    2014-01-01

    Full Text Available Acantholytic variant of penile squamous cell carcinoma (SCC is an exceedingly rare and associated with bad prognosis. Histologically it mimics angiosarcoma due to pseudovascular spaces. Vimentin immunopositivity in such cases represent epithelial to mesenchymal transition manifested by cellular discohesion. We describe a case of vimentin positive acantholytic penile SCC in a 55-year-old patient.

  13. Imatinib triggers mesenchymal-like conversion of CML cells associated with increased aggressiveness

    Institute of Scientific and Technical Information of China (English)

    Alexandre Puissant; Yann Chéli; Jill-Patrice Cassuto; Sophie Raynaud; Laurence Legros; Jean-Max Pasquet; Francois-Xavier Mahon; Frédéric Luciano; Patrick Auberger; Maeva Dufies; Nina Fenouille; Issam Ben Sahra; Arnaud Jacquel; Guillaume Robert; Thomas Cluzeau; Marcel Deckert; Mélanie Tichet

    2012-01-01

    Chronic myelogenous leukemia (CML) is a cytogenetic disorder resulting from the expression of p210BCR-ABL Imatinib,an inhibitor of BCR-ABL,has emerged as the leading compound to treat CML patients.Despite encouraging clinical results,resistance to imatinib represents a major drawback for therapy,as a substantial proportion of patients are refractory to this treatment.Recent publications have described the existence of a small cancer cell population with the potential to exhibit the phenotypic switch responsible for chemoresistance.To investigate the existence of such a chemoresistant cellular subpopulation in CML,we used a two-step approach of pulse and continuous selection by imatinib in different CML cell lines that allowed the emergence of a subpopulation of adherent cells (IM-R Adh) displaying an epithelial-mesenchymal transition (EMT)-like phenotype.Overexpression of several EMT markers was observed in this CML subpopulation,as well as in CD34+ CML primary cells from patients who responded poorly to imatinib treatment.In response to imatinib,this CD44high/CD24low IM-R Adh subpopulation exhibited increased adhesion,transmigration and invasion in vitro and in vivo through specific overexpression of the αVβ3 receptor.FAK/Akt pathway activation following integrin β3 (ITGβ3) engagement mediated the migration and invasion of IM-R Adh cells,whereas persistent activation of ERK counteracted BCR-ABL inhibition by imatinib,promoting cell adhesion-mediated resistance.

  14. Positioning.

    Science.gov (United States)

    Conone, Ruth M.

    The key to positioning is the creation of a clear benefit image in the consumer's mind. One positioning strategy is creating in the prospect's mind a position that takes into consideration the company's or agency's strengths and weaknesses as well as those of its competitors. Another strategy is to gain entry into a position ladder owned by…

  15. Fulvestrant radiosensitizes human estrogen receptor-positive breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jing, E-mail: wangstella5@163.com [Department of Breast Surgery, Qilu Hospital, Shandong Univeristy, Wenhua Xi Road 107, Shandong Province (China); Department of Oncology, Affiliated Hospital of Qingdao University Medical College, Shandong Province (China); Yang, Qifeng, E-mail: qifengy@gmail.com [Department of Breast Surgery, Qilu Hospital, Shandong Univeristy, Wenhua Xi Road 107, Shandong Province (China); Haffty, Bruce G., E-mail: hafftybg@umdnj.edu [Department of Radiation Oncology, UMDNJ-Robert Wood Johnson School of Medicine, Cancer Institute of New Jersey, NB (United States); Li, Xiaoyan, E-mail: xiaoyanli1219@gmail.com [Department of Oncology, Affiliated Hospital of Qingdao University Medical College, Shandong Province (China); Moran, Meena S., E-mail: meena.moran@yale.edu [Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT (United States)

    2013-02-08

    Highlights: ► Fulvestrant radiosensitizes MCF-7 cells. ► Fulvestrant increases G1 arrest and decreases S phase in MCF-7 cells. ► Fulvestrant down-regulates DNA-PKcs and RAD51 in MCF-7 cells. -- Abstract: The optimal sequencing for hormonal therapy and radiation are yet to be determined. We utilized fulvestrant, which is showing promise as an alternative to other agents in the clinical setting of hormonal therapy, to assess the cellular effects of concomitant anti-estrogen therapy (fulvestrant) with radiation (F + RT). This study was conducted to assess the effects of fulvestrant alone vs. F + RT on hormone-receptor positive breast cancer to determine if any positive or negative combined effects exist. The effects of F + RT on human breast cancer cells were assessed using MCF-7 clonogenic and tetrazolium salt colorimetric (MTT) assays. The assays were irradiated with a dose of 0, 2, 4, 6 Gy ± fulvestrant. The effects of F + RT vs. single adjuvant treatment alone on cell-cycle distribution were assessed using flow cytometry; relative expression of repair proteins (Ku70, Ku80, DNA-PKcs, Rad51) was assessed using Western Blot analysis. Cell growth for radiation alone vs. F + RT was 0.885 ± 0.013 vs. 0.622 ± 0.029 @2 Gy, 0.599 ± 0.045 vs. 0.475 ± 0.054 @4 Gy, and 0.472 ± 0.021 vs. 0.380 ± 0.018 @6 Gy RT (p = 0.003). While irradiation alone induced G2/M cell cycle arrest, the combination of F + RT induced cell redistribution in the G1 phase and produced a significant decrease in the proportion of cells in G2 phase arrest and in the S phase in breast cancer cells (p < 0.01). Furthermore, levels of repair proteins DNA-PKcs and Rad51 were significantly decreased in the cells treated with F + RT compared with irradiation alone. F + RT leads to a decrease in the surviving fraction, increased cell cycle arrest, down regulating of nonhomologous repair protein DNA-PKcs and homologous recombination repair protein RAD51. Thus, our findings suggest that F + RT

  16. FOXP3 positive regulatory T-cells in cutaneous and systemic CD30 positive T-cell lymphoproliferations

    DEFF Research Database (Denmark)

    Gjerdrum, Lise Mette; Woetmann, Anders; Ødum, Niels;

    2008-01-01

    -cells (Tregs) may be implicated. In this study, skin biopsies from lymphomatoid papulosis (LyP) (n = 14), primary cutaneous anaplastic large cells lymphoma (C-ALCL) (n = 13) and systemic anaplastic large cells lymphoma (S-ALCL) with (n = 9) or without (n = 6) ALK expression were examined by immunohistology...... for FOXP3 expression in tumour cells and tumour infiltrating Tregs. Labelling of a majority of the neoplastic cells was seen in one case of C-ALCL. Another three cases (one LyP and two C-ALCL) displayed weak labelling of very occasional atypical T-cells. In the remaining 38 cases the atypical lymphoid...... infiltrate was FOXP3 negative. By contrast, all biopsies contained tumour infiltrating FOXP3-positive Tregs. Significant higher numbers were recorded in ALK negative S-ALCL and LyP than in C-ALCL and S-ALCL positive for ALK. In conclusion, it is shown that FOXP3 expression in cutaneous and systemic CD30...

  17. Octreotide scintigraphy localizes somatostatin receptor-positive islet cell carcinomas

    International Nuclear Information System (INIS)

    Tyr-3-octreotide is a synthetic derivative of somatostatin and a somatostatin-receptor analogue. The iodine-123-labelled compound localizes somatostatin-receptor-positive tumours. In this paper two patients are reported in whom somatostatin receptors were demonstrated in vitro. In a 60-year-old female with an islet cell carcinoma of the pancreas, multiple liver metastases and previously uncrecognized bone metastases in the right acetabulum could be diagnosed as the reason for a persistent hypoglycaemia. In a 60-year-old male an islet cell carcinoma of the pancreas was localized with 123I-Tyr-3-octreotide. The somatostatin receptors were demonstrated in vitro and the tumour was successfully treated with somatostatin. These studies demonstrate that 123I-Tyr-3-octreotide offers the possibility of localizing somatostatin-receptor-positive tumours and their metastases. Moreover the method makes it possible to determine the receptor status of a tumour in vivo. (orig.)

  18. Segmentation of breast cancer cells positive 1+ and 3+ immunohistochemistry

    Science.gov (United States)

    Labellapansa, Ause; Muhimmah, Izzati; Indrayanti

    2016-03-01

    Breast cancer is a disease occurs as a result of uncontrolled cells growth. One examination method of breast cancer cells is using Immunohistochemistry (IHC) to determine status of Human Epidermal Growth Factor Receptor2 (HER2) protein. This study helps anatomic pathologist to determine HER2 scores using image processing techniques to obtain HER2 overexpression positive area percentages of 1+ and 3+ scores. This is done because the score of 0 is HER2 negative cells and 2+ scores have equivocal results, which means it could not be determined whether it is necessary to give targeted therapy or not. HER2 overexpression positive area percentage is done by dividing the area with a HER2 positive tumor area. To obtain better tumor area, repair is done by eliminating lymphocytes area which is not tumor area using morphological opening. Results of 10 images IHC scores of 1+ and 3+ and 10 IHC images testing without losing lymphocytes area in tumor area, has proven that the system has been able to provide an overall correct classification in accordance with the experts analysis. However by doing operation to remove non-tumor areas, classification can be done correctly 100% for scores of 3+ and 65% for scores of 1+.

  19. Isolated central nervous system relapse of chronic myeloid leukemia after allogeneic hematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Fuchs Mary

    2012-08-01

    Full Text Available Abstract Background This case report highlights the relevance of quantifying the BCR-ABL gene in cerebrospinal fluid of patients with suspected relapse of chronic myeloid leukemia in the central nervous system. Case presentation We report on a female patient with isolated central nervous system relapse of chronic myeloid leukemia (CML during peripheral remission after allogeneic hematopoietic stem cell transplantation. The patient showed a progressive cognitive decline as the main symptom. MRI revealed a hydrocephalus and an increase in cell count in the cerebrospinal fluid (CSF with around 50% immature blasts in the differential count. A highly elevated BCR-ABL/ ABL ratio was detected in the CSF, whilst the ratio for peripheral blood and bone marrow was not altered. On treatment of the malresorptive hydrocephalus with shunt surgery, the patient showed an initial cognitive improvement, followed by a secondary deterioration. At this time, the cranial MRI showed leukemic infiltration of lateral ventricles walls. Hence, intrathecal administration of cytarabine, methotrexate, and dexamethasone was initiated, which caused a significant decrease of cells in the CSF. Soon after, the patient demonstrated significant cognitive improvement with a good participation in daily activities. At a later time point, after the patient had lost the major molecular response of CML, therapy with dasatinib was initiated. In a further follow-up, the patient was neurologically and hematologically stable. Conclusions In patients with treated CML, the rare case of an isolated CNS blast crisis has to be taken into account if neurological symptoms evolve. The analysis of BCR-ABL in the CSF is a further option for the reliable detection of primary isolated relapse of CML in these patients.

  20. Embryonic and foetal Islet-1 positive cells in human hearts are also positive to c-Kit

    Directory of Open Access Journals (Sweden)

    C. Serradifalco

    2011-12-01

    Full Text Available During embryogenesis, the mammalian heart develops from a primitive heart tube originating from two bilateral primary heart fields located in the lateral plate mesoderm. Cells belongings to the pre-cardiac mesoderm will differentiate into early cardiac progenitors, which express early transcription factors which are also common to the Isl-1 positive cardiac progenitor cells isolated from the developing pharyngeal mesoderm and the foetal and post-natal mice hearts. A second population of cardiac progenitor cells positive to c-Kit has been abundantly isolated from adult hearts. Until now, these two populations have been considered two different sets of progenitor cells present in the heart in different stages of an individual life. In the present study we collected embryonic, foetal and infant hearts, and we tested the hypotheses that c-Kit positive cells, usually isolated from the adult heart, are also present in the intra-uterine life and persist in the adult heart after birth, and that foetal Isl-1 positive cells are also positive to c-Kit. Using immunohistochemistry we studied the temporal distribution of Isl-1 positive and c-Kit/CD105 double positive cells, and by immunofluorescence and confocal analysis we studied the co-localization of c-Kit and Isl-1 positive cells. The results indicated that cardiomyocytes and interstitial cells were positive for c-Kit from the 9th to the 19th gestational week, that cells positive for both c-Kit and CD105 appeared in the interstitium at the 17th gestational week and persisted in the postnatal age, and that the Isl-1 positive cells were a subset of the c-Kit positive population.

  1. Impaired DNA replication within progenitor cell pools promotes leukemogenesis.

    Directory of Open Access Journals (Sweden)

    Ganna Bilousova

    2005-12-01

    Full Text Available Impaired cell cycle progression can be paradoxically associated with increased rates of malignancies. Using retroviral transduction of bone marrow progenitors followed by transplantation into mice, we demonstrate that inhibition of hematopoietic progenitor cell proliferation impairs competition, promoting the expansion of progenitors that acquire oncogenic mutations which restore cell cycle progression. Conditions that impair DNA replication dramatically enhance the proliferative advantage provided by the expression of Bcr-Abl or mutant p53, which provide no apparent competitive advantage under conditions of healthy replication. Furthermore, for the Bcr-Abl oncogene the competitive advantage in contexts of impaired DNA replication dramatically increases leukemogenesis. Impaired replication within hematopoietic progenitor cell pools can select for oncogenic events and thereby promote leukemia, demonstrating the importance of replicative competence in the prevention of tumorigenesis. The demonstration that replication-impaired, poorly competitive progenitor cell pools can promote tumorigenesis provides a new rationale for links between tumorigenesis and common human conditions of impaired DNA replication such as dietary folate deficiency, chemotherapeutics targeting dNTP synthesis, and polymorphisms in genes important for DNA metabolism.

  2. A cell cycle timer for asymmetric spindle positioning.

    Directory of Open Access Journals (Sweden)

    Erin K McCarthy Campbell

    2009-04-01

    Full Text Available The displacement of the mitotic spindle to one side of a cell is important for many cells to divide unequally. While recent progress has begun to unveil some of the molecular mechanisms of mitotic spindle displacement, far less is known about how spindle displacement is precisely timed. A conserved mitotic progression mechanism is known to time events in dividing cells, although this has never been linked to spindle displacement. This mechanism involves the anaphase-promoting complex (APC, its activator Cdc20/Fizzy, its degradation target cyclin, and cyclin-dependent kinase (CDK. Here we show that these components comprise a previously unrecognized timer for spindle displacement. In the Caenorhabditis elegans zygote, mitotic spindle displacement begins at a precise time, soon after chromosomes congress to the metaphase plate. We found that reducing the function of the proteasome, the APC, or Cdc20/Fizzy delayed spindle displacement. Conversely, inactivating CDK in prometaphase caused the spindle to displace early. The consequence of experimentally unlinking spindle displacement from this timing mechanism was the premature displacement of incompletely assembled components of the mitotic spindle. We conclude that in this system, asymmetric positioning of the mitotic spindle is normally delayed for a short time until the APC inactivates CDK, and that this delay ensures that the spindle does not begin to move until it is fully assembled. To our knowledge, this is the first demonstration that mitotic progression times spindle displacement in the asymmetric division of an animal cell. We speculate that this link between the cell cycle and asymmetric cell division might be evolutionarily conserved, because the mitotic spindle is displaced at a similar stage of mitosis during asymmetric cell divisions in diverse systems.

  3. Umbilical Cord Blood Transplant, Cyclophosphamide, Fludarabine Phosphate, and Total-Body Irradiation in Treating Patients With Hematologic Disease

    Science.gov (United States)

    2016-10-04

    Acute Biphenotypic Leukemia; Acute Myeloid Leukemia Arising From Previous Myelodysplastic Syndrome; Acute Myeloid Leukemia in Remission; Adult Acute Lymphoblastic Leukemia in Complete Remission; Aggressive Non-Hodgkin Lymphoma; Burkitt Lymphoma; Childhood Acute Lymphoblastic Leukemia in Complete Remission; Chronic Phase Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Lymphoblastic Lymphoma; Mantle Cell Lymphoma; Myelofibrosis; Pancytopenia; Plasma Cell Myeloma; Prolymphocytic Leukemia; Recurrent Childhood Acute Myeloid Leukemia; Recurrent Chronic Lymphocytic Leukemia; Recurrent Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Recurrent Follicular Lymphoma; Recurrent Lymphoplasmacytic Lymphoma; Recurrent Marginal Zone Lymphoma; Recurrent Small Lymphocytic Lymphoma; Refractory Anemia With Excess Blasts

  4. Small molecule ErbB inhibitors decrease proliferative signaling and promote apoptosis in philadelphia chromosome-positive acute lymphoblastic leukemia.

    Directory of Open Access Journals (Sweden)

    Mary E Irwin

    Full Text Available The presence of the Philadelphia chromosome in patients with acute lymphoblastic leukemia (Ph(+ALL is a negative prognostic indicator. Tyrosine kinase inhibitors (TKI that target BCR/ABL, such as imatinib, have improved treatment of Ph(+ALL and are generally incorporated into induction regimens. This approach has improved clinical responses, but molecular remissions are seen in less than 50% of patients leaving few treatment options in the event of relapse. Thus, identification of additional targets for therapeutic intervention has potential to improve outcomes for Ph+ALL. The human epidermal growth factor receptor 2 (ErbB2 is expressed in ~30% of B-ALLs, and numerous small molecule inhibitors are available to prevent its activation. We analyzed a cohort of 129 ALL patient samples using reverse phase protein array (RPPA with ErbB2 and phospho-ErbB2 antibodies and found that activity of ErbB2 was elevated in 56% of Ph(+ALL as compared to just 4.8% of Ph(-ALL. In two human Ph+ALL cell lines, inhibition of ErbB kinase activity with canertinib resulted in a dose-dependent decrease in the phosphorylation of an ErbB kinase signaling target p70S6-kinase T389 (by 60% in Z119 and 39% in Z181 cells at 3 µM. Downstream, phosphorylation of S6-kinase was also diminished in both cell lines in a dose-dependent manner (by 91% in both cell lines at 3 µM. Canertinib treatment increased expression of the pro-apoptotic protein Bim by as much as 144% in Z119 cells and 49% in Z181 cells, and further produced caspase-3 activation and consequent apoptotic cell death. Both canertinib and the FDA-approved ErbB1/2-directed TKI lapatinib abrogated proliferation and increased sensitivity to BCR/ABL-directed TKIs at clinically relevant doses. Our results suggest that ErbB signaling is an additional molecular target in Ph(+ALL and encourage the development of clinical strategies combining ErbB and BCR/ABL kinase inhibitors for this subset of ALL patients.

  5. Leukemic stem cell persistence in chronic myeloid leukemia patients in deep molecular response induced by tyrosine kinase inhibitors and the impact of therapy discontinuation

    Science.gov (United States)

    Chomel, Jean Claude; Bonnet, Marie Laure; Sorel, Nathalie; Sloma, Ivan; Bennaceur-Griscelli, Annelise; Rea, Delphine; Legros, Laurence; Marfaing-Koka, Anne; Bourhis, Jean-Henri; Ame, Shanti; Guerci-Bresler, Agnès; Rousselot, Philippe; Turhan, Ali G.

    2016-01-01

    During the last decade, the use of tyrosine kinase inhibitor (TKI) therapy has modified the natural history of chronic myeloid leukemia (CML) allowing an increase of the overall and disease-free survival, especially in patients in whom molecular residual disease becomes undetectable. However, it has been demonstrated that BCR-ABL1- expressing leukemic stem cells (LSCs) persist in patients in deep molecular response. It has also been shown that the discontinuation of Imatinib leads to a molecular relapse in the majority of cases. To determine a possible relationship between these two phenomena, we have evaluated by clonogenic and long-term culture initiating cell (LTC-IC) assays, the presence of BCR-ABL1-expressing LSCs in marrow samples from 21 patients in deep molecular response for three years after TKI therapy (mean duration seven years). LSCs were detected in 4/21 patients. Discontinuation of TKI therapy in 13/21 patients led to a rapid molecular relapse in five patients (4 without detectable LSCs and one with detectable LSCs). No relapse occurred in the eight patients still on TKI therapy, whether LSCs were detectable or not. Thus, this study demonstrates for the first time the in vivo efficiency of TKIs, both in the progenitor and the LSC compartments. It also confirms the persistence of leukemic stem cells in patients in deep molecular response, certainly at the origin of relapses. Finally, it emphasizes the difficulty of detecting residual LSCs due to their rarity and their low BCR-ABL1 mRNA expression. PMID:27167108

  6. Leukemic stem cell persistence in chronic myeloid leukemia patients in deep molecular response induced by tyrosine kinase inhibitors and the impact of therapy discontinuation.

    Science.gov (United States)

    Chomel, Jean Claude; Bonnet, Marie-Laure; Sorel, Nathalie; Sloma, Ivan; Bennaceur-Griscelli, Annelise; Rea, Delphine; Legros, Laurence; Marfaing-Koka, Anne; Bourhis, Jean-Henri; Ame, Shanti; Guerci-Bresler, Agnès; Rousselot, Philippe; Turhan, Ali G

    2016-06-01

    During the last decade, the use of tyrosine kinase inhibitor (TKI) therapy has modified the natural history of chronic myeloid leukemia (CML) allowing an increase of the overall and disease-free survival, especially in patients in whom molecular residual disease becomes undetectable. However, it has been demonstrated that BCR-ABL1- expressing leukemic stem cells (LSCs) persist in patients in deep molecular response. It has also been shown that the discontinuation of Imatinib leads to a molecular relapse in the majority of cases. To determine a possible relationship between these two phenomena, we have evaluated by clonogenic and long-term culture initiating cell (LTC-IC) assays, the presence of BCR-ABL1-expressing LSCs in marrow samples from 21 patients in deep molecular response for three years after TKI therapy (mean duration seven years). LSCs were detected in 4/21 patients. Discontinuation of TKI therapy in 13/21 patients led to a rapid molecular relapse in five patients (4 without detectable LSCs and one with detectable LSCs). No relapse occurred in the eight patients still on TKI therapy, whether LSCs were detectable or not. Thus, this study demonstrates for the first time the in vivo efficiency of TKIs, both in the progenitor and the LSC compartments. It also confirms the persistence of leukemic stem cells in patients in deep molecular response, certainly at the origin of relapses. Finally, it emphasizes the difficulty of detecting residual LSCs due to their rarity and their low BCR-ABL1 mRNA expression. PMID:27167108

  7. Identification of uPAR-positive chemoresistant cells in small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Margarita Gutova

    Full Text Available BACKGROUND: The urokinase plasminogen activator (uPA and its receptor (uPAR/CD87 are major regulators of extracellular matrix degradation and are involved in cell migration and invasion under physiological and pathological conditions. The uPA/uPAR system has been of great interest in cancer research because it is involved in the development of most invasive cancer phenotypes and is a strong predictor of poor patient survival. However, little is known about the role of uPA/uPAR in small cell lung cancer (SCLC, the most aggressive type of lung cancer. We therefore determined whether uPA and uPAR are involved in generation of drug resistant SCLC cell phenotype. METHODS AND FINDINGS: We screened six human SCLC cell lines for surface markers for putative stem and cancer cells. We used fluorescence-activated cell sorting (FACS, fluorescence microscopy and clonogenic assays to demonstrate uPAR expression in a subpopulation of cells derived from primary and metastatic SCLC cell lines. Cytotoxic assays were used to determine the sensitivity of uPAR-positive and uPAR-negative cells to chemotherapeutic agents. The uPAR-positive cells in all SCLC lines demonstrated multi-drug resistance, high clonogenic activity and co-expression of CD44 and MDR1, putative cancer stem cell markers. CONCLUSIONS: These data suggest that uPAR-positive cells may define a functionally important population of cancer cells in SCLC, which are resistant to traditional chemotherapies, and could serve as critical targets for more effective therapeutic interventions in SCLC.

  8. Spindle Positioning and Cell Division in Caenorhabditis elegans

    NARCIS (Netherlands)

    Voet, M. van der

    2010-01-01

    During cell division a cell duplicates its genetic material and segregates one intact copy into each daughter cell. However, cell division has many aspects in addition to the propagation of the genome. For instance, some cells divide asymmetrically, which contributes to the generation of cell divers

  9. Potentiation of antileukemic therapies by the dual PI3K/PDK-1 inhibitor, BAG956: effects on BCR-ABL– and mutant FLT3-expressing cells

    Science.gov (United States)

    Weisberg, Ellen; Banerji, Lolita; Wright, Renee D.; Barrett, Rosemary; Ray, Arghya; Moreno, Daisy; Catley, Laurence; Jiang, Jingrui; Hall-Meyers, Elizabeth; Sauveur-Michel, Maira; Stone, Richard; Galinsky, Ilene; Fox, Edward; Kung, Andrew L.

    2008-01-01

    Mediators of PI3K/AKT signaling have been implicated in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). Studies have shown that inhibitors of PI3K/AKT signaling, such as wortmannin and LY294002, are able to inhibit CML and AML cell proliferation and synergize with targeted tyrosine kinase inhi-bitors. We investigated the ability of BAG956, a dual PI3K/PDK-1 inhibitor, to be used in combination with inhibitors of BCR-ABL and mutant FLT3, as well as with the mTOR inhibitor, rapamycin, and the rapamycin derivative, RAD001. BAG956 was shown to block AKT phosphorylation induced by BCR-ABL–, and induce apoptosis of BCR-ABL–expressing cell lines and patient bone marrow cells at concentrations that also inhibit PI3K signaling. Enhancement of the inhibitory effects of the tyrosine kinase inhibitors, imatinib and nilotinib, by BAG956 was demonstrated against BCR-ABL expressing cells both in vitro and in vivo. We have also shown that BAG956 is effective against mutant FLT3-expressing cell lines and AML patient bone marrow cells. Enhancement of the inhibitory effects of the tyrosine kinase inhibitor, PKC412, by BAG956 was demonstrated against mutant FLT3-expressing cells. Finally, BAG956 and rapamycin/RAD001 were shown to combine in a nonantagonistic fashion against BCR-ABL– and mutant FLT3-expressing cells both in vitro and in vivo. PMID:18184863

  10. Peripheral canine CD4(+)CD8(+) double-positive T cells - unique amongst others.

    Science.gov (United States)

    von Buttlar, Heiner; Bismarck, Doris; Alber, Gottfried

    2015-12-15

    T lymphocytes co-expressing CD4 and CD8 ("double-positive T cells") are commonly associated with a thymic developmental stage of T cells. Their first description in humans and pigs as extrathymic T cells with a memory phenotype almost 30 years ago came as a surprise. Meanwhile peripheral double-positive T cells have been described in a growing number of different species. In this review we highlight novel data from our very recent studies on canine peripheral double-positive T cells which point to unique features of double-positive T cells in the dog. In contrast to porcine CD4(+)CD8(+) T cells forming a homogenous cellular population based on their expression of CD4 and CD8α, canine CD4(+)CD8(+) T cells can be divided into three different cellular subsets with distinct expression levels of CD4 and CD8α. Double-positive T cells expressing CD8β are present in humans and dogs but absent in swine. Moreover, canine CD4(+)CD8(+) T cells can not only develop from CD4(+) single-positive T cells but also from CD8(+) single-positive T cells. Together, this places canine CD4(+)CD8(+) T cells closer to their human than porcine counterparts since human double-positive T cells also appear to be heterogeneous in their CD4 and CD8α expression and have both CD4(+) and CD8(+) T cells as progenitor cells. However, CD4(+) single-positive T cells are the more potent progenitors for canine double-positive T cells, whereas CD8(+) single-positive T cells are more potent progenitors for human double-positive T cells. Canine double-positive T cells have an activated phenotype and may have as yet unrecognized roles in vivo in immunity to infection or in inflammatory diseases such as chronic infection, autoimmunity, allergy, or cancer.

  11. Local positive feedback regulation determines cell shape in root hair cells.

    Science.gov (United States)

    Takeda, Seiji; Gapper, Catherine; Kaya, Hidetaka; Bell, Elizabeth; Kuchitsu, Kazuyuki; Dolan, Liam

    2008-02-29

    The specification and maintenance of growth sites are tightly regulated during cell morphogenesis in all organisms. ROOT HAIR DEFECTIVE 2 reduced nicotinamide adenine dinucleotide phosphate (RHD2 NADPH) oxidase-derived reactive oxygen species (ROS) stimulate a Ca2+ influx into the cytoplasm that is required for root hair growth in Arabidopsis thaliana. We found that Ca2+, in turn, activated the RHD2 NADPH oxidase to produce ROS at the growing point in the root hair. Together, these components could establish a means of positive feedback regulation that maintains an active growth site in expanding root hair cells. Because the location and stability of growth sites predict the ultimate form of a plant cell, our findings demonstrate how a positive feedback mechanism involving RHD2, ROS, and Ca2+ can determine cell shape.

  12. FGF7 and cell density are required for final differentiation of pancreatic amylase-positive cells from human ES cells.

    Science.gov (United States)

    Takizawa-Shirasawa, Sakiko; Yoshie, Susumu; Yue, Fengming; Mogi, Akimi; Yokoyama, Tadayuki; Tomotsune, Daihachiro; Sasaki, Katsunori

    2013-12-01

    The major molecular signals of pancreatic exocrine development are largely unknown. We examine the role of fibroblast growth factor 7 (FGF7) in the final induction of pancreatic amylase-containing exocrine cells from induced-pancreatic progenitor cells derived from human embryonic stem (hES) cells. Our protocol consisted in three steps: Step I, differentiation of definitive endoderm (DE) by activin A treatment of hES cell colonies; Step II, differentiation of pancreatic progenitor cells by re-plating of the cells of Step I onto 24-well plates at high density and stimulation with all-trans retinoic acid; Step III, differentiation of pancreatic exocrine cells with a combination of FGF7, glucagon-like peptide 1 and nicotinamide. The expression levels of pancreatic endodermal markers such as Foxa2, Sox17 and gut tube endoderm marker HNF1β were up-regulated in both Step I and II. Moreover, in Step III, the induced cells expressed pancreatic markers such as amylase, carboxypeptidase A and chymotrypsinogen B, which were similar to those in normal human pancreas. From day 8 in Step III, cells immunohistochemically positive for amylase and for carboxypeptidase A, a pancreatic exocrine cell product, were induced by FGF7. Pancreatic progenitor Pdx1-positive cells were localized in proximity to the amylase-positive cells. In the absence of FGF7, few amylase-positive cells were identified. Thus, our three-step culture protocol for human ES cells effectively induces the differentiation of amylase- and carboxypeptidase-A-containing pancreatic exocrine cells.

  13. Guggulsterone of Commiphora mukul resin reverses drug resistance in imatinib-resistant leukemic cells by inhibiting cyclooxygenase-2 and P-glycoprotein.

    Science.gov (United States)

    Xu, Hong-Bin; Xu, Lu-Zhong; Mao, Xia-Ping; Fu, Jun

    2014-06-15

    The purpose of this study was to investigate the effects of guggulsterone on cyclooxygenase-2 and P-glycoprotein mediated drug resistance in imatinib-resistant K562 cells (K562/IMA). MTT cytotoxicity assay, flow cytometry, western blot analysis, and ELISA were performed to investigate the anti-proliferative effect, the reversal action of drug resistance, and the inhibitory effect on cyclooxygenase-2, P-glycoprotein, BCR/ABL kinase, and PGE2 release in K562/IMA cells by guggulsterone. The results showed that co-administration of guggulsterone resulted in a significant increase in chemo-sensitivity of K562/IMA cells to imatinib, compared with imatinib treatment alone. Rhodamine123 accumulation in K562/IMA cells was significantly enhanced after incubation with guggulsterone (60, 120 μM), compared with untreated K562/IMA cells (pP-glycoprotein and prostaglandin E2. However, guggulsterone had little inhibitory effect on the activity of BCR/ABL kinase. The present study indicates guggulsterone induces apoptosis by inhibiting cyclooxygenase-2 and down-regulating P-glycoprotein expression in K562/IMA cells.

  14. ADAR1 Activation Drives Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis.

    Science.gov (United States)

    Zipeto, Maria Anna; Court, Angela C; Sadarangani, Anil; Delos Santos, Nathaniel P; Balaian, Larisa; Chun, Hye-Jung; Pineda, Gabriel; Morris, Sheldon R; Mason, Cayla N; Geron, Ifat; Barrett, Christian; Goff, Daniel J; Wall, Russell; Pellecchia, Maurizio; Minden, Mark; Frazer, Kelly A; Marra, Marco A; Crews, Leslie A; Jiang, Qingfei; Jamieson, Catriona H M

    2016-08-01

    Post-transcriptional adenosine-to-inosine RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) promotes cancer progression and therapeutic resistance. However, ADAR1 editase-dependent mechanisms governing leukemia stem cell (LSC) generation have not been elucidated. In blast crisis chronic myeloid leukemia (BC CML), we show that increased JAK2 signaling and BCR-ABL1 amplification activate ADAR1. In a humanized BC CML mouse model, combined JAK2 and BCR-ABL1 inhibition prevents LSC self-renewal commensurate with ADAR1 downregulation. Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1(E912A) mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing and LIN28B upregulation. A small-molecule tool compound antagonizes ADAR1's effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis. Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling. PMID:27292188

  15. CD4+/CD8+ double-positive T cells

    DEFF Research Database (Denmark)

    Overgaard, Nana H; Jung, Ji-Won; Steptoe, Raymond J;

    2015-01-01

    CD4(+)/CD8(+) DP thymocytes are a well-described T cell developmental stage within the thymus. However, once differentiated, the CD4(+) lineage or the CD8(+) lineage is generally considered to be fixed. Nevertheless, mature CD4(+)/CD8(+) DP T cells have been described in the blood and peripheral...... cells, CD4(+)/CD8(+) T cell populations, outside of the thymus, have recently been described to express concurrently ThPOK and Runx3. Considerable heterogeneity exists within the CD4(+)/CD8(+) DP T cell pool, and the function of CD4(+)/CD8(+) T cell populations remains controversial, with conflicting...... reports describing cytotoxic or suppressive roles for these cells. In this review, we describe how transcriptional regulation, lineage of origin, heterogeneity of CD4 and CD8 expression, age, species, and specific disease settings influence the functionality of this rarely studied T cell population....

  16. Plastid position in Arabidopsis columella cells is similar in microgravity and on a random-positioning machine

    Science.gov (United States)

    Kraft, T. F.; van Loon, J. J.; Kiss, J. Z.

    2000-01-01

    In order to study gravity effects on plant structure and function, it may become necessary to remove the g-stimulus. On Earth, various instruments such as clinostats have been used by biologists in an attempt to neutralize the effects of gravity. In this study, the position of amyloplasts was assayed in columella cells in the roots of Arabidopsis thaliana (L.) Heynh. seedlings grown in the following conditions: on Earth, on a two-dimensional clinostat at 1 rpm, on a three-dimensional clinostat (also called a random-positioning machine, or an RPM), and in space (true microgravity). In addition, the effects of these gravity treatments on columella cell area and plastid area also were measured. In terms of the parameters measured, only amyloplast position was affected by the gravity treatments. Plastid position was not significantly different between spaceflight and RPM conditions but was significantly different between spaceflight and the classical two-dimensional clinostat treatments. Flanking columella cells showed a greater susceptibility to changes in gravity compared to the central columella cells. In addition, columella cells of seedlings that were grown on the RPM did not exhibit deleterious effects in terms of their ultrastructure as has been reported previously for seedlings grown on a two-dimensional clinostat. This study supports the hypothesis that the RPM provides a useful simulation of weightlessness.

  17. CD163 positive subsets of blood dendritic cells

    DEFF Research Database (Denmark)

    Maniecki, Maciej Bogdan; Møller, Holger Jon; Moestrup, Søren Kragh;

    2006-01-01

    expression in dendritic cells (DCs) was investigated using multicolor flow cytometry in peripheral blood from 31 healthy donors and 15 HIV-1 patients in addition to umbilical cord blood from 5 newborn infants. Total RNA was isolated from MACS purified DCs and CD163 mRNA was determined with real-time reverse...... transcriptase polymerase chain reaction. The effect of glucocorticoid and phorbol ester stimulation on monocyte and dendritic cell CD163 and CD91 expression was investigated in cell culture of mononuclear cells using multicolor flow cytometry. We identified two CD163+ subsets in human blood with dendritic cell...

  18. The Culture Repopulation Ability (CRA) Assay and Incubation in Low Oxygen to Test Antileukemic Drugs on Imatinib-Resistant CML Stem-Like Cells.

    Science.gov (United States)

    Cheloni, Giulia; Tanturli, Michele

    2016-01-01

    Chronic myeloid leukemia (CML) is a stem cell-driven disorder caused by the BCR/Abl oncoprotein, a constitutively active tyrosine kinase (TK). Chronic-phase CML patients are treated with impressive efficacy with TK inhibitors (TKi) such as imatinib mesylate (IM). However, rather than definitively curing CML, TKi induces a state of minimal residual disease, due to the persistence of leukemia stem cells (LSC) which are insensitive to this class of drugs. LSC persistence may be due to different reasons, including the suppression of BCR/Abl oncoprotein. It has been shown that this suppression follows incubation in low oxygen under appropriate culture conditions and incubation times.Here we describe the culture repopulation ability (CRA) assay, a non-clonogenic assay capable - together with incubation in low oxygen - to reveal in vitro stem cells endowed with marrow repopulation ability (MRA) in vivo. The CRA assay can be used, before moving to animal tests, as a simple and reliable method for the prescreening of drugs potentially active on CML and other leukemias with respect to their activity on the more immature leukemia cell subsets. PMID:27581140

  19. Synthetic Quorum Sensing and Cell-Cell Communication in Gram-Positive Bacillus megaterium.

    Science.gov (United States)

    Marchand, Nicholas; Collins, Cynthia H

    2016-07-15

    The components of natural quorum-sensing (QS) systems can be used to engineer synthetic communication systems that regulate gene expression in response to chemical signals. We have used the machinery from the peptide-based agr QS system from Staphylococcus aureus to engineer a synthetic QS system in Bacillus megaterium to enable autoinduction of a target gene at high cell densities. Growth and gene expression from these synthetic QS cells were characterized in both complex and minimal media. We also split the signal production and sensing components between two strains of B. megaterium to produce sender and receiver cells and characterized the resulting communication in liquid media and on semisolid agar. The system described in this work represents the first synthetic QS and cell-cell communication system that has been engineered to function in a Gram-positive host, and it has the potential to enable the generation of dynamic gene regulatory networks in B. megaterium and other Gram-positive organisms. PMID:26203497

  20. Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells

    OpenAIRE

    Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H

    2013-01-01

    Summary Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cel...

  1. Conductivity and Dielectric Dispersion of Gram-Positive Bacterial Cells

    Science.gov (United States)

    van der Wal A; Minor; Norde; Zehnder; Lyklema

    1997-02-01

    The conductivity of bacterial cell suspensions has been studied over a wide range of ionic strengths and is interpreted in terms of their cell wall properties. The experimental data have been analyzed after improving the high kappaa double-layer theory of Fixman, by accounting for ionic mobility in the hydrodynamically stagnant layer, i.e., in the bacterial wall. Static conductivity and dielectric dispersion measurements both show that the counterions in the porous gel-like cell wall give rise to a considerable surface conductance. From a comparison of the mobile charge with the total cell wall charge it is inferred that the mobilities of the ions in the bacterial wall are of the same order but somewhat lower than those in the bulk electrolyte solution. The occurrence of surface conductance reduces the electrophoretic mobility in electrophoresis studies. If this effect is not taken into account, the zeta-potential will be underestimated, especially at low electrolyte concentrations.

  2. Conductivity and Dielectric Dispersion of Gram-Positive Bacterial Cells

    Science.gov (United States)

    van der Wal A; Minor; Norde; Zehnder; Lyklema

    1997-02-01

    The conductivity of bacterial cell suspensions has been studied over a wide range of ionic strengths and is interpreted in terms of their cell wall properties. The experimental data have been analyzed after improving the high kappaa double-layer theory of Fixman, by accounting for ionic mobility in the hydrodynamically stagnant layer, i.e., in the bacterial wall. Static conductivity and dielectric dispersion measurements both show that the counterions in the porous gel-like cell wall give rise to a considerable surface conductance. From a comparison of the mobile charge with the total cell wall charge it is inferred that the mobilities of the ions in the bacterial wall are of the same order but somewhat lower than those in the bulk electrolyte solution. The occurrence of surface conductance reduces the electrophoretic mobility in electrophoresis studies. If this effect is not taken into account, the zeta-potential will be underestimated, especially at low electrolyte concentrations. PMID:9056304

  3. Nivolumab and Dasatinib in Treating Patients With Relapsed or Refractory Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

    Science.gov (United States)

    2016-08-25

    B Acute Lymphoblastic Leukemia With t(9;22)(q34;q11.2); BCR-ABL1; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Childhood Acute Lymphoblastic Leukemia; Refractory Adult Acute Lymphoblastic Leukemia; Refractory Childhood Acute Lymphoblastic Leukemia

  4. Predomination of IL-17-producing tryptase-positive/chymase-positive mast cells in azoospermic chronic testicular inflammation.

    Science.gov (United States)

    Chen, S-J; Duan, Y-G; Haidl, G; Allam, J-P

    2016-08-01

    Chronic testicular inflammation and infection have been regarded as important factors in the pathogenesis of azoospermia. As key effector cells in innate and adaptive immune system, mast cells (MCs) were observed in inflammation and autoimmune disease. Furthermore, increased expression of tryptase-positive MCs has been reported in testicular disorders associated with male infertility/subfertility. However, little is known about the potential relationship between MCs and chronic testicular inflammation in azoospermic patients. Moreover, the preferential expression of MCs' subtypes in testis of these patients is still far from being understood. Thus, this study aimed to investigate characteristics of testicular MCs as well as their subtypes in azoospermic men with chronic testicular inflammation (AZI, n = 5) by immunohistochemical techniques. Our results showed significant increase of MCs in AZI, and more importantly, considerable numbers of tryptase-positive/chymase-positive MCs could also be demonstrated in AZI, when compared to control groups representing azoospermia without chronic testicular inflammation (AZW, n = 5) and normal spermatogenesis (NT, n = 5) respectively. Most interestingly, immunofluorescence staining revealed autoimmune-associated interleukin (IL)-17-producing MCs in AZI, whereas co-expression of MC markers with tumour necrosis factor (TNF)-α, IL-10 and IL-1β could not be detected. In conclusion, AZI is associated with significant increase of tryptase-positive/chymase-positive MCs expressing IL-17, and these MCs might contribute to the pathogenesis of AZI. PMID:26420243

  5. Octreotide scintigraphy localizes somatostatin receptor-positive islet cell carcinomas

    NARCIS (Netherlands)

    W. Becker (W.); J. Marienhagen (J.); R. Scheubel (R.); A. Saptogino (A.); W.H. Bakker (Willem); W.A.P. Breeman (Wouter); F. Wolf (F.)

    1991-01-01

    textabstractTyr-3-Octreotide is a synthetic derivative of somatostatin and a somatostatin-receptor analogue. The iodine-123-labelled compound localizes somatostatin-receptor-positive tumours. In this paper two patients are reported in whom somatostatin receptors were demonstrated in vitro. In a 60-y

  6. Increased intratumoral FOXP3-positive regulatory immune cells during interleukin-2 treatment in metastatic renal cell carcinoma

    DEFF Research Database (Denmark)

    Jensen, Hanne Krogh; Donskov, Frede; Nordsmark, Marianne;

    2009-01-01

    tumor-infiltrating immune cells at baseline and during treatment (P 180 cells/mm2) of on-treatment FOXP3-positive intratumoral immune cells were dead within 22 months (n = 11), whereas patients with low numbers (cells/mm2) of on-treatment......PURPOSE: The administration of interleukin-2 (IL-2) may increase the frequency of peripherally circulating FOXP3-positive regulatory immune cells, thus potentially compromising this treatment option for patients with metastatic renal cell carcinoma. The impact of IL-2-based therapy...... on the accumulation of FOXP3-positive immune cells in the tumor microenvironment in metastatic renal cell carcinoma is unknown. EXPERIMENTAL DESIGN: Baseline (n = 58) and on-treatment (n = 42) tumor core biopsies were prospectively obtained from patients with clear cell metastatic renal cell carcinoma before...

  7. Isolation of dendritic-cell-like S100β-positive cells in rat anterior pituitary gland.

    Science.gov (United States)

    Horiguchi, Kotaro; Fujiwara, Ken; Yoshida, Saishu; Higuchi, Masashi; Tsukada, Takehiro; Kanno, Naoko; Yashiro, Takashi; Tateno, Kozue; Osako, Shunji; Kato, Takako; Kato, Yukio

    2014-07-01

    S100β-protein-positive cells in the anterior pituitary gland appear to possess multifunctional properties. Because of their pleiotropic features, S100β-positive cells are assumed to be of a heterogeneous or even a non-pituitary origin. The observation of various markers has allowed these cells to be classified into populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. The isolation and characterization of each heterogeneous population is a prerequisite for clarifying the functional character and origin of the cells. We attempt to isolate two of the subpopulations of S100β-positive cells from the anterior lobe. First, from transgenic rats that express green fluorescent protein (GFP) driven by the S100β protein promoter, we fractionate GFP-positive cells with a cell sorter and culture them so that they can interact with laminin, a component of the extracellular matrix. We observe that one morphological type of GFP-positive cells possesses extended cytoplasmic processes and shows high adhesiveness to laminin (process type), whereas the other is round in shape and exhibits low adherence to laminin (round type). We successfully isolate cells of the round type from the cultured GFP-positive cells by taking advantage of their low affinity to laminin and then measure mRNA levels of the two cell types by real-time polymerase chain reaction. The resultant data show that the process type expresses vimentin (mesenchymal cell marker) and glial fibrillary acidic protein (astrocyte marker). The round type expresses dendritic cell markers, CD11b and interleukin-6. Thus, we found a method for isolating dendritic-cell-like S100β-positive cells by means of their property of adhering to laminin.

  8. Clinical Utility of Circulating Tumor Cells in ALK-Positive Non-Small-Cell Lung Cancer.

    Science.gov (United States)

    Faugeroux, Vincent; Pailler, Emma; Auger, Nathalie; Taylor, Melissa; Farace, Françoise

    2014-01-01

    The advent of rationally targeted therapies such as small-molecule tyrosine kinase inhibitors (TKIs) has considerably transformed the therapeutic management of a subset of patients with non-small-cell lung cancer (NSCLC) harboring defined molecular abnormalities. When such genetic molecular alterations are detected the use of specific TKI has demonstrated better results (overall response rate, progression free survival) compared to systemic therapy. However, the detection of such molecular abnormalities is complicated by the difficulty in obtaining sufficient tumor material, in terms of quantity and quality, from a biopsy. Here, we described how circulating tumor cells (CTCs) can have a clinical utility in anaplastic lymphoma kinase (ALK) positive NSCLC patients to diagnose ALK-EML4 gene rearrangement and to guide therapeutic management of these patients. The ability to detect genetic abnormalities such ALK rearrangement in CTCs shows that these cells could offer new perspectives both for the diagnosis and the monitoring of ALK-positive patients eligible for treatment with ALK inhibitors. PMID:25414829

  9. Clinical Utility of Circulating Tumor Cells in ALK-Positive Non-Small-Cell Lung Cancer

    Science.gov (United States)

    Faugeroux, Vincent; Pailler, Emma; Auger, Nathalie; Taylor, Melissa; Farace, Françoise

    2014-01-01

    The advent of rationally targeted therapies such as small-molecule tyrosine kinase inhibitors (TKIs) has considerably transformed the therapeutic management of a subset of patients with non-small-cell lung cancer (NSCLC) harboring defined molecular abnormalities. When such genetic molecular alterations are detected the use of specific TKI has demonstrated better results (overall response rate, progression free survival) compared to systemic therapy. However, the detection of such molecular abnormalities is complicated by the difficulty in obtaining sufficient tumor material, in terms of quantity and quality, from a biopsy. Here, we described how circulating tumor cells (CTCs) can have a clinical utility in anaplastic lymphoma kinase (ALK) positive NSCLC patients to diagnose ALK-EML4 gene rearrangement and to guide therapeutic management of these patients. The ability to detect genetic abnormalities such ALK rearrangement in CTCs shows that these cells could offer new perspectives both for the diagnosis and the monitoring of ALK-positive patients eligible for treatment with ALK inhibitors. PMID:25414829

  10. Thymoproteasomes produce unique peptide motifs for positive selection of CD8(+) T cells.

    Science.gov (United States)

    Sasaki, Katsuhiro; Takada, Kensuke; Ohte, Yuki; Kondo, Hiroyuki; Sorimachi, Hiroyuki; Tanaka, Keiji; Takahama, Yousuke; Murata, Shigeo

    2015-01-01

    Positive selection in the thymus provides low-affinity T-cell receptor (TCR) engagement to support the development of potentially useful self-major histocompatibility complex class I (MHC-I)-restricted T cells. Optimal positive selection of CD8(+) T cells requires cortical thymic epithelial cells that express β5t-containing thymoproteasomes (tCPs). However, how tCPs govern positive selection is unclear. Here we show that the tCPs produce unique cleavage motifs in digested peptides and in MHC-I-associated peptides. Interestingly, MHC-I-associated peptides carrying these tCP-dependent motifs are enriched with low-affinity TCR ligands that efficiently induce the positive selection of functionally competent CD8(+) T cells in antigen-specific TCR-transgenic models. These results suggest that tCPs contribute to the positive selection of CD8(+) T cells by preferentially producing low-affinity TCR ligand peptides.

  11. Deletions of Immunoglobulin heavy chain and T cell receptor gene regions are uniquely associated with lymphoid blast transformation of chronic myeloid leukemia

    Directory of Open Access Journals (Sweden)

    Valgañon Mikel

    2010-01-01

    Full Text Available Abstract Background Chronic myelogenous leukemia (CML results from the neoplastic transformation of a haematopoietic stem cell. The hallmark genetic abnormality of CML is a chimeric BCR/ABL1 fusion gene resulting from the Philadelphia chromosome rearrangement t(9;22(q34;q11. Clinical and laboratory studies indicate that the BCR/ABL1 fusion protein is essential for initiation, maintenance and progression of CML, yet the event(s driving the transformation from chronic phase to blast phase are poorly understood. Results Here we report multiple genome aberrations in a collection of 78 CML and 14 control samples by oligonucleotide array comparative genomic hybridization. We found a unique signature of genome deletions within the immunoglobulin heavy chain (IGH and T cell receptor regions (TCR, frequently accompanied by concomitant loss of sequences within the short arm regions of chromosomes 7 and 9, including IKZF1, HOXA7, CDKN2A/2B, MLLT3, IFNA/B, RNF38, PAX5, JMJD2C and PDCD1LG2 genes. Conclusions None of these genome losses were detected in any of the CML samples with myeloid transformation, chronic phase or controls, indicating that their presence is obligatory for the development of a malignant clone with a lymphoid phenotype. Notably, the coincidental deletions at IGH and TCR regions appear to precede the loss of IKZF1 and/or p16 genes in CML indicating a possible involvement of RAG in these deletions.

  12. The equatorial position of the metaphase plate ensures symmetric cell divisions.

    Science.gov (United States)

    Tan, Chia Huei; Gasic, Ivana; Huber-Reggi, Sabina P; Dudka, Damian; Barisic, Marin; Maiato, Helder; Meraldi, Patrick

    2015-01-01

    Chromosome alignment in the middle of the bipolar spindle is a hallmark of metazoan cell divisions. When we offset the metaphase plate position by creating an asymmetric centriole distribution on each pole, we find that metaphase plates relocate to the middle of the spindle before anaphase. The spindle assembly checkpoint enables this centering mechanism by providing cells enough time to correct metaphase plate position. The checkpoint responds to unstable kinetochore-microtubule attachments resulting from an imbalance in microtubule stability between the two half-spindles in cells with an asymmetric centriole distribution. Inactivation of the checkpoint prior to metaphase plate centering leads to asymmetric cell divisions and daughter cells of unequal size; in contrast, if the checkpoint is inactivated after the metaphase plate has centered its position, symmetric cell divisions ensue. This indicates that the equatorial position of the metaphase plate is essential for symmetric cell divisions. PMID:26188083

  13. EBV-positive diffuse large B-cell lymphoma of the elderly

    NARCIS (Netherlands)

    C.Y. Ok (Chi Young); T.G. Papathomas (Thomas ); L.J. Medeiros (L. Jeffrey); K.H. Young (Ken)

    2013-01-01

    textabstractEpstein-Barr virus (EBV) positive diffuse large B-cell lymphoma (DLBCL) of the elderly, initially described in 2003, is a provisional entity in the 2008World Health Organization classification system and is defined as an EBV-positive monoclonal large B-cell proliferation that occurs in p

  14. Blue light-dependent nuclear positioning in Arabidopsis thaliana leaf cells.

    Science.gov (United States)

    Iwabuchi, Kosei; Sakai, Tatsuya; Takagi, Shingo

    2007-09-01

    The plant nucleus changes its intracellular position not only upon cell division and cell growth but also in response to environmental stimuli such as light. We found that the nucleus takes different intracellular positions depending on blue light in Arabidopsis thaliana leaf cells. Under dark conditions, nuclei in mesophyll cells were positioned at the center of the bottom of cells (dark position). Under blue light at 100 mumol m(-2) s(-1), in contrast, nuclei were located along the anticlinal walls (light position). The nuclear positioning from the dark position to the light position was fully induced within a few hours of blue light illumination, and it was a reversible response. The response was also observed in epidermal cells, which have no chloroplasts, suggesting that the nucleus has the potential actively to change its position without chloroplasts. Light-dependent nuclear positioning was induced specifically by blue light at >50 mumol m(-2) s(-1). Furthermore, the response to blue light was induced in phot1 but not in phot2 and phot1phot2 mutants. Unexpectedly, we also found that nuclei as well as chloroplasts in phot2 and phot1phot2 mutants took unusual intracellular positions under both dark and light conditions. The lack of the response and the unusual positioning of nuclei and chloroplasts in the phot2 mutant were recovered by externally introducing the PHOT2 gene into the mutant. These results indicate that phot2 mediates the blue light-dependent nuclear positioning and the proper positioning of nuclei and chloroplasts. This is the first characterization of light-dependent nuclear positioning in spermatophytes.

  15. Human papillomavirus 52 positive squamous cell carcinoma of the conjunctiva

    Directory of Open Access Journals (Sweden)

    Silvia Olivia Salceanu

    2015-01-01

    Full Text Available Human papillomavirus (HPV infection is strongly associated with several human cancers; the most known genotypes involved being HPV 16 and HPV 18. We report the detection of HPV 52 in a sample taken from a 47-year-old patient with squamous cell carcinoma of the conjunctiva of the left eye. The method used for the detection of HPV was real time polymerase chain reaction. The evolution was favorable after surgical removal of the tumor and the patient was explained that long-term follow-up is essential to avoid recurrence.

  16. Anaplastic lymphoma kinase-positive primary cutaneous anaplastic large cell lymphoma- Is it a new variant?

    Directory of Open Access Journals (Sweden)

    Muthu Sendhil Kumaran

    2012-01-01

    Full Text Available Anaplastic large cell cutaneous lymphomas are clinically and pathologically heterogeneous, CD30 + (Ki-1 lymphoproliferative disorders. The importance of anaplastic lymphoma kinase (ALK positivity is well known in the prognosis of primary systemic anaplastic large cell cutaneous lymphomas; however, the same in primary cutaneous anaplastic large cell cutaneous lymphomas is not much clear. Herein we report a 65-year-old male with an 18-month history of minimally pruritic localized nodulo-plaque lesion over lower back. Histology revealed cutaneous large cell lymphoma and immunohistochemical staining showed positivity for CD30, CD3 and ALK. The role of ALK positivity in pcALCL is discussed in this article.

  17. T cells expressing CD19-specific Engager Molecules for the Immunotherapy of CD19-positive Malignancies

    OpenAIRE

    Mireya Paulina Velasquez; David Torres; Kota Iwahori; Sunitha Kakarla; Caroline Arber; Tania Rodriguez-Cruz; Arpad Szoor; Bonifant, Challice L.; Claudia Gerken; Cooper, Laurence J.N.; Xiao-Tong Song; Stephen Gottschalk

    2016-01-01

    T cells expressing chimeric antigen receptors (CARs) or the infusion of bispecific T-cell engagers (BITEs) have shown antitumor activity in humans for CD19-positive malignancies. While BITEs redirect the large reservoir of resident T cells to tumors, CAR T cells rely on significant in vivo expansion to exert antitumor activity. We have shown that it is feasible to modify T cells to secrete solid tumor antigen-specific BITEs, enabling T cells to redirect resident T cells to tumor cells. To ada...

  18. Positional information is reprogrammed in blastema cells of the regenerating limb of the axolotl (Ambystoma mexicanum).

    Science.gov (United States)

    McCusker, Catherine D; Gardiner, David M

    2013-01-01

    The regenerating region of an amputated salamander limb, known as the blastema, has the amazing capacity to replace exactly the missing structures. By grafting cells from different stages and regions of blastemas induced to form on donor animals expressing Green Fluorescent Protein (GFP), to non-GFP host animals, we have determined that the cells from early stage blastemas, as well as cells at the tip of late stage blastemas are developmentally labile such that their positional identity is reprogrammed by interactions with more proximal cells with stable positional information. In contrast, cells from the adjacent, more proximal stump tissues as well as the basal region of late bud blastemas are positionally stable, and thus form ectopic limb structures when grafted. Finally, we have found that a nerve is required to maintain the blastema cells in a positionally labile state, thus indicating a role for reprogramming cues in the blastema microenvironment. PMID:24086768

  19. Positional information is reprogrammed in blastema cells of the regenerating limb of the axolotl (Ambystoma mexicanum.

    Directory of Open Access Journals (Sweden)

    Catherine D McCusker

    Full Text Available The regenerating region of an amputated salamander limb, known as the blastema, has the amazing capacity to replace exactly the missing structures. By grafting cells from different stages and regions of blastemas induced to form on donor animals expressing Green Fluorescent Protein (GFP, to non-GFP host animals, we have determined that the cells from early stage blastemas, as well as cells at the tip of late stage blastemas are developmentally labile such that their positional identity is reprogrammed by interactions with more proximal cells with stable positional information. In contrast, cells from the adjacent, more proximal stump tissues as well as the basal region of late bud blastemas are positionally stable, and thus form ectopic limb structures when grafted. Finally, we have found that a nerve is required to maintain the blastema cells in a positionally labile state, thus indicating a role for reprogramming cues in the blastema microenvironment.

  20. Positional information is reprogrammed in blastema cells of the regenerating limb of the axolotl (Ambystoma mexicanum).

    Science.gov (United States)

    McCusker, Catherine D; Gardiner, David M

    2013-01-01

    The regenerating region of an amputated salamander limb, known as the blastema, has the amazing capacity to replace exactly the missing structures. By grafting cells from different stages and regions of blastemas induced to form on donor animals expressing Green Fluorescent Protein (GFP), to non-GFP host animals, we have determined that the cells from early stage blastemas, as well as cells at the tip of late stage blastemas are developmentally labile such that their positional identity is reprogrammed by interactions with more proximal cells with stable positional information. In contrast, cells from the adjacent, more proximal stump tissues as well as the basal region of late bud blastemas are positionally stable, and thus form ectopic limb structures when grafted. Finally, we have found that a nerve is required to maintain the blastema cells in a positionally labile state, thus indicating a role for reprogramming cues in the blastema microenvironment.

  1. Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria.

    OpenAIRE

    Mistou, Michel-Yves; Sutcliffe, Iain; van Sorge, Nina

    2016-01-01

    The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall te...

  2. Cell wall sorting signals in surface proteins of gram-positive bacteria.

    OpenAIRE

    Schneewind, O; Mihaylova-Petkov, D; Model, P

    1993-01-01

    Staphylococcal protein A is anchored to the cell wall, a unique cellular compartment of Gram-positive bacteria. The sorting signal sufficient for cell wall anchoring consists of an LPXTG motif, a C-terminal hydrophobic domain and a charged tail. Homologous sequences are found in many surface proteins of Gram-positive bacteria and we explored the universality of these sequences to serve as cell wall sorting signals. We show that several signals are able to anchor fusion proteins to the staphyl...

  3. [KI-1-positive, anaplastic, large-cell lymphoma related to Hodgkin's disease].

    Science.gov (United States)

    Veiga, M; Fresno, M F; Pérez del Río, M J; García, I; Madrigal, B; Herrero, A

    1997-02-01

    We report a case of lymphoma associated with lung carcinoma that shows morphological and immunohistochemical features of anaplastic large cell Ki-1 positive lymphoma and Hodgkin's disease, with positivity for Ki-1 (CD-30) (characteristic of both lymphomas) and Leu-M1 (CD-15) (normally dosent absent in anaplastic lymphoma). This subtype of lymphoma is designated anaplastic large-cell Hodgkin's related lymphoma (ALCL related to HD) and is considered by some authors as a secondary anaplastic large-cell lymphoma.

  4. Nestin expression in osteosarcomas and derivation of nestin/CD133 positive osteosarcoma cell lines

    International Nuclear Information System (INIS)

    Nestin was originally identified as a class VI intermediate filament protein that is expressed in stem cells and progenitor cells in the mammalian CNS during development. This protein is replaced in the adult organism by other intermediate filament proteins; however, nestin may be re-expressed under certain pathological conditions such as ischemia, inflammation, brain injury, and neoplastic transformation. Nestin has been detected in many kinds of tumors, especially in tumors derived from the CNS. Co-expression of nestin and the CD133 surface molecule is considered to be a marker for cancer stem cells in neurogenic tumors. Our work was aimed at a detailed study of nestin expression in osteosarcomas and osteosarcoma-derived cell lines. Using immunodetection methods, we examined nestin in tumor tissue samples from 18 patients with osteosarcomas. We also successfully established permanent cell lines from the tumor tissue of 4 patients and immunodetection of nestin and CD133 was performed on these cell lines. Nestin-positive tumor cells were immunohistochemically detected in all of the examined osteosarcomas, but the proportion of these cells that were positively stained as well as the intensity of staining varied. Nestin-positive cells were rarely observed in 2 tumor samples, and the remaining 16 tumor samples showed various nestin expression patterns ranging from very sporadic occurrence to an overwhelming proportion of cells with strong positive staining. Three of the established osteosarcoma cell lines were demonstrated to be nestin-positive, and only one cell line showed no expression of nestin; this finding corresponds with the rare occurrence of nestin-positive cells in the respective tumor sample. Moreover, three of these osteosarcoma cell lines were undoubtedly proven to be Nes+/CD133+. Our results represent the first evidence of nestin expression in osteosarcomas and suggest the possible occurrence of cells with a stem-like phenotype in these tumors

  5. Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum.

    Science.gov (United States)

    Lázaro-Diéguez, Francisco; Ispolatov, Iaroslav; Müsch, Anne

    2015-04-01

    All known mechanisms of mitotic spindle orientation rely on astral microtubules. We report that even in the absence of astral microtubules, metaphase spindles in MDCK and HeLa cells are not randomly positioned along their x-z dimension, but preferentially adopt shallow β angles between spindle pole axis and substratum. The nonrandom spindle positioning is due to constraints imposed by the cell cortex in flat cells that drive spindles that are longer and/or wider than the cell's height into a tilted, quasidiagonal x-z position. In rounder cells, which are taller, fewer cortical constraints make the x-z spindle position more random. Reestablishment of astral microtubule-mediated forces align the spindle poles with cortical cues parallel to the substratum in all cells. However, in flat cells, they frequently cause spindle deformations. Similar deformations are apparent when confined spindles rotate from tilted to parallel positions while MDCK cells progress from prometaphase to metaphase. The spindle disruptions cause the engagement of the spindle assembly checkpoint. We propose that cell rounding serves to maintain spindle integrity during its positioning. PMID:25657320

  6. A comparative proteomic study identified LRPPRC and MCM7 as putative actors in imatinib mesylate cross-resistance in Lucena cell line

    Directory of Open Access Journals (Sweden)

    Corrêa Stephany

    2012-03-01

    Full Text Available Abstract Background Although chronic myeloid leukemia (CML treatment has improved since the introduction of imatinib mesylate (IM, cases of resistance have been reported. This resistance has been associated with the emergence of multidrug resistance (MDR phenotype, as a BCR-ABL independent mechanism. The classic pathway studied in MDR promotion is ATP-binding cassette (ABC family transporters expression, but other mechanisms that drive drug resistance are largely unknown. To better understand IM therapy relapse due to the rise of MDR, we compared the proteomic profiles of K562 and Lucena (K562/VCR cells. Results The use of 2-DE coupled with a MS approach resulted in the identification of 36 differentially expressed proteins. Differential mRNA levels of leucine-rich PPR motif-containing (LRPPRC protein, minichromosome maintenance complex component 7 (MCM7 and ATP-binding cassette sub-family B (MDR/TAP member 1 (ABCB1 were capable of defining samples from CML patients as responsive or resistant to therapy. Conclusions Through the data presented in this work, we show the relevance of MDR to IM therapy. In addition, our proteomic approach identified candidate actors involved in resistance, which could lead to additional information on BCR-ABL-independent molecular mechanisms.

  7. Kinasen der Src-Familie vermitteln die zytoplasmatische Retention von aktiviertem STAT5A in BCR-ABL positiven Leukämiezellen

    OpenAIRE

    Chatain, Nicolas

    2012-01-01

    The transcription factor STAT5 belongs to the family of Signal Transducer and Activator of Transcription and subdivides into STAT5A and STAT5B which share 96% sequence homology on the amino acid level. STAT5A is involved in hematopoiesis, immunity and mammary differentiation. The phosphorylation of a conserved tyrosine is essential for the activation of STAT transcription factors leading to the dimerization of two STAT proteins through reciprocal SH2 domain-phosphotyrosine interactions. The p...

  8. BCR-ABL isoforms associated with intrinsic or acquired resistance to imatinib : more heterogeneous than just ABL kinase domain point mutations?

    NARCIS (Netherlands)

    Gruber, Franz X.; Lundan, Tuija; Goll, Rasmus; Silye, Aleksandra; Mikkola, Ingvild; Rekvig, Ole Petter; Knuutila, Sakari; Remes, Kari; Gedde-Dahl, Tobias; Porkka, Kimmo; Hjorth-Hansen, Henrik

    2012-01-01

    Imatinib, a small molecule inhibitor of ABL, PDGFR and C-KIT, has revolutionized treatment of chronic myeloid leukaemia (CML). However, resistance to treatment is of increasing importance and often is due to point mutations in the Abl kinase domain (Abl KD). Here, we analysed clinical outcome and mu

  9. Surface position, not signaling from surrounding maternal tissues, specifies aleurone epidermal cell fate in maize.

    Science.gov (United States)

    Gruis, Darren Fred; Guo, Hena; Selinger, David; Tian, Qing; Olsen, Odd-Arne

    2006-07-01

    Maize (Zea mays) endosperm consists of an epidermal-like surface layer of aleurone cells, an underlying body of starchy endosperm cells, and a basal layer of transfer cells. To determine whether surrounding maternal tissues perform a role in specifying endosperm cell fates, a maize endosperm organ culture technique was established whereby the developing endosperm is completely removed from surrounding maternal tissues. Using cell type-specific fluorescence markers, we show that aleurone cell fate specification occurs exclusively in response to surface position and does not require specific, continued maternal signal input. The starchy endosperm and aleurone cell fates are freely interchangeable throughout the lifespan of the endosperm, with internalized aleurone cells converting to starchy endosperm cells and with starchy endosperm cells that become positioned at the surface converting to aleurone cells. In contrast to aleurone and starchy endosperm cells, transfer cells fail to develop in in vitro-grown endosperm, supporting earlier indications that maternal tissue interaction is required to fully differentiate this cell type. Several parameters confirm that the maize endosperm organ cultures described herein retain the main developmental features of in planta endosperm, including fidelity of aleurone mutant phenotypes, temporal and spatial control of cell type-specific fluorescent markers, specificity of cell type transcripts, and control of mitotic cell divisions.

  10. Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Seon Young; Jang, Soo Hwa [Seoul National University, Laboratory of Veterinary Pharmacology, College of Veterinary Medicine and Institute for Veterinary Science (Korea, Republic of); Park, Jin; Jeong, Saeromi; Park, Jin Ho; Ock, Kwang Su [Soongsil University, Department of Chemistry (Korea, Republic of); Lee, Kangtaek [Yonsei University, Department of Chemical and Biomolecular Engineering (Korea, Republic of); Yang, Sung Ik [Kyung Hee University, College of Environment and Applied Chemistry (Korea, Republic of); Joo, Sang-Woo, E-mail: sjoo@ssu.ac.kr [Soongsil University, Department of Chemistry (Korea, Republic of); Ryu, Pan Dong; Lee, So Yeong, E-mail: leeso@snu.ac.kr [Seoul National University, Laboratory of Veterinary Pharmacology, College of Veterinary Medicine and Institute for Veterinary Science (Korea, Republic of)

    2012-12-15

    Cellular uptake, cytotoxicity, and mechanisms of cytotoxicity of the positively charged Au nanoparticles (NPs) were examined in A549 cells, which are one of the most characterized pulmonary cellular systems. Positively charged Au NPs were prepared by chemical reduction using chitosan. The dimension and surface charge of Au NPs were examined by transmission electron microscopy (TEM), dynamic light scattering, and zeta potential measurements. The uptake of Au NPs into A549 cells was also monitored using TEM and dark-field microscopy (DFM) and z-stack confocal microRaman spectroscopy. DFM live cell imaging was also performed to monitor the entry of chitosan Au NPs in real time. The cytotoxic assay, using both methylthiazol tetrazolium and lactate dehydrogenase assays revealed that positively charged Au NPs decreased cell viability. Flow cytometry, DNA fragmentation, real-time PCR, and western blot analysis suggest that positively charged chitosan Au NPs provoke cell damage through both apoptotic and necrotic pathways.

  11. Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells

    International Nuclear Information System (INIS)

    Cellular uptake, cytotoxicity, and mechanisms of cytotoxicity of the positively charged Au nanoparticles (NPs) were examined in A549 cells, which are one of the most characterized pulmonary cellular systems. Positively charged Au NPs were prepared by chemical reduction using chitosan. The dimension and surface charge of Au NPs were examined by transmission electron microscopy (TEM), dynamic light scattering, and zeta potential measurements. The uptake of Au NPs into A549 cells was also monitored using TEM and dark-field microscopy (DFM) and z-stack confocal microRaman spectroscopy. DFM live cell imaging was also performed to monitor the entry of chitosan Au NPs in real time. The cytotoxic assay, using both methylthiazol tetrazolium and lactate dehydrogenase assays revealed that positively charged Au NPs decreased cell viability. Flow cytometry, DNA fragmentation, real-time PCR, and western blot analysis suggest that positively charged chitosan Au NPs provoke cell damage through both apoptotic and necrotic pathways.

  12. Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells.

    Science.gov (United States)

    Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H

    2013-09-30

    Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cells contract, actin cables associate laterally with the nuclei, in some cases inducing nuclear turning so that actin cables become partially wound around the nuclei. Our data suggest that a perinuclear actin meshwork connects actin cables to nuclei via actin-crosslinking proteins such as the filamin Cheerio. We provide a revised model for how actin structures position nuclei in nurse cells, employing evolutionary conserved machinery.

  13. Berbamine exhibits potent antitumor effects on imatinib-resistant CML cells in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Yan-lin WEI; Lei XU; Yun LIANG; Xiao-hua XU; Xiao-ying ZHAO

    2009-01-01

    Aim: The aim of this study was to explore the effects and mechanism of berbamine on imatinib-resistant BCR-ABL-positive human leukemia K562 (K562-r) cells in vitro and in vivo. Methods: Cell viability was measured by MTT assay, and apoptotic morphology changes were detected by fluorescence microscopy. The apoptosis rate was measured by flow cytometric assay, mdr-1 mRNA levels were determined by RT-PCR. Bcl-2 family proteins, cytochrome c( cyt C), poly (ADP-ribose) polymerase (PARP), and P-glycoprotein were detected by Western blot. BALB/c nu/nu mice were injected with K562-r ceils subcutaneously. Tumor-bearing mice were treated intravenously with berbamine.Results: MTT tests revealed that berbamine significantly inhibited K562-r cell proliferation and increased the chemo-sensitivity of Kf62-r cells to imatirtib. The apoptosis rate was significantly increased following treatment with 21.2 μmol/L berbamine; formation of typical apoptotic blebs was apparent, as observed by fluorescence microscopy. Expression levels of mdr-1 mRNA and P-gp protein were high in untreated K562-r cells and significantly down-regulated by berbamine treatment. Berbamine-treated K562-r cells also exhibited down-regulated expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, up-regulated expression of the apoptotic proteins Box and cytoplasmic cyt C, and stimulated proteolytic cleavage of PARP. In addition, berbamine also suppressed the growth of K562-r xenotransplanted tumors in vivo. Conclusion: Berbamine inhibited proliferation of K562-r cells both in vitro and in vivo. Berbamine-induced apoptosis in K562-r cells appeared to occur through a mechanism involving Bcl-2 family proteins, as well as mdr-1 mRNA and P-gp pro- tein. 13erbamine in combination with imatirtib restored the chemo-sensitivity of K562-r cells to imatinib. Our findings sug-gest that berbamine may be useful in treating imatinib-resistant CML patients.

  14. 酪氨酸激酶抑制剂联合异基因造血干细胞移植治疗费城染色体阳性急性淋巴细胞白血病的研究进展%Research progress of tyrosine kinase inhibitor combining with allogeneic hematopoietic stem cell transplantation in treatment of Philadelphia chromosome positive acute lymphoblastic leukemia

    Institute of Scientific and Technical Information of China (English)

    王静静; 江明

    2016-01-01

    费城染色体阳性(Ph+)急性淋巴细胞白血病(ALL)是成年人ALL的常见类型.在酪氨酸激酶抑制剂(TKI)问世前,ph+是成年人ALL的不良预后因素之一.在TKI应用于Ph+ ALL治疗后显著改善其疗效,完全缓解(CR)率可达90%以上,缓解持续时间较前明显延长,从而使更多Ph+ ALL患者能够行异基因造血干细胞移植(allo-HSCT).采用以TKI为基础化疗获得CR后行allo-HSCT是Ph+ ALL患者的标准治疗方案.目前,移植前微小残留疾病(MRD)检测是否对该病的治疗具有临床指导意义,尚存争议.替代供者移植治疗Ph+ ALL在临床取得理想疗效,但需要前瞻性随机对照试验来证实其疗效.移植后联合TKI治疗可明显改善Ph+ ALL患者的长期生存,但其具体使用方案仍需多中心、大样本量的临床随机对照实验证实,而移植后复发仍是Ph+ ALL患者面临的难题之一.%Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) is the most common type of adult acute lymphoblastic leukemia(ALL).Before the advent of tyrosine kinase inhibitors (TKI),Ph+ ALL carried a dismal prognosis.Outcomes for patients with Ph+ ALL improved substantially with the administration of TKI,and the TKI induced complete remissions(CR) in more than 90% patients,with remission duration prolonging,making more patients able to undergo allogeneic hematopoietic stem cell transplantation(allo-HSCT).Currently,TKI-based chemotherapy following allo-HSCT is established as the first-line strategy.Breakpoint cluster region-abelson leukemia virus(BCR-ABL) monitoring appears to relate to prognostic relevance and may be as guiding treatment before allo-HSCT,but clinical benefit is controversial in the TKI era.Alternative donor transplantation obtains promising effect in clinical practice,but needs more research.The administration of TKI after allo-HSCT may improve quality of life and prolong life span,but relapse remains a major problem of treatment failure

  15. IKZF1 deletion is an independent prognostic marker in childhood B-cell precursor acute lymphoblastic leukemia, and distinguishes patients benefiting from pulses during maintenance therapy: results of the EORTC Children's Leukemia Group study 58951.

    Science.gov (United States)

    Clappier, E; Grardel, N; Bakkus, M; Rapion, J; De Moerloose, B; Kastner, P; Caye, A; Vivent, J; Costa, V; Ferster, A; Lutz, P; Mazingue, F; Millot, F; Plantaz, D; Plat, G; Plouvier, E; Poirée, M; Sirvent, N; Uyttebroeck, A; Yakouben, K; Girard, S; Dastugue, N; Suciu, S; Benoit, Y; Bertrand, Y; Cavé, H

    2015-11-01

    The added value of IKZF1 gene deletion (IKZF1(del)) as a stratifying criterion in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is still debated. We performed a comprehensive analysis of the impact of IKZF1(del) in a large cohort of children (n=1223) with BCR-ABL1-negative BCP-ALL treated in the EORTC-CLG trial 58951. Patients with IKZF1(del) had a lower 8-year event-free survival (EFS, 67.7% versus 86.5%; hazard ratio (HR)=2.41; 95% confidence interval (CI)=1.75-3.32; P<0.001). Importantly, despite association with high-risk features such as high minimal residual disease, IKZF1(del) remained significantly predictive in multivariate analyses. Analysis by genetic subtype showed that IKZF1(del) increased risk only in the high hyperdiploid ALLs (HR=2.57; 95% CI=1.19-5.55; P=0.013) and in 'B-other' ALLs, that is, lacking classifying genetic lesions (HR=2.22; 95% CI=1.45-3.39; P<0.001), the latter having then a dramatically low 8-year EFS (56.4; 95% CI=44.6-66.7). Among IKZF1(del)-positive patients randomized for vincristine-steroid pulses during maintenance, those receiving pulses had a significantly higher 8-year EFS (93.3; 95% CI=61.3-99.0 versus 42.1; 95% CI=20.4-62.5). Thus, IKZF1(del) retains independent prognostic significance in the context of current risk-adapted protocols, and is associated with a dismal outcome in 'B-other' ALL. Addition of vincristine-steroid pulses during maintenance may specifically benefit to IKZF1(del) patients in preventing relapses.

  16. Protein transport across the cell wall of monoderm Gram-positive bacteria

    OpenAIRE

    Forster, Brian M.; Marquis, Hélène

    2012-01-01

    In monoderm (single membrane) Gram-positive bacteria, the majority of secreted proteins are first translocated across the cytoplasmic membrane into the inner wall zone. For a subset of these proteins, final destination is within the cell envelope either as membrane-anchored or cell wall-anchored proteins, whereas another subset of proteins is destined to be transported across the cell wall into the extracellular milieu. Although the cell wall is a porous structure, there is evidence that, for...

  17. Interphase chromosome positioning in in vitro porcine cells and ex vivo porcine tissues

    Directory of Open Access Journals (Sweden)

    Foster Helen A

    2012-11-01

    Full Text Available Abstract Background In interphase nuclei of a wide range of species chromosomes are organised into their own specific locations termed territories. These chromosome territories are non-randomly positioned in nuclei which is believed to be related to a spatial aspect of regulatory control over gene expression. In this study we have adopted the pig as a model in which to study interphase chromosome positioning and follows on from other studies from our group of using pig cells and tissues to study interphase genome re-positioning during differentiation. The pig is an important model organism both economically and as a closely related species to study human disease models. This is why great efforts have been made to accomplish the full genome sequence in the last decade. Results This study has positioned most of the porcine chromosomes in in vitro cultured adult and embryonic fibroblasts, early passage stromal derived mesenchymal stem cells and lymphocytes. The study is further expanded to position four chromosomes in ex vivo tissue derived from pig kidney, lung and brain. Conclusions It was concluded that porcine chromosomes are also non-randomly positioned within interphase nuclei with few major differences in chromosome position in interphase nuclei between different cell and tissue types. There were also no differences between preferred nuclear location of chromosomes in in vitro cultured cells as compared to cells in tissue sections. Using a number of analyses to ascertain by what criteria porcine chromosomes were positioned in interphase nuclei; we found a correlation with DNA content.

  18. Multifunctionalized iron oxide nanoparticles for selective drug delivery to CD44-positive cancer cells

    Science.gov (United States)

    Aires, Antonio; Ocampo, Sandra M.; Simões, Bruno M.; Josefa Rodríguez, María; Cadenas, Jael F.; Couleaud, Pierre; Spence, Katherine; Latorre, Alfonso; Miranda, Rodolfo; Somoza, Álvaro; Clarke, Robert B.; Carrascosa, José L.; Cortajarena, Aitziber L.

    2016-02-01

    Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro.

  19. Kidney specific protein-positive cells derived from embryonic stem cells reproduce tubular structures in vitro and differentiate into renal tubular cells.

    Directory of Open Access Journals (Sweden)

    Ryuji Morizane

    Full Text Available Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.

  20. Kidney specific protein-positive cells derived from embryonic stem cells reproduce tubular structures in vitro and differentiate into renal tubular cells.

    Science.gov (United States)

    Morizane, Ryuji; Monkawa, Toshiaki; Fujii, Shizuka; Yamaguchi, Shintaro; Homma, Koichiro; Matsuzaki, Yumi; Okano, Hideyuki; Itoh, Hiroshi

    2014-01-01

    Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP)-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.

  1. Autocrine regulation of cell proliferation by estrogen receptor-alpha in estrogen receptor-alpha-positive breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Pan Zhongzong

    2009-01-01

    Full Text Available Abstract Background Estrogen receptor-α (ERα is essential for mammary gland development and is a major oncogene in breast cancer. Since ERα is not colocalized with the cell proliferation marker Ki-67 in the normal mammary glands and the majority of primary breast tumors, it is generally believed that paracrine regulation is involved in ERα mediated cell proliferation. In the paracrine model, ERα-positive cells don't proliferate but will release some paracrine growth factors to stimulate the neighboring cells to proliferate. In a subpopulation of cancer cells in some primary breast tumors, however, ERα does colocalize with the cell proliferation marker Ki-67, suggesting an autocrine regulation by ERα in some primary breast tumors. Methods Colocalization of ERα with Ki-67 in ERα-positive breast cancer cell lines (MCF-7, T47D, and ZR75-1 was evaluated by immunofluorescent staining. Cell cycle phase dependent expression of ERα was determined by co-immunofluorescent staining of ERα and the major cyclins (D, E, A, B, and by flow cytometry analysis of ERαhigh cells. To further confirm the autocrine action of ERα, MCF-7 cells were growth arrested by ICI182780 treatment, followed by treatment with EGFR inhibitor, before estrogen stimulation and analyses for colocalization of Ki-67 and ERα and cell cycle progression. Results Colocalization of ERα with Ki-67 was present in all three ERα-positive breast cancer cell lines. Unlike that in the normal mammary glands and the majority of primary breast tumors, ERα is highly expressed throughout the cell cycle in MCF-7 cells. Without E2 stimulation, MCF-7 cells released from ICI182780 treatment remain at G1 phase. E2 stimulation of ICI182780 treated cells, however, promotes the expression and colocalization of ERα and Ki-67 as well as the cell cycle progressing through the S and G2/M phases. Inhibition of EGFR signaling does not inhibit the autocrine action of ERα. Conclusion Our data indicate

  2. [Research progress in developing reporter systems for the enrichment of positive cells with targeted genome modification].

    Science.gov (United States)

    Bai, Yichun; Xu, Kun; Wei, Zehui; Ma, Zheng; Zhang, Zhiying

    2016-01-01

    Targeted genome editing technology plays an important role in studies of gene function, gene therapy and transgenic breeding. Moreover, the efficiency of targeted genome editing is increased dramatically with the application of recently developed artificial nucleases such as ZFNs, TALENs and CRISPR/Cas9. However, obtaining positive cells with targeted genome modification is restricted to some extent by nucleases expression plasmid transfection efficiency, nucleases expression and activity, and repair efficiency after genome editing. Thus, the enrichment and screening of positive cells with targeted genome modification remains a problem that need to be solved. Surrogate reporter systems could be used to reflect the efficiency of nucleases indirectly and enrich genetically modified positive cells effectively, which may increase the efficiency of the enrichment and screening of positive cells with targeted genome modification. In this review, we mainly summarized principles and applications of reporter systems based on NHEJ and SSA repair mechanisms, which may provide references for related studies in future.

  3. Allo-hematopoietic stem cell transplantation is a potential treatment for a patient with a combined disorder of hereditary spherocytosis

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiao-hui; WANG Yu; ZHAO Ting; LIU Kai-yan; HUANG Xiao-jun; FU Hai-xia; XU Lan-ping; LIU Dai-hong; CHEN Huan; HAN Wei; CHEN Yu-hong; WANG Feng-rong; WANG Jing-zhi

    2012-01-01

    Both human hereditary spherocytosis (HS) and chronic myelogenous leukemia (CML) are life threatening.Herein we have reported the case of a woman with a combined disorder of HS and CML who underwent the matched sibling allogeneic stem cell transplantation.The complete donor erythroid cells were obtained.The red blood cell counts significantly improved throughout life comparing with pre-hematopoietic stem cell transplantation (HSCT).Reticulocyte counts normalized,and BCR-ABL was cleared away.The total bilirubin level was also corrected in this recipient.Our case is a rare example with a combined disorder of HS and CML following allogeneic stem cell transplantation.HS was not a contraindication for patient in the matched sibling transplant setting.

  4. Design of a Single-Cell Positioning Controller Using Electroosmotic Flow and Image Processing

    OpenAIRE

    Jhong-Yin Chen; Chao-Wang Young; Chyung Ay

    2013-01-01

    The objective of the current research was not only to provide a fast and automatic positioning platform for single cells, but also improved biomolecular manipulation techniques. In this study, an automatic platform for cell positioning using electroosmotic flow and image processing technology was designed. The platform was developed using a PCI image acquisition interface card for capturing images from a microscope and then transferring them to a computer using human-machine interface softwar...

  5. Aurora A kinase regulates proper spindle positioning in C. elegans and in human cells.

    Science.gov (United States)

    Kotak, Sachin; Afshar, Katayon; Busso, Coralie; Gönczy, Pierre

    2016-08-01

    Accurate spindle positioning is essential for error-free cell division. The one-cell Caenorhabditis elegans embryo has proven instrumental for dissecting mechanisms governing spindle positioning. Despite important progress, how the cortical forces that act on astral microtubules to properly position the spindle are modulated is incompletely understood. Here, we report that the PP6 phosphatase PPH-6 and its associated subunit SAPS-1, which positively regulate pulling forces acting on spindle poles, associate with the Aurora A kinase AIR-1 in C. elegans embryos. We show that acute inactivation of AIR-1 during mitosis results in excess pulling forces on astral microtubules. Furthermore, we uncover that AIR-1 acts downstream of PPH-6-SAPS-1 in modulating spindle positioning, and that PPH-6-SAPS-1 negatively regulates AIR-1 localization at the cell cortex. Moreover, we show that Aurora A and the PP6 phosphatase subunit PPP6C are also necessary for spindle positioning in human cells. There, Aurora A is needed for the cortical localization of NuMA and dynein during mitosis. Overall, our work demonstrates that Aurora A kinases and PP6 phosphatases have an ancient function in modulating spindle positioning, thus contributing to faithful cell division. PMID:27335426

  6. Proteomic analysis of imatinib-resistant CML-T1 cells reveals calcium homeostasis as a potential therapeutic target

    Science.gov (United States)

    Toman, O.; Kabickova, T.; Vit, O.; Fiser, R.; Polakova, K. Machova; Zach, J.; Linhartova, J.; Vyoral, D.; Petrak, J.

    2016-01-01

    Chronic myeloid leukemia (CML) therapy has markedly improved patient prognosis after introduction of imatinib mesylate for clinical use. However, a subset of patients develops resistance to imatinib and other tyrosine kinase inhibitors (TKIs), mainly due to point mutations in the region encoding the kinase domain of the fused BCR-ABL oncogene. To identify potential therapeutic targets in imatinib-resistant CML cells, we derived imatinib-resistant CML-T1 human cell line clone (CML-T1/IR) by prolonged exposure to imatinib in growth media. Mutational analysis revealed that the Y235H mutation in BCR-ABL is probably the main cause of CML-T1/IR resistance to imatinib. To identify alternative therapeutic targets for selective elimination of imatinib-resistant cells, we compared the proteome profiles of CML-T1 and CML-T1/IR cells using 2-DE-MS. We identified eight differentially expressed proteins, with strongly upregulated Na+/H+ exchanger regulatory factor 1 (NHERF1) in the resistant cells, suggesting that this protein may influence cytosolic pH, Ca2+ concentration or signaling pathways such as Wnt in CML-T1/IR cells. We tested several compounds including drugs in clinical use that interfere with the aforementioned processes and tested their relative toxicity to CML-T1 and CML-T1/IR cells. Calcium channel blockers, calcium signaling antagonists and modulators of calcium homeostasis, namely thapsigargin, ionomycin, verapamil, carboxyamidotriazole and immunosuppressive drugs cyclosporine A and tacrolimus (FK-506) were selectively toxic to CML-T1/IR cells. The putative cellular targets of these compounds in CML-T1/IR cells are postulated in this study. We propose that Ca2+ homeostasis can be a potential therapeutic target in CML cells resistant to TKIs. We demonstrate that a proteomic approach may be used to characterize a TKI-resistant population of CML cells enabling future individualized treatment options for patients. PMID:27430982

  7. Aromatase expression increases the survival and malignancy of estrogen receptor positive breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Keya De Mukhopadhyay

    Full Text Available In postmenopausal women, local estrogen produced by adipose stromal cells in the breast is believed to support estrogen receptor alpha (ERα positive breast cancer cell survival and growth. This raises the question of how the ERα positive metastatic breast cancer cells survive after they enter blood and lymph circulation, where estrogen level is very low in postmenopausal women. In this study, we show that the aromatase expression increased when ERα positive breast cancer cells were cultured in suspension. Furthermore, treatment with the aromatase substrate, testosterone, inhibited suspension culture-induced apoptosis whereas an aromatase inhibitor attenuated the effect of testosterone suggesting that suspended circulating ERα positive breast cancer cells may up-regulate intracrine estrogen activity for survival. Consistent with this notion, a moderate level of ectopic aromatase expression rendered a non-tumorigenic ERα positive breast cancer cell line not only tumorigenic but also metastatic in female nude mice without exogenous estrogen supplementation. The increased malignant phenotype was confirmed to be due to aromatase expression as the growth of orthotopic tumors regressed with systemic administration of an aromatase inhibitor. Thus, our study provides experimental evidence that aromatase plays an important role in the survival of metastatic ERα breast cancer cells by suppressing anoikis.

  8. High expression of hTERT and stemness genes in BORIS/CTCFL positive cells isolated from embryonic cancer cells.

    Directory of Open Access Journals (Sweden)

    Loredana Alberti

    Full Text Available BORIS/CTCFL is a member of cancer testis antigen family normally expressed in germ cells. In tumors, it is aberrantly expressed although its functions are not completely well-defined. To better understand the functions of BORIS in cancer, we selected the embryonic cancer cells as a model. Using a molecular beacon, which specifically targets BORIS mRNA, we demonstrated that BORIS positive cells are a small subpopulation of tumor cells (3-5% of total. The BORIS-positive cells isolated using BORIS-molecular beacon, expressed higher telomerase hTERT, stem cell (NANOG, OCT4, SOX2 and cancer stem cell marker genes (CD44 and ALDH1 compared to the BORIS-negative tumor cells. In order to define the functional role of BORIS, stable BORIS-depleted embryonic cancer cells were generated. BORIS silencing strongly down-regulated the expression of hTERT, stem cell and cancer stem cell marker genes. Moreover, the BORIS knockdown increased cellular senescence in embryonic cancer cells, revealing a putative role of BORIS in the senescence biological program. Our data indicate an association of BORIS expressing cells subpopulation with the expression of stemness genes, highlighting the critical role played by BORIS in embryonic neoplastic disease.

  9. Surface cell differentiation controls tissue surface tension and tissue positioning during zebrafish gastrulation

    Science.gov (United States)

    Krens, S. F. G.

    2011-03-01

    Differences in tissue surface tension (TST) between different tissue types are thought to guide tissue organization and cell sorting in development. Measurements of TST have been useful to predict the outcome of in vitro cell sorting and envelopment experiments. However, the outcome of cell sorting experiments in vitro often substantially differs from tissue positioning in vivo, raising questions as to the actual contribution of TST to tissue positioning within the developing embryo. Here, we show that surface tension of germ layer tissues during zebrafish gastrulation critically relies on the differentiation of their surface cells. We also show that surface differentiation of the different germ layer tissues varies and is considerably different between the situation in vitro and in vivo, explaining the apparent dissimilar outcome of cell segregation between these two situations. To analyze germ layer TST as a function of surface cell differentiation, we interfere with surface cell properties of germ layer aggregates by misexpressing genes involved in surface cell differentiation specifically within surface cells using the GAL4-UAS system, and measure tissue surface tension using both parallel plate compression and micropipette aspiration techniques. Our data provides evidence in favor of a critical function of surface cell differentiation in modulating TST and subsequently tissue positioning within the developing embryo.

  10. Protamine-induced permeabilization of cell envelopes of gram-positive and gram-negative bacteria

    DEFF Research Database (Denmark)

    Johansen, Charlotte; Verheul, A.; Gram, Lone;

    1997-01-01

    carboxyfluorescein and ATP after 2 to 5 min. Maximum antibacterial activity was reached at alkaline pH and in the absence of divalent cations. The efficient permeabilization of cell envelopes of both gram-positive and gram-negative bacteria suggests that protamine causes a general disruption of the cell envelope...

  11. 3D Image-Guided Automatic Pipette Positioning for Single Cell Experiments in vivo

    OpenAIRE

    Brian Long; Lu Li; Ulf Knoblich; Hongkui Zeng; Hanchuan Peng

    2015-01-01

    We report a method to facilitate single cell, image-guided experiments including in vivo electrophysiology and electroporation. Our method combines 3D image data acquisition, visualization and on-line image analysis with precise control of physical probes such as electrophysiology microelectrodes in brain tissue in vivo. Adaptive pipette positioning provides a platform for future advances in automated, single cell in vivo experiments.

  12. From elasticity to inelasticity in cancer cell mechanics: A loss of scale-invariance

    Science.gov (United States)

    Laperrousaz, B.; Drillon, G.; Berguiga, L.; Nicolini, F.; Audit, B.; Satta, V. Maguer; Arneodo, A.; Argoul, F.

    2016-08-01

    Soft materials such as polymer gels, synthetic biomaterials and living biological tissues are generally classified as viscoelastic or viscoplastic materials, because they behave neither as pure elastic solids, nor as pure viscous fluids. When stressed beyond their linear viscoelastic regime, cross-linked biopolymer gels can behave nonlinearly (inelastically) up to failure. In living cells, this type of behavior is more frequent because their cytoskeleton is basically made of cross-linked biopolymer chains with very different structural and flexibility properties. These networks have high sensitivity to stress and great propensity to local failure. But in contrast to synthetic passive gels, they can "afford" these failures because they have ATP driven reparation mechanisms which often allow the recovery of the original texture. A cell pressed in between two plates for a long period of time may recover its original shape if the culture medium brings all the nutrients for keeping it alive. When the failure events are too frequent or too strong, the reparation mechanisms may abort, leading to an irreversible loss of mechanical homeostasis and paving the way for chronic diseases such as cancer. To illustrate this discussion, we consider a model of immature cell transformation during cancer progression, the chronic myelogenous leukemia (CML), where the formation of the BCR-ABL oncogene results from a single chromosomal translocation t(9; 22). Within the assumption that the cell response to stress is scale invariant, we show that the power-law exponent that characterizes their mechanosensitivity can be retrieved from AFM force indentation curves. Comparing control and BCR-ABL transduced cells, we observe that in the later case, one month after transduction, a small percentage the cancer cells no longer follows the control cell power law, as an indication of disruption of the initial cytoskeleton network structure.

  13. Decreased PD-1 positive blood follicular helper T cells in patients with psoriasis.

    Science.gov (United States)

    Shin, Dongyun; Kim, Dae Suk; Kim, Sung Hee; Je, Jung Hwan; Kim, Hee Ju; Young Kim, Do; Kim, Soo Min; Lee, Min-Geol

    2016-10-01

    Follicular helper T (Tfh) cells are recently characterized subset of helper T cells, which are initially found in the germinal centers of B cell follicles. The major role of Tfh cells is helping B cell activation and antibody production during humoral immunity. Recently, blood Tfh cells were shown to be associated with autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, bullous pemphigoid and psoriasis. There is only one study which investigated Tfh cells in psoriasis patients. Therefore, in this study, we evaluated and analyzed blood Tfh cells in Korean patients with psoriasis. A total of 28 psoriasis patients and 16 healthy controls were enrolled. The frequency and absolute number of CXCR5(+)PD-1(+) Tfh cells were decreased in patients with psoriasis compared to healthy controls. CD4(+)CXCR5(+) T cells and CXCR5(+)ICOS(+) Tfh cells did not show differences. The frequency and absolute number of CXCR5(+)PD-1(+) Tfh cells in psoriasis patients negatively correlated with erythrocyte sedimentation rate and positively correlated with disease duration. The absolute number of CXCR5(+)ICOS(+) Tfh cells also showed positive correlation with disease duration. However, the subpopulations of Tfh cells did not correlate with Psoriasis Area and Severity Index. Serum interleukin-21 level was significantly increased in psoriasis patients compared to healthy controls, however, its level did not correlate with clinical and experimental parameters of psoriasis patients. These findings suggest the decreased function of Tfh cells in psoriasis, which could result in attenuated B cell immune responses in the pathogenesis of psoriasis. However, further investigations are necessary to confirm the function of Tfh cells in psoriasis vulgaris. PMID:27501809

  14. Synergistic apoptosis induction in leukemic cells by the phosphatase inhibitor salubrinal and proteasome inhibitors.

    Directory of Open Access Journals (Sweden)

    Hannes C A Drexler

    Full Text Available BACKGROUND: Cells adapt to endoplasmic reticulum (ER-stress by arresting global protein synthesis while simultaneously activating specific transcription factors and their downstream targets. These processes are mediated in part by the phosphorylation-dependent inactivation of the translation initiation factor eIF2alpha. Following restoration of homeostasis protein synthesis is resumed when the serine/threonine-protein phosphatase PP1 dephosphorylates and reactivates eIF2alpha. Proteasome inhibitors, used to treat multiple myeloma patients evoke ER-stress and apoptosis by blocking the ER-associated degradation of misfolded proteins (ERAD, however, the role of eIF2alpha phosphorylation in leukemic cells under conditions of proteasome inhibitor-mediated ER stress is currently unclear. METHODOLOGY AND PRINCIPAL FINDINGS: Bcr-Abl-positive and negative leukemic cell lines were used to investigate the functional implications of PP1-related phosphatase activities on eIF2alpha phosphorylation in proteasome inhibitor-mediated ER stress and apoptosis. Rather unexpectedly, salubrinal, a recently identified PP1 inhibitor capable to protect against ER stress in various model systems, strongly synergized with proteasome inhibitors to augment apoptotic death of different leukemic cell lines. Salubrinal treatment did not affect the phosphorlyation status of eIF2alpha. Furthermore, the proapoptotic effect of salubrinal occurred independently from the chemical nature of the proteasome inhibitor, was recapitulated by a second unrelated phosphatase inhibitor and was unaffected by overexpression of a dominant negative eIF2alpha S51A variant that can not be phosphorylated. Salubrinal further aggravated ER-stress and proteotoxicity inflicted by the proteasome inhibitors on the leukemic cells since characteristic ER stress responses, such as ATF4 and CHOP synthesis, XBP1 splicing, activation of MAP kinases and eventually apoptosis were efficiently abrogated by the

  15. Phototheranostics of CD44-positive cell populations in triple negative breast cancer

    Science.gov (United States)

    Jin, Jiefu; Krishnamachary, Balaji; Mironchik, Yelena; Kobayashi, Hisataka; Bhujwalla, Zaver M.

    2016-01-01

    Triple-negative breast cancer (TNBC) is one of the most lethal subtypes of breast cancer that has limited treatment options. Its high rates of recurrence and metastasis have been associated, in part, with a subpopulation of breast cancer stem-like cells that are resistant to conventional therapies. A compendium of markers such as CD44high/CD24low, and increased expression of the ABCG2 transporter and increased aldehyde dehydrogenase (ALDH1), have been associated with these cells. We developed a CD44-targeted monoclonal antibody photosensitizer conjugate for combined fluorescent detection and photoimmunotherapy (PIT) of CD44 expressing cells in TNBC. The CD44-targeted conjugate demonstrated acute cell killing of breast cancer cells with high CD44 expression. This cell death process was dependent upon CD44-specific cell membrane binding combined with near-infrared irradiation. The conjugate selectively accumulated in CD44-positive tumors and caused dramatic tumor shrinkage and efficient elimination of CD44-positive cell populations following irradiation. This novel phototheranostic strategy provides a promising opportunity for the destruction of CD44-positive populations that include cancer stem-like cells, in locally advanced primary and metastatic TNBC. PMID:27302409

  16. Islet Cells Serve as Cells of Origin of Pancreatic Gastrin-Positive Endocrine Tumors

    DEFF Research Database (Denmark)

    Bonnavion, Rémy; Teinturier, Romain; Jaafar, Rami;

    2015-01-01

    The cells of origin of pancreatic gastrinomas remain an enigma, since no gastrin-expressing cells are found in the normal adult pancreas. It was proposed that the cellular origin of pancreatic gastrinomas may come from either the pancreatic cells themselves or gastrin-expressing cells which have ...

  17. Cytokines inducing bone marrow SCA+ cells migration into pancreatic islet and conversion into insulin-positive cells in vivo.

    Directory of Open Access Journals (Sweden)

    LuGuang Luo

    Full Text Available We hypothesize that specific bone marrow lineages and cytokine treatment may facilitate bone marrow migration into islets, leading to a conversion into insulin producing cells in vivo. In this study we focused on identifying which bone marrow subpopulations and cytokine treatments play a role in bone marrow supporting islet function in vivo by evaluating whether bone marrow is capable of migrating into islets as well as converting into insulin positive cells. We approached this aim by utilizing several bone marrow lineages and cytokine-treated bone marrow from green fluorescent protein (GFP positive bone marrow donors. Sorted lineages of Mac-1(+, Mac-1(-, Sca(+, Sca(-, Sca(-/Mac-1(+ and Sca(+/Mac-1(- from GFP positive mice were transplanted to irradiated C57BL6 GFP negative mice. Bone marrow from transgenic human ubiquitin C promoter GFP (uGFP, with strong signal C57BL6 mice was transplanted into GFP negative C57BL6 recipients. After eight weeks, migration of GFP positive donor' bone marrow to the recipient's pancreatic islets was evaluated as the percentage of positive GFP islets/total islets. The results show that the most effective migration comes from the Sca(+/Mac(- lineage and these cells, treated with cytokines for 48 hours, were found to have converted into insulin positive cells in pancreatic islets in vivo. This study suggests that bone marrow lineage positive cells and cytokine treatments are critical factors in determining whether bone marrow is able to migrate and form insulin producing cells in vivo. The mechanisms causing this facilitation as well as bone marrow converting to pancreatic beta cells still need to be investigated.

  18. A positive feedback regulation of ISL-1 in DLBCL but not in pancreatic β-cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qiao, E-mail: zhangqiao200824@126.com [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences (Ministry of Education), Peking University, 38 Xueyuan Road, 100191 Beijing (China); Yang, Zhe, E-mail: zheyang@bjmu.edu.cn [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences (Ministry of Education), Peking University, 38 Xueyuan Road, 100191 Beijing (China); Wang, Weiping, E-mail: wwp@bjmu.edu.cn [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences (Ministry of Education), Peking University, 38 Xueyuan Road, 100191 Beijing (China); Guo, Ting, E-mail: luckyguoting@bjmu.edu.cn [Department of Gastrointestinal Translation Research, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital, 52 Fucheng Road, 100142 Beijing (China); Jia, Zhuqing, E-mail: zhuqingjia@126.com [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences (Ministry of Education), Peking University, 38 Xueyuan Road, 100191 Beijing (China); Ma, Kangtao, E-mail: makangtao11@126.com [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences (Ministry of Education), Peking University, 38 Xueyuan Road, 100191 Beijing (China); Zhou, Chunyan, E-mail: chunyanzhou@bjmu.edu.cn [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences (Ministry of Education), Peking University, 38 Xueyuan Road, 100191 Beijing (China)

    2014-07-04

    Highlights: • ISL-1 is highly expressed in human pancreatic β-cells and DLBCL. • ISL-1 accelerates the tumorigenesis of DLBCL in vivo. • c-Myc positively regulates ISL-1 expression in DLBCL but not in pancreatic β-cells. • ISL-1 and c-Myc forms an ISL-1/c-Myc transcriptional complex only in DLBCL. • Positive feedback regulation of ISL-1 does not exist in normal pancreatic β-cell. - Abstract: Insulin enhancer binding protein-1 (ISL-1), a LIM-homeodomain transcription factor, has been reported to play essential roles in promoting adult pancreatic β-cells proliferation. Recent studies indicate that ISL-1 may also involve in the occurrence of a variety of tumors. However, whether ISL-1 has any functional effect on tumorigenesis, and what are the differences on ISL-1 function in distinct conditions, are completely unknown. In this study, we found that ISL-1 was highly expressed in human pancreatic β-cells, as well as in diffuse large B cell lymphoma (DLBCL), but to a much less extent in other normal tissues or tumor specimens. Further study revealed that ISL-1 promoted the proliferation of pancreatic β-cells and DLBCL cells, and also accelerated the tumorigenesis of DLBCL in vivo. We also found that ISL-1 could activate c-Myc transcription not only in pancreatic β-cells but also in DLBCL cells. However, a cell-specific feedback regulation was detectable only in DLBCL cells. This auto-regulatory loop was established by the interaction of ISL-1 and c-Myc to form an ISL-1/c-Myc transcriptional complex, and synergistically to promote ISL-1 transcription through binding on the ISL-1 promoter. Taken together, our results demonstrate a positive feedback regulation of ISL-1 in DLBCL but not in pancreatic β-cells, which might result in the functional diversities of ISL-1 in different physiological and pathological processes.

  19. T Cell Interstitial Migration: Motility Cues from the Inflamed Tissue for Micro- and Macro-Positioning

    Science.gov (United States)

    Gaylo, Alison; Schrock, Dillon C.; Fernandes, Ninoshka R. J.; Fowell, Deborah J.

    2016-01-01

    Effector T cells exit the inflamed vasculature into an environment shaped by tissue-specific structural configurations and inflammation-imposed extrinsic modifications. Once within interstitial spaces of non-lymphoid tissues, T cells migrate in an apparent random, non-directional, fashion. Efficient T cell scanning of the tissue environment is essential for successful location of infected target cells or encounter with antigen-presenting cells that activate the T cell’s antimicrobial effector functions. The mechanisms of interstitial T cell motility and the environmental cues that may promote or hinder efficient tissue scanning are poorly understood. The extracellular matrix (ECM) appears to play an important scaffolding role in guidance of T cell migration and likely provides a platform for the display of chemotactic factors that may help to direct the positioning of T cells. Here, we discuss how intravital imaging has provided insight into the motility patterns and cellular machinery that facilitates T cell interstitial migration and the critical environmental factors that may optimize the efficiency of effector T cell scanning of the inflamed tissue. Specifically, we highlight the local micro-positioning cues T cells encounter as they migrate within inflamed tissues, from surrounding ECM and signaling molecules, as well as a requirement for appropriate long-range macro-positioning within distinct tissue compartments or at discrete foci of infection or tissue damage. The central nervous system (CNS) responds to injury and infection by extensively remodeling the ECM and with the de novo generation of a fibroblastic reticular network that likely influences T cell motility. We examine how inflammation-induced changes to the CNS landscape may regulate T cell tissue exploration and modulate function. PMID:27790220

  20. A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable.

    Science.gov (United States)

    Tian, Hua; Biehs, Brian; Warming, Søren; Leong, Kevin G; Rangell, Linda; Klein, Ophir D; de Sauvage, Frederic J

    2011-10-13

    The small intestine epithelium renews every 2 to 5 days, making it one of the most regenerative mammalian tissues. Genetic inducible fate mapping studies have identified two principal epithelial stem cell pools in this tissue. One pool consists of columnar Lgr5-expressing cells that cycle rapidly and are present predominantly at the crypt base. The other pool consists of Bmi1-expressing cells that largely reside above the crypt base. However, the relative functions of these two pools and their interrelationship are not understood. Here we specifically ablated Lgr5-expressing cells in mice using a human diphtheria toxin receptor (DTR) gene knocked into the Lgr5 locus. We found that complete loss of the Lgr5-expressing cells did not perturb homeostasis of the epithelium, indicating that other cell types can compensate for the elimination of this population. After ablation of Lgr5-expressing cells, progeny production by Bmi1-expressing cells increased, indicating that Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells. Indeed, lineage tracing showed that Bmi1-expressing cells gave rise to Lgr5-expressing cells, pointing to a hierarchy of stem cells in the intestinal epithelium. Our results demonstrate that Lgr5-expressing cells are dispensable for normal intestinal homeostasis, and that in the absence of these cells, Bmi1-expressing cells can serve as an alternative stem cell pool. These data provide the first experimental evidence for the interrelationship between these populations. The Bmi1-expressing stem cells may represent both a reserve stem cell pool in case of injury to the small intestine epithelium and a source for replenishment of the Lgr5-expressing cells under non-pathological conditions. PMID:21927002

  1. Reversible immortalization of Nestin-positive precursor cells from pancreas and differentiation into insulin-secreting cells

    International Nuclear Information System (INIS)

    Highlights: ► The NPPCs from mouse pancreas were isolated. ► Tet-on system for SV40 large in NPPCs was used to get RINPPCs. ► The RINPPCs can undergo at least 80 population doublings without senescence. ► The RINPPCs can be induced to differentiate into insulin-producing cells. ► The combination of GLP-1 and sodium butyrate promoted the differentiation process. -- Abstract: Pancreatic stem cells or progenitor cells posses the ability of directed differentiation into pancreatic β cells. However, these cells usually have limited proliferative capacity and finite lifespan in vitro. In the present study, Nestin-positive progenitor cells (NPPCs) from mouse pancreas that expressed the pancreatic stem cells or progenitor cell marker Nestin were isolated to obtain a sufficient number of differentiated pancreatic β cells. Tet-on system for SV40 large T-antigen expression in NPPCs was used to achieve reversible immortalization. The reversible immortal Nestin-positive progenitor cells (RINPPCs) can undergo at least 80 population doublings without senescence in vitro while maintaining their biological and genetic characteristics. RINPPCs can be efficiently induced to differentiate into insulin-producing cells that contain a combination of glucagon-like peptide-1 (GLP-1) and sodium butyrate. The results of the present study can be used to explore transplantation therapy of type I diabetes mellitus.

  2. Reversible immortalization of Nestin-positive precursor cells from pancreas and differentiation into insulin-secreting cells

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Pei; Li, Li; Qi, Hui [The Clinical Medical Research Center, The Second Clinical Medical College (Shenzhen People' s Hospital), Jinan University, 518020 Shenzhen (China); Zhou, Han-xin [Department of General Surgery, First Hospital (Shenzhen Second People' s Hospital) of Shenzhen University, 518020 Shenzhen (China); Deng, Chun-yan [The Clinical Medical Research Center, The Second Clinical Medical College (Shenzhen People' s Hospital), Jinan University, 518020 Shenzhen (China); Li, Fu-rong, E-mail: frli62@yahoo.com [The Clinical Medical Research Center, The Second Clinical Medical College (Shenzhen People' s Hospital), Jinan University, 518020 Shenzhen (China); Shenzhen Institution of Gerontology, 518020 Shenzhen (China)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer The NPPCs from mouse pancreas were isolated. Black-Right-Pointing-Pointer Tet-on system for SV40 large in NPPCs was used to get RINPPCs. Black-Right-Pointing-Pointer The RINPPCs can undergo at least 80 population doublings without senescence. Black-Right-Pointing-Pointer The RINPPCs can be induced to differentiate into insulin-producing cells. Black-Right-Pointing-Pointer The combination of GLP-1 and sodium butyrate promoted the differentiation process. -- Abstract: Pancreatic stem cells or progenitor cells posses the ability of directed differentiation into pancreatic {beta} cells. However, these cells usually have limited proliferative capacity and finite lifespan in vitro. In the present study, Nestin-positive progenitor cells (NPPCs) from mouse pancreas that expressed the pancreatic stem cells or progenitor cell marker Nestin were isolated to obtain a sufficient number of differentiated pancreatic {beta} cells. Tet-on system for SV40 large T-antigen expression in NPPCs was used to achieve reversible immortalization. The reversible immortal Nestin-positive progenitor cells (RINPPCs) can undergo at least 80 population doublings without senescence in vitro while maintaining their biological and genetic characteristics. RINPPCs can be efficiently induced to differentiate into insulin-producing cells that contain a combination of glucagon-like peptide-1 (GLP-1) and sodium butyrate. The results of the present study can be used to explore transplantation therapy of type I diabetes mellitus.

  3. Isolation and analysis of SSEA-4 positive cells derived from fetal marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    LIU Daqing; PEI Xuetao; YANG Yinxiang; GAO Yanhong; YUAN Hongfeng; QIN Lipeng; WANG Yunfang; NAN Xue; SHI Shuangshuang; YUE Wen

    2006-01-01

    A big issue in stem cell research is to derive prospective totipotential stem cells. In this study, fMSC-SSEA-4 cells expressing SSEA-4 antigen were isolated from fetal marrow masenchymal stem cells (fMSCs) using immunomagnetic bead sorting technique. The totipotent cells were identified and their biological characteristics were further studied. The expression of Oct-4 and SSEA-4, carcino- genicity, and the ability to differentiation of fMSC- SSEA-4 cells were evaluated to verify the totipotent potential. fMSC-SSEA-4 cells were isolated successfully from fMSCs (2.5% among fMSCs), while no obvious differences were seen in morphology, growth curve, cell cycle and immunophenotype, Oct-4 and SSEA-4 expression between fMSC-SSEA-4 cells and fMSCs. fMSC-SSEA-4 cells showed normal diploid chromosome karyotype and no carcinoma was induced after inoculation into nude mice. fMSC- SSEA-4 cells could be induced to fat cells, osteogenic cells and neuron-like cells in vitro with different induced factors. The results indicated that there may be a few totipotent cells among the fMSCs and it may offer the experimental basis for the further study and application of fMSCs.

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    Lifescience Database Archive (English)

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  12. Phosphatidylserine-positive particles in the apical domain of sensory hair cells

    Institute of Scientific and Technical Information of China (English)

    SHI Xiao-rui; Alfred Nuttall

    2006-01-01

    Apical membrane recycling has been proposed to be important for normal hair cell function. The current study reports an in vitro work that demonstrates the presence of phosphatidylserine (PS) and PS-positive vesicles labeled by Annexin V in the apical portion of hair cells. The following characteristics of the PS-positive vesicles were noticed using scanning confocal fluorescence microscopy: (1) variable sizes around 200 nm; (2)variable distribution patterns (either uniformly along individual stereocilia in the hair bundle or irregular) in the stereocilia from cell to cell; (3) variable sizes and numbers at locations along the border of the cuticular plate (CP),with a large number of them located at the vestigal kinocilial location; (4) motility with some of the vesicles during the observation period; (5) increase in PS labeling and the number of PS-positive vesicles after loud sound stimulation; and (6) decreased PS labeling and PS-positive vesicle numbers following treatment with LY-294002, a PI3 -kinase inhibitor. These results suggest that the presence of PS-positive vesicles at the apical area of hair cells may be indicative of vesicle shedding or transportation of a protein or rafts.

  13. Biochanin A Modulates Cell Viability, Invasion, and Growth Promoting Signaling Pathways in HER-2-Positive Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Vikas Sehdev

    2009-01-01

    Full Text Available Overexpression of HER-2 receptor is associated with poor prognosis and aggressive forms of breast cancer. Scientific literature indicates a preventive role of isoflavones in cancer. Since activation of HER-2 receptor initiates growth-promoting events in cancer cells, we studied the effect of biochanin A (an isoflavone on associated signaling events like receptor activation, downstream signaling, and invasive pathways. HER-2-positive SK-BR-3 breast cancer cells, MCF-10A normal breast epithelial cells, and NIH-3T3 normal fibroblast cells were treated with biochanin A (2–100 μM for 72 hours. Subsequently cell viability assay, western blotting and zymography were carried out. The data indicate that biochanin A inhibits cell viability, signaling pathways, and invasive enzyme expression and activity in SK-BR-3 cancer cells. Biochanin A did not inhibit MCF-10A and NIH-3T3 cell viability. Therefore, biochanin A could be a unique natural anticancer agent which can selectively target cancer cells and inhibit multiple signaling pathways in HER-2-positive breast cancer cells.

  14. Immunohistochemical staining of Langerhans cells in HPV-positive and HPV-negative cases of oral squamous cells carcinoma

    Directory of Open Access Journals (Sweden)

    Karuza Maria Alves Pereira

    2011-08-01

    Full Text Available The Human Papillomavirus (HPV has been strongly implicated in development of some cases of oral squamous cell carcinoma (OSCC. However, the immunological system somehow reacts against the presence of this virus. Among the cells involved in such mechanism of defense Langerhans cells (LC stand out, which are responsible for processing and presenting antigens. OBJECTIVES: The purposes of this study were to investigate the presence of HPV DNA and to evaluate the immunohistochemical reactivity for Langerhans cells between HPV-positive and HPV-negative OSCC. Twenty-seven cases of OSSC were evaluated. MATERIAL AND METHODS: DNA was extracted from paraffin-embedded tissue samples and amplified by Polymerase Chain Reaction (PCR for the detection of HPV DNA. Viral typing was performed by dot blot hybridization. Immunohistochemistry was performed by the Streptavidin-biotin technique. RESULTS: From the 27 cases, 9 (33.3% were HPV-positive and 18 (66.0% HPV-negative. HPV 18 was the most prevalent viral type (100% cases and infection with HPV-16 (co-infection was detected in only 1 case. In the OSCC specimens examined, immunoreactivity to S-100 antibody was detected in all cases, with a mean number of 49.48±30.89 Langerhans cells positive for immunostaining. The mean number of immunostained Langerhans cells was smaller in the HPV-positive cases (38 cells/case than in the HPV-negative cases (42.5 cells/case, but this difference was not significant (p=0.38. CONCLUSIONS: The low frequency of detection of HPV DNA in OSCC indicates a possible participation of the virus in the development and progression of only a subgroup of these tumors. There was no association between the immunohistochemical labeling for Langerhans cells (S-100+ and HPV infection of in OSSC. These findings suggest that the presence of HPV in such OSCC cases could not alter the immunological system, particularly the Langerhans cells.

  15. PDGFBB promotes PDGFR{alpha}-positive cell migration into artificial bone in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Shigeyuki [Department of Dentistry and Oral Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Iwasaki, Ryotaro; Kawana, Hiromasa [Department of Dentistry and Oral Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Miyauchi, Yoshiteru [Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Integrated Bone Metabolism and Immunology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Hoshi, Hiroko; Miyamoto, Hiroya; Mori, Tomoaki [Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Kanagawa, Hiroya [Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Katsuyama, Eri; Fujie, Atsuhiro [Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Hao, Wu [Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); and others

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer We examined effects of PDGFBB in PDGFR{alpha} positive cell migration in artificial bones. Black-Right-Pointing-Pointer PDGFBB was not expressed in osteoblastic cells but was expressed in peripheral blood cells. Black-Right-Pointing-Pointer PDGFBB promoted PDGFR{alpha} positive cell migration into artificial bones but not osteoblast proliferation. Black-Right-Pointing-Pointer PDGFBB did not inhibit osteoblastogenesis. -- Abstract: Bone defects caused by traumatic bone loss or tumor dissection are now treated with auto- or allo-bone graft, and also occasionally by artificial bone transplantation, particularly in the case of large bone defects. However, artificial bones often exhibit poor affinity to host bones followed by bony union failure. Thus therapies combining artificial bones with growth factors have been sought. Here we report that platelet derived growth factor bb (PDGFBB) promotes a significant increase in migration of PDGF receptor {alpha} (PDGFR{alpha})-positive mesenchymal stem cells/pre-osteoblastic cells into artificial bone in vivo. Growth factors such as transforming growth factor beta (TGF{beta}) and hepatocyte growth factor (HGF) reportedly inhibit osteoblast differentiation; however, PDGFBB did not exhibit such inhibitory effects and in fact stimulated osteoblast differentiation in vitro, suggesting that combining artificial bones with PDGFBB treatment could promote host cell migration into artificial bones without inhibiting osteoblastogenesis.

  16. Identification and analysis of CXCR4-positive synovial sarcoma-initiating cells.

    Science.gov (United States)

    Kimura, T; Wang, L; Tabu, K; Tsuda, M; Tanino, M; Maekawa, A; Nishihara, H; Hiraga, H; Taga, T; Oda, Y; Tanaka, S

    2016-07-28

    Synovial sarcoma accounts for almost 10% of all soft tissue sarcomas, and its prognosis is poor with 5-year survival rates at 36%. Thus, new treatments and therapeutic targets for synovial sarcoma are required. Tumor-initiating cells have been defined by the ability for self-renewal and multipotent differentiation, and they exhibit higher tumorigenic capacity, chemoresistance and radiation resistance, expecting to be a new therapeutic target. In synovial sarcoma, the presence of such stemness remains largely unclear; thus, we analyzed whether synovial sarcoma possessed tumor-initiating cells and explored specific markers, and we discovered that synovial sarcoma cell lines possessed heterogeneity by way of containing a sphere-forming subpopulation highly expressing NANOG, OCT4 and SOX2. By expression microarray analysis, CXCR4 was identified to be highly expressed in the sphere subpopulation and correlated with stem-cell-associated markers. Inhibition of CXCR4 suppressed the cell proliferation of synovial sarcoma cell lines in vitro. The tumor-initiating ability of CXCR4-positive cells was demonstrated by xenograft propagation assay. CXCR4-positive cells showed higher tumorigenicity than negative ones and possessed both self-renewal and multipotent differentiation ability. Immunohistochemical analysis of 39 specimens of synovial sarcoma patients revealed that CXCR4 strongly correlated with poor prognosis of synovial sarcoma. Thus, we conclude that CXCR4 is the marker of synovial sarcoma-initiating cells, a new biomarker for prognosis and a new potential therapeutic target. PMID:26640147

  17. Positional variation of antipodal cells in polyembryonic rice ApⅢ before and after fertilization

    Institute of Scientific and Technical Information of China (English)

    YUAN Liangchen; CHEN Yongzhe; MU Xijin; LIN Jinxing

    2003-01-01

    The variation in the position of antipodal cells before and after fertilization in polyembryonic rice strain ApⅢ was studied by using conventional sectioning technique. It was shown that initially the three antipodal cells lie at the chalazal end of the young embryo sacs. Along with the development of embryo sac, the antipodal cells proliferate into a multicellular structure containing up to about 20 cells. Most of the cells migrate to its dorsal side when the embryo sacs turn into mature. In a few embryo sacs the antipodal cells are situated in the positions adjacent to the egg apparatus at the micropylar end, while in other sacs, they spread from the midst of the embryo sac to the micropylar end clinging to the nucellar tissue. Furthermore, it is reported for the first time that cells of some egg apparatus or proembryos disorganize when antipodal cells lie at the micropylar end, indicating that the function of the antipodal cells may vary during the embryogenesis in rice ApⅢ.

  18. Robust Cell Size Checkpoint from Spatiotemporal Positive Feedback Loop in Fission Yeast

    Directory of Open Access Journals (Sweden)

    Jie Yan

    2013-01-01

    Full Text Available Cells must maintain appropriate cell size during proliferation. Size control may be regulated by a size checkpoint that couples cell size to cell division. Biological experimental data suggests that the cell size is coupled to the cell cycle in two ways: the rates of protein synthesis and the cell polarity protein kinase Pom1 provide spatial information that is used to regulate mitosis inhibitor Wee1. Here a mathematical model involving these spatiotemporal regulations was developed and used to explore the mechanisms underlying the size checkpoint in fission yeast. Bifurcation analysis shows that when the spatiotemporal regulation is coupled to the positive feedback loops (active Cdc2 promotes its activator, Cdc25, and suppress its inhibitor, Wee1, the mitosis-promoting factor (MPF exhibits a bistable steady-state relationship with the cell size. The switch-like response from the positive feedback loops naturally generates the cell size checkpoint. Further analysis indicated that the spatial regulation provided by Pom1 enhances the robustness of the size checkpoint in fission yeast. This was consistent with experimental data.

  19. Activating β-catenin signaling in CD133-positive dermal papilla cells increases hair inductivity.

    Science.gov (United States)

    Zhou, Linli; Yang, Kun; Xu, Mingang; Andl, Thomas; Millar, Sarah E; Boyce, Steven; Zhang, Yuhang

    2016-08-01

    Bioengineering hair follicles using cells isolated from human tissue remains a difficult task. Dermal papilla (DP) cells are known to guide the growth and cycling activities of hair follicles by interacting with keratinocytes. However, DP cells quickly lose their inductivity during in vitro passaging. Rodent DP cell cultures need external addition of growth factors, including WNT and BMP molecules, to maintain the hair inductive property. CD133 is expressed by a subpopulation of DP cells that are capable of inducing hair follicle formation in vivo. We report here that expression of a stabilized form of β-catenin promoted clonal growth of CD133-positive (CD133+) DP cells in in vitro three-dimensional hydrogel culture while maintaining expression of DP markers, including alkaline phosphatase (AP), CD133, and integrin α8. After a 2-week in vitro culture, cultured CD133+ DP cells with up-regulated β-catenin activity led to an accelerated in vivo hair growth in reconstituted skin compared to control cells. Further analysis showed that matrix cell proliferation and differentiation were significantly promoted in hair follicles when β-catenin signaling was up-regulated in CD133+ DP cells. Our data highlight an important role for β-catenin signaling in promoting the inductive capability of CD133+ DP cells for in vitro expansion and in vivo hair follicle regeneration, which could potentially be applied to cultured human DP cells. PMID:27312243

  20. [Ultrastructure of the cell walls and septa in glucuronate-positive species of Candida].

    Science.gov (United States)

    Golubev, V I; Loginova, T M; Tiurin, V S

    1980-01-01

    According to the ultrastructure of cell walls, glucuronate-positive species of the genus Candida include both ascomycetous organisms (C. ciferrii, C. incommunis, C. steatolytica) and basidiomycetous organisms (C. bogoriensis, C. curiosa, C. diffluens, C. javanica, C. marina). The character of budding and the structure of septa suggest that the perfect forms of glucuronate-positive ascomycetous Candida species should be looked for within the family Ascoideaceae.

  1. Antibody to a molecular marker of cell position inhibits synapse formation in retina.

    OpenAIRE

    Trisler, D.; Bekenstein, J; Daniels, M P

    1986-01-01

    A topographic gradient of TOP molecules in retina can be used to identify neuron position. Antibody to TOP from hybridoma cells that were injected into in vivo embryo eyes diffused into the retina and bound in a topographic gradient of [antibody.TOP] ([Ab.TOP]) complexes. Synapse formation in retina was inhibited in the presence of anti-TOP antibody. This suggests that TOP is involved in synapse formation and that recognition of position by neurons is necessary for normal synapse formation.

  2. Cytokeratin-positive interstitial reticulum cell tumors of lymph nodes: a case report and review of literature

    Institute of Scientific and Technical Information of China (English)

    DONG Ying-chun; WU Bo; SHENG Zhen; WANG Jian-dong; ZHOU Hang-bo; ZHOU Xiao-jun

    2008-01-01

    @@ Cytokeratin-positive interstitial reticulum cells(CIRCs)are considered to represent a subset of fibroblastic reticulum cells(FBRCs)belonging to accessory dendritic cells in lymph nodes,the spleen and tonsils.1-3

  3. Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes

    Directory of Open Access Journals (Sweden)

    Kyung Hoon Lee

    2016-01-01

    Full Text Available Enrichment of spermatogonial stem cells is important for studying their self-renewal and differentiation. Although germ cell-derived colonies (GDCs have been successfully cultured from neonatal pig testicular cells under 31°C conditions, the short period of in vitro maintenance (<2 months limited their application to further investigations. To develop a culture method that allows for in vitro maintenance of GDCs for long periods, we subcultured the GDCs with freshly prepared somatic cells from neonatal pig testes as feeder cells. The subcultured GDCs were maintained up to passage 13 with the fresh feeder cells (FFCs and then frozen. Eight months later, the frozen GDCs could again form the colonies on FFCs as shown in passages 1 to 13. Immunocytochemistry data revealed that the FFCs expressed GATA-binding protein 4 (GATA4, which is also detected in the cells of neonatal testes and total testicular cells, and that the expression of GATA4 was decreased in used old feeder cells. The subcultured GDCs in each passage had germ and stem cell characteristics, and flow cytometric analyses revealed that ~60% of these cells were GFRα-1 positive. In conclusion, neonatal pig testes-derived GDCs can be maintained for long periods with GATA4-expressing testicular somatic cells.

  4. Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes.

    Science.gov (United States)

    Lee, Kyung Hoon; Lee, Won Young; Kim, Jin Hoi; Park, Chan Kyu; Do, Jeong Tae; Kim, Jae Hwan; Choi, Young Suk; Kim, Nam Hyung; Song, Hyuk

    2016-01-01

    Enrichment of spermatogonial stem cells is important for studying their self-renewal and differentiation. Although germ cell-derived colonies (GDCs) have been successfully cultured from neonatal pig testicular cells under 31°C conditions, the short period of in vitro maintenance (subcultured the GDCs with freshly prepared somatic cells from neonatal pig testes as feeder cells. The subcultured GDCs were maintained up to passage 13 with the fresh feeder cells (FFCs) and then frozen. Eight months later, the frozen GDCs could again form the colonies on FFCs as shown in passages 1 to 13. Immunocytochemistry data revealed that the FFCs expressed GATA-binding protein 4 (GATA4), which is also detected in the cells of neonatal testes and total testicular cells, and that the expression of GATA4 was decreased in used old feeder cells. The subcultured GDCs in each passage had germ and stem cell characteristics, and flow cytometric analyses revealed that ~60% of these cells were GFRα-1 positive. In conclusion, neonatal pig testes-derived GDCs can be maintained for long periods with GATA4-expressing testicular somatic cells.

  5. Combinatorial treatment of mammospheres with trastuzumab and salinomycin efficiently targets HER2-positive cancer cells and cancer stem cells.

    Science.gov (United States)

    Oak, Prajakta S; Kopp, Florian; Thakur, Chitra; Ellwart, Joachim W; Rapp, Ulf R; Ullrich, Axel; Wagner, Ernst; Knyazev, Pjotr; Roidl, Andreas

    2012-12-15

    A major obstacle in the successful treatment of cancer is the occurrence of chemoresistance. Cancer cells surviving chemotherapy and giving rise to a recurrence of the tumor are termed cancer stem cells and can be identified by elevated levels of certain stem cell markers. Eradication of this cell population is a priority objective in cancer therapy. Here, we report elevated levels of stem cell markers in MCF-7 mammospheres. Likewise, an upregulation of HER2 and its differential expression within individual cells of mammospheres was observed. Sorting for HER2(high) and HER2(low) cells revealed an upregulation of stem cell markers NANOG, OCT4 and SOX2 in the HER2(low) cell fraction. Accordingly, HER2(low) cells also showed reduced proliferation, ductal-like outgrowths and an increased number of colonies in matrigel. Xenografts from subcutaneously injected HER2(low) sorted cells exihibited earlier onset but slower growth of tumors and an increase in stem cell markers compared to tumors developed from the HER2(high) fraction. Treatment of mammospheres with salinomycin reduced the expression of SOX2 indicating a selective targeting of cancer stem cells. Trastuzumab however, did not reduce the expression of SOX2 in mammospheres. Furthermore, a combinatorial treatment of mammospheres with trastuzumab and salinomycin was superior to single treatment with each drug. Thus, targeting HER2 expressing tumors with anti-HER2 therapies will not necessarily eliminate cancer stem cells and may lead to a more aggressive cancer cell phenotype. Our study demonstrates efficient killing of both HER2 positive cells and cancer stem cells, hence opening a possibility for a new combinatorial treatment strategy. PMID:22511343

  6. Design of a Single-Cell Positioning Controller Using Electroosmotic Flow and Image Processing

    Directory of Open Access Journals (Sweden)

    Jhong-Yin Chen

    2013-05-01

    Full Text Available The objective of the current research was not only to provide a fast and automatic positioning platform for single cells, but also improved biomolecular manipulation techniques. In this study, an automatic platform for cell positioning using electroosmotic flow and image processing technology was designed. The platform was developed using a PCI image acquisition interface card for capturing images from a microscope and then transferring them to a computer using human-machine interface software. This software was designed by the Laboratory Virtual Instrument Engineering Workbench, a graphical language for finding cell positions and viewing the driving trace, and the fuzzy logic method for controlling the voltage or time of an electric field. After experiments on real human leukemic cells (U-937, the success of the cell positioning rate achieved by controlling the voltage factor reaches 100% within 5 s. A greater precision is obtained when controlling the time factor, whereby the success rate reaches 100% within 28 s. Advantages in both high speed and high precision are attained if these two voltage and time control methods are combined. The control speed with the combined method is about 5.18 times greater than that achieved by the time method, and the control precision with the combined method is more than five times greater than that achieved by the voltage method.

  7. Tuberculosis-specific CD8 cells in HLA A*02-positive TB- and LTBI patients

    DEFF Research Database (Denmark)

    Fløe, Andreas; Brix, Liselotte; Wejse, Christian;

    Background: Understanding the CD8+ response against Mycobacterium tuberculosis (MTB) may be a key to improved TB diagnostics and vaccine development. Aims and Objectives: To detect a CD8+ T-cell response against Mycobacterium tuberculosis (MTB) in active tuberculosis (TB) and latent TB (LTBI), in...... candidates, from which we constructed MHC multimers (Dextramers). Peripheral blood mononuclear cells (PBMC) from 7 TB-patients, 16 LTBI patients and 8 MTB-exposed, IGRA-negative, healthy subjects (HE), all HLA A*02 positive, were stained with the Dextramers and with anti-CD8 and anti-CD3, and analyzed on a...... flow cytometer. The MTB epitopes were analyzed in 5 pools (3-7 epitopes each). Positive responses included >0.001 % of CD8+, CD3+ cells, supported by inspection of flow cytometry plots. Results: MTB-specific CD8+ T-cells were detected more often in TB patients (57%) than in LTBI patients (41%) and in...

  8. Retinal Targets ALDH Positive Cancer Stem Cell and Alters the Phenotype of Highly Metastatic Osteosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Xiaodong Mu

    2015-01-01

    Full Text Available Aldehyde dehydrogenase (ALDH is a cancer stem cell marker. Retinoic acid has antitumor properties, including the induction of apoptosis and inhibition of proliferation. Retinal, the precursor of retinoic acid, can be oxidized to retinoic acid by dehydrogenases, including ALDH. We hypothesized that retinal could potentially be transformed to retinoic acid with higher efficiency by cancer stem cells, due to the higher ALDH activity. We previously observed that ALDH activity is greater in highly metastatic K7M2 osteosarcoma (OS cells than in nonmetastatic K12 OS cells. We also demonstrated that ALDH activity correlates with clinical metastases in bone sarcoma patients, suggesting that ALDH may be a therapeutic target specific to cells with high metastatic potential. Our current results demonstrated that retinal preferentially affected the phenotypes of ALDH-high K7M2 cells in contrast to ALDH-low K12 cells, which could be mediated by the more efficient transformation of retinal to retinoic acid by ALDH in K7M2 cells. Retinal treatment of highly metastatic K7M2 cells decreased their proliferation, invasion capacity, and resistance to oxidative stress. Retinal altered the expression of metastasis-related genes. These observations indicate that retinal may be used to specifically target metastatic cancer stem cells in OS.

  9. Depletion of γ-catenin by Histone Deacetylase Inhibition Confers Elimination of CML Stem Cells in Combination with Imatinib.

    Science.gov (United States)

    Jin, Yanli; Yao, Yiwu; Chen, Li; Zhu, Xiaohui; Jin, Bei; Shen, Yingying; Li, Juan; Du, Xin; Lu, Yuhong; Jiang, Sheng; Pan, Jingxuan

    2016-01-01

    Quiescent leukemia stem cells (LSCs) that are insensitive to BCR-ABL tyrosine kinase inhibitors confer resistance to imatinib in chronic myelogenous leukemia (CML). Identifying proteins to regulate survival and stemness of LSCs is urgently needed. Although histone deacetylase inhibitors (HDACis) can eliminate quiescent LSCs in CML, little is known about the underlying mechanism that HDACis kill LSCs. By fishing with a biotin-labeled probe, we identified that HDACi JSL-1 bound to the protein γ-catenin. γ-Catenin expression was higher in LSCs from CML patients than normal hematopoietic stem cells. Silencing γ-catenin in human CML CD34(+) bone-marrow (BM) cells sufficiently eliminated LSCs, which suggests that γ-catenin is required for survival of CML LSCs. Pharmacological inhibition of γ-catenin thwarted survival and self-renewal of human CML CD34(+) cells in vitro, and of murine LSCs in BCR-ABL-driven CML mice. γ-Catenin inhibition reduced long-term engraftment of human CML CD34(+) cells in NOD.Cg-Prkdc (scid) II2rg (tm1Sug)/JicCrl (NOG) mice. Silencing γ-catenin by shRNA in human primary CD34(+) cells did not alter β-catenin, implying a β-catenin-independent role of γ-catenin in survival and self-renewal of CML LSCs. Taken together, our findings validate that γ-catenin may be a novel therapeutic target of LSCs, and suppression of γ-catenin by HDACi may explain elimination of CML LSCs. PMID:27570562

  10. A positive feedback synapse from retinal horizontal cells to cone photoreceptors.

    Directory of Open Access Journals (Sweden)

    Skyler L Jackman

    2011-05-01

    Full Text Available Cone photoreceptors and horizontal cells (HCs have a reciprocal synapse that underlies lateral inhibition and establishes the antagonistic center-surround organization of the visual system. Cones transmit to HCs through an excitatory synapse and HCs feed back to cones through an inhibitory synapse. Here we report that HCs also transmit to cone terminals a positive feedback signal that elevates intracellular Ca(2+ and accelerates neurotransmitter release. Positive and negative feedback are both initiated by AMPA receptors on HCs, but positive feedback appears to be mediated by a change in HC Ca(2+, whereas negative feedback is mediated by a change in HC membrane potential. Local uncaging of AMPA receptor agonists suggests that positive feedback is spatially constrained to active HC-cone synapses, whereas the negative feedback signal spreads through HCs to affect release from surrounding cones. By locally offsetting the effects of negative feedback, positive feedback may amplify photoreceptor synaptic release without sacrificing HC-mediated contrast enhancement.

  11. Proteomic Profiling of Ex Vivo Expanded CD34-Positive Haematopoetic Cells Derived from Umbilical Cord Blood

    Directory of Open Access Journals (Sweden)

    Heiner Falkenberg

    2013-01-01

    Full Text Available Ex vivo expansion of haematopoetic cells by application of specific cytokines is one approach to overcome boundaries in cord blood transplantation due to limited numbers of haematopoetic stem cells. While many protocols describe an effective increase of total cell numbers and the amount of CD34-positive cells, it still remains unclear if and how the procedure actually affects the cells’ properties. In the presented publications, CD34-positive cells were isolated from cord blood and expanded for up to 7 days in media supplemented with stem cell factor (SCF, thrombopoietin (THPO, interleukin 6 (IL-6, and fms-related tyrosine kinase 3 ligand (FLT3lg. At days 3 and 7, expanded cells were harvested and analyzed by flow cytometry and quantitative proteomics. 2970 proteins were identified, whereof proteomic analysis showed 440 proteins significantly changed in abundance during ex vivo expansion. Despite the fact that haematopoetic cells still expressed CD34 on the surface after 3 days, major changes in regard to the protein profile were observed, while further expansion showed less effect on the proteome level. Enrichment analysis of biological processes clearly showed a proteomic change toward a protein biosynthesis phenotype already within the first three days of expression.

  12. Positional cues specify and maintain aleurone cell fate in maize endosperm development.

    Science.gov (United States)

    Becraft, P W; Asuncion-Crabb, Y

    2000-09-01

    A genetic analysis of maize aleurone development was conducted. Cell lineage was examined by simultaneously marking cells with C1 for anthocyanin pigmentation in the aleurone and wx1 for amylose synthesis in the starchy endosperm. The aleurone and starchy endosperm share a common lineage throughout development indicating that positional cues specify aleurone fate. Mutants in dek1 block aleurone formation at an early stage and cause peripheral endosperm cells to develop as starchy endosperm. Revertant sectors of a transposon-induced dek1 allele showed that peripheral endosperm cells remain competent to differentiate as aleurone cells until late in development. Ds-induced chromosome breakage was used to generate Dek1 loss-of-function sectors. Events occurring until late development caused aleurone cells to switch fate to starchy endosperm indicating that cell fate is not fixed. Thus, positional cues are required to specify and maintain aleurone fate and Dek1 function is required to respond to these cues. An analysis of additional mutants that disrupt aleurone differentiation suggests a hierarchy of gene functions to specify aleurone cell fate and then control aleurone differentiation. These mutants disrupt aleurone differentiation in reproducible patterns suggesting a relationship to endosperm pattern formation.

  13. Positive and negative selection of T cells in T-cell receptor transgenic mice expressing a bcl-2 transgene.

    OpenAIRE

    Strasser, A.; Harris, A W; von Boehmer, H; Cory, S

    1994-01-01

    To explore the role of bcl-2 in T-cell development, a bcl-2 transgene was introduced into mice expressing a T-cell receptor (TCR) transgene encoding reactivity for the mouse male antigen HY presented by the H-2Db class I antigen of the major histocompatibility complex (MHC). Normal thymic development is contingent on the ability of immature thymocytes to interact with self-MHC molecules presented by thymic stroma (positive selection). Thus, thymocyte numbers are low in femal...

  14. Generation model of positional values as cell operation during the development of multicellular organisms.

    Science.gov (United States)

    Ogawa, Ken-ichiro; Miyake, Yoshihiro

    2011-03-01

    Many conventional models have used the positional information hypothesis to explain each elementary process of morphogenesis during the development of multicellular organisms. Their models assume that the steady concentration patterns of morphogens formed in an extracellular environment have an important property of positional information, so-called "robustness". However, recent experiments reported that a steady morphogen pattern, the concentration gradient of the Bicoid protein, during early Drosophila embryonic development is not robust for embryo-to-embryo variability. These reports encourage a reconsideration of a long-standing problem in systematic cell differentiation: what is the entity of positional information for cells? And, what is the origin of the robust boundary of gene expression? To address these problems at a cellular level, in this article we pay attention to the re-generative phenomena that show another important property of positional information, "size invariance". In view of regenerative phenomena, we propose a new mathematical model to describe the generation mechanism of a spatial pattern of positional values. In this model, the positional values are defined as the values into which differentiable cells transform a spatial pattern providing positional information. The model is mathematically described as an associative algebra composed of various terms, each of which is the multiplication of some fundamental operators under the assumption that the operators are derived from the remarkable properties of cell differentiation on an amputation surface in regenerative phenomena. We apply this model to the concentration pattern of the Bicoid protein during the anterior-posterior axis formation in Drosophila, and consider the conditions needed to establish the robust boundary of the expression of the hunchback gene. PMID:21167904

  15. Activity of cells in the lateral vestibular nucleus as a function of head position

    Science.gov (United States)

    Fujita, Y.; Rosenberg, Jay; Segundo, J. P.

    1968-01-01

    1. The spike activity of cells in the lateral vestibular nucleus was recorded in cats anaesthetized with pentobarbital sodium. Natural labyrinthine stimulation was applied by fixing the animal at different positions reached through roations about a longitudinal or transverse axis. 2. The majority of cells responded to rotations only about the longitudinal axis. Two types of response were found. The first was characterized by a transient change in activity which occurred only during the movement. The second type had an initial transient component and a subsequent steady component that persisted as long as the head remained fixed. 3. The interspike interval means, standard deviations, histograms and autocorrelograms of the steady response components of cells sensitive to lateral tilt were calculated. In every cell the relation between the head position with respect to gravity and the mean interspike interval of the steady discharge showed two main features. (a) `Directional sensitivity': the mean interval increased following rotation in one sense, and decreased following rotation in the other. In twenty-two out of thirty-three cells, the mean increased when the recording side was raised. The remaining cells showed the opposite relation. (b) `Multivaluedness': each particular position is associated with several different values of mean interval and these values had a relatively wide scatter. The curve that resulted from joining points in the order in which they occurred during the experiment was either closed, open, or combined closed and open portions. 4. The standard deviations, histograms and autocorrelograms also showed directional sensitivity and multivaluedness with respect to position. Several types of interspike interval histograms and autocorrelograms characterized lateral vestibular activity. The forms of the histogram and the autocorrelogram of the discharge from each cell usually remained unchanged during stimulation. 5. The extensive spread of the

  16. Measuring relative utilization of aerobic glycolysis in breast cancer cells by positional isotopic discrimination.

    Science.gov (United States)

    Yang, Da-Qing; Freund, Dana M; Harris, Benjamin R E; Wang, Defeng; Cleary, Margot P; Hegeman, Adrian D

    2016-09-01

    The ability of cancer cells to produce lactate through aerobic glycolysis is a hallmark of cancer. In this study, we established a positional isotopic labeling and LC-MS-based method that can specifically measure the conversion of glucose to lactate in glycolysis. We show that the rate of aerobic glycolysis is closely correlated with glucose uptake and lactate production in breast cancer cells. We also found that the production of [3-(13) C]lactate is significantly elevated in metastatic breast cancer cells and in early stage metastatic mammary tumors in mice. Our findings may enable the development of a biomarker for the diagnosis of aggressive breast cancer. PMID:27531463

  17. Positioning of polarity formation by extracellular signaling during asymmetric cell division.

    Science.gov (United States)

    Seirin Lee, Sungrim

    2016-07-01

    Anterior-posterior (AP) polarity formation of cell membrane proteins plays a crucial role in determining cell asymmetry, which ultimately generates cell diversity. In Caenorhabditis elegans, a single fertilized egg cell (P0), its daughter cell (P1), and the germline precursors (P2 and P3 cells) form two exclusive domains of different PAR proteins on the membrane along the anterior-posterior axis. However, the phenomenon of polarity reversal has been observed in which the axis of asymmetric cell division of the P2 and P3 cells is formed in an opposite manner to that of the P0 and P1 cells. The extracellular signal MES-1/SRC-1 has been shown to induce polarity reversal, but the detailed mechanism remains elusive. Here, using a mathematical model, I explore the mechanism by which MES-1/SRC-1 signaling can induce polarity reversal and ultimately affect the process of polarity formation. I show that a positive correlation between SRC-1 and the on-rate of PAR-2 is the essential mechanism underlying polarity reversal, providing a mathematical basis for the orientation of cell polarity patterns. PMID:27086039

  18. Lgr5 positive stem cells sorted from small intestines of diabetic mice differentiate into higher proportion of absorptive cells and Paneth cells in vitro.

    Science.gov (United States)

    Zhong, Xian-Yang; Yu, Tao; Zhong, Wa; Li, Jie-Yao; Xia, Zhong-Sheng; Yuan, Yu-Hong; Yu, Zhong; Chen, Qi-Kui

    2015-08-01

    Intestinal epithelial stem cells (IESCs) can differentiate into all types of intestinal epithelial cells (IECs) and Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is a marker for IESC. Previous studies reported enhanced proliferation of IECs in diabetic mice. In this study, the in vitro differentiation of Lgr5 positive IESCs sorted from diabetic mice was further investigated. The diabetic mouse model was induced by streptozotocin (STZ), and crypt IECs were isolated from small intestines. Subsequently, Lgr5 positive IESCs were detected by flow cytometry (FCM) and sorted by magnetic activated cell sorting (MACS). Differentiation of the sorted IESCs was investigated by detecting the IEC markers in the diabetic mice using immunostaining, quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR), and Western blot analysis, which was compared with normal mice. We found that the proportion of Lgr5 positive cells in the crypt IECs of diabetic mice was higher than that of control mice (P absorptive cell marker sucrase-isomaltase (SI) and the Paneth cell marker lysozyme 1 (Lyz1) were more highly expressed in the differentiated cells derived from Lgr5 positive IESCs of diabetic mice in vitro (P small intestines of STZ-induced diabetic mice. Lgr5 positive IESCs sorted from the diabetic mice can differentiate into a higher proportion of absorptive cells and Paneth cells in vitro. We characterized the expression of Lgr5 in the small intestine of diabetic mice, and sorted Lgr5 positive intestinal epithelial stem cells (IESCs) for investigating their differentiation in vitro. We proved that the quantity of Lgr5 positive IESCs was significantly increased in the small intestines of diabetic mice. IESCs sorted from the diabetic mice can differentiate into a higher proportion of absorptive cells and Paneth cells in vitro.

  19. Prevalence of t(12;21)[ETV6-RUNX1]-positive cells in healthy neonates

    DEFF Research Database (Denmark)

    Lausten-Thomsen, Ulrik; Madsen, Hans O.; Vestergaard, Therese Risom;

    2011-01-01

    t(12;21)(p13;q22)[ETV6-RUNX1] is the most common chromosomal translocation in childhood acute lymphoblastic leukemia, and it can often be backtracked to Guthrie cards supporting prenatal initiation and high levels of circulating t(12;21)-positive cells at birth. To explore the prevalence of ETV6...

  20. Global Attractivity of Positive Periodic Solutions for a Survival Model of Red Blood Cells

    Institute of Scientific and Technical Information of China (English)

    Xin-min Wu; Jing-wen Li

    2007-01-01

    In this paper, we deal with a model for the survival of red blood cells with periodic coefficients x′(t)=- μ(t)x(t)+P(t)e-γ(t)x(t-(τ))>0. (*)A new sufficient condition for global attractivity of positive periodic solutions of Eq.(*) is obtained. Our criterion improves corresponding result obtained by Li and Wang in 2005.

  1. Bacterial vaginosis (clue cell-positive discharge) : diagnostic, ultra-structural and therapeutic aspects

    NARCIS (Netherlands)

    W.I. van der Meijden (Willem)

    1987-01-01

    textabstractThis thesis deals with several aspects of (abnormal) vaginal discharge, focusing especially on clue cell-positive discharge (bacterial vaginosis, nonspecific vaginitis). It reports data on epidemiology and clinical features, pathogenesis, and treatment of this vaginal disease entity, as

  2. Leukemic phase of anaplastic lymphoma kinase positive, anaplastic large cell lymphoma

    Directory of Open Access Journals (Sweden)

    Vijaya S Gadage

    2011-01-01

    Full Text Available Anaplastic large cell lymphoma (ALCL is a distinct type of CD30+ T/null-cell non-Hodgkin′s lymphoma that frequently involves nodal and extranodal sites. The presence of leukemic phase in ALCL is extremely rare and occurs exclusively with ALK1-positive ALCL. We describe two patients with ALK1-positive ALCL who developed a leukemic phase with rapid progression of the disease. Immunophenotypic pattern assessed on peripheral blood by flow cytometry revealed CD45, CD30, and CD25 positivity in both cases but NPM-ALK1 was expressed in only one case. Both patients developed leukemic phase as a terminal event of the disease and we share the immunophenotypic features of both cases.

  3. False Positive B-Cells Crossmatch after Prior Rituximab Exposure of the Kidney Donor

    Directory of Open Access Journals (Sweden)

    Judith Desoutter

    2016-01-01

    Full Text Available Crossmatching is essential prior to kidney transplantation to confirm compatibility between the donor and the recipient, particularly to prevent acute antibody-mediated rejection. An unexpected positive crossmatch may be obtained in recipients with an autoimmune disease or preexisting antibodies not detected by single-antigen bead array due to complement interference or who have been previously treated by desensitization protocols such as rituximab, antithymocyte globulin, or intravenous immunoglobulins. We report donor and recipient investigations that revealed unexpected positive B-cells crossmatch, probably due to donor cells, as the donor had received rituximab therapy shortly before organ harvesting, in a context of severe idiopathic thrombocytopenic purpura. We consequently detected unexpected Class II IgG complement-dependent cytotoxicity for all sera tested. Other laboratory investigations failed to elucidate the reasons for this recipient-related positivity.

  4. Identification of alpha beta and gamma delta T cell receptor-positive cells

    DEFF Research Database (Denmark)

    Geisler, C; Larsen, J K; Plesner, T

    1988-01-01

    distribution and function of these different T cells. In immunofluorescence studies gamma delta TCR+ cells have been identified as CD3+WT-31- or CD3+CD4-CD8- cells. However, this may not be the optimal procedure because gamma delta TCR+ cells are weakly WT-31+, and some are CD8+. The aim of this study......Two lineages of T lymphocytes bearing the CD3 antigen can be defined on the basis of the nature of the heterodimeric receptor chain (alpha beta or gamma delta T cell receptor (TCR) expressed. Precise identification of alpha beta and gamma delta TCR+ cells is essential when studying the tissue...... was to evaluate a panel of monoclonal antibodies (MoAb) directed against different chains of the TCR-T3 complex for a more precise identification of alpha beta+ and gamma delta TCR+ cells in flow cytometric studies. We found that the MoAb anti-Ti-gamma A and delta-TCS-1, recognizing the TCR-gamma and the TCR...

  5. Caffeine Positively Modulates Ferritin Heavy Chain Expression in H460 Cells: Effects on Cell Proliferation

    Science.gov (United States)

    Battaglia, Anna Martina; Faniello, Maria Concetta; Cuda, Giovanni; Costanzo, Francesco

    2016-01-01

    Both the methylxanthine caffeine and the heavy subunit of ferritin molecule (FHC) are able to control the proliferation rate of several cancer cell lines. While caffeine acts exclusively as a negative modulator of cell proliferation, FHC might reduce or enhance cell viability depending upon the different cell type. In this work we have demonstrated that physiological concentrations of caffeine reduce the proliferation rate of H460 cells: along with the modulation of p53, pAKT and Cyclin D1, caffeine also determines a significant FHC up-regulation through the activation of its transcriptional efficiency. FHC plays a central role in the molecular pathways modulated by caffeine, ending in a reduced cell growth, since its specific silencing by siRNA almost completely abolishes caffeine effects on H460 cell proliferation. These results allow the inclusion of ferritin heavy subunits among the multiple molecular targets of caffeine and open the way for studying the relationship between caffeine and intracellular iron metabolism. PMID:27657916

  6. A Case of p63 Positive Diffuse Large B Cell Lymphoma of the Bladder

    Science.gov (United States)

    Jones, Carol

    2016-01-01

    Diffuse large B cell lymphoma (DLBCL), currently the most common type of non-Hodgkin lymphoma (NHL), is an aggressive B cell neoplasm that typically presents in older adults as a rapidly enlarging mass. The enlarging mass typically represents a lymph node, although extranodal disease can occur in a significant percentage (40%) of cases. The most common extranodal sites of involvement include the gastrointestinal tract and skin; primary bladder lymphoma represents only 0.2% of extranodal non-Hodgkin lymphomas. We report a case of diffuse large B cell lymphoma occurring in the bladder of an 83-year-old gentleman with an initial presentation of hematuria. This neoplasm displayed large, atypical cells with vesicular chromatin and prominent nucleoli that involved the bladder mucosa with invasion into muscularis propria, prostate, and urethra. Positive staining for p63 initially raised suspicion for poorly differentiated urothelial carcinoma; however, lack of staining for pancytokeratin and positive staining for LCA, CD20, CD79a, and PAX-5 confirmed the diagnosis of diffuse large B cell lymphoma. Though it does not occur in all cases, p63 can be positive in a significant percentage of cases of DLBCL; therefore, a diagnosis of lymphoma remains an important entity on the differential diagnosis of aggressive and particularly poorly differentiated neoplasms. PMID:27648316

  7. CCL21/IL21-armed oncolytic adenovirus enhances antitumor activity against TERT-positive tumor cells.

    Science.gov (United States)

    Li, Yang; Li, Yi-Fei; Si, Chong-Zhan; Zhu, Yu-Hui; Jin, Yan; Zhu, Tong-Tong; Liu, Ming-Yuan; Liu, Guang-Yao

    2016-07-15

    Multigene-armed oncolytic adenoviruses are capable of efficiently generating a productive antitumor immune response. The chemokine (C-C motif) ligand 21 (CCL21) binds to CCR7 on naïve T cells and dendritic cells (DCs) to promote their chemoattraction to the tumor and resultant antitumor activity. Interleukin 21 (IL21) promotes survival of naïve T cells while maintaining their CCR7 surface expression, which increases their capacity to transmigrate in response to CCL21 chemoattraction. IL21 is also involved in NK cell differentiation and B cell activation and proliferation. The generation of effective antitumor immune responses is a complex process dependent upon coordinated interactions of various subsets of effector cells. Using the AdEasy system, we aimed to construct an oncolytic adenovirus co-expressing CCL21 and IL21 that could selectively replicate in TERTp-positive tumor cells (Ad-CCL21-IL21 virus). The E1A promoter of these oncolytic adenoviruses was replaced by telomerase reverse transcriptase promoter (TERTp). Ad-CCL21-IL21 was constructed from three plasmids, pGTE-IL21, pShuttle-CMV-CCL21 and AdEasy-1 and was homologously recombined and propagated in the Escherichia coli strain BJ5183 and the packaging cell line HEK-293, respectively. Our results showed that our targeted and armed oncolytic adenoviruses Ad-CCL21-IL21 can induce apoptosis in TERTp-positive tumor cells to give rise to viral propagation, in a dose-dependent manner. Importantly, we confirm that these modified oncolytic adenoviruses do not replicate efficiently in normal cells even under high viral loads. Additionally, we investigate the role of Ad-CCL21-IL21 in inducing antitumor activity and tumor specific cytotoxicity of CTLs in vitro. This study suggests that Ad-CCL21-IL21 is a promising targeted tumor-specific oncolytic adenovirus. PMID:27157859

  8. Isolation and characterization of portal branch ligation-stimulated Hmga2-positive bipotent hepatic progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Sakai, Hiroshi [Department of Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 B51, Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Tagawa, Yoh-ichi, E-mail: ytagawa@bio.titech.ac.jp [Frontier Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 B51, Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012 (Japan); Tamai, Miho [Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 B51, Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Motoyama, Hiroaki [Department of Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Ogawa, Shinichiro [Department of Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); McEwen Center for Regenerative Medicine, University Health Network, 190 Elizabeth Street, Toronto, Ont., Canada M5G 2C4 (Canada); Soeda, Junpei; Nakata, Takenari; Miyagawa, Shinichi [Department of Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan)

    2010-12-17

    Research highlights: {yields} Hepatic progenitor cells were isolated from the portal branch-ligated liver of mice. {yields} Portal branch ligation-stimulated hepatic progenitor cells (PBLHCs) express Hmga2. {yields} PBLHCs have bidirectional differentiation capability in vitro. -- Abstract: Hepatic stem/progenitor cells are one of several cell sources that show promise for restoration of liver mass and function. Although hepatic progenitor cells (HPCs), including oval cells, are induced by administration of certain hepatotoxins in experimental animals, such a strategy would be inappropriate in a clinical setting. Here, we investigated the possibility of isolating HPCs in a portal branch-ligated liver model without administration of any chemical agents. A non-parenchymal cell fraction was prepared from the portal branch-ligated or non-ligated lobe, and seeded onto plates coated with laminin. Most of the cells died, but a small number were able to proliferate. These proliferating cells were cloned as portal branch ligation-stimulated hepatic cells (PBLHCs) by the limiting dilution method. The PBLHCs expressed cytokeratin19, albumin, and Hmga2. The PBLHCs exhibited metabolic functions such as detoxification of ammonium ions and synthesis of urea on Matrigel-coated plates in the presence of oncostatin M. In Matrigel mixed with type I collagen, the PBLHCs became rearranged into cystic and tubular structures. Immunohistochemical staining demonstrated the presence of Hmga2-positive cells around the interlobular bile ducts in the portal branch-ligated liver lobes. In conclusion, successful isolation of bipotent hepatic progenitor cell clones, PBLHCs, from the portal branch-ligated liver lobes of mice provides the possibility of future clinical application of portal vein ligation to induce hepatic progenitor cells.

  9. Cell to cell communication by autoinducing in gram-positive bacteria

    NARCIS (Netherlands)

    Sturme, M.H.J.; Kleerebezem, M.; Nakayama, J.; Akkermans, A.D.L.; Vaughan, E.E.; Vos, de W.M.

    2002-01-01

    While intercellular communication systems in Gram-negative bacteria are often based on homoserine lactones as signalling molecules, it has been shown that autoinducing peptides are involved in intercellular communication in Gram-positive bacteria. Many of these peptides are exported by dedicated sys

  10. Reciprocal positive regulation between TRPV6 and NUMB in PTEN-deficient prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sung-Young; Hong, Chansik; Wie, Jinhong [Department of Physiology, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Kim, Euiyong [Department of Physiology, College of Medicine, Inje University, Busan 614-735 (Korea, Republic of); Kim, Byung Joo [Division of Longevity and Biofunctional Medicine, Pusan National University School of Korean Medicine, Yangsan 626-870 (Korea, Republic of); Ha, Kotdaji [Department of Physiology, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Cho, Nam-Hyuk; Kim, In-Gyu [Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Jeon, Ju-Hong [Department of Physiology, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); So, Insuk, E-mail: insuk@snu.ac.kr [Department of Physiology, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

    2014-04-25

    Highlights: • TRPV6 interacts with tumor suppressor proteins. • Numb has a selective effect on TRPV6, depending on the prostate cancer cell line. • PTEN is a novel regulator of TRPV6–Numb complex. - Abstract: Calcium acts as a second messenger and plays a crucial role in signaling pathways involved in cell proliferation. Recently, calcium channels related to calcium influx into the cytosol of epithelial cells have attracted attention as a cancer therapy target. Of these calcium channels, TRPV6 is overexpressed in prostate cancer and is considered an important molecule in the process of metastasis. However, its exact role and mechanism is unclear. NUMB, well-known tumor suppressor gene, is a novel interacting partner of TRPV6. We show that NUMB and TRPV6 have a reciprocal positive regulatory relationship in PC-3 cells. We repeated this experiment in two other prostate cancer cell lines, DU145 and LNCaP. Interestingly, there were no significant changes in TRPV6 expression following NUMB knockdown in DU145. We revealed that the presence or absence of PTEN was the cause of NUMB–TRPV6 function. Loss of PTEN caused a positive correlation of TRPV6–NUMB expression. Collectively, we determined that PTEN is a novel interacting partner of TRPV6 and NUMB. These results demonstrated a novel relationship of NUMB–TRPV6 in prostate cancer cells, and show that PTEN is a novel regulator of this complex.

  11. Synergy of photoacoustic and fluorescence flow cytometry of circulating cells with negative and positive contrasts.

    Science.gov (United States)

    Nedosekin, Dmitry A; Sarimollaoglu, Mustafa; Galanzha, Ekaterina I; Sawant, Rupa; Torchilin, Vladimir P; Verkhusha, Vladislav V; Ma, Jie; Frank, Markus H; Biris, Alexandru S; Zharov, Vladimir P

    2013-05-01

    In vivo photoacoustic (PA) and fluorescence flow cytometry were previously applied separately using pulsed and continuous wave lasers respectively, and positive contrast detection mode only. This paper introduces a real-time integration of both techniques with positive and negative contrast modes using only pulsed lasers. Various applications of this new tool are summarized, including detection of liposomes loaded with Alexa-660 dye, red blood cells labeled with Indocyanine Green, B16F10 melanoma cells co-expressing melanin and green fluorescent protein (GFP), C8161-GFP melanoma cells targeted by magnetic nanoparticles, MTLn3 adenocarcinoma cells expressing novel near-infrared iRFP protein, and quantum dot-carbon nanotube conjugates. Negative contrast flow cytometry provided label-free detection of low absorbing or weakly fluorescent cells in blood absorption and autofluorescence background, respectively. The use of pulsed laser for time-resolved discrimination of objects with long fluorescence lifetime (e.g., quantum dots) from shorter autofluorescence background (e.g., blood plasma) is also highlighted in this paper. The supplementary nature of PA and fluorescence detection increased the versatility of the integrated method for simultaneous detection of probes and cells having various absorbing and fluorescent properties, and provided verification of PA data using a more established fluorescence based technique. PMID:22903924

  12. Light-dependent intracellular positioning of mitochondria in Arabidopsis thaliana mesophyll cells.

    Science.gov (United States)

    Islam, Md Sayeedul; Niwa, Yasuo; Takagi, Shingo

    2009-06-01

    Mitochondria, the power house of the cell, are one of the most dynamic cell organelles. Although there are several reports on actin- or microtubule-dependent movement of mitochondria in plant cells, intracellular positioning and motility of mitochondria under different light conditions remain open questions. Mitochondria were visualized in living Arabidopsis thaliana leaf cells using green fluorescent protein fused to a mitochondrion-targeting signal. In darkness, mitochondria were distributed randomly in palisade cells. In contrast, mitochondria accumulated along the periclinal walls, similar to the accumulation response of chloroplasts, when treated with weak blue light (470 nm, 4 micromol m(-2) s(-1)). Under strong blue light (100 micromol m(-2) s(-1)), mitochondria occupied the anticlinal positions similar to the avoidance response of chloroplasts and nuclei. While strong red light (660 nm, 100 micromol m(-2) s(-1)) induced the accumulation of mitochondria along the inner periclinal walls, green light exhibited little effect on the distribution of mitochondria. In addition, the mode of movement of individual mitochondria along the outer periclinal walls under different light conditions was precisely analyzed by time-lapse fluorescence microscopy. A gradual increase in the number of static mitochondria located in the vicinity of chloroplasts with a time period of blue light illumination clearly demonstrated the accumulation response of mitochondria. Light-induced co-localization of mitochondria with chloroplasts strongly suggested their mutual metabolic interactions. This is the first characterization of the light-dependent redistribution of mitochondria in plant cells.

  13. Distinct and conserved prominin-1/CD133-positive retinal cell populations identified across species.

    Directory of Open Access Journals (Sweden)

    József Jászai

    Full Text Available Besides being a marker of various somatic stem cells in mammals, prominin-1 (CD133 plays a role in maintaining the photoreceptor integrity since mutations in the PROM1 gene are linked with retinal degeneration. In spite of that, little information is available regarding its distribution in eyes of non-mammalian vertebrates endowed with high regenerative abilities. To address this subject, prominin-1 cognates were isolated from axolotl, zebrafish and chicken, and their retinal compartmentalization was investigated and compared to that of their mammalian orthologue. Interestingly, prominin-1 transcripts--except for the axolotl--were not strictly restricted to the outer nuclear layer (i.e., photoreceptor cells, but they also marked distinct subdivisions of the inner nuclear layer (INL. In zebrafish, where the prominin-1 gene is duplicated (i.e., prominin-1a and prominin-1b, a differential expression was noted for both paralogues within the INL being localized either to its vitreal or scleral subdivision, respectively. Interestingly, expression of prominin-1a within the former domain coincided with Pax-6-positive cells that are known to act as progenitors upon injury-induced retino-neurogenesis. A similar, but minute population of prominin-1-positive cells located at the vitreal side of the INL was also detected in developing and adult mice. In chicken, however, prominin-1-positive cells appeared to be aligned along the scleral side of the INL reminiscent of zebrafish prominin-1b. Taken together our data indicate that in addition to conserved expression of prominin-1 in photoreceptors, significant prominin-1-expressing non-photoreceptor retinal cell populations are present in the vertebrate eye that might represent potential sources of stem/progenitor cells for regenerative therapies.

  14. Nitric oxide is a positive regulator of the Warburg effect in ovarian cancer cells.

    Science.gov (United States)

    Caneba, C A; Yang, L; Baddour, J; Curtis, R; Win, J; Hartig, S; Marini, J; Nagrath, D

    2014-01-01

    Ovarian cancer (OVCA) is among the most lethal gynecological cancers leading to high mortality rates among women. Increasing evidence indicate that cancer cells undergo metabolic transformation during tumorigenesis and growth through nutrients and growth factors available in tumor microenvironment. This altered metabolic rewiring further enhances tumor progression. Recent studies have begun to unravel the role of amino acids in the tumor microenvironment on the proliferation of cancer cells. One critically important, yet often overlooked, component to tumor growth is the metabolic reprogramming of nitric oxide (NO) pathways in cancer cells. Multiple lines of evidence support the link between NO and tumor growth in some cancers, including pancreas, breast and ovarian. However, the multifaceted role of NO in the metabolism of OVCA is unclear and direct demonstration of NO's role in modulating OVCA cells' metabolism is lacking. This study aims at indentifying the mechanistic links between NO and OVCA metabolism. We uncover a role of NO in modulating OVCA metabolism: NO positively regulates the Warburg effect, which postulates increased glycolysis along with reduced mitochondrial activity under aerobic conditions in cancer cells. Through both NO synthesis inhibition (using L-arginine deprivation, arginine is a substrate for NO synthase (NOS), which catalyzes NO synthesis; using L-Name, a NOS inhibitor) and NO donor (using DETA-NONOate) analysis, we show that NO not only positively regulates tumor growth but also inhibits mitochondrial respiration in OVCA cells, shifting these cells towards glycolysis to maintain their ATP production. Additionally, NO led to an increase in TCA cycle flux and glutaminolysis, suggesting that NO decreases ROS levels by increasing NADPH and glutathione levels. Our results place NO as a central player in the metabolism of OVCA cells. Understanding the effects of NO on cancer cell metabolism can lead to the development of NO targeting drugs

  15. Distribution of S-100 positive dendritic cells in bovine pharynx,tonsils, and retropharyngeal lymph nodes

    Institute of Scientific and Technical Information of China (English)

    Jiaxin WANG; Haixia BIAN; Wei SHI; Zhanjun LU

    2008-01-01

    Dendritic cells (DCs) are professional antigen-presenting cells. However, the distribution of bovine DCs in the pharynx, tonsil, and retropharyngeal lymph nodes has not yet been documented. To address this issue, immunohistochemistry was conducted using S-100 pro-tein as a marker for DCs. It was observed that S-100 positive Langerhans cells (LCs) were primarily found in the basal layer of the pharyngeal epithelium. Some DCs were found in the outer layer of the epithelium and their dendrites extended out towards the epithelial surface. In the tonsil, S-100 positive DCs were found either in follicular germinal centers or in the T-cell areas. It is worth noting that the S-100 positive DCs were not only distributed in the cortex, but also in the medulla of bovine retropharyngeal lymph nodes. The distribution patterns of bovine DCs in the pharynx, tonsil, and retropharyngeal lymph nodes have an important implica-tion for our understanding of the interaction between pathogens and host.

  16. Ethical aspects of human embryonic stem cell research in the islamic world: positions and reflections.

    Science.gov (United States)

    Ilkilic, Ilhan; Ertin, Hakan

    2010-06-01

    Rapid technological developments in human embryonic stem cell research are holding promises of future new medical treatment for a range of currently incurable chronic diseases. At the same time, stem cell research using human embryos raises radically new, previously unimaginable ethical issues posing a dramatic challenge to humankind. By analysing the discourses on these ethical issues we can show that the cultural values and religious convictions of all stakeholders involved play a decisive role in formulating ethical positions. In the Islamic world, too, stem cell research using human embryos provokes new discussions about the moral status of the embryo according to Islamic ethical norms. In our paper we describe the theological and philosophical criteria used in this debate and discuss some ethical positions vis-à-vis embryonic stem cell research formulated in the Islamic world, including official regulations existing in some Muslim countries. While most of the existing literature in this field is primarily descriptive, the present paper endeavours to examine not only the arguments and their historical conditions as such; in addition, we will for the first time provide a critical reflection on the methodology underlying commonly held positions. In our view, this reflection is of paramount importance in establishing a straightforward constructive dialogue between different cultures and academic disciplines.

  17. Controlled Positioning of Cells in Biomaterials—Approaches Towards 3D Tissue Printing

    Directory of Open Access Journals (Sweden)

    Sandra Hofmann

    2011-08-01

    Full Text Available Current tissue engineering techniques have various drawbacks: they often incorporate uncontrolled and imprecise scaffold geometries, whereas the current conventional cell seeding techniques result mostly in random cell placement rather than uniform cell distribution. For the successful reconstruction of deficient tissue, new material engineering approaches have to be considered to overcome current limitations. An emerging method to produce complex biological products including cells or extracellular matrices in a controlled manner is a process called bioprinting or biofabrication, which effectively uses principles of rapid prototyping combined with cell-loaded biomaterials, typically hydrogels. 3D tissue printing is an approach to manufacture functional tissue layer-by-layer that could be transplanted in vivo after production. This method is especially advantageous for stem cells since a controlled environment can be created to influence cell growth and differentiation. Using printed tissue for biotechnological and pharmacological needs like in vitro drug-testing may lead to a revolution in the pharmaceutical industry since animal models could be partially replaced by biofabricated tissues mimicking human physiology and pathology. This would not only be a major advancement concerning rising ethical issues but would also have a measureable impact on economical aspects in this industry of today, where animal studies are very labor-intensive and therefore costly. In this review, current controlled material and cell positioning techniques are introduced highlighting approaches towards 3D tissue printing.

  18. Positive association of long telomeres with the invasive capacity of hepatocellular carcinoma cells.

    Science.gov (United States)

    Ko, Eunkyong; Jung, Guhung

    2014-05-01

    Invasion, the representative feature of malignant tumors, leads to an increase in mortality. The malignant liver tumor - hepatocellular carcinoma (HCC) - has an enhanced invasive capacity that results in increased patient mortality. Moreover, this enhanced invasive capacity is due to the up-regulation of invasion promoters such as zinc finger protein SNAI1 (Snail) and matrix metalloproteinases (MMPs), and the down-regulation of invasion suppressor molecules such as E-cadherin. Telomerase reverse transcriptase (TERT), which encodes the catalytic subunit of telomerase, is highly expressed in a variety of invasive cancers, including HCC. Telomerase activation induces telomere elongation, thereby leading to cell immortalization during malignant tumor progression. However, the relationship between telomere length and invasion is yet to be experimentally corroborated. In this paper, we revealed that invasive HCC cells passing through the Matrigel display significantly longer telomeres than non-invasive HCC cells. Moreover, we established a method that can distinguish and sort cells containing long telomeres and short telomeres. Using this system, we observed that the HCC cells containing long telomeres had a high-level expression of invasion-promoting genes and a low-level expression of invasion-suppressing E-cadherin. Furthermore, HCC cells containing long telomeres exhibited a higher invasive capacity than HCC cells containing short telomeres. Taken together, our findings suggest that long telomeres are positively associated with the invasive capacity of HCC cells and may be a potent target for malignant liver cancer treatment. PMID:24732358

  19. Cell position during larval development affects postdiapause development in Megachile rotundata (Hymenoptera: Megachilidae).

    Science.gov (United States)

    Yocum, George D; Rinehart, Joseph P; Kemp, William P

    2014-08-01

    Megachile rotundata (F.) (Hymenoptera: Megachilidae) is the primary pollinator of alfalfa in the northwestern United States and western Canada and provides pollination services for onion, carrot, hybrid canola, various legumes, and other specialty crops. M. rotundata females are gregarious, nest in cavities either naturally occurring or in artificial nesting blocks, where they construct a linear series of brood cells. Because of the physical layout of the nest, the age of the larvae within the nest and the microenvironment the individual larvae experience will vary. These interacting factors along with other maternal inputs affect the resulting phenotypes of the nest mates. To further our understanding of in-nest physiology, gender and developmental rates were examined in relationship to cell position within the nest. Eighty-two percent of the females were located within the first three cells, those furthest from the nest entrance. For those individuals developing in cells located in the deepest half of the nest, the sex of the previous bee had a significant effect on the female decision of the gender of the following nest mate. Removing the prepupae from the nest and rearing them under identical conditions demonstrated that position within the nest during larval development had a significant effect on the postdiapause developmental rates, with males whose larval development occurred deeper in the nest developing more slowly than those toward the entrance. No positional effect on postdiapause developmental rates was noted for the females. The cell position effect on male postdiapause developmental rate demonstrates that postdiapause development is not a rigid physiological mechanism uniform in all individuals, but is a dynamic plastic process shaped by past environmental conditions. PMID:24914676

  20. Indium-111-oxine labeled leukocyte uptake in Ki-1-positive anaplastic large cell lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Chiu, W.; Amodio, J.B.; Scharf, S.C. (New York Univ., NY (United States). Dept. of Radiology); Rivlin, K.A. (Department of Hematology and Oncology, New York Univ. Medical Center, NY (United States)); Desai, P. (Department of Pathology, Hospital for Joint Diseases-Orthopaedic Inst., New York, NY (United States)); Breuer, F. (Department of Pathology, Lenox Hill Hospital, New York, NY (United States))

    1999-08-01

    Indium-111-oxine labeled leukocyte ([sup 111]In-WBC) scintigraphy is well known for its ability to localize in areas of active infection, but not in areas of lymphomatous involvement. We present a case of Ki-1-positive anaplastic large-cell lymphoma that was initially thought to be a case of multifocal osteomyelitis because of positive uptake on a [sup 111]In-WBC scan. The areas of abnormal uptake on the indium scan were demonstrated histopathologically to be sites of lymphomatous involvement in bone. (orig.) With 4 figs., 3 refs.

  1. Monocyte cell surface glycosaminoglycans positively modulate IL-4-induced differentiation toward dendritic cells.

    NARCIS (Netherlands)

    Dekker, E. den; Grefte, S.; Huijs, T.; Dam, G.B. ten; Versteeg, E.M.M.; Berk, L.C.J. van den; Bladergroen, B.A.; Kuppevelt, A.H.M.S.M. van; Figdor, C.G.; Torensma, R.

    2008-01-01

    IL-4 induces the differentiation of monocytes toward dendritic cells (DCs). The activity of many cytokines is modulated by glycosaminoglycans (GAGs). In this study, we explored the effect of GAGs on the IL-4-induced differentiation of monocytes toward DCs. IL-4 dose-dependently up-regulated the expr

  2. Leukemia Mediated Endothelial Cell Activation Modulates Leukemia Cell Susceptibility to Chemotherapy through a Positive Feedback Loop Mechanism.

    Directory of Open Access Journals (Sweden)

    Bahareh Pezeshkian

    Full Text Available In acute myeloid leukemia (AML, the chances of achieving disease-free survival are low. Studies have demonstrated a supportive role of endothelial cells (ECs in normal hematopoiesis. Here we show that similar intercellular relationships exist in leukemia. We demonstrate that leukemia cells themselves initiate these interactions by directly modulating the behavior of resting ECs through the induction of EC activation. In this inflammatory state, activated ECs induce the adhesion of a sub-set of leukemia cells through the cell adhesion molecule E-selectin. These adherent leukemia cells are sequestered in a quiescent state and are unaffected by chemotherapy. The ability of adherent cells to later detach and again become proliferative following exposure to chemotherapy suggests a role of this process in relapse. Interestingly, differing leukemia subtypes modulate this process to varying degrees, which may explain the varied response of AML patients to chemotherapy and relapse rates. Finally, because leukemia cells themselves induce EC activation, we postulate a positive-feedback loop in leukemia that exists to support the growth and relapse of the disease. Together, the data defines a new mechanism describing how ECs and leukemia cells interact during leukemogenesis, which could be used to develop novel treatments for those with AML.

  3. Spatial distribution of prominin-1 (CD133-positive cells within germinative zones of the vertebrate brain.

    Directory of Open Access Journals (Sweden)

    József Jászai

    Full Text Available BACKGROUND: In mammals, embryonic neural progenitors as well as adult neural stem cells can be prospectively isolated based on the cell surface expression of prominin-1 (CD133, a plasma membrane glycoprotein. In contrast, characterization of neural progenitors in non-mammalian vertebrates endowed with significant constitutive neurogenesis and inherent self-repair ability is hampered by the lack of suitable cell surface markers. Here, we have investigated whether prominin-1-orthologues of the major non-mammalian vertebrate model organisms show any degree of conservation as for their association with neurogenic geminative zones within the central nervous system (CNS as they do in mammals or associated with activated neural progenitors during provoked neurogenesis in the regenerating CNS. METHODS: We have recently identified prominin-1 orthologues from zebrafish, axolotl and chicken. The spatial distribution of prominin-1-positive cells--in comparison to those of mice--was mapped in the intact brain in these organisms by non-radioactive in situ hybridization combined with detection of proliferating neural progenitors, marked either by proliferating cell nuclear antigen or 5-bromo-deoxyuridine. Furthermore, distribution of prominin-1 transcripts was investigated in the regenerating spinal cord of injured axolotl. RESULTS: Remarkably, a conserved association of prominin-1 with germinative zones of the CNS was uncovered as manifested in a significant co-localization with cell proliferation markers during normal constitutive neurogenesis in all species investigated. Moreover, an enhanced expression of prominin-1 became evident associated with provoked, compensatory neurogenesis during the epimorphic regeneration of the axolotl spinal cord. Interestingly, significant prominin-1-expressing cell populations were also detected at distinct extraventricular (parenchymal locations in the CNS of all vertebrate species being suggestive of further, non

  4. Cytotoxicity and apoptosis induced by a plumbagin derivative in estrogen positive MCF-7 breast cancer cells

    KAUST Repository

    Sagar, Sunil

    2014-01-31

    Plumbagin [5-hydroxy- 2-methyl-1, 4-naphthaquinone] is a well-known plant derived anticancer lead compound. Several efforts have been made to synthesize its analogs and derivatives in order to increase its anticancer potential. In the present study, plumbagin and its five derivatives have been evaluated for their antiproliferative potential in one normal and four human cancer cell lines. Treatment with derivatives resulted in dose- and time-dependent inhibition of growth of various cancer cell lines. Prescreening of compounds led us to focus our further investigations on acetyl plumbagin, which showed remarkably low toxicity towards normal BJ cells and HepG2 cells. The mechanisms of apoptosis induction were determined by APOPercentage staining, caspase-3/7 activation, reactive oxygen species production and cell cycle analysis. The modulation of apoptotic genes (p53, Mdm2, NF-kB, Bad, Bax, Bcl-2 and Casp-7) was also measured using real time PCR. The positive staining using APOPercentage dye, increased caspase-3/7 activity, increased ROS production and enhanced mRNA expression of proapoptotic genes suggested that acetyl plumbagin exhibits anticancer effects on MCF-7 cells through its apoptosis-inducing property. A key highlighting point of the study is low toxicity of acetyl plumbagin towards normal BJ cells and negligible hepatotoxicity (data based on HepG2 cell line). Overall results showed that acetyl plumbagin with reduced toxicity might have the potential to be a new lead molecule for testing against estrogen positive breast cancer. 2014 Bentham Science Publishers.

  5. The distribution and number of Leu-7 (CD57) positive cells in lung tissue from patients with pulmonary fibrosis.

    OpenAIRE

    Yamanouchi H; Ohtsuki Y; Fujita J; Bandoh S; Yoshinouchi T; Ishida T

    2002-01-01

    Leu-7 positive lymphocytes, including natural killer cells, play an important role in the immune system's surveillance function to prevent the development of cancer. The incidence of lung cancer is significantly high in patients with end-stage pulmonary fibrosis. We hypothesized that the number of Leu-7 positive cells may be decreased in areas of severe pulmonary fibrosis. To demonstrate this, Leu-7 positive cells were immunohistochemically stained in 41 lung specimens obtained from patients ...

  6. Activated WNT signaling in postnatal SOX2-positive dental stem cells can drive odontoma formation.

    Science.gov (United States)

    Xavier, Guilherme M; Patist, Amanda L; Healy, Chris; Pagrut, Ankita; Carreno, Gabriela; Sharpe, Paul T; Martinez-Barbera, Juan Pedro; Thavaraj, Selvam; Cobourne, Martyn T; Andoniadou, Cynthia L

    2015-09-28

    In common with most mammals, humans form only two dentitions during their lifetime. Occasionally, supernumerary teeth develop in addition to the normal complement. Odontoma represent a small group of malformations containing calcified dental tissues of both epithelial and mesenchymal origin, with varying levels of organization, including tooth-like structures. The specific cell type responsible for the induction of odontoma, which retains the capacity to re-initiate de novo tooth development in postnatal tissues, is not known. Here we demonstrate that aberrant activation of WNT signaling by expression of a non-degradable form of β-catenin specifically in SOX2-positive postnatal dental epithelial stem cells is sufficient to generate odontoma containing multiple tooth-like structures complete with all dental tissue layers. Genetic lineage-tracing confirms that odontoma form in a similar manner to normal teeth, derived from both the mutation-sustaining epithelial stem cells and adjacent mesenchymal tissues. Activation of the WNT pathway in embryonic SOX2-positive progenitors results in ectopic expression of secreted signals that promote odontogenesis throughout the oral cavity. Significantly, the inductive potential of epithelial dental stem cells is retained in postnatal tissues, and up-regulation of WNT signaling specifically in these cells is sufficient to promote generation and growth of ectopic malformations faithfully resembling human odontoma.

  7. Activated WNT signaling in postnatal SOX2-positive dental stem cells can drive odontoma formation.

    Science.gov (United States)

    Xavier, Guilherme M; Patist, Amanda L; Healy, Chris; Pagrut, Ankita; Carreno, Gabriela; Sharpe, Paul T; Martinez-Barbera, Juan Pedro; Thavaraj, Selvam; Cobourne, Martyn T; Andoniadou, Cynthia L

    2015-01-01

    In common with most mammals, humans form only two dentitions during their lifetime. Occasionally, supernumerary teeth develop in addition to the normal complement. Odontoma represent a small group of malformations containing calcified dental tissues of both epithelial and mesenchymal origin, with varying levels of organization, including tooth-like structures. The specific cell type responsible for the induction of odontoma, which retains the capacity to re-initiate de novo tooth development in postnatal tissues, is not known. Here we demonstrate that aberrant activation of WNT signaling by expression of a non-degradable form of β-catenin specifically in SOX2-positive postnatal dental epithelial stem cells is sufficient to generate odontoma containing multiple tooth-like structures complete with all dental tissue layers. Genetic lineage-tracing confirms that odontoma form in a similar manner to normal teeth, derived from both the mutation-sustaining epithelial stem cells and adjacent mesenchymal tissues. Activation of the WNT pathway in embryonic SOX2-positive progenitors results in ectopic expression of secreted signals that promote odontogenesis throughout the oral cavity. Significantly, the inductive potential of epithelial dental stem cells is retained in postnatal tissues, and up-regulation of WNT signaling specifically in these cells is sufficient to promote generation and growth of ectopic malformations faithfully resembling human odontoma. PMID:26411543

  8. Improved accuracy of cell surface shaving proteomics in Staphylococcus aureus using a false-positive control

    DEFF Research Database (Denmark)

    Solis, Nestor; Larsen, Martin Røssel; Cordwell, Stuart J

    2010-01-01

    with either trypsin or proteinase-K combined with LC-MS/MS. Trypsin-derived data were controlled using a "false-positive" strategy where cells were incubated without protease, removed by centrifugation and the resulting supernatants digested. Peptides identified in this fraction most likely result from cell...... lysis and were removed from the trypsin-shaved data set. We identified 42 predicted S. aureus COL surface proteins from 260 surface-exposed peptides. Trypsin and proteinase-K digests were highly complementary with ten proteins identified by both, 16 specific to proteinase-K treatment, 13 specific...

  9. Cytokeratin-positive cells in preoperative peripheral blood and bone marrow aspirates of patients with colorectal cancer

    DEFF Research Database (Denmark)

    Werther, K; Normark, M; Brünner, N;

    2002-01-01

    Detection of cytokeratin-positive cells in bone marrow and peripheral blood may have prognostic significance in cancer patients. Furthermore, a correlation between uPAR expression on micrometastases and patient prognosis has been suggested. However, in patients with colorectal cancer, preoperative...... cancer, were immunocytochemically screened for cytokeratin-positive cells. Where cytokeratin-positive cells were observed, an additional microslide was double immunostained for simultaneous detection of cytokeratin and uPAR/CD87. RESULTS: Cytokeratin-positive cells were observed in 4 out of 41 bone...... preoperatively obtained bone marrow aspirates or peripheral blood from patients with colorectal cancer....

  10. Clinical utility of Circulating Tumor Cells in ALK-Positive Non-Small-Cell Lung Cancer

    OpenAIRE

    Vincent eFaugeroux; Emma ePailler; Nathalie eAuger; Meilissa eTaylor; Françoise eFarace

    2014-01-01

    The advent of rationally targeted therapies such as small-molecule tyrosine kinase inhibitors (TKIs) has considerably transformed the therapeutic management of a subset of patients with non-small cell lung cancer (NSCLC) harboring defined molecular abnormalities. When such genetic molecular alterations are detected the use of specific TKI has demonstrated better results (overall response rate (ORR), progression free survival (PFS)) compared to systemic therapy. However the detection of such m...

  11. Clinical Utility of Circulating Tumor Cells in ALK-Positive Non-Small-Cell Lung Cancer

    OpenAIRE

    Faugeroux, Vincent; Pailler, Emma; Auger, Nathalie; Taylor, Melissa; Farace, Françoise

    2014-01-01

    The advent of rationally targeted therapies such as small-molecule tyrosine kinase inhibitors (TKIs) has considerably transformed the therapeutic management of a subset of patients with non-small-cell lung cancer (NSCLC) harboring defined molecular abnormalities. When such genetic molecular alterations are detected the use of specific TKI has demonstrated better results (overall response rate, progression free survival) compared to systemic therapy. However, the detection of such molecular ab...

  12. Autoimmune pancreatitis metachronously associated with retroperitoneal fibrosis with IgG4-positive plasma cell infiltration

    Institute of Scientific and Technical Information of China (English)

    Terumi Kamisawa; Pong Yui Chen; Yuyang Tu; Hitoshi Nakajima; Naoto Egawa

    2006-01-01

    Retroperitoneal fibrosis is an uncommon disorder characterized by the formation of a dense plaque of fibrous tissue in the retroperitoneum, and its etiology remains unknown. Autoimmune pancreatitis is a rare type of chronic pancreatitis characterized by fibrosis with abundant infiltration of IgG4-positive plasma cells and lymphocytes and obliterative phlebitis in the pancreas. We present a case of autoimmune pancreatitis that developed 10 mo after the occurrence of retroperitoneal fibrosis. Histological findings of the resected retroperitoneal mass were marked periureteral fibrosis with abundant infiltration of IgG4-positive plasma cells and lymphocytes and obliterative phlebitis.These findings suggest a common pathophysiological mechanism for retroperitoneal fibrosis and autoimmune pancreatitis in this case. Some cases of retroperitoneal fibrosis might be a retroperitoneal lesion of IgG4-related sclerosing disease.

  13. Oral lichen sclerosus expressing extracellular matrix proteins and IgG4-positive plasma cells.

    Science.gov (United States)

    De Aquino Xavier, Flavia Calo; Prates, Alisio Alves; Gurgel, Clarissa Araujo; De Souza, Tulio Geraldo; Andrade, Rodrigo Guimaraes; Goncalves Ramos, Eduardo Antonio; Pedreira Ramalho, Luciana Maria; Dos Santos, Jean Nunes

    2014-09-16

    Lichen sclerosus (LS) is a mucocutaneous disease with uncommon oral involvement. The etiology is not yet well understood, but LS has been associated with autoimmune, genetic, and immunological factors. We report a 47-year-old man with LS that exhibited an asymptomatic white plaque with red patches on the maxillary alveolar mucosa extending to the labial mucosa. He had no other skin disease. Positive immunostaining for tenascin and scarcity of fibronectin suggested extracellular matrix reorganization. Elastin immunostaining indicated a reduction of elastic fibers. Immunoexpression of collagen IV in blood vessels and its absence in the epithelial basement membrane, together with diffuse MMP-9 immunoexpression, suggested altered proteolytic activity. Mast cell staining bordering areas of sclerosis indicated a possible role in the synthesis of collagen. IgG4 positivity in plasma cells suggested a role in the fibrogenesis. This is an unusual presentation of oral LS and we discuss immunohistochemical findings regarding cellular and extracellular matrix components.

  14. Cell cultivation under different gravitational loads using a novel random positioning incubator.

    Science.gov (United States)

    Benavides Damm, Tatiana; Walther, Isabelle; Wüest, Simon L; Sekler, Jörg; Egli, Marcel

    2014-06-01

    Important in biotechnology is the establishment of cell culture methods that reflect the in vivo situation accurately. One approach for reaching this goal is through 3D cell cultivation that mimics tissue or organ structures and functions. We present here a newly designed and constructed random positioning incubator (RPI) that enables 3D cell culture in simulated microgravity (0 g). In addition to growing cells in a weightlessness-like environment, our RPI enables long-duration cell cultivation under various gravitational loads, ranging from close to 0 g to almost 1 g. This allows the study of the mechanotransductional process of cells involved in the conversion of physical forces to an appropriate biochemical response. Gravity is a type of physical force with profound developmental implications in cellular systems as it modulates the resulting signaling cascades as a consequence of mechanical loading. The experiments presented here were conducted on mouse skeletal myoblasts and human lymphocytes, two types of cells that have been shown in the past to be particularly sensitive to changes in gravity. Our novel RPI will expand the horizon at which mechanobiological experiments are conducted. The scientific data gathered may not only improve the sustainment of human life in space, but also lead to the design of alternative countermeasures against diseases related to impaired mechanosensation and downstream signaling processes on earth.

  15. Refractory gastric ulcer with abundant IgG4-positive plasma cell infiltration: A case report

    Institute of Scientific and Technical Information of China (English)

    Takayoshi; Fujita; Takafumi; Ando; Masatoshi; Sakakibara; Waki; Hosoda; Hidemi; Goto

    2010-01-01

    We describe a 77-year-old man with refractory gastric ulcer that worsened after Helicobacter pylori eradication therapy.Pathology showed marked infiltration of IgG4-positive plasma cells in the gastric lesions,which led us to suspect IgG4-related sclerosing disease.To the best of our knowledge,this is the first report of IgG4-related gastric ulcer without the main manifestation of autoimmune pancreatitis.

  16. Enriched environment induces higher CNPase positive cells in aged rat hippocampus.

    Science.gov (United States)

    Zhao, Yuan-Yu; Shi, Xiao-Yan; Zhang, Lei; Wu, Hong; Chao, Feng-Lei; Huang, Chun-Xia; Gao, Yuan; Qiu, Xuan; Chen, Lin; Lu, Wei; Tang, Yong

    2013-10-25

    It had been reported that enriched environment was beneficial for the brain cognition and for the neurons and synapses in hippocampus. Previous study reported that the oligodendrocyte density in hippocampus was increased when the rats were reared in the enriched environment from weaning to adulthood. However, biological conclusions based on density were difficult to interpret because the changes in density could be due to an alteration of total quantity and/or an alteration in the reference volume. In the present study, we used unbiased stereological methods to investigate the effect of enriched environment on the total number of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) positive cells in CA1 and dentate gyrus (DG) of the hippocampus in aged rats. Our results indicated that there was significant difference in the total numbers of CNPase positive cells in both CA1 and DG between enriched environment group and standard environment group. The present study provided the first evidence for the protective effects of enriched environment on the CNPase positive cells in aged hippocampus.

  17. Rare Association of Anti-Hu Antibody Positive Paraneoplastic Neurological Syndrome and Transitional Cell Bladder Carcinoma

    Directory of Open Access Journals (Sweden)

    S. Lukacs

    2012-01-01

    Full Text Available Introduction. Paraneoplastic encephalomyelitis (PEM and subacute sensory neuronopathy (SSN are remote effects of cancer, usually associated with small-cell lung carcinoma and positive anti-Hu antibody. We describe the rare association of bladder transitional cell carcinoma (TCC with anti-Hu antibody positivity resulting in this paraneoplastic neurological syndrome. Patient. A 76-year-old female presented with bilateral muscle weakness and paraesthesia of the upper and lower limbs in a length-dependent “glove and stocking” distribution. Central nervous system symptoms included cognitive problems, personality change, and truncal ataxia. Case notes and the literature were reviewed. Result. Autoantibody screening was positive for anti-Hu antibody (recently renamed antineuronal nuclear antibody 1, ANNA-1. The diagnosis of PEM and SSN was supported by MRI and lumbar puncture results. A superficial bladder TCC was demonstrated on CT and subsequently confirmed on histology. No other primary neoplasm was found on full-body imaging. The neurological symptoms were considered to be an antibody-mediated paraneoplastic neurological syndrome and improved after resection of the tumour. Discussion. The association of anti-Hu positive paraneoplastic neurological syndrome and TCC has not been described in the literature previously. We emphasize the need for detailed clinical examination and the importance of a multidisciplinary thought process and encourage further awareness of this rare association.

  18. Effects of soft X-ray irradiation on NK cell activity and the percentage of asialo GM1-positive cells in spleen cells of mice

    International Nuclear Information System (INIS)

    Effects of soft X-ray irradiation on the natural killer (NK) cell activity and the percentage of asialo GM1-positive cells of spleen cells in mice were investigated by using a soft X-ray generator intended for non-destructive radiological examination. Soft X-ray irradiation in graded doses of more than 25.8 mC/kg indicated dose dependent reductions in the NK cell activity in the spleen of irradiated mice. Significant reductions in the population of asialo GM1-positive cells in spleen cells were also observed. These results suggest that a soft X-ray generator could also be useful in immuno-irradiation studies. (author)

  19. Galpha/LGN-mediated asymmetric spindle positioning does not lead to unequal cleavage of the mother cell in 3-D cultured MDCK cells

    OpenAIRE

    Xiao, Zhuoni; Wan, Qingwen; Du, Quansheng; Zheng, Zhen

    2012-01-01

    The position of the mitotic spindle plays a key role in spatial control of cell division. It is generally believed that when a spindle is positioned asymmetrically in a dividing cell, the resulting daughter cells are usually unequal in size due to eccentric cleavage of the mother cell. Molecular mechanisms underlying the generation of unequal sized daughter cells have been extensively studied in Drosophila neuroblast and C elegans zygote where the Gα subunit of the heterotrimeric G proteins a...

  20. TCR affinity for thymoproteasome-dependent positively selecting peptides conditions antigen responsiveness in CD8(+) T cells.

    Science.gov (United States)

    Takada, Kensuke; Van Laethem, Francois; Xing, Yan; Akane, Kazuyuki; Suzuki, Haruhiko; Murata, Shigeo; Tanaka, Keiji; Jameson, Stephen C; Singer, Alfred; Takahama, Yousuke

    2015-10-01

    In the thymus, low-affinity T cell antigen receptor (TCR) engagement facilitates positive selection of a useful T cell repertoire. Here we report that TCR responsiveness of mature CD8(+) T cells is fine tuned by their affinity for positively selecting peptides in the thymus and that optimal TCR responsiveness requires positive selection on major histocompatibility complex class I-associated peptides produced by the thymoproteasome, which is specifically expressed in the thymic cortical epithelium. Thymoproteasome-independent positive selection of monoclonal CD8(+) T cells results in aberrant TCR responsiveness, homeostatic maintenance and immune responses to infection. These results demonstrate a novel aspect of positive selection, in which TCR affinity for positively selecting peptides produced by thymic epithelium determines the subsequent antigen responsiveness of mature CD8(+) T cells in the periphery.

  1. Hypoxia inducible factor-1 alpha expression is increased in infected positive HPV16 DNA oral squamous cell carcinoma and positively associated with HPV16 E7 oncoprotein

    Directory of Open Access Journals (Sweden)

    Di Fede Olga

    2011-10-01

    Full Text Available Abstract Background There is increasing evidence for the role of High Risk (HR Human PapillomaVirus (HPV in the pathogenesis of Oral Squamous Cell Carcinoma (OSCC. The E6 and E7 oncogenes from HR HPVs are responsible for the deregulation of p53 and pRB proteins involved in cell cycle and apoptotic pathways. In cell lines experiments, the HPV E7 protein seems to be able to enhance Hypoxia Inducible Factor-1 alpha (HIF-1α activity, normally involved in the response to hypoxia and able to enhance angiogenesis. Results We studied tumor specimens from 62 OSCC; a higher prevalence of tumors in TNM stage II and also in pT2 class between OSCC infected positive HPV16 DNA than non-infected ones was observed. HIF-1α positivity was detected throughout the analysed fields, not associated with areas of necrosis and also observed in cells immediately adjacent to blood vessels. A significant increase in mean values of the HIF-1α labeling indexes was observed for pT1-T2, as well for stage I-II, in the infected positive HPV16 DNA tumors than non-infected ones. HIF-1α and HPV16 E7 labeling indexes showed a significantly positive correlation which suggested a positive association between HPV16 E7 and HIF-1α expression. Conclusions In our specimens HIF-1α immunoreactivity hints for an O2-independent regulatory mechanism in infected positive HPV16 DNA tumors, especially for pT1-T2 and stage I-II tumors, suggesting a very early involvement in the development of HPV-induced OSCC. HIF-1α and HPV16 E7 labeling indexes suggest also a positive association between the two proteins in infected positive HPV16 DNA OSCC.

  2. Graft-versus-leukemia effects of Wilms' tumor 1 protein-specific cytotoxic T lymphocytes in patients with chronic myeloid leukemia after allogeneic hematopoietic stem cell transplantation

    Institute of Scientific and Technical Information of China (English)

    WANG Zhi-dong; LI Dan; HUANG Xiao-jun

    2010-01-01

    Background The role of Wilms' tumor 1 protein (WT1)-specific cytotoxic T cells (CTL) in eradicating chronic myeloid leukemia (CML) cells is to be established. The aim of this study was to determine whether WT1 contributed to the graft-versus-leukemia effects (GVLE) for CML following allogeneic hematopoietic stem cell transplantation (HSCT). Methods High-resolution human leukocyte antigen (HLA) class I genotyping was performed by sequence-specific polymerase chain reaction (PCR). Fifteen HLA-A~*2402 patients with CML who underwent allogeneic HSCT were enrolled in this study. We monitored the frequency of WT1-specific CTL by pentamer assay and the molecular minimal residual disease by real-time quantitative PCR.Results A CD8~+ T-cell response to WT1 was observed in 14 of 15 patients after HSCT. The median frequencies of WT1-CTL were 0.54%, 0.62%, 0.81% and 1.28% (%CD8) on days 30, 60, 90 and 180, respectively. The median frequency of WT1-CTL (1.38%) in patients with molecular remission (MoR) was significantly higher than that in those without MoR (0.38%) on day 30, while no significant differences between them were detected on days 60, 90 and 180. The increase of WT1-CTL was associated with a decrease in bcr-abl expression and MoR; and the decrease of WT1-CTL was associated with an increase in bcr-abl expression, suggesting a WT1 -driven GVL effect. WT1-CTL had a predominant effector-memory phenotype (CD45RO~+CD27~-CD57~+).Conclusions The emergence of WT1-CTL with an effector-memory phenotype is associated with GVLE in CML patients after HSCT. This will pave the way for the WT1 vaccines to enhance GVLE after HSCT in CML.

  3. STEREOLOGIC ESTIMATION OF KI-67, CASPASE 3 AND GSTP1 POSITIVE CELLS IN PROSTATE LESIONS

    Directory of Open Access Journals (Sweden)

    Luis Santamaría

    2011-05-01

    Full Text Available Cell proliferation, caspase 3 and pi-form of glutathione S transferase (GSTP1 were evaluated in prostate carcinoma (PCA, proliferative inflammatory atrophy (PIA and prostate intraepithelial neoplasia (PIN. Forty biopsies were classified as: without morphological lesions (controls: CTR, PIA, PIN and PCA. Ki67, caspase3 and GSTP1 were immunostained. The following estimates were performed: Numerical densities of Ki67+ cells (NVEPKi67, of all epithelial cells (NVEPtotal and of GSTP1+ cells (NVEPGSTP1; labelling index for Ki67 (LIKi67; volume fraction to caspase 3 positive tissue (VVcaspase 3 and of GSTP1 positive tissue (VVGSTP1. ANOVA was performed to compare the groups. NVEPtotal and NVEPKi67 were increased in PIA. LIKi67 was only increased in PCA. VVcaspase 3 was decreased in PIN and PCA. VVEGSTP1 was decreased in PCA. In our results PIA lacks the characteristics of a premalignant lesion. The result may be explained by the use of unbiased quantitative methods, the inadequate definition of PIA and the scarce inflammation observed in the samples with PIA included in this study.

  4. Quality Indicator Development for Positive Screen Follow-up for Sickle Cell Disease and Trait.

    Science.gov (United States)

    Faro, Elissa Z; Wang, C Jason; Oyeku, Suzette O

    2016-07-01

    Extensive variation exists in the follow-up of positive screens for sickle cell disease. Limited quality indicators exist to measure if the public health goals of screening-early initiation of treatment and enrollment to care-are being achieved. This manuscript focuses on the development of quality indicators related to the follow-up care for individuals identified with sickle cell disease and trait through screening processes. The authors used a modified Delphi method to develop the indicators. The process included a comprehensive literature review with rating of the evidence followed by ratings of draft indicators by an expert panel held in September 2012. The expert panel was nominated by leaders of various professional societies, the Health Resources and Services Administration, and the National Heart, Lung, and Blood Institute and met face to face to discuss and rate each indicator. The panel recommended nine quality indicators focused on key aspects of follow-up care for individuals with positive screens for sickle cell disease and trait. Public health programs and healthcare institutions can use these indicators to assess the quality of follow-up care and provide a basis for improvement efforts to ensure appropriate family education, early initiation of treatment, and appropriate referral to care for individuals identified with sickle cell disease and trait. PMID:27320465

  5. TALE activators regulate gene expression in a position- and strand-dependent manner in mammalian cells.

    Science.gov (United States)

    Uhde-Stone, Claudia; Cheung, Edna; Lu, Biao

    2014-01-24

    Transcription activator-like effectors (TALEs) are a class of transcription factors that are readily programmable to regulate gene expression. Despite their growing popularity, little is known about binding site parameters that influence TALE-mediated gene activation in mammalian cells. We demonstrate that TALE activators modulate gene expression in mammalian cells in a position- and strand-dependent manner. To study the effects of binding site location, we engineered TALEs customized to recognize specific DNA sequences located in either the promoter or the transcribed region of reporter genes. We found that TALE activators robustly activated reporter genes when their binding sites were located within the promoter region. In contrast, TALE activators inhibited the expression of reporter genes when their binding sites were located on the sense strand of the transcribed region. Notably, this repression was independent of the effector domain utilized, suggesting a simple blockage mechanism. We conclude that TALE activators in mammalian cells regulate genes in a position- and strand-dependent manner that is substantially different from gene activation by native TALEs in plants. These findings have implications for optimizing the design of custom TALEs for genetic manipulation in mammalian cells.

  6. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells

    Science.gov (United States)

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-06-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ~95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes.

  7. Genistein inhibits the proliferation of human HER2-positive cancer cells by downregulating HER2 receptor

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    Guodong Shen

    2013-07-01

    Full Text Available Functional Foods in Health and Disease 2013; 3(7:291-299Research Article Open AccessGenistein inhibits the proliferation of human HER2-positive cancer cells by downregulating HER2 receptorGuodong Shen, Haiying Yu, Geng Bian, Min Gao, Lingqing Liu, Min Cheng, Gan Shen, Shilian HuGeriatrics Department, Gerontology Institute, Anhui Provincial Hospital; Anhui Provincial Key Laboratory of Tumor Immunotherapy and Nutrition Therapy, Hefei 230001, ChinaCorresponding Author: Shilian Hu, Department of Geriatrics, Anhui Provincial Hospital, No. 17 Lujiang Road, Hefei 230001, China Submission date: June 9, 2013; Acceptance date: July 19, 2013; Publication date: July 20, 2013ABSTRACTBackground: It was well studied that HER2/ErbB2/p185 overexpression in human malignant cancers correlates with poor prognosis and chemo-resistance. Meanwhile, Genistein (4,5,7-trihydroxyisoflavone, a major isoflavone component of soybeans and other leguminous plants, has been shown to exhibit a potent anti-proliferative effect on some sex hormone dependent cancers. Objective: The effects of genistein on the proliferation of human HER2-overexpressing breast and ovarian cancer cell lines were investigated, and the action mechanism was explored.Methods: Western blotting, fluorescence-activated cell sorting (FACS and immunofluorescence methods, cell proliferation assay kit from Promega and cell apoptosis assay kit from Biolegend were used. The dose- or time-response relationship of genistein were observed on the HER2-negative breast cancer cell line MCF-7 or HER2-positive breast cancer cell lines BT-474 and MCF-7/Her2 derived from MCF-7, and ovarian cancer cell line SKOV-3.Results: The addition of genistein ranged from 1-10g/ml in the medium for 48 hours had a marked inhibition on the proliferation of HER2-positive cancer cell lines MCF-7/Her2, BT-474 and SKOV-3, compared with tamoxifen and DMSO control (P<0.01, and a dose-dependent response was presented. However, genistein

  8. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells

    Science.gov (United States)

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-01-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes. PMID:27251117

  9. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells.

    Science.gov (United States)

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-01-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes. PMID:27251117

  10. Notch and TGFβ form a positive regulatory loop and regulate EMT in epithelial ovarian cancer cells.

    Science.gov (United States)

    Zhou, Jiesi; Jain, Saket; Azad, Abul K; Xu, Xia; Yu, Hai Chuan; Xu, Zhihua; Godbout, Roseline; Fu, YangXin

    2016-08-01

    Epithelial-mesenchymal transition (EMT) plays a critical role in the progression of epithelial ovarian cancer (EOC). However, the mechanisms that regulate EMT in EOC are not fully understood. Here, we report that activation of Notch1 induces EMT in EOC cells as evidenced by downregulation of E-cadherin and cytokeratins, upregulation of Slug and Snail, as well as morphological changes. Interestingly, activation of Notch1 increases TGFβ/Smad signaling by upregulating the expression of TGFβ and TGFβ type 1 receptor. Time course experiments demonstrate that inhibition of Notch by DAPT (a γ-secretase inhibitor) decreases TGFβ-induced phosphorylation of receptor Smads at late, but not at early, timepoints. These results suggest that Notch activation plays a role in sustaining TGFβ/Smad signaling in EOC cells. Furthermore, inhibition of Notch by DAPT decreases TGFβ induction of Slug and repression of E-cadherin and knockdown of Notch1 decreases TGFβ-induced repression of E-cadherin, indicating that Notch is required, at least in part, for TGFβ-induced EMT in EOC cells. On the other hand, TGFβ treatment increases the expression of Notch ligand Jagged1 and Notch target gene HES1 in EOC cells. Functionally, the combination of Notch1 activation and TGFβ treatment is more potent in promoting motility and migration of EOC cells than either stimulation alone. Taken together, our results indicate that Notch and TGFβ form a reciprocal positive regulatory loop and cooperatively regulate EMT and promote EOC cell motility and migration. PMID:27075926

  11. Effect of Weight Reduction on Cardiovascular Risk Factors and CD34-positive Cells in Circulation

    Directory of Open Access Journals (Sweden)

    Nina A Mikirova, Joseph J Casciari, Ronald E Hunninghake, Margaret M Beezley

    2011-01-01

    Full Text Available Being overweight or obese is associated with an increased risk for the development of non-insulin-dependent diabetes mellitus, hypertension, and cardiovascular disease. Dyslipidemia of obesity is characterized by elevated fasting triglycerides and decreased high-density lipoprotein-cholesterol concentrations. Endothelial damage and dysfunction is considered to be a major underlying mechanism for the elevated cardiovascular risk associated with increased adiposity. Alterations in endothelial cells and stem/endothelial progenitor cell function associated with overweight and obesity predispose to atherosclerosis and thrombosis.In our study, we analyzed the effect of a low calorie diet in combination with oral supplementation by vitamins, minerals, probiotics and human chorionic gonadotropin (hCG, 125-180 IUs on the body composition, lipid profile and CD34-positive cells in circulation.During this dieting program, the following parameters were assessed weekly for all participants: fat free mass, body fat, BMI, extracellular/intracellular water, total body water and basal metabolic rate. For part of participants blood chemistry parameters and circulating CD34-positive cells were determined before and after dieting.The data indicated that the treatments not only reduced body fat mass and total mass but also improved the lipid profile. The changes in body composition correlated with the level of lipoproteins responsible for the increased cardiovascular risk factors. These changes in body composition and lipid profile parameters coincided with the improvement of circulatory progenitor cell numbers.As the result of our study, we concluded that the improvement of body composition affects the number of stem/progenitor cells in circulation.

  12. Quantitative analysis of infiltrating immune cells and bovine papillomavirus type 1 E2-positive cells in equine sarcoids.

    Science.gov (United States)

    Geisshüsler, H; Marti, E; Stoffel, M H; Kühni, K; Stojiljkovic, A; von Tscharner, C; Vidondo, B; Gerber, V; Koch, C

    2016-10-01

    Sarcoids are the most frequently observed skin tumours in equids and consist of cutaneous accumulations of transformed fibroblasts. Their aetiopathogenesis is closely linked to a presumably abortive infection by bovine papillomavirus (BPV) types 1 and 2. In cattle, dermal fibropapillomas induced by BPV1/2 usually regress spontaneously due to a local, cell-mediated, immune response; however, equids appear to lack an effective immune response to BPV1/2 and mechanisms of immune evasion have been postulated. As a consequence, equine sarcoids tend to persist and are prone to recur. In this study, cryosections were analysed by immunofluorescent staining and a high content analysis system to determine the presence and distribution of CD4(+), CD8(+), FoxP3(+), RORγt(-), CD206(+) and CD14(+) cells, along with expression of the BPV1 early regulatory protein E2. A higher density of cells was positive for BPV1 E2(+) within the transformed tissue than in perilesional tissue or normal skin of horses with sarcoids and control horses. The proportion of CD8(+) cytotoxic T cells was significantly increased in perilesional and lesional tissues, whereas CD4(+) T helper cells were present in higher density only in lesional tissue compared to normal skin from horses with and without sarcoids. The proportion of pro-inflammatory CD4(+)FoxP3(+)RORγt(+) regulatory T cells was decreased in sarcoid tissue compared to perilesional, distant and control tissue. There were no significant differences in densities of CD4(+)FoxP3(+) RORγt(-) regulatory T cells between sarcoids and control tissues. Equine sarcoids are characterised by infiltrations of CD8(+) and CD4(+) T cells, with decreased representation by pro-inflammatory CD4(+)FoxP3(+)RORγt(+) regulatory T cells. PMID:27687925

  13. In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

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    Morteza Abouzaripour

    2016-02-01

    Full Text Available Objective: Bone marrow has recently been recognized as a novel source of stem cells for the treatment of wide range of diseases. A number of studies on murine bone marrow have shown a homogenous population of rare stage-specific embryonic antigen 1 (SSEA-1 positive cells that express markers of pluripotent stem cells. This study focuses on SSEA-1 positive cells isolated from murine bone marrow in an attempt to differentiate them into insulin-secreting cells (ISCs in order to investigate their differentiation potential for future use in cell therapy. Materials and Methods: This study is an experimental research. Mouse SSEA-1 positive cells were isolated by Magnetic-activated cell sorting (MACS followed by characterization with flow cytometry. Induced SSEA-1 positive cells were differentiated into ISCs with specific differentiation media. In order to evaluate differentiation quality and analysis, dithizone (DTZ staining was use, followed by reverse transcription polymerase chain reaction (RT-PCR, immunocytochemistry and insulin secretion assay. Statistical results were analyzed by one-way ANOVA. Results: The results achieved in this study reveal that mouse bone marrow contains a population of SSEA-1 positive cells that expresses pluripotent stem cells markers such as SSEA-1, octamer-binding transcription factor 4 (OCT-4 detected by immunocytochemistry and C-X-C chemokine receptor type 4 (CXCR4 and stem cell antigen-1 (SCA-1 detected by flow cytometric analysis. SSEA-1 positive cells can differentiate into ISCs cell clusters as evidenced by their DTZ positive staining and expression of genes such as Pdx1 (pancreatic transcription factors, Ngn3 (endocrine progenitor marker, Insulin1 and Insulin2 (pancreaticβ-cell markers. Additionally, our results demonstrate expression of PDX1 and GLUT2 protein and insulin secretion in response to a glucose challenge in the differentiated cells. Conclusion: Our study clearly demonstrates the potential of SSEA-1

  14. Significance of a positive Clostridium difficile toxin test after hematopoietic stem cell transplantation.

    Science.gov (United States)

    Akahoshi, Yu; Kimura, Shun-Ichi; Nakano, Hirofumi; Harada, Naonori; Kameda, Kazuaki; Ugai, Tomotaka; Wada, Hidenori; Yamasaki, Ryoko; Ishihara, Yuko; Kawamura, Koji; Sakamoto, Kana; Ashizawa, Masahiro; Sato, Miki; Terasako-Saito, Kiriko; Nakasone, Hideki; Kikuchi, Misato; Yamazaki, Rie; Kanda, Junya; Kako, Shinichi; Nishida, Junji; Kanda, Yoshinobu

    2016-06-01

    Patients with hematological malignancies show a high prevalence of asymptomatic colonization with Clostridium difficile (CD colonization). Therefore, it is difficult to distinguish CD colonization with diarrhea induced by a conditioning regimen from true Clostridium difficile infection (CDI) in hematopoietic stem cell transplantation (HSCT) recipients. We retrospectively analyzed 308 consecutive patients who underwent a CD toxin A/B enzyme immunoassay test for diarrhea within 100 d after HSCT from November 2007 to May 2014. Thirty patients (9.7%) had positive CD toxin results, and 11 of these had positive results in subsequent tests after an initial negative result. Allogeneic HSCT, total body irradiation, stem cell source, acute leukemia, and the duration of neutropenia were significantly correlated with positive CD toxin results. In a logistic regression model, allogeneic HSCT was identified as a significant risk factor (odds ratio 18.6, p < 0.01). In an analysis limited to within 30 d after the conditioning regimen, the duration of neutropenia was the sole risk factor (odds ratio 10.4, p < 0.01). There were no distinctive clinical features for CDI, including the onset or duration of diarrhea. In conclusion, although CDI may be overdiagnosed in HSCT recipients, it is difficult to clinically distinguish between CDI and CD colonization. PMID:27019071

  15. Mesotheliomas show higher hyaluronan positivity around tumor cells than metastatic pulmonary adenocarcinomas.

    Science.gov (United States)

    Törrönen, Kari; Soini, Ylermi; Pääkkö, Paavo; Parkkinen, Jyrki; Sironen, Reijo; Rilla, Kirsi

    2016-10-01

    Hyaluronan is a unique glycosaminoglycan of the extracellular matrix, abundant in normal connective tissues but highly increased in many pathological conditions like cancer. Mesothelioma, one of the most malignant cancer types, is associated with high content of hyaluronan, with elevated levels of hyaluronan in pleural effusions and serum of the patients. Metastatic lung adenocarcinomas are typically less aggressive and have a better prognosis as compared to mesotheliomas, a reason why it is highly important to find reliable tools to differentiate these cancer types. The main purpose of this study was to evaluate the amount of hyaluronan, hyaluronan producing synthases (HAS's) and hyaluronan receptor CD44, in mesothelioma and metastatic lung adenocarcinomas. Furthermore, we wanted to clarify the role of hyaluronan, CD44 and HAS's as putative markers for differentiating malignant mesothelioma from metastatic lung adenocarcinomas. The main finding of this study was that mesotheliomas are significantly more positive for hyaluronan staining than metastatic adenocarcinomas. Unexceptionally, a trend of CD44 positivity of stromal cells was higher in adenocarcinomas as compared to mesotheliomas. However, no statistically significant differences were found between the staining of any of the HAS isoenzymes either in tumor cells or stromal cells of different groups of cases. The results show that there are significant differences in hyaluronan content between metastatic lung adenocarcinomas and mesotheliomas. However, as previous studies have suggested, hyaluronan alone is not a sufficient independent marker for diagnostic differentiation of these cancer types, but could be utilized as a combination together with other specific markers. PMID:26912058

  16. [Novel calretinin-positive cells with polymorphous spines in the mouse forebrain during early postnatal ontogenesis].

    Science.gov (United States)

    Revishchin, A V; Okhotin, V E; Pavlova, G V

    2009-01-01

    Using an immunocytochemical method for calretinin (CR) detection, we have earlier described (Morfologiya, 2009 v. 135. No. 3, p. 7-19) the population of previously unknown mono- and bipolar cells with polymorphous spines (PS) covering their cell bodies and processes, in adult mice forebrain structures adjacent to anterior horn of lateral ventricle. CR-positive spiny (CR+PS) cells were negative to GAD67 and were detected in the white matter and in layers V and VI of frontal area of dorsomedial cortex close to the cingulum, in in rostro-dorsal part of the caudate nucleus-putamen complex, anterior olfactory nucleus and in subependymal layer of the dorso-lateral angle of the lateral ventricle. In this work, the distribution of these cells in 7-day-old mice was studied. Comparative topographical analysis of definitive and early CR+PS cells demonstrated that in 7-day-old mice CR+PS cells were absent from the areas of their localization in adult animals - anterior olfactory nucleus, cortical plate and inner portion of neostriatum. Meanwhile, some CR+PS-like cells were detected in 7-day-old mice inside the rostral migratory route, close to neostriatum anterior boundary, along the dorsal border between neostriatum and corpus callosum, subependymal layer of lateral wall of the lateral ventricle, and in the cingulum area. These findings are indicative of the possible postnatal appearance of CR+PS cells. To test this hypothesis, the experiments were conducted in which bromodeoxyuridine (BrdU) was administered to the mice on their postnatal days 2-4 with the subsequent study of the brain sections of these animals sacrificed on their postnatal day 20. Double immunolabeling of these sections for CR and BrdU has detected the presence of CR+PS cells that contained postnatally administered BrdU. These results strongly suggest that, at least, some portion of CR+PS cells have their mitosis postnatally. It may be assumed, that CR+PS cells migrate to the sites of their distribution in

  17. Artificially introduced aneuploid chromosomes assume a conserved position in colon cancer cells.

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    Kundan Sengupta

    Full Text Available BACKGROUND: Chromosomal aneuploidy is a defining feature of carcinomas. For instance, in colon cancer, an additional copy of Chromosome 7 is not only observed in early pre-malignant polyps, but is faithfully maintained throughout progression to metastasis. These copy number changes show a positive correlation with average transcript levels of resident genes. An independent line of research has also established that specific chromosomes occupy a well conserved 3D position within the interphase nucleus. METHODOLOGY/PRINCIPAL FINDINGS: We investigated whether cancer-specific aneuploid chromosomes assume a 3D-position similar to that of its endogenous homologues, which would suggest a possible correlation with transcriptional activity. Using 3D-FISH and confocal laser scanning microscopy, we show that Chromosomes 7, 18, or 19 introduced via microcell-mediated chromosome transfer into the parental diploid colon cancer cell line DLD-1 maintain their conserved position in the interphase nucleus. CONCLUSIONS: Our data is therefore consistent with the model that each chromosome has an associated zip code (possibly gene density that determines its nuclear localization. Whether the nuclear localization determines or is determined by the transcriptional activity of resident genes has yet to be ascertained.

  18. New calretinin-positive cells with polymorphous spines in the mouse forebrain during early postnatal ontogeny.

    Science.gov (United States)

    Revishchin, A V; Okhotin, V E; Pavlova, G V

    2010-10-01

    Immunohistochemical studies of calretinin (CR) in forebrain structures adjacent to the anterior horn of the lateral ventricle in adult mice allowed us to detect a population of previously unknown mono- and bipolar cells whose bodies and processes were coated with polymorphous spines (PS) (Morfologiya, 135, No. 3, 7-19 (2009)). CR-positive spiny (CR(+)PS) cells did not contain GAD67 and were located in the white matter and layers V-VI of the frontal area of the dorsomedial cortex close to the cingulum, the rostrodorsal part of the caudate-putamen, the anterior olfactory nucleus, and the subependyma of the dorsolateral angle of the lateral ventricle. We report here studies of the distribution of these cells in seven-day-old mice. Comparative topographic analysis of definitive and early CR(+)PS cells showed that in seven-day-old mice, CR(+)PS cells were absent from the sites at which they were seen in adults, i.e., the anterior olfactory cortex, the cortical plate, and the inner part of the neostriatum. In addition, small numbers of CR(+)PS-like cells were seen at this age within the dorsal migration pathway, at the anterior margin of the neostriatum, along the dorsal border of the neostriatum with the corpus callosum, in the subependymal layer of the lateral wall of the lateral ventricle, and in the cingulum area. These data demonstrate that CR(+)PS cells may have a postnatal origin. Experiments to verify this hypothesis were performed using postnatal administration of bromodeoxyuridine (BrdU) to mice aged 2-4 days, followed by assessment of brain sections fixed at age 20 days. Double immunolabeling of sections for CR and BrdU demonstrated the presence of CR(+)PS cells containing postnatally supplied BrdU. These data provide evidence that at least some CR(+)PS cells undergo mitosis at postnatal age. In all probability, during the period from 7 to 20 days of postnatal development, CR(+)PS cells migrate to the sites that they occupy in adult animals. PMID:20721693

  19. False positive indium-111 white blood cell scan in a closed clavicle fracture

    International Nuclear Information System (INIS)

    Aggressive treatment of the multiply injured patient often requires early fixation of many fractures, some of which may be open. Often, patients develop postoperative fevers requiring a thorough workup to rule out infection. Recently, indium-111 white blood cell (WBC) imaging has become a valuable adjunct in the diagnosis of acute infection. The patient described had a simple, closed clavicle fracture with markedly increased activity on an indium-111 WBC scan obtained for fever workup. This subsequently proved to be a normal, healing, noninfected fracture by other diagnostic techniques. Noninfected, simple closed fractures should be added to the list of causes for a false-positive indium-111 WBC scan

  20. BRAF V600E-Positive Multisite Langerhans Cell Histiocytosis in a Preterm Neonate

    Directory of Open Access Journals (Sweden)

    Sara V. Bates

    2013-10-01

    Full Text Available Hemorrhagic pustules with a “blueberry muffin” appearance accompanied by respiratory failure in a neonate present a challenging differential diagnosis that includes infections and neoplasms. We present a case of multiorgan, multisite Langerhans cell histiocytosis (LCH, positive for the oncogenic BRAF V600E mutation, in a preterm neonate. Infants with LCH pose a diagnostic challenge due to their heterogeneous presentations. This case is unusual in that the newborn presented with severe multiorgan involvement. Due to the rare incidence, wide spectrum of clinical manifestations, and high mortality rate, clinicians must maintain a high index of suspicion for LCH.

  1. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells*

    Science.gov (United States)

    Matheson, Nicholas J.; Wals, Kim; Antrobus, Robin; Göttgens, Berthold; Dougan, Gordon; Dawson, Mark A.; Lehner, Paul J.

    2015-01-01

    Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A non-lethal forward genetic screen in near-haploid KBM7 cells identified the Human Silencing Hub (HUSH), a complex of three poorly-characterised proteins, TASOR, MPP8, and periphilin, which is absent from Drosophila but conserved from fish to humans. Loss of HUSH subunits resulted in decreased H3K9me3 at both endogenous genomic loci and retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing. PMID:26022416

  2. GENE SILENCING. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells.

    Science.gov (United States)

    Tchasovnikarova, Iva A; Timms, Richard T; Matheson, Nicholas J; Wals, Kim; Antrobus, Robin; Göttgens, Berthold; Dougan, Gordon; Dawson, Mark A; Lehner, Paul J

    2015-06-26

    Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A nonlethal forward genetic screen in near-haploid KBM7 cells identified the HUSH (human silencing hub) complex, comprising three poorly characterized proteins, TASOR, MPP8, and periphilin; this complex is absent from Drosophila but is conserved from fish to humans. Loss of HUSH components resulted in decreased H3K9me3 both at endogenous genomic loci and at retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing.

  3. Position-effect variegation revisited: HUSHing up heterochromatin in human cells.

    Science.gov (United States)

    Timms, Richard T; Tchasovnikarova, Iva A; Lehner, Paul J

    2016-04-01

    Much of what we understand about heterochromatin formation in mammals has been extrapolated from forward genetic screens for modifiers of position-effect variegation (PEV) in the fruit fly Drosophila melanogaster. The recent identification of the HUSH (Human Silencing Hub) complex suggests that more recent evolutionary developments contribute to the mechanisms underlying PEV in human cells. Although HUSH-mediated repression also involves heterochromatin spreading through the reading and writing of the repressive H3K9me3 histone modification, clear orthologues of HUSH subunits are not found in Drosophila but are conserved in vertebrates. Here we compare the insights into the mechanisms of PEV derived from genetic screens in the fly, the mouse and in human cells, review what is currently known about the HUSH complex and discuss the implications of HUSH-mediated silencing for viral latency. Future studies will provide mechanistic insight into HUSH complex function and reveal the relationship between HUSH and other epigenetic silencing complexes.

  4. Anti-oxidative therapy with oral dapsone improved HCV antibody positive annular elastolytic giant cell granuloma.

    Science.gov (United States)

    Igawa, K; Maruyama, R; Katayama, I; Nishioka, K

    1997-05-01

    A 72-year-old fisherman who was positive for the HCV antibody developed an annular, erythematous, infiltrated lesions on sun-exposed areas. The lesions were diagnosed as annular elastolytic giant cell granuloma both clinically and histologically. Topical corticosteroid and cryotherapy with liquid nitrogen for several months failed to improve the lesions. We then started dapsone, a known anti-oxidant, at 50 mg/day. A month later, the margins of the erythematous lesions faded, and the infiltration gradually decreased. No recurrence has been observed for one year after the start of the therapy. Anti-oxidative therapy appears to be effective for annular elastolytic giant cell granuloma and could be an alternate therapy for refractory granulomatous disease. PMID:9198323

  5. Lymphotoxin signals from positively selected thymocytes regulate the terminal differentiation of medullary thymic epithelial cells.

    Science.gov (United States)

    White, Andrea J; Nakamura, Kyoko; Jenkinson, William E; Saini, Manoj; Sinclair, Charles; Seddon, Benedict; Narendran, Parth; Pfeffer, Klaus; Nitta, Takeshi; Takahama, Yousuke; Caamano, Jorge H; Lane, Peter J L; Jenkinson, Eric J; Anderson, Graham

    2010-10-15

    The thymic medulla represents a key site for the induction of T cell tolerance. In particular, autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) provide a spectrum of tissue-restricted Ags that, through both direct presentation and cross-presentation by dendritic cells, purge the developing T cell repertoire of autoimmune specificities. Despite this role, the mechanisms of Aire(+) mTEC development remain unclear, particularly those stages that occur post-Aire expression and represent mTEC terminal differentiation. In this study, in mouse thymus, we analyze late-stage mTEC development in relation to the timing and requirements for Aire and involucrin expression, the latter a marker of terminally differentiated epithelium including Hassall's corpuscles. We show that Aire expression and terminal differentiation within the mTEC lineage are temporally separable events that are controlled by distinct mechanisms. We find that whereas mature thymocytes are not essential for Aire(+) mTEC development, use of an inducible ZAP70 transgenic mouse line--in which positive selection can be temporally controlled--demonstrates that the emergence of involucrin(+) mTECs critically depends upon the presence of mature single positive thymocytes. Finally, although initial formation of Aire(+) mTECs depends upon RANK signaling, continued mTEC development to the involucrin(+) stage maps to activation of the LTα-LTβR axis by mature thymocytes. Collectively, our results reveal further complexity in the mechanisms regulating thymus medulla development and highlight the role of distinct TNFRs in initial and terminal differentiation stages in mTECs.

  6. [Cells in the system of multicelular organisms from positions of non-linear dynamics].

    Science.gov (United States)

    Kotolupov, V A; Isaeva, V V

    2012-01-01

    The organism physiological systems forming a hierarchic network with mutual dependence and subordination can be considered as systems with non-linear dynamics including positive and negative feedbacks. In the course of evolution there occurred selection of robust, flexible, modular systems capable for adaptive self-organization by non-linear interaction of components, which leads to formation of the ordered in space and time robust and plastic organization of the whole. Cells of multicellular organisms are capable for coordinated "social" behavior with formation of ordered cell assemblies, which provides a possibility of morphological and functional variability correlating with manifestations of the large spectrum of adaptive reactions. The multicellular organism is the multilevel system with hierarchy of numerous subsystems capable for adaptive self-organization; disturbance of their homeostasis can lead to pathological changes. The healthy organism regulates homeostasis, self-renewal, differentiation, and apoptosis of cells serving its parts and construction blocks by preserving its integrity and controlling behavior of cells. The systemic approach taking into account biological regularities of the appearance and development of functions in evolution of multicellular organisms opens new possibilities for diagnostics and treatment of many diseases.

  7. ALK-positive anaplastic large cell lymphoma with soft tissue involvement in a young woman

    Directory of Open Access Journals (Sweden)

    Gao KH

    2016-07-01

    Full Text Available Kehai Gao, Hongtao Li, Caihong Huang, Huazhuang Li, Jun Fang, Chen Tian Department of Orthopaedics, Yidu Central Hospital, Shandong, People’s Republic of China Introduction: Anaplastic large cell lymphoma (ALCL is a type of non-Hodgkin lymphoma that has strong expression of CD30. ALCL can sometimes involve the bone marrow, and in advanced stages, it can produce destructive extranodal lesions. But anaplastic large cell lymphoma kinase (ALK+ ALCL with soft tissue involvement is very rare.Case report: A 35-year-old woman presented with waist pain for over 1 month. The biopsy of soft tissue lesions showed that these cells were positive for ALK-1, CD30, TIA-1, GranzymeB, CD4, CD8, and Ki67 (90%+ and negative for CD3, CD5, CD20, CD10, cytokeratin (CK, TdT, HMB-45, epithelial membrane antigen (EMA, and pan-CK, which identified ALCL. After six cycles of Hyper-CVAD/MA regimen, she achieved partial remission. Three months later, she died due to disease progression.Conclusion: This case illustrates the unusual presentation of ALCL in soft tissue with a bad response to chemotherapy. Because of the tendency for rapid progression, ALCL in young adults with extranodal lesions are often treated with high-grade chemotherapy, such as Hyper-CVAD/MA. Keywords: anaplastic large cell lymphoma, ALK+, soft tissue involvement, Hyper-CVAD/MA

  8. Gram-positive bacterial cell envelopes: The impact on the activity of antimicrobial peptides.

    Science.gov (United States)

    Malanovic, Nermina; Lohner, Karl

    2016-05-01

    A number of cationic antimicrobial peptides, effectors of innate immunity, are supposed to act at the cytoplasmic membrane leading to permeabilization and eventually membrane disruption. Thereby, interaction of antimicrobial peptides with anionic membrane phospholipids is considered to be a key factor in killing of bacteria. Recently, evidence was provided that killing takes place only when bacterial cell membranes are completely saturated with peptides. This adds to an ongoing debate, which role cell wall components such as peptidoglycan, lipoteichoic acid and lipopolysaccharide may play in the killing event, i.e. if they rather entrap or facilitate antimicrobial peptides access to the cytoplasmic membrane. Therefore, in this review we focused on the impact of Gram-positive cell wall components for the mode of action and activity of antimicrobial peptides as well as in innate immunity. This led us to conclude that interaction of antimicrobial peptides with peptidoglycan may not contribute to a reduction of their antimicrobial activity, whereas interaction with anionic lipoteichoic acids may reduce the local concentration of antimicrobial peptides on the cytoplasmic membrane necessary for sufficient destabilization of the membranes and bacterial killing. Further affinity studies of antimicrobial peptides toward the different cell wall as well as membrane components will be needed to address this problem on a quantitative level. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

  9. Surface Position, Not Signaling from Surrounding Maternal Tissues, Specifies Aleurone Epidermal Cell Fate in Maize[OA

    Science.gov (United States)

    Gruis, Darren (Fred); Guo, Hena; Selinger, David; Tian, Qing; Olsen, Odd-Arne

    2006-01-01

    Maize (Zea mays) endosperm consists of an epidermal-like surface layer of aleurone cells, an underlying body of starchy endosperm cells, and a basal layer of transfer cells. To determine whether surrounding maternal tissues perform a role in specifying endosperm cell fates, a maize endosperm organ culture technique was established whereby the developing endosperm is completely removed from surrounding maternal tissues. Using cell type-specific fluorescence markers, we show that aleurone cell fate specification occurs exclusively in response to surface position and does not require specific, continued maternal signal input. The starchy endosperm and aleurone cell fates are freely interchangeable throughout the lifespan of the endosperm, with internalized aleurone cells converting to starchy endosperm cells and with starchy endosperm cells that become positioned at the surface converting to aleurone cells. In contrast to aleurone and starchy endosperm cells, transfer cells fail to develop in in vitro-grown endosperm, supporting earlier indications that maternal tissue interaction is required to fully differentiate this cell type. Several parameters confirm that the maize endosperm organ cultures described herein retain the main developmental features of in planta endosperm, including fidelity of aleurone mutant phenotypes, temporal and spatial control of cell type-specific fluorescent markers, specificity of cell type transcripts, and control of mitotic cell divisions. PMID:16698897

  10. CD1A-positive cells and HSP60 (HSPD1) levels in keratoacanthoma and squamous cell carcinoma.

    Science.gov (United States)

    Cabibi, Daniela; Conway de Macario, Everly; Ingrao, Sabrina; Porcasi, Rossana; Zucco, Francesco; Macario, Alberto J L; Cappello, Francesco; Rappa, Francesca

    2016-01-01

    CD1a is involved in presentation to the immune system of lipid antigen derived from tumor cells with subsequent T cell activation. Hsp60 is a molecular chaperone implicated in carcinogenesis by, for instance, modulating the immune reaction against the tumor. We have previously postulated a synergism between CD1a and Hsp60 as a key factor in the activation of an effective antitumor immune response in squamous epithelia. Keratoacantomas (KAs) are benign tumors that however can transform into squamous cell carcinomas (SCCs), but the reasons for this malignization are unknown. In a previous study, we found that CD1a-positive cells are significantly more numerous in KA than in SCC. In this study, we analyzed a series of KAs and SCCs by immunohistochemistry for CD1a and Hsp60. Our results show that the levels of both are significantly lower in KA than in SCC and support the hypothesis that KA may evolve towards SCC if there is a failure of the local modulation of the antitumor immune response. The data also show that immunohistochemistry for CD1a and Hsp60 can be of help in differential diagnosis between KAs and well-differentiated forms of SCC.

  11. Real-time single-ion hit position detecting system for cell irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Satoh, Takahiro, E-mail: satoh.takahiro37@jaea.go.jp [Takasaki Advanced Radiation Research Institute, Japan Atomic Energy Agency (JAEA), 1233 Watanuki-machi, Takasaki, Gunma 370-1292 (Japan); Koka, Masahi [Takasaki Advanced Radiation Research Institute, Japan Atomic Energy Agency (JAEA), 1233 Watanuki-machi, Takasaki, Gunma 370-1292 (Japan); Kada, Wataru [Faculty of Science and Technology, Gunma University, 1-5-1 Tenjin-machi, Kiryu, Gunma 376-8515 (Japan); Yokoyama, Akihito; Kamiya, Tomihiro [Takasaki Advanced Radiation Research Institute, Japan Atomic Energy Agency (JAEA), 1233 Watanuki-machi, Takasaki, Gunma 370-1292 (Japan)

    2014-08-01

    We have developed a real-time single-ion hit position detecting system to replace a CR-39 solid-state nuclear-track detector for cell irradiation experiments because the CR-39 takes several minutes for off-line etching. The new real-time system consists of a 500-μm-thick CaF{sub 2}(Eu) scintillator, an optical microscope with a 10× objective lens, and a high-gain charge-coupled device camera. Each of the 260-MeV neon ions passing through a 100-μm-thick CR-39 sheet was detected using the real-time system in a performance test for the spatial resolution. The full width at half maxima (FWHMs) of the distances between positions detected by the real-time system and the centers of the etch pits on CR-39 were 6.5 and 6.9 μm in the x and y directions, respectively. The result shows that the system is useful for typical cultured cells of a few tens of micrometers in size.

  12. Interferon-α/β enhances temozolomide activity against MGMT-positive glioma stem-like cells.

    Science.gov (United States)

    Shen, Dong; Guo, Cheng-Cheng; Wang, Jing; Qiu, Zhi-Kun; Sai, Ke; Yang, Qun-Ying; Chen, Yin-Sheng; Chen, Fu-Rong; Wang, Jie; Panasci, Lawrence; Chen, Zhong-Ping

    2015-11-01

    Glioma is one of the most common primary tumors of the central nervous system in adults. Glioblastoma (GBM) is the most lethal type of glioma, whose 5-year survival is 9.8% at best. Glioma stem-like cells (GSCs) play an important role in recurrence and treatment resistance. MGMT is a DNA repair protein that removes DNA adducts and therefore attenuates treatment efficiency. It has been reported that interferon-α/β (IFN-α/β) downregulates the level of MGMT and sensitizes glioma cells to temozolomide. In the present study, we assessed whether IFN-α/β is able to sensitize GSCs to temozolomide by modulating MGMT expression. Upon the treatment of IFN-α/β, the efficacy of temozolomide against MGMT‑positive GSCs was markedly enhanced by combination treatment with IFN-α/β when compared with the temozolomide single agent group, and MGMT expression was markedly decreased at the same time. Further mechanistic study showed that IFN-α/β suppressed the NF-κB activity, which further mediated the sensitization of MGMT‑positive GSCs to temozolomide. Our data therefore demonstrated that the application of IFN-α/β is a promising agent with which to enhance temozolomide efficiency and reduce drug resistance, and our findings shed light on improving clinical outcomes and prolonging the survival of patients with malignant gliomas. PMID:26329778

  13. SPOT14-Positive Neural Stem/Progenitor Cells in the Hippocampus Respond Dynamically to Neurogenic Regulators

    Directory of Open Access Journals (Sweden)

    Marlen Knobloch

    2014-11-01

    Full Text Available Proliferation of neural stem/progenitor cells (NSPCs in the adult brain is tightly controlled to prevent exhaustion and to ensure proper neurogenesis. Several extrinsic stimuli affect NSPC regulation. However, the lack of unique markers led to controversial results regarding the in vivo behavior of NSPCs to different stimuli. We recently identified SPOT14, which controls NSPC proliferation through regulation of de novo lipogenesis, selectively in low-proliferating NSPCs. Whether SPOT14-expressing (SPOT14+ NSPCs react in vivo to neurogenic regulators is not known. We show that aging is accompanied by a marked disappearance of SPOT14+ NSPCs, whereas running, a positive neurogenic stimulus, increases proliferation of SPOT14+ NSPCs. Furthermore, transient depletion of highly proliferative cells recruits SPOT14+ NSPCs into the proliferative pool. Additionally, we have established endogenous SPOT14 protein staining, reflecting transgenic SPOT14-GFP expression. Thus, our data identify SPOT14 as a potent marker for adult NSPCs that react dynamically to positive and negative neurogenic regulators.

  14. A highly restricted T-cell receptor dominates the CD8+ T-cell response to parvovirus B19 infection in HLA-A*2402-positive individuals

    DEFF Research Database (Denmark)

    Kasprowicz, V; Jeffery, K; Broliden, K;

    2006-01-01

    Six of seven HLA-A*2402-positive individuals with acute parvovirus B19 infections made vigorous CD8-positive cytotoxic T-cell (CTL) responses to the viral epitope FYTPLADQF. All responders showed highly focused T-cell receptor (TCR) usage, using almost exclusively BV5.1. The BV5.1 TCR dominated...

  15. Relationship between nucleosome positioning and progesterone-induced alternative splicing in breast cancer cells.

    Science.gov (United States)

    Iannone, Camilla; Pohl, Andy; Papasaikas, Panagiotis; Soronellas, Daniel; Vicent, Guillermo P; Beato, Miguel; ValcáRcel, Juan

    2015-03-01

    Splicing of mRNA precursors can occur cotranscriptionally and it has been proposed that chromatin structure influences splice site recognition and regulation. Here we have systematically explored potential links between nucleosome positioning and alternative splicing regulation upon progesterone stimulation of breast cancer cells. We confirm preferential nucleosome positioning in exons and report four distinct profiles of nucleosome density around alternatively spliced exons, with RNA polymerase II accumulation closely following nucleosome positioning. Hormone stimulation induces switches between profile classes, correlating with a subset of alternative splicing changes. Hormone-induced exon inclusion often correlates with higher nucleosome occupancy at the exon or the preceding intronic region and with higher RNA polymerase II accumulation. In contrast, exons skipped upon hormone stimulation display low nucleosome densities even before hormone treatment, suggesting that chromatin structure primes alternative splicing regulation. Skipped exons frequently harbor binding sites for hnRNP AB, a hormone-induced splicing regulator whose knock down prevents some hormone-induced skipping events. Collectively, our results argue that a variety of chromatin architecture mechanisms can influence alternative splicing decisions.

  16. Analysis of the position of robotic cell components and its impact on energy consumption by robot

    Science.gov (United States)

    Banas, W.; Gwiazda, A.; Monica, Z.; Sekala, A.; Foit, K.

    2016-08-01

    Location elements in the robot cell is very important must provide reasonable access to technological points. This is a basic condition, but it is possible to shift these elements worth considering over other criteria. One of them can be energy consumption. This is an economic parameter and in most cases its improvement make shorten the working time an industrial robot. In most conventional mechanical systems you do not need to consume power in standby mode only for a move. Robot because of its construction, even if it does not move has enabled engines and is ready to move. In this case, the servo speed is zero. During this stop servo squeak. Low-speed motors cause the engine torque is reduced and increases power consumption. In larger robots are installed brakes that when the robot does not move mechanically hold the position. Off the robot has enabled brakes and remembers the position servo drives. Brakes must be released when the robot wants to move and drives hold the position.

  17. Proliferation and survival signaling from both Jak2-V617F and Lyn involving GSK3 and mTOR/p70S6K/4EBP1 in PVTL-1 cell line newly established from acute myeloid leukemia transformed from polycythemia vera.

    Directory of Open Access Journals (Sweden)

    Toshikage Nagao

    Full Text Available The gain of function mutation JAK2-V617F is very frequently found in myeloproliferative neoplasms (MPNs and is strongly implicated in pathogenesis of these and other hematological malignancies. Here we report establishment of a new leukemia cell line, PVTL-1, homozygous for JAK2-V617F from a 73-year-old female patient with acute myeloid leukemia (AML transformed from MPN. PVTL-1 is positive for CD7, CD13, CD33, CD34, CD117, HLA-DR, and MPO, and has complex karyotypic abnormalities, 44,XX,-5q,-7,-8,add(11(p11.2,add(11(q23,-16,+21,-22,+mar1. Sequence analysis of JAK2 revealed only the mutated allele coding for Jak2-V617F. Proliferation of PVTL-1 was inhibited and apoptosis was induced by the pan-Jak inhibitor Jak inhibitor-1 (JakI-1 or dasatinib, which inhibits the Src family kinases as well as BCR/ABL. Consistently, the Src family kinase Lyn was constitutively activated with phosphorylation of Y396 in the activation loop, which was inhibited by dasatinib but not by JakI-1. Further analyses with JakI-1 and dasatinib indicated that Jak2-V617F phosphorylated STAT5 and SHP2 while Lyn phosphorylated SHP1, SHP2, Gab-2, c-Cbl, and CrkL to induce the SHP2/Gab2 and c-Cbl/CrkL complex formation. In addition, JakI-1 and dasatinib inactivated the mTOR/p70S6K/4EBP1 pathway and reduced the inhibitory phosphorylation of GSK3 in PVTL-1 cells, which correlated with their effects on proliferation and survival of these cells. Furthermore, inhibition of GSK3 by its inhibitor SB216763 mitigated apoptosis induced by dasatinib but not by JakI-1. Together, these data suggest that apoptosis may be suppressed in PVTL-1 cells through inactivation of GSK3 by Lyn as well as Jak2-V617F and additionally through activation of STAT5 by Jak2-V617F. It is also speculated that activation of the mTOR/p70S6K/4EBP1 pathway may mediate proliferation signaling from Jak2-V617F and Lyn. PVTL-1 cells may provide a valuable model system to elucidate the molecular mechanisms involved in

  18. MBP-Positive and CD11c-Positive Cells Are Associated with Different Phenotypes of Korean Patients with Non-Asthmatic Chronic Rhinosinusitis

    Science.gov (United States)

    Chang, Dong-Yeop; Eun, Kyung Mi; Shin, Hyun-Woo; Mo, Ji-Hun; Shin, Eui-Cheol; Jin, Hong Ryul; Shin, Sue; Roh, Eun Youn; Han, Doo Hee; Kim, Dae Woo

    2014-01-01

    Background Asthmatic nasal polyps primarily exhibit eosinophilic infiltration. However, the identities of the immune cells that infiltrate non-asthmatic nasal polyps remain unclear. Thus, we thought to investigate the distribution of innate immune cells and its clinical relevance in non-asthmatic chronic rhinosinusitis (CRS) in Korea. Methods Tissues from uncinate process (UP) were obtained from controls (n = 18) and CRS without nasal polyps (CRSsNP, n = 45). Nasal polyps (NP) and UP were obtained from CRS with nasal polyps (CRSwNP, n = 56). The innate immune cells was evaluated by immunohistochemistry such as, eosinophil major basic protein (MBP), tryptase, CD68, CD163, CD11c, 2D7, human neutrophil elastase (HNE) and its distribution was analyzed according to clinical parameters. Results In comparisons between UP from each group, CRSwNP had a higher number of MPB+, CD68+, and CD11c+ cells relative to CRSsNP. Comparisons between UP and NP from CRSwNP indicated that NP have a higher infiltrate of MBP+, CD163+, CD11c+, 2D7+ and HNE+ cells, whereas fewer CD68+ cells were found in NP. In addition, MBP+ and CD11c+ cells were increased from UP of CRSsNP, to UP of CRSwNP, and to NP of CRSwNP. Moreover, in UP from CRSwNP, the number of MBP+ and CD11c+ cells positively correlated with CT scores. In the analysis of CRSwNP phenotype, allergic eosinophilic polyps had a higher number of MBP+, tryptase+, CD11c+, 2D7+ cells than others, whereas allergic non-eosinophilic polyps showed mainly infiltration of HNE+ and 2D7+ cells. Conclusions The infiltration of MBP+ and CD11c+ innate immune cells show a significant association with phenotype and disease extent of CRS and allergic status also may influences cellular phenotype in non-asthmatic CRSwNP in Korea. PMID:25361058

  19. Detection of (Leu-7)-positive cells with NK activity in human gingival tissues from patients with periodontitis

    Energy Technology Data Exchange (ETDEWEB)

    Komiyama, K.; Hirsch, H.Z.; Mestecky, J.; Moro, I.

    1986-03-05

    Natural killer (NK) cells have been identified in peripheral blood, lymphoid tissue and more recently in gut mucosa and may be involved in the regulation of immunoglobulin synthesis. They have assayed gingival tissues obtained from 25 periodontitis patients, for the presence and activity of NK cells. Routine histological techniques demonstrated an inflammatory infiltrate dominated by plasma cells and B lymphocytes. Indirect staining procedures with a biotin-labeled mouse anti-human, Leu-7 antibody revealed the presence of numerous positive cells accompanying the inflammatory cellular infiltrate in perivascular areas. Several specimens demonstrated positive-staining cells in the epithelium as well. Few cells were observed in histologically uninflammed areas. Single cell suspension obtained by collagenase digestion of 5 gingival samples were used in /sup 51/Cr release cytotoxicity assay against K562 cells. Three of the five samples were positive in this assay. The finding of Leu-7-positive cells in areas of intense plasma cell foci but not in uninflammed areas, may support a role for these cells in the regulation of immunoglobulin synthesis in oral mucosal tissues.

  20. Vitamin D status is positively correlated with regulatory T cell function in patients with multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Joost Smolders

    Full Text Available BACKGROUND: In several autoimmune diseases, including multiple sclerosis (MS, a compromised regulatory T cell (Treg function is believed to be critically involved in the disease process. In vitro, the biologically active metabolite of vitamin D has been shown to promote Treg development. A poor vitamin D status has been linked with MS incidence and MS disease activity. In the present study, we assess a potential in vivo correlation between vitamin D status and Treg function in relapsing remitting MS (RRMS patients. METHODOLOGY/PRINCIPAL FINDINGS: Serum levels of 25-hydroxyvitamin D (25(OHD were measured in 29 RRMS patients. The number of circulating Tregs was assessed by flow-cytometry, and their functionality was tested in vitro in a CFSE-based proliferation suppression assay. Additionally, the intracellular cytokine profile of T helper cells was determined directly ex-vivo by flow-cytometry. Serum levels of 25(OHD correlated positively with the ability of Tregs to suppress T cell proliferation (R = 0.590, P = 0.002. No correlation between 25(OHD levels and the number of Tregs was found. The IFN-gamma/IL-4 ratio (Th1/Th2-balance was more directed towards IL-4 in patients with favourable 25(OHD levels (R = -0.435, P = 0.023. CONCLUSIONS/SIGNIFICANCE: These results show an association of high 25(OHD levels with an improved Treg function, and with skewing of the Th1/Th2 balance towards Th2. These findings suggest that vitamin D is an important promoter of T cell regulation in vivo in MS patients. It is tempting to speculate that our results may not only hold for MS, but also for other autoimmune diseases. Future intervention studies will show whether modulation of vitamin D status results in modulation of the T cell response and subsequent amelioration of disease activity.

  1. Low dose irradiation of thyroid cells reveals a unique transcriptomic and epigenetic signature in RET/PTC-positive cells

    Energy Technology Data Exchange (ETDEWEB)

    Abou-El-Ardat, Khalil, E-mail: kabouela@sckcen.be [Radiobiology Unit, Molecular and Cellular Biology, GKD Building, Studiecentrum voor Kernenergie - Centre d' Etude de l' Energie Nucleaire (SCK-CEN), Boeretang 200, 2400 Mol (Belgium); Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); Monsieurs, Pieter [Radiobiology Unit, Molecular and Cellular Biology, GKD Building, Studiecentrum voor Kernenergie - Centre d' Etude de l' Energie Nucleaire (SCK-CEN), Boeretang 200, 2400 Mol (Belgium); Anastasov, Natasa; Atkinson, Mike [Department of Radiation Sciences, Helmholtz Zentrum Muenchen, Munich (Germany); Derradji, Hanane [Radiobiology Unit, Molecular and Cellular Biology, GKD Building, Studiecentrum voor Kernenergie - Centre d' Etude de l' Energie Nucleaire (SCK-CEN), Boeretang 200, 2400 Mol (Belgium); De Meyer, Tim [Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); Department of Applied Mathematics, Biometrics and Process Control, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); Bekaert, Sofie [Clinical Research Center, Faculty for Medicine and Health Sciences, Universiteit Gent, 185 De Pintelaan, 9000 Ghent (Belgium); Van Criekinge, Wim [Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Universiteit Gent, 9000 Ghent (Belgium); and others

    2012-03-01

    The high doses of radiation received in the wake of the Chernobyl incident and the atomic bombing of Hiroshima and Nagasaki have been linked to the increased appearance of thyroid cancer in the children living in the vicinity of the site. However, the data gathered on the effect of low doses of radiation on the thyroid remain limited. We have examined the genome wide transcriptional response of a culture of TPC-1 human cell line of papillary thyroid carcinoma origin with a RET/PTC1 translocation to various doses (0.0625, 0.5, and 4 Gy) of X-rays and compared it to response of thyroids with a RET/PTC3 translocation and against wild-type mouse thyroids irradiated with the same doses using Affymetrix microarrays. We have found considerable overlap at a high dose of 4 Gy in both RET/PTC-positive systems but no common genes at 62.5 mGy. In addition, the response of RET/PTC-positive system at all doses was distinct from the response of wild-type thyroids with both systems signaling down different pathways. Analysis of the response of microRNAs in TPC-1 cells revealed a radiation-responsive signature of microRNAs in addition to dose-responsive microRNAs. Our results point to the fact that a low dose of X-rays seems to have a significant proliferative effect on normal thyroids. This observation should be studied further as opposed to its effect on RET/PTC-positive thyroids which was subtle, anti-proliferative and system-dependent.

  2. Low dose irradiation of thyroid cells reveals a unique transcriptomic and epigenetic signature in RET/PTC-positive cells.

    Science.gov (United States)

    Abou-El-Ardat, Khalil; Monsieurs, Pieter; Anastasov, Nataša; Atkinson, Mike; Derradji, Hanane; De Meyer, Tim; Bekaert, Sofie; Van Criekinge, Wim; Baatout, Sarah

    2012-03-01

    The high doses of radiation received in the wake of the Chernobyl incident and the atomic bombing of Hiroshima and Nagasaki have been linked to the increased appearance of thyroid cancer in the children living in the vicinity of the site. However, the data gathered on the effect of low doses of radiation on the thyroid remain limited. We have examined the genome wide transcriptional response of a culture of TPC-1 human cell line of papillary thyroid carcinoma origin with a RET/PTC1 translocation to various doses (0.0625, 0.5, and 4Gy) of X-rays and compared it to response of thyroids with a RET/PTC3 translocation and against wild-type mouse thyroids irradiated with the same doses using Affymetrix microarrays. We have found considerable overlap at a high dose of 4Gy in both RET/PTC-positive systems but no common genes at 62.5mGy. In addition, the response of RET/PTC-positive system at all doses was distinct from the response of wild-type thyroids with both systems signaling down different pathways. Analysis of the response of microRNAs in TPC-1 cells revealed a radiation-responsive signature of microRNAs in addition to dose-responsive microRNAs. Our results point to the fact that a low dose of X-rays seems to have a significant proliferative effect on normal thyroids. This observation should be studied further as opposed to its effect on RET/PTC-positive thyroids which was subtle, anti-proliferative and system-dependent. PMID:22027090

  3. GATA Factor-Dependent Positive-Feedback Circuit in Acute Myeloid Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Koichi R. Katsumura

    2016-08-01

    Full Text Available The master regulatory transcription factor GATA-2 triggers hematopoietic stem and progenitor cell generation. GATA2 haploinsufficiency is implicated in myelodysplastic syndrome (MDS and acute myeloid leukemia (AML, and GATA2 overexpression portends a poor prognosis for AML. However, the constituents of the GATA-2-dependent genetic network mediating pathogenesis are unknown. We described a p38-dependent mechanism that phosphorylates GATA-2 and increases GATA-2 target gene activation. We demonstrate that this mechanism establishes a growth-promoting chemokine/cytokine circuit in AML cells. p38/ERK-dependent GATA-2 phosphorylation facilitated positive autoregulation of GATA2 transcription and expression of target genes, including IL1B and CXCL2. IL-1β and CXCL2 enhanced GATA-2 phosphorylation, which increased GATA-2-mediated transcriptional activation. p38/ERK-GATA-2 stimulated AML cell proliferation via CXCL2 induction. As GATA2 mRNA correlated with IL1B and CXCL2 mRNAs in AML-M5 and high expression of these genes predicted poor prognosis of cytogenetically normal AML, we propose that the circuit is functionally important in specific AML contexts.

  4. Interleukin 13-positive mast cells are increased in immunoglobulin G4-related sialadenitis.

    Science.gov (United States)

    Takeuchi, Mai; Ohno, Kyotaro; Takata, Katsuyoshi; Gion, Yuka; Tachibana, Tomoyasu; Orita, Yorihisa; Yoshino, Tadashi; Sato, Yasuharu

    2015-01-01

    Interleukin (IL)-13 is a T helper 2 (Th2) cytokine that plays important roles in the pathogenesis of asthma. IL-13 induces hypersensitivity of the airways, increased mucous production, elevated serum immunoglobulin (Ig) E levels, and increased numbers of eosinophils. Many patients with IgG4-related disease have allergic backgrounds and show elevated serum IgE levels and an increase in the number of eosinophils. Upregulation of Th2/regulatory T (Treg) cytokines, including IL-13, has been detected in affected tissues of patients with IgG4-related disease. We previously reported that mast cells might be responsible for the production of the Th2/Treg cytokines IL-4, IL-10, and transforming growth factor (TGF)-β1 in IgG4-related disease. In this study, immunohistochemical analysis showed increased numbers of IL-13-positive mast cells in IgG4-related disease, which suggests that mast cells also produce IL-13 and contribute to elevation of serum IgE levels and eosinophil infiltration in IgG4-related disease. PMID:25571893

  5. p75 neurotrophin receptor positive dental pulp stem cells: new hope for patients with neurodegenerative disease and neural injury%p75 neurotrophin receptor positive dental pulp stem cells:new hope for patients with neurodegenerative disease and neural injury

    Institute of Scientific and Technical Information of China (English)

    DAI Jie-wen; YUAN Hao; SHEN Shun-yao; LU Jing-ting; ZHU Xiao-fang; YANG Tong; ZHANG Jiang-fei

    2013-01-01

    Neurodegenerative diseases and neural injury are 2 of the most feared disorders that afflict humankind by leading to permanent paralysis and loss of sensation.Cell based treatment for these diseases had gained special interest in recent years.Previous studies showed that dental pulp stem cells (DPSCs) could differentiate toward functionally active neurons both in vitro and in vivo,and could promote neuranagenesis through both cell-autonomous and paracrine neuroregenerative activities.Some of these neuroregenerative activities were unique to tooth-derived stem cells and superior to bone marrow stromal cells.However,DPSCs used in most of these studies were mixed and unfractionated dental pulp cells that contain several types of cells,and most were fibroblast cells while just contain a small portion of DPSCs.Thus,there might be weaker ability of neuranagenesis and more side effects from the fibroblast cells that cannot differentiate into neural cells.p75 neurotrophin receptor (p75NTR) positive DPSCs subpopulation was derived from migrating cranial neural crest cells and had been isolated from DPSCs,which had capacity of differentiation into neurons and repairing neural system.In this article,we hypothesize that p75NTR positive DPSCs simultaneously have greater propensity for neuronal differentiation and fewer side effects from fibroblast,and in vivo transptantation of autologous p75NTR positive DPSCs is a novel method for neuranagenesis.This will bring great hope to patients with neurodegenerative disease and neural injury.

  6. CAR-pNK Cell Immunotherapy in CD7 Positive Leukemia and Lymphoma

    Science.gov (United States)

    2016-07-11

    Acute Myeloid Leukemia; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; T-cell Prolymphocytic Leukemia; T-cell Large Granular Lymphocytic Leukemia; Peripheral T-cell Lymphoma, NOS; Angioimmunoblastic T-cell Lymphoma; Extranodal NK/T-cell Lymphoma, Nasal Type; Enteropathy-type Intestinal T-cell Lymphoma; Hepatosplenic T-cell Lymphoma

  7. Microchannel-connected SU-8 honeycombs by single-step projection photolithography for positioning cells on silicon oxide nanopillar arrays

    International Nuclear Information System (INIS)

    We report on the fabrication, functionalization and testing of SU-8 microstructures for cell culture and positioning over large areas. The microstructure consists of a honeycomb arrangement of cell containers interconnected by microchannels and centered on nanopillar arrays designed for promoting cell positioning. The containers have been dimensioned to trap single cells and, with a height of 50 µm, prevent cells from escaping. The structures are fabricated using a single ultraviolet photolithography exposure with focus depth in the lower part of the SU-8 resist. With optimized process parameters, microchannels of various aspect ratios are thus produced. The cell containers and microchannels serve for the organization of axonal growth between neurons. The roughly 2 µm-high and 500 nm-wide nanopillars are made of silicon oxide structured by deep reactive ion etching. In future work, beyond their cell positioning purpose, the nanopillars could be functionalized as sensors. The proof of concept of the novel microstructure for organized cell culture is given by the successful growth of interconnected PC12 cells. Promoted by the honeycomb geometry, a dense network of interconnections between the cells has formed and the intended intimate contact of cells with the nanopillar arrays was observed by scanning electron microscopy. This proves the potential of these new devices as tools for the controlled cell growth in an interconnected container system with well-defined 3D geometry. (paper)

  8. Without peripheral interference, thymic deletion is mediated in a cohort of double-positive cells without classical activation

    Science.gov (United States)

    Zhan, Yifan; Purton, Jared F.; Godfrey, Dale I.; Cole, Timothy J.; Heath, William R.; Lew, Andrew M.

    2003-01-01

    Peripheral activation can cause bystander thymocyte death by eliciting a “cytokine storm.” This event complicates in vivo studies using exogenous ligand-induced models of negative selection. A stable transgenic model that selectively eliminates peripheral CD4 cells has allowed us to analyze negative selection as direct cognate events in two T cell receptor transgenic mice, OT-II and DO11. Whereas cognate peptide induced a massive deletion in double-positive (DP) cells in mice with peripheral CD4 cells, this DP deletion was modest in mice lacking peripheral CD4 cells. Using BrdUrd and annexin V staining, we found that negative selection primarily occurs in a cohort of DP cells and the absence of single-positive (SP) cells is largely caused by reduction in the cohort of DP precursors. Moreover, the fates of DP cells and SP cells after antigen exposure were vastly different. Whereas SP cells up-regulated uniformly their CD69 and CD44 levels, increased their cell size, and survived after antigen exposure, DP cells had less CD69 and CD44 up-regulation, no size change, and promptly died. Thus, negative selection represents an “abortive” activation different from activation-induced cell death of mature T cells. PMID:12538873

  9. Evaluation of CD25-positive cells in relation to the subtypes and prognoses in various lymphoid tumours in dogs.

    Science.gov (United States)

    Mizutani, Noriyuki; Goto-Koshino, Yuko; Tsuboi, Masaya; Kagawa, Yumiko; Ohno, Koichi; Uchida, Kazuyuki; Tsujimoto, Hajime

    2016-05-01

    Interleukin-2 receptor alpha chain (CD25) expression has been reported in human lymphoid tumours and suggested to correlate with the prognosis. In this study, we detected CD25-positive cells in various types of lymphoid tumours in dogs. Immunohistochemical analyses of the tissues from diffuse large B-cell lymphoma (DLBCL) (n = 6), T-zone lymphoma (TZL) (n = 5), and follicular lymphoma (FL) (n = 2) revealed that cells strongly positive for CD25 were observed generally in accordance with lymphoma cell localization. CD25-positive cells were consistently detected in TZL and FL cases; however, the number of CD25-positive cells was variable among DLBCL cases. Furthermore, we evaluated the rate of CD25-positive cells by flow cytometric analysis in 29 dogs with lymphoid malignancies, including high-grade B-cell lymphoma (n = 17), TZL (n = 5), FL (n = 2), cutaneous lymphoma (n=2), and acute lymphoblastic leukaemia (ALL) (n = 3). CD25-positivity in the lymph node cells was significantly higher in dogs with high-grade B-cell lymphoma (mean ± SD, 49.6 ± 31.3%) or TZL (mean ± SD, 80.2 ± 10.0%) than that in healthy dogs (mean ± SD, 9.8 ± 2.8%). In prognostic analysis of 15 cases with high-grade B-cell lymphoma, the progression-free survival was significantly shorter in CD25-high group than that in CD25-low group. The results obtained in this study are useful for subtype differentiation and prognostic analysis of canine lymphomas and future development of molecular-targeted therapy directed at CD25.

  10. [Research Progress on CALR Mutation in the Myeloproliterative Neoplasm -Review].

    Science.gov (United States)

    Yuan, Jun; Hao, Hong-Ling; Li, Yan; Wang, Rui-Cang; Zhang, Xiao-Xia

    2016-08-01

    There is no gold diagnostic standard for BCR-ABL fusion gene negative chronic myeloproliterative neoplasm(cMPN). The following detection methods such as comprehensive bone marrow cell morphology, bone marrow pathology, genetic mutation, flow cytometry and immunohistochemical are needed to diagnose the BCR-ABL fusion gene positive cMPN. The JAK2 mutation can be used as a specific diagnostic criteria for polycythemia vera (PV), but there is no specific and sensitive indication for the JAK2 mutation-negative MPN. CALR mutation would be an indication in a certain extent. In this review, the CALR mutation detection, detection mean and its correlation with disease diagnosis and prognosis etc were summarized. PMID:27531810

  11. The distribution and number of Leu-7 (CD57 positive cells in lung tissue from patients with pulmonary fibrosis.

    Directory of Open Access Journals (Sweden)

    Yamanouchi H

    2002-04-01

    Full Text Available Leu-7 positive lymphocytes, including natural killer cells, play an important role in the immune system's surveillance function to prevent the development of cancer. The incidence of lung cancer is significantly high in patients with end-stage pulmonary fibrosis. We hypothesized that the number of Leu-7 positive cells may be decreased in areas of severe pulmonary fibrosis. To demonstrate this, Leu-7 positive cells were immunohistochemically stained in 41 lung specimens obtained from patients with idiopathic pulmonary fibrosis and pulmonary fibrosis associated with collagen vascular disorders. The number of Leu-7 positive cells was evaluated according to the pathological findings. In pathologically normal lung, Leu-7 positive cells were mostly found within the capillaries of the septa and rarely in the alveolar space or the stroma. The number of Leu-7 positive cells was 0.69 +/- 0.15 in areas of advanced fibrosis (n = 41, 2.39 +/- 0.60 in areas that had newly developeing fibrosis (n = 41, 1.14 +/- 0.57 in bronchiolitis obliterans organizing pneumonia (n = 9, and 1.35 +/- 0.87 in diffuse alveolar damage (DAD (n = 11. The number of Leu-7 positive cells in areas of newly developing fibrosis (2.39 +/- 0.60 was significantly higher than that in areas of established fibrosis (0.69 +/- 0.15, P < 0.05. Our present study demonstrates a significant decrease in the number of Leu-7 positive cells in areas of advanced fibrosis. This evidence may partly explain the high incidence of lung cancer associated with pulmonary fibrosis.

  12. Different Characters of Spleen OX-62 Positive Dendritic Cells between Fischer and Lewis Rats

    Institute of Scientific and Technical Information of China (English)

    Linsong Yang; Yayi Hou

    2006-01-01

    The phenotype, DNA-binding activities of NF-κB, cytokine production, endocytosis and stimulatory capacity of spleen OX-62-positive dendritc cells (SDCs) from Fischer rats were compared with those from Lewis rats. Results showed that the expressions of CD11b, MHC-Ⅱ, CD8, CD45RA, CD54 and CD86 on SDCs were significantly higher in Fischer than those in Lewis rats. The levels of IL-2, IL-4, IL-10 and IFN-γ in SDCs from Fischer rats were distinctly higher than those from Lewis. Both stimulatory capacity and DNA-binding activities of NF-κB in SDCs were all lower in Fischer than those in Lewis rats. These differences may partly contribute to rat strainspecificity in susceptibility to chronic inflammatory stimuli.

  13. p75 neurotrophin receptor positive dental pulp stem cells: new hope for patients with neurodegenerative disease and neural injury.

    Science.gov (United States)

    Dai, Jie-wen; Yuan, Hao; Shen, Shun-yao; Lu, Jing-ting; Zhu, Xiao-fang; Yang, Tong; Zhang, Jiang-fei; Shen, Guo-fang

    2013-08-01

    Neurodegenerative diseases and neural injury are 2 of the most feared disorders that afflict humankind by leading to permanent paralysis and loss of sensation. Cell based treatment for these diseases had gained special interest in recent years. Previous studies showed that dental pulp stem cells (DPSCs) could differentiate toward functionally active neurons both in vitro and in vivo, and could promote neuranagenesis through both cell-autonomous and paracrine neuroregenerative activities. Some of these neuroregenerative activities were unique to tooth-derived stem cells and superior to bone marrow stromal cells. However, DPSCs used in most of these studies were mixed and unfractionated dental pulp cells that contain several types of cells, and most were fibroblast cells while just contain a small portion of DPSCs. Thus, there might be weaker ability of neuranagenesis and more side effects from the fibroblast cells that cannot differentiate into neural cells. p75 neurotrophin receptor (p75NTR) positive DPSCs subpopulation was derived from migrating cranial neural crest cells and had been isolated from DPSCs, which had capacity of differentiation into neurons and repairing neural system. In this article, we hypothesize that p75NTR positive DPSCs simultaneously have greater propensity for neuronal differentiation and fewer side effects from fibroblast, and in vivo transptantation of autologous p75NTR positive DPSCs is a novel method for neuranagenesis. This will bring great hope to patients with neurodegenerative disease and neural injury.

  14. Hyperfractionated high-dose total body irradiation in bone marrow transplantation for Ph{sup 1}-positive acute lymphoblastic leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Kikuchi, Akira; Ebihara, Yasuhiro; Mitsui, Tetsuo [Tokyo Univ. (Japan). Hospital of the Institute of Medical Science] [and others

    1998-12-01

    In two cases of Philadelphia-positive childhood acute lymphoblastic leukemia (Ph{sup 1} ALL), we performed allogeneic bone marrow transplantation (AlloBMT) with preconditioning regimen, including hyperfractionated high-dose total body irradiation (TBI) (13.5 Gy, in 9 fractions). Their disease statuses at BMT were hematological relapse in case 1 and molecular relapse in case 2. Bone marrow donors were unrelated in case 1, and HLA was a partially mismatched mother in case 2. Regimen-related toxicity was tolerable in both cases. Hematological recovery was rapid, and engraftment was obtained on day 14 in case 1 and on day 12 in case 2. BCR/ABL message in bone marrow disappeared on day 89 in case 1 and on day 19 in case 2 and throughout their subsequent clinical courses. Although short-term MTX and Cy-A continuous infusion were used for GVHD prophylaxis, grade IV GVHD was observed in case 1 and grade III in case 2. Both cases experienced hemorrhagic cystitis because of adenovirus type 11 infection. Although case 1 died of interstitial pneumonitis on day 442, case 2 has been free of disease through day 231. AlloBMT for Ph{sup 1} ALL with preconditioning regimen including hyperfractionated high-dose TBI is considered to be worth further investigation. (author)

  15. Squamous cell carcinoma of the anus-an opportunistic cancer in HIV-positive male homosexuals

    Institute of Scientific and Technical Information of China (English)

    Pascal Gervaz; Alexandra Calmy; Ymer Durmishi; Abdelkarim S Allal; Philippe Morel

    2011-01-01

    Squamous cell carcinoma of the anus (SCCA) is a common cancer in the human immunodeficiency virus (HIV)- infected population, and its incidence continues to increase in male homosexuals. Combined chemoradiation with mitomycin C and 5-fluorouracil was poorly tolerated by severely immunocompromised patients in the early 1990s. In the era of highly active antiretroviral therapy (HAART), however, recent data indicate that: (1) most HIV patients with anal cancer can tolerate standard chemotherapy regimens; and (2) this approach is associated with survival rates similar to those of HIV-negative patients. However, HIV-positive patients with SCCA are much younger, more likely to develop local tumor recurrence, and ultimately die from anal cancer than immune competent patients. Taken together, these findings suggest that anal cancer is an often fatal neoplasia in middle- aged HIV-positive male homosexuals. In this population, SCCA is an opportunistic disease resulting in patients with suboptimal immune function from persistent papillomaviruses (HPVs). Large-scale cancer-prevention strategies (routine anuscopy and anal papanicolaou testing) should be implemented in this population. In addition, definitive eradication of oncogenic HPVs within the anogenital mucosa of high-risk individuals might require a proactive approach with repeated vaccination.

  16. Quantitative trait gene Slit2 positively regulates murine hematopoietic stem cell numbers.

    Science.gov (United States)

    Waterstrat, Amanda; Rector, Kyle; Geiger, Hartmut; Liang, Ying

    2016-01-01

    Hematopoietic stem cells (HSC) demonstrate natural variation in number and function. The genetic factors responsible for the variations (or quantitative traits) are largely unknown. We previously identified a gene whose differential expression underlies the natural variation of HSC numbers in C57BL/6 (B6) and DBA/2 (D2) mice. We now report the finding of another gene, Slit2, on chromosome 5 that also accounts for variation in HSC number. In reciprocal chromosome 5 congenic mice, introgressed D2 alleles increased HSC numbers, whereas B6 alleles had the opposite effect. Using gene array and quantitative polymerase chain reaction, we identified Slit2 as a quantitative trait gene whose expression was positively correlated with the number of HSCs. Ectopic expression of Slit2 not only increased the number of the long-term colony forming HSCs, but also enhanced their repopulation capacity upon transplantation. Therefore, Slit2 is a novel quantitative trait gene and a positive regulator of the number and function of murine HSCs. This finding suggests that Slit2 may be a potential therapeutic target for the effective in vitro and in vivo expansion of HSCs without compromising normal hematopoiesis. PMID:27503415

  17. Quantitative trait gene Slit2 positively regulates murine hematopoietic stem cell numbers

    Science.gov (United States)

    Waterstrat, Amanda; Rector, Kyle; Geiger, Hartmut; Liang, Ying

    2016-01-01

    Hematopoietic stem cells (HSC) demonstrate natural variation in number and function. The genetic factors responsible for the variations (or quantitative traits) are largely unknown. We previously identified a gene whose differential expression underlies the natural variation of HSC numbers in C57BL/6 (B6) and DBA/2 (D2) mice. We now report the finding of another gene, Slit2, on chromosome 5 that also accounts for variation in HSC number. In reciprocal chromosome 5 congenic mice, introgressed D2 alleles increased HSC numbers, whereas B6 alleles had the opposite effect. Using gene array and quantitative polymerase chain reaction, we identified Slit2 as a quantitative trait gene whose expression was positively correlated with the number of HSCs. Ectopic expression of Slit2 not only increased the number of the long-term colony forming HSCs, but also enhanced their repopulation capacity upon transplantation. Therefore, Slit2 is a novel quantitative trait gene and a positive regulator of the number and function of murine HSCs. This finding suggests that Slit2 may be a potential therapeutic target for the effective in vitro and in vivo expansion of HSCs without compromising normal hematopoiesis. PMID:27503415

  18. Quantitative trait gene Slit2 positively regulates murine hematopoietic stem cell numbers.

    Science.gov (United States)

    Waterstrat, Amanda; Rector, Kyle; Geiger, Hartmut; Liang, Ying

    2016-08-09

    Hematopoietic stem cells (HSC) demonstrate natural variation in number and function. The genetic factors responsible for the variations (or quantitative traits) are largely unknown. We previously identified a gene whose differential expression underlies the natural variation of HSC numbers in C57BL/6 (B6) and DBA/2 (D2) mice. We now report the finding of another gene, Slit2, on chromosome 5 that also accounts for variation in HSC number. In reciprocal chromosome 5 congenic mice, introgressed D2 alleles increased HSC numbers, whereas B6 alleles had the opposite effect. Using gene array and quantitative polymerase chain reaction, we identified Slit2 as a quantitative trait gene whose expression was positively correlated with the number of HSCs. Ectopic expression of Slit2 not only increased the number of the long-term colony forming HSCs, but also enhanced their repopulation capacity upon transplantation. Therefore, Slit2 is a novel quantitative trait gene and a positive regulator of the number and function of murine HSCs. This finding suggests that Slit2 may be a potential therapeutic target for the effective in vitro and in vivo expansion of HSCs without compromising normal hematopoiesis.

  19. Positive feedback can lead to dynamic nanometer-scale clustering on cell membranes

    Energy Technology Data Exchange (ETDEWEB)

    Wehrens, Martijn; Rein ten Wolde, Pieter [FOM Institute AMOLF, Science Park 104, 1098 XG Amsterdam (Netherlands); Mugler, Andrew, E-mail: amugler@purdue.edu [FOM Institute AMOLF, Science Park 104, 1098 XG Amsterdam (Netherlands); Department of Physics, Purdue University, West Lafayette, Indiana 47907 (United States)

    2014-11-28

    Clustering of molecules on biological membranes is a widely observed phenomenon. A key example is the clustering of the oncoprotein Ras, which is known to be important for signal transduction in mammalian cells. Yet, the mechanism by which Ras clusters form and are maintained remains unclear. Recently, it has been discovered that activated Ras promotes further Ras activation. Here we show using particle-based simulation that this positive feedback is sufficient to produce persistent clusters of active Ras molecules at the nanometer scale via a dynamic nucleation mechanism. Furthermore, we find that our cluster statistics are consistent with experimental observations of the Ras system. Interestingly, we show that our model does not support a Turing regime of macroscopic reaction-diffusion patterning, and therefore that the clustering we observe is a purely stochastic effect, arising from the coupling of positive feedback with the discrete nature of individual molecules. These results underscore the importance of stochastic and dynamic properties of reaction diffusion systems for biological behavior.

  20. Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria.

    Science.gov (United States)

    Catalão, Maria João; Figueiredo, Joana; Henriques, Mafalda X; Gomes, João Paulo; Filipe, Sérgio R

    2014-01-01

    The understanding of how Gram-positive bacteria divide and ensure the correct localization of different molecular machineries, such as those involved in the synthesis of the bacterial cell surface, is crucial to design strategies to fight bacterial infections. In order to determine the correct subcellular localization of fluorescent proteins in Streptococcus pneumoniae, we have previously described tools to express derivatives of four fluorescent proteins, mCherry, Citrine, CFP and GFP, to levels that allow visualization by fluorescence microscopy, by fusing the first ten amino acids of the S. pneumoniae protein Wze (the i-tag), upstream of the fluorescent protein. Here, we report that these tools can also be used in other Gram-positive bacteria, namely Lactococcus lactis, Staphylococcus aureus and Bacillus subtilis, possibly due to optimized translation rates. Additionally, we have optimized the i-tag by testing the effect of the first ten amino acids of other pneumococcal proteins in the increased expression of the fluorescent protein Citrine. We found that manipulating the structure and stability of the 5' end of the mRNA molecule, which may influence the accessibility of the ribosome, is determinant to ensure the expression of a strong fluorescent signal.

  1. A double mechanism for the mesenchymal stem cells' positive effect on pancreatic islets.

    Directory of Open Access Journals (Sweden)

    Arianna Scuteri

    Full Text Available The clinical usability of pancreatic islet transplantation for the treatment of type I diabetes, despite some encouraging results, is currently hampered by the short lifespan of the transplanted tissue. In vivo studies have demonstrated that co-transplantation of Mesenchymal Stem Cells (MSCs with transplanted pancreatic islets is more effective with respect to pancreatic islets alone in ensuring glycemia control in diabetic rats, but the molecular mechanisms of this action are still unclear. The aim of this study was to elucidate the molecular mechanisms of the positive effect of MSCs on pancreatic islet functionality by setting up direct, indirect and mixed co-cultures. MSCs were both able to prolong the survival of pancreatic islets, and to directly differentiate into an "insulin-releasing" phenotype. Two distinct mechanisms mediated these effects: i the survival increase was observed in pancreatic islets indirectly co-cultured with MSCs, probably mediated by the trophic factors released by MSCs; ii MSCs in direct contact with pancreatic islets started to express Pdx1, a pivotal gene of insulin production, and then differentiated into insulin releasing cells. These results demonstrate that MSCs may be useful for potentiating pancreatic islets' functionality and feasibility.

  2. Positioning of follicular dendritic cells within the spleen controls prion neuroinvasion.

    Science.gov (United States)

    Prinz, Marco; Heikenwalder, Mathias; Junt, Tobias; Schwarz, Petra; Glatzel, Markus; Heppner, Frank L; Fu, Yang-Xin; Lipp, Martin; Aguzzi, Adriano

    2003-10-30

    Peripheral infection is the natural route of transmission in most prion diseases. Peripheral prion infection is followed by rapid prion replication in lymphoid organs, neuroinvasion and progressive neurological disease. Both immune cells and nerves are involved in pathogenesis, but the mechanisms of prion transfer from the immune to the nervous system are unknown. Here we show that ablation of the chemokine receptor CXCR5 juxtaposes follicular dendritic cells (FDCs) to major splenic nerves, and accelerates the transfer of intraperitoneally administered prions into the spinal cord. Neuroinvasion velocity correlated exclusively with the relative locations of FDCs and nerves: transfer of CXCR5-/- bone marrow to wild-type mice induced perineural FDCs and enhanced neuroinvasion, whereas reciprocal transfer to CXCR5-/- mice abolished them and restored normal efficiency of neuroinvasion. Suppression of lymphotoxin signalling depleted FDCs, abolished splenic infectivity, and suppressed acceleration of pathogenesis in CXCR5-/- mice. This suggests that prion neuroimmune transition occurs between FDCs and sympathetic nerves, and relative positioning of FDCs and nerves controls the efficiency of peripheral prion infection. PMID:14562059

  3. CD8 positive T cells express IL-17 in patients with chronic obstructive pulmonary disease

    Directory of Open Access Journals (Sweden)

    Eidelman David H

    2011-04-01

    Full Text Available Abstract Background Chronic obstructive pulmonary disease (COPD is a progressive and irreversible chronic inflammatory disease of the lung. The nature of the immune reaction in COPD raises the possibility that IL-17 and related cytokines may contribute to this disorder. This study analyzed the expression of IL-17A and IL-17F as well as the phenotype of cells producing them in bronchial biopsies from COPD patients. Methods Bronchoscopic biopsies of the airway were obtained from 16 COPD subjects (GOLD stage 1-4 and 15 control subjects. Paraffin sections were used for the investigation of IL-17A and IL-17F expression in the airways by immunohistochemistry, and frozen sections were used for the immunofluorescence double staining of IL-17A or IL-17F paired with CD4 or CD8. In order to confirm the expression of IL-17A and IL-17F at the mRNA level, a quantitative RT-PCR was performed on the total mRNA extracted from entire section or CD8 positive cells selected by laser capture microdissection. Results IL-17F immunoreactivity was significantly higher in the bronchial biopsies of COPD patients compared to control subjects (P P P P Conclusion These findings support the notion that Th17 cytokines could play important roles in the pathogenesis of COPD, raising the possibility of using this mechanism as the basis for novel therapeutic approaches.

  4. The p16(INK4A)/pRb pathway and telomerase activity define a subgroup of Ph+ adult Acute Lymphoblastic Leukemia associated with inferior outcome.

    Science.gov (United States)

    Chien, Wei W; Catallo, Régine; Chebel, Amel; Baranger, Laurence; Thomas, Xavier; Béné, Marie-Christine; Gerland, Luc M; Schmidt, Aline; Beldjord, Kheira; Klein, Nathalie; Escoffre-Barbe, Martine; Leguay, Thibaut; Huguet, Françoise; Larosa, Fabrice; Hayette, Sandrine; Plesa, Adriana; Ifrah, Norbert; Dombret, Hervé; Salles, Gilles; Chassevent, Agnès; Ffrench, Martine

    2015-04-01

    Adult Acute Lymphoblastic Leukemia (ALL) therapies have been improved by pediatric-like approaches. However, treatment failures and relapses are common and new markers are needed to identify patients with poor prognosis in prospective trials. The p16(INK4A)/CDK4-6/pRb pathway and telomerase activity, which are implicated in cell activation and aging, were analyzed to identify new prognostic markers. Proteins of the p16(INK4A)/CDK4-6/pRb pathway and telomerase activity were analyzed in 123 adult B-cell precursor (BCP) ALL cases included in the GRAALL/GRAAPH trials. We found a significantly increased expression of p16(INK4A) in BCP-ALLs with MLL rearrangement. Telomerase activity was significantly lower in Philadelphia chromosome-negative/IKAROS-deleted (BCR-ABL1(-)/IKAROS(del)) cases compared to Philadelphia chromosome-positive (BCR-ABL1+) BCP-ALLs. In BCR-ABL1+ ALLs, high CDK4 expression, phosphorylated pRb (p-pRb) and telomerase activity were significantly associated with a shorter disease-free survival (DFS) and event-free survival (EFS). Enhanced p16(INK4A) expression was only related to a significantly shorter DFS. In vitro analyses of normal stimulated lymphocytes after short- and long-term cultures demonstrated that the observed protein variations of poor prognosis in BCR-ABL1+ ALLs may be related to cell activation but not to cell aging. For these patients, our findings argue for the development of therapeutic strategies including the addition of new lymphocyte activation inhibitors to current treatments.

  5. Niemann-Pick disease, type B with TRAP-positive storage cells and secondary sea blue histiocytosis

    Directory of Open Access Journals (Sweden)

    R. Saxena

    2009-09-01

    Full Text Available We present 2 cases of Niemann Pick disease, type B with secondary sea-blue histiocytosis. Strikingly, in both cases the Pick cells were positive for tartrate resistant acid phosphatase, a finding hitherto described only in Gaucher cells. This report highlights the importance of this finding as a potential cytochemical diagnostic pitfall in the diagnosis of Niemann Pick disease.

  6. Niemann-Pick disease, type B with TRAP-positive storage cells and secondary sea blue histiocytosis

    OpenAIRE

    R. Saxena; Pati, H. P.; Dutta, S.; Kar, R.; Sharma, P.

    2009-01-01

    We present 2 cases of Niemann Pick disease, type B with secondary sea-blue histiocytosis. Strikingly, in both cases the Pick cells were positive for tartrate resistant acid phosphatase, a finding hitherto described only in Gaucher cells. This report highlights the importance of this finding as a potential cytochemical diagnostic pitfall in the diagnosis of Niemann Pick disease.

  7. The MYB23 gene provides a positive feedback loop for cell fate specification in the Arabidopsis root epidermis.

    Science.gov (United States)

    Kang, Yeon Hee; Kirik, Victor; Hulskamp, Martin; Nam, Kyoung Hee; Hagely, Katherine; Lee, Myeong Min; Schiefelbein, John

    2009-04-01

    The specification of cell fates during development requires precise regulatory mechanisms to ensure robust cell type patterns. Theoretical models of pattern formation suggest that a combination of negative and positive feedback mechanisms are necessary for efficient specification of distinct fates in a field of differentiating cells. Here, we examine the role of the R2R3-MYB transcription factor gene, AtMYB23 (MYB23), in the establishment of the root epidermal cell type pattern in Arabidopsis thaliana. MYB23 is closely related to, and is positively regulated by, the WEREWOLF (WER) MYB gene during root epidermis development. Furthermore, MYB23 is able to substitute for the function of WER and to induce its own expression when controlled by WER regulatory sequences. We also show that the MYB23 protein binds to its own promoter, suggesting a MYB23 positive feedback loop. The localization of MYB23 transcripts and MYB23-green fluorescent protein (GFP) fusion protein, as well as the effect of a chimeric MYB23-SRDX repressor construct, links MYB23 function to the developing non-hair cell type. Using mutational analyses, we find that MYB23 is necessary for precise establishment of the root epidermal pattern, particularly under conditions that compromise the cell specification process. These results suggest that MYB23 participates in a positive feedback loop to reinforce cell fate decisions and ensure robust establishment of the cell type pattern in the Arabidopsis root epidermis.

  8. Erythropoietin Receptor Positive Circulating Progenitor Cells and Endothelial Progenitor Cells in Patients with Different Stages of Diabetic Retinopathy

    Institute of Scientific and Technical Information of China (English)

    Liu-mei Hu; Guo-xu Xu; Guo-tong XU; Wei-ye Li; Xia Lei; Bo Ma; Yu Zhang; Yan Yan; Ya-lan Wu; Ge-zhi Xu; Wen Ye; Ling Wang

    2011-01-01

    Objective To investigate the possible involvement of erythropoietin (EPO)/erythropoietin receptor(EPOR) system in neovascularization and vascular regeneration in diabetic retinopathy (DR).Methods EPOR positive circulating progenitor cells (CPCs: CD34+) and endothelial progenitor cells (EPCs: CD34+KDR+) were assessed by flow cytometry in type 2 diabetic patients with different stages of DR. The cohort consisted of age- and sex-matched control patients without diabetes (n=7), non-prolif-erative DR (NPDR, n=7), proliferative DR (PDR, n=8), and PDR complicated with diabetic nephropathy (PDR-DN, n=7). Results The numbers of EPOR+ CPCs and EPOR+ EPCs were reduced remarkably in NPDR compared with the control group (both P<0.01), whereas rebounded in PDR and PDR-DN groups in varying degrees. Similar changes were observed in respect of the proportion of EPOR+ CPCs in CPCs (NPDR vs.control, P< 0.01) and that of EPOR+ EPCs in EPCs (NPDR vs. control, P< 0.05). Conclusion Exogenous EPO, mediated via the EPO/EPOR system of EPCs, may alleviate the im-paired vascular regeneration in NPDR, whereas it might aggravate retinal neovascularization in PDR due to a rebound of EPOR+ EPCs associated with ischemia.

  9. Spontaneous Remission of Epstein-Barr Virus-Positive Diffuse Large B-Cell Lymphoma of the Elderly

    Directory of Open Access Journals (Sweden)

    T. Mizuno

    2013-05-01

    Full Text Available A 94-year-old female patient presented with anorexia and left axillar lymphadenopathy on admission. Her past history was angina pectoris at 83 years of age and total gastrectomy due to gastric cancer at 87 years. The family history revealed that her son had had a malignant lymphoma, the histopathological diagnosis of which was diffuse large B-cell lymphoma. A physical examination showed both cervical, axillar, and inguinal lymphadenopathy without tenderness. She had elevated lactate dehydrogenase, ferritin, and soluble interleukin-2 receptor (sIL-2R. Whole-body computed tomography confirmed the cervical, axillary, and inguinal lymphadenopathy. Gallium-68 imaging revealed positive accumulation in these superficial lymph nodes. A right inguinal lymph node biopsy showed features of Epstein-Barr virus-associated lymphoproliferative disorder. Immunohistological studies on this lymph node biopsy showed CD20-positive large cells, CD3-positive small cells, and CD30-partly-positive large cells. In situ hybridization showed Epstein-Barr virus-positive, LMP-partly-positive, and EBNA2-negative cells. She refused chemotherapy as her son had died from hematemesis during chemotherapy. She received intravenous hyperalimentation for 1 month after admission. No palpable lymph nodes were identified by physical examination or computed tomography 3 months after admission, and regression of lactate dehydrogenase, ferritin, and sIL-2R was observed. She recovered from anorexia and was discharged. She died from pneumonia 10 months later after initial symptoms of anorexia. The autopsy showed no superficial lymphadenopathy.

  10. Without peripheral interference, thymic deletion is mediated in a cohort of double-positive cells without classical activation

    OpenAIRE

    Zhan, Yifan; Purton, Jared F.; Godfrey, Dale I.; Cole, Timothy J.; Heath, William R.; Lew, Andrew M.

    2003-01-01

    Peripheral activation can cause bystander thymocyte death by eliciting a “cytokine storm.” This event complicates in vivo studies using exogenous ligand-induced models of negative selection. A stable transgenic model that selectively eliminates peripheral CD4 cells has allowed us to analyze negative selection as direct cognate events in two T cell receptor transgenic mice, OT-II and DO11. Whereas cognate peptide induced a massive deletion in double-positive (DP) cells in mice with peripheral ...

  11. Positional Isomers of Aspirin Are Equally Potent in Inhibiting Colon Cancer Cell Growth: Differences in Mode of Cyclooxygenase Inhibition

    OpenAIRE

    Kodela, Ravinder; Chattopadhyay, Mitali; Goswami, Satindra; Gan, Zong Yuan; Rao, Praveen P.N.; Nia, Kamran V.; Velázquez-Martínez, Carlos A.; Kashfi, Khosrow

    2013-01-01

    We compared the differential effects of positional isomers of acetylsalicylic acid (o-ASA, m-ASA, and p-ASA) on cyclooxygenase (COX) inhibition, gastric prostaglandin E2 (PGE2), malondialdehyde, tumor necrosis factor-alpha (TNF-α) levels, superoxide dismutase (SOD) activity, human adenocarcinoma colon cancer cell growth inhibition, cell proliferation, apoptosis, and cell-cycle progression. We also evaluated the gastric toxicity exerted by ASA isomers. All ASA isomers inhibit COX enzymes, but ...

  12. High Level of Tregs Is a Positive Prognostic Marker in Patients with HPV-Positive Oral and Oropharyngeal Squamous Cell Carcinomas

    Directory of Open Access Journals (Sweden)

    E. Lukesova

    2014-01-01

    Full Text Available Background. Human papillomaviruses (HPVs have been proved as one of the etiological factors of oropharyngeal squamous cell carcinoma (OPSCC. Patients with tumors of viral etiology have a lower recurrence rate and better prognosis. OPSCC is linked to an alteration in the immune system. Only a limited number of studies have correlated both the immunological parameters and HPV status with patient prognosis. The aim of this study was to determine whether HPV infection and the immunological status influence patient prognosis individually or in concurrence. Material and Methods. Sixty patients with oral and oropharyngeal carcinomas were enrolled. They were divided into HPV-positive and HPV-negative groups based on the expression of HPV 16 E6 mRNA. Basic lymphocyte subpopulations were determined in the peripheral blood by means of flow cytometry. Results. Significantly better disease-specific survival (DSS was observed in patients with HPV-positive tumors. Nodal status, tumor grade, recurrence, and CD8+/Tregs ratio were identified as factors influencing DSS. A higher level of Tregs and a lower ratio of CD8/Tregs influenced overall survival (OS independently of HPV status and age. Patients with HPV-positive tumors and high levels of Tregs survived significantly better than patients from the other groups. Conclusion. Better survival is associated with HPV positivity and elevated Tregs levels. Our data suggest that HPV infection and Tregs do not influence patient prognosis in concurrence.

  13. 亲缘异基因造血干细胞移植联合甲磺酸伊马替尼治疗Ph阳性白血病%Hematopoietic stem cell transplantation in combination with imatinib mesylate for treatment of Philadelphia-positive leukemia

    Institute of Scientific and Technical Information of China (English)

    李梦醒; 王季石; 张燕; 孙志强; 赵鹏; 卢英豪

    2014-01-01

    BACKGROUND:Hematopoietic stem cel transplantation is recognized as the only method of curing Philadelphia-positive leukemia. Imatinib mesylate is a competitive inhibitor of the BCR/ABL tyrosine kinase, which has been used more and more in the treatment of Philadelphia-positive leukemia. OBJECTIVE:To observe the curative effect of imatinib mesylate combined with alogeneic hematopoietic stem cel transplantation for treatment of Philadelphia-positive leukemia. METHODS: We retrospectively analyzed the clinical effect of 12 patients with Philadelphia-positive leukemia who were treated with the combined therapy of imatinib mesylate and alogeneic hematopoietic stem cel transplantation from January 2011 to October 2012, and reviewed the relevant literatures. RESULTS AND CONCLUSION: Hematopoietic reconstitution was achieved in al 12 patients and the median times of neutrophil recovery and platelet recovery were 15 and 18 days, respectively. Of the 12 patients, 7 patients developed acute graft-versus-host disease II, 1 developed acute graft-versus-host disease III, 7 developed localized chronic graft-versus-host disease, and 3 developed extensive chronic graft-versus-host disease. Leukemia-free survival rate was 66.7%, and transplant-related mortality was 25.0%. The overal survival rate of HLA-matched sibling donors was 75.0%. The mean disease-free survival period was 8.5 months (7-17 months), and the time needed for BCR/ABL becoming negative was 2-5 months. As an effective and safe method for Philadelphia-positive leukemia, alogeneic hematopoietic stem cel transplantation combined with imatinib mesylate before and after transplantation reduces the leukemia cel load before transplantation, inhibits the proliferation of residual leukemia cels, and promotes ful chimerism change.%背景:造血干细胞移植是可以治愈Ph+白血病有效方法,甲磺酸伊马替尼是一种高度特异的酪氨酸激酶抑制剂,能抑制BCR/ABL酪氨酸激酶活性,在Ph+白血病

  14. Autoimmune pancreatitis results from loss of TGFβ signalling in S100A4-positive dendritic cells

    Science.gov (United States)

    Boomershine, C S; Chamberlain, A; Kendall, P; Afshar-Sharif, A-R; Huang, H; Washington, M K; Lawson, W E; Thomas, J W; Blackwell, T S; Bhowmick, N A

    2009-01-01

    Background and aims: Autoimmune pancreatitis (AIP) is a poorly understood human disease affecting the exocrine pancreas. The goal of the present study was to elucidate the pathogenic mechanisms underlying pancreatic autoimmunity in a murine disease model. Methods: A transgenic mouse with an S100A4/fibroblast-specific protein 1 (FSP1) Cre-mediated conditional knockout of the transforming growth factor β (TGFβ) type II receptor, termed Tgfbr2fspKO, was used to determine the direct role of TGFβ in S100A4+ cells. Immunohistochemical studies suggested that Tgfbr2fspKO mice develop mouse AIP (mAIP) characterised by interlobular ductal inflammatory infiltrates and pancreatic autoantibody production. Fluorescence-activated cell sorting (FACS)-isolated dendritic cells (DCs) from diseased pancreata were verified to have S100A4-Cre-mediated DNA recombination. Results: The Tgfbr2fspKO mice spontaneously developed mAIP by 6 weeks of age. DCs were confirmed to express S100A4, a previously reported protein expressed by fibroblasts. Adoptive transfer of bone marrow-derived DCs from Tgfbr2fspKO mice into 2-week-old syngenic wild-type C57BL/6 mice resulted in reproduction of pancreatitis within 6 weeks. Similar adoptive transfer of wild-type DCs had no effect on pancreas pathology of the host mice. The inability to induce pancreatitis by adoptive transfer of Tgfbr2fspKO DCs in adult mice suggested a developmental event in mAIP pathogenesis. Tgfbr2fspKO DCs undergo elevated maturation in response to antigen and increased activation of naïve CD4-positive T cells. Conclusion: The development of mAIP in the Tgfbr2fspKO mouse model illustrates the role of TGFβ in maintaining myeloid DC immune tolerance. The loss of immune tolerance in myeloid S100A4+ DCs can mediate mAIP and may explain some aspects of AIP disease pathogenesis. PMID:19625278

  15. A novel fixative for immunofluorescence staining of CD133-positive glioblastoma stem cells

    OpenAIRE

    Sherman, Jonathan H.; Redpath, Gerard T.; Redick, Jan A.; Purow, Benjamin W.; Laws, Edward R.; Jane, John A.; Shaffrey, Mark E.; Hussaini, Isa M.

    2011-01-01

    Isolation of glioblastoma stem cells requires incubation of tumor cells in a neural stem cell media. Neurospheres containing these glioblastoma stem cells are formed after approximately a five-day period. These cells can then be analyzed for the presence of stem cell markers. Immunofluorescence staining for these markers can serve as a valuable tool for analyzing the intact neurosphere directly in stem cell media. Here we present the use of a novel fixative (1,4-benzoquinone) for immunofloure...

  16. The ER membrane-anchored ubiquitin ligase Hrd1 is a positive regulator of T-cell immunity

    Science.gov (United States)

    Xu, Yuanming; Zhao, Fang; Qiu, Quan; Chen, Kun; Wei, Juncheng; Kong, Qingfei; Gao, Beixue; Melo-Cardenas, Johanna; Zhang, Bin; Zhang, Jinping; Song, Jianxun; Zhang, Donna D.; Zhang, Jianing; Fan, Yunping; Li, Huabin; Fang, Deyu

    2016-01-01

    Identification of positive regulators of T-cell immunity induced during autoimmune diseases is critical for developing novel therapies. The endoplasmic reticulum resident ubiquitin ligase Hrd1 has recently emerged as a critical regulator of dendritic cell antigen presentation, but its role in T-cell immunity is unknown. Here we show that genetic deletion of Hrd1 in mice inhibits T-cell proliferation, production of IL-2, and differentiation of Th1 and Th17 cells, and consequently protects mice from experimental autoimmune encephalomyelitis. Hrd1 facilitates T-cell proliferation by the destruction of cyclin-dependent kinase inhibitor p27kip1, and deletion of p27kip1 in Hrd1-null T-cells rescues proliferative capacity but not the production of cytokines, including IL-2, IFN-γ and IL-17. T-cell expression of Hrd1 is higher in patients with multiple sclerosis than in healthy individuals, and knockdown of Hrd1 in human CD4+ T cells inhibits activation and differentiation to Th1 and Th17 cells. Our study identifies Hrd1 as a previously unappreciated positive regulator of T cells and implies that Hrd1 is a potential therapeutic target for autoimmune diseases. PMID:27417417

  17. Combination of CD34-positive cell subsets with infarcted myocardium-like matrix stiffness: a potential solution to cell-based cardiac repair.

    Science.gov (United States)

    Zhang, Shuning; Ma, Xin; Yao, Kang; Zhu, Hong; Huang, Zheyong; Shen, Li; Qian, Juying; Zou, Yunzeng; Sun, Aijun; Ge, Junbo

    2014-06-01

    Detection of the optimal cell transplantation strategy for myocardial infarction (MI) has attracted a great deal of attention. Commitment of engrafted cells to angiogenesis within damaged myocardium is regarded as one of the major targets in cell-based cardiac repair. Bone marrow-derived CD34-positive cells, a well-characterized population of stem cells, might represent highly functional endothelial progenitor cells and result in the formation of new blood vessels. Recently, physical microenvironment (extracellular matrix stiffness) around the engrafted cells was found to exert an essential impact on their fate. Stem cells are able to feel and respond to the tissue-like matrix stiffness to commit to a relevant lineage. Notably, the infarct area after MI experiences a time-dependent stiffness change from flexible to rigid. Our previous observations demonstrated myocardial stiffness-dependent differentiation of the unselected bone marrow-derived mononuclear cells (BMMNCs) along endothelial lineage cells. Myocardial stiffness (~42 kPa) within the optimal time domain of cell engraftment (at week 1 to 2) after MI provided a more favourable physical microenvironment for cell specification and cell-based cardiac repair. However, the difference in tissue stiffness-dependent cell differentiation between the specific cell subsets expressing and no expressing CD34 phenotype remains uncertain. We presumed that CD34-positive cell subsets facilitated angiogenesis and subsequently resulted in cardiac repair under induction of infarcted myocardium-like matrix stiffness compared with CD34-negative cells. If the hypothesis were true, it would contribute greatly to detect the optimal cell subsets for cell therapy and to establish an optimized therapy strategy for cell-based cardiac repair.

  18. Value of counting positive PHH3 cells in the diagnosis of uterine smooth muscle tumors.

    Science.gov (United States)

    Pang, Shu-Jie; Li, Cheng-Cheng; Shen, Yan; Liu, Yian-Zhu; Shi, Yi-Quan; Liu, Yi-Xin

    2015-01-01

    The diagnosis of uterine smooth muscle tumors including leiomyosarcomas (LMS), smooth muscle tumors of uncertain malignant potential (STUMP), bizarre (atypical) leiomyoma (BLM), mitotically active leiomyoma (MAL) and leiomyoma (LM) depends on a combination of microscopic features, such as mitoses, cytologic atypia, and coagulative tumor cell necrosis. However, a small number of these tumors still pose difficult diagnostic challenges. The assessment of accurate mitotic figures (MF) is one of the major parameters in the proper classification of uterine smooth muscle tumors. This assessment can be hampered by the presence of increased number of apoptotic bodies or pyknotic nuclei, which frequently mimic mitoses. Phospho-histone H3 (PHH3) is a recently described immunomarker specific for cells undergoing mitoses. In our study, we collected 132 cases of uterine smooth muscle tumors, including 26 LMSs, 16 STUMPs, 30 BLMs, 30 MALs and 30 LMs. We used mitosis specific marker PHH3 to count mitotic indexes (MI) of uterine smooth muscle tumors and compared with the mitotic indexes of hematoxylin and eosin (H&E). There is a positive correlation with the number of mitotic figures in H&E-stained sections and PHH3-stained sections (r=0.944, P0.05). The counting value of PHH3 in LMSs have significantly higher than STUMPs, BLMs, MALs and LMs (Pnumber of mitotic indexes in H&E. To conclude, our results show that counting PHH3 is a useful index in the diagnosis of uterine smooth muscle tumors and it can provide a more accurate index instead of the time-honored mitotic figure counts at a certain ratio.

  19. P-glycoprotein is expressed and causes resistance to chemotherapy in EBV-positive T-cell lymphoproliferative diseases.

    Science.gov (United States)

    Yoshimori, Mayumi; Takada, Honami; Imadome, Ken-Ichi; Kurata, Morito; Yamamoto, Kouhei; Koyama, Takatoshi; Shimizu, Norio; Fujiwara, Shigeyoshi; Miura, Osamu; Arai, Ayako

    2015-10-01

    Epstein-Barr virus-positive T-cell lymphoproliferative diseases (EBV-T-LPDs) are rare lymphomas with poor prognosis. Although chemotherapeutic strategies such as CHOP have been often selected, they have exhibited only limited efficacy. To clarify the mechanism of chemoresistance, we examined P-glycoprotein (P-gp) expression. P-gp acts as an energy-dependent efflux pump that excretes drugs from the cytoplasm, resulting in low-intracellular drug concentrations and poor sensitivity to chemotherapy. We examined P-gp expression in EBV-positive cells by immunohistochemistry staining in three patients of EBV-T-LPDs and the expression was detected in all patients. We also examined mdr1 mRNA expression by reverse-transcriptase polymerase-chain reaction (RT-PCR) in EBV-positive tumor cells from these patients and additional three patients. The expression was detected in all examined patients. In five EBV-T-LPDs patients, P-gp function was detected by Rhodamine-123 efflux assay in these cells. The efflux was inhibited by treatment with a P-gp inhibitor, cyclosporine A (CsA). We also examined and detected P-gp expression in EBV-positive T-cell lines SNT8 and SNT16 established from EBV-T-LPDs patients, by RT-PCR and western blotting. The function was also detected by Rhodamine-123 efflux in these cell lines. Inhibition and knock down of P-gp by CsA and siRNA, respectively, enhanced etoposide- and doxorubicin-induced cell death in the EBV-positive T-cell lines. Finally, we infected the T-cell line MOLT4 with EBV, and found that mdr1 mRNA expression and Rhodamine 123 efflux were upregulated after infection. These results indicated that enhanced P-gp expression contributed to the chemoresistance of EBV-T-LPDs.

  20. P-glycoprotein is expressed and causes resistance to chemotherapy in EBV-positive T-cell lymphoproliferative diseases

    International Nuclear Information System (INIS)

    Epstein–Barr virus-positive T-cell lymphoproliferative diseases (EBV-T-LPDs) are rare lymphomas with poor prognosis. Although chemotherapeutic strategies such as CHOP have been often selected, they have exhibited only limited efficacy. To clarify the mechanism of chemoresistance, we examined P-glycoprotein (P-gp) expression. P-gp acts as an energy-dependent efflux pump that excretes drugs from the cytoplasm, resulting in low-intracellular drug concentrations and poor sensitivity to chemotherapy. We examined P-gp expression in EBV-positive cells by immunohistochemistry staining in three patients of EBV-T-LPDs and the expression was detected in all patients. We also examined mdr1 mRNA expression by reverse-transcriptase polymerase-chain reaction (RT-PCR) in EBV-positive tumor cells from these patients and additional three patients. The expression was detected in all examined patients. In five EBV-T-LPDs patients, P-gp function was detected by Rhodamine-123 efflux assay in these cells. The efflux was inhibited by treatment with a P-gp inhibitor, cyclosporine A (CsA). We also examined and detected P-gp expression in EBV-positive T-cell lines SNT8 and SNT16 established from EBV-T-LPDs patients, by RT-PCR and western blotting. The function was also detected by Rhodamine-123 efflux in these cell lines. Inhibition and knock down of P-gp by CsA and siRNA, respectively, enhanced etoposide- and doxorubicin-induced cell death in the EBV-positive T-cell lines. Finally, we infected the T-cell line MOLT4 with EBV, and found that mdr1 mRNA expression and Rhodamine 123 efflux were upregulated after infection. These results indicated that enhanced P-gp expression contributed to the chemoresistance of EBV-T-LPDs

  1. Position of fuel cells in Italy; Situation des piles a combustible en Italie

    Energy Technology Data Exchange (ETDEWEB)

    Janot-Giorgetti, M.; Mottini, N.

    2000-02-01

    The main researches concerning the fuel cells in Italy are the PEFC (Polymer Electrolyte Fuel Cell) and the MCFC (Molten Carbonate Fuel Cell). This reports takes stock of these two techniques in Italy, explaining the running of these two types of cells and relating the Italian situation (development and research program, development programs of fuel cells vehicles). (O.M.)

  2. WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning.

    Science.gov (United States)

    Lee, M M; Schiefelbein, J

    1999-11-24

    The formation of the root epidermis of Arabidopsis provides a simple and elegant model for the analysis of cell patterning. A novel gene, WEREWOLF (WER), is described here that is required for position-dependent patterning of the epidermal cell types. The WER gene encodes a MYB-type protein and is preferentially expressed within cells destined to adopt the non-hair fate. Furthermore, WER is shown to regulate the position-dependent expression of the GLABRA2 homeobox gene, to interact with a bHLH protein, and to act in opposition to the CAPRICE MYB. These results suggest a simple model to explain the specification of the two root epidermal cell types, and they provide insight into the molecular mechanisms used to control cell patterning.

  3. File list: Unc.Bld.20.AllAg.CD4_CD8_double_positive_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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