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Sample records for bcr-abl positive cells

  1. Combination of bortezomib and mitotic inhibitors down-modulate Bcr-Abl and efficiently eliminates tyrosine-kinase inhibitor sensitive and resistant Bcr-Abl-positive leukemic cells.

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    Bucur, Octavian; Stancu, Andreea Lucia; Goganau, Ioana; Petrescu, Stefana Maria; Pennarun, Bodvael; Bertomeu, Thierry; Dewar, Rajan; Khosravi-Far, Roya

    2013-01-01

    Emergence of resistance to Tyrosine-Kinase Inhibitors (TKIs), such as imatinib, dasatinib and nilotinib, in Chronic Myelogenous Leukemia (CML) demands new therapeutic strategies. We and others have previously established bortezomib, a selective proteasome inhibitor, as an important potential treatment in CML. Here we show that the combined regimens of bortezomib with mitotic inhibitors, such as the microtubule-stabilizing agent Paclitaxel and the PLK1 inhibitor BI2536, efficiently kill TKIs-resistant and -sensitive Bcr-Abl-positive leukemic cells. Combined treatment activates caspases 8, 9 and 3, which correlate with caspase-induced PARP cleavage. These effects are associated with a marked increase in activation of the stress-related MAP kinases p38MAPK and JNK. Interestingly, combined treatment induces a marked decrease in the total and phosphorylated Bcr-Abl protein levels, and inhibits signaling pathways downstream of Bcr-Abl: downregulation of STAT3 and STAT5 phosphorylation and/or total levels and a decrease in phosphorylation of the Bcr-Abl-associated proteins CrkL and Lyn. Moreover, we found that other mitotic inhibitors (Vincristine and Docetaxel), in combination with bortezomib, also suppress the Bcr-Abl-induced pro-survival signals and result in caspase 3 activation. These results open novel possibilities for the treatment of Bcr-Abl-positive leukemias, especially in the imatinib, dasatinib and nilotinib-resistant CML cases.

  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

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    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. Activity of the Aurora kinase inhibitor VX-680 against Bcr/Abl-positive acute lymphoblastic leukemias.

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    Fei, Fei; Stoddart, Sonia; Groffen, John; Heisterkamp, Nora

    2010-05-01

    The emergence of resistance to tyrosine kinase inhibitors due to point mutations in Bcr/Abl is a challenging problem for Philadelphia chromosome-positive (Ph-positive) acute lymphoblastic leukemia (ALL) patients, especially for those with the T315I mutation, against which neither nilotinib or dasatinib shows significant activity. VX-680 is a pan-Aurora kinase inhibitor active against all Bcr/Abl proteins but has not been extensively examined in preclinical models of Ph-positive ALL. Here, we have tested VX-680 for the treatment of Bcr/Abl-positive ALL when leukemic cells are protected by the presence of stroma. Under these conditions, VX-680 showed significant effects on primary human Ph-positive ALL cells both with and without the T315I mutation, including ablation of tyrosine phosphorylation downstream of Bcr/Abl, decreased viability, and induction of apoptosis. However, drug treatment of human Ph-positive ALL cells for 3 days followed by drug removal allowed the outgrowth of abnormal cells 21 days later, and on culture of mouse Bcr/Abl ALL cells on stroma with lower concentrations of VX-680, drug-resistant cells emerged. Combined treatment of human ALL cells lacking the T315I mutation with both VX-680 and dasatinib caused significantly more cytotoxicity than each drug alone. We suggest that use of VX-680 together with a second effective drug as first-line treatment for Ph-positive ALL is likely to be safer and more useful than second-line treatment with VX-680 as monotherapy for drug-resistant T315I Ph-positive ALL.

  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. Characterization of the CDR3 structure of the Vβ21 T cell clone in patients with P210(BCR-ABL)-positive chronic myeloid leukemia and B-cell acute lymphoblastic leukemia.

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    Zha, Xianfeng; Chen, Shaohua; Yang, Lijian; Li, Bo; Chen, Yu; Yan, Xiaojuan; Li, Yangqiu

    2011-10-01

    The clonally expanded T cells identified in most cancer patients that respond to tumor-associated antigen such as P210(BCR-ABL) protein have definite, specific antitumor cytotoxicity. T cell receptor (TCR) Vβ CDR3 repertoire diversity was analyzed in patients with chronic myeloid leukemia (CML) and BCR-ABL(+) B-cell acute lymphoblastic leukemia (B-ALL) by GeneScan. A high frequency of oligoclonal expansion of the TCR Vβ21 subfamily was observed in the peripheral blood of CML and B-ALL patients. These clonally expanded Vβ21 T cells were correlated with the pathophysiologic process of CML. A conserved amino acid motif (SLxxV) was observed within the CDR3 region in only 3 patients with CML. Preferential usage of the Jβ segments was also observed in a minority of patients. The 3-dimensional structures of the CDR3 region containing the same motif or using the same Jβ segment displayed low similarity; on the contrary, the conformation of the CDR3 region containing no conserved motif in some T cell clones was highly similar. In conclusion, our findings indicate a high frequency of TCR Vβ21 subfamily expansion in p210(BCR-ABL)-positive CML and B-ALL patients. The characterization of the CDR3 structure was complex. Regrettably, at this time it was not possible to confirm that the Vβ21 T cell clones were derived from the stimulation of p210(BCR-ABL) protein.

  7. 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.

  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.

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    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.

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    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. The synthetic heat shock protein 90 (Hsp90) inhibitor EC141 induces degradation of Bcr-Abl p190 protein and apoptosis of Ph-positive acute lymphoblastic leukemia cells.

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    Tong, Wei-Gang; Estrov, Zeev; Wang, Yongtao; O'Brien, Susan; Faderl, Stefan; Harris, David M; Van Pham, Quin; Hazan-Halevy, Inbal; Liu, Zhiming; Koch, Patricia; Kantarjian, Hagop; Keating, Michael J; Ferrajoli, Alessandra

    2011-12-01

    The prognosis of patients with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) is poor. Chemotherapy is rarely curative and tyrosine kinase inhibitors (TKIs) induce only transient responses. Heat shock protein 90 (Hsp90) is a chaperone protein that is important in signal transduction, cell cycle control, and transcription regulation in both normal and leukemia cells. In the current study, we tested the growth inhibitory and apoptotic effects of a novel Hsp90 inhibitor, EC141 on the Ph+ ALL lines Z-119, Z-181, and Z-33, as well as primary bone marrow-derived blasts from patients with newly diagnosed Ph+ ALL. We found that EC141 inhibited the growth of Ph+ ALL cells in a concentration-dependent manner with IC(50) ranged from 1 to 10 nM. EC141 also inhibited the proliferation of primary bone marrow-derived blasts using the ALL blast colony assay. EC141 down-regulated Hsp90 and up-regulated Hsp70 protein levels, inhibited CrkL phosphorylation, and induced degradation of Bcr-Abl p190 protein through ubiquitin-dependent proteasomal pathway. Furthermore, exposure of Ph+ ALL cells to EC141 resulted in activation of caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and induction of apoptosis. In conclusion, our data suggest that EC141 is a potent Hsp90 inhibitor with activity against Ph+ ALL. Further studies to investigate the anticancer effect of EC141 either as a single agent, or in combination in Ph+ ALL and other hematological malignancies are warranted.

  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. Antileukemia effects of xanthohumol in Bcr/Abl-transformed cells involve nuclear factor-kappaB and p53 modulation.

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    Monteghirfo, Stefano; Tosetti, Francesca; Ambrosini, Claudia; Stigliani, Sara; Pozzi, Sarah; Frassoni, Francesco; Fassina, Gianfranco; Soverini, Simona; Albini, Adriana; Ferrari, Nicoletta

    2008-09-01

    The oncogenic Bcr-Abl tyrosine kinase activates various signaling pathways including phosphoinositide 3-kinase/Akt and nuclear factor-kappaB that mediate proliferation, transformation, and apoptosis resistance in Bcr-Abl+ myeloid leukemia cells. The hop flavonoid xanthohumol inhibits tumor growth by targeting the nuclear factor-kappaB and Akt pathways and angiogenesis. Here, we show that xanthohumol has in vitro activity against Bcr-Abl+ cells and clinical samples and retained its cytotoxicity when imatinib mesylate-resistant K562 cells were examined. Xanthohumol inhibition of K562 cell viability was associated with induction of apoptosis, increased p21 and p53 expression, and decreased survivin levels. We show that xanthohumol strongly inhibited Bcr-Abl expression at both mRNA and protein levels and show that xanthohumol caused elevation of intracellular reactive oxygen species and that the antioxidant N-acetylcysteine blunted xanthohumol-induced events. Further, we observed that xanthohumol inhibits leukemia cell invasion, metalloprotease production, and adhesion to endothelial cells, potentially preventing in vivo life-threatening complications of leukostasis and tissue infiltration by leukemic cells. As structural mutations and/or gene amplification in Bcr-Abl can circumvent an otherwise potent anticancer drug such as imatinib, targeting Bcr-Abl expression as well as its kinase activity could be a novel additional therapeutic approach for the treatment of Bcr-Abl+ myeloid leukemia.

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

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    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.

  16. 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.

  17. BCR-ABL1 compound mutations combining key kinase domain positions confer clinical resistance to ponatinib in Ph chromosome-positive leukemia.

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    Zabriskie, Matthew S; Eide, Christopher A; Tantravahi, Srinivas K; Vellore, Nadeem A; Estrada, Johanna; Nicolini, Franck E; Khoury, Hanna J; Larson, Richard A; Konopleva, Marina; Cortes, Jorge E; Kantarjian, Hagop; Jabbour, Elias J; Kornblau, Steven M; Lipton, Jeffrey H; Rea, Delphine; Stenke, Leif; Barbany, Gisela; Lange, Thoralf; Hernández-Boluda, Juan-Carlos; Ossenkoppele, Gert J; Press, Richard D; Chuah, Charles; Goldberg, Stuart L; Wetzler, Meir; Mahon, Francois-Xavier; Etienne, Gabriel; Baccarani, Michele; Soverini, Simona; Rosti, Gianantonio; Rousselot, Philippe; Friedman, Ran; Deininger, Marie; Reynolds, Kimberly R; Heaton, William L; Eiring, Anna M; Pomicter, Anthony D; Khorashad, Jamshid S; Kelley, Todd W; Baron, Riccardo; Druker, Brian J; Deininger, Michael W; O'Hare, Thomas

    2014-09-08

    Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-ABL1 single mutants in Philadelphia chromosome-positive (Ph(+)) leukemia, including the recalcitrant BCR-ABL1(T315I) mutant. However, emergence of compound mutations in a BCR-ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1 compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib, dasatinib, ponatinib, rebastinib, and bosutinib. T315I-inclusive compound mutants confer high-level resistance to TKIs, including ponatinib. In vitro resistance profiling was predictive of treatment outcomes in Ph(+) leukemia patients. Structural explanations for compound mutation-based resistance were obtained through molecular dynamics simulations. Our findings demonstrate that BCR-ABL1 compound mutants confer different levels of TKI resistance, necessitating rational treatment selection to optimize clinical outcome.

  18. Targeting of heme oxygenase-1 attenuates the negative impact of Ikaros isoform 6 in adult BCR-ABL1-positive B-ALL.

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    Lin, Xiaojing; Zou, Xingli; Wang, Ziming; Fang, Qin; Chen, Shuya; Huang, Jun; Zhe, Nana; Yu, Meisheng; Zhang, Yaming; Wang, Jishi

    2016-08-16

    The correlation between Heme oxygenase-1 (HO-1) and dominant-negative Ikaros isoform 6 (IK6) is unclear. Firstly, we detected that IK6 existed in 20 of 42 (47.6%) adult BCR-ABL1-positive B-lineage acute lymphoblastic leukemia (BCR-ABL1-positive B-ALL) by using reverse transcribed polymerase chain reaction (PCR) and nucleotide sequencing. IK6-positive patients had an unfavorable outcome compared with IK6-negative ones. Further study showed that the level of HO-1 expression was higher in IK6-positive patients' samples than that in IK6-negative ones. And there was a strong correlation between the expression of IK6 and HO-1. The growth of primary CD34+ leukemic cells derived from our IK6-positive patients' pool was prohibited by silencing HO-1, further promoting their apoptosis. Furthermore, primary CD34+ leukemic cells derived from IK6-positive patients shown poor responses to imatinib in comparison with wild-type (IK1) patients, suggesting that the expression of IK6 resisted to imatinib in adult BCR-ABL1-positive B-ALL. Importantly, inhibition of HO-1 also increased their sensitivity to tyrosine kinase inhibitors (TKIs). Finally, we found that IK6 activated downstream STAT5, and HO-1 was one of the downstream target genes of STAT5. In conclusion, HO-1 is an essential survival factor in BCR-ABL1-positive B-ALL with IK6, and targeting HO-1 can attenuate the negative impact of IK6.

  19. Rapid detection of BCR-ABL fusion genes using a novel combined LUX primer, in-cell RT-PCR and flow cytometric method.

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    Shi, Yan; Li, Li-Zhen; Sun, Jian-Zhi; Zhang, Ti; Peng, Jun; Xu, Cong-Gao

    2008-01-01

    Currently, quantitative and semiquantitative assays for minimal residual disease detection include fluorescence in situ hybridisation, multiparameter flow cytometric immunophenotyping and real-time quantitative polymerase chain reaction (RQ-PCR). We have developed a new approach to detect hybrid breakpoint cluster region and Abelson proto-oncogene (BCR-ABL) transcripts inside suspension cells using in situ RT-PCR and light upon extension (LUX) primer, followed by rapid quantitative analysis with flow cytometry. After cellular permeabilization and fixation of single cell suspension, the neoplastic mRNA was reverse transcribed and amplified by PCR with LUX primer. The results demonstrated that a strong positive yellow-green signal was observed in 99-100% cells of K562 cell line, only the red nucleus was detected in NB4 cell line and normal controls. The technique has been utilised to study 12 patients with chronic myeloid leukemia, and the results were compared with those of BCR-ABL fusion mRNA by RT-PCR and BCR-ABL fusion gene of the interphase cells by fluorescence in situ hybridization (FISH). In the five diagnosed patients, 90-98% cells were strongly positive. Four patients, including three patients treated with interferon-alpha and hydroxyurea and one patient treated with imatinib mesylate, had 26-82.5% positive cells. Three patients treated with imatinib mesylate were negative. The in situ RT-PCR results demonstrated complete concordance with the results of I-FISH and RT-PCR. A fluorescence signal was detectable at 1/10(4) cells and became negative below this threshold with flow cytometry. The results of the present study suggest that (1) LUX primers can be used to efficiently detect BCR-ABL fusion mRNA by in-cell RT-PCR; (2) the novel technique is a specific and sensitive way of detecting fusion gene with potential clinical usefulness.

  20. CCR7 is involved in BCR-ABL/STAP-2-mediated cell growth in hematopoietic Ba/F3 cells.

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    Kubo, Kaori; Iwakami, Masashi; Muromoto, Ryuta; Inagaki, Takuya; Kitai, Yuichi; Kon, Shigeyuki; Sekine, Yuichi; Oritani, Kenji; Matsuda, Tadashi

    2015-08-07

    Chronic myeloid leukemia is a clonal disease characterized by the presence of the Philadelphia chromosome and its oncogenic product, BCR-ABL, which activates multiple pathways involved in cell survival, growth promotion, and disease progression. We previously reported that in murine hematopoietic Ba/F3 cells, signal transducing adaptor protein-2 (STAP-2) binds to BCR-ABL and up-regulates BCR-ABL phosphorylation, leading to enhanced activation of its downstream signaling molecules. The binding of STAP-2 to BCR-ABL also influenced the expression levels of chemokine receptors, such as CXCR4 and CCR7. For the induction of CCR7 expression, signals mediated by the MAPK/ERK pathway were critical in Ba/F3 cells expressing BCR-ABL and STAP-2. In addition, STAP-2 cooperated with BCR-ABL to induce the production of CCR7 ligands, CCL19 and CCL21. Our results demonstrate a contribution of CCR7 to STAP-2-dependent enhancement of BCR-ABL-mediated cell growth in Ba/F3 cells.

  1. Pristimerin induces apoptosis in imatinib-resistant chronic myelogenous leukemia cells harboring T315I mutation by blocking NF-κB signaling and depleting Bcr-Abl

    Directory of Open Access Journals (Sweden)

    Cao Qi

    2010-05-01

    Full Text Available Abstract Background Chronic myelogenous leukemia (CML is characterized by the chimeric tyrosine kinase Bcr-Abl. Bcr-Abl-T315I is the notorious point mutation that causes resistance to imatinib and the second generation tyrosine kinase inhibitors, leading to poor prognosis. CML blasts have constitutive p65 (RelA NF-κB transcriptional activity, and NF-κB may be a potential target for molecular therapies in CML that may also be effective against CML cells with Bcr-Abl-T315I. Results In this report, we discovered that pristimerin, a quinonemethide triterpenoid isolated from Celastraceae and Hippocrateaceae, inhibited growth and induced apoptosis in CML cells, including the cells harboring Bcr-Abl-T315I mutation. Additionally, pristimerin inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Pristimerin blocked the TNFα-induced IκBα phosphorylation, translocation of p65, and expression of NF-κB-regulated genes. Pristimerin inhibited two steps in NF-κB signaling: TAK1→IKK and IKK→IκBα. Pristimerin potently inhibited two pairs of CML cell lines (KBM5 versus KBM5-T315I, 32D-Bcr-Abl versus 32D-Bcr-Abl-T315I and primary cells from a CML patient with acquired resistance to imatinib. The mRNA and protein levels of Bcr-Abl in imatinib-sensitive (KBM5 or imatinib-resistant (KBM5-T315I CML cells were reduced after pristimerin treatment. Further, inactivation of Bcr-Abl by imatinib pretreatment did not abrogate the TNFα-induced NF-κB activation while silencing p65 by siRNA did not affect the levels of Bcr-Abl, both results together indicating that NF-κB inactivation and Bcr-Abl inhibition may be parallel independent pathways. Conclusion To our knowledge, this is the first report to show that pristimerin is effective in vitro and in vivo against CML cells, including those with the T315I mutation. The mechanisms may involve inhibition of NF-κB and Bcr-Abl. We concluded that pristimerin could be a lead compound for

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

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    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

  3. 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.

    Science.gov (United States)

    van Kooten Niekerk, Peter B; Petersen, Charlotte C; Nyvold, Charlotte G; Ommen, Hans B; Roug, Anne S; Nederby, Line; Hokland, Peter; Kjeldsen, Eigil

    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 this, CD34(+) stem cell and progenitor cell subpopulations were isolated by fluorescence-activated cell sorting (FACS), and their content of residual Philadelphia positive (Ph(+) ) cells was evaluated in 17 CML patients (major molecular response, n = 6; 4-log reduction in BCR-ABL1 expression (MR(4) ), 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 cells in these patients. This approach demonstrates a clinically applicable manner of assessing residual disease at the stem cell level in CML patients in MR(4) , and may enable early and safe identification of candidates for tyrosine kinase inhibitor withdrawal.

  4. Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS).

    Science.gov (United States)

    Breitkopf, Susanne B; Yuan, Min; Pihan, German A; Asara, John M

    2012-10-02

    Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.

  5. 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.

  6. Blockade of Y177 and Nuclear Translocation of Bcr-Abl Inhibits Proliferation and Promotes Apoptosis in Chronic Myeloid Leukemia Cells.

    Science.gov (United States)

    Li, Qianyin; Huang, Zhenglan; Gao, Miao; Cao, Weixi; Xiao, Qin; Luo, Hongwei; Feng, Wenli

    2017-03-02

    The gradual emerging of resistance to imatinib urgently calls for the development of new therapy for chronic myeloid leukemia (CML). The fusion protein Bcr-Abl, which promotes the malignant transformation of CML cells, is mainly located in the cytoplasm, while the c-Abl protein which is expressed in the nucleus can induce apoptosis. Based on the hetero-dimerization of FKBP (the 12-kDa FK506- and rapamycin-binding protein) and FRB (the FKBP-rapamycin binding domain of the protein kinase, mTOR) mediated by AP21967, we constructed a nuclear transport system to induce cytoplasmic Bcr-Abl into nuclear. In this study, we reported the construction of the nuclear transport system, and we demonstrated that FN3R (three nuclear localization signals were fused to FRBT2098L with a FLAG tag), HF2S (two FKBP domains were in tandem and fused to the SH2 domain of Grb2 with an HA tag) and Bcr-Abl form a complexus upon AP21967. Bcr-Abl was imported into the nucleus successfully by the nuclear transport system. The nuclear transport system inhibited CML cell proliferation through mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 5 (STAT5) pathways mainly by HF2S. It was proven that nuclear located Bcr-Abl induced CML cell (including imatinib-resistant K562G01 cells) apoptosis by activation of p73 and its downstream molecules. In summary, our study provides a new targeted therapy for the CML patients even with Tyrosine Kinase Inhibitor (TKI)-resistance.

  7. Blockade of Y177 and Nuclear Translocation of Bcr-Abl Inhibits Proliferation and Promotes Apoptosis in Chronic Myeloid Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Qianyin Li

    2017-03-01

    Full Text Available The gradual emerging of resistance to imatinib urgently calls for the development of new therapy for chronic myeloid leukemia (CML. The fusion protein Bcr-Abl, which promotes the malignant transformation of CML cells, is mainly located in the cytoplasm, while the c-Abl protein which is expressed in the nucleus can induce apoptosis. Based on the hetero-dimerization of FKBP (the 12-kDa FK506- and rapamycin-binding protein and FRB (the FKBP-rapamycin binding domain of the protein kinase, mTOR mediated by AP21967, we constructed a nuclear transport system to induce cytoplasmic Bcr-Abl into nuclear. In this study, we reported the construction of the nuclear transport system, and we demonstrated that FN3R (three nuclear localization signals were fused to FRBT2098L with a FLAG tag, HF2S (two FKBP domains were in tandem and fused to the SH2 domain of Grb2 with an HA tag and Bcr-Abl form a complexus upon AP21967. Bcr-Abl was imported into the nucleus successfully by the nuclear transport system. The nuclear transport system inhibited CML cell proliferation through mitogen-activated protein kinase (MAPK and signal transducer and activator of transcription 5 (STAT5 pathways mainly by HF2S. It was proven that nuclear located Bcr-Abl induced CML cell (including imatinib-resistant K562G01 cells apoptosis by activation of p73 and its downstream molecules. In summary, our study provides a new targeted therapy for the CML patients even with Tyrosine Kinase Inhibitor (TKI-resistance.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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

  13. Patan hospital experience in treating philadelphia chromosome/BCR-ABL1 positive chronic myeloid leukemia patients with gleevec (imatinib mesylate; the first generation specific tyrosine kinase inhibitor

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    Zimmerman Mark

    2010-12-01

    Full Text Available Abstract Background Chronic Myeloid Leukemia (CML is caused by the abnormal fusion protein BCR-ABL1, a constitutively active tyrosine kinase and product of the Philadelphia chromosome. Gleevec (Imatinib mesylate is a selective inhibitor of this kinase. Treatment with this agent is known to result in hematologic, cytogenetic, and molecular responses. Patan hospital (Patan, Nepal is one of the Gleevec International Patient Assistance Program (GIPAP centers for patients with CML. Methods A total of 106 Philadelphia positive CML patients were enrolled in our center between Feb 2003 and Jun 2008, and 103 of them were eligible for cytogenetic and/or hematologic response analyses. Results Out of 103 patients, 27% patients underwent cytogenetic analysis. Imatinib induced major cytogenetic responses in 89% and complete hematologic responses in almost 100% of the patients with confirmed CML. After a mean follow up of 27 months, an estimated 90% of the patients on imatinib remained in hematologic remission and more than 90% of the patients are still alive. About 30% of patients developed some form of manageable myelosuppression. A few patients developed non-hematologic toxic side effects such as edema and hepatotoxicity. Conclusions Our study demonstrates that imatinib is safe to use in a developing country. Furthermore, we demonstrate that imatinib is very effective and induced long lasting responses in a high proportion of patients with Ph chromosome/BCR-ABL1 positive CML. Imatinib is well tolerated by our patients. The lack of cytogenetic analysis in the majority of our patients hindered our ability to detect inadequate responses to imatinib and adjust therapy appropriately.

  14. 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.

  15. 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

  16. Depletion of SAM50 Specifically Targets BCR-ABL-Expressing Leukemic Stem and Progenitor Cells by Interfering with Mitochondrial Functions

    NARCIS (Netherlands)

    Capala, Marta E; Pruis, Maurien; Vellenga, Edo; Schuringa, Jan Jacob

    2016-01-01

    A high proliferation rate of malignant cells requires an increased energy production, both by anaerobic glucose metabolism and mitochondrial respiration. Moreover, increased levels of mitochondria-produced reactive oxygen species (ROS) promote survival of transformed cells and contribute to the dise

  17. A natural-like synthetic small molecule impairs bcr-abl signaling cascades and induces megakaryocyte differentiation in erythroleukemia cells.

    Directory of Open Access Journals (Sweden)

    Silvia Turroni

    Full Text Available Over the past years, we synthesized a series of new molecules that are hybrids of spirocyclic ketones as complexity-bearing cores with bi- and ter-phenyls as privileged fragments. Some of these newly-shaped small molecules showed antiproliferative, pro-apoptotic and differentiating activity in leukemia cell lines. In the present study, to investigate more in depth the mechanisms of action of these molecules, the protein expression profiles of K562 cells treated with or without the compounds IND_S1, MEL_T1, IND_S7 and MEL_S3 were analyzed using two-dimensional gel electrophoresis coupled with mass spectrometry. Proteome comparisons revealed several differentially expressed proteins, mainly related to cellular metabolism, chaperone activity, cytoskeletal organization and RNA biogenesis. The major results were validated by Western blot and qPCR. To attempt integrating findings into a cellular signaling context, proteomic data were explored using MetaCore. Network analysis highlighted relevant relationships between the identified proteins and additional potential effectors. Notably, qPCR validation of central hubs showed that the compound MEL_S3 induced high mRNA levels of the transcriptional factors EGR1 and HNF4-alpha; the latter to our knowledge is reported here for the first time to be present in K562 cells. Consistently with the known EGR1 involvement in the regulation of differentiation along megakaryocyte lineage, MEL_S3-treated leukemia cells showed a marked expression of glycoprotein IIb/IIIa (CD41 and glycoprotein Ib (CD42, two important cell markers in megakaryocytic differentiation, together with morphological aspects of megakaryoblasts and megakaryocytes.

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

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    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.

  19. Molecular measurement of BCR-ABL transcript variations in chronic myeloid leukemia patients in cytogenetic remission

    Directory of Open Access Journals (Sweden)

    Costa Juliana

    2010-11-01

    Full Text Available Abstract Background The monitoring of BCR-ABL transcript levels by real-time quantitative polymerase chain reaction (RT-qPCR has become important to assess minimal residual disease (MRD and standard of care in the treatment of chronic myeloid leukemia (CML. In this study, we performed a prospective, sequential analysis using RT-qPCR monitoring of BCR-ABL gene rearrangements in blood samples from 91 CML patients in chronic phase (CP who achieved complete cytogenetic remission (CCyR and major molecular remission (MMR throughout imatinib treatment. Methods The absolute level of BCR-ABL transcript from peripheral blood was serially measured every 4 to 12 weeks by RT-qPCR. Only level variations > 0.5%, according to the international scale, was considered positive. Sequential cytogenetic analysis was also performed in bone marrow samples from all patients using standard protocols. Results Based on sequential analysis of BCR-ABL transcripts, the 91 patients were divided into three categories: (A 57 (62.6% had no variation on sequential analysis; (B 30 (32.9% had a single positive variation result obtained in a single sample; and (C 4 (4.39% had variations of BCR-ABL transcripts in at least two consecutive samples. Of the 34 patients who had elevated levels of transcripts (group B and C, 19 (55.8% had a BCR-ABL/BCR ratio, 13 (38.2% patients had a 1% to 10% increase and 2 patients had a >10% increase of RT-qPCR. The last two patients had lost a CCyR, and none of them showed mutations in the ABL gene. Transient cytogenetic alterations in Ph-negative cells were observed in five (5.5% patients, and none of whom lost CCyR. Conclusions Despite an increase levels of BCR-ABL/BCR ratio variations by RT-qPCR, the majority of CML patients with MMR remained in CCyR. Thus, such single variations should neither be considered predictive of subsequent failure and nor an indication for altering imatinib dose or switching to second generation therapy. Changing of

  20. Persistence of BCR/ABL mRNA-expressing bone-marrow cells in patients with chronic myelogenous leukemia in complete cytogenetic remission induced by interferon-alpha therapy.

    Science.gov (United States)

    Eberlé, F; Toiron, Y; Camerlo, J; Lafage, M; Sainty, D; Arnoulet, C; Raineri, V; Gabert, J; Maraninchi, D; Mannoni, P

    1995-06-01

    Complete hematologic and cytogenetic responses can be obtained with interferon-alpha (IFN-alpha) in 15-25% of the patients with chronic myelogenous leukemia (CML). In these patients, reverse-transcription polymerase chain reaction (RT-PCR) can be used to evaluate minimal residual disease. We studied 12 patients who remained Philadelphia-negative for a median period of 21 months on IFN-alpha therapy. Using RT-PCR, the specific transcript was found in all bone marrow (BM) samples. Ten patients still exhibiting a persistent residual clone remained in cytogenetic remission for a median period of 14 months. As we observed a dissociation between bcr-abl expression in BM and peripheral blood (PB) cells, and given the known fluctuations of the bcr-abl PCR results, we suggest that PB negative results should be confirmed on BM specimens. Alternatively, it remains to be demonstrated whether longitudinal monitoring of residual disease would benefit from quantitative PCR or double fluorescence in situ hybridization.

  1. 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.

  2. Efficacy of Dasatinib in Treatment of Imatinib-Resistant BCR/ABL Positive Leukemia%达沙替尼治疗伊马替尼耐药的BCR/ABL阳性白血病的临床研究

    Institute of Scientific and Technical Information of China (English)

    朱雨; 潘良琴; 钱思轩; 宋萍; 于慧; 张苏江; 葛峥; 洪鸣; 田甜

    2013-01-01

    本研究评价达沙替尼治疗原发或继发伊马替尼耐药的BCR/ABL阳性白血病的疗效和安全性.对27例原发或继发伊马替尼耐药的慢性髓系白血病(CML)或Ph阳性急性淋巴细胞白血病(Ph+ ALL)患者,给予达沙替尼100-140 mg/d口服治疗,评估疗效、总体生存和耐受情况.结果表明:27例伊马替尼耐药的BCR/ABL阳性白血病中位达沙替尼治疗时间8(1-66)个月,中位随访时间54(3-75)个月.27例接受达沙替尼治疗的患者中,88.8%获得完全血液学反应(CHR),44.4%获得主要细胞遗传学反应(mCyR),37%获得完全遗传学反应(CCyR),18.5%获得主要分子学反应(MMR).伊马替尼耐药的进展期(CML-AP、CML-BC、骨髓复发Ph+ ALL)患者接受达沙替尼治疗获CCyR率低于疾病稳定期(CML-CP、骨髓缓解Ph+ ALL)患者(P=0.0377),且3-4级不良反应发生率明显增高.达沙替尼治疗后获得CCyR的患者生存期(OS)较未达CCyR者明显延长(63个月vs9个月,P=0.0126).达沙替尼治疗后最常见的3-4级不良反应包括血液学反应如血小板减少(51.8%)、中性粒细胞减少(48.1%)、贫血(33.3%)和非血液学反应如胸腔积液(18.5%)、肺部感染(18.5%)、心包积液(11.1%).3-4级不良反应主要发生在疾病进展期时改服达沙替尼者,均发生于服药12个月内.结论:达沙替尼治疗伊马替尼耐药的BCR/ABL阳性白血病有效,且在疾病稳定期改服达沙替尼疗效和耐受性更好.%This study was aimed to evaluate the efficacy and safety of dasatinib in BCR/ABL positive leukemia patients with primary or secondary resistance to imatinib.27 patients with primary or secondary imatinib-resistant chronic myelogenous leukemia (CML) or Philadelphia chromosome positive acute lymphocytic leukemia (Ph + ALL) received 100-140 mg/d dasatinib orally.Their overall survival and tolerance were evaluated.The results showed that the median duration of dasatinib therapy was 8 (1-66) months in the 27

  3. Detection of residual bcr/abl translocation by polymerase chain reaction in chronic myeloid leukaemia patients after bone-marrow transplantation.

    Science.gov (United States)

    Gabert, J; Thuret, I; Lafage, M; Carcassonne, Y; Maraninchi, D; Mannoni, P

    1989-11-11

    The polymerase chain reaction was used to evaluate minimum residual disease in chronic myelogenous leukaemia (CML) patients after bone-marrow transplantation, by amplification of the transcript of the specific bcr/abl hybrid gene. Strict precautions were taken to avoid contamination. Peripheral blood cells from 22 patients transplanted for haematological malignant disorders were analysed. The results were clearcut for positive controls (patients with CML in relapse) and negative controls (patients with malignant disorders other than CML). In 11 of 12 CML patients in clinical and cytogenetic remission the bcr/abl transcript was detected 3 months to 6 years after transplantation. Thus, it appears that cells expressing the bcr/abl mRNA are not eradicated from the blood of CML patients in complete clinical remission even years after bone-marrow transplantation.

  4. 达沙替尼治疗伊马替尼耐药的 BCR/ABL阳性白血病%Dasatinib in treatment of imatinib-resistant BCR/ABL positive leukemia

    Institute of Scientific and Technical Information of China (English)

    严红; 赵海军

    2014-01-01

    This study was aimed to evaluate the efficacy and safety of dasatinib in BCR /ABL positive leukemia patients with resistance to imatinib.Method 9 patients with imatinib -resistant chronic myelogenous leukemia ( CML) or Philadelphia chromosome positive acute lymphocytic leukemia (Ph+ALL) received 100~140 mg· d-1 dasatinib orally.Their overall survival and tolerance were evalua-ted.Results After the dasatinib treatment ,2 patients with CML in chronic phase achieved complete hematologic response ( CHR) ,one of them achieved complete cytogenetic response (CCyR);4 of 5 patients with CML in blast crisis achieved CHR and partly cytogenetic response (PCyR);one patient had no response .Treated with dasatinib,one patient with Ph +ALL,presented with E255V mutation,a-chieved CHR and PCyR .The other died of infection .Conlusion Receiving therapy of dasatinib , patients with resistance to imatinib could achieve CHR ,even PCyR .%目的:评价达沙替尼治疗伊马替尼耐药的BCR/ABL阳性白血病的疗效和安全性。方法对9例伊马替尼耐药的慢性髓系白血病( CML)或Ph阳性急性淋巴细胞白血病( Ph+ALL)患者,给予达沙替尼100~140 mg· d-1口服治疗,评估疗效和耐受情况。结果9例伊马替尼耐药的BCR/ABL阳性白血病,2例CML-CP患者均获得CHR,1例达CCyR;5例CML-BC患者中4例获得CHR和PCyR,1例NR;2例Ph+ALL患者中1例检测到E255V突变,采用达沙替尼治疗达CHR和PCyR,1例诱导缓解时,同时行VDP方案化疗,继发严重感染死亡。结论达沙替尼治疗伊马替尼耐药的BCR/ABL阳性白血病患者可获得血液学甚至细胞遗传学缓解,且耐受性好。

  5. 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

  6. 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.

  7. Improved coiled-coil design enhances interaction with Bcr-Abl and induces apoptosis.

    Science.gov (United States)

    Dixon, Andrew S; Miller, Geoffrey D; Bruno, Benjamin J; Constance, Jonathan E; Woessner, David W; Fidler, Trevor P; Robertson, James C; Cheatham, Thomas E; Lim, Carol S

    2012-01-01

    The oncoprotein Bcr-Abl drives aberrant downstream activity through trans-autophosphorylation of homo-oligomers in chronic myelogenous leukemia (CML).(1, 2) The formation of Bcr-Abl oligomers is achieved through the coiled-coil domain at the N-terminus of Bcr.(3, 4) We have previously reported a modified version of this coiled-coil domain, CCmut2, which exhibits disruption of Bcr-Abl oligomeric complexes and results in decreased proliferation of CML cells and induction of apoptosis.(5) A major contributing factor to these enhanced capabilities is the destabilization of the CCmut2 homodimers, increasing the availability to interact with and inhibit Bcr-Abl. Here, we included an additional mutation (K39E) that could in turn further destabilize the mutant homodimer. Incorporation of this modification into CCmut2 (C38A, S41R, L45D, E48R, Q60E) generated what we termed CCmut3, and resulted in further improvements in the binding properties with the wild-type coiled-coil domain representative of Bcr-Abl [corrected]. A separate construct containing one revert mutation, CCmut4, did not demonstrate improved oligomeric properties and indicated the importance of the L45D mutation. CCmut3 demonstrated improved oligomerization via a two-hybrid assay as well as through colocalization studies, in addition to showing similar biologic activity as CCmut2. The improved binding between CCmut3 and the Bcr-Abl coiled-coil may be used to redirect Bcr-Abl to alternative subcellular locations with interesting therapeutic implications.

  8. 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.

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. A Non-ATP-Competitive Dual Inhibitor of JAK2 and BCR-ABL Kinases: Elucidation of a Novel Therapeutic Spectrum Based on Substrate Competitive Inhibition.

    Science.gov (United States)

    Jatiani, Shashidhar S; Cosenza, Stephen C; Reddy, M V Ramana; Ha, Ji Hee; Baker, Stacey J; Samanta, Ajoy K; Olnes, Matthew J; Pfannes, Loretta; Sloand, Elaine M; Arlinghaus, Ralph B; Reddy, E Premkumar

    2010-04-01

    Here we report the discovery of ON044580, an α-benzoyl styryl benzyl sulfide that possesses potent inhibitory activity against two unrelated kinases, JAK2 and BCR-ABL, and exhibits cytotoxicity to human tumor cells derived from chronic myelogenous leukemia (CML) and myelodysplasia (MDS) patients or cells harboring a mutant JAK2 kinase. This novel spectrum of activity is explained by the non-ATP-competitive inhibition of JAK2 and BCR-ABL kinases. ON044580 inhibits mutant JAK2 kinase and the proliferation of JAK2(V617F)-positive leukemic cells and blocks the IL-3-mediated phosphorylation of JAK2 and STAT5. Interestingly, this compound also directly inhibits the kinase activity of both wild-type and imatinib-resistant (T315I) forms of the BCR-ABL kinase. Finally, ON044580 effectively induces apoptosis of imatinib-resistant CML patient cells. The apparently unrelated JAK2 and BCR-ABL kinases share a common substrate, STAT5, and such substrate competitive inhibitors represent an alternative therapeutic strategy for development of new inhibitors. The novel mechanism of kinase inhibition exhibited by ON044580 renders it effective against mutant forms of kinases such as the BCR-ABL(T315I) and JAK2(V617F). Importantly, ON044580 selectively reduces the number of aneuploid cells in primary bone marrow samples from monosomy 7 MDS patients, suggesting another regulatory cascade amenable to this agent in these aberrant cells. Data presented suggest that this compound could have multiple therapeutic applications including monosomy 7 MDS, imatinib-resistant CML, and myeloproliferative neoplasms that develop resistance to ATP-competitive agents.

  12. Oridonin Triggers Chaperon-mediated Proteasomal Degradation of BCR-ABL in Leukemia

    Science.gov (United States)

    Huang, Huilin; Weng, Hengyou; Dong, Bowen; Zhao, Panpan; Zhou, Hui; Qu, Lianghu

    2017-01-01

    Inducing degradation of oncoproteins by small molecule compounds has the potential to avoid drug resistance and therefore deserves to be exploited for new therapies. Oridonin is a natural compound with promising antitumor efficacy that can trigger the degradation of oncoproteins; however, the direct cellular targets and underlying mechanisms remain unclear. Here we report that oridonin depletes BCR-ABL through chaperon-mediated proteasomal degradation in leukemia. Mechanistically, oridonin poses oxidative stress in cancer cells and directly binds to cysteines of HSF1, leading to the activation of this master regulator of the chaperone system. The resulting induction of HSP70 and ubiquitin proteins and the enhanced binding to CHIP E3 ligase hence target BCR-ABL for ubiquitin-proteasome degradation. Both wild-type and mutant forms of BCR-ABL can be efficiently degraded by oridonin, supporting its efficacy observed in cultured cells as well as mouse tumor xenograft assays with either imatinib-sensitive or -resistant cells. Collectively, our results identify a novel mechanism by which oridonin induces rapid degradation of BCR-ABL as well as a novel pharmaceutical activator of HSF1 that represents a promising treatment for leukemia. PMID:28128329

  13. PESV对K562细胞BCR/ABL融合基因及凋亡调控因子Bcl-2、Bad表达的影响%The Effects of PESV on the Expression of BCR/ABL Fusion Gene and Bcl-2, Bad of Apoptosis Regulators on the K562 Cells

    Institute of Scientific and Technical Information of China (English)

    于文俊; 杨文华; 杨向东; 史哲新; 王兴丽; 郝征; 张佳

    2012-01-01

    Objective: To investigate the PESV of K562 cells BCR / ABL fusion gene and apoptosis regulators bcl-2 and bad expression. Methods: K562 cells were cultured in vitro, by PESV for different times, the apoptosis rate by flow cytometry, fluorescence quantitative RT-PCR detection of BCR / ABL, Bcl-2, Bad mRNA level changes. Results: Compared with the control group, PESV treated K562 cells, apoptosis increased, BCR / ABL fusion gene reduced expression, anti-apoptotic gene Bcl-2 mRNA expression decreased, pro-apoptotic gene Bad mRNA expression. Conclusion: PESV reduced in K562 cells can reduce the BCR / ABL fusion gene, may regulate the expression of Bcl-2 and Bad, inhibit proliferation of K562 cells and promote their apoptosis.%目的:探讨PESV对K562细胞BCR/ABL融合基因及凋亡调控因子bcl-2和bad表达的影响.方法:将体外培养K562细胞,经PESV处理不同时间后,流式细胞术检测细胞凋亡率,荧光定量RT-PCR检测BCR/ABL、Bcl-2、Bad mRNA水平变化.结果:与对照组相比,PESV处理后K562细胞,凋亡率增加,BCR/ABL融合基因表达降低,抗凋亡相关基因Bcl-2 mRNA表达降低,促凋亡基因Bad mRNA表达增加.结论:PESV能降低降低K562细胞BCR/ABL融合基因的表达,可能通过调节Bcl-2和Bad表达,抑制K562细胞增殖,促进其凋亡.

  14. shRNA library screening identifies nucleocytoplasmic transport as a mediator of BCR-ABL1 kinase-independent resistance.

    Science.gov (United States)

    Khorashad, Jamshid S; Eiring, Anna M; Mason, Clinton C; Gantz, Kevin C; Bowler, Amber D; Redwine, Hannah M; Yu, Fan; Kraft, Ira L; Pomicter, Anthony D; Reynolds, Kimberly R; Iovino, Anthony J; Zabriskie, Matthew S; Heaton, William L; Tantravahi, Srinivas K; Kauffman, Michael; Shacham, Sharon; Chenchik, Alex; Bonneau, Kyle; Ullman, Katharine S; O'Hare, Thomas; Deininger, Michael W

    2015-03-12

    The mechanisms underlying tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML) patients lacking explanatory BCR-ABL1 kinase domain mutations are incompletely understood. To identify mechanisms of TKI resistance that are independent of BCR-ABL1 kinase activity, we introduced a lentiviral short hairpin RNA (shRNA) library targeting ∼5000 cell signaling genes into K562(R), a CML cell line with BCR-ABL1 kinase-independent TKI resistance expressing exclusively native BCR-ABL1. A customized algorithm identified genes whose shRNA-mediated knockdown markedly impaired growth of K562(R) cells compared with TKI-sensitive controls. Among the top candidates were 2 components of the nucleocytoplasmic transport complex, RAN and XPO1 (CRM1). shRNA-mediated RAN inhibition or treatment of cells with the XPO1 inhibitor, KPT-330 (Selinexor), increased the imatinib sensitivity of CML cell lines with kinase-independent TKI resistance. Inhibition of either RAN or XPO1 impaired colony formation of CD34(+) cells from newly diagnosed and TKI-resistant CML patients in the presence of imatinib, without effects on CD34(+) cells from normal cord blood or from a patient harboring the BCR-ABL1(T315I) mutant. These data implicate RAN in BCR-ABL1 kinase-independent imatinib resistance and show that shRNA library screens are useful to identify alternative pathways critical to drug resistance in CML.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Frequency of BCR-ABL Transcript Types in Syrian CML Patients

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    Sulaf Farhat-Maghribi

    2016-01-01

    Full Text Available Background. In Syria, CML patients are started on tyrosine kinase inhibitors (TKIs and monitored until complete molecular response is achieved. BCR-ABL mRNA transcript type is not routinely identified, contrary to the recommendations. In this study we aimed to identify the frequency of different BCR-ABL transcripts in Syrian CML patients and highlight their significance on monitoring and treatment protocols. Methods. CML patients positive for BCR-ABL transcripts by quantitative RT-PCR were enrolled. BCR-ABL transcript types were investigated using a home-made PCR method that was adapted from published protocols and optimized. The transcript types were then confirmed using a commercially available research kit. Results. Twenty-four transcripts were found in 21 patients. The most common was b2a2, followed by b3a2, b3a3, and e1a3 present solely in 12 (57.1%, 3 (14.3%, 2 (9.5%, and 1 (4.8%, respectively. Three samples (14.3% contained dual transcripts. While b3a2 transcript was apparently associated with warning molecular response to imatinib treatment, b2a2, b3a3, and e1a3 transcripts collectively proved otherwise (P=0.047. Conclusion. It might be advisable to identify the BCR-ABL transcript type in CML patients at diagnosis, using an empirically verified method, in order to link the detected transcript with the clinical findings, possible resistance to treatment, and appropriate monitoring methods.

  17. Fluorescent in-situ hybridization (FISH for BCR/ABL in chronic myeloid leukemia after bone marrow transplantation

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    Maria de Lourdes Lopes Ferrari Chauffaille

    2001-01-01

    Full Text Available CONTEXT: Identification of Philadelphia chromosome or BCR/ABL gene rearrangement in chronic myeloid leukemia is important at diagnosis as well as after treatment. OBJECTIVE: To compare the results of karyotyping using fluorescent in-situ hybridization (FISH upon diagnosis and 1 year after bone marrow transplantation in 12 patients. TYPE OF STUDY: Diagnostic test and residual disease detection. SETTING: Hematology and Hemotherapy Department, Federal University of São Paulo/Escola Paulista de Medicina, São Paulo, Brazil. SAMPLE: 12 patients with chronic myeloid leukemia at diagnosis and 1 year after bone marrow transplantation. DIAGNOSTIC TEST: Karyotyping was done in the usual way and the BCR/ABL gene-specific probe was used for FISH. MAIN MEASUREMENTS: Disease at diagnosis and residual. RESULTS: At diagnosis, 10 patients presented t(9;22(q34.1;q11 as well as positive FISH. Two cases did not have metaphases but FISH was positive. After bone marrow transplantation, 8 patients presented normal karyotype, 1 had persistence of identifiable Philadelphia chromosome and 3 had no metaphases. Two cases showed complete chimera and 2 had donor and host cells simultaneously. FISH was possible in all cases after bone marrow transplantation and confirmed the persistence of identifiable Philadelphia chromosome clone in one patient, and identified another that did not present metaphases for analysis. Cases that showed mixed chimera in karyotype were negative for BCR/ABL by FISH. CONCLUSION: The applicability of FISH is clear, particularly for residual disease detection. Classical and molecular cytogenetics are complementary methods.

  18. Efficacy of Retinoids in IKZF1-Mutated BCR-ABL1 Acute Lymphoblastic Leukemia.

    Science.gov (United States)

    Churchman, Michelle L; Low, Jonathan; Qu, Chunxu; Paietta, Elisabeth M; Kasper, Lawryn H; Chang, Yunchao; Payne-Turner, Debbie; Althoff, Mark J; Song, Guangchun; Chen, Shann-Ching; Ma, Jing; Rusch, Michael; McGoldrick, Dan; Edmonson, Michael; Gupta, Pankaj; Wang, Yong-Dong; Caufield, William; Freeman, Burgess; Li, Lie; Panetta, John C; Baker, Sharyn; Yang, Yung-Li; Roberts, Kathryn G; McCastlain, Kelly; Iacobucci, Ilaria; Peters, Jennifer L; Centonze, Victoria E; Notta, Faiyaz; Dobson, Stephanie M; Zandi, Sasan; Dick, John E; Janke, Laura; Peng, Junmin; Kodali, Kiran; Pagala, Vishwajeeth; Min, Jaeki; Mayasundari, Anand; Williams, Richard T; Willman, Cheryl L; Rowe, Jacob; Luger, Selina; Dickins, Ross A; Guy, R Kiplin; Chen, Taosheng; Mullighan, Charles G

    2015-09-14

    Alterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. Janus kinase 2 mutations in cases with BCR-ABL-negative chronic myeloproliferative disorders from Turkey

    Science.gov (United States)

    Yildiz, Ismail; Yokuş, Osman; Gedik, Habip

    2017-01-01

    Objective: We aimed to investigate the frequency of Janus kinase 2 (JAK2) mutations in cases with chronic myeloproliferative disorders (CMDs), and the relationship between the presence of JAK2 mutation and leukocytosis and splenomegaly, retrospectively. Materials and Methods: Patients, who were diagnosed with BCR-ABL-negative CMDs according to diagnosis criteria of the World Health Organization and followed up at the hematology clinic between 2013 and 2015, were investigated in terms of the frequency of JAK2 mutation in cases with CMDs, and the relationship between the presence of JAK2 mutation and leukocytosis and splenomegaly, retrospectively. Results: In total, 100 patients, who were diagnosed with BCR-ABL-negative CMDs, were evaluated retrospectively. The mean age of the patients with JAK2 positivity was significantly higher compared to patients with negative. JAK2-positivity rates in the age groups were significantly different. Gender, diagnosis, splenomegaly, and leukocytosis were not statistically different for JAK2 positivity between the groups. Conclusion: JAK2 V617F mutation is more commonly seen in older age as a risk for complications related to CDMS. Splenomegaly and leukocytosis are not associated with JAK2 V617F mutation. PMID:28182037

  4. 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.

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

    OpenAIRE

    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

    2011-01-01

    Summary 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 hig...

  6. 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.

  7. 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.

  8. Detection of p190BCR-ABL AND p210BCR-ABL fusion transcripts in patients with chronic myeloid leukemia (CML using qualitative RT-PCR

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    Aya Bonilla, Carlos Alberto

    2014-10-01

    Full Text Available Introduction: Chronic myelogenous leukemia (CML is characterized by the presence of the Philadelphia chromosome (Ph, resulting from the balanced reciprocal translocation t(9;22(q34;q11. This marker chromosome is found less frequently in patients suffering from acute lymphoblastic leukemia. Objective: To determine the frequency of BCR-ABL gene fusions encoding the p210BCR-ABL y p190 BCR-ABL transcripts in Colombian patients diagnosed with CML in different stages of the disease and/or its treatment. Materials and methods: Cross sectional, descriptive study of thirty one CML patients (aged 15-78. Analysis was carried out through qualitative nested PCR for the isoforms P210 BCR-ABL (b3a2 e b2a2 and P190 BCR-ABL (e1a2, and based on peripheral blood samples. Results: In 29 of the 31 patients (93.6% transcript p210BCR-ABL was detected; b2a2 and b3a2 gene fusions and the coexpression b3a2 y b2a2 were identified in 55.2% (16/29, 34.5% (10/29 and 10.3% (3/29 of the cases, respectively. Conclusion: b2a2 gene fusion was the most frequent in this CML population.

  9. Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface.

    Science.gov (United States)

    Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

    2016-04-15

    Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl.

  10. 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.

  11. ON012380: A Non-ATP Competitive Inhibitor of BCR-ABL for the Therapy of Imatinib-Resistant CMLs

    Science.gov (United States)

    2010-05-01

    diverse client proteins. Biopolymers 2009. 41. Tsuruma R, Ohbayashi N, Kamitani S, et al. Physical and functional interactions between STAT3 and...compounds to significantly inhibit the proliferation of K562 and Ba/F3:JAK2V617F cells suggests that this scaffold could be a promising lead for the... scaffold could be a promising lead for the development of anticancer agents that are able to block BCR-ABL phosphorylation in leuke- mic cells. 2010

  12. Immunologic evaluation of peptides derived from BCR/ABL-out-of-frame fusion protein in HLA A2.1 transgenic mice.

    Science.gov (United States)

    Casnici, Claudia; Volpe, Gisella; Crotta, Katia; Lattuada, Donatella; Saglio, Giuseppe; Marelli, Ornella

    2012-05-01

    Philadelphia chromosome-positive chronic myelogenous leukemia and acute lymphocytic leukemia express, besides the main BCR/ABL transcripts, novel BCR/ABL transcripts derived from alternative splicing between BCR exons 1, 13, or 14 with ABL exons 4 and 5. Their translational products present at C-terminus an amino acid portion derived from out-of-frame (OOF) reading of the ABL gene. The presence of OOF-peptide-specific T cells in chronic myelogenous leukemia patients was demonstrated and a first study in in vivo model demonstrated that OOF ABL portion was immunogenic in human leukcocyte antigen (HLA)-A2.1 transgenic mice. Here we immunized HLA A2.1 mice with novel peptides designed on the ABL OOF sequence, containing epitopes with high affinity for HLA A2.1 molecule. The specific immune response, cellular and humoral, obtained ex vivo against HLA A2.1-positive human chronic myelogenous leukemia cells using peptide 22-53 and the cytotoxic activity induced by peptide 32mer confirm the possibility to use the ABL OOF portion as target to evoke a specific and multiple immune response in Philadelphia positive leukemic patients in cytogenetic remission.

  13. 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.

  14. BCR/ABL阴性骨髓增生性疾病患者JAK2-V617F点突变与外周血三系变化的相关性%Correlation between JAK2-V617F mutations and variation of peripheral blood cells among BCR/ABL-negative MPD patients

    Institute of Scientific and Technical Information of China (English)

    叶远馨; 宋兴勃; 周易; 应斌武; 陆小军

    2012-01-01

    目的 探讨BCR/ABL阴性骨髓增生性疾病患者JAK2-V617F突变的阳性率以及突变量与外周血三系变化的相关性.方法 对191例确诊为BCR/ABL阴性的骨髓增生性疾病患者进行JAK2-V617F突变检测,分别以外周血中红细胞、白细胞、血小板计数进行分类统计,分析不同类型的标本JAK2-V617F基因点突变的发生率及相对定量变化.结果 外周血红细胞、白细胞、血小板计数不同范围的标本JAK2-V617F突变阳性率有统计学差异.随着外周血红细胞、血小板计数增高,JAK2-V617F突变阳性率也随之增高,且以三系均增多者JAK2-V617F突变率为最高(92.86%).结论 BCR/ABL阴性的骨髓增生性疾病JAK2-V617F点突变发生率与外周血三系变化存在明显的相关性.外周血三系变化、尤其是红细胞计数与BCR/ABL阴性的骨髓增生性疾病JAK2-V617F突变的阳性率以及突变相对定量存在一定的相关性.%Objective To assess the correlation between JAK2-V617F mutation and complete blood counts among patients with BCR/ABL-negative myeloproliferative diseases (MPD).Methods One hundred and ninety one patients were recruited.Retrospectively,their laboratory data were analyzed for the counts of red blood cells (RBC),white blood cells (WBC) and platelets (PLT).And the incidence of JAK2-V617F mutation was determined.Results There was significant difference in the incidence of JAK2-V617F mutation between patients with different cell counts (P<0.01).The incidence of JAK2-V617F mutation has increased with the counts of RBC and PLT,which was the highest (92.86%) among those featuring simultaneous increase in all three series.Conclusion The incidence of JAK2-V617F mutation seems to be strongly associated with variation of peripheral blood cell counts among patients with BCR/ABL-negative MPD.Variation of peripheral blood cells,particularly RBC,may be correlated with the rate of JAK2-V617F mutation.

  15. A Non-ATP Competitive Inhibitor of BCR-ABL for the Therapy of Imatinib-Resistant Cmls

    Science.gov (United States)

    2008-05-01

    10% SDS-PAGE and analyzed for indicated proteins by infrared labeled secondary antibodies and scanning with Odyssey scanner ( LiCor Technology). pBcr...proteins by infrared labeled secondary antibodies and scanning with Odyssey scanner ( LiCor Technology). 10 treated 32D/T315I-BCR-ABL cells with increasing...with Odyssey scanner ( LiCor Technology). V G 0.5 1.0 2.5 5.0 0.5 1.0 2.5 5.0 (µM

  16. Papel da P190 BCR-ABL como parâmetro de recaída na leucemia mielóide crônica P190 BCR-ABL role in myeloid chronic leukemia relapse

    Directory of Open Access Journals (Sweden)

    Gabriela V. Andrade

    2008-08-01

    Full Text Available A leucemia mielóide crônica (LMC é uma desordem hematológica mieloproloferativa clonal causada por uma mutação em uma célula-tronco pluripotente, resultando na proliferação e acúmulo de células mielóides e seus progenitores. O cromossomo Philadelphia é o resultado de uma translocação recíproca de material genético entre os genes abl (Abelson murine leukemia no cromossomo 9, e o bcr (breakpoint cluster region no cromossomo 22, resultando na formação do gene quimérico BCR-ABL. Neste trabalho, 45 pacientes foram acompanhados de forma seqüencial, de acordo com as avaliações periódicas individuais, e 360 amostras foram selecionadas e analisadas pela técnica da reação em cadeia da polimerase (PCR através da técnica qualitativa para as isoformas características da P210BCR-ABL (b3a2 e b2a2 e P190BCR-ABL (e1a2. No nosso estudo no pré-TMO, observou-se uma prevalência das isoformas características da LMC (b3a2 e/ou b2a2, fato imprescindível para que o paciente fosse acompanhado dentro do protocolo da LMC. A isoforma e1a2, característica da LLA, foi detectada em 11 pacientes em conjunto com essas isoformas. A detecção do transcrito e1a2 foi avaliada quanto ao seu provável papel na LMC, e foi um dos parâmetros de avaliação deste estudo.Chronic myeloid leukemia (CML is a myeloproliferative clonal disorder caused by a mutation in a stem cell, resulting in the proliferation and accumulation of myeloid cells and their progenitors. The Philadelphia chromosome (Ph1 is a result of a mutual translocation of genetic material between the abl gene (Abelson murine leukemia in chromosome 9 and bcr gene (breakpoint cluster region in chromosome 22, resulting in the formation of the chimerical gene, BCR-ABL. In this work 45 patients were sequentially followed up in individual periodic evaluations, and 360 samples were selected and analyzed by PCR using the qualitative technique for isoforms characteristic of P210CR-ABL (b3a2 and b2a2

  17. LMC y detección del gen de fusión BCR/ABL

    Directory of Open Access Journals (Sweden)

    C. G. Artigas Allaire

    2006-04-01

    Full Text Available La anormalidad citogenética más común en la leucemia mieloide crónica (LMC es el cromosoma Philadelphia producida por la translocación t(9:22, cuya expresión molecular es el gen de fusión BCR-ABL, que codifica una proteína con actividad tirosinquinasa.

  18. 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.

  19. 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.

  20. 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

  1. 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...

  2. 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

  3. 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.

  4. 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.

    Science.gov (United States)

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

    2014-07-07

    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 minimal interactions with ABC drug transporters. Three-dimensional pharmacophore modeling and quantitative structure-activity relationship (QSAR) studies were carried out on a series of nilotinib analogues to identify chemical features that contribute to inhibitory activity of nilotinib against BCR-ABL kinase activity, P-gp, and ABCG2. Twenty-five derivatives of nilotinib were synthesized and were then tested to measure their activity to inhibit BCR-ABL kinase and to inhibit the function of ABC drug transporters. A set of in vitro experiments including kinase activity and cell-based transport assays and photolabeling of P-gp and ABCG2 with a transport substrate, [(125)I]-iodoarylazido-prazosin (IAAP), were carried out in isolated membranes to evaluate the potency of the derivatives to inhibit the function of ABC drug transporters and BCR-ABL kinase. Sixteen, fourteen, and ten compounds were selected as QSAR data sets, respectively, to generate PHASE v3.1 pharmacophore models for BCR-ABL kinase, ABCG2, and P-gp inhibitors. The IC50 values of these derivatives against P-gp, ABCG2, or BCR-ABL kinase were used to generate pharmacophore features required for optimal interactions with these targets. A seven-point pharmacophore (AADDRRR) for BCR-ABL kinase inhibitory activity, a six-point pharmacophore (ADHRRR) for ABCG2 inhibitory activity, and a seven-point pharmacophore (AADDRRR) for P-gp inhibitory activity were generated. The derived models clearly demonstrate high predictive power

  5. Colorimetric assessment of BCR-ABL1 transcripts in clinical samples via gold nanoprobes.

    Science.gov (United States)

    Vinhas, Raquel; Correia, Cláudia; Ribeiro, Patricia; Lourenço, Alexandra; Botelho de Sousa, Aida; Fernandes, Alexandra R; Baptista, Pedro V

    2016-07-01

    Gold nanoparticles functionalized with thiolated oligonucleotides (Au-nanoprobes) have been used in a range of applications for the detection of bioanalytes of interest, from ions to proteins and DNA targets. These detection strategies are based on the unique optical properties of gold nanoparticles, in particular, the intense color that is subject to modulation by modification of the medium dieletric. Au-nanoprobes have been applied for the detection and characterization of specific DNA sequences of interest, namely pathogens and disease biomarkers. Nevertheless, despite its relevance, only a few reports exist on the detection of RNA targets. Among these strategies, the colorimetric detection of DNA has been proven to work for several different targets in controlled samples but demonstration in real clinical bioanalysis has been elusive. Here, we used a colorimetric method based on Au-nanoprobes for the direct detection of the e14a2 BCR-ABL fusion transcript in myeloid leukemia patient samples without the need for retro-transcription. Au-nanoprobes directly assessed total RNA from 38 clinical samples, and results were validated against reverse transcription-nested polymerase chain reaction (RT-nested PCR) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The colorimetric Au-nanoprobe assay is a simple yet reliable strategy to scrutinize myeloid leukemia patients at diagnosis and evaluate progression, with obvious advantages in terms of time and cost, particularly in low- to medium-income countries where molecular screening is not routinely feasible. Graphical abstract Gold nanoprobe for colorimetric detection of BCR-ABL1 fusion transcripts originating from the Philadelphia chromosome.

  6. The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia.

    Science.gov (United States)

    García-Tuñón, Ignacio; Hernández-Sánchez, María; Ordoñez, José Luis; Alonso-Pérez, Veronica; Álamo-Quijada, Miguel; Benito, Rocio; Guerrero, Carmen; Hernández-Rivas, Jesús María; Sánchez-Martín, Manuel

    2017-02-09

    CRISPR/Cas9 technology was used to abrogate p210 oncoprotein expression in the Boff-p210 cell line, a pro-B line derived from interlukin-3-dependent Baf/3, that shows IL-3-independence arising from the constitutive expression of BCR-ABL p210. Using this approach, pools of Boff-p210-edited cells and single edited cell-derived clones were obtained and functionally studied in vitro. The loss of p210 expression in Boff-p210 cells resulted in the loss of ability to grow in the absence of IL-3, as the Baf/3 parental line, showing significantly increased apoptosis levels. Notably, in a single edited cell-derived clone carrying a frame-shift mutation that prevents p210 oncoprotein expression, the effects were even more drastic, resulting in cell death. These edited cells were injected subcutaneously in immunosuppressed mice and tumor growth was followed for three weeks. BCR/ABL-edited cells developed smaller tumors than those originating from unedited Boff-p210 parental cells. Interestingly, the single edited cell-derived clone was unable to develop tumors, similar to what is observed with the parental Baf/3 cell line.CRISPR/Cas9 genomic editing technology allows the ablation of the BCR/ABL fusion gene, causing an absence of oncoprotein expression, and blocking its tumorigenic effects in vitro and in the in vivo xenograft model of CML. The future application of this approach in in vivo models of CML will allow us to more accurately assess the value of CRISPR/Cas9 technology as a new therapeutic tool that overcomes resistance to the usual treatments for CML patients.

  7. 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.

  8. [Guidelines for molecular monitoring of BCR-ABL1 in chronic myeloid leukemia patients by RT-qPCR].

    Science.gov (United States)

    Larripa, Irene; Ruiz, María Sol; Gutiérrez, Marina; Bianchini, Michele

    2017-01-01

    Current clinical guidelines for managing chronic myeloid leukemia include molecular monitoring of BCR-ABL1 transcript quantitative reverse-transcription PCR. Despite the proven prognostic significance of molecular response, it is not widely appreciated that quantitative reverse-transcription PCR potentially produces highly variable data, which may affect the validity of results, making comparability between different laboratories difficult. Therefore, standardized reporting of BCR-ABL1 measurements is needed for optimal clinical management. An approach to achieve comparable BCR-ABL1 values is the use of an international reporting scale. Conversion to the international scale is achieved by the application of laboratory specific conversion factor that is obtained by using validated secondary reference calibrators. Moreover, with the aim to mitigate the interlaboratory imprecision of quantitative BCR-ABL1 measurements and to facilitate local laboratory results interpretation and reporting, we decide to prepare laboratory guidelines that will further facilitate interlaboratory comparative studies and independent quality-assessment programs, which are of paramount importance for worldwide standardization of BCR-ABL1 monitoring results, in particular for those most isolated laboratories, with not easy access to commercial kits or sample interchange programs.

  9. Guidelines for molecular monitoring of BCR-ABL1 in chronic myeloid leukemia patients by RT-qPCR

    Directory of Open Access Journals (Sweden)

    Irene Larripa

    2017-02-01

    Full Text Available Current clinical guidelines for managing chronic myeloid leukemia include molecular monitoring of BCR-ABL1 transcript quantitative reverse-transcription PCR. Despite the proven prognostic significance of molecular response, it is not widely appreciated that quantitative reverse-transcription PCR potentially produces highly variable data, which may affect the validity of results, making comparability between different laboratories difficult. Therefore, standardized reporting of BCR-ABL1 measurements is needed for optimal clinical management. An approach to achieve comparable BCR-ABL1 values is the use of an international reporting scale. Conversion to the international scale is achieved by the application of laboratory specific conversion factor that is obtained by using validated secondary reference calibrators. Moreover, with the aim to mitigate the interlaboratory imprecision of quantitative BCR-ABL1 measurements and to facilitate local laboratory results interpretation and reporting, we decide to prepare laboratory guidelines that will further facilitate interlaboratory comparative studies and independent quality-assessment programs, which are of paramount importance for worldwide standardization of BCR-ABL1 monitoring results, in particular for those most isolated laboratories, with not easy access to commercial kits or sample interchange programs

  10. 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

    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......), 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...

  11. Attomolar electrochemical detection of the BCR/ABL fusion gene based on an amplifying self-signal metal nanoparticle-conducting polymer hybrid composite.

    Science.gov (United States)

    Avelino, Karen Y P S; Frias, Isaac A M; Lucena-Silva, Norma; Gomes, Renan G; de Melo, Celso P; Oliveira, Maria D L; Andrade, César A S

    2016-12-01

    In the last ten years, conjugated polymers started to be used in the immobilization of nucleic acids via non-covalent interactions. In the present study, we describe the construction and use of an electrochemical DNA biosensor based on a nanostructured polyaniline-gold composite, specifically developed for the detection of the BCR/ABL chimeric oncogene. This chromosome translocation is used as a biomarker to confirm the clinical diagnosis of both chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). The working principle of the biosensor rests on measuring the conductivity resulting from the non-covalent interactions between the hybrid nanocomposite and the DNA probe. The nanostructured platform exhibits a large surface area that enhances the conductivity. Positive cases, which result from the hybridization between DNA probe and targeted gene, induce changes in the amperometric current and in the charge transfer resistance (RCT) responses. Atomic force microscopy (AFM) images showed changes in the genosensor surface after exposure to cDNA sample of patient with leukemia, evidencing the hybridization process. This new hybrid sensing-platform displayed high specificity and selectivity, and its detection limit is estimated to be as low as 69.4 aM. The biosensor showed excellent analytical performance for the detection of the BCR/ABL oncogene in clinical samples of patients with leukemia. Hence, this electrochemical sensor appears as a simple and attractive tool for the molecular diagnosis of the BCR/ABL oncogene even in early-stage cases of leukemia and for the monitoring of minimum levels of residual disease.

  12. 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.

  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. 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.

  15. Sensitivity of imatinib-resistant T315I BCR-ABL CML to a synergistic combination of ponatinib and forskolin treatment.

    Science.gov (United States)

    Oaxaca, Derrick M; Yang-Reid, Sun Ah; Ross, Jeremy A; Rodriguez, Georgialina; Staniswalis, Joan G; Kirken, Robert A

    2016-09-01

    Tyrosine kinase inhibitors (TKIs) have dramatically improved the life expectancy of patients suffering from chronic myeloid leukemia (CML); however, patients will eventually develop resistance to TKI therapy or adverse side effects due to secondary off-target mechanisms associated with TKIs. CML patients exhibiting TKI resistance are at greater risk of developing an aggressive and drug-insensitive disease. Drug-resistant CML typically arises in response to spontaneous mutations within the drug binding sites of the targeted oncoproteins. To better understand the mechanism of drug resistance in TKI-resistant CML patients, the BCR-ABL transformed cell line KCL22 was grown with increasing concentrations of imatinib for a period of 6 weeks. Subsequently, a drug-resistant derivative of the parental KCL22 cell line harboring the T315I gatekeeper mutation was isolated and investigated for TKI drug sensitivity via multi-agent drug screens. A synergistic combination of ponatinib- and forskolin-reduced cell viability was identified in this clinically relevant imatinib-resistant CML cell line, which also proved efficacious in other CML cell lines. In summary, this study provides new insight into the biological underpinnings of BCR-ABL-driven CML and potential rationale for investigating novel treatment strategies for patients with T315I CML.

  16. Detection of BCR/ABL Fusion Gene by Fluorescence In Situ Hybridization and Its Clinical Application%荧光原位杂交技术检测BCR/ABL融合基因的临床应用

    Institute of Scientific and Technical Information of China (English)

    周瑞莲; 莫耀禧; 蓝梅; 林金盈

    2011-01-01

    This study was aimed to investigate the clinical value of detecting BCR/ABL fusion gene by fluorescence in situ hybridization (FISH). The conventional cytogenetic test and detection of BCR/ABL fusion gene by FISH for bone marrow of patients with newly diagnosed chronic myeloproliferative disease or myelodysplastic and myeloproliferative disorders, acute lymphocytic leukemia and chronic myelogeneous leukemia (CML) after allogeneic hematopoietic stem cell transplantation were carried out. The results showed that ( 1 ) out of 46 newly diagnosed as chronic myeloproliferative dsease or myelodysplastic and myeloproliferative disorders, 22 cases were diagnosed as CML, the FISH detection showed all positive (100% ), while cytogenetic test showed 86.4% (19/22) positive, in the other 24 patients who were diagnosed as other chronic myeloproliferative disease or myelodysplastic and myeloproliferative disorders, BCR/ABL fusion gene all were be detected as regative 100% by FISH, while the cytogenetic test of bone marrow in 3 cases supported the diagnosis of CML, and the diagnosis of myelodysplastic disorder in 1 case; (2) in 3 out of 7 acute lymphocytic leukemia cases the BCR/ABL fusion gene could not be detected by FISH; (3) the BCR/ABL fusion gene could be detected by FISH in 2 cases of CML received allogeneic hematopoietic stem cell transplantation, with abnormal threshold 6.5% and 1. 2% respectively. It is concluded that the detection of BCR/ABL fusion gene by FISH is sensitive and reliable, which is very important for the diagnosis and differential dignosis of chronic myeloproliferative disorders, myelodysplastic and myeloproliferative disease, as well as definite diagnosis of Ph* acute lymphoblastic leukemia. This method also has an important significance for monitor of minimal residual disease in CML patients received allogeneic hematopoietic stem cell transplantation.%本研究探讨应用荧光原位杂交技术( FISH)检测BCR/ABL融合基因在临床中的应用价

  17. Combinations of Novel Histone Deacetylase and Bcr-Abl Inhibitors in the Therapy of Imatinib Mesylate-Sensitive and -Refractory Bcr-Abl Expressing Leukemia

    Science.gov (United States)

    2008-12-01

    changes to the principles of treatment for both basal cell and squamous cell skin cancers and changes in the consideration of radiotherapy . 550...including those that contain FLT-3 mutations, while relatively sparing NBMCs.26,28,29 On the basis of the strong rationale generated by these observations...neutropenia (14 percent) were mainly grade 3 or 4 and appeared to increase with increasing doses of the drug. Pruritus, rash, and dry skin were almost

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. Determination of lactate dehydrogenase (LDH and Bcr-Abl transcript in the follow-up of patients with chronic myeloid leukemia - doi: 10.4025/actascihealthsci.v32i2.6408 Determination of lactate dehydrogenase (LDH and Bcr-Abl transcript in the follow-up of patients with chronic myeloid leukemia - doi: 10.4025/actascihealthsci.v32i2.6408

    Directory of Open Access Journals (Sweden)

    Thiago Cezar Fujita

    2010-09-01

    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.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

  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. 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.

  7. 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

  8. BCR/ABL DCDF-FISH信号特征与染色体核型的关系及其在慢性粒细胞白血病中的意义%Significance of BCR/ABL DCDF-FISH signal pattern and karyotype in chronic myeloid leukemia

    Institute of Scientific and Technical Information of China (English)

    杜庆华; 应逸; 陈晓燕

    2010-01-01

    目的 探讨慢性粒细胞白血病(CML)BCR/ABL DCCF-FISH的信号特点及其与染色体核型的关系.结果 使用BCR/ABL的DCDF探针对65例慢性粒细胞白血病骨髓标本进行荧光原位杂交及染色体核型检查.结果 65例CML核型43例Ph阳性,1例阴性,其余21例未行核性检查或无可分析分裂相;FISH结果65例均为BCR/ABL阳性,其中9例伴ASS基因缺失,5例复杂易位,1例+Ph伴ASS基因缺失.结论 传统核型与DCDF-FISH,应互相结合,方可对遗传学特征作出更准确分析.%Objective To investigate the relation between the BCR/ABL DCDF-FISH signal pattern and karyotype in chronic myeloid leukemia(CML). Methods 65 cases of CML were performed FISH and karyotyping. Results In 65 cases CML. 43 cases were Ph positive, 1 case was Ph negative, 21 cases had no performed karyotyping analysis. All of 65 cases were FISH positive,9 cases with ASS gene deletion, 5 cases with complex variant translocation, 1 case with (+) Ph and ASS gene deletion. Conclusion More precise analysis can be got by combining the result of FISH and karyotyping.

  9. Do endothelial cells belong to the primitive stem leukemic clone in CML? Role of extracellular vesicles.

    Science.gov (United States)

    Ramos, Teresa L; Sánchez-Abarca, Luis Ignacio; López-Ruano, Guillermo; Muntión, Sandra; Preciado, Silvia; Hernández-Ruano, Montserrat; Rosado, Belén; de las Heras, Natalia; Chillón, M Carmen; Hernández-Hernández, Ángel; González, Marcos; Sánchez-Guijo, Fermín; Del Cañizo, Consuelo

    2015-08-01

    The expression of BCR-ABL in hematopoietic stem cells is a well-defined primary event in chronic myeloid leukemia (CML). Some reports have described the presence of BCR-ABL on endothelial cells from CML patients, suggesting the origin of the disease in a primitive hemangioblastic cell. On the other hand, extracellular vesicles (EVs) released by CML leukemic cells are involved in the angiogenesis modulation process. In the current work we hypothesized that EVs released from BCR-ABL(+) cells may carry inside the oncogene that can be transferred to endothelial cells leading to the expression of both BCR-ABL transcript and the oncoprotein. EVs from K562 cells and plasma of newly diagnosed CML patients were isolated by ultracentrifugation. RT-PCR analysis detected the presence of BCR-ABL RNA in the EVs isolated from both K562 cells and plasma of CML patients. The incorporation of these EVs into endothelial cells was demonstrated by flow cytometry and fluorescence microscopy showed that after 24h of incubation most EVs were incorporated. BCR-ABL transcripts were detected in all experiments on endothelial cells incubated with EVs from both sources. The presence of BCR-ABL on endothelial cells incubated with Philadelphia(+) EVs was also confirmed by Western blot assays. In summary, endothelial cells acquire BCR-ABL RNA and the oncoprotein after incubation with EVs released from Ph(+) positive cells (either from K562 cells or from plasma of newly diagnosed CML patients). This results challenge the hypothesis that endothelial cells may be part of the Philadelphia(+) clone in CML.

  10. 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.

  11. 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.

  12. 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).

  13. “Preleukemic or smoldering” chronic myelogenous leukemia (CML:BCR-ABL1 positive: A brief case report

    Directory of Open Access Journals (Sweden)

    John M. Bennett

    2015-01-01

    The most common feature of CML is an elevated WBC count, usually above 25×103/µL, and frequently above 100×103/µL. We report a case of confirmed Ph+CML with a normal CBC detected because of the presence of rare myelocytes and 2% basophils [Fig. 1]. Previous leukocyte counts for the preceding eight years were normal with the exception of one done four months prior to his presentation that showed an abnormal differential with 1% basophils, 2% metamyelocytes and 2% myelocytes.

  14. [Standardization of quantitative detection of BCR-ABL gene expression by RQ-PCR in patients with chronic myeloid leukemia in cooperation with European Leukemia Net].

    Science.gov (United States)

    Sacha, Tomasz; Zawada, Magdalena; Czekalska, Sylwia; Florek, Izabela; Mueller, Martin; Gniot, Michał; Jaźwiec, Bozena; Kyrcz-Krzemień, Sławomira; Leszczyńska, Aleksandra; Lewandowski, Krzysztof; Matiakowska, Karolina; Solarska, Iwona; Stokłosa, Tomasz; Skotnicki, Aleksander B

    2010-01-01

    Monitoring of chronic myeloid leukemia treatment efficacy requires very sensitive methods of BCR-ABL gene detection based on polymerase chain reaction (PCR). The lack of comparability of BCR-ABL mRNA quantification results generated by various methodologies in different laboratories was the cause of an international multicenter trial initiation with the participation of 133 laboratories in 24 European countries cooperating within the "EUTOS for CML" project. Pracownia Diagnostyki Molekularnej Kliniki Hematologii is taking part in standardisation rounds organised since 2005. The compatibility of methodology used in Pracownia with European Leukemia Net (ELN) standards was confirmed, and correction factor for the expression of RQ-PCR results in an international scale was calculated. Pracownia was charge by ELN with a task of conducting the standardisation in polish molecular biology laboratories. Test probes were prepared and sent to eight cooperating laboratories. The results obtained in six laboratories were concordant with results from laboratory in Krakow after conversion to international scale, therefore it was possible to calculate individual correction factors. The participation of polish laboratories in international standardization process created the opportunity for unification of BCR-ABL quantification methodologies with recommendations of international experts, and showed that the quality of analyses performed in majority of them was satisfactory enough to calculate correction factor and to express the RQ-PCR results in widely accepted international scale.

  15. 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.

  16. 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.

  17. 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

  18. A BCR-ABL1 cutoff of 1.5% at 3 months, determined by the GeneXpert system, predicts an optimal response in patients with chronic myeloid leukemia

    Science.gov (United States)

    García-Gutiérrez, Valentín; Gómez-Casares, María T.; Puerta, José M.; Alonso-Domínguez, Juan M.; Osorio, Santiago; Hernández-Boluda, Juan C.; Collado, Rosa; Ramírez, María J.; Ibáñez, Fátima; Martín, María L.; Rodríguez-Gambarte, Juan D.; Martínez-Laperche, Carolina; Gómez, Montse; Fiallo, Dolly V.; Redondo, Sara; Rodríguez, Alicia; Ruiz-Nuño, Concepción; Steegmann, Juan L.

    2017-01-01

    In chronic myeloid leukemia (CML) patients, 3-month BCR-ABL1 levels have consistently been correlated with further outcomes. Monitoring molecular responses in CML using the GeneXpert (Cepheid) platform has shown an optimal correlation with standardized RQ-PCR (IS) when measuring BCR-ABL1 levels lower than 10%, as it is not accurate for values over 10%. The aim of the present study was to determine the predictive molecular value at three months on different outcome variables using the Xpert BCR-ABL1 MonitorTM assay (Xpert BCR-ABL1). We monitored 125 newly diagnosed consecutive CML patients in the chronic phase (CML-CP) using an automated method: Xpert BCR-ABL1. Only 5% of patients did not achieve an optimal response at 3 months, and the 10% BCR-ABL1 cutoff defined by RQ-PCR (IS) methods was unable to identify significant differences in the probabilities of achieving a complete cytogenetic response (CCyR) (50% vs. 87%, p = 0.1) or a major molecular response (MMR) (60% vs. 80%, p = 0.29) by 12 months. In contrast, a cutoff of 1.5% more accurately identified differences in the probabilities of achieving CCyR (98% vs. 54%, p<0.001) and MMR (88% vs. 56%, p<0.001) by 12 months, as well as probabilities of treatment changes (p = 0.005). Therefore, when using the Xpert BCR-ABL1 assay, a cutoff of 1.5% at 3 months could with high probability identify patients able to achieve an optimal response at 12 months. PMID:28278193

  19. 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

  20. BCR-ABL1: Test

    Science.gov (United States)

    ... molecular genetic test, and/or fluorescence in situ hybridization (FISH) . These help establish the initial diagnosis of ... Health Professionals ©2001 - by American Association for Clinical Chemistry • Contact Us | Terms of Use | Privacy We comply ...

  1. 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.

  2. Application of BCR-ABL in chronic myelogenous leukemia%BCR-ABL探针在慢性粒细胞白血病中的应用

    Institute of Scientific and Technical Information of China (English)

    李娟; 陆莉; 蒲育栋; 杨柳; 赵丽

    2009-01-01

    目的 用常规细胞遗传学(conventional cytogenetics,CC)和荧光原位杂交(fluorescence chromosomal in situ hybridization,FISH)技术检测Ph染色体.方法 常规细胞遗传学分析(CC),荧光原位杂交(FISH)技术.结果 7例患者4例Ph染色体阴性,其中2例分别伴有t(18;22)和t(17;22)异常,其余2例为异基因造血干细胞移植后正常核型.一例培养后无中期分裂相.2例Ph染色体阳性,FISH结果bcr/abl(+)细胞检出率分别为63.87%,84.51%,7.56%,4.0%,74.45%,67%,47%.结论 常规细胞遗传学与荧光原位杂交技术相结合对CML患者诊断治疗过程中肿瘤负荷动态检测有显著意义.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

    Aurora kinase overexpression has been observed in patients with hematologic malignancies. MK-0457, a pan-aurora kinase inhibitor that also inhibits the ABL T315I mutant, was evaluated to treat patients with chronic myelogenous leukemia (CML) or Philadelphia chromosome (Ph+) acute lymphoblastic...

  8. 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

  9. Imatinib Mesylate and Combination Chemotherapy in Treating Patients With Newly Diagnosed Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

    Science.gov (United States)

    2016-12-28

    B Acute Lymphoblastic Leukemia With t(9;22)(q34.1;q11.2); BCR-ABL1; BCR-ABL1 Fusion Protein Expression; Minimal Residual Disease; Philadelphia Chromosome Positive; T Acute Lymphoblastic Leukemia; Untreated Adult Acute Lymphoblastic Leukemia; Untreated Childhood Acute Lymphoblastic Leukemia

  10. The HDAC inhibitor SB939 overcomes resistance to BCR-ABL kinase Inhibitors conferred by the BIM deletion polymorphism in chronic myeloid leukemia.

    Science.gov (United States)

    Rauzan, Muhammad; Chuah, Charles T H; Ko, Tun Kiat; Ong, S Tiong

    2017-01-01

    Chronic myeloid leukemia (CML) treatment has been improved by tyrosine kinase inhibitors (TKIs) such as imatinib mesylate (IM) but various factors can cause TKI resistance in patients with CML. One factor which contributes to TKI resistance is a germline intronic deletion polymorphism in the BCL2-like 11 (BIM) gene which impairs the expression of pro-apoptotic splice isoforms of BIM. SB939 (pracinostat) is a hydroxamic acid based HDAC inhibitor with favorable pharmacokinetic, physicochemical and pharmaceutical properties, and we investigated if this drug could overcome BIM deletion polymorphism-induced TKI resistance. We found that SB939 corrects BIM pre-mRNA splicing in CML cells with the BIM deletion polymorphism, and induces apoptotic cell death in CML cell lines and primary cells with the BIM deletion polymorphism. More importantly, SB939 both decreases the viability of CML cell lines and primary CML progenitors with the BIM deletion and restores TKI-sensitivity. Our results demonstrate that SB939 overcomes BIM deletion polymorphism-induced TKI resistance, and suggest that SB939 may be useful in treating CML patients with BIM deletion-associated TKI resistance.

  11. The HDAC inhibitor SB939 overcomes resistance to BCR-ABL kinase Inhibitors conferred by the BIM deletion polymorphism in chronic myeloid leukemia

    Science.gov (United States)

    Rauzan, Muhammad; Chuah, Charles T. H.; Ko, Tun Kiat; Ong, S. Tiong

    2017-01-01

    Chronic myeloid leukemia (CML) treatment has been improved by tyrosine kinase inhibitors (TKIs) such as imatinib mesylate (IM) but various factors can cause TKI resistance in patients with CML. One factor which contributes to TKI resistance is a germline intronic deletion polymorphism in the BCL2-like 11 (BIM) gene which impairs the expression of pro-apoptotic splice isoforms of BIM. SB939 (pracinostat) is a hydroxamic acid based HDAC inhibitor with favorable pharmacokinetic, physicochemical and pharmaceutical properties, and we investigated if this drug could overcome BIM deletion polymorphism-induced TKI resistance. We found that SB939 corrects BIM pre-mRNA splicing in CML cells with the BIM deletion polymorphism, and induces apoptotic cell death in CML cell lines and primary cells with the BIM deletion polymorphism. More importantly, SB939 both decreases the viability of CML cell lines and primary CML progenitors with the BIM deletion and restores TKI-sensitivity. Our results demonstrate that SB939 overcomes BIM deletion polymorphism-induced TKI resistance, and suggest that SB939 may be useful in treating CML patients with BIM deletion-associated TKI resistance. PMID:28301600

  12. siRNA-cell-penetrating peptides complexes as a combinatorial therapy against chronic myeloid leukemia using BV173 cell line as model.

    Science.gov (United States)

    Freire, João Miguel; Rego de Figueiredo, Inês; Valle, Javier; Veiga, Ana Salomé; Andreu, David; Enguita, Francisco J; Castanho, Miguel A R B

    2017-01-10

    Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by a single gene mutation, a reciprocal translocation that originates the Bcr-Abl gene with constitutive tyrosine kinase activity. As a monogenic disease, it is an optimum target for RNA silencing therapy. We developed a siRNA-based therapeutic approach in which the siRNA is delivered by pepM or pepR, two cell-penetrating peptides (CPPs) derived from the dengue virus capsid protein. These peptides have a dual role: siRNA delivery into cells and direct action as bioportides, i.e. intracellularly bioactive CPPs, targetting cancer-related signaling processes. Both pepM and pepR penetrate the positive Bcr-Abl(+) Cell Line (BV173). Five in silico designed anti-Bcr-Abl siRNA were selected for in vitro analysis after thorough screening. The Bcr-Abl downregulation kinetics (48h to 168h) was followed by quantitative PCR. The bioportide action of the peptide vectors was evaluated by genome-wide microarray analysis and further validated by testing BV173 cell cycle and cell proliferation monitoring different genes involved in housekeeping/cell stress (RPL13A, HPRT1), cell proliferation (ki67), cell apoptosis (Caspase 3 and Caspase 9) and cell cycle steps (CDK2, CCDN2, CDKN1A). Assays with a commercial transfection agent were carried out for comparison purposes. Maximal Bcr-Abl gene knockdown was observed for one of the siRNA when delivered by pepM at 120h. Both pepM and pepR showed downregulation effects on proliferative CML-related signaling pathways having direct impact on BV173 cell cycle and proliferation, thus reinforcing the siRNA effect by acting as anticancer molecules. With this work we show the therapeutic potential of a CPP shuttle that combines intrinsic anticancer properties with the ability to deliver functional siRNA into CML cell models. By such combination, the pepM-siRNA conjugates lowered Bcr-Abl gene expression levels more extensively than conventional siRNA delivery technologies and

  13. 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

  14. 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

  15. Efficacy of the polo-like kinase inhibitor rigosertib, alone or in combination with Abelson tyrosine kinase inhibitors, against break point cluster region-c-Abelson-positive leukemia cells.

    Science.gov (United States)

    Okabe, Seiichi; Tauchi, Tetsuzo; Tanaka, Yuko; Sakuta, Juri; Ohyashiki, Kazuma

    2015-08-21

    The potency of Abelson (ABL) tyrosine kinase inhibitors (TKIs) against chronic myeloid leukemia (CML) has been demonstrated. However, ABL TKI resistance can develop. In this study, we investigated the efficacy of a combination therapy including rigosertib (ON 01910.Na), a polo-like kinase (PLK) and phosphoinositide 3-kinase (PI3K) inhibitor, and ABL TKIs. A 72-h rigosertib treatment was found to inhibit cell growth, induce apoptosis, reduce phosphorylation of the breakpoint cluster region-c (BCR)-ABL and its substrate Crk-L, and increase the activities of caspase 3 and poly (ADP-ribose) polymerase (PARP). This combination therapy also exerted a synergistic inhibitory effect on Philadelphia chromosome (Ph)-positive cell proliferation and reduced the phosphorylation of BCR-ABL and Crk-L while increasing that of cleaved PARP and the H2A.X histone. Rigosertib also potently inhibited the growth of ABL TKI-resistant cells, and cotreatment with ABL TKIs and rigosertib induced higher cytotoxicity. These results indicate that rigosertib treatment may be a powerful strategy against ABL TKI-resistant cells and could enhance the cytotoxic effects of ABL TKIs.

  16. 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.

  17. 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

  18. The in-vitro antiproliferative effect of PRI-2191 and imatinib applied in combined treatment with cisplatin, idarubicin, or docetaxel on human leukemia cells.

    Science.gov (United States)

    Switalska, Marta; Nasulewicz-Goldeman, Anna; Opolska, Aleksandra; Maciejewska, Magdalena; Kutner, Andrzej; Wietrzyk, Joanna

    2012-01-01

    Imatinib mesylate (Gleevec, STI571) is a specific inhibitor of the Bcr/Abl fusion tyrosine kinase that exhibits potent antileukemic effects in chronic myelogenous leukemia. Bcr/Abl-positive K562 and Bcr/Abl-negative HL-60 human leukemia cells were used to investigate the effect of PRI-2191, a calcitriol analog, on the biological effects of imatinib combined with other anticancer drugs. The results show that PRI-2191 enhances the antiproliferative effect of imatinib on HL-60 cells. When these two agents together are applied with either docetaxel or cisplatin, but not with idarubicin, the antiproliferative effect could still be enhanced. Moreover, when the interaction between the chemotherapy agents was antagonistic or additive, PRI-2191 could even shift it to synergism. This effect correlated with an accumulation of HL-60 cells in the G0/G1 phase of the cell cycle and a decrease in the percentage of cells in the G2/M and S stage in the ternary combinations used. PRI-2191 did not influence apoptosis induced by imatinib alone or in ternary combinations with all the chemotherapy agents used. These results may suggest that the stronger antiproliferative effect of the combined treatment with PRI-2191 on HL-60 cells is related to cell cycle arrest rather than to the induction of apoptosis.

  19. Triomics Analysis of Imatinib-Treated Myeloma Cells Connects Kinase Inhibition to RNA Processing and Decreased Lipid Biosynthesis.

    Science.gov (United States)

    Breitkopf, Susanne B; Yuan, Min; Helenius, Katja P; Lyssiotis, Costas A; Asara, John M

    2015-11-03

    The combination of metabolomics, lipidomics, and phosphoproteomics that incorporates triple stable isotope labeling by amino acids in cell culture (SILAC) protein labeling, as well as (13)C in vivo metabolite labeling, was demonstrated on BCR-ABL-positive H929 multiple myeloma cells. From 11 880 phosphorylation sites, we confirm that H929 cells are primarily signaling through the BCR-ABL-ERK pathway, and we show that imatinib treatment not only downregulates phosphosites in this pathway but also upregulates phosphosites on proteins involved in RNA expression. Metabolomics analyses reveal that BCR-ABL-ERK signaling in H929 cells drives the pentose phosphate pathway (PPP) and RNA biosynthesis, where pathway inhibition via imatinib results in marked PPP impairment and an accumulation of RNA nucleotides and negative regulation of mRNA. Lipidomics data also show an overall reduction in lipid biosynthesis and fatty acid incorporation with a significant decrease in lysophospholipids. RNA immunoprecipitation studies confirm that RNA degradation is inhibited with short imatinib treatment and transcription is inhibited upon long imatinib treatment, validating the triomics results. These data show the utility of combining mass spectrometry-based "-omics" technologies and reveals that kinase inhibitors may not only downregulate phosphorylation of their targets but also induce metabolic events via increased phosphorylation of other cellular components.

  20. 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

  1. 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

  2. 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

  3. Leucemia Linfoblástica Aguda Filadélfia positiva Phyladelphia positive acute lymphoblastic leukemia

    Directory of Open Access Journals (Sweden)

    Rony Schaffel

    2008-04-01

    Full Text Available O cromossomo Filadélfia (Ph1 é a alteração citogenética mais comum da Leucemia Linfoblástica Aguda do adulto (LLA. Esta alteração citogenética predomina nos adultos com mais de 50 anos e na LLA de origem na célula B, principalmente CD10 positiva. O diagnóstico requer a análise citogenética e a pesquisa do mRNA do gene BCR-ABL no sangue periférico ou na medula óssea. A LLA Ph1 apresenta uma sobrevida global em cinco anos inferior a 20% quando tratada com protocolos para LLA. Os poucos casos de cura ocorrem nos pacientes submetidos ao transplante alogênico de medula óssea (TMO. A adição do imatinibe à quimioterapia resultou em melhora na taxa de remissão completa, maior taxa de remissão molecular completa, maior número de pacientes aptos para realizar o TMO, uma maior sobrevida livre de eventos e maior sobrevida global, embora o tempo de seguimento seja ainda muito curto. Entretanto, a taxa de recaídas e o aparecimento de mutações do BCR-ABL resistentes ao imatinibe ainda são preocupantes. No futuro, novos inibidores de tirosina quinase poderão ser incorporados ao tratamento da LLA Ph1.The Philadelphia chromosome (Ph1 is the most frequent abnormality in acute adult lymphoblastic leukemia (ALL. Ph1 positive ALL is more frequent in over 50-year-old adults, in B-cell ALL and CD10-positive ALL. Diagnosis is based on the identification of the BCR-ABL gene mRNA in peripheral blood or bone marrow. The 5-year overall survival of patients with Ph1 positive ALL treated with chemotherapy alone is less than 20%. A few cases may be cured by allogeneic stem cell transplantation. The addition of imatinib to the chemotherapeutic treatment has resulted in more complete remissions, more complete molecular responses, more patients able to perform stem cell transplantation, better event-free survival and better overall survival, although the study follow-up period is very short so far. High relapse rates and the emergence of BCR-ABL

  4. 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

  5. Real-time analysis of imatinib- and dasatinib-induced effects on chronic myelogenous leukemia cell interaction with fibronectin.

    Directory of Open Access Journals (Sweden)

    Adam Obr

    Full Text Available Attachment of stem leukemic cells to the bone marrow extracellular matrix increases their resistance to chemotherapy and contributes to the disease persistence. In chronic myelogenous leukemia (CML, the activity of the fusion BCR-ABL kinase affects adhesion signaling. Using real-time monitoring of microimpedance, we studied in detail the kinetics of interaction of human CML cells (JURL-MK1, MOLM-7 and of control BCR-ABL-negative leukemia cells (HEL, JURKAT with fibronectin-coated surface. The effect of two clinically used kinase inhibitors, imatinib (a relatively specific c-ABL inhibitor and dasatinib (dual ABL/SRC family kinase inhibitor, on cell binding to fibronectin is described. Both imatinib and low-dose (several nM dasatinib reinforced CML cell interaction with fibronectin while no significant change was induced in BCR-ABL-negative cells. On the other hand, clinically relevant doses of dasatinib (100 nM had almost no effect in CML cells. The efficiency of the inhibitors in blocking the activity of BCR-ABL and SRC-family kinases was assessed from the extent of phosphorylation at autophosphorylation sites. In both CML cell lines, SRC kinases were found to be transactivated by BCR-ABL. In the intracellular context, EC50 for BCR-ABL inhibition was in subnanomolar range for dasatinib and in submicromolar one for imatinib. EC50 for direct inhibition of LYN kinase was found to be about 20 nM for dasatinib and more than 10 µM for imatinib. Cells pretreated with 100 nM dasatinib were still able to bind to fibronectin and SRC kinases are thus not necessary for the formation of cell-matrix contacts. However, a minimal activity of SRC kinases might be required to mediate the increase in cell adhesivity induced by BCR-ABL inhibition. Indeed, active (autophosphorylated LYN was found to localize in cell adhesive structures which were visualized using interference reflection microscopy.

  6. Successful Peripheral Blood Stem Cells Collection in Imatinib Pretreated and Nilotinib-Treated Chronic Myeloid Leukemia Patient

    Directory of Open Access Journals (Sweden)

    Samuel Vokurka

    2010-01-01

    Full Text Available We report a case of a successful mobilization and harvest of the peripheral blood stem cells (PBSCs in imatinib-pretreated and nilotinib treated 52-year-old woman diagnosed with Philadelphia chromosome-positive and BCR-ABL (b2a2 positive chronic phase CML in 2/2002. She failed interferon-alfa and imatinib treatment. She achieved her first complete molecular remission after 16 months of nilotinib treatment and later on was mobilized with filgrastim at a dose of 10 ug/kg/day applied subcutaneously once daily. The total number of 2.98×106 CD34+ cells/kg was harvested on the fourth day of the mobilization. The autologous graft of the stem cells was cryopreserved and tested for the residual disease: the FISH revealed negative results and the RT-PCR was positive (BCR-ABL/ABL ratio 0,0017 in RQ-PCR. To our knowledge, this is the first report of successful PBSC harvest in a patient significantly pretreated with imatinib and nilotinib.

  7. Dasatinib treatment based on BCR-ABL mutation detection in imatinib-resistant patients with chronic myeloid leukemia%BCR-ABL突变检测指导下的达沙替尼治疗伊马替尼耐药的慢性髓性白血病疗效分析

    Institute of Scientific and Technical Information of China (English)

    江倩; 秦亚溱; 赖悦云; 江浩; 石红霞

    2016-01-01

    Objective To evaluate the efficiency of dasatinib as the second-or third-line tyrosine kinase inhibitor(TKI)in imatinib-resistant patients with chronic myeloid leukemia(CML)based on BCR-ABL mutation detection. Methods 122 CML patients received dasatinib treatment, including 83 with imatinib-resistance and 39 with both imatinib-and nilotinib-resistance, 55 in the chronic-phase(CP), 21 in the accelerated-phase (AP) and 46 in the blast-phase (BP). Those harboring dasatinib highly-resistant mutations(T315I/A, F317L/V/C and V299L)were excluded based on BCR-ABL kinase domain mutation screening by Sanger sequencing at baseline. Hematologic, cytogenetic and molecular responses were evaluated regularly, and rates of progression-free-survival(PFS)and overall survival(OS)were analyzed. BCR-ABL mutation detection was performed once the patients failed on dasatinib. Results In the CP patients, the rates of complete hematological response (CHR), complete cytogenetic response (CCyR), major molecular response(MMR)and molecular response 4.5(MR4.5)were 92.7%, 53.7%, 29.6%and 14.8%, respectively. 4-year PFS and OS rates were 84.4%and 89.5%, respectively. In the AP patients, HR and CCyR rates were 81.0%and 35.0%;and 3-year PFS and OS rates were 56.1%and 59.3%, respectively. In the BP patients, HR and CCyR rates were 63.0%and 21.4%;and 1-year PFS and OS rates were 43.6%and 61.8%, respectively. Outcomes were similar when dasatinib was used as the second-line TKI or thethird-line TKI. Of the 75 patients who were resistant to dasatinib, 37(48.7%)developed new mutation(s), and T315I(59.5%)was the most common mutation type. The patients who already harbored mutation(s) before dasatinib therapy achieved similar responses and outcomes to those with no mutation at baseline. However, they had higher likelihood of developing additional mutations associated with resistance to dasatinib(65.7%vs 34.1%, P=0.006). Conclusions Dasatinib was proved to be effective in the treatment of imatinib

  8. Treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia in pregnancy.

    Science.gov (United States)

    Mainor, Candace B; Duffy, Alison P; Atkins, Kristin L; Kimball, Amy S; Baer, Maria R

    2016-04-01

    BCR-ABL inhibitors administered in conjunction with chemotherapy have significantly improved outcomes in Philadelphia chromosome-positive acute lymphoblastic leukemia but, for patients diagnosed during pregnancy, data on risks to the fetus are limited. We report a woman treated with chemotherapy and imatinib mesylate who delivered a healthy baby at 30 weeks, and we discuss available data.

  9. 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.

  10. [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.

  11. 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...

  12. 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

  13. 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.

  14. 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.

  15. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  17. 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.

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

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    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.

  19. 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.

  20. Growth Arrest Specific 2 Is Up-Regulated in Chronic Myeloid Leukemia Cells and Required for Their Growth

    OpenAIRE

    Haixia Zhou; Yue Ge; Lili Sun; Wenjuan Ma; Jie Wu; Xiuyan Zhang; Xiaohui Hu; Eaves, Connie J; Depei Wu; Yun Zhao

    2014-01-01

    Although the generation of BCR-ABL is the molecular hallmark of chronic myeloid leukemia (CML), the comprehensive molecular mechanisms of the disease remain unclear yet. Growth arrest specific 2 (GAS2) regulates multiple cellular functions including cell cycle, apoptosis and calpain activities. In the present study, we found GAS2 was up-regulated in CML cells including CD34+ progenitor cells compared to their normal counterparts. We utilized RNAi and the expression of dominant negative form o...

  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. Targeted destruction of HIV-positive cells

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    Jyoti R Sharma

    2014-11-01

    Full Text Available Introduction: HIV/AIDS is now a global epidemic that has become the leading infectious killer of adults worldwide. Although antiretroviral (ARV therapy has dramatically improved the quality of life and increased the life expectancy of those infected with HIV but frequency of dosing and drug toxicity as well as the development of viral resistance pose additional limitations. The rapidly expanding field of nanotechnology has vast potential to radically advance the treatment and prevention of HIV/AIDS. Nanoparticles can provide improved drug delivery, by virtue of their small size, robustness, safety, multimodality or multifunctionality. Aims and objectives: Since HIV primarily infects CD4+ cells; we aim to use CD4 as a selectable target to deliver a pro-apoptotic protein to HIV-infected cells using nanoparticles as carriers. The aim of study was to develop a nanotechnology-based death inducing delivery system for the destruction of CD4+HIV infected cells through the activation of caspase-3. Methodology: A modified caspase-3 protein (Mut-3 was engineered, which is cleavable only by HIV-1 protease. Mut-3 can activate apoptosis in the presence of HIV-1 protease, consequently killing HIV-positive cells. Mut-3 protein was conjugated to gold nanoparticles together with a CD4-targeting peptide. The efficacy of the gold nanoparticles was tested on CHO cells that were genetically engineered to express GFP labelled CD4 and HIV-1 protease. Results: Mut-3 was expressed in bacterial cells and purified. CHO cells that stably over express CD4-GFP and HIV-1 protease were selected using Fluorescence Activated Cell Sorting. Dose response cell culture experiments showed that gold nanoparticles without Mut-3 and CD4-targeting peptide did not induce cell death in CHO cells, while gold nanoparticles that was conjugated with Mut-3 and the CD4-targeting peptide rapidly induced cell death in CHO cells. Conclusions: Our results suggest that gold nanoparticles conjugated

  3. A palmitoyl conjugate of insect pentapeptide Yamamarin arrests cell proliferation and respiration

    OpenAIRE

    2010-01-01

    A palmitoyl conjugate of an insect pentapeptide that occurs in diapausing insects causes a reversible cell-cycle arrest and suppresses mitochondrial respiration. This peptide compound also causes growth arrest in murine leukemic cell line expressing human gene Bcr/Abl and a farnesoyl peptide induces embryonic diapause in Bombyx mori. These results demonstrate that the insect peptide compounds can lead to the understanding of a common pathway in developmental arrest in animals and may provide ...

  4. Ubiquitin-positive inclusions in ependymal cells.

    Science.gov (United States)

    Kawanishi, Ryuta; Mizutani, Tomohiko; Yamada, Hiroshi; Minami, Masayuki; Kakimi, Shigeo; Yamada, Tsutomu; Hatori, Tsutomu; Akima, Michio

    2003-08-01

    Ubiquitin-positive inclusions (UbIs) have not been well studied in ependymal cells. Since we detected such UbIs in the central canals of the medulla and spinal cord while investigating UbIs in neurodegenerative diseases, we studied UbIs in the entire ependymal system of 42 patients with various neurological diseases and of 10 non-neurological controls. UbIs were located in the cytoplasm of the ependymal cells, and were round to oval in shape, measuring 4-11 microm in diameter. The UbIs were non-argyrophilic and undetectable by hematoxylin and eosin staining, but mildly reactive to periodic acid-Schiff staining with and without digestion. The UbIs were variably immunoreactive for anti-epithelial membrane antigen (EMA) antibody, but did not react with several other antibodies. The co-existence of ubiquitin and EMA was confirmed by confocal laser microscopy. Throughout the ependymal system, UbIs were variably found in ependymal cells as well as in subependymal cells. There was no significant difference in the overall incidence of either ependymal or subependymal UbIs between the patients with neurological diseases and controls. However, ependymal UbIs in the central canal were more frequent in the neurological disease patients than in controls, although there was no disease specificity. This is the first comprehensive report to show common occurrence of UbIs in the ependymal cells of adult human brains.

  5. p53 modulates the effect of ribosomal protein S6 kinase1 (S6K1) on cisplatin toxicity in chronic myeloid leukemia cells.

    Science.gov (United States)

    Xiao, Ling-Yi; Kan, Wai-Ming

    2017-03-14

    Chronic myeloid leukemia (CML) is characterized by the expression of the oncoprotein, BCR-ABL. BCR-ABL inhibitors revolutionized CML chemotherapy while blast crisis (BC) CML patients are less responsive. Since suppression of ribosomal protein S6 kinase1 (S6K1) phosphorylation reverses the resistance to BCR-ABL inhibitor in CML cells and S6K1 inhibitors augment cisplatin toxicity in lung cancer cells, we speculated that combination of S6K1 inhibitor and cisplatin may be beneficial for eliminating BC CML cells. To our surprise, S6K1 inhibition decreased cisplatin-induced DNA damage and cell death only in p53(-/-) BC CML cells but not in p53(+/+) BC CML cells. During the progression of CML, p53 expression either decreases or mutates. Moreover, the expression of p53 affects drug response of CML cells. Our results confirmed that S6K1 inhibition reversed cisplatin toxicity is dependent on p53 expression in CML cells. Moreover, p53 attenuated the phosphorylation and localization of S6K1 via attenuating 3-phosphoinositide dependent protein kinase-1 (PDK1) phosphorylation. Furthermore, S6K1 acts via DNA-PKcs to regulate H2AX phosphorylation and PARP cleavage, respectively. Taken together, our results suggest that p53/PDK1/S6K1 is a novel pathway regulating cisplatin toxicity in BC CML cells.

  6. 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.

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

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    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. EFFECTS OF IFN-a COMBINED WITH IL-6 ON GROWTH AND EXPRESSION OF THE GENES RELATED TO CELL-GROWTH AND APOPTOSIS OF BONE MARROW CELLS FROM PATIENTS WITH CML

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To investigate the effects of interferon-a (IFN-a ) and IFN-a combined with interleukin-6 (IL-6) on growth and expression of bcr-abl, bcl-2 and c-myc genes in the mononuclear cells (MNCs) from bone marrow (BM) of patients with chronic myelogenous leukemia (CML). Methods: MNCs were collected from BM of the patients with CML in chronic phase by centrifugation in lymphocyte separation medium and cultured in liquid with IFN-a (200U/ml) or IFN-a (200U/ml) plus IL-6 (100 ng/ml). The growing cells were counted every day. The expression levels of b -actin, bcr-abl, bcl-2 and c-myc genes in the MNCs incubated for 24 h were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and relatively quantitative analysis of the amplified fragments by optical density scanning for the bands on gel. Results: The cell growth was markedly suppressed by IFN-a but the degree of cell-growth inhibition was slightly decreased by IL-6 on the basis of IFN-a effect. The expression of bcr-abl chimeric gene was intensely inhibited by IFN-a or IFN-a plus IL-6. The expression of bcl-2 gene was suppressed by either IFN-a or IFN-a plus IL-6, whereas that of c-myc gene was also inhibited by IFN-a but strongly elevated by IL-6 on the basis of IFN-a action. Conclusions: Both IFN-a and IFN-a plus IL-6 can inhibit the expression of anti-apoptosis gene such as bcr-abl and bcl-2 and regulate the expression of the gene related to cell proliferation and differentiation such as c-myc. Either IFN-a or IFN-a combined with IL-6 will serve as a trustful strategy of clinical treatment for CML.

  9. 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.

  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. 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.

  12. 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

  13. Detection of MRD may predict the outcome of patients with Philadelphia chromosome-positive ALL treated with tyrosine kinase inhibitors plus chemotherapy.

    Science.gov (United States)

    Ravandi, Farhad; Jorgensen, Jeffrey L; Thomas, Deborah A; O'Brien, Susan; Garris, Rebecca; Faderl, Stefan; Huang, Xuelin; Wen, Sijin; Burger, Jan A; Ferrajoli, Alessandra; Kebriaei, Partow; Champlin, Richard E; Estrov, Zeev; Challagundla, Pramoda; Wang, Sa A; Luthra, Rajyalakshmi; Cortes, Jorge E; Kantarjian, Hagop M

    2013-08-15

    From 2001 to 2011, 122 patients with newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia were treated with chemotherapy + imatinib (n = 54) or + dasatinib (n = 68). One hundred fifteen (94%) achieved complete remission (CR) including 101 patients who achieved it with only 1 induction course and had at least 1 minimal residual disease (MRD) assessment; 25 patients underwent an allogeneic stem cell transplant in first CR and were excluded, leaving 76 patients as the subject of this report. MRD monitoring by multiparameter flow cytometry (MFC) and real-time quantitative polymerase chain reaction (PCR) was performed at the end of induction and at ~3-month intervals thereafter. Median age was 54 years (range, 21-84 years). There was no difference in survival by achievement of at least a major molecular response (MMR; BCR-ABL/ABL < 0.1%) at CR (P = .22). Patients achieving MMR at 3, 6, 9, and 12 months had a better survival (P = .02, .04, .05, and .01, respectively). Negative MFC at CR did not predict for improved survival (P = .2). At 3 and 12 months, negative MRD by MFC was associated with improved survival (P = .04 and .001). MRD monitoring by PCR and MFC identifies patients who benefit from treatment intensification in first CR.

  14. Detection of MRD may predict the outcome of patients with Philadelphia chromosome–positive ALL treated with tyrosine kinase inhibitors plus chemotherapy

    Science.gov (United States)

    Jorgensen, Jeffrey L.; Thomas, Deborah A.; O’Brien, Susan; Garris, Rebecca; Faderl, Stefan; Huang, Xuelin; Wen, Sijin; Burger, Jan A.; Ferrajoli, Alessandra; Kebriaei, Partow; Champlin, Richard E.; Estrov, Zeev; Challagundla, Pramoda; Wang, Sa A.; Luthra, Rajyalakshmi; Cortes, Jorge E.; Kantarjian, Hagop M.

    2013-01-01

    From 2001 to 2011, 122 patients with newly diagnosed Philadelphia chromosome–positive acute lymphoblastic leukemia were treated with chemotherapy + imatinib (n = 54) or + dasatinib (n = 68). One hundred fifteen (94%) achieved complete remission (CR) including 101 patients who achieved it with only 1 induction course and had at least 1 minimal residual disease (MRD) assessment; 25 patients underwent an allogeneic stem cell transplant in first CR and were excluded, leaving 76 patients as the subject of this report. MRD monitoring by multiparameter flow cytometry (MFC) and real-time quantitative polymerase chain reaction (PCR) was performed at the end of induction and at ∼3-month intervals thereafter. Median age was 54 years (range, 21-84 years). There was no difference in survival by achievement of at least a major molecular response (MMR; BCR-ABL/ABL < 0.1%) at CR (P = .22). Patients achieving MMR at 3, 6, 9, and 12 months had a better survival (P = .02, .04, .05, and .01, respectively). Negative MFC at CR did not predict for improved survival (P = .2). At 3 and 12 months, negative MRD by MFC was associated with improved survival (P = .04 and .001). MRD monitoring by PCR and MFC identifies patients who benefit from treatment intensification in first CR. PMID:23836561

  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. Hydroxychavicol, a Piper betle leaf component, induces apoptosis of CML cells through mitochondrial reactive oxygen species-dependent JNK and endothelial nitric oxide synthase activation and overrides imatinib resistance.

    Science.gov (United States)

    Chakraborty, Jayashree B; Mahato, Sanjit K; Joshi, Kalpana; Shinde, Vaibhav; Rakshit, Srabanti; Biswas, Nabendu; Choudhury Mukherjee, Indrani; Mandal, Labanya; Ganguly, Dipyaman; Chowdhury, Avik A; Chaudhuri, Jaydeep; Paul, Kausik; Pal, Bikas C; Vinayagam, Jayaraman; Pal, Churala; Manna, Anirban; Jaisankar, Parasuraman; Chaudhuri, Utpal; Konar, Aditya; Roy, Siddhartha; Bandyopadhyay, Santu

    2012-01-01

    Alcoholic extract of Piper betle (Piper betle L.) leaves was recently found to induce apoptosis of CML cells expressing wild type and mutated Bcr-Abl with imatinib resistance phenotype. Hydroxy-chavicol (HCH), a constituent of the alcoholic extract of Piper betle leaves, was evaluated for anti-CML activity. Here, we report that HCH and its analogues induce killing of primary cells in CML patients and leukemic cell lines expressing wild type and mutated Bcr-Abl, including the T315I mutation, with minimal toxicity to normal human peripheral blood mononuclear cells. HCH causes early but transient increase of mitochondria-derived reactive oxygen species. Reactive oxygen species-dependent persistent activation of JNK leads to an increase in endothelial nitric oxide synthase-mediated nitric oxide generation. This causes loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, cleavage of caspase 9, 3 and poly-adenosine diphosphate-ribose polymerase leading to apoptosis. One HCH analogue was also effective in vivo in SCID mice against grafts expressing the T315I mutation, although to a lesser extent than grafts expressing wild type Bcr-Abl, without showing significant bodyweight loss. Our data describe the role of JNK-dependent endothelial nitric oxide synthase-mediated nitric oxide for anti-CML activity of HCH and this molecule merits further testing in pre-clinical and clinical settings.

  17. 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.

  18. 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.

  19. In Vitro Sensitivity Profiling Of Neuroblastoma Cells Against A Comprehensive Small Molecule Kinase Inhibitor Library To Identify Agents For Future Therapeutic Studies.

    Science.gov (United States)

    Singh, Anjali; Meier-Stephenson, Vanessa; Jayanthan, Aarthi; Narendran, Aru

    2016-11-22

    Solid tumors represent one of the most widespread causes of death in children across the world. Neuroblastoma (NB) constitutes about 8% of all childhood tumors, yet accounts for more than 15% of death, with an unacceptable overall survival rate. Despite the current multimodal therapeutic approaches involving surgery, radiation, chemotherapy with myeloablative therapy and hematopoietic stem cell rescue, there is growing realization of the limitations of conventional agents to improve the outcome in high risk metastatic disease. Hence, efforts have intensified to identify new targets and novel therapeutic approaches to improve cure rates in these children. Among the significant number of new therapeutics that are being evaluated for cancer each year, the agents that have been developed for common adult malignancies have the added advantage of having usable toxicity data already available for consideration. To identify potential therapeutic targets, we screened a small molecule library of 151 small kinase inhibitors against NB cell lines. Based on our initial screening data, we further examined the potential of Bcr-Abl targeting small molecule inhibitors to affect the growth and survival of NB cells. Our findings confirm the diversity in activity among the currently available Bcr-Abl inhibitors, possibly reflecting the molecular heterogeneity and off-target activity in each combination. In depth analyses of ponatinib, an orally bioavailable multi-target kinase inhibitor and an effective agent in the treatment of refractory Philadelphia chromosome (Ph) positive leukemia, show growth inhibition at sub-micromolar concentrations. In addition, we also identified the potential of this agent to interfere with insulin-like growth factor-1 receptor (IGF-1R) signaling pathways and Src activity. Ponatinib also induced apoptosis, indicated by caspase-9 and PARP cleavage. Furthermore, at sub-lethal conditions ponatinib significantly inhibited the ability of these cells to migrate

  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. 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

  2. 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...... using a panel of antibodies directed against CD117/c-kit, CD34, CD31, S100, tryptase, neurofilament, NSE, Factor-VIII and GFAP. A striking finding was an interstitial type of cell which is CD34 immunoreactive (CD34-ir) but CD117/c-kit negative. The cells have a tentacular morphology, enveloping...... and intermingling with individual muscle fasicles. Morphologically and immunohistochemically, they show no neurogenic, endothelial or mast cell differentiation. Transmission electron microscopy (TEM) showed the presence of interstitial cells with a round-to-oval nucleus, sparse perinuclear cytoplasm and long...

  3. Ph+, BCR-ABL+ chronic myeloid leukemia (CML) in early chronic phase | EU Clinical Trials Register [EU Clinical Trials Register

    Lifescience Database Archive (English)

    Full Text Available ALT ≤ 2.5 x valore massimo della normalità o ≤ 5.0 x valore massimo della normalità se considerato secondario alla leucemia...o venga considerato secondario alla leucemia - Bilirubina diretta ≤ 1.5 x valore massimo della normalità, sa

  4. HLA-DR-positive cells in large plaque (atrophic) parapsoriasis.

    Science.gov (United States)

    McMillan, E M; Wasik, R; Everett, M A

    1981-10-01

    The development of a monoclonal antibody directed against HLA DR (Ia-like) antigens of B cells and monocytes but not against normal peripheral human T cells suggested that this antibody might be used as a marker of B cells and monocytes in tissue sections. The T cell nature of large plaque (atrophic) parapsoriasis has recently been demonstrated by the immunoperoxidase technic. Immunoperoxidase examination of serial sections of tissues from two cases of large plaque parapsoriasis with one T cell antiserum, two monoclonal T cell antibodies, and one monoclonal reagent directed against HLA DR indicated that T cells in the cutaneous infiltrates were also HLA DR-positive. Evidence is accumulating that HLA DR positivity may be expressed by activated T cells. The findings here therefore suggest that many of the T lymphoid cells in two cases of large plaque (atrophic) parapsoriasis examined were activated in nature, and that HLA DR may not be a specific marker for B cells and monocytes in certain pathologic conditions. Caution should therefore presently be exercised in attempting to use this marker for the specific identification of B cells and monocytes in pathologic specimens, without simultaneous testing for T cell markers.

  5. 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

  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. 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.

  8. 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.

  9. Increased peroxisome proliferator-activated receptor-gamma activity reduces imatinib uptake and efficacy in chronic myeloid leukemia mononuclear cells.

    Science.gov (United States)

    Wang, Jueqiong; Lu, Liu; Kok, Chung H; Saunders, Verity A; Goyne, Jarrad M; Dang, Phuong; Leclercq, Tamara M; Hughes, Timothy P; White, Deborah L

    2017-02-02

    Imatinib is actively transported by OCT-1 influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Here we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor gamma agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor gamma antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to Bcr-Abl kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor gamma-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor gamma transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; preceptor gamma activation has a negative impact on the intracellular uptake of imatinib and consequent Bcr-Abl kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor gamma activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor gamma agonist pioglitazone was reported to act synergistically with imatinib targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor gamma ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor gamma activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in patients with high peroxisome proliferator-activated receptor gamma

  10. 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.

  11. Caveolin-1 contributes to realgar nanoparticle therapy in human chronic myelogenous leukemia K562 cells

    Directory of Open Access Journals (Sweden)

    Shi D

    2016-11-01

    Full Text Available Dan Shi,1,* Yan Liu,1,* Ronggang Xi,1 Wei Zou,2 Lijun Wu,3 Zhiran Zhang,1 Zhongyang Liu,1 Chao Qu,1 Baoli Xu,1 Xiaobo Wang1 1Department of Pharmacy, The 210th Hospital of People’s Liberation Army, 2College of Life Science, Liaoning Normal University, Dalian, Liaoning, 3Department of Pharmaceutics, College of Pharmacy, Harbin Medical University, Harbin, Heilongjiang, People’s Republic of China *These authors contributed equally to this work Abstract: Chronic myelogenous leukemia (CML is characterized by the t(9;22 (q34;q11-associated Bcr-Abl fusion gene, which is an essential element of clinical diagnosis. As a traditional Chinese medicine, realgar has been widely used for the treatment of various diseases for >1,500 years. Inspired by nano-drug, realgar nanoparticles (NPs have been prepared with an average particle size of <100 nm in a previous work. Compared with coarse realgar, the realgar NPs have higher bioavailability. As a principal constituent protein of caveolae, caveolin-1 (Cav-1 participates in regulating various cellular physiological and pathological processes including tumorigenesis and tumor development. In previous studies, it was found that realgar NPs can inhibit several types of tumor cell proliferation. However, the therapeutic effect of realgar NPs on CML has not been fully elucidated. In the present paper, it was demonstrated that realgar NPs can inhibit the proliferation of K562 cells and degrade Bcr-Abl fusion protein effectively. Both apoptosis and autophagy were activated in a dose-dependent manner in realgar NPs treated cells, and the induction of autophagy was associated with class I phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathway. Morphological analysis indicated that realgar NPs induced differentiation effectively in CML cells. Furthermore, it was identified that Cav-1 might play a crucial role in realgar NP therapy. In order to study the effects of Cav-1 on K562 cells during

  12. A palmitoyl conjugate of insect pentapeptide Yamamarin arrests cell proliferation and respiration.

    Science.gov (United States)

    Sato, Yosinori; Yang, Ping; An, Ying; Matsukawa, Kazushige; Ito, Kikukatsu; Imanishi, Shigeo; Matsuda, Hirokazu; Uchiyama, Yusuke; Imai, Kunio; Ito, Shigeki; Ishida, Yoji; Suzuki, Koichi

    2010-05-01

    A palmitoyl conjugate of an insect pentapeptide that occurs in diapausing insects causes a reversible cell-cycle arrest and suppresses mitochondrial respiration. This peptide compound also causes growth arrest in murine leukemic cell line expressing human gene Bcr/Abl and a farnesoyl peptide induces embryonic diapause in Bombyx mori. These results demonstrate that the insect peptide compounds can lead to the understanding of a common pathway in developmental arrest in animals and may provide a new peptidominetic analog in the development of biopharmaceuticals and pest management.

  13. 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.

  14. Positional identity of adult stem cells in salamander limb regeneration.

    Science.gov (United States)

    Kumar, Anoop; Gates, Phillip B; Brockes, Jeremy P

    2007-01-01

    Limb regeneration in larval and adult salamanders proceeds from a mound of mesenchymal stem cells called the limb blastema. The blastema gives rise just to those structures distal to its level of origin, and this property of positional identity is reset to more proximal values by treatment with retinoic acid. We have identified a cell surface protein, called Prod1/CD59, which appears to be a determinant of proximodistal identity. Prod1 is expressed in an exponential gradient in an adult limb as determined by detection of both mRNA and immunoreactive protein. Prod1 protein is up-regulated after treatment of distal blastemas with RA and this is particularly marked in cells of the dermis. These cells have previously been implicated in pattern formation during limb regeneration.

  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. Micro-magnet arrays for specific single bacterial cell positioning

    Science.gov (United States)

    Pivetal, Jérémy; Royet, David; Ciuta, Georgeta; Frenea-Robin, Marie; Haddour, Naoufel; Dempsey, Nora M.; Dumas-Bouchiat, Frédéric; Simonet, Pascal

    2015-04-01

    In various contexts such as pathogen detection or analysis of microbial diversity where cellular heterogeneity must be taken into account, there is a growing need for tools and methods that enable microbiologists to analyze bacterial cells individually. One of the main challenges in the development of new platforms for single cell studies is to perform precise cell positioning, but the ability to specifically target cells is also important in many applications. In this work, we report the development of new strategies to selectively trap single bacterial cells upon large arrays, based on the use of micro-magnets. Escherichia coli bacteria were used to demonstrate magnetically driven bacterial cell organization. In order to provide a flexible approach adaptable to several applications in the field of microbiology, cells were magnetically and specifically labeled using two different strategies, namely immunomagnetic labeling and magnetic in situ hybridization. Results show that centimeter-sized arrays of targeted, isolated bacteria can be successfully created upon the surface of a flat magnetically patterned hard magnetic film. Efforts are now being directed towards the integration of a detection tool to provide a complete micro-system device for a variety of microbiological applications.

  17. 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+.

  18. 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

  19. Micro-magnet arrays for specific single bacterial cell positioning

    Energy Technology Data Exchange (ETDEWEB)

    Pivetal, Jérémy, E-mail: jeremy.piv@netcmail.com [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Royet, David [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Ciuta, Georgeta [Univ. Grenoble Alpes, Inst NEEL, F-38042 Grenoble (France); CNRS, Inst NEEL, F-38042 Grenoble (France); Frenea-Robin, Marie [Université de Lyon, Université Lyon 1, CNRS UMR 5005, Laboratoire Ampère, F-69622 Villeurbanne (France); Haddour, Naoufel [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Dempsey, Nora M. [Univ. Grenoble Alpes, Inst NEEL, F-38042 Grenoble (France); CNRS, Inst NEEL, F-38042 Grenoble (France); Dumas-Bouchiat, Frédéric [Univ Limoges, CNRS, SPCTS UMR 7513, 12 Rue Atlantis, F-87068 Limoges (France); Simonet, Pascal [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France)

    2015-04-15

    In various contexts such as pathogen detection or analysis of microbial diversity where cellular heterogeneity must be taken into account, there is a growing need for tools and methods that enable microbiologists to analyze bacterial cells individually. One of the main challenges in the development of new platforms for single cell studies is to perform precise cell positioning, but the ability to specifically target cells is also important in many applications. In this work, we report the development of new strategies to selectively trap single bacterial cells upon large arrays, based on the use of micro-magnets. Escherichia coli bacteria were used to demonstrate magnetically driven bacterial cell organization. In order to provide a flexible approach adaptable to several applications in the field of microbiology, cells were magnetically and specifically labeled using two different strategies, namely immunomagnetic labeling and magnetic in situ hybridization. Results show that centimeter-sized arrays of targeted, isolated bacteria can be successfully created upon the surface of a flat magnetically patterned hard magnetic film. Efforts are now being directed towards the integration of a detection tool to provide a complete micro-system device for a variety of microbiological applications. - Highlights: 1.We report a new approach to selectively micropattern bacterial cells individually upon micro-magnet arrays. 2.Permanent micro-magnets of a size approaching that of bacteria could be fabricated using a Thermo-Magnetic Patterning process. 3.Bacterial cells were labeled using two different magnetic labeling strategies providing flexible approach adaptable to several applications in the field of microbiology.

  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. 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.

  2. A cell cycle timer for asymmetric spindle positioning.

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    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. 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

  4. Stem cell populations in the heart and the role of Isl1 positive cells

    Directory of Open Access Journals (Sweden)

    V. Di Felice

    2013-05-01

    Full Text Available Cardiac progenitor cells are multipotent stem cells isolated from both embryonic and adult hearts in several species and are able to differentiate at least into smooth muscle cells, endothelial cells and cardiomyocytes. The embryonic origin of these cells has not yet been demonstrated, but it has been suggested that these cells may derive from the first and secondary heart fields and from the neural crest. In the last decade, two diffe-rent populations of cardiac progenitor or stem cells have been identified and isolated, i.e., the Islet1 positive (Isl1+ and c-Kit positive (c-Kit+/Stem Cell Antigen-1 positive (Sca-1+ cells. Until 2012, these two populations have been considered two separate entities with different roles and a different origin, but new evidence now suggests a con-nection between the two populations and that the two populations may represent two subpopulations of a unique pool of cardiac stem cells, derived from a common immature primitive cell. To find a common consensus on this concept is very important in furthe-ring the application of stem cells to cardiac tissue engineering.

  5. Stem cell populations in the heart and the role of Isl1 positive cells.

    Science.gov (United States)

    Di Felice, V; Zummo, G

    2013-05-09

    Cardiac progenitor cells are multipotent stem cells isolated from both embryonic and adult hearts in several species and are able to differentiate at least into smooth muscle cells, endothelial cells and cardiomyocytes. The embryonic origin of these cells has not yet been demonstrated, but it has been suggested that these cells may derive from the first and secondary heart fields and from the neural crest. In the last decade, two diffe-rent populations of cardiac progenitor or stem cells have been identified and isolated, i.e., the Islet1 positive (Isl1+) and c-Kit positive (c-Kit+)/Stem Cell Antigen-1 positive (Sca-1+) cells. Until 2012, these two populations have been considered two separate entities with different roles and a different origin, but new evidence now suggests a con-nection between the two populations and that the two populations may represent two subpopulations of a unique pool of cardiac stem cells, derived from a common immature primitive cell. To find a common consensus on this concept is very important in furthe-ring the application of stem cells to cardiac tissue engineering.

  6. 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

  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. 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.

  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. FLASH and NPAT positive but not Coilin positive Cajal Bodies correlate with cell ploidy.

    Science.gov (United States)

    Bongiorno-Borbone, Lucilla; De Cola, Antonella; Vernole, Patrizia; Finos, Livio; Barcaroli, Daniela; Knight, Richard A; Melino, Gerry; De Laurenzi, Vincenzo

    2008-08-01

    Cajal Bodies are one of many specialised organelles contained in the eukaryotic cell nucleus, and are involved in a number of functions, including regulation of replication-dependent histone gene transcription. In normal diploid cells their number varies between 0 and 4 depending on the cell cycle phase, although in cancer cell lines their number is extremely variable and it has been suggested that it correlates with cell ploidy. Here we show that in mammalian cells, as in Drosophila, two distinct though functionally related bodies exist: a histone gene locus body and a Cajal Body. The first one can be detected using FLASH or NPAT as markers while the second is labelled using antibodies against Coilin. Only the number of FLASH/NPAT histone gene locus bodies correlates with ploidy and only these organelles appear to be regulated during the cell cycle. Finally, we show that the two organelles completely co-localize during the S phase of the cell cycle.

  12. 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.

  13. 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.

  14. CD34-positive interstitial cells of the human detrusor

    DEFF Research Database (Denmark)

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

    2007-01-01

    and intermingling with individual muscle fasicles. Morphologically and immunohistochemically, they show no neurogenic, endothelial or mast cell differentiation. Transmission electron microscopy (TEM) showed the presence of interstitial cells with a round-to-oval nucleus, sparse perinuclear cytoplasm and long...

  15. 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...

  16. Apoptotic HPV positive cancer cells exhibit transforming properties.

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    Emilie Gaiffe

    Full Text Available Previous studies have shown that DNA can be transferred from dying engineered cells to neighboring cells through the phagocytosis of apoptotic bodies, which leads to cellular transformation. Here, we provide evidence of an uptake of apoptotic-derived cervical cancer cells by human mesenchymal cells. Interestingly, HeLa (HPV 18+ or Ca Ski (HPV16+ cells, harboring integrated high-risk HPV DNA but not C-33 A cells (HPV-, were able to transform the recipient cells. Human primary fibroblasts engulfed the apoptotic bodies effectively within 30 minutes after co-cultivation. This mechanism is active and involves the actin cytoskeleton. In situ hybridization of transformed fibroblasts revealed the presence of HPV DNA in the nucleus of a subset of phagocytosing cells. These cells expressed the HPV16/18 E6 gene, which contributes to the disruption of the p53/p21 pathway, and the cells exhibited a tumorigenic phenotype, including an increased proliferation rate, polyploidy and anchorage independence growth. Such horizontal transfer of viral oncogenes to surrounding cells that lack receptors for HPV could facilitate the persistence of the virus, the main risk factor for cervical cancer development. This process might contribute to HPV-associated disease progression in vivo.

  17. 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

  18. 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...... lymphoid tissues of numerous species, as well as in numerous disease settings, including cancer. The expression of CD4 and CD8 is regulated by a very strict transcriptional program involving the transcription factors Runx3 and ThPOK. Initially thought to be mutually exclusive within CD4(+) and CD8(+) T...... 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...

  19. 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

    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 accumul......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......-treatment FOXP3-positive cells (n = 31) had a 5-year survival rate of 19% (hazard ratio, 2.2; confidence interval, 1.03-4.5; P = 0.043). All long-term survivors were characterized by low-baseline FOXP3-positive cells and a modest absolute rise in FOXP3-positive cells. CONCLUSION: Intratumoral FOXP3-positive...

  20. Transcriptome profiling of LGR5 positive colorectal cancer cells

    Directory of Open Access Journals (Sweden)

    Daniela Hirsch

    2014-12-01

    Full Text Available The concept of cancer stem cells (CSCs claims that colorectal carcinomas (CRCs, like normal colorectal epithelium, are organized hierarchically and contain a subpopulation of qualitatively distinct cancer cells. The expression of distinctive surface markers or of certain enzymes is a prerequisite for the isolation and characterization of the CSC population. With respect to CRCs, putative CSCs can be identified by leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5, also known as G-protein-coupled receptor 49, Gpr49. However, the precise function of the intestinal stem cell marker Lgr5 in CRCs remains largely unknown. We silenced LGR5 expression in SW480 CRC cells via lentiviral shRNA constructs. This led to the depletion of a morphologically distinct subpopulation of SW480 CRC cells. Microarray gene expression profiling revealed a down-regulation of NOTCH signaling upon LGR5 silencing that could be confirmed by immunohistochemistry. Furthermore, we induced inflammation-driven colon tumors in Lgr5-EGFP-IRES-Cre-ERT2 mice via administration of azoxymethane and dextrane sodium sulfate. The induced tumors were flow-sorted into fractions of epithelial cells that expressed high or low levels of Lgr5 and were characterized using gene expression profiling. Lgr5 high tumor cells showed higher levels of several stem cell-associated genes and higher Wnt signaling than Lgr5 low tumor cells and Lgr5 high normal stem cells. Here we provide a thorough description of our two gene expression datasets including quality control checks uploaded to Gene Expression Omnibus database (data accession number: GSE46200. The analysis and interpretation of our gene expression data and related results have been published recently by Hirsch and colleagues in Carcinogenesis in 2014.

  1. Visualization of proteolytic activity associated with the apoptotic response in cancer cells

    Science.gov (United States)

    Tice, Brian George

    Caspases execute programmed cell death, where low levels of caspase activity are linked to cancer. Chemotherapies utilize induction of apoptosis as a key mechanism for cancer treatment, where caspase-3 is a major player involved in dismantling these aberrant cells. The ability to sensitively measure the initial caspase-3 cleavage events during apoptosis is important for understanding the initiation of this complex cellular process, however, current ensemble methods are not sensitive enough to measure single cleavage events in cells. By utilizing the optical properties of plasmon coupling, peptide-linked gold nanoparticles were developed to enable single molecule imaging of caspase-3 activity in two different cancer systems. Au crown nanoparticles were assembled in a multimeric fashion to overcome the high and heterogeneous background scattering of live cells. In a colon cancer (SW620) cell line challenged with tumor necrosis factor-alpha (TNF-alpha), single molecule trajectories show early stage caspase-3 activation within minutes, which was not detectable by ensemble assays until 23 hours. Variability in caspase-3 activation among the population of cells was identified and likely a result of each cell's specific resistance to death receptor-induced apoptosis. Following these studies, improvements by way of sensitivity and selectivity were tailored into an improved nanosensor construct. Au nanoshell dimers were prepared as a comparably bright construct with 1) reduced heterogeneity compared to the synthesis of the crown nanoparticles and 2) a peptide sequence highly selective for caspase-3. Chronic myeloid leukemia (CML) K562 cells were assessed for their early apoptotic response upon treatment with dasatinib, a clinically approved tyrosine kinase inhibitor that specifically targets BCR-ABL. It has been demonstrated that inhibition of BCR-ABL by dasatinib commits K562 cells to apoptosis. Single molecule experiments with Au nanoshell dimers show caspase-3 activation

  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.

  3. 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...

  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.

  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. 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.

  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. 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. Small cell variant of anaplastic large cell lymphoma with positive immunoreactivity for CD99.

    Science.gov (United States)

    Shiran, M S; Tan, G C; Sabariah, A R; Chye, P C; Pathmanathan, R

    2008-06-01

    A 13 year old boy presented with a huge mass on his right arm of 6 months duration. Histopathological examination revealed sheets of malignant small round blue cells with immunopositivity for LCA, CD43, CD45Ro, CD30, EMA, ALK-1 and CD99, and negativity for CD20, TdT, myogenin, myoD1, NSE, bcl-6, bcl-2 and CD10. Fluorescent In-Situ Hybridization (FISH) testing excluded the diagnosis of Ewing's sarcoma/PNET. Pathologists need to be aware of the diagnosis of a small cell variant of ALCL, as well as of the fact that CD99 expression commonly occurs in cases of ALK-positive ALCL, in order to distinguish this entity from Ewing's sarcoma/PNET.

  10. Increased numbers of P63-positive/CD117-positive cells in advanced adenoid cystic carcinoma give a poorer prognosis

    Directory of Open Access Journals (Sweden)

    Zhou Quan

    2012-09-01

    Full Text Available Abstract Objectives This study consisted of two parts. One part was to analyze the survival rates of adenoid cystic carcinoma (ACC in Chinese and explain the difference between our data and the literature. The other was to analyze the relationship between the expression of CD117 and the histological grade and the prognosis. Methods A retrospective study of 80 ACC patients was performed. Clinical data were collected, and p63, CD117 were detected by immunohistochemical staining. Results Eighty patients received follow-ups 3 to 216 months after initial diagnosis. ACC occurred in the lacrimal gland (26.3%, n = 21, nasal cavity and parasinus (33.8%, n = 27 and other sites (40.0%, n = 33. The 5-year and 10-year survival rates were 66.41% and 10.16%, respectively. Over expression of CD117 was detected in p63-negative cells in 94.3% of cases and in p63-positive cells in 45.8%. The expression of CD117 in p63-positive cells was significantly associated with the histological grade (P Conclusions ACC had a good 5-year survival but poor 10-year survival in Chinese, which differed from the occidental data. More p63+/CD117+ cells were associated with a higher histological grade and poorer outcome. Virtual slides The virtual slide(s for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1701457278762097

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Metal-sulfur type cell having improved positive electrode

    Science.gov (United States)

    Dejonghe, Lutgard C.; Visco, Steven J.; Mailhe, Catherine C.; Armand, Michel B.

    1988-03-01

    A novel metal-sulfur type cell operable at a temperature of 200 C or less with an energy density of 150 Whrs/Kg or better is disclosed characterized by an organo-sulfur cathode formed from an organic-sulfur compound having the general formula, in its charged state, of (R(S) sub y) n wherein y = 1 to 6; n = 2 to 20; and R is one or more different aliphatic or aromatic organic moieties having 1 to 20 carbon atoms, which may include one or more oxygen, sulfur, or nitrogen heteroatoms when R comprises one or more aromatic rings, or one or more oxygen, sulfur, nitrogen, or fluorine atoms associated with the chain when R comprises an aliphatic chain, wherein the aliphatic group may be linear or branched, saturated or unsaturated, and wherein either the aliphatic chain or the aromatic ring may have substituted groups thereon.

  16. Homologous recombination in human telomerase-positive and ALT cells occurs with the same frequency

    OpenAIRE

    Bechter, Oliver E.; Zou, Ying; Shay, Jerry W.; Woodring E. Wright

    2003-01-01

    Homologous recombination is thought to be the molecular mechanism for maintaining telomere length in alternative lengthening of telomeres (ALT) cells. We used a recombination reporter system to show that the frequency of homologous recombination is the same for ALT- and telomerase-positive cells, suggesting that if ALT cells have a recombination defect it specifically involves telomeric sequences. We compared internal and telomere-adjacent positions of our ...

  17. [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.

  18. Role of cell-to-cell variability in activating a positive feedback antiviral response in human dendritic cells.

    Directory of Open Access Journals (Sweden)

    Jianzhong Hu

    Full Text Available In the first few hours following Newcastle disease viral infection of human monocyte-derived dendritic cells, the induction of IFNB1 is extremely low and the secreted type I interferon response is below the limits of ELISA assay. However, many interferon-induced genes are activated at this time, for example DDX58 (RIGI, which in response to viral RNA induces IFNB1. We investigated whether the early induction of IFNBI in only a small percentage of infected cells leads to low level IFN secretion that then induces IFN-responsive genes in all cells. We developed an agent-based mathematical model to explore the IFNBI and DDX58 temporal dynamics. Simulations showed that a small number of early responder cells provide a mechanism for efficient and controlled activation of the DDX58-IFNBI positive feedback loop. The model predicted distributions of single cell responses that were confirmed by single cell mRNA measurements. The results suggest that large cell-to-cell variation plays an important role in the early innate immune response, and that the variability is essential for the efficient activation of the IFNB1 based feedback loop.

  19. Understanding positional cues in salamander limb regeneration: implications for optimizing cell-based regenerative therapies.

    Science.gov (United States)

    McCusker, Catherine D; Gardiner, David M

    2014-06-01

    Regenerative medicine has reached the point where we are performing clinical trials with stem-cell-derived cell populations in an effort to treat numerous human pathologies. However, many of these efforts have been challenged by the inability of the engrafted populations to properly integrate into the host environment to make a functional biological unit. It is apparent that we must understand the basic biology of tissue integration in order to apply these principles to the development of regenerative therapies in humans. Studying tissue integration in model organisms, where the process of integration between the newly regenerated tissues and the 'old' existing structures can be observed and manipulated, can provide valuable insights. Embryonic and adult cells have a memory of their original position, and this positional information can modify surrounding tissues and drive the formation of new structures. In this Review, we discuss the positional interactions that control the ability of grafted cells to integrate into existing tissues during the process of salamander limb regeneration, and discuss how these insights could explain the integration defects observed in current cell-based regenerative therapies. Additionally, we describe potential molecular tools that can be used to manipulate the positional information in grafted cell populations, and to promote the communication of positional cues in the host environment to facilitate the integration of engrafted cells. Lastly, we explain how studying positional information in current cell-based therapies and in regenerating limbs could provide key insights to improve the integration of cell-based regenerative therapies in the future.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Nestin-positive cells in the spinal cord: a potential source of neural stem cells.

    Science.gov (United States)

    Xu, Renshi; Wu, Chengsi; Tao, Yuhui; Yi, Juan; Yang, Yunzhu; Zhang, Xiong; Liu, Rugao

    2008-11-01

    Some literatures have reported neural precursor cells (NPCs) exist in spinal cord of adult mammal, however, the NPCs distribution feature in spinal cord of adult mice so far is not described in detail. In order to observe and compare the distribution feature of NPCs in various spinal cord regions of adult mice, to research a potential source of neural stem cells (NSCs), we obtained NPCs distribution feature by analyzing the distribution of the nestin-containing cells (NCCs) in spinal cord of adult nestin second-intron enhancer controlled LacZ reporter transgenic mice (pNes-Tg) with LacZ staining and positive cell quantification. The results showed that: NCCs were observed in various regions of spinal cord of adult mice, but amount of NCCs was different in distinct region, the rank order of NCCs amount in various spinal cord regions was dorsal horn region greater than central canal greater than the ventral and lateral horn. NCCs in dorsal horn region mainly distributed in substantia gelatinosa, NCCs in central canal mainly distributed in ependymal zone, on the contrary, NCCs in ventral, lateral horn, medullae, nucleus regions of spinal cord were comparatively less. The rank order of NCCs amount in various spinal cord segments was cervical segment greater than lumbar sacral segment greater than thoracic segment. There was no significantly difference between NCCs amount in the left and right sides, and within cervical 1-7, thoracic 1-12, lumbar 1-5, sacral segment of spinal cord in adult mice. These data collectively indicate that NPCs extensively distribute in various regions of spinal cord of adult mice, especially in substantia gelatinosa and ependymal zone. NPCs in cervical segment are abundant, NPCs in thoracic segment are the least while compared the different spinal cord segment, the NPCs in various regions of spinal cord of adult mice are a potential source of NSCs.

  5. Biological properties of leukemia cell-derived exosome and anti-leukemia effects of its sensitized DCs%白血病细胞来源胞外体的生物学特性及其致敏DC的抗白血病效应

    Institute of Scientific and Technical Information of China (English)

    姚烨; 王椿; 沈畅; 陈琳军; 邓晓辉; 郝思国

    2013-01-01

    目的:探索白血病细胞来源的胞外体(leukemia cell-derived exosome,LEX)的生物学特性及其致敏DC的抗白血病免疫效应.方法:超速离心分离纯化BCR-ABL阳性的白血病K562细胞来源的胞外体(LEXK562),采用免疫电镜及Western blotting检测LEXK562中热激蛋白70(heat shock protein 70,HSP70)及BCR-ABL的表达,采用激光扫描共聚焦显微镜及流式细胞术检测LEXK562靶向结合DC的动力学,采用LDH释放法以及小鼠模型体内实验检测LEX及其致敏DC的抗白血病免疫效应.结果:与其他细胞来源的胞外体相似,K562细胞来源的胞外体LEXK562为直径50 ~ 100 nm的囊状结构,且表达K562细胞特异性的BCR-ABL和HSP70分子.LEXK562在体外可靶向结合DC,3~4h到达高峰,并能在DC中稳定存在72 h以上.LEXK562致敏DC(DC/LEXK562)诱导的细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)可有效杀伤K562靶细胞,效靶比为50:1时,其杀伤活性显著高于LEXK562诱导的CTL[(68.6±5.7)%vs(22.5±2.9)%,P<0.01];体内实验进一步证实,白血病L1210细胞来源的胞外体(LEXL1210)免疫后小鼠接种L1210细胞的成瘤率明显高于LEXL1210致敏DC(DC/LEXL1210)免疫小鼠的成瘤率[(54.17±8.33)%vs(16.67±4.18)%,P<0.05].结论:LEX表达白血病细胞相关抗原,LEX体外可靶向结合DC,其致敏的DC能诱导更强的抗白血病免疫效应.%Objective: To explore the biological properties of leukemia cell-derived exosomes (LEXs) and the anti-leukemia immunological effect of LEX-sensitized dendritic cells ( DCs). Methods: LEX from BCR-ABL positive leukemia K562 cells was separated and purified by ultracentrifugation. The expressions of heat shock protein 70 ( HSP70) and BCR-ABL were investigated by immuno-electron microscopy and Western blotting. The kinetics of LEXk562 targeted binding to DCs was examined by laser scanning confocal microscopy and flow cytometry. The anti-leukemia immunological effect of LEX and its sensitized DCs was

  6. Nestin-positive progenitor cells isolated from human fetal pancreas have phenotypic markers identical to mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Ling Zhang; Tian-Pei Hong; Jiang Hu; Yi-Nan Liu; Yong-Hua Wu; Ling-Song Li

    2005-01-01

    AIM: To isolate nestin-positive progenitor cells from human fetal pancreas and to detect their surface markers and their capability of proliferation and differentiation into pancreatic islet endocrine cells in vitro.METHODS: Islet-like cell clusters (ICCs) were isolated from human fetal pancreas by using collagenase digestion. The free-floating ICCs were handpicked and cultured in a new dish. After the ICCs developed into monolayer epithelium-like cells, they were passaged and induced for differentiation. Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence stain, fluorescenceactivated cell sorting (FACS) and radioimmunoassay (RIA)were used to detect the expression of cell markers. RESULTS: (1) The monolayer epithelium-like cells had highly proliferative potential and could be passaged more than 16 timesin vitro; (2) RT-PCR analysis and immunofluorescence stain showed that these cells expressed both nestin and ABCG2, two of stem cellmarkers; (3) FACS analysis revealed that CD44, CD90and CD147 were positive, whereas CD34, CD38, CD45, CD71, CD117, CD133 and HLA-DR were negative on the nestin-positive cells; (4) RT-PCR analysis showed that the mRNA expression of insulin, glucagon and pancreaticduodenal homeobox gene-1 was detected, whereas the expression of nestin and neurogenin 3 disappeared in these cells treated with serum-free media supplemented with the cocktail of growth factors. Furthermore, the intracellular insulin content was detected by RIA after the induction culture.CONCLUSION: Nestin-positive cells isolated from human fetal pancreas possess the characteristics of pancreatic progenitor cells since they have highly proliferative potential and the capability of differentiation into insulinproducing cells in vitro. Interestingly, the nestin-positive pancreatic progenitor cells share many phenotypic markers with mesenchymal stem cells derived from bone marrow.

  7. 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.

  8. Therapeutic Potential of Adipose-Derived SSEA-3-Positive Muse Cells for Treating Diabetic Skin Ulcers.

    Science.gov (United States)

    Kinoshita, Kahori; Kuno, Shinichiro; Ishimine, Hisako; Aoi, Noriyuki; Mineda, Kazuhide; Kato, Harunosuke; Doi, Kentaro; Kanayama, Koji; Feng, Jingwei; Mashiko, Takanobu; Kurisaki, Akira; Yoshimura, Kotaro

    2015-02-01

    Stage-specific embryonic antigen-3 (SSEA-3)-positive multipotent mesenchymal cells (multilineage differentiating stress-enduring [Muse] cells) were isolated from cultured human adipose tissue-derived stem/stromal cells (hASCs) and characterized, and their therapeutic potential for treating diabetic skin ulcers was evaluated. Cultured hASCs were separated using magnetic-activated cell sorting into positive and negative fractions, a SSEA-3+ cell-enriched fraction (Muse-rich) and the remaining fraction (Muse-poor). Muse-rich hASCs showed upregulated and downregulated pluripotency and cell proliferation genes, respectively, compared with Muse-poor hASCs. These cells also released higher amounts of certain growth factors, particularly under hypoxic conditions, compared with Muse-poor cells. Skin ulcers were generated in severe combined immunodeficiency (SCID) mice with type 1 diabetes, which showed delayed wound healing compared with nondiabetic SCID mice. Treatment with Muse-rich cells significantly accelerated wound healing compared with treatment with Muse-poor cells. Transplanted cells were integrated into the regenerated dermis as vascular endothelial cells and other cells. However, they were not detected in the surrounding intact regions. Thus, the selected population of ASCs has greater therapeutic effects to accelerate impaired wound healing associated with type 1 diabetes. These cells can be achieved in large amounts with minimal morbidity and could be a practical tool for a variety of stem cell-depleted or ischemic conditions of various organs and tissues.

  9. 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.

  10. 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

    2016-02-12

    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.

  11. 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.

  12. [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.

  13. 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

  14. 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.

  15. Two distinct subpopulations of nestin-positive cells in adult mouse dentate gyrus.

    Science.gov (United States)

    Fukuda, Satoshi; Kato, Fusao; Tozuka, Yusuke; Yamaguchi, Masahiro; Miyamoto, Yusei; Hisatsune, Tatsuhiro

    2003-10-15

    Neurogenesis in the dentate gyrus of the adult mammalian hippocampus has been proven in a series of studies, but the differentiation process toward newborn neurons is still unclear. In addition to the immunohistochemical study, electrophysiological membrane recordings of precursor cells could provide an alternative view to address this differentiation process. In this study, we performed green fluorescent protein (GFP)-guided selective recordings of nestin-positive progenitor cells in adult dentate gyrus by means of nestin-promoter GFP transgenic mice, because nestin is a typical marker for precursor cells in the adult dentate gyrus. The patch-clamp recordings clearly demonstrated the presence of two distinct subpopulations (type I and type II) of nestin-positive cells. Type I cells had a lower input resistance value of 77.1 M(Omega) (geometric mean), and their radial processes were stained with anti-glial fibrillary acidic protein antibody. On the other hand, type II nestin-positive cells had a higher input resistance value of 2110 MOmega and expressed voltage-dependent sodium current. In most cases, type II cells were stained with anti-polysialylated neural cell adhesion molecule. Taken together with a bromodeoxyuridine pulse-chase analysis, our results may reflect a rapid and dynamic cell conversion of nestin-positive progenitor, from type I to type II, at an early stage of adult neurogenesis in the dentate gyrus.

  16. 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.

  17. Restoration of spermatogenesis after transplantation of c-Kit positive testicular cells in the fowl.

    Science.gov (United States)

    Trefil, Pavel; Bakst, Murray R; Yan, Haifeng; Hejnar, Jiří; Kalina, Jiří; Mucksová, Jitka

    2010-12-01

    Transplantation of male germ line cells into sterilized recipients has been used in mammals for conventional breeding as well as for transgenesis. We have previously adapted this approach for the domestic chicken and we present now an improvement of the germ cell transplantation technique by using an enriched subpopulation of c-Kit-positive spermatogonia as donor cells. Dispersed c-Kit positive testicular cells from 16 to 17 week-old pubertal donors were transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient's testes with c-Kit positive donor testicular cells, which resulted in the production of functional heterologous spermatozoa. Using manual semen collection, the first sperm production in the recipient males was observed about nine weeks after the transplantation. The full reproduction cycle was accomplished by artificial insemination of hens and hatching of chickens.

  18. 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.

  19. Critical cell wall hole size for lysis in Gram-positive bacteria

    Science.gov (United States)

    Mitchell, Gabriel; Wiesenfeld, Kurt; Nelson, Daniel; Weitz, Joshua

    2013-03-01

    Gram-positive bacteria transport molecules necessary for their survival through holes in their cell wall. The holes in cell walls need to be large enough to let critical nutrients pass through. However, the cell wall must also function to prevent the bacteria's membrane from protruding through a large hole into the environment and lysing the cell. As such, we hypothesize that there exists a range of cell wall hole sizes that allow for molecule transport but prevent membrane protrusion. Here we develop and analyze a biophysical theory of the response of a Gram-positive cell's membrane to the formation of a hole in the cell wall. We predict a critical hole size in the range 15-24nm beyond which lysis occurs. To test our theory, we measured hole sizes in Streptococcus pyogenes cells undergoing enzymatic lysis via transmission electron microscopy. The measured hole sizes are in strong agreement with our theoretical prediction. Together, the theory and experiments provide a means to quantify the mechanisms of death of Gram-positive cells via enzymatically mediated lysis and provides insight into the range of cell wall hole sizes compatible with bacterial homeostasis.

  20. Calpain-Mediated Positional Information Directs Cell Wall Orientation to Sustain Plant Stem Cell Activity, Growth and Development.

    Science.gov (United States)

    Liang, Zhe; Brown, Roy C; Fletcher, Jennifer C; Opsahl-Sorteberg, Hilde-Gunn

    2015-09-01

    Eukaryotic development and stem cell control depend on the integration of cell positional sensing with cell cycle control and cell wall positioning, yet few factors that directly link these events are known. The DEFECTIVE KERNEL1 (DEK1) gene encoding the unique plant calpain protein is fundamental for development and growth, being essential to confer and maintain epidermal cell identity that allows development beyond the globular embryo stage. We show that DEK1 expression is highest in the actively dividing cells of seeds, meristems and vasculature. We further show that eliminating Arabidopsis DEK1 function leads to changes in developmental cues from the first zygotic division onward, altered microtubule patterns and misshapen cells, resulting in early embryo abortion. Expression of the embryonic marker genes WOX2, ATML1, PIN4, WUS and STM, related to axis organization, cell identity and meristem functions, is also altered in dek1 embryos. By monitoring cell layer-specific DEK1 down-regulation, we show that L1- and 35S-induced down-regulation mainly affects stem cell functions, causing severe shoot apical meristem phenotypes. These results are consistent with a requirement for DEK1 to direct layer-specific cellular activities and set downstream developmental cues. Our data suggest that DEK1 may anchor cell wall positions and control cell division and differentiation, thereby balancing the plant's requirement to maintain totipotent stem cell reservoirs while simultaneously directing growth and organ formation. A role for DEK1 in regulating microtubule-orchestrated cell wall orientation during cell division can explain its effects on embryonic development, and suggests a more general function for calpains in microtubule organization in eukaryotic cells.

  1. Tuberculosis-specific CD8 cells in HLA A*02-positive TB- and LTBI patients

    DEFF Research Database (Denmark)

    Fløe, Andreas; Brix, Liselotte; Wejse, Christian;

    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...

  2. Restoration of spermatogenesis after transplantation of c-Kit positive testicular cells in the fowl

    Science.gov (United States)

    Transplantation of male germ line cells into sterilized recipients has been used in mammals for conventional breeding as well as for transgenesis. This study presents an improvement in the approach for germ cell transplantation between fowl males by using an enriched subpopulation of c-Kit positive ...

  3. Presence of Gumprecht shadows (smudge cells) in bovine leukemia virus-positive cattle.

    Science.gov (United States)

    Panei, Carlos Javier; Larsen, Alejandra; González, Ester Teresa; Echeverría, María Gabriela

    2013-11-01

    Enzootic Bovine Leukosis is a chronic disease caused by the bovine leukemia virus (BLV). Smudge cells, also known as Gumprecht shadows, are not simple artifacts of slide preparation, but ragged lymphoid cells found mainly in peripheral blood smears from human patients with chronic lymphocytic leukemia. In this study, we report the presence of Gumprecht shadows in peripheral blood from BLV-positive cattle.

  4. 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...

  5. 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.

  6. Understanding positional cues in salamander limb regeneration: implications for optimizing cell-based regenerative therapies

    Directory of Open Access Journals (Sweden)

    Catherine D. McCusker

    2014-06-01

    Full Text Available Regenerative medicine has reached the point where we are performing clinical trials with stem-cell-derived cell populations in an effort to treat numerous human pathologies. However, many of these efforts have been challenged by the inability of the engrafted populations to properly integrate into the host environment to make a functional biological unit. It is apparent that we must understand the basic biology of tissue integration in order to apply these principles to the development of regenerative therapies in humans. Studying tissue integration in model organisms, where the process of integration between the newly regenerated tissues and the ‘old’ existing structures can be observed and manipulated, can provide valuable insights. Embryonic and adult cells have a memory of their original position, and this positional information can modify surrounding tissues and drive the formation of new structures. In this Review, we discuss the positional interactions that control the ability of grafted cells to integrate into existing tissues during the process of salamander limb regeneration, and discuss how these insights could explain the integration defects observed in current cell-based regenerative therapies. Additionally, we describe potential molecular tools that can be used to manipulate the positional information in grafted cell populations, and to promote the communication of positional cues in the host environment to facilitate the integration of engrafted cells. Lastly, we explain how studying positional information in current cell-based therapies and in regenerating limbs could provide key insights to improve the integration of cell-based regenerative therapies in the future.

  7. Anchor cell signaling and vulval precursor cell positioning establish a reproducible spatial context during C. elegans vulval induction.

    Science.gov (United States)

    Grimbert, Stéphanie; Tietze, Kyria; Barkoulas, Michalis; Sternberg, Paul W; Félix, Marie-Anne; Braendle, Christian

    2016-08-01

    How cells coordinate their spatial positioning through intercellular signaling events is poorly understood. Here we address this topic using Caenorhabditis elegans vulval patterning during which hypodermal vulval precursor cells (VPCs) adopt distinct cell fates determined by their relative positions to the gonadal anchor cell (AC). LIN-3/EGF signaling by the AC induces the central VPC, P6.p, to adopt a 1° vulval fate. Exact alignment of AC and VPCs is thus critical for correct fate patterning, yet, as we show here, the initial AC-VPC positioning is both highly variable and asymmetric among individuals, with AC and P6.p only becoming aligned at the early L3 stage. Cell ablations and mutant analysis indicate that VPCs, most prominently 1° cells, move towards the AC. We identify AC-released LIN-3/EGF as a major attractive signal, which therefore plays a dual role in vulval patterning (cell alignment and fate induction). Additionally, compromising Wnt pathway components also induces AC-VPC alignment errors, with loss of posterior Wnt signaling increasing stochastic vulval centering on P5.p. Our results illustrate how intercellular signaling reduces initial spatial variability in cell positioning to generate reproducible interactions across tissues.

  8. 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.

  9. Neuronal Dystroglycan Is Necessary for Formation and Maintenance of Functional CCK-Positive Basket Cell Terminals on Pyramidal Cells.

    Science.gov (United States)

    Früh, Simon; Romanos, Jennifer; Panzanelli, Patrizia; Bürgisser, Daniela; Tyagarajan, Shiva K; Campbell, Kevin P; Santello, Mirko; Fritschy, Jean-Marc

    2016-10-05

    Distinct types of GABAergic interneurons target different subcellular domains of pyramidal cells, thereby shaping pyramidal cell activity patterns. Whether the presynaptic heterogeneity of GABAergic innervation is mirrored by specific postsynaptic factors is largely unexplored. Here we show that dystroglycan, a protein responsible for the majority of congenital muscular dystrophies when dysfunctional, has a function at postsynaptic sites restricted to a subset of GABAergic interneurons. Conditional deletion of Dag1, encoding dystroglycan, in pyramidal cells caused loss of CCK-positive basket cell terminals in hippocampus and neocortex. PV-positive basket cell terminals were unaffected in mutant mice, demonstrating interneuron subtype-specific function of dystroglycan. Loss of dystroglycan in pyramidal cells had little influence on clustering of other GABAergic postsynaptic proteins and of glutamatergic synaptic proteins. CCK-positive terminals were not established at P21 in the absence of dystroglycan and were markedly reduced when dystroglycan was ablated in adult mice, suggesting a role for dystroglycan in both formation and maintenance of CCK-positive terminals. The necessity of neuronal dystroglycan for functional innervation by CCK-positive basket cell axon terminals was confirmed by reduced frequency of inhibitory events in pyramidal cells of dystroglycan-deficient mice and further corroborated by the inefficiency of carbachol to increase IPSC frequency in these cells. Finally, neurexin binding seems dispensable for dystroglycan function because knock-in mice expressing binding-deficient T190M dystroglycan displayed normal CCK-positive terminals. Together, we describe a novel function of dystroglycan in interneuron subtype-specific trans-synaptic signaling, revealing correlation of presynaptic and postsynaptic molecular diversity.

  10. RON confers lapatinib resistance in HER2-positive breast cancer cells.

    Science.gov (United States)

    Wang, Quanren; Quan, Haitian; Zhao, Jie; Xie, Chengying; Wang, Lei; Lou, Liguang

    2013-10-28

    Lapatinib-resistance is a major problem for HER2-positive breast cancer treatment. SK-BR-3-LR, a lapatinib-resistant cell clone, was established from HER2-positive SK-BR-3 breast cancer cells following chronic exposure to lapatinib. The PI3K/AKT signaling pathway was demonstrated to be resistant to HER2 inhibition in SK-BR-3-LR cells. However, both small-molecular Recepteur d'Origine Nantais (RON) inhibitors and RON-targeted small interfering RNA (siRNA) effectively restored lapatinib sensitivity in these cells by inhibiting PI3K/AKT activation. Our results demonstrate for the first time the important role of RON in mediating lapatinib resistance and suggest that RON-targeted therapy may become a novel, promising therapeutic strategy after the failure of lapatinib treatment in patients with HER2-positive breast cancer.

  11. Effects of Herceptin on circulating tumor cells in HER2 positive early breast cancer.

    Science.gov (United States)

    Zhang, J-L; Yao, Q; Chen Y Wang, J-H; Wang, H; Fan, Q; Ling, R; Yi, J; Wang, L

    2015-03-20

    The objective of this study was to determine the changes in peripheral blood circulating tumor cells in HER2-positive early breast cancer before and after Herceptin therapy, and to explore the effects of the HER2 gene and Herceptin on circulating tumor cells. CK19 mRNA expression in peripheral blood was evaluated by qRT-PCR as an index of circulating tumor cells in 15 cases of HER-2-positive breast cancer and 18 cases of HER2-negative breast cancer before, and after chemotherapy as well. Ten cases of HER2-positive breast cancer continued on Herceptin therapy for 3 months after chemotherapy, and their peripheral blood was again drawn and assayed for CK-19 mRNA expression. Preoperatively, all cases of HER2-positive cancer were positive for CK19 mRNA in peripheral blood, but 6 cases of HER2-negative breast cancer were positive (33.3%), where there was a substantial difference between the two groups. After 6 cycles of adjuvant chemotherapy, CK19 positive rates in cases of HER2-positive and -negative breast cancer reduced by 93.3 and 11.1%, respectively, with a significant difference still existing. After 3 months of Herceptin therapy, expression of CK19 mRNA declined considerably in 10 cases of HER2 positive breast cancer (113.66 ± 88.65 vs 63.35 ± 49.27, P = 0.025). HER-2 gene expression closely correlated with circulating tumor cells in peripheral blood of early breast cancer patients. Moreover, Herceptin, a monoclonal antibody for HER2, can reduce the number of circulating tumor cells, which can be an early predictive factor for Herceptin therapy effectiveness against breast cancer.

  12. Differential positioning of chloroplasts in C4 mesophyll and bundle sheath cells.

    Science.gov (United States)

    Maai, Eri; Miyake, Hiroshi; Taniguchi, Mitsutaka

    2011-08-01

    Chloroplast photorelocation movement is extensively studied in C3 but not C4 plants. C4 plants have 2 types of photosynthetic cells: mesophyll and bundle sheath cells. Mesophyll chloroplasts are randomly distributed along cell walls, whereas bundle sheath chloroplasts are located close to the vascular tissues or mesophyll cells depending on the plant species. The cell-specific C 4 chloroplast arrangement is established during cell maturation, and is maintained throughout the life of the cell. However, only mesophyll chloroplasts can change their positions in response to environmental stresses. The migration pattern is unique to C4 plants and differs from that of C3 chloroplasts. In this mini-review, we highlight the cell-specific disposition of chloroplasts in C4 plants and discuss the possible physiological significances.

  13. Higher positive identification of malignant CSF cells using the cytocentrifuge than the Suta chamber

    Directory of Open Access Journals (Sweden)

    Sérgio Monteiro de Almeida

    Full Text Available ABSTRACT Objective To define how to best handle cerebrospinal fluid (CSF specimens to obtain the highest positivity rate for the diagnosis of malignancy, comparing two different methods of cell concentration, sedimentation and cytocentrifugation. Methods A retrospective analysis of 411 CSF reports. Results This is a descriptive comparative study. The positive identification of malignant CSF cells was higher using the centrifuge than that using the Suta chamber (27.8% vs. 19.0%, respectively; p = 0.038. Centrifuge positively identified higher numbers of malignant cells in samples with a normal concentration of white blood cells (WBCs (< 5 cells/mm3 and with more than 200 cells/mm3, although this was not statistically significant. There was no lymphocyte loss using either method. Conclusions Cytocentrifugation positively identified a greater number of malignant cells in the CSF than cytosedimentation with the Suta chamber. However, there was no difference between the methods when the WBC counts were within the normal range.

  14. Type 1 collagen as a potential niche component for CD133-positive glioblastoma cells.

    Science.gov (United States)

    Motegi, Hiroaki; Kamoshima, Yuuta; Terasaka, Shunsuke; Kobayashi, Hiroyuki; Houkin, Kiyohiro

    2014-08-01

    Cancer stem cells are thought to be closely related to tumor progression and recurrence, making them attractive therapeutic targets. Stem cells of various tissues exist within niches maintaining their stemness. Glioblastoma stem cells (GSCs) are located at tumor capillaries and the perivascular niche, which are considered to have an important role in maintaining GSCs. There were some extracellular matrices (ECM) on the perivascular connective tissue, including type 1 collagen. We here evaluated whether type 1 collagen has a potential niche for GSCs. Imunohistochemical staining of type 1 collagen and CD133, one of the GSCs markers, on glioblastoma (GBM) tissues showed CD133-positive cells were located in immediate proximity to type 1 collagen around tumor vessels. We cultured human GBM cell lines, U87MG and GBM cells obtained from fresh surgical tissues, T472 and T555, with serum-containing medium (SCM) or serum-free medium with some growth factors (SFM) and in non-coated (Non-coat) or type 1 collagen-coated plates (Col). The RNA expression levels of CD133 and Nestin as stem cell markers in each condition were examined. The Col condition not only with SFM but SCM made GBM cells more enhanced in RNA expression of CD133, compared to Non-coat/SCM. Semi-quantitative measurement of CD133-positive cells by immunocytochemistry showed a statistically significant increase of CD133-positive cells in Col/SFM. In addition, T472 cell line cultured in the Col/SFM had capabilities of sphere formation and tumorigenesis. Type 1 collagen was found in the perivascular area and showed a possibility to maintain GSCs. These findings suggest that type 1 collagen could be one important niche component for CD133-positive GSCs and maintain GSCs in adherent culture.

  15. 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.

  16. 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

  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. 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.

  19. 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.

  20. Liver tryptase-positive mast cells and fibrosis in children with hepatic echinococcosis

    Directory of Open Access Journals (Sweden)

    Gulubova Maya

    2005-01-01

    Full Text Available The hepatic echinococcosis in children is a serious surgical problem. The aim of this study is to investigate the participation of mast cells in liver inflammatory reactions triggered by echinococcal cysts. Liver biopsy samples were collected from the tissue surrounding the cysts from 16 sick children (11 boys and 5 girls in the course of abdominal surgery and from 5 controls. Light and ultrastructural immunocytochemistry was performed using monoclonal antibody against tryptase. Light microscopical immunocytochemistry revealed abundance of tryptase-positive (MCT mast cells in the capsules of the cysts (43.58 cells/mm2. There were also observed greatly increased numbers of mast cells in portal tracts surrounding the cyst, compared to those of control biopsies (26.49 vs. 1.78 cells/mm2, p=0.0009, Mann-Whitney U test. Based on the ultrastructural appearance of tryptase-positive mast cell granules, morphological sings of activation of most of the mast cells were distinguished. In conclusion, we suggest that the accumulated and activated tryptase-positive mast cells in liver tissues surrounding the echinococcal cysts play a crucial role in modulation of the inflammatory liver response and could induce chronic inflammation and fibrogenesis, resulting in serious liver injury such as nonspecific reactive hepatitis.

  1. 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.

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    Lifescience Database Archive (English)

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  8. File list: ALL.Bld.20.AllAg.CD45_positive_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Bld.20.AllAg.CD45_positive_cells mm9 All antigens Blood CD45 positive cells SRX...472651,SRX472653,SRX472652 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Bld.20.AllAg.CD45_positive_cells.bed ...

  9. File list: Unc.Bld.20.AllAg.CD45_positive_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Bld.20.AllAg.CD45_positive_cells mm9 Unclassified Blood CD45 positive cells htt...p://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Bld.20.AllAg.CD45_positive_cells.bed ...

  10. 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

    and beta cells. Interestingly, Men1 disruption in both Ngn3 progenitors and beta and alpha cells resulted in the development of pancreatic gastrin-expressing tumors, suggesting that the latter developed from islet cells. Finally, we detected gastrin expression using three human cohorts with pancreatic......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...... migrated from the duodenum. In the current study, we further characterized previously described transient pancreatic gastrin-expressing cells using cell lineage tracing in a pan-pancreatic progenitor and a pancreatic endocrine progenitor model. We provide evidence showing that pancreatic gastrin...

  11. Porcine reproductive and respiratory syndrome virus infection is associated with an increased number of Sn-positive and CD8-positive cells in the maternal-fetal interface.

    Science.gov (United States)

    Karniychuk, U U; De Spiegelaere, W; Nauwynck, H J

    2013-09-01

    It is already known that porcine reproductive and respiratory syndrome virus (PRRSV) infection in lungs changes a local cell pattern and cytokine profile. However, there is no information about cellular and immunological events upon PRRSV infection in the maternal-fetal interface yet. The altered number and/or function of macrophages and NK cells in the maternal-fetal interface during infection may have a functional importance for virus replication. In addition, local cellular and immunological disbalance may also disrupt fragile homeostasis and contribute to the PRRSV-related reproductive disorders. Sialoadhesin (Sn)-positive macrophages are target cells for PRRSV and Sn overexpression has been observed upon chronic inflammatory and infectious diseases. It is also known that mouse Sn-positive macrophages in lymph nodes are able to closely interact with and activate NK cells in response to viral particles. Therefore, the main purpose of the present study was to examine if PRRSV infection is associated with altered Sn expression on endometrial and placental macrophages. In addition, CD8-positive cells (porcine endometrial NK cells were previously described as CD8(+)CD3(-) cells) were localized and quantified in the PRRSV-positive and control tissues. Tissue samples were obtained from three PRRSV-inoculated and three non-inoculated control sows at 100 days of gestation. Real-time RT-PCR showed a clear upregulation of Sn mRNA expression in the PRRSV-positive endometrium/placenta (pPRRSV-inoculated sows had a significantly higher number of Sn-positive macrophages in the endometrium and placenta due to de novo Sn expression on local CD163-positive macrophages. Along with the increased number of Sn-positive macrophages an increased number of CD8-positive cells, which were mostly CD3-negative, was observed in the PRRSV-positive endometrium. The effects of the observed cellular changes on virus replication and potential contribution to placental damage and reproductive

  12. Retargeting T cells for HER2-positive tumor killing by a bispecific Fv-Fc antibody.

    Directory of Open Access Journals (Sweden)

    Lei Wang

    Full Text Available To exploit the biological and pharmacological properties of immunoglobulin constant domain Fc fragment and increase the killing efficacy of T cells, a single chain variable fragment specific to CD3 was fused with Fcab (Fc antigen binding, a mutant Fc fragment with specificity against Human epidermal growth factor receptor 2 (HER2 developed by F-star. The bispecific fusion named as FcabCD3 was expressed by transient transfection in HEK-293T cells and purified by affinity chromatography. Specific cytolytic activity of retargeted T cells to kill HER2 positive SKBR3 cell line was evaluated in vitro. FcabCD3 was able to retarget T cells to kill both Herceptin insensitive Colo205-luc cell line and HER2 low expression MDA-MB-231-luc cell line. Furthermore, FcabCD3 was effective in eliminating the Colo205 tumor established on BALB/c nu/nu mice.

  13. Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria.

    Science.gov (United States)

    Kinjo, Yuki; Illarionov, Petr; Vela, José Luis; Pei, Bo; Girardi, Enrico; Li, Xiangming; Li, Yali; Imamura, Masakazu; Kaneko, Yukihiro; Okawara, Akiko; Miyazaki, Yoshitsugu; Gómez-Velasco, Anaximandro; Rogers, Paul; Dahesh, Samira; Uchiyama, Satoshi; Khurana, Archana; Kawahara, Kazuyoshi; Yesilkaya, Hasan; Andrew, Peter W; Wong, Chi-Huey; Kawakami, Kazuyoshi; Nizet, Victor; Besra, Gurdyal S; Tsuji, Moriya; Zajonc, Dirk M; Kronenberg, Mitchell

    2011-09-04

    Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.

  14. 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.

  15. Bmi1-positive cells in the lingual epithelium could serve as cancer stem cells in tongue cancer.

    Science.gov (United States)

    Tanaka, Toshihiro; Atsumi, Naho; Nakamura, Naohiro; Yanai, Hirotsugu; Komai, Yoshihiro; Omachi, Taichi; Tanaka, Kiyomichi; Ishigaki, Kazuhiko; Saiga, Kazuho; Ohsugi, Haruyuki; Tokuyama, Yoko; Imahashi, Yuki; Hisha, Hiroko; Yoshida, Naoko; Kumano, Keiki; Okazaki, Kazuichi; Ueno, Hiroo

    2016-12-22

    We recently reported that the polycomb complex protein Bmi1 is a marker for lingual epithelial stem cells (LESCs), which are involved in the long-term maintenance of lingual epithelial tissue in the physiological state. However, the precise role of LESCs in generating tongue tumors and Bmi1-positive cell lineage dynamics in tongue cancers are unclear. Here, using a mouse model of chemically (4-nitroquinoline-1-oxide: 4-NQO) induced tongue cancer and the multicolor lineage tracing method, we found that each unit of the tumor was generated by a single cell and that the assembly of such cells formed a polyclonal tumor. Although many Bmi1-positive cells within the tongue cancer specimens failed to proliferate, some proliferated continuously and supplied tumor cells to the surrounding area. This process eventually led to the formation of areas derived from single cells after 1-3 months, as determined using the multicolor lineage tracing method, indicating that such cells could serve as cancer stem cells. These results indicate that LESCs could serve as the origin for tongue cancer and that cancer stem cells are present in tongue tumors.

  16. Co-expression of the CBFβ-MYH11 and BCR-ABL fusion genes in chronic myeloid leukaemia / Coexistenţa genelor de fuziune CBFβ-MYH11 şi BCR-ABL în leucemia mieloidă cronică

    Directory of Open Access Journals (Sweden)

    Popescu Roxana

    2015-06-01

    Full Text Available Coexistenţa t(9;22 şi a inv(16 a fost descrisă într-un număr limitat de cazuri de LMC, LAM de novo sau LAM post chimioterapie. Raportăm un pacient cu LMC care a prezentat atât inversie de 16 cât şi cromozom Philadelphia şi care a evoluat spre criză blastică sub tratament cu Imatinib. Diagnosicul de laborator şi monitorizarea s-a realizat prin citometrie în flux, citogenetică convenţională şi tehnici de genetică moleculară. Inv(16, detectată prin cariotipare în clona Philadelphia pozitivă la momentul transformării blastice, a fost evaluată retrospective prin metoda real-time PCR, şi s-a dovedit a fi fost prezentă încă de la diagnostic. Biopsia de măduvă osoasă, efectuată în faza blastică a LMC, a confirmat prezenţa blaştilor aparţinând liniei mieloide, cu indicii de diferenţiere monocitoidă, frecvent asociată cu inv (16. De asemenea, cazul a asociat şi mutaţia F359V în domeniul kinazic al ABL, care determină rezistenţă intermediară la Imatinib şi Nilotinib, ceea ce a impus schimbarea terapiei cu Dasatinib. În cazul prezentat evoluţia a fost progresivă, urmată de deces ca urmare a lipsei de răspuns la inhibitorii de tirozin kinază, la 18 luni de la diagnosticare. Coexistenţa t(9; 22 şi inv(16 în LMC pare a fi asociată cu o evoluţie clinică agresivă şi rezistenţă la terapia cu inhibitori de tirozin kinază. Având în vedere numărul foarte mic de cazuri descrise în literatura de specialitate, deciziile terapeutice în cazul pacienţilor care prezintă aceste anomalii sunt încă dificile

  17. AFM study shows prominent physical changes in elasticity and pericellular layer in human acute leukemic cells due to inadequate cell-cell communication

    Science.gov (United States)

    Guz, Nataliia V.; Patel, Sapan J.; Dokukin, Maxim E.; Clarkson, Bayard; Sokolov, Igor

    2016-12-01

    Biomechanical properties of single cells in vitro or ex vivo and their pericellular interfaces have recently attracted a lot of attention as a potential biophysical (and possibly prognostic) marker of various diseases and cell abnormalities. At the same time, the influence of the cell environment on the biomechanical properties of cells is not well studied. Here we use atomic force microscopy to demonstrate that cell-cell communication can have a profound effect on both cell elasticity and its pericellular coat. A human pre-B p190BCR/ABL acute lymphoblastic leukemia cell line (ALL3) was used in this study. Assuming that cell-cell communication is inversely proportional to the distance between cells, we study ALL3 cells in vitro growing at different cell densities. ALL3 cells demonstrate a clear density dependent behavior. These cells grow very well if started at a relatively high cell density (HD, >2 × 105 cells ml-1) and are poised to grow at low cell density (LD, communication must be taken into account when studying biomechanics of cells, in particular, correlating cell phenotype and its biophysical properties.

  18. Human NK cells positively regulate gammadelta T cells in response to Mycobacterium tuberculosis.

    Science.gov (United States)

    Zhang, Ruijun; Zheng, Xiaodong; Li, Baiqing; Wei, Haiming; Tian, Zhigang

    2006-02-15

    The decrease in NK cell activity and the loss of gammadelta T cells in active pulmonary tuberculosis patients have been reported. In this study, we observed that the proliferating response of gammadelta T cells to the heat-treated Ags of Mycobacterium tuberculosis from different individuals was noted to be dependent on the content or function of NK cells in PBMC in a population study. We also found that NK cells were directly rapidly activated by the heat-treated Ags from M. tuberculosis (H37Ra) in vitro; in turn, the activated NK cells improved gammadelta T cell proliferation both by CD54-mediated cell-cell contact through the forming immune synapse and by soluble factors TNF-alpha, GM-CSF, and IL-12, but not IFN-gamma. Our results demonstrated that an interaction between NK cells and gammadelta T cells existed in antituberculosis immunity. Up-regulating the function of NK cells might be beneficial to the prevention and control of pulmonary tuberculosis.

  19. 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.

  20. 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.

  1. Case-positive versus case-negative designs for low-rate lithium thionyl chloride cells

    Science.gov (United States)

    Mahy, T. X.

    1982-01-01

    Case polarity design choices are discussed. Two examples of case-negative designs are presented. One battery is thionyl chloride limited and the other is lithium limited. The case-positive design is thionyl chloride limited. It is found that the case-positive/case-negative design consideration does not seem to have much bearing on storage. However, during low rate discharge, the case-negative cells show a steadily decreasing capacity as you go to lower and lower rates.

  2. [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.

  3. Steroid induction of therapy-resistant cytokeratin-5-positive cells in estrogen receptor-positive breast cancer through a BCL6-dependent mechanism.

    Science.gov (United States)

    Goodman, C R; Sato, T; Peck, A R; Girondo, M A; Yang, N; Liu, C; Yanac, A F; Kovatich, A J; Hooke, J A; Shriver, C D; Mitchell, E P; Hyslop, T; Rui, H

    2016-03-17

    Therapy resistance remains a major problem in estrogen receptor-α (ERα)-positive breast cancer. A subgroup of ERα-positive breast cancer is characterized by mosaic presence of a minor population of ERα-negative cancer cells expressing the basal cytokeratin-5 (CK5). These CK5-positive cells are therapy resistant and have increased tumor-initiating potential. Although a series of reports document induction of the CK5-positive cells by progestins, it is unknown if other 3-ketosteroids share this ability. We now report that glucocorticoids and mineralocorticoids effectively expand the CK5-positive cell population. CK5-positive cells induced by 3-ketosteroids lacked ERα and progesterone receptors, expressed stem cell marker, CD44, and displayed increased clonogenicity in soft agar and broad drug-resistance in vitro and in vivo. Upregulation of CK5-positive cells by 3-ketosteroids required induction of the transcriptional repressor BCL6 based on suppression of BCL6 by two independent BCL6 small hairpin RNAs or by prolactin. Prolactin also suppressed 3-ketosteroid induction of CK5+ cells in T47D xenografts in vivo. Survival analysis with recursive partitioning in node-negative ERα-positive breast cancer using quantitative CK5 and BCL6 mRNA or protein expression data identified patients at high or low risk for tumor recurrence in two independent patient cohorts. The data provide a mechanism by which common pathophysiological or pharmacologic elevations in glucocorticoids or other 3-ketosteroids may adversely affect patients with mixed ERα+/CK5+ breast cancer. The observations further suggest a cooperative diagnostic utility of CK5 and BCL6 expression levels and justify exploring efficacy of inhibitors of BCL6 and 3-ketosteroid receptors for a subset of ERα-positive breast cancers.

  4. Chronic ketamine produces altered distribution of parvalbumin-positive cells in the hippocampus of adult rats.

    Science.gov (United States)

    Sabbagh, Jonathan J; Murtishaw, Andrew S; Bolton, Monica M; Heaney, Chelcie F; Langhardt, Michael; Kinney, Jefferson W

    2013-08-29

    The underlying mechanisms of schizophrenia pathogenesis are not well understood. Increasing evidence supports the glutamatergic hypothesis that posits a hypofunction of the N-methyl D-aspartate (NMDA) receptor on specific gamma amino-butyric acid (GABA)-ergic neurons may be responsible for the disorder. Alterations in the GABAergic system have been observed in schizophrenia, most notably a change in the expression of parvalbumin (PV) in the cortex and hippocampus. Several reports also suggest abnormal neuronal migration may play a role in the etiology of schizophrenia. The current study examined the positioning and distribution of PV-positive cells in the hippocampus following chronic treatment with the NMDA receptor antagonist ketamine. A robust increase was found in the number of PV-positive interneurons located outside the stratum oriens (SO), the layer where most of these cells are normally localized, as well as an overall numerical increase in CA3 PV cells. These results suggest ketamine leads to an abnormal distribution of PV-positive cells, which may be indicative of aberrant migratory activity and possibly related to the Morris water maze deficits observed. These findings may also be relevant to alterations observed in schizophrenia populations.

  5. 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

  6. 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.

  7. 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.

  8. Prognostic value of CD66b positive tumor-infiltrating neutrophils in testicular germ cell tumor

    OpenAIRE

    2016-01-01

    Background Prognostic value of immune cells is not clear in testicular germ cell tumors (TGCTs). We aimed to investigate the prognostic value of tumor-infiltrating neutrophils in TGCTs. Methods A total of 102 patients who underwent orchiectomy for TGCT were investigated for CD66b positive tumor-infiltrating neutrophils (CD66b + TINs). Immmunostaining for CD66b was performed in 102 sections as described. Clinicopathological parameters as well as cancer specific survival and overall survival we...

  9. Patterns of expression of keratin 17 in human epithelia: dependency on cell position.

    Science.gov (United States)

    Troyanovsky, S M; Guelstein, V I; Tchipysheva, T A; Krutovskikh, V A; Bannikov, G A

    1989-07-01

    By immunomorphology, using keratin 17-specific monoclonal antibody, it has been shown that this keratin is expressed only in the basal cells of a group of complex epithelia: glandular epithelium with myoepithelial component, transitional and pseudostratified epithelia. Immunolocalization of keratin 17 provides evidence that the expression of this keratin strongly depends on the cell position within epithelial structures. The topographical character of the keratin expression suggests that these proteins may be implicated in the generation of spatial organization of epithelial tissues.

  10. MOLECULAR BIOLOGICAL CHARACTERISTICS OF ALK-POSITIVE ANAPLASTIC LARGE CELL LYMPHOMA

    Directory of Open Access Journals (Sweden)

    E. V. Chernyshova

    2016-01-01

    Full Text Available ALK-positive anaplastic large cell lymphoma is a heterogeneous group of mature T-cell non-Hodgkin lymphoma, and is characterized by CD30/Ki-1 expression. Recently, value of various prognostic factors is investigated. These include clinical, histological and molecular genetic changes associated with different signaling pathways activation. Some features of the mechanism of action of anaplastic lymphoma kinases and targeted therapies possibilities addressed in this review.

  11. 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.

  12. Positioning of human chromosomes in murine cell hybrids according to synteny.

    Science.gov (United States)

    Meaburn, Karen J; Newbold, Robert F; Bridger, Joanna M

    2008-12-01

    Chromosomes occupy non-random spatial positions in interphase nuclei. It remains unclear what orchestrates this high level of organisation. To determine how the nuclear environment influences the spatial positioning of chromosomes, we utilised a panel of stable mouse hybrid cell lines carrying a single, intact human chromosome. Eleven of 22 human chromosomes revealed an alternative location in hybrid nuclei compared to that of human fibroblasts, with the majority becoming more internally localised. Human chromosomes in mouse nuclei position according to neither their gene density nor size, but rather the position of human chromosomes in hybrid nuclei appears to mimic that of syntenic mouse chromosomes. These results suggest that chromosomes adopt the behaviour of their host species chromosomes and that the nuclear environment is an important determinant of the interphase positioning of chromosomes.

  13. 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.

  14. 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.

  15. Developments in stem cell research and therapeutic cloning: Islamic ethical positions, a review.

    Science.gov (United States)

    Fadel, Hossam E

    2012-03-01

    Stem cell research is very promising. The use of human embryos has been confronted with objections based on ethical and religious positions. The recent production of reprogrammed adult (induced pluripotent) cells does not - in the opinion of scientists - reduce the need to continue human embryonic stem cell research. So the debate continues. Islam always encouraged scientific research, particularly research directed toward finding cures for human disease. Based on the expectation of potential benefits, Islamic teachings permit and support human embryonic stem cell research. The majority of Muslim scholars also support therapeutic cloning. This permissibility is conditional on the use of supernumerary early pre-embryos which are obtained during infertility treatment in vitro fertilization (IVF) clinics. The early pre-embryos are considered in Islamic jurisprudence as worthy of respect but do not have the full sanctity offered to the embryo after implantation in the uterus and especially after ensoulment. In this paper the Islamic positions regarding human embryonic stem cell research and therapeutic cloning are reviewed in some detail, whereas positions in other religious traditions are mentioned only briefly. The status of human embryonic stem cell research and therapeutic cloning in different countries, including the USA and especially in Muslim countries, is discussed.

  16. 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.

  17. Utilization of a cell separation technique to evaluate patients with a positive direct antiglobulin test.

    Science.gov (United States)

    Wallas, C H; Tanley, P C; Gorrell, L P

    1982-01-01

    Density distribution curves of red blood cells (RBC) from patients with a positive direct antiglobulin test (DAT) were compared to a standardized curve constructed from cell column measurements of centrifuged microcapillary tubes filled with RBC and phthalate ester mixtures encompassing a specific gravity range of 1.078 to 1.114. Shortened survival resulted in a loss of older RBC and a shift of the curve to the right over the high specific gravity ester range. Reticulocytosis resulted in a downward shift of the curve over the low specific gravity range. In patients with a positive DAT due to an autoantibody or drug, the density distribution curve was either normal or demonstrated evidence of shortened RBC survival. In patients with a positive DAT due to an alloantibody, however, evidence of shortened survival was not seen. The distribution of antibody on young and old RBC harvested from the appropriate microcapillary tubes depended upon the etiology of that antibody. In patients with a positive DAT due to to an alloantibody or drug, both the young and old RBC gave an equally reactive DAT, while in patients with a positive DAT due to an alloantibody the young cells were weakly reactive or nonreactive and the older cells were more strongly reactive. When used together, the position of the density distribution curve and the pattern of distribution of antibody coating on young and old RBC provide important diagnostic information about the etiology and clinical status in a patient with a positive DAT and allow for the recognition of an alloantibody and autoantibody when both are present in the same patient.

  18. 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

  19. 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

  20. 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...

  1. 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.

  2. Sox2-positive dermal papilla cells specify hair follicle type in mammalian epidermis.

    Science.gov (United States)

    Driskell, Ryan R; Giangreco, Adam; Jensen, Kim B; Mulder, Klaas W; Watt, Fiona M

    2009-08-01

    The dermal papilla comprises the specialised mesenchymal cells at the base of the hair follicle. Communication between dermal papilla cells and the overlying epithelium is essential for differentiation of the hair follicle lineages. We report that Sox2 is expressed in all dermal papillae at E16.5, but from E18.5 onwards expression is confined to a subset of dermal papillae. In postnatal skin, Sox2 is only expressed in the dermal papillae of guard/awl/auchene follicles, whereas CD133 is expressed both in guard/awl/auchene and in zigzag dermal papillae. Using transgenic mice that express GFP under the control of the Sox2 promoter, we isolated Sox2(+) (GFP(+)) CD133(+) cells and compared them with Sox2(-) (GFP(-)) CD133(+) dermal papilla cells. In addition to the 'core' dermal papilla gene signature, each subpopulation expressed distinct sets of genes. GFP(+) CD133(+) cells had upregulated Wnt, FGF and BMP pathways and expressed neural crest markers. In GFP(-) CD133(+) cells, the hedgehog, IGF, Notch and integrin pathways were prominent. In skin reconstitution assays, hair follicles failed to form when dermis was depleted of both GFP(+) CD133(+) and GFP(-) CD133(+) cells. In the absence of GFP(+) CD133(+) cells, awl/auchene hairs failed to form and only zigzag hairs were found. We have thus demonstrated a previously unrecognised heterogeneity in dermal papilla cells and shown that Sox2-positive cells specify particular hair follicle types.

  3. Positioning of polarity formation by extracellular signaling during asymmetric cell division.

    Science.gov (United States)

    Seirin Lee, Sungrim

    2016-07-07

    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.

  4. CD13-positive anaplastic large cell lymphoma of T-cell origin--a diagnostic and histogenetic problem.

    Science.gov (United States)

    Popnikolov, N K; Payne, D A; Hudnall, S D; Hawkins, H K; Kumar, M; Norris, B A; Elghetany, M T

    2000-12-01

    The expression of myelomonocytic-associated antigens in anaplastic large cell lymphomas (ALCLs), particularly those presenting in extranodal sites, can make their distinction from extramedullary myeloid cell tumors (EMCTs) or histiocytic tumors problematic. Yet, this distinction is clinically significant because of its therapeutic and prognostic implications. Herein, we describe a case of extranodal anaplastic lymphoma kinase-positive CD30-positive ALCL of T-cell origin in a 12-year-old boy, which was initially called an EMCT because of the expression of CD13 and HLA-DR detected by flow cytometry and the absence of other T-cell-related surface markers. However, the detection of cytoplasmic CD3 by flow cytometry prompted further studies. The tumor was composed of large cells with abundant slightly eosinophilic vacuolated cytoplasm and ovoid or reniform nuclei with a few small nucleoli. Using immunohistochemistry, the tumor was positive for CD45, CD30, CD45RO, and CD43 with a strong cytoplasmic and nuclear anaplastic lymphoma kinase stain. The tumor cells showed a T-cell clonal genotype. Electron microscopy revealed no ultrastructural features of myelomonocytic or histiocytic origin. The patient responded well to the chemotherapy and was in complete remission for 10 months at the time of submission of this manuscript. Review of the literature showed inconsistencies regarding the diagnosis, nomenclature, and, therefore, treatment and prognosis of these tumors. In addition, the CD13 expression in ALCL raises some histogenetic questions and may indicate origin from a pluripotent stem cell, misprogramming during malignant transformation, or a microenvironmental effect on lymphoid cell expression of surface antigens. Therefore, ALCL should be considered in the differential diagnosis of EMCTs or histiocytic tumors, particularly when surface marker lineage assignment is ambiguous.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. Modeling shear-induced CHO cell damage in a rotary positive displacement pump.

    Science.gov (United States)

    Kamaraju, Hari; Wetzel, Kenneth; Kelly, William J

    2010-01-01

    Rotary lobe pumps are commonly used in the biotechnology industry for a variety of purposes. Shear damage to animal cells within the rotary lobe pump can adversely affect the product yield or purity during, for example, cell concentration via cross-flow filtration. In this research, CHO cells grown in 20-L bioreactors were fed to a rotary lobe pump in both single pass and recycle experiments were conducted at different RPMs and "slip" conditions. The results indicate that the slip flow rate more severely impacts the viability of the CHO cells than the pump RPM. A novel mathematical modeling approach is presented that predicts shear rates in all of the positive displacement pump's slip regions, and then predicts cell death vs. operating conditions. This model accounts for the complex flow situation that results from changes to RPM, backpressure and pump geometry (i.e., clearances).

  10. Estrogen-Mediated Upregulation of Noxa Is Associated with Cell Cycle Progression in Estrogen Receptor-Positive Breast Cancer Cells

    OpenAIRE

    2011-01-01

    Noxa is a Bcl-2-homology domain (BH3)-only protein reported to be a proapoptotic member of the Bcl-2 family. Estrogen has been well documented to stimulate cell growth and inhibit apoptosis in estrogen receptor (ER)-positive breast cancer cells. Intriguingly, recent reports have shown that 17β-estradiol (E2) induces Noxa expression, although the mechanisms underlying E2-mediated induction of Noxa and its functional significance are unknown. Using MCF7 human breast cancer cells as an experimen...

  11. Programming of donor T cells using allogeneic δ-like ligand 4-positive dendritic cells to reduce GVHD in mice.

    Science.gov (United States)

    Mochizuki, Kazuhiro; Meng, Lijun; Mochizuki, Izumi; Tong, Qing; He, Shan; Liu, Yongnian; Purushe, Janaki; Fung, Henry; Zaidi, M Raza; Zhang, Yanyun; Reshef, Ran; Blazar, Bruce R; Yagita, Hideo; Mineishi, Shin; Zhang, Yi

    2016-06-23

    Alloreactive T cells play a critical role in eliminating hematopoietic malignant cells but are also the mediators of graft-versus-host disease (GVHD), a major complication that subverts the success of allogeneic hematopoietic stem cell transplantation (HSCT). However, induction of alloreactive T cells does not necessarily lead to GVHD. Here we report the development of a cellular programming approach to render alloreactive T cells incapable of causing severe GVHD in both major histocompatibility complex (MHC)-mismatched and MHC-identical but minor histocompatibility antigen-mismatched mouse models. We established a novel platform that produced δ-like ligand 4-positive dendritic cells (Dll4(hi)DCs) from murine bone marrow using Flt3 ligand and Toll-like receptor agonists. Upon allogeneic Dll4(hi)DC stimulation, CD4(+) naïve T cells underwent effector differentiation and produced high levels of interferon γ (IFN-γ) and interleukin-17 in vitro, depending on Dll4 activation of Notch signaling. Following transfer, allogeneic Dll4(hi)DC-induced T cells were unable to mediate severe GVHD but preserved antileukemic activity, significantly improving the survival of leukemic mice undergoing allogeneic HSCT. This effect of Dll4(hi)DC-induced T cells was associated with their impaired expansion in GVHD target tissues. IFN-γ was important for Dll4(hi)DC programming to reduce GVHD toxicities of alloreactive T cells. Absence of T-cell IFN-γ led to improved survival and expansion of Dll4(hi)DC-induced CD4(+) T cells in transplant recipients and caused lethal GVHD. Our findings demonstrate that Dll4(hi)DC programming can overcome GVHD toxicity of donor T cells and produce leukemia-reactive T cells for effective immunotherapy.

  12. The positional control of mitosis and cytokinesis in higher-plant cells.

    Science.gov (United States)

    Giménez-Abián, M I; Utrilla, L; Cánovas, J L; Giménez-Martín, G; Navarrete, M H; De la Torre, C

    1998-01-01

    The present work establishes a correlation between cell length and patterns of mitotic microtubular assemblies in Allium cepa L. root meristems. Binucleate cells were formed by a short caffeine treatment which aborted the formation of the phragmoplast during telophase. The largest binucleate cells (about 50 microns in length) behaved as two contiguous mononucleate cells in their next mitosis: they developed two preprophase bands (PPBs), one around each nucleus, where two spindles and two phragmoplasts were subsequently formed. On the other hand, the shortest binucleate cells (about 36 microns in length) formed a single PPB at the site of the aborted phragmoplast and, in the medium-sized cells (about 44 microns) in which the single PPB formed around the nucleus possessing the largest cytoplasmic environment, the two mitotic spindles and the new phragmoplasts moved to, or were assembled in the position of the phragmoplast that had been aborted one cycle earlier. Some rules derive from these observations. First of all, the aborted phragmoplast left a signal for microtubule positioning which was still operative one cycle later, in two-thirds of the bimitoses. Also, that formation of the PPB is dispensable. Moreover, its development does not always predict the future division plane, because of the presence of competing old signals which are stronger than those shed by the PPB in the same mitosis, but which fade away with distance. Finally, the positional signals were reinforced when the ratio of monomeric to fibrillar actin was increased by cytochalasin D during their shedding. When this drug was given simultaneously with caffeine, the frequency of bimitoses which, one cycle later, developed spindles and phragmoplasts in the positions of the old phragmoplast increased. On the other hand, those frequencies dropped in relation to control when the cytochalasin D treatment took place during bimitosis, indicating that at this time the treatment reinforced the signals

  13. 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.

  14. Proteome Analysis of Thyroid Cancer Cells After Long-Term Exposure to a Random Positioning Machine

    Science.gov (United States)

    Pietsch, Jessica; Bauer, Johann; Weber, Gerhard; Nissum, Mikkel; Westphal, Kriss; Egli, Marcel; Grosse, Jirka; Schönberger, Johann; Eilles, Christoph; Infanger, Manfred; Grimm, Daniela

    2011-11-01

    Annulling gravity during cell culturing triggers various types of cells to change their protein expression in a time dependent manner. We therefore decided to determine gravity sensitive proteins and their period of sensitivity to the effects of gravity. In this study, thyroid cancer cells of the ML-1 cell line were cultured under normal gravity (1 g) or in a random positioning machine (RPM), which simulated near weightlessness for 7 and 11 days. Cells were then sonicated and proteins released into the supernatant were separated from those that remained attached to the cell fragments. Subsequently, both types of proteins were fractionated by free-flow isoelectric focussing (FF-IEF). The fractions obtained were further separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to which comparable FF-IEF fractions derived from cells cultured either under 1 g or on the RPM had been applied side by side. The separation resulted in pairs of lanes, on which a number of identical bands were observed. Selected gel pieces were excised and their proteins determined by mass spectrometry. Equal proteins from cells cultured under normal gravity and the RPM, respectively, were detected in comparable gel pieces. However, many of these proteins had received different Mascot scores. Quantifying heat shock cognate 71 kDa protein, glutathione S-transferase P, nucleoside diphosphate kinase A and annexin-2 by Western blotting using whole cell lysates indicated usefulness of Mascot scores for selecting the most efficient antibodies.

  15. Autophagic vesicles on mature human reticulocytes explain phosphatidylserine-positive red cells in sickle cell disease.

    Science.gov (United States)

    Mankelow, Tosti J; Griffiths, Rebecca E; Trompeter, Sara; Flatt, Joanna F; Cogan, Nicola M; Massey, Edwin J; Anstee, David J

    2015-10-08

    During maturation to an erythrocyte, a reticulocyte must eliminate any residual organelles and reduce its surface area and volume. Here we show this involves a novel process whereby large, intact, inside-out phosphatidylserine (PS)-exposed autophagic vesicles are extruded. Cell surface PS is a well-characterized apoptotic signal initiating phagocytosis. In peripheral blood from patients after splenectomy or in patients with sickle cell disease (SCD), the number of circulating red cells exposing PS on their surface is elevated. We show that in these patients PS is present on the cell surface of red cells in large (∼1.4 µm) discrete areas corresponding to autophagic vesicles. The autophagic vesicles found on reticulocytes are identical to those observed on red cells from splenectomized individuals and patients with SCD. Our data suggest the increased thrombotic risk associated with splenectomy, and patients with hemoglobinopathies is a possible consequence of increased levels of circulating mature reticulocytes expressing inside-out PS-exposed autophagic vesicles because of asplenia.

  16. 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.

  17. 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.

  18. 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.

  19. Flow cytometric analysis of hemetopoietic progenitor cells in peripheral blood stem cell harvest from patients with CD34 positive acute leukemia.

    Science.gov (United States)

    Miyazaki, T; Matsuda, I; Oguri, M; Amaya, H; Kiyosaki, M; Hamada, A; Tamaki, S; Tashiro, E; Kudo, Y; Taniguchi, O; Nakamura, T; Tomoyasu, S

    2001-01-01

    We analyzed CD34 positive cells in peripheral blood stem cell harvest (PBSCH) using flow cytometry. PBSCH from CD34 positive acute myelogeous leukemia (AML-M2) patient contained 1.87% CD34 positive cells, of which 1.21% was represented by MRD.PBSCH from CD34 positive acute lymphoblast leukemia (ALL) patient contained 3.14% CD34 positive cells, of which 0.11% was accounted for by minimal residual disease (MRD). If PBSCH from CD34 positive acute leukemia patient is analyzed for CD34 monoclonal antibody alone, the presence of CD34 positive MRD may escape attention so that CD34 positive hematopoietic progenitor cells may be overestimated. To avoid this risk, it is necessary to analyze PBSCH using both CD34 monoclonal antibody and characteristic markers of leukemia cells that were found pre-treatment.

  20. Recurrence rate of basal cell carcinoma with positive histopathological margins and related risk factors*

    Science.gov (United States)

    Lara, Fernanda; Santamaría, Jesus Rodriguez; Garbers, Luiz Eduardo Fabricio de Melo

    2017-01-01

    BACKGROUND The best way to approach surgically removed basal cell carcinoma with positive histopathological margins is a controversial issue. Some authors believe that the more appropriate treatment is an immediate reoperation while others prefer a periodic follow up. The rates of recurrence are variable in literature, between 10% and 67%. OBJECTIVE To define the recurrence rate of basal cell carcinoma with positive margins after surgery. Secondarily, identify morphological aspects that can suggest a more frequent tumoral recurrence. METHODS This was a retrospective and observational study made by analysis of medical records of 487 patients between January 2003 and December 2009 in Hospital de Clínicas da Universidade Federal do Paraná (HC-UFPR). From 402 basal cell carcinomas surgically treated, 41 fulfilled inclusion criteria and were evaluated for five years or more. Recurrence rate of these tumors was analyzed in all patients and clinical characteristics such as sex, age, tumor size, tumor site, ulceration, and histological type were evaluated in order to find if they were related to more common tumoral recurrence. RESULTS The rate of positive margins after surgery was 12.18%. There were five cases of tumoral recurrence in the observation group and three cases in the re-excision group. Tumor size, site, histological type, ulceration and type of positive margin did not differ statistically between groups. It was not possible to consider if these factors were important in recurrence rates. STUDY LIMITATIONS Ideally, a prospective study with a larger sample would be more accurate. CONCLUSION The treatment of choice in basal cell carcinoma with positive margins must be individualized to reduce recurrence rates. PMID:28225958

  1. 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.

  2. 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.

  3. Estrogen-mediated upregulation of Noxa is associated with cell cycle progression in estrogen receptor-positive breast cancer cells.

    Science.gov (United States)

    Liu, Wensheng; Swetzig, Wendy M; Medisetty, Rajesh; Das, Gokul M

    2011-01-01

    Noxa is a Bcl-2-homology domain (BH3)-only protein reported to be a proapoptotic member of the Bcl-2 family. Estrogen has been well documented to stimulate cell growth and inhibit apoptosis in estrogen receptor (ER)-positive breast cancer cells. Intriguingly, recent reports have shown that 17β-estradiol (E2) induces Noxa expression, although the mechanisms underlying E2-mediated induction of Noxa and its functional significance are unknown. Using MCF7 human breast cancer cells as an experimental model, we show that Noxa is upregulated by E2 via p53-independent processes that involve c-Myc and ERα. Experiments using small interfering ribonucleic acids (siRNA) to specifically knock down p53, c-Myc, and ERα demonstrated that c-Myc and ERα, but not p53, are involved in the transcriptional upregulation of Noxa following E2 treatment. Furthermore, while E2 promoted the recruitment of c-Myc and ERα to the NOXA promoter in chromatin immunoprecipitation (ChIP) assays, E2 did not induce p53 recruitment. Interestingly, E2-mediated upregulation of Noxa was not associated with apoptosis. However, siRNA-mediated knockdown of Noxa resulted in cell cycle arrest in G(0)/G(1)-phase and significantly delayed the G(1)-to-S-phase transition following E2 treatment, indicating that Noxa expression is required for cell cycle progression in ER-positive breast cancer cells.

  4. Estrogen-mediated upregulation of Noxa is associated with cell cycle progression in estrogen receptor-positive breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Wensheng Liu

    Full Text Available Noxa is a Bcl-2-homology domain (BH3-only protein reported to be a proapoptotic member of the Bcl-2 family. Estrogen has been well documented to stimulate cell growth and inhibit apoptosis in estrogen receptor (ER-positive breast cancer cells. Intriguingly, recent reports have shown that 17β-estradiol (E2 induces Noxa expression, although the mechanisms underlying E2-mediated induction of Noxa and its functional significance are unknown. Using MCF7 human breast cancer cells as an experimental model, we show that Noxa is upregulated by E2 via p53-independent processes that involve c-Myc and ERα. Experiments using small interfering ribonucleic acids (siRNA to specifically knock down p53, c-Myc, and ERα demonstrated that c-Myc and ERα, but not p53, are involved in the transcriptional upregulation of Noxa following E2 treatment. Furthermore, while E2 promoted the recruitment of c-Myc and ERα to the NOXA promoter in chromatin immunoprecipitation (ChIP assays, E2 did not induce p53 recruitment. Interestingly, E2-mediated upregulation of Noxa was not associated with apoptosis. However, siRNA-mediated knockdown of Noxa resulted in cell cycle arrest in G(0/G(1-phase and significantly delayed the G(1-to-S-phase transition following E2 treatment, indicating that Noxa expression is required for cell cycle progression in ER-positive breast cancer cells.

  5. Local circuitry involving parvalbumin-positive basket cells in the CA2 region of the hippocampus.

    Science.gov (United States)

    Mercer, Audrey; Eastlake, Karen; Trigg, Hayley L; Thomson, Alex M

    2012-01-01

    There is a growing recognition that the CA2 region of the hippocampus has its own distinctive properties, inputs, and pathologies. The dendritic and axonal patterns of some interneurons in this region are also strikingly different from those described previously in CA1 and CA3. The local circuitry in this region, however, had yet to be studied in detail. Accordingly, using dual intracellular recordings and biocytin-filling, excitatory and inhibitory connections involving CA2 parvalbumin-positive basket cells were characterized for the first time. CA2 basket cells targeted neighboring pyramidal cells and received excitatory inputs from them. CA2 basket cells that resembled those in CA1 with a fast spiking behavior and dendritic tree confined to the region of origin received depressing excitatory postsynaptic potentials (EPSPs). In contrast, unlike CA1 basket cells but like CA1 Oriens-Lacunosum Moleculare (OLM) cells, the majority of CA2 basket cells had horizontally oriented dendrites in Stratum Oriens (SO), which extended into all three CA subfields, had an adapting firing pattern, presented a "sag" in their voltage responses to hyperpolarizing current injection, and received facilitating EPSPs. The expression of I(h) did not influence the EPSP time courses and paired pulse ratios (PPR). Estimates of the probability of release (p) for the depressing and facilitating EPSPs were correlated with the PPR. Connections with low probabilities of release had higher PPR. Quantal amplitude (q) for the facilitating connections was larger than q at depressing inputs onto fast spiking basket cells.

  6. Positive Regulation of Interleukin-2 Expression by a Pseudokinase, Tribbles 1, in Activated T Cells.

    Science.gov (United States)

    Miyajima, Chiharu; Itoh, Yuka; Inoue, Yasumichi; Hayashi, Hidetoshi

    2015-01-01

    Tribbles 1 (TRB1), a member of the Tribbles family, is a pseudokinase that is conserved among species and implicated in various human diseases including leukemia, cardiovascular diseases, and metabolic disorders. However, the role of TRB1 in the immune response is not understood. To evaluate this role, we examined regulation of TRB1 expression and the function of TRB1 in interleukin-2 (IL-2) induction in Jurkat cells, a human acute T cell leukemia cell line. We found that TRB1 was strongly induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin in these cells. IL-2 expression was induced in Jurkat cells activated by PMA and ionomycin; however, knockdown of TRB1 resulted in decreased induction of IL-2. TRB1 null Jurkat cells established using the CRISPR/Cas9 system also showed reduction of IL-2 expression on PMA/ionomycin stimulation. TRB1 knockdown also markedly inhibited IL-2 promoter activation. To determine the mechanism of the stimulatory effect on IL-2 induction, we focused on histone deacetylases (HDACs), and found that HDAC1 preferentially interacts with TRB1. TRB1 suppressed the interaction of HDAC1 with nuclear factor of activated T cells 2 (NFAT2), which is a crucial transcription factor for IL-2 induction. These results indicate that TRB1 is a positive regulator of IL-2 induction in activated T cells.

  7. 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.

  8. Decreased infiltration of macrophage scavenger receptor-positive cells in initial negative biopsy specimens is correlated with positive repeat biopsies of the prostate.

    Science.gov (United States)

    Nonomura, Norio; Takayama, Hitoshi; Kawashima, Atsunari; Mukai, Masatoshi; Nagahara, Akira; Nakai, Yasutomo; Nakayama, Masashi; Tsujimura, Akira; Nishimura, Kazuo; Aozasa, Katsuyuki; Okuyama, Akihiko

    2010-06-01

    Macrophage scavenger receptor (MSR)-positive inflammatory cells and tumor-associated macrophages (TAMs) have been reported to regulate the growth of various cancers. In this study, the infiltration of MSR-positive cells and TAMs was analyzed to predict the outcome of repeat biopsy in men diagnosed as having no malignancy at the first prostate biopsy. Repeat biopsy of the prostate was carried out in 92 patients who were diagnosed as having no malignancy at the first biopsy. Of these, 30 patients (32.6%) were positive for prostate cancer at the repeat biopsy. Tumor-associated macrophages and MSR-positive cells were immunohistochemically stained with mAbs CD68 and CD204, respectively. Six ocular measuring fields were chosen randomly under a microscope at x400 power in the initial negative biopsy specimens, and the mean TAM and MSR counts for each case were determined. No difference in TAM count was found between the cases with or without prostate cancer. By contrast, the MSR count in patients with cancer was significantly lower than that in patients without cancer at the repeat biopsy (P biopsies, or TAM count. Decreased infiltration of MSR-positive cells in negative first biopsy specimens was correlated with positive findings in the repeat biopsy. The MSR count might be a good indicator for avoiding unnecessary repeat biopsies.

  9. 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

  10. Sox2 regulates cholinergic amacrine cell positioning and dendritic stratification in the retina.

    Science.gov (United States)

    Whitney, Irene E; Keeley, Patrick W; St John, Ace J; Kautzman, Amanda G; Kay, Jeremy N; Reese, Benjamin E

    2014-07-23

    The retina contains two populations of cholinergic amacrine cells, one positioned in the ganglion cell layer (GCL) and the other in the inner nuclear layer (INL), that together comprise ∼1/2 of a percent of all retinal neurons. The present study examined the genetic control of cholinergic amacrine cell number and distribution between these two layers. The total number of cholinergic amacrine cells was quantified in the C57BL/6J and A/J inbred mouse strains, and in 25 recombinant inbred strains derived from them, and variations in their number and ratio (GCL/INL) across these strains were mapped to genomic loci. The total cholinergic amacrine cell number was found to vary across the strains, from 27,000 to 40,000 cells, despite little variation within individual strains. The number of cells was always lower within the GCL relative to the INL, and the sizes of the two populations were strongly correlated, yet there was variation in their ratio between the strains. Approximately 1/3 of that variation in cell ratio was mapped to a locus on chromosome 3, where Sex determining region Y box 2 (Sox2) was identified as a candidate gene due to the presence of a 6-nucleotide insertion in the protein-coding sequence in C57BL/6J and because of robust and selective expression in cholinergic amacrine cells. Conditionally deleting Sox2 from the population of nascent cholinergic amacrine cells perturbed the normal ratio of cells situated in the GCL versus the INL and induced a bistratifying morphology, with dendrites distributed to both ON and OFF strata within the inner plexiform layer.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells

    OpenAIRE

    Tchasovnikarova, Iva A.; Timms, Richard T.; Matheson, Nicholas J.; Wals, Kim; Antrobus, Robin; Göttgens, Berthold; Dougan, Gordon; Dawson, Mark A.; Lehner, Paul J.

    2015-01-01

    This is the author accepted manuscript. The final version is available from AAAS via http://dx.doi.org/10.1126/science.aaa7227 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 (...

  16. 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.

  17. 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.

  18. CD45/CD11b Positive Subsets of Adult Lung Anchorage-Independent Cells Harness Epithelial Stem Cells

    Science.gov (United States)

    Peter, Yakov; Sen, Namita; Levantini, Elena; Keller, Steven; Ingenito, Edward P; Ciner, Aaron; Sackstein, Robert; Shapiro, Steven D

    2015-01-01

    Compensatory growth is mediated by multiple cell types that interact during organ repair. To elucidate the relationship between the stem/progenitor cells that proliferate or differentiate and the somatic cells of lung, we utilized a novel ex vivo pneumoexplant system. Applying this technique, we identified a sustained culture of repopulating adult progenitors in the form of free floating anchorage-independent cells (AICs). AICs did not express integrin proteins α5, β3, and β7, and constituted 37% of the total culture at day 14, yielding a mixed yet conserved population that recapitulated RNA expression patterns of the healthy lung. AICs exhibited rapid proliferation manifested by a marked 60-fold increase in cell numbers by day 21. Over 50% of the AIC population was cKit+ or double-positive for CD45+ and CD11b+ antigenic determinants, consistent with cells of hematopoietic origin. The latter subset was found to be enriched with prosurfactant protein-C and SCGB1A1 expressing putative stem cells and with aquaporin-5 producing cells, characteristic of terminally differentiated alveolar epithelial type-1 pneumocytes. AICs undergo remodeling to form a cellular lining at the air/gel interface, and TGFβ1 treatment modifies protein expression, implying direct-differentiation of this population. These data confirm the active participation of clonogenic hematopietic stem cells in a mammalian model of lung repair and validate mixed stem/somatic cell cultures, which embrace sustained cell viability, proliferation, and differentiation, for use in studies of compensatory pulmonary growth. PMID:22585451

  19. Trastuzumab-mediated selective delivery for platinum drug to HER2-positive breast cancer cells.

    Science.gov (United States)

    Huang, Rong; Sun, Yu; Gao, Qihe; Wang, Qiucui; Sun, Baiwang

    2015-10-01

    Oxaliplatin is used widely as an anticancer drug for clinical treatment. However, its applications are limited because of its poor selectivity. In this work, we described the design, synthesis, and characterization of conjugates combining trastuzumab with a platinum (IV) analog of oxaliplatin, in which the trastuzumab acted as an active targeting agent for HER2-positive cancer cells. Indirect enzyme-linked immunosorbent assay and immunofluorescence study indicated the platinum (IV)-trastuzumab conjugates retained specific binding activity to HER2 overexpressed SK-BR-3 cells. In the presence of ascorbic acid, platinum (IV)-trastuzumab conjugates were reduced to platinum (II) analogs, which could bind to and unwind PUC19 DNA in a manner similar to oxaliplatin. The cytotoxic study was tested on three breast cell lines: SK-BR-3, MCF-7, and MDA-MB-231. Platinum (IV)-trastuzumab conjugates showed promising antiproliferative activity against SK-BR-3 cells, but significantly decreased the inhibition to MDA-MB-231 and MCF-7 cells. The flow cytometric analysis showed that the conjugates arrested the cell cycle mainly at the G2/M phase and killed the cells through an apoptotic pathway.

  20. 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.

  1. Fate study of water-borne gram positive vegetative bacterial cells with Raman microscopy

    Science.gov (United States)

    Guicheteau, Jason; Tripathi, Ashish; Minter, Jennifer; Wilcox, Phillip; Christesen, Steven

    2010-04-01

    We present an initial bacterial fate study of Gram positive vegetative cells suspended in water and stored at ambient room temperature via Raman spectroscopy monitoring. Two types of cells were considered for this study: vegetative cells of Bacillus cereus, Bacillus thuringiensis which contain the polyhydroxybutyric acid (PHBA) as an energy storage compound and Bacillus subtlilis cells which do not. The cells were cultured specifically for this project. Immediately following the culturing phase, the bacteria were extracted, cleaned and at the onset of the study were suspended in de-ionized water and stored at room temperature. Aliquots of suspensions were deposited onto aluminum slides at different times and allowed to dry for Raman analysis. Spectra from multiple regions of each dried spot and each deposit time were acquired along with the bright-field and fluorescence images. Results were examined to investigate the effect of suspension time on the spectral signatures as well as the fate behavior of the three types of cells investigated. The cells were monitored daily for over a 14 period during which time the onset of starvation induced sporulation was observed.

  2. Novel insights into the flexibility of cell and positional identity during urodele limb regeneration.

    Science.gov (United States)

    Kragl, M; Knapp, D; Nacu, E; Khattak, S; Schnapp, E; Epperlein, H-H; Tanaka, E M

    2008-01-01

    The ability of diverse metazoans to regenerate whole-body structures was first described systematically by Spallanzani in 1768 and continues to fascinate biologists today. Given the current interest in stem cell biology and its therapeutic potential, examples of vertebrate regeneration garner strong interest. Among regeneration-competent vertebrates such as the fish, frog, and salamander, the salamander is particularly impressive because it can regenerate the entire limb and tail as well as various internal organs as an adult (Goss 1969). This spectacular natural phenomenon leads us to ask what cellular properties allow regeneration and what prevents this phenomenon in other vertebrates. From this perspective, it is imperative to know whether the stem cells in regenerating limbs harbor particularly special traits such as a higher plasticity in cell fate compared to tissue stem cells in other organisms. Flexibility in cell fate needs to be considered with respect not only to tissue identity, but also to patterning because limb amputation causes cells in a particular limb segment to form more distal limb elements. How positional identity is encoded in stem cells and how it is controlled to produce only the missing portion of the limb are also questions of fundamental importance.

  3. Socioeconomic position and surgery for early-stage non-small-cell lung cancer

    DEFF Research Database (Denmark)

    Starr, Laila Kærgaard; Osler, Merete; Steding-Jessen, Marianne;

    2012-01-01

    AIM: To examine possible associations between socioeconomic position and surgical treatment of patients with early-stage non-small-cell lung cancer (NSCLC). METHODS: In a register-based clinical cohort study, patients with early-stage (stages I-IIIa) NSCLC were identified in the Danish Lung Cancer...... in a health care system with free, equal access to health services, disadvantaged groups are less likely to receive surgery for lung cancer....

  4. Mice lacking TrkB in parvalbumin-positive cells exhibit sexually dimorphic behavioral phenotypes

    OpenAIRE

    Lucas, Elizabeth K.; Jegarl, Anita; Clem, Roger L.

    2014-01-01

    Activity-dependent brain-derived neurotrophic factor (BDNF) signaling through receptor tyrosine kinase B (TrkB) is required for cued fear memory consolidation and extinction. Although BDNF is primarily secreted from glutamatergic neurons, TrkB is expressed by other genetically defined cells whose contributions to the behavioral effects of BDNF remain poorly understood. Parvalbumin (PV)-positive interneurons, which are highly enriched in TrkB, are emerging as key regulators of fear memory expr...

  5. 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.

  6. Pseudocarcinomatous hyperplasia mimicking squamous cell carcinoma in a case of CD56-positive cytotoxic T-cell lymphoma.

    Science.gov (United States)

    Ginsberg, David; Hill, Hilary; Wilson, Barbara; Plaza, Jose A; Schieke, Stefan M

    2015-03-01

    We present the case of an 84-year-old patient with a cutaneous CD56 positive cytotoxic T-cell lymphoma associated with substantial pseudocarcinomatous hyperplasia mimicking squamous cell carcinoma (SCC). The patient presented with a 7-month history of several progressive, ulcerated plaques on his right forearm. An initial biopsy showed changes consistent with a diagnosis of SCC for which the patient underwent surgical treatment. Several months later, the patient developed recurrent ulcerated plaques on the right forearm of which several biopsies were performed. The biopsies repeatedly showed marked pseudocarcinomatous hyperplasia resembling SCC. Deeper punch biopsies, however, showed a dense superficial and deep infiltrate of markedly atypical lymphocytes. Immunohistochemical analysis revealed strong positive staining for CD3, CD8, CD56 with negative stains for CD30 and Epstein-Barr virus-encoded small non-polyadenylated RNAs (EBER). Staining for beta F1 and gamma-delta T-cell receptor (γδ TCR) were both negative. This constellation was most consistent with a diagnosis of cutaneous peripheral T-cell lymphoma, unspecified in association with marked pseudocarcinomatous hyperplasia. Our case adds cutaneous peripheral T-cell lymphoma, unspecified to the list of conditions associated with pseudocarcinomatous hyperplasia (PCH) and illustrates once again the potential pitfalls of distinguishing marked pseudocarcinomatous hyperplasia from SCC.

  7. Cytokeratin positive anaplastic large cell lymphoma: Difficulty in differentiation from metastatic carcinoma

    Directory of Open Access Journals (Sweden)

    Nishat Afroz

    2015-01-01

    Full Text Available Cytokeratin and epithelial membrane antigen (EMA are usually included in the first panel of immunomarkers used to differentiate metastatic carcinoma from lymphoma in cases presenting with enlarged lymph nodes. While carcinomas are cytokeratin and EMA positive, most lymphomas are negative for the above. However, recently few cases of cytokeratin positive lymphomas have also been reported. Here, we describe a very rare case of cytokeratin positive anaplastic large cell lymphoma (ALCL masquerading as a poorly differentiated carcinoma. Simultaneously, we also discuss the differential diagnosis and difficulty in differentiation from metastatic carcinoma in such a scenario. Review of literature shows that this is probably the first case report of anaplastic lymphoma kinase negative-ALCL seen in a young adult.

  8. Atrophy, fibrosis, and increased PAX7-positive cells in pharyngeal muscles of oculopharyngeal muscular dystrophy patients.

    Science.gov (United States)

    Gidaro, Teresa; Negroni, Elisa; Perié, Sophie; Mirabella, Massimiliano; Lainé, Jeanne; Lacau St Guily, Jean; Butler-Browne, Gillian; Mouly, Vincent; Trollet, Capucine

    2013-03-01

    Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomal dominant inherited dystrophy caused by an abnormal trinucleotide repeat expansion in the poly(A)-binding-protein-nuclear 1 (PABPN1) gene. Primary muscular targets of OPMD are the eyelid elevator and pharyngeal muscles, including the cricopharyngeal muscle (CPM), the progressive involution of which leads to ptosis and dysphagia, respectively. To understand the consequences of PABPN1 polyalanine expansion in OPMD, we studied muscle biopsies from 14 OPMD patients, 3 inclusion body myositis patients, and 9 healthy controls. In OPMD patient CPM (n = 6), there were typical dystrophic features with extensive endomysial fibrosis and marked atrophy of myosin heavy-chain IIa fibers. There were more PAX7-positive cells in all CPM versus other muscles (n = 5, control; n = 3, inclusion body myositis), and they were more numerous in OPMD CPM versus control normal CPM without any sign of muscle regeneration. Intranuclear inclusions were present in all OPMD muscles but unaffected OPMD patient muscles (i.e. sternocleidomastoid, quadriceps, or deltoid; n = 14) did not show evidence of fibrosis, atrophy, or increased PAX7-positive cell numbers. These results suggest that the specific involvement of CPM in OPMD might be caused by failure of the regenerative response with dysfunction of PAX7-positive cells and exacerbated fibrosis that does not correlate with the presence of PABPN1 inclusions.

  9. Mice lacking TrkB in parvalbumin-positive cells exhibit sexually dimorphic behavioral phenotypes.

    Science.gov (United States)

    Lucas, Elizabeth K; Jegarl, Anita; Clem, Roger L

    2014-11-01

    Activity-dependent brain-derived neurotrophic factor (BDNF) signaling through receptor tyrosine kinase B (TrkB) is required for cued fear memory consolidation and extinction. Although BDNF is primarily secreted from glutamatergic neurons, TrkB is expressed by other genetically defined cells whose contributions to the behavioral effects of BDNF remain poorly understood. Parvalbumin (PV)-positive interneurons, which are highly enriched in TrkB, are emerging as key regulators of fear memory expression. We therefore hypothesized that activity-dependent BDNF signaling in PV-interneurons may modulate emotional learning. To test this hypothesis, we utilized the LoxP/Cre system for conditional deletion of TrkB in PV-positive cells to examine the impact of cell-autonomous BDNF signaling on Pavlovian fear conditioning and extinction. However, behavioral abnormalities indicative of vestibular dysfunction precluded the use of homozygous conditional knockouts in tests of higher cognitive functioning. While vestibular dysfunction was apparent in both sexes, female conditional knockouts exhibited an exacerbated phenotype, including extreme motor hyperactivity and circling behavior, compared to their male littermates. Heterozygous conditional knockouts were spared of vestibular dysfunction. While fear memory consolidation was unaffected in heterozygotes of both sexes, males exhibited impaired extinction consolidation compared to their littermate controls. Our findings complement evidence from human and rodent studies suggesting that BDNF signaling promotes consolidation of extinction and point to PV-positive neurons as a discrete population that mediates these effects in a sex-specific manner.

  10. 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.

  11. 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.

  12. Positioning of centrioles is a conserved readout of Frizzled planar cell polarity signalling.

    Science.gov (United States)

    Carvajal-Gonzalez, Jose Maria; Roman, Angel-Carlos; Mlodzik, Marek

    2016-03-29

    Planar cell polarity (PCP) signalling is a well-conserved developmental pathway regulating cellular orientation during development. An evolutionarily conserved pathway readout is not established and, moreover, it is thought that PCP mediated cellular responses are tissue-specific. A key PCP function in vertebrates is to regulate coordinated centriole/cilia positioning, a function that has not been associated with PCP in Drosophila. Here we report instructive input of Frizzled-PCP (Fz/PCP) signalling into polarized centriole positioning in Drosophila wings. We show that centrioles are polarized in pupal wing cells as a readout of PCP signalling, with both gain and loss-of-function Fz/PCP signalling affecting centriole polarization. Importantly, loss or gain of centrioles does not affect Fz/PCP establishment, implicating centriolar positioning as a conserved PCP-readout, likely downstream of PCP-regulated actin polymerization. Together with vertebrate data, these results suggest a unifying model of centriole/cilia positioning as a common downstream effect of PCP signalling from flies to mammals.

  13. Differences in immune cell function between tuberculosis positive and negative Asian elephants.

    Science.gov (United States)

    Landolfi, Jennifer A; Miller, Michele; Maddox, Carol; Zuckermann, Federico; Langan, Jennifer N; Terio, Karen A

    2014-07-01

    Tuberculosis is an important health concern for Asian elephant (Elephas maximus) populations worldwide, however, mechanisms underlying susceptibility to Mycobacterium tuberculosis are unknown. Proliferative responses assessed via brominated uridine incorporation and cytokine expression measured by real-time RT-PCR were evaluated in peripheral blood mononuclear cell (PBMC) cultures from 8 tuberculosis negative and 8 positive Asian elephants. Cultures were stimulated with Mycobacterium bovis purified protein derivative (PPD-B), M. tuberculosis culture filtrate protein (CFP)-10, and Mycobacterium avium PPD (PPD-A). Following stimulation with PPD-B, proliferation was higher (α = 0.005) in positive samples; no significant differences were detected following CFP-10 or PPD-A stimulation. Tumor necrosis factor (TNF)-α, interleukin (IL)-12, and interferon (IFN)-γ expression was greater in samples from positive elephants following stimulation with PPD-B (α = 0.025) and CFP-10 (α = 0.025 TNF-α and IL-12; α = 0.005 IFN-γ). Stimulation with PPD-A also produced enhanced IL-12 expression in positive samples (α = 0.025). Findings suggested that differences in immune cell function exist between tuberculosis positive and negative elephants. Proliferative responses and expression of TNF-α, IL-12, and IFN-γ in response to stimulation with PPD-B and CFP-10 differ between tuberculosis positive and negative elephants, suggesting these parameters may be important to tuberculosis immunopathogenesis in this species.

  14. 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.

  15. BANA-Positive Plaque Samples Are Associated with Oral Hygiene Practices and Not CD4+ T Cell Counts in HIV-Positive Patients

    Directory of Open Access Journals (Sweden)

    Cathy Nisha John

    2012-01-01

    Full Text Available Background. The “red complex” microorganisms, namely, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia are considered as potential pathogens causing HIV-associated periodontal diseases. Moreover, it has been recognized that an association exists between CD4+ T cell counts and periodontal disease progression. Objective. To establish whether CD4+ T cell counts or oral hygiene plays a greater role in producing BANA-positive results in HIV-associated periodontal disease. Materials and Methods. One hundred and twenty HIV-positive patients participated in the study, and their CD4+ T cell counts were obtained from their medical records. The six Ramfjord teeth were used for evaluating periodontal clinical indices and subgingival plaque sampling. BANA test was used for the detection and prevalence of the “red complex” bacteria in plaque samples. Results. A majority of 69.17% HIV-positive patients were BANA-positive. No significant associations were found between BANA and CD4+ T cell counts. A highly significant association was found between BANA with probing depth and clinical attachment level (P≤0.0001 and between BANA and the use of interdental aids (P=0.0168. Conclusion. HIV-associated periodontal diseases are strongly related to oral hygiene practices rather than the effect of CD4+ T cell counts, and the use of interdental aids was marked as a significant predictor of BANA-negative plaque samples.

  16. 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.

  17. 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.

  18. Genistein inhibits the proliferation of human HER2-positive cancer cells by downregulating HER2 receptor

    Directory of Open Access Journals (Sweden)

    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

  19. Positive feedback between Dia1, LARG, and RhoA regulates cell morphology and invasion.

    Science.gov (United States)

    Kitzing, Thomas M; Sahadevan, Arul S; Brandt, Dominique T; Knieling, Helga; Hannemann, Sebastian; Fackler, Oliver T; Grosshans, Jörg; Grosse, Robert

    2007-06-15

    The RhoA-effector Dia1 controls actin-dependent processes such as cytokinesis, SRF transcriptional activity, and cell motility. Dia1 polymerizes actin through its formin homology (FH) 2 domain. Here we show that Dia1 acts upstream of RhoA independently of its effects on actin assembly. Dia1 binds to the leukemia-associated Rho-GEF (LARG) through RhoA-dependent release of Dia1 autoinhibition. The FH2 domain stimulates the guanine nucleotide exchange activity of LARG in vitro. Our results reveal that Dia1 is necessary for LPA-stimulated Rho/ROCK signaling and bleb-associated cancer cell invasion. Thus, Dia1-dependent RhoA activation constitutes a positive feedback mechanism to modulate cell behavior.

  20. Acellbia® and Mabtera® are recognize CD20-positive cells with equal efficiency

    Directory of Open Access Journals (Sweden)

    V. A. Misyurin

    2015-01-01

    Full Text Available In present study were compared characteristics of rituximab produced by Hoffmann–La Roche (Mabtera® and first domestic biosimilar Acellbia® from Biocad Company. Concentration of protein was measured using Bradford, s method. According to our results, protein concentration in formulations was the same. We electrophoresed formulations in denaturing conditions. Protein from formulations was denatured into fragments. Heavy and light chains of immunoglobulin were observed in gel. Finally, we performed flow cytometry where rituximab was used as primary antibody to detect CD20-positive B-cells of patients with B-cell chronic lymphocytic leukemia. Both Mabtera® and Acellbia® recognized the same number of cells. Thus, assays performed in vitro submitted identity of Mabtera® and Acellbia® characteristics.

  1. 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.

  2. 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

  3. Proteome Analysis of Human Follicular Thyroid Cancer Cells Exposed to the Random Positioning Machine.

    Science.gov (United States)

    Bauer, Johann; Kopp, Sascha; Schlagberger, Elisabeth Maria; Grosse, Jirka; Sahana, Jayashree; Riwaldt, Stefan; Wehland, Markus; Luetzenberg, Ronald; Infanger, Manfred; Grimm, Daniela

    2017-03-03

    Several years ago, we detected the formation of multicellular spheroids in experiments with human thyroid cancer cells cultured on the Random Positioning Machine (RPM), a ground-based model to simulate microgravity by continuously changing the orientation of samples. Since then, we have studied cellular mechanisms triggering the cells to leave a monolayer and aggregate to spheroids. Our work focused on spheroid-related changes in gene expression patterns, in protein concentrations, and in factors secreted to the culture supernatant during the period when growth is altered. We detected that factors inducing angiogenesis, the composition of integrins, the density of the cell monolayer exposed to microgravity, the enhanced production of caveolin-1, and the nuclear factor kappa B p65 could play a role during spheroid formation in thyroid cancer cells. In this study, we performed a deep proteome analysis on FTC-133 thyroid cancer cells cultured under conditions designed to encourage or discourage spheroid formation. The experiments revealed more than 5900 proteins. Their evaluation confirmed and explained the observations mentioned above. In addition, we learned that FTC-133 cells growing in monolayers or in spheroids after RPM-exposure incorporate vinculin, paxillin, focal adhesion kinase 1, and adenine diphosphate (ADP)-ribosylation factor 6 in different ways into the focal adhesion complex.

  4. Fisetin inhibits migration, invasion and epithelial-mesenchymal transition of LMP1-positive nasopharyngeal carcinoma cells.

    Science.gov (United States)

    Li, Rong; Zhao, Yinhai; Chen, Jin; Shao, Songjun; Zhang, Xiujuan

    2014-02-01

    Fisetin (3,3',4',7-tetrahydroxyflavone) has been reported to possess certain anticancer properties. It may inhibit tumor cell proliferation, metastasis and induce apoptosis. However, the effects of fisetin in preventing the metastasis of nasopharyngeal carcinoma (NPC) cells remain to be determined. The epithelial-mesenchymal transition (EMT) is involved in several metastatic malignancies including NPC. It has been reported that the Epstein-Barr virus latent membrane protein-1 (LMP1) induced EMT and is associated with the metastasis of NPC. The aim of this study was to examine the effects of fisetin in preventing the migration and invasion of LMP1-expressing NPC cells (CNE1-LMP1 cells), as well as to investigate whether fisetin may inhibit the molecular changes associated with EMT induced by LMP1. The investigation demonstrated that fisetin suppressed the migration and invasion of CNE1-LMP1 cells under non-cytotoxic concentrations. Fisetin inhibited molecular changes associated with EMT induced by LMP1, upregulated the epithelial marker, E-cadherin protein, and downregulated the mesenchymal marker, vimentin protein, levels. Fisetin also significantly reduced the levels of Twist protein, an EMT regulator. The investigation suggested that fisetin inhibits the migration and invasion of LMP1-positive NPC cells, and the molecular mechanism involves fisetin reversing the EMT induced by LMP1 and downregulates the expression of Twist. This study indicated that fisetin serves as a potential candidate for the treatment of cancer metastasis.

  5. 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.

  6. Effect of Weight Reduction on Cardiovascular Risk Factors and CD34-positive Cells in Circulation

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    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.

  7. 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

  8. Glycone-rich Soy Isoflavone Extracts Promote Estrogen Receptor Positive Breast Cancer Cell Growth.

    Science.gov (United States)

    Johnson, Kailee A; Vemuri, Sravan; Alsahafi, Sameerh; Castillo, Rudy; Cheriyath, Venugopalan

    2016-01-01

    Due to the association of hormone replacement therapy (HRT) with breast cancer risk, estrogenically active soy isoflavones are considered as an HRT alternative to alleviate menopausal symptoms. However, several recent reports challenged the health benefits of soy isoflavones and associated them with breast cancer promotion. While glyconic isoflavones are the major constituents of soybean seeds, due to their low cell permeability, they are considered to be biologically inactive. The glyconic isoflavones may exert their effects on membrane-bound estrogen receptors or could be converted to aglycones by extracellular β-glucosidases. Therefore, we hypothesized that despite their low cell permeability, soybean cultivars with high glyconic isoflavones may promote breast cancer cell growth. To test this, composition and estrogenic activity of isoflavones from 54 commercial soybean cultivars were determined. Soybean seeds produced in identical climate and growth conditions were used to minimize the effects of extraneous factors on isoflavone profile and concentrations. The glyconic daidzin concentration negatively correlated with genistin and with other aglycones. Relative to control, isoflavone extracts from 51 cultivars were estrogenic and promoted the growth of estrogen receptor positive (ER+) breast cancer cell line MCF-7 from 1.14 to 4.59 folds and other three cultivars slightly reduced the growth. Among these, extracts from three cultivars were highly estrogenic and promoted MCF-7 cell growth by 2.59-4.64 folds (Psoy isoflavone extracts may exert estrogenic effects and promote ER+ breast cancer growth.

  9. 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.

  10. BRAF V600E-Positive Multisite Langerhans Cell Histiocytosis in a Preterm Neonate

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    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.

  11. 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

  12. 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.

  13. 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.

  14. Serum-Free Generation of Multipotent Mesoderm (Kdr-positive) Progenitor Cells in Mouse Embryonic Stem Cells For Functional Genomics Screening

    OpenAIRE

    2012-01-01

    This unit describes a robust protocol for producing multipotent Kdr-expressing mesoderm progenitor cells in serum-free conditions and functional genomics screening using these cells. Kdr-positive cells are known to be able to differentiate into a wide array of mesoderm derivatives including, vascular endothelial cells, cardiomyocytes, hematopietic progenitors and smooth muscle cells. The efficient generation of such progenitor cells is of particular interest because it permits subsequent step...

  15. Nests with numerous SOX10 and MiTF-positive cells in lichenoid inflammation: pseudomelanocytic nests or authentic melanocytic proliferation?

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    Silva, Claudine Yap; Goldberg, Lynne J; Mahalingam, Meera; Bhawan, Jag; Wolpowitz, Deon

    2011-10-01

    Pseudomelanocytic nests in the setting of lichenoid inflammation can mimic atypical melanocytic proliferations. Both melanocytic and cytokeratin immunohistochemical stains may be utilized to differentiate these entities. Unlike true melanocytic nests, pseudomelanocytic nests contain Melanoma Antigen Recognized by T-cells 1 (MART-1)/ Melan-A-positive cells and cells positive for pan-cytokeratins, CD3 and/or CD68. Recently, rare (1-2 cells/nest) microphthalmia- associated transcription factor (MiTF)-positive cells were also reported in pseudomelanocytic nests. We present a 48-year-old man with a 2 × 3 cm violaceous to hyperpigmented, non-blanching, polygonal patch on the neck. Histopathology showed focal epidermal atrophy, irregularly distributed junctional nests and a lichenoid infiltrate with colloid bodies. Immunoperoxidase studies revealed occasional pan-cytokeratin and MART-1/Melan-A-positive staining in nests as well as focal S-100 protein-positive cells. Importantly, the majority of nests showed numerous cells positive for MiTF and SOX10 (>2 cells/nest and some the majority of cells). This combined staining pattern confounds the above-described immunohistochemical distinction between pseudo and true melanocytic nests. Clinically felt to represent unilateral lichen planus pigmentosus/erythema dyschromicum perstans and not malignant melanoma in situ, this lesion highlights the importance of clinicopathologic correlation and suggests either a new melanocytic entity or a novel pattern of benign melanocytic reorganization in a subset of lichenoid dermatitides.

  16. 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.

  17. Satellite Cells CD44 Positive Drive Muscle Regeneration in Osteoarthritis Patients

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    Manuel Scimeca

    2015-01-01

    Full Text Available Age-related bone diseases, such as osteoarthritis and osteoporosis, are strongly associated with sarcopenia and muscle fiber atrophy. In this study, we analyzed muscle biopsies in order to demonstrate that, in osteoarthritis patients, both osteophytes formation and regenerative properties of muscle stem cells are related to the same factors. In particular, thanks to immunohistochemistry, transmission electron microscopy, and immunogold labeling we investigated the role of BMP-2 in muscle stem cells activity. In patients with osteoarthritis both immunohistochemistry and transmission electron microscopy allowed us to note a higher number of CD44 positive satellite muscle cells forming syncytium. Moreover, the perinuclear and cytoplasmic expression of BMP-2 assessed by in situ molecular characterization of satellite cells syncytia suggest a very strict correlation between BMP-2 expression and muscle regeneration capability. Summing up, the higher BMP-2 expression in osteoarthritic patients could explain the increased bone mineral density as well as decreased muscle atrophy in osteoarthrosic patients. In conclusion, our results suggest that the control of physiological BMP-2 balance between bone and muscle tissues may be considered as a potential pharmacological target in bone-muscle related pathology.

  18. [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.

  19. ALK-positive anaplastic large cell lymphoma with soft tissue involvement in a young woman

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    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

  20. Assembly and positioning of actomyosin rings by contractility and planar cell polarity.

    Science.gov (United States)

    Sehring, Ivonne M; Recho, Pierre; Denker, Elsa; Kourakis, Matthew; Mathiesen, Birthe; Hannezo, Edouard; Dong, Bo; Jiang, Di

    2015-10-21

    The actomyosin cytoskeleton is a primary force-generating mechanism in morphogenesis, thus a robust spatial control of cytoskeletal positioning is essential. In this report, we demonstrate that actomyosin contractility and planar cell polarity (PCP) interact in post-mitotic Ciona notochord cells to self-assemble and reposition actomyosin rings, which play an essential role for cell elongation. Intriguingly, rings always form at the cells' anterior edge before migrating towards the center as contractility increases, reflecting a novel dynamical property of the cortex. Our drug and genetic manipulations uncover a tug-of-war between contractility, which localizes cortical flows toward the equator and PCP, which tries to reposition them. We develop a simple model of the physical forces underlying this tug-of-war, which quantitatively reproduces our results. We thus propose a quantitative framework for dissecting the relative contribution of contractility and PCP to the self-assembly and repositioning of cytoskeletal structures, which should be applicable to other morphogenetic events.

  1. 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.

  2. Effects of diverse dietary phytoestrogens on cell growth, cell cycle and apoptosis in estrogen-receptor-positive breast cancer cells.

    Science.gov (United States)

    Sakamoto, Takako; Horiguchi, Hyogo; Oguma, Etsuko; Kayama, Fujio

    2010-09-01

    Phytoestrogens have attracted attention as being safer alternatives to hormone replacement therapy (HRT) and as chemopreventive reagents for breast cancer because dietary soy isoflavone intake has been correlated with reduction in risk. To identify safe and effective phytoestrogen candidates for HRT and breast cancer prevention, we investigated the effects of daidzein, genistein, coumestrol, resveratrol and glycitein on cell growth, cell cycle, cyclin D1 expression, apoptosis, Bcl-2/Bax expression ratio and p53-dependent or NF-kappaB-dependent transcriptional activity in MCF-7 breast cancer cells. Phytoestrogens, except for glycitein, significantly enhanced estrogen-response-element-dependent transcriptional activity up to a level similar to that of 17beta-estradiol (E(2)). E(2) increased cell growth significantly, coumestrol increased cell growth moderately, and resveratrol and glycitein reduced cell growth. Phytoestrogens, except for glycitein, stimulated the promotion of cells to G(1)/S transition in cell cycle analysis, similar to E(2). This stimulation was accompanied by transient up-regulation of cyclin D1. While genistein, resveratrol and glycitein all increased apoptosis and reduced the Bcl-2/Bax ratio, resveratrol reduced this ratio more than either genistein or glycitein. Moreover, resveratrol significantly enhanced p53-dependent transcriptional activity, but slightly reduced NF-kappaB-dependent transcriptional activity. On knockdown analysis, genistein, resveratrol and glycitein all reduced the Bcl-2/Bax ratio in the presence of apoptosis-inducing stimuli, and estrogen receptor (ER) alpha silencing had no effect on these reductions. In contrast, in the absence of apoptosis-inducing stimuli, only resveratrol reduced the ratio, and ERalpha silencing abolished this reduction. Thus, resveratrol might be the most promising candidate for HRT and chemoprevention of breast cancer due to its estrogenic activity and high antitumor activity.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. The scaffold protein MEK Partner 1 is required for the survival of estrogen receptor positive breast cancer cells.

    Science.gov (United States)

    Marina, Mihaela; Wang, Limin; Conrad, Susan E

    2012-07-09

    MEK Partner 1 (MP1 or MAPKSP1) is a scaffold protein that has been reported to function in multiple signaling pathways, including the ERK, PAK and mTORC pathways. Several of these pathways influence the biology of breast cancer, but MP1's functional significance in breast cancer cells has not been investigated. In this report, we demonstrate a requirement for MP1 expression in estrogen receptor (ER) positive breast cancer cells. MP1 is widely expressed in both ER-positive and negative breast cancer cell lines, and in non-tumorigenic mammary epithelial cell lines. However, inhibition of its expression using siRNA duplexes resulted in detachment and apoptosis of several ER-positive breast cancer cell lines, but not ER-negative breast cancer cells or non-tumorigenic mammary epithelial cells. Inhibition of MP1 expression in ER-positive MCF-7 cells did not affect ERK activity, but resulted in reduced Akt1 activity and reduced ER expression and activity. Inhibition of ER expression did not result in cell death, suggesting that decreased ER expression is not the cause of cell death. In contrast, pharmacological inhibition of PI3K signaling did induce cell death in MCF-7 cells, and expression of a constitutively active form of Akt1 partially rescued the cell death observed when the MP1 gene was silenced in these cells. Together, these results suggest that MP1 is required for pro-survival signaling from the PI3K/Akt pathway in ER-positive breast cancer cells.

  11. 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...

  12. 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.

  13. 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.

  14. Photoperiodic variation in CD45-positive cells and cell proliferation in the mediobasal hypothalamus of the Soay sheep.

    Science.gov (United States)

    Hazlerigg, D G; Wyse, C A; Dardente, H; Hanon, E A; Lincoln, G A

    2013-05-01

    The Earth's solar orbit induces annual climatic changes challenging to survival. Many animals have evolved to cope with seasonal variability through compensatory annual changes in their physiology and behavior, which involve innate long-term timing and photoperiodic synchronization to anticipate the environmental seasonal cycles. Here we considered the potential involvement of cyclical histogenesis in seasonal timing mechanisms in the sheep. Adult Soay rams were established in three distinctive seasonal states by controlled photoperiod exposure. A first group, representing the condition in late spring (long-photoperiod [LP] group), was taken indoors in May and exposed to 4 wks of 16 h light/day (LP). A second group was exposed to 20 wks of LP to establish a late-summer/long-day refractory condition (LPR group). A third group of animals was brought indoors in August and exposed to 4 wks of LP followed by 4 wks of 8 h light/day (short photoperiod [SP]) to establish an autumn-like condition (SP group). At the end of these regimes, we injected 5-bromo-2-deoxyuridine (BrdU), and animals were killed 24 h or 4 wks later. When BrdU was administered 24 h before death, more BrdU-immunopositive cells were detected in the hilus of the hippocampus in LP compared with SP animals, indicative of a higher proliferation rate. When BrdU was administered 4 wks before death, more BrdU-positive cells were detected in the hippocampus under LP, compared with SP, indicating increased cell survival. These mitotic cells were occasionally seen to adopt a neuronal phenotype in the hippocampus, but not in the hypothalamus. Approximately 10% of BrdU-positive cells in the basal hypothalamus coexpressed the pan-leukocytic marker CD45, and showed morphological features and regional distribution consistent with ameboid microglia. Increased numbers of these cells were detected in the region of the median eminence and tuberoinfundibular sulcus of animals kept in SP compared with LP or LPR. These data

  15. Management of Recurrent and Metastatic HPV-Positive Oropharyngeal Squamous Cell Carcinoma after Transoral Robotic Surgery.

    Science.gov (United States)

    Sims, John R; Van Abel, Kathryn; Martin, Eliot J; Lohse, Christine M; Price, Daniel L; Olsen, Kerry D; Moore, Eric J

    2017-03-01

    Objective To describe management and oncologic outcomes for patients who develop locoregional recurrence (LRR) or distant metastasis (DM) following transoral robotic surgery for human papilloma virus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC). Study Design Case series with chart review. Setting Tertiary care referral center. Subjects and Methods A total of 286 patients with HPV-positive OPSCC who underwent transoral robotic surgery-based treatment from May 2007 to May 2015. Results Of 286 patients (12.2%), 35 met inclusion criteria. Of these, 19 experienced an LRR and 16 developed a DM; 2 patients with LRR subsequently developed DM. In those patients with an LRR, 79% had T1/T2 tumors, and 47% had N0/N1 nodal disease, compared with 75% and 6% in the DM group, respectively. The median time to LRR or DM was 0.6 years (interquartile range [IQR], 0.4-1.0) and 1.8 years (IQR, 1.0-2.1), respectively. Salvage treatment with intent to cure was attempted in 23 patients (16 LRR, 7 DM). The median time from LRR or DM to last follow-up for the 18 patients who were still alive after salvage was 1.9 years (IQR, 0.4-3.8; range, 7 days-6.2 years). Estimated cancer-specific survival rates at 3 years following intent-to-cure treatment were 63% (95% CI, 39-100; number still at risk, 5) in the LRR group and 100% (95% CI, 100-100; number still at risk, 2) in the DM group. Conclusion Overall, LRR and DM for HPV-positive OPSCC following transoral robotic surgery-based therapy are infrequent. In our subset of patients who underwent intent-to-cure treatment, cancer-specific survival rates were favorable. Therefore, aggressive salvage treatment for LRR and DM for HPV-positive OPSCC should be recommended for appropriate candidates.

  16. 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.

  17. 荧光原位杂交三探针联合检测在儿童急性B淋巴细胞白血病诊断中的应用%Application of three-probe fluorescence in situ hybridization panel in the diagnosis of pediatric B cell acute lymphoblastic leukemia

    Institute of Scientific and Technical Information of China (English)

    范婧; 李承文; 赵佳炜; 贡金英; 郑迎春; 汝昆

    2014-01-01

    Objective To evaluate the three-probe fluorescence in situ hybridization (FISH) panel in the diagnosis of pediatric B cell acute lymphoblastic leukemia (B-ALL).Methods Three-probe (TEL-AML1,BCR-ABL and MLL) FISH and conventional cytogenetic analysis were performed in 207 children with B-ALL.Results In 207 B-ALL children,the three-probe FISH panel assay showed that 151 cases carried genetic aberrancies with a positive rate of 72.9%,including 44 cases with typical positive signal patterns and 148 cases with atypical signal patterns (among them 41 cases have multiprobe abberancy).The conventional cytogenetic analysis detected 53 cases chromosomal abnormality with a positive rate of 25.6%.Conclusion The detection rate of genetic abnormalities in newly-diagnosed pediatric B-ALL could be significantly improved by using three-probe FISH panel upon conventional cytogenetic analysis.%目的 探讨荧光原位杂交(FISH)三探针联合检测在儿童急性B淋巴细胞白血病(B-ALL)诊断中的应用价值.方法 对207例B-ALL患儿全部行三探针(TEL-AML1、BCR-ABL、MLL) FISH联合检测和常规染色体核型分析.结果 207例B-ALL患儿经FISH三探针联合检测,151例(72.9%)检出异常,包括典型阳性信号44例、非典型信号148例(其中41例为多探针同时检出异常).常规染色体核型分析技术检出染色体异常53例(25.6%).结论 对初诊的儿童B-ALL患者采用三探针联合FISH检测可以有效提高遗传学异常检出率.

  18. 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.

  19. 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.

  20. CAR-pNK Cell Immunotherapy in CD7 Positive Leukemia and Lymphoma

    Science.gov (United States)

    2016-12-04

    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

  1. Ascorbic acid kills Epstein-Barr virus positive Burkitt lymphoma cells and Epstein-Barr virus transformed B-cells in vitro, but not in vivo.

    Science.gov (United States)

    Shatzer, Amber N; Espey, Michael Graham; Chavez, Mayra; Tu, Hongbin; Levine, Mark; Cohen, Jeffrey I

    2013-05-01

    Ascorbic acid has been shown to kill various cancer cell lines at pharmacologic concentrations. We found that Epstein-Barr virus (EBV)-positive Burkitt lymphoma (BL) cells were more susceptible to ascorbic acid-induced cell killing than EBV-negative BL cells or EBV-transformed lymphoblastoid cells (LCLs). Ascorbic acid did not induce apoptosis in any of the tested cells but did induce the production of reactive oxygen species and cell death. Previously, we showed that bortezomib, a proteasome inhibitor, induces cell death in LCLs and EBV-positive BL cells. We found that ascorbic acid is strongly antagonistic for bortezomib-induced cell death in LCLs and EBV-positive BL cells. Finally, ascorbic acid did not prolong survival of severe combined immunodefiency mice inoculated with LCLs either intraperitoneally or subcutaneously. Thus, while ascorbic acid was highly effective at killing EBV-positive BL cells and LCLs in vitro, it antagonized cell killing by bortezomib and was ineffective in an animal model.

  2. 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.

  3. Resistance to hypoxia-induced, BNIP3-mediated cell death contributes to an increase in a CD133-positive cell population in human glioblastomas in vitro.

    Science.gov (United States)

    Kahlert, Ulf Dietrich; Maciaczyk, Donata; Dai, Fangping; Claus, Rainer; Firat, Elke; Doostkam, Soroush; Bogiel, Tomasz; Carro, Maria Stella; Döbrössy, Mate; Herold-Mende, Christel; Niedermann, Gabriele; Prinz, Marco; Nikkhah, Guido; Maciaczyk, Jaroslaw

    2012-12-01

    In addition to intrinsic regulatory mechanisms, brain tumor stemlike cells (BTSCs), a small subpopulation of malignant glial tumor-derived cells, are influenced by environmental factors. Previous reports showed that lowering oxygen tension induced an increase of BTSCs expressing CD133 and other stem cell-related genes and more pronounced clonogenic capacity in vitro. We investigated the mechanisms responsible for hypoxia-dependent induction of CD133-positive BTSCs in glioblastomas. We confirmed that cultures exposed to lowered oxygen levels showed a severalfold increase of CD133-positive BTSCs. Both the increase of CD133-positive cells and deceleration of the growth kinetics were reversible after transfer to normoxic conditions. Exposure to hypoxia induced BNIP3 (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3)-dependent apoptosis preferentially in CD133-negative cells. In contrast, CD133-positive cells proved to be more resistant to hypoxia-induced programmed cell death. Application of the demethylating agent 5'-azacitidine resulted in an increase of BNIP3 expression levels in CD133-positive cells. Thus, epigenetic modifications led to their better survival in lowered oxygen tension. Moreover, the, hypoxia-induced increase of CD133-positive cells was inhibited after 5'-azacitidine treatment. These results suggest the possible efficacy of a novel therapy for glioblastoma focused on eradication of BTSCs by modifications of epigenetic regulation of gene expression.

  4. Chaperone-rich tumor cell lysate-mediated activation of antigen-presenting cells resists regulatory T cell suppression.

    Science.gov (United States)

    Larmonier, Nicolas; Cantrell, Jessica; Lacasse, Collin; Li, Gang; Janikashvili, Nona; Situ, Elaine; Sepassi, Marjan; Andreansky, Samita; Katsanis, Emmanuel

    2008-04-01

    CD4(+)CD25(+) regulatory T lymphocytes (Tregs) critically contribute to the mechanisms of cancer-induced tolerance. These cells suppress anti-tumoral CD8(+) and CD4(+) T lymphocytes and can also restrain the function of APCs. We have previously documented the immunostimulatory effects of a chaperone-rich cell lysate (CRCL) anti-cancer vaccine. Tumor-derived CRCL induces tumor immunity in vivo, partly by promoting dendritic cell (DC) and macrophage activation. In the current study, we evaluated the effects of CD4(+)CD25(+)forkhead box P3(+) Tregs isolated from mice bearing 12B1 bcr-abl(+) leukemia on DC and macrophages that had been activated by 12B1-derived CRCL. CRCL-activated DC and macrophages resisted Treg suppression, as the production of proinflammatory cytokines, the activation of transcription factor NF-kappaB, and their immunostimulatory potential was unaffected by Tregs. Our results thus highlight CRCL as a powerful adjuvant endowed with the capacity to overcome tumor-induced Treg-inhibitory effects on APCs.

  5. 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.

  6. Effects of gram-positive microorganisms and their products on in vivo survival of hemopoietic clonogenic cells.

    Science.gov (United States)

    Proskuryakov, S Ya; Konoplyannikov, A G; Konoplyannikova, O A; Tsyb, A F; Logunov, D Yu; Naroditsky, B S; Gintsburg, A L

    2008-04-01

    The effects of gram-positive bacterial strains (Lactobacillus acidophilus and Lactobacillus rhamnosus) and their subcellular components on the survival of hemopoietic clonogenic cells were evaluated by the formation of endogenous splenic colonies. The effects of these preparations on NO production were studied by the spin-trap paramagnetic resonance spectroscopy. Bacterial preparations from gram-positive bacteria stimulated survival of hemopoietic clonogenic cells, but did not induce NO production in contrast to E. coli LPS.

  7. 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.

  8. The Mitotic Spindle in the One-Cell C. elegans Embryo Is Positioned with High Precision and Stability

    Science.gov (United States)

    Pécréaux, Jacques; Redemann, Stefanie; Alayan, Zahraa; Mercat, Benjamin; Pastezeur, Sylvain; Garzon-Coral, Carlos; Hyman, Anthony A.; Howard, Jonathon

    2016-10-01

    Precise positioning of the mitotic spindle is important for specifying the plane of cell division, which in turn determines how the cytoplasmic contents are partitioned into the daughter cells, and how the daughters are positioned within the tissue. During metaphase in the early C. elegans embryo, the spindle is aligned and centered on the anterior-posterior axis by a microtubule-dependent machinery that exerts restoring forces when the spindle is displaced from the center. To investigate the accuracy and stability of centering, we tracked the position and orientation of the mitotic spindle during the first cell division with high temporal and spatial resolution. We found that the precision is remarkably high: the cell-to-cell variation in the transverse position of the center of the spindle during metaphase, as measured by the standard deviation, was only 1.5% of the length of the short axis of the cell. Spindle position is also very stable: the standard deviation of the fluctuations in transverse spindle position during metaphase was only 0.5% of the short axis of the cell. Assuming that stability is limited by fluctuations in the number of independent motor elements such as microtubules or dyneins underlying the centering machinery, we infer that the number is on the order of one thousand, consistent with the several thousand of astral microtubules in these cells. Astral microtubules grow out from the two spindle poles, make contact with the cell cortex, and then shrink back shortly thereafter. The high stability of centering can be accounted for quantitatively if, while making contact with the cortex, the astral microtubules buckle as they exert compressive, pushing forces. We thus propose that the large number of microtubules in the asters provides a highly precise mechanism for positioning the spindle during metaphase while assembly is completed prior to the onset of anaphase.

  9. Identification of orexin A- and orexin type 2 receptor-positive cells in the gastrointestinal tract of neonatal dogs

    Directory of Open Access Journals (Sweden)

    C Dall’Aglio

    2009-08-01

    Full Text Available The presence and distribution of cells positive to orexin A (OXA and to orexin type 2 receptor (OX2R were investigated in the gastrointestinal tract of neonatal dogs by means of immunohistochemical techniques. The orexin A-positive cells were identified with some of the endocrine cells in the stomach and in the duodenum; they were both of the open and closed type and were lacking in the large intestine. In the stomach, a large subset of orexin A-positive cells also showed gastrin-like immunoreactivity while, in the duodenum, many of them seemed to store serotonin. The orexin type 2 receptor-positive cells were evidenced all along the gastrointestinal tract examined, also in the large intestine, and they showed the same morphological characteristics as those positive to orexin A. Moreover, the immunohistochemical techniques revealed intense positivity for both orexin A and orexin type 2 receptor in the neurons and fibers of the enteric nervous system. A large subset of orexin A-positive neurons seemed to store substance P.

  10. 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.

  11. Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria.

    Directory of Open Access Journals (Sweden)

    Maria João Catalão

    Full Text Available 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.

  12. EGFR mutation positive stage IV non-small-cell lung cancer : Treatment beyond progression

    Directory of Open Access Journals (Sweden)

    Katrijn eVan Assche

    2014-12-01

    Full Text Available Non-small-cell lung cancer (NSCLC is the leading cause of death from cancer for both men en women. Chemotherapy is the mainstay of treatment in advanced disease, but is only marginally effective. In about 30% of patients with advanced NSCLC in East Asia and in 10-15% in Western countries, EGFR mutations are found. In this population, first-line treatment with the tyrosine kinase inhibitors (TKI erlotinib, gefitinib or afatinib is recommended. The treatment beyond progression is less well-defined. In this paper we present 3 patients, EGFR mutation positive, with local progression after an initial treatment with TKI. These patients were treated with local radiotherapy. TKI was temporarily stopped and restarted after radiotherapy. We give an overview of the literature and discuss the different treatment options in case of progression after TKI: TKI continuation with or without chemotherapy, TKI continuation with local therapy, alternative dosing or switch to next-generation TKI or combination therapy. There are different options for treatment beyond progression in EGFR mutation positive metastatic NSCLC, but the optimal strategy is still to be defined. Further research on this topic is ongoing.

  13. 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.

  14. Detection of circulating prostate tumor cells: alternative spliced variant of PSM induced false-positive result.

    Science.gov (United States)

    Hisatomi, Hisashi; Nagao, Kumi; Kawakita, Mutsuji; Matsuda, Tadashi; Hirata, Hiroyuki; Yamamoto, Shigeki; Nakamoto, Takaaki; Harasawa, Hiroshi; Kaneko, Noboru; Hikiji, Kazumasa; Tsukada, Yutaka

    2002-11-01

    RT-nested PCR has been introduced as a highly specific and sensitive assay method to detect the prostate-specific membrane antigen (PSM) mRNA in peripheral blood. However, appreciable percentages of false-positive cases have been reported. Additionally, primer sets reported previously could not discriminate between PSM and PSM', an alternatively spliced variant, mRNA. These isoforms can be produced from a single gene. Switches in alternative splicing patterns are often controlled with strict cell-type or developmental-stage specificity. Therefore, it is most important to discriminate between PSM mRNA and PSM' mRNA. Using our highly specific primer sets, PSM mRNA was detected in 3 of 24 peripheral blood samples of normal male volunteers (12.5%) and was not detected in peripheral blood of 11 normal female volunteers. PSM' mRNA was detected in 5 of 24 peripheral blood samples of normal male volunteers (20.8%) and in 4 of 11 of normal female volunteers (36.4%). PSM' mRNA induced false-positive results, it is important for genetic diagnosis of prostate cancer to discriminate between PSM and PSM' using our primer sets with high specificity. The advances in the uniquely designed primer sets may allow researchers to detect a real PSM mRNA without PSM' mRNA.

  15. 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.

  16. 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.

  17. The positive effects of Midazolam on functional activity of white rat brain cells in conditions of halothane anesthesia.

    Science.gov (United States)

    Vadachkoria, Z; Dzidziguri, L; Bakuradze, E; Dzidziguri, D

    2009-05-01

    For the elimination of postoperative complications, which are evident in infants with congenital palatine and upper lip cleft after halothane anesthesia during standard premedication (Atropine, Dimedrol) the usage of agents of benzodiazepine group with anti-hypoxic effects is advised. The latter may modulate the blocked ion channels by neuromediators, which are activated by GABA and GABA receptor function. The neuro-protective ability of halothane is demonstrated. To reveal the mechanisms of positive effects using benzodiazepine group for premedication we have investigated the effects of midazolam of brain cell functional activity of experimental animals (white rats) in conditions of halothane anesthesia. For the estimation of white rat brain cells functional activity the nuclear transcriptional activity was studied (based on the intensity of [14C]-UTF inclusion), also the glutamic acid decarboxilaze activity (GAD65/67) using immunohistochemistry. It is estimated that halothane inhibits the transcription in rat brain cells. During midazolam premedication the halothane inhibitory effect on RNA synthesis is not revealed. After an hour of pseudo-operation halothane also induces proved decrease of quantity of GAD65/67 positive cells in CA3 hippocampal field. At the same time the quantity of similar cells are increased in CA1 field. The increase of GAD65/67 positive cells in CA1 is more evident during midazolam premedication. Based on the data received we can conclude that the positive effect of midazolam results in increase of GAD65/67 positive cells in CA1 hippocampal field.

  18. 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.

  19. RB inactivation in keratin 18 positive thymic epithelial cells promotes non-cell autonomous T cell hyperproliferation in genetically engineered mice

    Science.gov (United States)

    Song, Yurong; Sullivan, Teresa; Klarmann, Kimberly; Gilbert, Debra; O’Sullivan, T. Norene; Lu, Lucy; Wang, Sophie; Haines, Diana C.; Van Dyke, Terry; Keller, Jonathan R.

    2017-01-01

    Thymic epithelial cells (TEC), as part of thymic stroma, provide essential growth factors/cytokines and self-antigens to support T cell development and selection. Deletion of Rb family proteins in adult thymic stroma leads to T cell hyperplasia in vivo. To determine whether deletion of Rb specifically in keratin (K) 18 positive TEC was sufficient for thymocyte hyperplasia, we conditionally inactivated Rb and its family members p107 and p130 in K18+ TEC in genetically engineered mice (TgK18GT121; K18 mice). We found that thymocyte hyperproliferation was induced in mice with Rb inactivation in K18+ TEC, while normal T cell development was maintained; suggesting that inactivation of Rb specifically in K18+ TEC was sufficient and responsible for the phenotype. Transplantation of wild type bone marrow cells into mice with Rb inactivation in K18+ TEC resulted in donor T lymphocyte hyperplasia confirming the non-cell autonomous requirement for Rb proteins in K18+ TEC in regulating T cell proliferation. Our data suggests that thymic epithelial cells play an important role in regulating lymphoid proliferation and thymus size. PMID:28158249

  20. Dendritic cells in uninfected infants born to hepatitis B virus-positive mothers.

    Science.gov (United States)

    Koumbi, Lemonica J; Papadopoulos, Nikolaos G; Anastassiadou, Vassiliki; Machaira, Maria; Kafetzis, Dimitris A; Papaevangelou, Vassiliki

    2010-07-01

    Plasmacytoid dendritic cells (pDCs) play a central role in antiviral immunity, detecting viruses via Toll-like receptors (TLR) and producing in response vast amounts of type I interferons (IFNs). Hepatitis B virus (HBV) causes chronic infection after vertical transmission. This study investigated whether an HBV-infected maternal environment might influence DC numbers and pDC function in uninfected infants. Blood was collected from inactive HBsAg carrier and control mothers and their infants at birth and 1 and 6 months of age. HBV DNA was measured in maternal and neonatal perinatal sera using real-time PCR. The circulating frequencies of myeloid DCs (mDCs) and pDCs were determined in the babies by flow cytometry. Peripheral blood mononuclear cells (PBMCs) and cord blood pDCs were stimulated with resiquimod, and alpha interferon (IFN-alpha) production and the pDC phenotype were assessed. The effect of the common-cold virus, rhinovirus (RV), on resiquimod stimulation was also determined. HBV DNA was detected in 62.3% of the mothers and 41% of their infants. DC numbers and pDC functions were similar between subjects and controls and were not correlated with maternal or neonatal viremia. RV infection did not induce pDC maturation until the age of 6 months, and it reduced TLR7-dependent resiquimod-induced IFN-alpha production similarly in both groups. Although the DC system is immature at birth, DCs of uninfected neonates of HBV-positive mothers are competent to initiate and maintain T-cell responses. RV is a weak inducer of IFN-alpha production until the age of 6 months and inhibits IFN-alpha responses triggered by the TLR7 pathway.