WorldWideScience

Sample records for bcr-abl positive cells

  1. The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias.

    Science.gov (United States)

    Kamitsuji, Y; Kuroda, J; Kimura, S; Toyokuni, S; Watanabe, K; Ashihara, E; Tanaka, H; Yui, Y; Watanabe, M; Matsubara, H; Mizushima, Y; Hiraumi, Y; Kawata, E; Yoshikawa, T; Maekawa, T; Nakahata, T; Adachi, S

    2008-11-01

    Bcr-Abl tyrosine kinase (TK) inhibitors are promising therapeutic agents for Bcr-Abl-positive (Bcr-Abl(+)) leukemias. Although they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we demonstrated that INNO-406, a second-generation Bcr-Abl TK inhibitor, induces programmed cell death (PCD) in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways. The latter pathways include caspase-independent apoptosis (CIA) and necrosis-like cell death (CIND), and the cell lines varied regarding which mechanism was elicited upon INNO-406 treatment. We also observed that the propensity toward CIA or CIND in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity. Cells that undergo CIND have a high apoptosome activity potential whereas cells that undergo CIA tend to have a lower potential. Moreover, we found that INNO-406 promotes autophagy. When autophagy was inhibited with chloroquine or gene knockdown of beclin1 by shRNA, INNO-406-induced cell death was enhanced, which indicates that the autophagic response of the tumor cells is protective. These findings suggest new insights into the biology and therapy of Bcr-Abl(+) leukemias.

  2. BCR-ABL-positive acute myeloid leukemia: a new entity? Analysis of clinical and molecular features.

    Science.gov (United States)

    Neuendorff, Nina Rosa; Burmeister, Thomas; Dörken, Bernd; Westermann, Jörg

    2016-08-01

    BCR-ABL-positive acute myeloid leukemia (AML) is a rare subtype of AML that is now included as a provisional entity in the 2016 revised WHO classification of myeloid malignancies. Since a clear distinction between de novo BCR-ABL+ AML and chronic myeloid leukemia (CML) blast crisis is challenging in many cases, the existence of de novo BCR-ABL+ AML has been a matter of debate for a long time. However, there is increasing evidence suggesting that BCR-ABL+ AML is in fact a distinct subgroup of AML. In this study, we analyzed all published cases since 1975 as well as cases from our institution in order to present common clinical and molecular features of this rare disease. Our analysis shows that BCR-ABL predominantly occurs in AML-NOS, CBF leukemia, and AML with myelodysplasia-related changes. The most common BCR-ABL transcripts (p190 and p210) are nearly equally distributed. Based on the analysis of published data, we provide a clinical algorithm for the initial differential diagnosis of BCR-ABL+ AML. The prognosis of BCR-ABL+ AML seems to depend on the cytogenetic and/or molecular background rather than on BCR-ABL itself. A therapy with tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib, or nilotinib is reasonable, but-due to a lack of systematic clinical data-their use cannot be routinely recommended in first-line therapy. Beyond first-line treatment of AML, the use of TKI remains an individual decision, both in combination with intensive chemotherapy and/or as a bridge to allogeneic stem cell transplantation. In each single case, potential benefits have to be weighed against potential risks.

  3. Regulation of hTERT by BCR-ABL at multiple levels in K562 cells

    International Nuclear Information System (INIS)

    Chai, Juin Hsien; Zhang, Yong; Tan, Wei Han; Chng, Wee Joo; Li, Baojie; Wang, Xueying

    2011-01-01

    The cytogenetic characteristic of Chronic Myeloid Leukemia (CML) is the formation of the Philadelphia chromosome gene product, BCR-ABL. Given that BCR-ABL is the specific target of Gleevec in CML treatment, we investigated the regulation of the catalytic component of telomerase, hTERT, by BCR-ABL at multiple levels in K562 cells. Molecular techniques such as over expression, knockdown, real-time PCR, immunoprecipitation, western blotting, reporter assay, confocal microscopy, telomerase assays and microarray were used to suggest that hTERT expression and activity is modulated by BCR-ABL at multiple levels. Our results suggest that BCR-ABL plays an important role in regulating hTERT in K562 (BCR-ABL positive human leukemia) cells. When Gleevec inhibited the tyrosine kinase activity of BCR-ABL, phosphorylation of hTERT was downregulated, therefore suggesting a positive correlation between BCR-ABL and hTERT. Gleevec treatment inhibited hTERT at mRNA level and significantly reduced telomerase activity (TA) in K562 cells, but not in HL60 or Jurkat cells (BCR-ABL negative cells). We also demonstrated that the transcription factor STAT5a plays a critical role in hTERT gene regulation in K562 cells. Knockdown of STAT5a, but not STAT5b, resulted in a marked downregulation of hTERT mRNA level, TA and hTERT protein level in K562 cells. Furthermore, translocation of hTERT from nucleoli to nucleoplasm was observed in K562 cells induced by Gleevec. Our data reveal that BCR-ABL can regulate TA at multiple levels, including transcription, post-translational level, and proper localization. Thus, suppression of cell growth and induction of apoptosis by Gleevec treatment may be partially due to TA inhibition. Additionally, we have identified STAT5a as critical mediator of the hTERT gene expression in BCR-ABL positive CML cells, suggesting that targeting STAT5a may be a promising therapeutic strategy for BCR-ABL positive CML patients

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

    Science.gov (United States)

    Synowiec, Ewelina; Hoser, Grazyna; Wojcik, Katarzyna; Pawlowska, Elzbieta; Skorski, Tomasz; Błasiak, Janusz

    2015-01-01

    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. PMID:26251899

  5. 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......) 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....

  6. Protein Kinase CK2: A Targetable BCR-ABL Partner in Philadelphia Positive Leukemias

    Directory of Open Access Journals (Sweden)

    Alessandro Morotti

    2015-01-01

    Full Text Available BCR-ABL-mediated leukemias, either Chronic Myeloid Leukemia (CML or Philadelphia positive Acute Lymphoblastic Leukemia (ALL, are the paradigm of targeted molecular therapy of cancer due to the impressive clinical responses obtained with BCR-ABL specific tyrosine kinase inhibitors (TKIs. However, BCR-ABL TKIs do not allow completely eradicating both CML and ALL. Furthermore, ALL therapy is associated with much worse responses to TKIs than those observed in CML. The identification of additional pathways that mediate BCR-ABL leukemogenesis is indeed mandatory to achieve synthetic lethality together with TKI. Here, we review the role of BCR-ABL/protein kinase CK2 interaction in BCR-ABL leukemias, with potentially relevant implications for therapy.

  7. Differentiation status of primary chronic myeloid leukemia cells affects sensitivity to BCR-ABL1 inhibitors.

    Science.gov (United States)

    Pietarinen, Paavo O; Eide, Christopher A; Ayuda-Durán, Pilar; Potdar, Swapnil; Kuusanmäki, Heikki; Andersson, Emma I; Mpindi, John P; Pemovska, Tea; Kontro, Mika; Heckman, Caroline A; Kallioniemi, Olli; Wennerberg, Krister; Hjorth-Hansen, Henrik; Druker, Brian J; Enserink, Jorrit M; Tyner, Jeffrey W; Mustjoki, Satu; Porkka, Kimmo

    2017-04-04

    Tyrosine kinase inhibitors (TKI) are the mainstay treatment of BCR-ABL1-positive leukemia and virtually all patients with chronic myeloid leukemia in chronic phase (CP CML) respond to TKI therapy. However, there is limited information on the cellular mechanisms of response and particularly on the effect of cell differentiation state to TKI sensitivity in vivo and ex vivo/in vitro. We used multiple, independent high-throughput drug sensitivity and resistance testing platforms that collectively evaluated 295 oncology compounds to characterize ex vivo drug response profiles of primary cells freshly collected from newly-diagnosed patients with BCR-ABL1-positive leukemia (n = 40) and healthy controls (n = 12). In contrast to the highly TKI-sensitive cells from blast phase CML and Philadelphia chromosome-positive acute lymphoblastic leukemia, primary CP CML cells were insensitive to TKI therapy ex vivo. Despite maintaining potent BCR-ABL1 inhibitory activity, ex vivo viability of cells was unaffected by TKIs. These findings were validated in two independent patient cohorts and analysis platforms. All CP CML patients under study responded to TKI therapy in vivo. When CP CML cells were sorted based on CD34 expression, the CD34-positive progenitor cells showed good sensitivity to TKIs, whereas the more mature CD34-negative cells were markedly less sensitive. Thus in CP CML, TKIs predominantly target the progenitor cell population while the differentiated leukemic cells (mostly cells from granulocytic series) are insensitive to BCR-ABL1 inhibition. These findings have implications for drug discovery in CP CML and indicate a fundamental biological difference between CP CML and advanced forms of BCR-ABL1-positive leukemia.

  8. 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.

    Science.gov (United States)

    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. Copyright © 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  9. An asymptomatic 61-year-old man with BCR-ABL-positive bone marrow following autologous transplantation for multiple myeloma

    Science.gov (United States)

    Roper, Nitin; Deangelo, Daniel; Kuo, Frank; Cin, Paola dal; Ghobrial, Irene; Aster, Jon C.

    2010-01-01

    A 61-year-old man treated with an autologous transplant for multiple myeloma was incidentally found to have a high level of BCR-ABL fusion gene-positive cells in his bone marrow. We describe the clinical decision-making process that led us to initiate therapy with imatinib, despite the absence of any clinical evidence of chronic myelogenous leukemia or other BCR-ABL associated hematologic malignancy. PMID:20730794

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

    Science.gov (United States)

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

    2005-03-01

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

  11. [TEC promoter mediates P210(bcr/abl) gene expression in BaF3 cells].

    Science.gov (United States)

    Zhu, Yu-Feng; Wang, Yuan-Zhan; Meng, Fan-Yi

    2012-06-01

    P210(bcr/abl) transgene mouse is a good model to research the chronic myelogenous leukemia (CML), but the P210(bcr/abl) gene has a lethal effect on embryogenesis if driven by the constitutive promoter. So, the use of promoter which induces the special expression in hematopoietic tissue is the key to construct CML transgenic mice. This study was purposed to investigate the TEC promoter mediated P210(bcr/abl) gene expression in BaF3 cells. The CMVie promotes of IRES2-eGFP vector was replaced with the -364-+22 domain of TEC promoter cloned from mouse genome, and the P210(bcr/abl) gene was inserted into the EcoR I site of TEC-IRES2-eGFP vector. Then, the constructed vector was transfected into the BaF3 cells and 293 cells respectively. The expression levels of eGFP gene and P210(bcr/abl) gene in BaF3 and 293 cells were detected. The results showed that with fluorescent microscopy and flow cytometry, the eGFP gene was found to be expressed in the BaF3 cells, the expression rate was 7.10%, 23.35%, 64.61% at 6, 24, 72 h respectively after transfection, but the fluorescence was not seen in 293 cells. A 372 bp fragment of BCR/ABL mRNA was amplified by RT-PCR in BaF3 cells, but not in 293 cells. It is concluded that the -364-+22 domain of TEC promoter can mediate high-effective and specific expression of related genes in hematopoietic tissue, which can be used to construct P210(bcr/abl) transgene mice model.

  12. Improved FRET Biosensor for the Measurement of BCR-ABL Activity in Chronic Myeloid Leukemia Cells.

    Science.gov (United States)

    Horiguchi, Mika; Fujioka, Mari; Kondo, Takeshi; Fujioka, Yoichiro; Li, Xinxin; Horiuchi, Kosui; O Satoh, Aya; Nepal, Prabha; Nishide, Shinya; Nanbo, Asuka; Teshima, Takanori; Ohba, Yusuke

    2017-02-02

    Although the co-development of companion diagnostics with molecular targeted drugs is desirable, truly efficient diagnostics are limited to diseases in which chromosomal translocations or overt mutations are clearly correlated with drug efficacy. Moreover, even for such diseases, few methods are available to predict whether drug administration is effective for each individual patient whose disease is expected to respond to the drug(s). We have previously developed a biosensor based on the principle of Förster resonance energy transfer to measure the activity of the tyrosine kinase BCR-ABL and its response to drug treatment in patient-derived chronic myeloid leukemia cells. The biosensor harbors CrkL, one of the major substrates of BCR-ABL, and is therefore named Pickles after phosphorylation indicator of CrkL en substrate. The efficacy of this technique as a clinical test has been demonstrated, but the number of cells available for analysis is limited in a case-dependent manner, owing to the cleavage of the biosensor in patient-derived leukemia cells. Here, we describe an improved biosensor with an amino acid substitution and a nuclear export signal being introduced. Of the two predicted cleavage positions in CrkL, the mutations inhibited one cleavage completely and the other cleavage partially, thus collectively increasing the number of cells available for drug evaluation. This improved version of the biosensor holds promise in the future development of companion diagnostics to predict responses to tyrosine kinase inhibitors in patients with chronic myeloid leukemia.

  13. 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......-up was 52 months from mutation detection and 26 months from transplantation. At transplantation, 51.5% of patients with chronic myeloid leukemia were in the chronic phase and 4.5% were in advanced phases. Median overall survival after transplantation was 10.3 months (range 5.7 months to not reached [ie......, still alive]) for those with chronic myeloid leukemia in the blast phase and 7.4 months (range 1.4 months to not reached [ie, still alive]) for those with Philadelphia chromosome-positive acute lymphoblastic leukemia but has not yet been reached for those in the chronic and accelerated phases of chronic...

  14. A non-radioactive assay for precise determination of intracellular levels of imatinib and its main metabolite in Bcr-Abl positive cells

    Czech Academy of Sciences Publication Activity Database

    Mlejnek, P.; Novák, Ondřej; Doležel, P.

    2011-01-01

    Roč. 83, č. 5 (2011), s. 1466-1471 ISSN 0039-9140 R&D Projects: GA ČR GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : K562 cells * P-glycoprotein * Multidrug resistance * N-desmethyl imatinib * CGP 74588 Subject RIV: EF - Botanics Impact factor: 3.794, year: 2011

  15. Quantification of BCR-ABL transcripts in peripheral blood cells and ...

    African Journals Online (AJOL)

    Purpose: To investigate the feasibility of using peripheral blood plasma samples as surrogates for blood cell sampling for quantification of breakpoint cluster region-Abelson oncogene (BCR-ABL) transcript levels to monitor treatment responses in chronic myeloid leukemia (CML) patients. Methods: Peripheral blood samples ...

  16. Interphase FISH for BCR-ABL1 rearrangement on neutrophils: A decisive tool to discriminate a lymphoid blast crisis of chronic myeloid leukemia from a de novo BCR-ABL1 positive acute lymphoblastic leukemia.

    Science.gov (United States)

    Balducci, Estelle; Loosveld, Marie; Rahal, Ilhem; Boudjarane, John; Alazard, Emilie; Missirian, Chantal; Lafage-Pochitaloff, Marina; Michel, Gérard; Zattara, Hélène

    2018-02-01

    Discrimination between lymphoid blast crisis of chronic myeloid leukemia (CML) and de novo BCR-ABL1 positive acute lymphoblastic leukemia (ALL) represents a diagnostic challenge because this distinction has a major incidence on the management of patients. Here, we report an uncommon pediatric case of ALL with cryptic ins(22;9)(q11;q34q34) and p190-type BCR-ABL1 transcript. We performed interphase fluorescence in situ hybridization (FISH) for BCR-ABL1 rearrangement on blood neutrophils, which was positive consistent with the diagnosis of lymphoid blast crisis of CML. This case illustrates the major interest of interphase FISH for BCR-ABL1 rearrangement on blood neutrophils as a decisive method to discriminate a lymphoid blast crisis of CML from a de novo BCR-ABL1 positive ALL. Copyright © 2017 John Wiley & Sons, Ltd.

  17. Unique amplification of BCR-ABL1 gene fusion in a case of T-cell acute lymphoblastic leukemia.

    Science.gov (United States)

    Koka, Rima; Bade, Najeebah A; Sausville, Edward A; Ning, Yi; Zou, Ying

    2017-01-01

    ABL1 gene translocations can be seen in precursor T-acute lymphoblastic leukemia (T-ALL). The typical translocation partner is the NUP214 gene. BCR-ABL translocations are relatively rare in this entity. Furthermore, while there have been unique patterns of amplification noted among the NUP214-ABL fusion genes, there have been few such reports among cases with BCR-ABL fusion genes. Here we report a unique case of a 44-year old patient with T-ALL in which the blasts demonstrated a derivative chromosome 9 involving a 9;22 translocation and a dicentric Philadelphia chromosome 22 with a homogeneously staining region at the interface of the 9;22 translocation, leading to BCR-ABL1 gene amplification. Fluorescence in-situ hybridization (FISH) showed abnormal BCR/ABL1 fusions with the BCR-ABL1 gene amplification in 48% of the interphase cells analyzed. The translocation was confirmed by SNP array. We present a novel derivative chromosome 9 that shows BCR-ABL gene fusion along with a dicentric Philadelphia chromosome 22 with BCR-ABL1 gene amplification. This is a unique pattern of BCR-ABL fusion which has never been described in T-ALL. It is significant that the patient responded to standard treatment with the CALGB 10403 protocol and supplementation with a tyrosine kinase inhibitor. Identification of additional patients with this pattern of BCR-ABL fusion will allow for enhanced risk assessment and prognostication.

  18. c-Myb and its target Bmi1 are required for p190BCR/ABL leukemogenesis in mouse and human cells.

    Science.gov (United States)

    Waldron, T; De Dominici, M; Soliera, A R; Audia, A; Iacobucci, I; Lonetti, A; Martinelli, G; Zhang, Y; Martinez, R; Hyslop, T; Bender, T P; Calabretta, B

    2012-04-01

    Expression of c-Myb is required for normal hematopoiesis and for proliferation of myeloid leukemia blasts and a subset of T-cell leukemia, but its role in B-cell leukemogenesis is unknown. We tested the role of c-Myb in p190(BCR/ABL)-dependent B-cell leukemia in mice transplanted with p190(BCR/ABL)-transduced marrow cells with a c-Myb allele (Myb(f/d)) and in double transgenic p190(BCR/ABL)/Myb(w/d) mice. In both models, loss of a c-Myb allele caused a less aggressive B-cell leukemia. In p190(BCR/ABL)-expressing human B-cell leukemia lines, knockdown of c-Myb expression suppressed proliferation and colony formation. Compared with c-Myb(w/f) cells, expression of Bmi1, a regulator of stem cell proliferation and maintenance, was decreased in pre-B cells from Myb(w/d) p190(BCR/ABL) transgenic mice. Ectopic expression of a mutant c-Myb or Bmi1 enhanced the proliferation and colony formation of Myb(w/d) p190(BCR/ABL) B-cells; by contrast, Bmi1 downregulation inhibited colony formation of p190(BCR/ABL)-expressing murine B cells and human B-cell leukemia lines. Moreover, c-Myb interacted with a segment of the human Bmi1 promoter and enhanced its activity. In blasts from 19 Ph(1) adult acute lymphoblastic leukemia patients, levels of c-Myb and Bmi1 showed a positive correlation. Together, these findings support the existence of a c-Myb-Bmi1 transcription-regulatory pathway required for p190(BCR/ABL) leukemogenesis.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Chronic Myeloid Leukemia 2011: Successes, challenges, and strategies – Proceedings of the 5th Annual BCR-ABL1 positive and BCR-ABL1 negative myeloproliferative neoplasms workshop

    Science.gov (United States)

    Mughal, Tariq I; Radich, Jerald P; Van Etten, Richard A.; Quintás-Cardama, Alfonso; Skorski, Tomasz; Ravandi, Farhad; DeAngelo, Daniel J.; Gambacorti-Passerini, Carlo; Martinelli, Giovanni; Tefferi, Ayalew

    2012-01-01

    This report is based on the presentations and discussions at the 5th annual BCR-ABL1 positive and BCR-ABL1 negative myeloproliferative neoplasms (MPN) workshop, which took place immediately following the 52nd American Society of Hematology (ASH) meeting in Orlando, Florida on December 7th-8th, 2011. Relevant data which was presented at the ASH meeting as well as all other recent publications were presented and discussed at the workshop. This report covers front-line therapies of BCR-ABL1-positive leukemias, in addition to addressing some topical biological, pre-clinical and clinical issues, such as new insights into genomic instability and resistance to tyrosine kinase inhibitors (TKIs), risk stratification and optimizing molecular monitoring. A report pertaining to the new therapies and other pertinent preclinical and clinical issues in the BCR-ABL1 negative MPNs is published separately. PMID:21850662

  1. Combining the ABL1 kinase inhibitor ponatinib and the histone deacetylase inhibitor vorinostat: a potential treatment for BCR-ABL-positive leukemia.

    Science.gov (United States)

    Okabe, Seiichi; Tauchi, Tetsuzo; Kimura, Shinya; Maekawa, Taira; Kitahara, Toshihiko; Tanaka, Yoko; Ohyashiki, Kazuma

    2014-01-01

    Resistance to imatinib (Gleevec®) in cancer cells is frequently because of acquired point mutations in the kinase domain of BCR-ABL. Ponatinib, also known as AP24534, is an oral multi-targeted tyrosine kinase inhibitor (TKI), and it has been investigated in a pivotal phase 2 clinical trial. The histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid) has been evaluated for its significant clinical activity in hematological malignancies. Thus, treatments combining ABL TKIs with additional drugs may be a promising strategy in the treatment of leukemia. In the current study, we analyzed the efficacy of ponatinib and vorinostat treatment by using BCR-ABL-positive cell lines. Treatment with ponatinib for 72 h inhibited cell growth and induced apoptosis in K562 cells in a dose-dependent manner. We found that ponatinib potently inhibited the growth of Ba/F3 cells ectopically expressing BCR-ABL T315I mutation. Upon BCR-ABL phosphorylation, Crk-L was decreased, and poly (ADP-ribose) polymerase (PARP) was activated in a dose-dependent manner. Combined treatment of Ba/F3 T315I mutant cells with vorinostat and ponatinib resulted in significantly increased cytotoxicity. Additionally, the intracellular signaling of ponatinib and vorinostat was examined. Caspase 3 and PARP activation increased after combination treatment with ponatinib and vorinostat. Moreover, an increase in the phosphorylation levels of γH2A.X was observed. Previously established ponatinib-resistant Ba/F3 cells were also resistant to imatinib, nilotinib, and dasatinib. We investigated the difference in the efficacy of ponatinib and vorinostat by using ponatinib-resistant Ba/F3 cells. Combined treatment of ponatinib-resistant cells with ponatinib and vorinostat caused a significant increase in cytotoxicity. Thus, combined administration of ponatinib and vorinostat may be a powerful strategy against BCR-ABL mutant cells and could enhance the cytotoxic effects of ponatinib in those BCR-ABL

  2. 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.

  3. Expression of p210 BCR/ABl increases hematopoietic progenitor cell radiosensitivity

    International Nuclear Information System (INIS)

    Santucci, M.A.; Anklesaria, P.; Das, I.J.; Sakakeeny, M.A.; FitzGerald, T.J.; Greenberger, J.S.; Laneuville, P.

    1993-01-01

    The cytogenetic finding of the Ph1+ chromosome and its molecular biologic marker bcr/abl gene rearrangement in cells from patients with chronic myeloid leukemia are associated with a proliferative advantage of the Ph1+ clone in vivo. Although the transition to the acute terminal phase or blastic crisis is often associated with additional cytogenetic abnormalities, the molecular events which correlate the initial cytogenetic lesion with the terminal phase are poorly understood. Defective cellular DNA repair capacity is often associated with chromosomal instability, increased mutation frequency, and biologic alterations. The authors tested whether the protein product of the bcr/abl translocation (p210) could alter DNA repair after gamma-irradiation of murine cell lines expressing the bcr/abl cDNA. The 32D cl 3 parent, 32D cl 3 pYN (containing the control vector plasmid) and each of two sources of 32D cl 3 cells expressing p210 cDNA (32D-PC1 cell line and 32D-LG7 subclone) showed a D 0 of 1.62, 1.57, 1.16, and 1.27 Gy, respectively. Thus, expression of the p210 product induced a significant increase in radiosensitivity at the clinically relevant radiation therapy dose-rate. The increased radiosensitivity of p210-expressing cells persisted if cells were held before plating in a density-inhibited state for 8 hr after gamma-irradiation, indicating little effect on the repair of potentially lethal gamma-irradiation damage. The IL-3 dependent parent 32D cl 3 cells demonstrated programmed cell death in the absence of growth factor or following gamma-irradiation to 200 cGy. Expression of p210 cDNA in the 32D-PC1 and 32D-LG7 subclones abrogated IL-3 requirement of these cell lines and inhibited gamma-irradiation induced programmed cell death. These data suggest a role for p210 in amplifying gamma-irradiation DNA damage or broadly inhibiting DNA repair, conditions that may stimulate further cytogenetic alterations in hematopoietic cells. 43 refs., 3 figs., 1 tab

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-01-01

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

  5. The Role of Mitochondrial DNA Damage and Repair in the Resistance of BCR/ABL-Expressing Cells to Tyrosine Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Janusz Blasiak

    2013-08-01

    Full Text Available Chronic myeloid leukemia (CML is a hematological malignancy that arises from the transformation of stem hematopoietic cells by the fusion oncogene BCR/ABL and subsequent clonal expansion of BCR/ABL-positive progenitor leukemic cells. The BCR/ABL protein displays a constitutively increased tyrosine kinase activity that alters many regulatory pathways, leading to uncontrolled growth, impaired differentiation and increased resistance to apoptosis featured by leukemic cells. Current CML therapy is based on tyrosine kinase inhibitors (TKIs, primarily imatinib, which induce apoptosis in leukemic cells. However, some patients show primary resistance to TKIs while others develop it in the course of therapy. In both cases, resistance may be underlined by perturbations in apoptotic signaling in leukemic cells. As mitochondria may play an important role in such signaling, alteration in mitochondrial metabolism may change resistance to pro-apoptotic action of TKIs in BCR/ABL-positive cells. Because BCR/ABL may induce reactive oxygen species and unfaithful DNA repair, it may affect the stability of mitochondrial DNA, influencing mitochondrial apoptotic signaling and in this way change the sensitivity of CML cells to TKIs. Moreover, cancer cells, including BCR/ABL-positive cells, show an increased level of glucose metabolism, resulting from the shift from oxidative phosphorylation to glycolysis to supply ATP for extensive proliferation. Enhanced level of glycolysis may be associated with TKI resistance and requires change in the expression of several genes regulated mostly by hypoxia-inducible factor-1α, HIF-1α. Such regulation may be associated with the impaired mitochondrial respiratory system in CML cells. In summary, mitochondria and mitochondria-associated molecules and pathways may be attractive targets to overcome TKI resistance in CML.

  6. The role of mitochondrial DNA damage and repair in the resistance of BCR/ABL-expressing cells to tyrosine kinase inhibitors.

    Science.gov (United States)

    Glowacki, Sylwester; Synowiec, Ewelina; Blasiak, Janusz

    2013-08-07

    Chronic myeloid leukemia (CML) is a hematological malignancy that arises from the transformation of stem hematopoietic cells by the fusion oncogene BCR/ABL and subsequent clonal expansion of BCR/ABL-positive progenitor leukemic cells. The BCR/ABL protein displays a constitutively increased tyrosine kinase activity that alters many regulatory pathways, leading to uncontrolled growth, impaired differentiation and increased resistance to apoptosis featured by leukemic cells. Current CML therapy is based on tyrosine kinase inhibitors (TKIs), primarily imatinib, which induce apoptosis in leukemic cells. However, some patients show primary resistance to TKIs while others develop it in the course of therapy. In both cases, resistance may be underlined by perturbations in apoptotic signaling in leukemic cells. As mitochondria may play an important role in such signaling, alteration in mitochondrial metabolism may change resistance to pro-apoptotic action of TKIs in BCR/ABL-positive cells. Because BCR/ABL may induce reactive oxygen species and unfaithful DNA repair, it may affect the stability of mitochondrial DNA, influencing mitochondrial apoptotic signaling and in this way change the sensitivity of CML cells to TKIs. Moreover, cancer cells, including BCR/ABL-positive cells, show an increased level of glucose metabolism, resulting from the shift from oxidative phosphorylation to glycolysis to supply ATP for extensive proliferation. Enhanced level of glycolysis may be associated with TKI resistance and requires change in the expression of several genes regulated mostly by hypoxia-inducible factor-1α, HIF-1α. Such regulation may be associated with the impaired mitochondrial respiratory system in CML cells. In summary, mitochondria and mitochondria-associated molecules and pathways may be attractive targets to overcome TKI resistance in CML.

  7. [Construction of Eukaryotic Expression Vector of siRNA Specific for BCR/ABL Fusion Gene and Its Effects on K562 Cells].

    Science.gov (United States)

    Li, Ming; Wang, Bao-Lin; Wang, Li-Na; Xi, Ya-Ming

    2016-12-01

    To construct eukaryotic expression vector of siRNA specific for BCR/ABL and to investigate the effect of recombinant plasmid on BCR/ABL and P210 protein expression in K562 cells. siRNA(small interfering RNA)was designed according to the Tuschl's principle of Ai-based medicine, and was converted into cDNA coding expression of shRNA(small hairpin RNAs)of siRNA for BCR/ABL fusion gene. The cDNA was synthesized and inserted into plasmid pTER. The pTER117 and pTER363 of recombinant plasmid being eukaryotic expression vector was controlled by the H1 promoter of RNA polymerase III, and identified by the restriction map and the sequence analysis. The recombinant plasmid did not only have the screening resisting antibiotics, its expression but also are induced by tetracycline (tet). After steadily transfection into K562 cells by Lipofectamine, their positive mono-cell clones being resistant to Zeocin were isolated. TaqMan real-time quantitative RT-PCR (RQ-PCR) and Western blot respectively detected expression of BCR/ABL mRNA and P210 protein. Trypaum blue dying was used to analyze the proliferation of K562 cells. Cell apoptosis was observed by flow cytometer. the recombinant plasmid was steadily transfected into K562 cells by Lipofectamine 2000, Their positive mono-cell clones being resistant to Zeocin were isolated. The proliferation of K562 cells were remarkably inhibited by the recombinant plasmid induced gene expression by tetracycline. Tetracycline induced its expression for 48 h and 72 h. pTER117, pTER363 decreased the mRNA level of BCR/ABL 90%, 82% and 91.5%, 84%, respectively, P210 protein were almost measured in K562 cells. FCM analysis showed that the recombinant plasmid induced apoptosis in K562 cells, the apoptosis rate were respectively 34.4%, 58.1% in K562 cells treated by pTER117 for 48 h and 72 h, apoptosis rate were 31.8%, 54.6% by pTER363, but the control groups did not show these effects on K562 cells. The siRNA eukaryotic expression vector against BCR/ABL

  8. Deregulated expression of Cdc6 as BCR/ABL-dependent survival factor in chronic myeloid leukemia cells.

    Science.gov (United States)

    Zhang, Jia-Hua; He, Yan-Li; Zhu, Rui; Du, Wen; Xiao, Jun-Hua

    2017-06-01

    Chronic myeloid leukemia is characterized by the presence of the reciprocal translocation t(9;22) and the BCR/ABL oncogene. The BCR/ABL oncogene activates multiple signaling pathways and involves the dysregulation of oncogenes during the progression of chronic myeloid leukemia. The cell division cycle protein 6, an essential regulator of DNA replication, is elevated in some human cancer cells. However, the expression of cell division cycle protein 6 in chronic myeloid leukemia and the underlying regulatory mechanism remain to be elucidated. In this study, our data showed that cell division cycle protein 6 expression was significantly upregulated in primary chronic myeloid leukemia cells and the chronic myeloid leukemia cell line K562 cells, as compared to the normal bone marrow mononuclear cells. BCR/ABL kinase inhibitor STI571 or BCR/ABL small interfering RNA could significantly downregulate cell division cycle protein 6 messenger RNA expression in K562 cells. Moreover, phosphoinositide 3-kinase/AKT pathway inhibitor LY294002 and Janus kinase/signal transducer and activator of transcription pathway inhibitor AG490 could downregulate cell division cycle protein 6 expression in K562 cells, but not RAS/mitogen-activated protein kinase pathway inhibitor PD98059 had such effect. Cell division cycle protein 6 gene silencing by small interfering RNA effectively resulted in decrease of proliferation, increase of apoptosis, and arrest of cell cycle in K562 cells. These findings have demonstrated that cell division cycle protein 6 overexpression may contribute to the high proliferation and low apoptosis in chronic myeloid leukemia cells and can be regulated by BCR/ABL signal transduction through downstream phosphoinositide 3-kinase/Akt and Janus kinase/signal transducer and activator of transcription pathways, suggesting cell division cycle protein 6 as a potential therapeutic target in chronic myeloid leukemia.

  9. Disruption of Survivin in K562 cells elevates telomerase activity and protects cells against apoptosis induced by the Bcr-abl kinase inhibitor STI571

    OpenAIRE

    Wang, Zhanxiang; Pelus, Louis M.

    2008-01-01

    The Bcr-abl kinase inhibitor STI571 produces clinical responses in most patients with Chronic Myeloid Leukemia (CML); however, development of resistance limits utility. One strategy to overcome STI571 resistance is to decrease the level/activity of Bcr-abl. We reported that disruption of the anti-apoptotic protein Survivin promoted STI571-induced apoptosis in Bcr-abl+ K562 cells, through caspase-dependent Bcr-abl degradation. To investigate the utility of Survivin disruption in drug-resistant...

  10. BCR-ABL1-positive chronic myeloid leukemia emerging in a patient with secondary myelofibrosis harboring the JAK2-V617F mutation.

    Science.gov (United States)

    Amemiya, Ayae; Ito, Yoshikazu; Ishibashi, Yasunori; Saito, Yuu; Katagiri, Seiichiro; Suguro, Tamiko; Asano, Michiyo; Yoshizawa, Seiichiro; Akahane, Daigo; Tanaka, Yuko; Fujimoto, Hiroaki; Okabe, Seiichi; Gotoh, Moritaka; Tauchi, Tetsuzo; Ohyashiki, Kazuma

    2017-01-01

    A 53-year-old woman with a 27-year history of myeloproliferative neoplasms came to our hospital because of a marked white blood cell count increase and progressive anemia. Clinical examination demonstrated positivity for BCR-ABL1 and JAK2-V617F mutations. She was given a diagnosis of chronic myeloid leukemia. Using the international scale, a molecular response (MR) 4.5 was achieved after treatment with dasatinib, despite the persistence of marked splenomegaly. The pathological findings of myelofibrosis were demonstrated by bone marrow biopsy. After stopping dasatinib administration for 4 years and 5 months, treatment with ruxolitinib was started. Five months later, the size of her spleen was reduced. We speculated that translocation of BCR-ABL1 might have occurred in a sub-clone of the JAK2-V617F mutated tumor clone.

  11. Direct transcriptional regulation of Bim by FoxO3a mediates STI571-induced apoptosis in Bcr-Abl-expressing cells

    NARCIS (Netherlands)

    Essafi, A.; Mattos, S.F. de; Hassen, Y.A.M.; Soeiro, I.; Mufti, G.J.; Thomas, N.S.B.; Medema, R.H.; Lam, E.W.-F.

    2005-01-01

    In this study, we have used the human BV173 and the mouse BaF3/Bcr-Abl-expressing cell lines as model systems to investigate the molecular mechanisms whereby STI571 and FoxO3a regulate Bim expression and apoptosis. FoxO3a lies downstream of Bcr-Abl signalling and is constitutively

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

    Science.gov (United States)

    2010-01-01

    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 further drug development to

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

    Directory of Open Access Journals (Sweden)

    Axel Weber

    2015-03-01

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

  14. Bach2 regulates aberrant activation of B cell in systemic lupus erythematosus and can be negatively regulated by BCR-ABL/PI3K.

    Science.gov (United States)

    Zhu, Zhengwei; Yang, Chao; Wen, Leilei; Liu, Lu; Zuo, Xianbo; Zhou, Fusheng; Gao, Jinping; Zheng, Xiaodong; Shi, Yinjuan; Zhu, Caihong; Liang, Bo; Yin, Xianyong; Wang, Wenjun; Cheng, Hui; Shen, Songke; Tang, Xianfa; Tang, Huayang; Sun, Liangdan; Zhang, Anping; Yang, Sen; Cui, Yong; Zhang, Xuejun; Sheng, Yujun

    2018-04-01

    This study was aimed to explore the effect of Bach2 on B cells in systemic lupus erythematosus (SLE), as well as the underlying mechanisms. Expression of Bach2, phosphorylated-Bach2 (p-Bach2), Akt, p-Akt and BCR-ABL (p210) in B cells isolated from SLE patients and the healthy persons were assessed by Western blot. Immunofluorescence staining was performed to assess the localization of Bach2 in B cells. Enzyme-linked immunosorbent assay (ELISA) was employed to detect IgG produced by B cells. Cell counting kit-8 (CCK-8) and Annexin-V FITC/PI double staining assay were adopted to evaluate cell proliferation and apoptosis in B cells, respectively. Compared to the healthy controls, Bach2, p-Akt and p210 were significantly decreased, while nuclear translocation of Bach2, IgG, CD40 and CD86 obviously up-regulated in B cells from SLE patients. Bach2 significantly inhibited the proliferation, promoted apoptosis of B cells from SLE patients, whereas BCR-ABL dramatically reversed cell changes induced by Bach2. Besides, BCR-ABL also inhibited nuclear translocation of Bach2 in B cells from SLE patients. Further, LY294002 treatment had no effect on decreased expression of Bach2 induced by BCR-ABL, but significantly eliminated BCR-ABL-induced phosphorylation of Bach2 and restored reduced nuclear translocation of Bach2 induced by BCR-ABL in B cells from SLE. Bach2 may play a suppressive role in B cells from SLE, and BCR-ABL may inhibit the nuclear translocation of Bach2 via serine phosphorylation through the PI3K pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Quantification of BCR-ABL transcripts in peripheral blood cells and ...

    African Journals Online (AJOL)

    The disease status of CML patients was categorized according to the European Leukemia Net [10]. RNA extraction procedure,. Peripheral blood samples of study subjects were collected in ethylenediaminetetraacetate (EDTA) tubes. Plasma was separated from blood cells by centrifugation at 1200 x g for 10 minutes. RNA.

  16. Dehydrocostus Lactone Suppresses Proliferation of Human Chronic Myeloid Leukemia Cells Through Bcr/Abl-JAK/STAT Signaling Pathways.

    Science.gov (United States)

    Cai, Hong; Qin, Xiaosong; Yang, Chunhui

    2017-10-01

    This study evaluates the anticancer effects of dehydrocostus lactone, a plant-derived sesquiterpene lactone, on human chronic myeloid leukemia cells. Dehydrocostus lactone significantly inhibits cell proliferation by inducing cells to undergo cell cycle arrest, apoptosis, and differentiation. Dehydrocostus lactone suppresses the expression of cyclin B1, cyclin A, cyclin E, cyclin-dependent kinase 2 (CDK2), and cyclin-dependent kinase 1 (CDK1) and increases p21 expression, resulting in S-G2/M phase arrest in K562 cells. Dehydrocostus lactone also induces apoptosis by increasing the generation of reactive oxygen species (ROS), disruption of mitochondrial membrane potential (MMP), and modulating the protein levels of Bcl-2 family members. We also found that dehydrocostus lactone significantly inhibits the phosphorylation expression of Bcr/Abl, STAT5, JAK2, and STAT3 and downstream molecules including p-CrkL, Mcl-1, Bcl-XL, and Bcl-2 proteins in K562 cells. At a low concentration, dehydrocostus lactone significantly increased CD11b and CD14 expression on the surface of K562 cells, and induced cells to differentiate into monocytes or mature macrophages. Taken together, this study provides new insight into the molecular mechanisms of dehydrocostus lactone actions that may contribute to the chemoprevention of chronic myeloid leukemia. J. Cell. Biochem. 118: 3381-3390, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

    DEFF Research Database (Denmark)

    Järås, Marcus; Johnels, Petra; Agerstam, Helena

    2009-01-01

    OBJECTIVE: The P190 and P210 BCR/ABL1 fusion genes are mainly associated with different types of hematologic malignancies, but it is presently unclear whether they are functionally different following expression in primitive human hematopoietic cells. MATERIALS AND METHODS: We investigated...... and systematically compared the effects of retroviral P190 BCR/ABL1 and P210 BCR/ABL1 expression on cell proliferation, differentiation, and global gene expression in human CD34(+) cells from cord blood. RESULTS: Expression of either P190 BCR/ABL1 or P210 BCR/ABL1 resulted in expansion of erythroid cells...... and stimulated erythropoietin-independent burst-forming unit-erythroid colony formation. By using a lentiviral anti-signal transducer and activator of transcription 5 (STAT5) short-hairpin RNA, we found that both P190 BCR/ABL1- and P210 BCR/ABL1-induced erythroid cell expansion were STAT5-dependent. Under...

  18. 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.

  19. [Construction of genetically modified dendritic cell vaccine expressing bcr/abl fusion gene and inducing specific cytotoxic T lymphocytes to kill K562 cells in vitro].

    Science.gov (United States)

    Wang, Wen-Wen; Huang, Ren-Wei; Hu, Yuan; Li, Xu-Dong; Wang, Dong-Ning; He, Yi; Liu, Jia-Jun

    2009-06-01

    Specific immunological effect mediated by T lymphocytes plays an important role in treating chronic myelocytic leukemia (CML). Dendritic cells (DCs)-based immunotherapy has become popular in treating tumors. This study was to construct DC vaccines by transducing with replication-defective recombinant adenoviruses expressing bcr/abl fusion gene of CML, observe the lethal effects of specific cytotoxic T lymphocytes (CTLs) triggered by genetically modified DC vaccines expressing bcr/abl fusion gene against K562 cells in vitro. DNA fragment of bcr/abl fusion gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) to construct a recombinant adenovirus vector and produce recombinant adenoviruses. DCs were induced from peripheral blood monocytes in vitro, and transfected with recombinant adenoviruses or pulsed with peptide to induce specific CTLs. The lethal effect of CTLs against leukemic K562 cells in vitro was observed. We successfully constructed the replication-defective recombinant adenoviral vector expressing bcr/abl fusion gene. The recombinant adenoviruses we produced had a high virus titer of 2.0 x 10(10) pfu/mL. Transfection efficiency of DCs in vitro was 50%-60%. DC vaccines expressing bcr/abl fusion gene were successfully prepared and used to induce specific CTLs. With effector:target cell ratios of 40:1 and 20:1, the killing rates of K562 cells by CTLs were (47.6+/-4.7)% and (47.5+/-1.6)% in genetically modified DCs group, (25.8+/-4.4)% and (24.6+/-6.3)% in peptide-pulsed DCs group, and were (5.7+/-1.3)% and (4.5+/-1.6)% in control DCs group. The differences between every two groups were significant (all Pfusion gene has a stronger contribution than peptide-pulsed DCs in triggering specific CTLs against K562 cells.

  20. Philadelphia-positive acute lymphoblastic leukemia patients already harbor BCR-ABL kinase domain mutations at low levels at the time of diagnosis

    Science.gov (United States)

    Soverini, Simona; Vitale, Antonella; Poerio, Angela; Gnani, Alessandra; Colarossi, Sabrina; Iacobucci, Ilaria; Cimino, Giuseppe; Elia, Loredana; Lonetti, Annalisa; Vignetti, Marco; Paolini, Stefania; Meloni, Giovanna; di Maio, Valeria; Papayannidis, Cristina; Amabile, Marilina; Guarini, Anna; Baccarani, Michele; Martinelli, Giovanni; Foà, Robin

    2011-01-01

    Background In patients with Philadelphia-positive acute lymphoblastic leukemia, resistance to treatment with tyrosine kinase inhibitors is frequent and most often associated with the development of point mutations in the BCR-ABL kinase domain. We aimed to assess: (i) in how many patients BCR-ABL kinase domain mutations are already detectable at relatively low levels at the time of diagnosis, and (ii) whether mutation detection correlates with subsequent response to therapy. Design and Methods We retrospectively analyzed samples collected at diagnosis from 15 patients with Philadelphia-positive acute lymphoblastic leukemia who subsequently received tyrosine kinase inhibitor therapy (dasatinib) by cloning the BCR-ABL kinase domain in a bacterial vector and sequencing 200 independent clones per sample. Results Mutations at relatively low levels (2–4 clones out of 200) could be detected in all patients – eight who relapsed and seven who achieved persistent remission. Each patient had evidence of two to eight different mutations, the majority of which have never been reported in association with resistance to tyrosine kinase inhibitors. In two patients out of six who relapsed because of a mutation, the mutation (a T315I) was already detectable in a few clones at the time of diagnosis. On the other hand, a patient who was found to harbor an F317L mutation is in persistent remission on dasatinib. Conclusions Our results suggest that the BCR-ABL kinase domain is prone to randomly accumulate point mutations in Philadelphia-positive acute lymphoblastic leukemia, although the presence of these mutations in a relatively small leukemic subclone does not always preclude a primary response to tyrosine kinase inhibitors. PMID:21193419

  1. PLK1 inhibitors synergistically potentiate HDAC inhibitor lethality in imatinib mesylate-sensitive or -resistant BCR/ABL+ leukemia cells in vitro and in vivo.

    Science.gov (United States)

    Dasmahapatra, Girija; Patel, Hiral; Nguyen, Tri; Attkisson, Elisa; Grant, Steven

    2013-01-15

    To determine whether Polo-like kinase 1 (PLK1) inhibitors (e.g., BI2536) and histone deacetylase (HDAC) inhibitors (e.g., vorinostat) interact synergistically in the BCR/ABL(+) leukemia cells sensitive or resistant to imatinib mesylate (IM) in vitro and in vivo. K562 and LAMA84 cells sensitive or resistant to imatinib mesylate and primary CML cells were exposed to BI2536 and vorinostat. Effects on cell viability and signaling pathways were determined using flow cytometry, Western blotting, and gene transfection. K562 and BV173/E255K animal models were used to test in vivo efficacy. Cotreatment with BI2536 and vorinostat synergistically induced cell death in parental or imatinib mesylate-resistant BCR/ABL(+) cells and primary CD34(+) bone marrow cells but was minimally toxic to normal cells. BI2536/vorinostat cotreatment triggered pronounced mitochondrial dysfunction, inhibition of p-BCR/ABL, caspase activation, PARP cleavage, reactive oxygen species (ROS) generation, and DNA damage (manifest by increased expression of γH2A.X, p-ATM, p-ATR), events attenuated by the antioxidant TBAP. PLK1 short hairpin RNA (shRNA) knockdown significantly increased HDACI lethality, whereas HDAC1-3 shRNA knockdown reciprocally increased BI2536-induced apoptosis. Genetic interruption of the DNA damage linker H1.2 partially but significantly reduced PLK1/HDAC inhibitor-mediated cell death, suggesting a functional role for DNA damage in lethality. Finally, BI2536/vorinostat cotreatment dramatically reduced tumor growth in both subcutaneous and systemic BCR/ABL(+) leukemia xenograft models and significantly enhanced animal survival. These findings suggest that concomitant PLK1 and HDAC inhibition is active against imatinib mesylate-sensitive or refractory CML and ALL cells both in vitro and in vivo and that this strategy warrants further evaluation in the setting of BCR/ABL(+) leukemias. ©2012 AACR.

  2. BCR-ABL transcripts are early predictors for hematological relapse in chronic myeloid leukemia after hematopoietic cell transplantation with reduced intensity conditioning

    NARCIS (Netherlands)

    Lange, T; Deininger, M; Brand, R; Hegenbart, U; Al-Ali, H; Krahl, R; Poenisch, W; Uharek, L; Leiblein, S; Gentilini, C; Petersdorf, E; Storb, RF; Niederwieser, D

    Kinetics of BCR-ABL transcript elimination and its prognostic implications on relapse were analyzed in patients with chronic myeloid leukemia (CML) after reduced intensity hematopoietic cell transplantation (HCT). In all, 19 CML patients were conditioned with 2Gy total-body irradiation in

  3. Tyrosine kinase fusion genes in pediatric BCR-ABL1-like acute lymphoblastic leukemia.

    Science.gov (United States)

    Boer, Judith M; Steeghs, Elisabeth M P; Marchante, João R M; Boeree, Aurélie; Beaudoin, James J; Beverloo, H Berna; Kuiper, Roland P; Escherich, Gabriele; van der Velden, Vincent H J; van der Schoot, C Ellen; de Groot-Kruseman, Hester A; Pieters, Rob; den Boer, Monique L

    2017-01-17

    Approximately 15% of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by gene expression similar to that of BCR-ABL1-positive disease and unfavorable prognosis. This BCR-ABL1-like subtype shows a high frequency of B-cell development gene aberrations and tyrosine kinase-activating lesions. To evaluate the clinical significance of tyrosine kinase gene fusions in children with BCP-ALL, we studied the frequency of recently identified tyrosine kinase fusions, associated genetic features, and prognosis in a representative Dutch/German cohort. We identified 14 tyrosine kinase fusions among 77 BCR-ABL1-like cases (18%) and none among 76 non-BCR-ABL1-like B-other cases. Novel exon fusions were identified for RCSD1-ABL2 and TERF2-JAK2. JAK2 mutation was mutually exclusive with tyrosine kinase fusions and only occurred in cases with high CRLF2 expression. The non/late response rate and levels of minimal residual disease in the fusion-positive BCR-ABL1-like group were higher than in the non-BCR-ABL1-like B-others (pfusion-negative BCR-ABL1-like group. The 8-year cumulative incidence of relapse in the fusion-positive BCR-ABL1-like group (35%) was comparable with that in the fusion-negative BCR-ABL1-like group (35%), and worse than in the non-BCR-ABL1-like B-other group (17%, p=0.07). IKZF1 deletions, predominantly other than the dominant-negative isoform and full deletion, co-occurred with tyrosine kinase fusions. This study shows that tyrosine kinase fusion-positive cases are a high-risk subtype of BCP-ALL, which warrants further studies with specific kinase inhibitors to improve outcome.

  4. 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

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

    Directory of Open Access Journals (Sweden)

    Mian Afsar

    2012-09-01

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

  6. [Clinical and laboratorial analysis for 15 adult cases of mixed phenotypic acute leukemia with Ph chromosome and/or positive BCR-ABL].

    Science.gov (United States)

    Yan, Ling-Zhi; Chen, Su-Ning; Ping, Na-Na; Wang, Qin-Rong; Liu, Hong; Ding, Zi-Xuan; Zhu, Ming-Qing; Liang, Jian-Ying; Liu, Dan-Dan; Cen, Jian-Nong; Pan, Jin-Lan; Qiu, Hui-Ying; Sun, Ai-Ning; Wu, De-Pei

    2013-10-01

    The purpose of this study was to summary the clinical and laboratorial features in 15 adult cases of mixed phenotypic acute leukemia with Ph chromosome and/or BCR-ABL fusion gene positive (Ph(+)MPAL), 15 adult patients with Ph(+)MPAL were defined by WHO-2008 classification. The clinical characteristics, results of morphology, immunology, cytogenetics and molecular genetic detections and results of follow-up in 15 adult patients with Ph(+)MPAL were analyzed retrospectively. The results showed that 15 patients among 87 cases of MPAL demonstrated Ph(+)MPAL (17.2%; 15/87) (7 males and 8 females), their median age was 51 (range 16-81) year old and median WBC count at diagnosis was 69 (12.7-921)×10(9)/L. Based on FAB criteria, these patients showed different morphologic types, including AML (13.3%; 2/15), ALL (40.0%; 6/15), HAL (46.7%; 7/15). Immunologic analysis indicated that 15 cases of Ph(-)MPAL were all classified as B-lymphoid +myeloid mixed immunophenotype. Except one patient, all expressed CD34 antigen on the surface of leukemia cells with 64.3% strong positive, only Ph (53.3%; 8/15), Ph with additional chromosomal abnormalities (33.3%; 5/15) and normal karyotype (13.3%; 2/15) were cytogenetically identified. BCR-ABL fusion gene transcript positive were detected by multiplex reverse transcription PCR in all cases, with e1a2 subtype (p190) (40.0%; 6/15) and b2a2 or b3a2 (p210) subtype (60.0%; 9/15). Four out of 7 (57.1%) patients were found to have IKZF1 gene deletion, without other common gene mutations. Seven out of 10 cases (70.0%) achieved complete remission (CR) after one cycle of induction chemotherapy. In the induction stage, CR rate seemed higher when tyrosine kinase inhibitors (TKI) were added to chemotherapy (83.3%:50.0%; P = 0.206). Overall survival (OS) in 4 patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT) was longer than that in 4 patients received chemotherapy alone (P = 0.004). It is concluded that Ph(+)MPAL mainly

  7. A comprehensive target selectivity survey of the BCR-ABL kinase inhibitor INNO-406 by kinase profiling and chemical proteomics in chronic myeloid leukemia cells.

    Science.gov (United States)

    Rix, U; Remsing Rix, L L; Terker, A S; Fernbach, N V; Hantschel, O; Planyavsky, M; Breitwieser, F P; Herrmann, H; Colinge, J; Bennett, K L; Augustin, M; Till, J H; Heinrich, M C; Valent, P; Superti-Furga, G

    2010-01-01

    Resistance to the BCR-ABL tyrosine kinase inhibitor imatinib poses a pressing challenge in treating chronic myeloid leukemia (CML). This resistance is often caused by point mutations in the ABL kinase domain or by overexpression of LYN. The second-generation BCR-ABL inhibitor INNO-406 is known to inhibit most BCR-ABL mutants and LYN efficiently. Knowledge of its full target spectrum would provide the molecular basis for potential side effects or suggest novel therapeutic applications and possible combination therapies. We have performed an unbiased chemical proteomics native target profile of INNO-406 in CML cells combined with functional assays using 272 recombinant kinases thereby identifying several new INNO-406 targets. These include the kinases ZAK, DDR1/2 and various ephrin receptors. The oxidoreductase NQO2, inhibited by both imatinib and nilotinib, is not a relevant target of INNO-406. Overall, INNO-406 has an improved activity over imatinib but a slightly broader target profile than both imatinib and nilotinib. In contrast to dasatinib and bosutinib, INNO-406 does not inhibit all SRC kinases and most TEC family kinases and is therefore expected to elicit fewer side effects. Altogether, these properties may make INNO-406 a valuable component in the drug arsenal against CML.

  8. BCR-ABL1: Test

    Science.gov (United States)

    ... Thompson, E. (Revised 2009 July). Modalities of Cancer Therapy. Merck Manual for Healthcare Professionals [On-line information]. Available online at http://www.merck.com/mmpe/sec11/ch149/ch149b.html?qt=bcr-abl&alt=sh through ... 15). Current Status of Therapy for Chronic Myeloid Leukemia: A Review of Drug ...

  9. 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

  10. INNO-406, a novel BCR-ABL/Lyn dual tyrosine kinase inhibitor, suppresses the growth of Ph+ leukemia cells in the central nervous system, and cyclosporine A augments its in vivo activity.

    Science.gov (United States)

    Yokota, Asumi; Kimura, Shinya; Masuda, Satohiro; Ashihara, Eishi; Kuroda, Junya; Sato, Kiyoshi; Kamitsuji, Yuri; Kawata, Eri; Deguchi, Yasuyuki; Urasaki, Yoshimasa; Terui, Yasuhito; Ruthardt, Martin; Ueda, Takanori; Hatake, Kiyohiko; Inui, Ken-ichi; Maekawa, Taira

    2007-01-01

    Central nervous system (CNS) relapse accompanying the prolonged administration of imatinib mesylate has recently become apparent as an impediment to the therapy of Philadelphia chromosome-positive (Ph+) leukemia. CNS relapse may be explained by limited penetration of imatinib mesylate into the cerebrospinal fluid because of the presence of P-glycoprotein at the blood-brain barrier. To overcome imatinib mesylate-resistance mechanisms such as bcr-abl amplification, mutations within the ABL kinase domain, and activation of Lyn, we developed a dual BCR-ABL/Lyn inhibitor, INNO-406 (formerly NS-187), which is 25 to 55 times more potent than imatinib mesylate in vitro and at least 10 times more potent in vivo. The aim of this study was to investigate the efficacy of INNO-406 in treating CNS Ph+ leukemia. We found that INNO-406, like imatinib mesylate, is a substrate for P-glycoprotein. The concentrations of INNO-406 in the CNS were about 10% of those in the plasma. However, this residual concentration was enough to inhibit the growth of Ph+ leukemic cells which expressed not only wild-type but also mutated BCR-ABL in the murine CNS. Furthermore, cyclosporine A, a P-glycoprotein inhibitor, augmented the in vivo activity of INNO-406 against CNS Ph+ leukemia. These findings indicate that INNO-406 is a promising agent for the treatment of CNS Ph+ leukemia.

  11. PF-114, a potent and selective inhibitor of native and mutated BCR/ABL is active against Philadelphia chromosome-positive (Ph+) leukemias harboring the T315I mutation.

    Science.gov (United States)

    Mian, A A; Rafiei, A; Haberbosch, I; Zeifman, A; Titov, I; Stroylov, V; Metodieva, A; Stroganov, O; Novikov, F; Brill, B; Chilov, G; Hoelzer, D; Ottmann, O G; Ruthardt, M

    2015-05-01

    Targeting BCR/ABL with tyrosine kinase inhibitors (TKIs) is a proven concept for the treatment of Philadelphia chromosome-positive (Ph+) leukemias. Resistance attributable to either kinase mutations in BCR/ABL or nonmutational mechanisms remains the major clinical challenge. With the exception of ponatinib, all approved TKIs are unable to inhibit the 'gatekeeper' mutation T315I. However, a broad spectrum of kinase inhibition increases the off-target effects of TKIs and may be responsible for cardiovascular issues of ponatinib. Thus, there is a need for more selective options for the treatment of resistant Ph+ leukemias. PF-114 is a novel TKI developed with the specifications of (i) targeting T315I and other resistance mutations in BCR/ABL; (ii) achieving a high selectivity to improve safety; and (iii) overcoming nonmutational resistance in Ph+ leukemias. PF-114 inhibited BCR/ABL and clinically important mutants including T315I at nanomolar concentrations. It suppressed primary Ph+ acute lymphatic leukemia-derived long-term cultures that either displayed nonmutational resistance or harbor the T315I. In BCR/ABL- or BCR/ABL-T315I-driven murine leukemia as well as in xenograft models of primary Ph+ leukemia harboring the T315I, PF-114 significantly prolonged survival to a similar extent as ponatinib. Our work supports clinical evaluation of PF-114 for the treatment of resistant Ph+ leukemia.

  12. Patients with Philadelphia-positive leukemia with BCR-ABL kinase mutations before allogeneic transplantation predominantly relapse with the same mutation.

    Science.gov (United States)

    Egan, Daniel N; Beppu, Lan; Radich, Jerald P

    2015-01-01

    Despite the successes of tyrosine kinase inhibitors (TKIs) in improving outcomes in patients with chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL), allogeneic hematopoietic stem cell transplantation (HSCT) continues to be an important and potentially curative option for selected patients with either disease. After HSCT, TKIs are increasingly being used to treat or prevent disease relapse, and practice patterns suggest that these TKIs are often chosen empirically without regard to pre-HSCT mutation status. We investigated whether ABL kinase domain mutations persist after transplantation and, thus, whether pre-HSCT mutation status should inform the selection of post-HSCT TKIs in these patients. We retrospectively analyzed adults who underwent allogeneic HSCT for CML and Ph + ALL at our institution between 2000 and 2010, and we identified subjects who had detectable BCR-ABL transcripts by polymerase chain reaction (PCR), as well as available RNA for Sanger sequencing of the ABL kinase domain, in both the pre- and post-HSCT settings. In total, 95 CML and 20 Ph + ALL patients with positive PCR transcripts were identified, of which 10 (10.5%) and 4 (20.0%), respectively, were found to have pre-HSCT ABL kinase mutations known to confer TKI resistance. In 9 (64.2%) of these 14 patients, the same kinase mutation was also detectable at an average time of 191 days after HSCT. Seven (50.0%) of the 14 harboring mutations had relapsed/refractory disease by last follow-up, of which, in retrospect, 6 had received a predictably ineffective TKI within the first 100 days after transplantation based on our mutation analysis. These data support the idea that pre-existing mutations in the ABL kinase domain, frequently associated with resistance to TKIs and prevalent in a transplantation population, are persistently detectable in the majority of patients after transplantation. We propose that such resistance patterns should be considered

  13. 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.

  14. Disruption of Bcr-Abl coiled coil oligomerization by design.

    Science.gov (United States)

    Dixon, Andrew S; Pendley, Scott S; Bruno, Benjamin J; Woessner, David W; Shimpi, Adrian A; Cheatham, Thomas E; Lim, Carol S

    2011-08-05

    Oligomerization is an important regulatory mechanism for many proteins, including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity, suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges, resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations, which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl, reduced cell proliferation, and increased caspase-3/7 activity and DNA segmentation. Importantly, the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention.

  15. Nilotinib first-line therapy in patients with Philadelphia chromosome-negative/BCR-ABL-positive chronic myeloid leukemia in chronic phase: ENEST1st sub-analysis.

    Science.gov (United States)

    Hochhaus, Andreas; Mahon, Franҫois-Xavier; le Coutre, Philipp; Petrov, Ljubomir; Janssen, Jeroen J W M; Cross, Nicholas C P; Rea, Delphine; Castagnetti, Fausto; Hellmann, Andrzej; Rosti, Gianantonio; Gattermann, Norbert; Coronel, Maria Liz Paciello; Gutierrez, Maria Asuncion Echeveste; Garcia-Gutierrez, Valentin; Vincenzi, Beatrice; Dezzani, Luca; Giles, Francis J

    2017-07-01

    The ENEST1st sub-analysis presents data based on Philadelphia chromosome (Ph) status, i.e., Ph+ and Ph-/BCR-ABL1 + chronic myeloid leukemia. Patients received nilotinib 300 mg twice daily, up to 24 months. At screening, 983 patients were identified as Ph+ and 30 patients as Ph-/BCR-ABL + based on cytogenetic and RT-PCR assessment; 76 patients had unknown karyotype (excluded from this sub-analysis). In the Ph-/BCR-ABL1 + subgroup, no additional chromosomal aberrations were reported. In the Ph+ subgroup, 952 patients had safety and molecular assessments. In the Ph-/BCR-ABL1 + subgroup, 30 patients had safety assessments and 28 were followed up for molecular assessments. At 18 months, the molecular response (MR) 4 rate [MR 4 ; BCR-ABL1 ≤0.01% on International Scale (IS)] was similar in the Ph-/BCR-ABL1+ (39.3%) and Ph+ subgroups (38.1%). By 24 months, the cumulative rates of major molecular response (BCR-ABL1 IS ≤0.1%;), MR 4 , and MR 4.5 (BCR-ABL1 IS ≤0.0032%) were 85.7, 60.7, and 50.0%, respectively, in the Ph-/BCR-ABL1 + subgroup, and 80.3, 54.7, and 38.3%, respectively, in the Ph+ subgroup. In both Ph-/BCR-ABL1 + and Ph+ subgroups, rash (20 and 22%), pruritus (16.7 and 16.7%), nasopharyngitis (13.3 and 10.4%), fatigue (10 and 14.2%), headache (10 and 15.8%), and nausea (6.7 vs 11.4%) were frequent non-hematologic adverse events, whereas hypophosphatemia (23.3 and 6.8%), anemia (10 and 6.5%), and thrombocytopenia (3.3 and 10.2%) were the common hematologic/biochemical laboratory events. Based on similar molecular response and safety results in both subgroups, we conclude that Ph-/BCR-ABL1 + patients benefit from nilotinib in the same way as Ph+ patients.

  16. 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

  17. BCR-ABL fusion genes are inducible by X-irradiation in vitro

    International Nuclear Information System (INIS)

    Ito, Takashi; Seyama, Toshio; Mizuno, Terumi; Hayashi, Tomonori; Nakamura, Nori; Akiyama, Mitoshi; Dohi, Kiyohiko.

    1992-01-01

    The Philadelphia chromosome consists of a reciprocal translocation between the ABL oncogene at chromosome 9q34 and the BCR gene at chromosome 22q resulting in the expression of chimeric BCR-ABL mRNAs specific to chronic myelogenous leukemia (CML). The presence of the fusion genes can be detected with high specificity and sensitivity by means of reverse transcription and polymerase chain reaction. Using this assay, it was possible to detect BCR-ABL fusion genes induced among HL60 cells after 100 Gy of X-irradiation in vitro. A total of five fusion gene transcripts were obtained. These fusion genes contained not only CML-specific BCR-ABL rearrangements, but also other forms of BCR-ABL fusions. These latter genes had junctions of BCR exon 4/ABL exon 2 intervened by a segment of DNA of unknown origin, BCR exon 5/ABL exon 2, and BCR exon 4/ABL exon 2. The results appear to be the first evidence for the induction of the BCR-ABL fusion gene by X-irradiation. In terms of leukemogenesis, it is suggested that only those cells bearing certain CML-related BCR-ABL fusion genes are positively selected by virtue of a growth advantage in vivo. (author)

  18. Treating Philadelphia chromosome/BCR-ABL1 positive patients with Glivec (Imatinib mesylate): 10 years' experience at Patan Hospital, Nepal.

    Science.gov (United States)

    Kayastha, Gyan K; Ranjitkar, Nora; Gurung, Radha; Kc, Raj K; Karki, Sanjit; Shrestha, Roshan; Thapa, Raj K; Rajbhandari, Piyush; Poudyal, Buddhi; Acharya, Paras; Roberts, David J; Hayes, Bruce; Zimmerman, Mark; Basnyat, Buddha; Mansfield, Aaron

    2017-06-01

    The Glivec International Patient Assistance Programme makes Glivec (Imatinib mesylate) available to Philadelphia chromosome/BCR-ABL1 positive patients with chronic myeloid leukaemia (CML) in Lower and Middle Income Countries (LMIC). We have established a large cohort of 211 CML patients who are eligible for Imatinib, in Kathmandu, Nepal. Thirty-one patients were lost to follow-up. We report on 180 CML patients with a median age of 38 years (range 9-81). Of these 180 patients, 162 underwent cytogenetic testing and 110 were investigated by reverse transcription polymerase chain reaction. One hundred and thirty-nine of the 180 patients (77·2%) had at least one optimal response. Taken together, our cohort has a 95% overall survival rate and 78% of the patients were still taking Glivec at a median time of 48·8 months (range 3-140 months). The number of patients who actually failed therapy, as defined by the LeukaemiaNet 2013 criteria, was 39 (21·7%). While our cohort has some differences with those in North America or Europe, we have shown Glivec is effective in inducing an optimal response in our patients in Nepal and that it is possible to deliver a clinical service for CML patients using tyrosine kinase inhibitors in resource-poor settings. © 2017 John Wiley & Sons Ltd.

  19. Bcr-Abl Amplification Plays a Major Role in Resistance to Tyrosine Kinase Inhibitors in K-562 Cell Line

    OpenAIRE

    Czyżewski, Krzysztof; Skonieczka, Katarzyna; Różycki, Patryk; Kołodziej, Beata; Kuryło-Rafińska, Beata; Kubicka, Małgorzata; Matiakowska, Karolina; Mucha, Barbara; Haus, Olga; Wysocki, Mariusz; Styczyński, Jan

    2012-01-01

    An emerging problem in patients with chronic myeloid leukemia (CML) is increasing resistance to tyrosine kinase inhibitors (TKIs). To determine genetic and cellular mechanisms involved in the development of resistance to TKIs, nine imatinib-resistant cell lines were derived from K- 562 cell line followed by testing of drug sensitivity, multidrug resistance proteins and cytogenetic studies. In imatinib-resistant cell lines cross-resistance to daunorubicin, etoposide and cytarabine were observe...

  20. Mouse embryonic stem cells that express a NUP98-HOXD13 fusion protein are impaired in their ability to differentiate and can be complemented by BCR-ABL.

    Science.gov (United States)

    Slape, Christopher; Chung, Yang Jo; Soloway, Paul D.; Tessarollo, Lino; Aplan, Peter D

    2007-01-01

    NUP98-HOXD13 (NHD13) fusions have been identified in patients with myelodysplastic syndrome (MDS), acute myelogenous leukemia (AML) and chronic myeloid leukemia blast crisis (CML-BC). We generated “knock-in” mouse embryonic stem (ES) cells that express a NHD13 fusion gene from the endogenous murine NUP98 promoter, and used an in vitro differentiation system to differentiate the ES cells to haematopoietic colonies. Replating assays demonstrated that the partially differentiated NHD13 ES cells were immortal, and two of these cultures were transferred to liquid culture. These cell lines are partially differentiated immature haematopoietic cells, as determined by morphology, immunophenotype and gene expression profile. Despite these characteristics, they were unable to differentiate when exposed to high concentrations of Epo, G-CSF, or M-CSF. The cell lines are incompletely transformed, as evidenced by their dependence on IL3, and their failure to initiate tumours when injected into immunodeficient mice. We attempted genetic complementation of the NHD13 gene using IL3 independence and tumorigenicity in immunodeficient mice as markers of transformation, and found that BCR-ABL successfully transformed the cell lines. These findings support the hypothesis that expression of a NHD13 fusion gene impairs haematopoietic differentiation, and that these cell lines present a model system to study the nature of this impaired differentiation. PMID:17377591

  1. 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

    leukemia (ALL) with the T315I mutation. Adults with Ph+ chronic phase (CP)-, accelerated phase (AP)- or blast phase (BP)-CML, or ALL and documented BCR-ABL T315I mutation were treated with a 5-day continuous infusion of MK-0457 administered every 14 days at 40 mg/m(2)/h, 32 mg/m(2)/h or 24 mg/m(2)/h. Fifty...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-01-12

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

  3. MEK inhibition potentiates the antitumor effect of Arsenic Trioxide in Bcr-Abl+ Imatinib-resistant Chronic Myeloid Leukemia cells: preclinical in vitro and in vivo study

    OpenAIRE

    Mazzera, Laura

    2014-01-01

    La Leucemia Mieloide Cronica (LMC) è una malattia caratterizzata dalla presenza di una specifica anormalità cromosomica, il cromosoma Philadelphia, codificante per una proteina di fusione di peso molecolare 210 Kd (p210) chiamata Bcr-Abl, che è una tirosin-chinasi sempre attiva. Il farmaco d’elezione per il trattamento di questa patologia è il tirosin-chinasi inibitore (TKI) Imatinib (Gleevec) che riduce l'attività di Bcr-Abl. L’utilizzo dell’Imatinib in clinica è però limitato dall’insorgenz...

  4. Photodynamic treatment (ALA-PDT) suppresses the expression of the oncogenic Bcr-Abl kinase and affects the cytoskeleton organization in K562 cells

    Czech Academy of Sciences Publication Activity Database

    Pluskalová, M.; Pešlová, G.; Grebeňová, D.; Halada, Petr; Hrkal, Z.

    2006-01-01

    Roč. 83, - (2006), s. 205-212 ISSN 1011-1344 R&D Projects: GA MZd NL7681 Institutional research plan: CEZ:AV0Z50200510 Keywords : k562 * bcr -abl * photodynamic treatment Subject RIV: EE - Microbiology, Virology Impact factor: 1.909, year: 2006

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

    Directory of Open Access Journals (Sweden)

    Pallavi Sontakke

    Full Text Available 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 transduced with various oncogenes, including BCR-ABL, MLL-AF9, FLT3-ITD, NUP98-HOXA9, STAT5A and KRASG12V were analyzed in detail. Our data indicate that in particular BCR-ABL, KRASG12V and STAT5 could impose hypoxic signaling under normoxic conditions. This coincided with an upregulation of glucose importers SLC2A1/3, hexokinases and HIF1 and 2. NMR-based metabolic profiling was performed in CB CD34+ cells transduced with BCR-ABL versus controls, both cultured under normoxia and hypoxia. Lactate and pyruvate levels were increased in BCR-ABL-expressing cells even under normoxia, coinciding with enhanced glutaminolysis which occurred in an HIF1/2-dependent manner. Expression of the glutamine importer SLC1A5 was increased in BCR-ABL+ cells, coinciding with an increased susceptibility to the glutaminase inhibitor BPTES. Oxygen consumption rates also decreased upon BPTES treatment, indicating a glutamine dependency for oxidative phosphorylation. The current study suggests that BCR-ABL-positive cancer cells make use of enhanced glutamine metabolism to maintain TCA cell cycle activity in glycolytic cells.

  6. Nuclear topography and expression of the BCR/ABL fusion gene and its protein level influenced by cell differentiation and RNA interference

    Czech Academy of Sciences Publication Activity Database

    Bártová, Eva; Harničarová, Andrea; Pacherník, Jiří; Kozubek, Stanislav

    2005-01-01

    Roč. 29, č. 8 (2005), s. 901-913 ISSN 0145-2126 R&D Projects: GA AV ČR(CZ) 1QS500040508; GA ČR(CZ) GA202/04/0907; GA MZd NC6987; GA AV ČR(CZ) IAA5004306; GA MŠk(CZ) LC535 Institutional research plan: CEZ:AV0Z50040507 Keywords : BCR /ABL fusion gene * chromatin arrangement * gene expression Subject RIV: BO - Biophysics Impact factor: 2.372, year: 2005

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

    Directory of Open Access Journals (Sweden)

    Groffen John

    2007-10-01

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

  8. Detection of BCR-ABL and E2A-PBX1 fusion genes by RT-PCR in acute lymphoblastic leukaemia with failed or normal cytogenetics.

    Science.gov (United States)

    Devaraj, P E; Foroni, L; Kitra-Roussos, V; Secker-Walker, L M

    1995-02-01

    To evaluate the use of molecular analysis as a complement to karyotypic analysis in the detection of specific chromosomal abnormalities, the occurrence of t(1;19)(q23;p13) and t(9;22)(q34;q11) was investigated by RT-PCR in 43 diagnostic acute lymphoblastic leukaemia cases in whom cytogenetic investigations had failed (32 cases) or showed only a normal karyotype (> or = 20 normal metaphases, 11 cases). One child (aged 14 years) and five adults (aged 18-60 years) were BCR-ABL positive on first round for M-BCR-ABL (one case) or m-BCR-ABL (one case), or on nested PCR for m-BCR-ABL (three cases). Co-expression of M-BCR-ABL (first-round PCR) and m-BCR-ABL (nested PCR was seen in one case. One m-BCR-ABL-positive case also expressed the E2A-PBX1 fusion transcript. Patients positive for the transcript(s) were older, had higher white blood cell counts and a significantly poorer event-free survival (P < 0.001) than those negative for the transcript.

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

    Directory of Open Access Journals (Sweden)

    Shuit-Mun Wong

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

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

    Science.gov (United States)

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

    2016-01-01

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

  11. Distinct in vivo engraftment and growth patterns of t(1;19)+/E2A-PBX1+ and t(9;22)+/BCR-ABL+ human leukemia cells in SCID mice.

    Science.gov (United States)

    Waurzyniak, B J; Heerema, N; Sensel, M G; Gaynon, P S; Kraft, P; Sather, H N; Chelstrom, L; Reaman, G H; Uckun, F M

    1998-12-01

    The SCID mouse represents a valuable tool for assessing growth characteristics and drug sensitivity of human leukemic cells. We have examined differences in the engraftment patterns in SCID mice of primary human leukemic cells isolated from children (E2A-PBX1+ or t(9;22)+/BCR-ABL+ acute lymphoblastic leukemia. Leukemic cells from 13/24 t(1;19)+/E2A-PBX1+ patients caused overt leukemia in SCID mice. Macroscopic lesions were evident in 6/13 cases, with multiple sites involved in some mice: hepatomegaly,(3) splenomegaly(4), thymic enlargement; liver tumors(1), kidney tumors(1), abdominal tumors(1). Microscopic lesions in SCID mouse organs were present in all 13 cases and involved the bone marrow, brain, heart, gut, liver, kidney, lung, ovary, pancreas, skeletal muscle, spleen, and thymus. Leukemic cells from 5/20 t(9;22)+/BCR-ABL+ patients caused overt leukemia in SCID mice. Notably, macroscopic lesions (splenomegaly; leukemic bones; hepatic tumors) were observed in only 1 case. In all 5 cases, microscopic lesions were found in the mouse bone marrow. Additional microscopic lesions were restricted to skeletal muscle, spleen, and mesentery (1 case) or thymus (1 case). These findings differ markedly from those of t(1;19)+/E2A-PBX1+ leukemic cells due to the lack of involvement of major organs such as liver, pancreas, kidney, skin, or brain. These data illustrate the biological heterogeneity of childhood ALL and suggest that the differential risks associated with t(1;19)+/E2A-PBX1+ and t(9;22)+/BCR-ABL ALL might arise from unique engraftment and proliferation capabilities of the respective leukemic cell populations.

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

    International Nuclear Information System (INIS)

    Mundhada, Shailendra; Luthra, Rajyalakshmi; Cano, Pedro

    2004-01-01

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

  13. CD-200 induces apoptosis and inhibits Bcr-Abl signaling in imatinib-resistant chronic myeloid leukemia with T315I mutation.

    Science.gov (United States)

    Fang, Zhenghuan; Jung, Kyung Hee; Yan, Hong Hua; Kim, Soo Jung; Son, Mi Kwon; Rumman, Marufa; Lee, Hyunseung; Kim, Ki Woon; Yoo, Hye-Dong; Hong, Soon-Sun

    2015-07-01

    Chronic myeloid leukemia (CML) is characterized by a constitutively active Bcr-Abl tyrosine kinase. Although Imatinib has been proven to be an effective drug against CML, its resistance has been observed with disease relapse due to T315I predominant point mutation. Liriodendron tulipifera L., one of the fastest growing hardwood tree species, exerts antioxidant activity and anti-inflammatory effects. However, its anticancer effect has been minimally reported. In this study, we extracted CD-200 from Liriodendron tulipifera L. and investigated its effect on cell survival or apoptosis in CML cells with Bcr-Abl/T315I (BaF3/T315I) as well as wild-type Bcr-Abl (BaF3/WT). CD-200 inhibited cell proliferation in the BaF3/WT cells, and also in the BaF3/T315I cells with Imatinib resistance. Moreover, it strongly inhibited Bcr-Abl signaling pathways in a dose-dependent manner. Also, it significantly increased the sub-G1 phase and the expression of cleaved PARP and caspase-3, as well as the TUNEL-positive apoptotic cells. In addition, we observed that CD-200 induced apoptosis with a loss of mitochondrial membrane potential by decreasing the expression of Mcl-1 and survivin. Furthermore, CD-200 showed a significant inhibition in tumor growth, compared to Imatinib in BaF3/T315I mouse xenograft models. Taken together, our study demonstrates that CD-200 exhibits apoptosis induction and anti-proliferative effect by blocking the Bcr-Abl signaling pathways in the Bcr-Abl/T315I with resistance to Imatinib. We suggest that CD-200 may be a natural product to target Bcr-Abl and overcome Imatinib resistance in CML patients.

  14. Inhibition of BCR/ABL protein expression by miR-203 sensitizes for imatinib mesylate.

    Directory of Open Access Journals (Sweden)

    Yajuan Li

    Full Text Available Selective inhibition of BCR/ABL expression by RNA interference has been demonstrated as an effective strategy in CML treatment and a reversal to imatinib resistance. microRNAs (miRNAs are small regulatory RNAs involved in post-transcriptional gene regulation. miR-203 is supposed to directly regulate ABL and BCR/ABL expression, however, the role of miR-203 in imatinib-resistant cells is not clear. Here, we report that overexpression of miR-203 in BaF3-BCR/ABL cells with T315I mutant inhibited cell growth and colony formation ability. Furthermore, miR-203 increased sensitivity to imatinib in BaF3-BCR/ABL(T315I cells, thereby antagonizing the main mechanism of resistance to imatinib.

  15. A BCR-ABL Kinase Activity-Independent Signaling Pathway in Chronic Myelogenous Leukemia

    National Research Council Canada - National Science Library

    Li, Shaoguang

    2008-01-01

    .... We identified Src kinases as key molecules in this BCR- ABL kinase activity-independent pathway and they are essential for leukemic cells to survive imatinib treatment and for CML transition to lymphoid blast crisis...

  16. Increased expression of fibroblast growth factor receptor 3 in CD34+ BCR-ABL+ cells from patients with chronic myeloid leukemia

    Czech Academy of Sciences Publication Activity Database

    Dvořák, Petr; Dvořáková, D.; Doubek, M.; Faitová, Jitka; Pacholíková, J.; Hampl, Aleš; Mayer, J.

    2003-01-01

    Roč. 17, - (2003), s. 1-8 ISSN 0887-6924 R&D Projects: GA ČR GA301/03/1122; GA MŠk LN00A065 Institutional research plan: CEZ:AV0Z5039906; CEZ:MSM 00065269705; CEZ:VZ432100001 Keywords : CD34+cells, imatinib Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.116, year: 2003

  17. Allium Roseum L. Extract Exerts Potent Suppressive Activities on Chronic Myeloid Leukemia K562 Cell Viability Through the Inhibition of BCR-ABL, PI3K/Akt, and ERK1/2Pathways and the Abrogation of VEGF Secretion.

    Science.gov (United States)

    Souid, Soumaya; Najjaa, Hanen; Riahi-Chebbi, Ichrak; Haoues, Meriam; Neffati, Mohamed; Arnault, Ingrid; Auger, Jacques; Karoui, Habib; Essafi, Makram; Essafi-Benkhadir, Khadija

    2017-01-01

    Use of plant extracts, alone or combined to the current chemotherapy as chemosensitizers, has emerged as a promising strategy to overcome tumor drug resistance. Here, we investigated the anticancer activity of Allium roseum L. extracts, a wild edible species in North Africa, on human Chronic Myeloid Leukemia (CML) K562 cells. The dehydrated aqueous extract (DAE) disturbed the cell cycle progression and induced the apoptosis of K562 cells. Chemical analysis of DAE showed a diversity of organosulfur compounds S-alk(en)yl-cysteine sulfoxides (RCSO) and high amount of allicin, suggesting that such molecule may be behind its antitumor effect. DAE was efficient in inhibiting K562 cell viability. DAE inhibitory effect was associated with the dephosphorylation of the BCR-ABL kinase and interfered with ERK 1/2 , Akt, and STAT5 pathways. Furthermore, we found that DAE-induced inactivation of Akt kinase led to the activation of its target FOXO3 transcription factor, enhancing the expression of FOXO3-regulated proapoptotic effectors, Bim and Bax, and cell cycle inhibitor p27. Finally, we found that DAE reduced the secretion of vascular endothelial growth factor. Overall, our data suggest that A. roseum extract has great potential as a nontoxic cheap and effective alternative to conventional chemotherapy.

  18. BCR-ABL promotes the frequency of mutagenic single-strand annealing DNA repair

    Science.gov (United States)

    Fernandes, Margret S.; Reddy, Mamatha M.; Gonneville, Jeffrey R.; DeRoo, Scott C.; Podar, Klaus; Griffin, James D.; Weinstock, David M.

    2009-01-01

    Intracellular oxidative stress in cells transformed by the BCR-ABL oncogene is associated with increased DNA double-strand breaks. Imprecise repair of these breaks can result in the accumulation of mutations, leading to therapy-related drug resistance and disease progression. Using several BCR-ABL model systems, we found that BCR-ABL specifically promotes the repair of double-strand breaks through single-strand annealing (SSA), a mutagenic pathway that involves sequence repeats. Moreover, our results suggest that mutagenic SSA repair can be regulated through the interplay between BCR-ABL and extrinsic growth factors. Increased SSA activity required Y177 in BCR-ABL, as well as a functional PI3K and Ras pathway downstream of this site. Furthermore, our data hint at a common pathway for DSB repair whereby BCR-ABL, Tel-ABL, Tel-PDGFR, FLT3-ITD, and Jak2V617F all increase mutagenic repair. This increase in SSA may not be sufficiently suppressed by tyrosine kinase inhibitors in the stromal microenvironment. Therefore, drugs that target growth factor receptor signaling represent potential therapeutic agents to combat tyrosine kinase-induced genomic instability. PMID:19571320

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

    International Nuclear Information System (INIS)

    Manzotti, G; Mariani, S A; Corradini, F; Bussolari, R; Cesi, V; Vergalli, J; Ferrari-Amorotti, G; Fragliasso, V; Soliera, A R; Cattelani, S; Raschellà, G; Holyoake, T L; Calabretta, B

    2012-01-01

    The c-Myb gene encodes the p75 c-Myb isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is p c-Mybex9b , which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein–protein interactions and negative regulation. In hematopoietic cells, expression of p c-Mybex9b accounts for 10–15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of p c-Mybex9b and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75 c-Myb , p c-Mybex9b is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of p c-Mybex9b enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of p c-Mybex9b reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34 + cells, without affecting the levels of p75 c-Myb . Together, these studies indicate that expression of the low-abundance p c-Mybex9b isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells

  20. BCR-ABL1 transcript types showed distinct laboratory characteristics in patients with chronic myeloid leukemia.

    Science.gov (United States)

    Vasconcelos, A P; Azevedo, I F; Melo, F C B C; Neves, W B; Azevedo, A C A C; Melo, R A M

    2017-04-20

    In chronic myeloid leukemia (CML) two main types of messenger RNA (e14a2 and e13a2) can be produced by BCR-ABL1 gene rearrangement. Due to conflicting results, the clinical value of these transcripts remains controversial. The aim of this study was to identify associations of e14a2 and e13a2 transcripts with laboratory variables and also the response to treatment. This study included 203 adult patients with CML treated with Imatinib as first-line drug in a reference hematology center in Northeast Brazil. Clinical and laboratory data were obtained after informed consent. Samples were collected for RNA extraction and analyzed by reverse transcription-polymerase chain reaction (PCR), according to the international protocol BIOMED-1. The LeukemiaNet 2013 criteria were used to establish the molecular response. The frequency distribution of the BCR-ABL1 transcripts was e14a2 (64%), e13a2 (34%), and double positives (2%). The results showed a statistically significant association of the e14a2 transcript type with thrombocytosis (P = 0.0005) and the e13a2 with higher leukocyte count (P = 0.0491). In a subgroup of 44 patients, the molecular response to treatment with Imatinib was assessed by quantitative PCR at 3 months (BCR-ABL1 ≤ 10%), 6 months (BCR-ABL1 ≤ 1%), or 12 months (BCR-ABL1 ≤ 0.1%). Although patients with the transcript e14a2 showed higher frequency of good responses than patients with the transcript e13a2, this difference was not statistically significant. In agreement with published data, our results showed association of the BCR-ABL1 transcript e14a2 with thrombocytosis and the BCR-ABL1 transcript e13a2 with higher leukocytosis in patients with chronic myeloid leukemia.

  1. [Generation and identification of P210(T315I-BCR/ABL) transgenic mice].

    Science.gov (United States)

    Zhu, Yufeng; Wang, Yuanzhan; Meng, Fanyi

    2015-03-01

    To construct the P210(T315I-BCR/ABL) transgenic mice model. The transgenic vector in which the P210(T315I-BCR/ABL) gene and eGFP gene was derived by APN/CD13 promoter was constructed and microinjected into the single-cell fertilized eggs of C57 mice. Transgene integration was conformed by PCR genotyping and P210(T315I-BCR/ABL) expression levels was evaluated by RT-PCR. The CML phenotype was confirmed by blood routine examination, Wright's staining for peripheral blood and bone marrow smears, HE staining for organs of transgenic mice. Three transgenic mice lines with high expression of P210(T315I-BCR/ABL) gene and eGFP gene was selected. Compared with the wild type mice, the levels of WBC, platelet and neutrophil granulocyte of transgenic mice began to increase gradually at 2 months, and increase to 23.9×10⁹/L, 4 136×10⁹/L, and 74.6% respectively at 6 months. The remarkable hyperplasia of granulocytes was seen in the peripheral blood and bone marrow smears with splenomegaly infiltrated by leukemic cells. The P210(T315I-BCR/ABL) transgenic mice was constructed and provided a model to explore the mechanism of T315I CML and screen out the drug for T315 CML patient.

  2. 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.

  3. Drug resistance and BCR-ABL kinase domain mutations in Philadelphia chromosome-positive acute lymphoblastic leukemia from the imatinib to the second-generation tyrosine kinase inhibitor era: The main changes are in the type of mutations, but not in the frequency of mutation involvement.

    Science.gov (United States)

    Soverini, Simona; De Benedittis, Caterina; Papayannidis, Cristina; Paolini, Stefania; Venturi, Claudia; Iacobucci, Ilaria; Luppi, Mario; Bresciani, Paola; Salvucci, Marzia; Russo, Domenico; Sica, Simona; Orlandi, Ester; Intermesoli, Tamara; Gozzini, Antonella; Bonifacio, Massimiliano; Rigolin, Gian Matteo; Pane, Fabrizio; Baccarani, Michele; Cavo, Michele; Martinelli, Giovanni

    2014-04-01

    Patients with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) frequently relapse on imatinib with acquisition of BCR-ABL kinase domain (KD) mutations. To analyze the changes that second-generation tyrosine kinase inhibitors (TKIs) have brought in mutation frequency and type, a database review was undertaken of the results of all the BCR-ABL KD mutation analyses performed in the authors' laboratory from January 2004 to January 2013. Interrogation of the database retrieved 450 mutation analyses in 272 patients with Ph+ ALL. Prescreening of samples was performed with denaturing high-performance liquid chromatography (D-HPLC), followed by direct sequencing of D-HPLC-positive cases. BCR-ABL KD mutations were detected in 70% of imatinib-resistant patients, with T315I, E255K, and Y253H mutations accounting for 75% of cases. Seventy-eight percent of the patients reported to be resistant to second-generation TKIs after imatinib failure were positive for mutations, and 58% of them had multiple mutations. Analysis of patients relapsing on dasatinib revealed a newly acquired T315I mutation in almost two-thirds of the cases. Direct sequencing detected no mutations at diagnosis, even in patients who relapsed after a few months. Second-generation TKIs ensure a more rapid debulking of the leukemic clone and have much fewer insensitive mutations, but long-term disease control remains a problem, and the T315I mutation is revealed to be an even more frequent enemy. BCR-ABL KD mutation screening of patients with Ph+ ALL who are receiving imatinib or second-generation TKIs would be a precious ally for timely treatment optimization. In contrast, the clinical usefulness of conventional direct sequencing at diagnosis seems to be very low. American Cancer Society. © 2013 American Cancer Society.

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

    Directory of Open Access Journals (Sweden)

    Yanan Qin

    2013-01-01

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

  5. Effect of Thai saraphi flower extracts on WT1 and BCR/ABL protein ...

    African Journals Online (AJOL)

    In this study, the cytotoxic effects of crude ethanolic and fractional extracts including hexane, ethyl acetate, and methanol fractions from M. siamensis flowers were investigated in order to determine their effect on WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. Materials and Methods: The ...

  6. 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.

  7. Molecular detection of BCR/ABL fusion gene in Saudi acute lymphoblastic leukemia patients.

    Science.gov (United States)

    El-Sissy, Azza; El-Mashari, May; Bassuni, Wafaa; El-Swaayed, Aziza

    2006-06-01

    Molecular cytogenetics is becoming one of the most useful tools targeting some genes which are generally considered to lead to leukemic transformation (as well as for numerical abnormalities). A fraction of acute lymphoblastic leukemia (ALL) cases carry the translocation t(9;22) (q34;q11.2) which juxtaposes the ABL proto-oncogene to the BCR gene generating a chimeric gene, BCR/ABL. This aberration is more frequent in adult ALL (20%-40%) than in pediatric ALL (<5%), and predicts poor clinical outcome. AIM OF OUR WORK: Is to study BCR/ABL fusion gene in ALL cases using fluorescent in situ hybridization. Twenty newly diagnosed ALL patients, 16 adult and 4 paediatric cases, were included in the study, 11 cases (55%) were of precursor B phenotype, 8 cases (40%) belonged to T lineage, while one case was biphenotypic expressing mainly precursor B cell markers tether with CD13, CD33, CD117, Detection of BCR/ABL fusion gene was done using interphase FISH technique and was confirmed molecularly using the RT-PCR technique. BCR/ABL fusion gene was negative in all the examined cases, yet abnormality involving 9q34, ABL gene, either by addition or deletion was detected in three cases (15%). Two of these cases were associated with BCR gene extra copies (three and four copies, respectively). This may reflect the frequency of association of ABL gene and BCR gene abnormality in our cases, and that absence of fusion gene BCR/ABL does not exclude their role in the leukomogenic process, yet a larger study is required to confirm and detect the prevalence of these gene disturbances in ALL and their association.

  8. Chronic myeloid leukemia may be associated with several bcr-abl transcripts including the acute lymphoid leukemia-type 7 kb transcript

    NARCIS (Netherlands)

    Selleri, L.; von Lindern, M.; Hermans, A.; Meijer, D.; Torelli, G.; Grosveld, G.

    1990-01-01

    In the majority of Philadelphia (Ph)-positive chronic myeloid leukemia (CML) patients, the c-abl gene is fused to the bcr gene, resulting in the transcription of an 8.5 kb chimeric bcr-abl mRNA, which is translated into a p210bcr-abl fusion protein. In about 50% of the Ph-positive acute lymphoid

  9. Unleashing the Guardian: The Targetable BCR-ABL/HAUSP/PML/PTEN Network in Chronic Myeloid Leukemia.

    Science.gov (United States)

    Morotti, Alessandro; Torti, Davide; Carra, Giovanna; Panuzzo, Cristina; Crivellaro, Sabrina; Taulli, Riccardo; Fava, Carmen; Guerrasio, Angelo; Saglio, Giuseppe

    2017-01-01

    The complete eradication of Chronic Myeloid Leukemia is still challenging even in the era of highly selective and potent BCR-ABL tyrosine kinase inhibitors (TKIs). The 'Achilles heel' of TKI-based CML therapy is the inability of TKI to effectively target CML stem cells. Several pathways have been described to induce TKI insensitiveness in quiescent CML stem cells. In this review, we will describe the BCR-ABL/HAUSP/PML/PTEN network, whose signaling mediators converge to regulate the function of the tumor suppressor PTEN. We will also highlight the pharmacological strategies to modulate PTEN functions in order to sustain CML stem cell eradication. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

    Directory of Open Access Journals (Sweden)

    Qinghuang Chen

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

  11. Fluorescence in situ hybridization patterns of BCR/ABL1 fusion in chronic myelogenous leukemia at diagnosis

    Directory of Open Access Journals (Sweden)

    Poonam P Jain

    2012-01-01

    Full Text Available Background : Chronic myelogenous leukemia (CML is characterised by the t(9;22(q34;q11.2 which results in the formation of the BCR/ABL1 fusion gene. Occasionally, the t(9;22 may be associated with submicroscopic deletions of chromosomes 9 and/or 22 which appear to be associated with a worse prognosis. Three or four-way variant t(9;22 may also occur. All these changes as well as gain of the Philadelphia chromosome which represents disease progression can be detected by fluorescence in situ hybridization (FISH analysis. FISH analysis at presentation is used to determine the number of cells with BCR/ABL1 fusion and establish whether the patterns are typical or atypical. Response to therapy can then be monitored by serial testing. Patients and Methods : The study group consisted of all patients diagnosed or suspected to have CML who had interphase FISH analysis at presentation on peripheral blood/bone marrow using a commercially available BCR/ABL1 dual colour, dual fusion probe. The study was performed at a tertiary hospital in India between 2004 and 2010. Results: There were 1076 diagnostic samples which were positive for BCR/ABL1 fusion. Typical dual fusion signals (two fusions, one red and one green, 2F1R1G were seen in 801 cases (74 %. Atypical signal patterns were seen in 275 cases (26%. These were: 1F1R2G (4%, 1F2R1G (2.5% and 1F1R1G (11% representing deletions of the derivative 9 involving chromosome 9 sequences, chromosome 22 sequences, or both respectively; 3F1R1G (6.5% usually representing gain of an additional Philadelphia chromosome and 1F2R2G (1% representing a three- or four-way variant translocation. More than one signal pattern was seen in 1%. Conclusions: Our findings were similar to the literature with respect to the distribution of signal patterns except that we had a lower number of patients with variant translocations. While each signal pattern is typically associated with a particular abnormality, there can be more than one

  12. Loss of the xeroderma pigmentosum group B protein binding site impairs p210 BCR/ABL1 leukemogenic activity

    International Nuclear Information System (INIS)

    Pannucci, N L; Li, D; Sahay, S; Thomas, E K; Chen, R; Tala, I; Hu, T; Ciccarelli, B T; Megjugorac, N J; Adams III, H C; Rodriguez, P L; Fitzpatrick, E R; Lagunoff, D; Williams, D A; Whitehead, I P

    2013-01-01

    Previous studies have demonstrated that p210 BCR/ABL1 interacts directly with the xeroderma pigmentosum group B (XPB) protein, and that XPB is phosphorylated on tyrosine in cells that express p210 BCR/ABL1. In the current study, we have constructed a p210 BCR/ABL1 mutant that can no longer bind to XPB. The mutant has normal kinase activity and interacts with GRB2, but can no longer phosphorylate XPB. Loss of XPB binding is associated with reduced expression of c-MYC and reduced transforming potential in ex-vivo clonogenicity assays, but does not affect nucleotide excision repair in lymphoid or myeloid cells. When examined in a bone marrow transplantation (BMT) model for chronic myelogenous leukemia, mice that express the mutant exhibit attenuated myeloproliferation and lymphoproliferation when compared with mice that express unmodified p210 BCR/ABL1. Thus, the mutant-transplanted mice show predominantly neutrophilic expansion and altered progenitor expansion, and have significantly extended lifespans. This was confirmed in a BMT model for B-cell acute lymphoblastic leukemia, wherein the majority of the mutant-transplanted mice remain disease free. These results suggest that the interaction between p210 BCR/ABL1 and XPB can contribute to disease progression by influencing the lineage commitment of lymphoid and myeloid progenitors

  13. Overcoming imatinib resistance using Src inhibitor CGP76030, Abl inhibitor nilotinib and Abl/Lyn inhibitor INNO-406 in newly established K562 variants with BCR-ABL gene amplification.

    Science.gov (United States)

    Morinaga, Koji; Yamauchi, Takahiro; Kimura, Shinya; Maekawa, Taira; Ueda, Takanori

    2008-06-01

    Because imatinib (IM) resistance in chronic myeloid leukemia is primarily caused by the re-establishment of Abl kinase, new inhibitors may be efficacious. We evaluated 3 new agents against 2 new K562 variants, IM-R1 and IM-R2 cells, which were developed having 7- and 27-fold greater IM resistance, respectively, than the parental K562 cells. Both variants possessed BCR-ABL gene amplification along with elevated levels of its transcript and protein. Greater BCR-ABL gene amplification was observed in IM-R2 cells than in IM-R1 cells, which was consistent with the higher mRNA and protein levels of Bcr-Abl, and ultimately correlated with the greater IM resistance in IM-R2 cells. No mutation in the Abl kinase domain was detected in either variant. Despite the absence of Lyn overexpression, the Src kinase inhibitor CGP76030 showed positive cooperability with IM in inhibiting cell growth of not only K562 cells but also these 2 variants. This might be because of the augmented inhibition of Erk1/2 phosphorylation. The new Abl kinase inhibitor nilotinib was 10-fold more potent than IM in inhibiting the growth of K562 cells. Nilotinib inhibited the growth of IM-R1 and IM-R2 cells as potently as K562 cells. The combination of nilotinib with CGP76030 showed little additivity, because the potency of nilotinib masked the efficacy of CGP76030. The new dual Abl/Lyn inhibitor INNO-406 (formerly NS-187) was slightly more potent than nilotinib in inhibiting the growth of all 3 cell lines. Because BCR-ABL gene amplification occurs in blast crisis, these inhibitors might overcome IM resistance in such patients' leukemia. (c) 2008 Wiley-Liss, Inc.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Characterization of a novel variant BCR-ABL1 fusion transcript in a patient with chronic myeloid leukemia: Implications for molecular monitoring.

    Science.gov (United States)

    Crampe, Mireille; Haslam, Karl; Kelly, Johanna; Conneally, Eibhlin; Langabeer, Stephen E

    2017-06-01

    Molecular monitoring of BCR-ABL1 transcript levels using quantitative polymerase chain reaction is an essential part of the modern management of chronic myeloid leukemia patients treated with tyrosine kinase inhibitors. Establishing the diagnostic BCR-ABL1 fusion transcript is necessary in order to select appropriate primers and probes for such monitoring. A case is described in which quantitative polymerase chain reaction failed to detect the presence of BCR-ABL1 fusion transcript in a Philadelphia chromosome-positive chronic myeloid leukemia patient. Further investigation demonstrated a novel in-frame BCR-ABL1 fusion transcript with a breakpoint in BCR exon 13 and insertion of a sequence of ABL1 intron 1, therefore enabling subsequent molecular monitoring. This case highlights the requirement for characterization of the BCR-ABL1 transcript type at chronic myeloid leukemia diagnosis. Issues concerning standardized methodological approaches and interpretation of transcript levels in such rare cases are discussed. Copyright © 2016 King Faisal Specialist Hospital & Research Centre. Published by Elsevier B.V. All rights reserved.

  16. Expression of BCR-ABL, E2A-PBX1, and MLL-AF4 fusion transcripts in newly diagnosed children with acute lymphoblastic leukemia: a Children's Cancer Group initiative.

    Science.gov (United States)

    Gaynon, P S; Crotty, M L; Sather, H N; Bostrom, B C; Nachman, J B; Steinherz, P G; Heerema, N A; Sarquis, M; Tuel-Ahlgren, L; Uckun, F M

    1997-06-01

    We used reverse transcriptase polymerase chain reaction (RT-PCR) assays to examine primary leukemic cells in on-study diagnostic bone marrow specimens from 642 children with newly diagnosed acute lymphoblastic leukemia (ALL) for the expression of MLL-AF4, E2A-PBX1, and BCR-ABL fusion transcripts. All PCR assays were performed centrally in the Children's Cancer Group ALL Biology Reference Laboratory. MLL-AF4 transcript was found in only 0.7% of the study population which excluded infants. E2A-PBX1 transcript was found in 2.5% of the study population and 3.3% of B-precursor cases. Expression was associated with massive hepatomegaly. BCR-ABL transcript was found in 2.3% of cases and correlated with older age, induction failure, and inferior event-free survival (EFS). RT-PCR assays allow rapid identification of patients with MLL-AF4 and BCR-ABL positive ALL. These patients have a poor outcome with contemporary therapy and rapid identification facilitates timely allocation to innovative treatment programs.

  17. A novelBCR-ABL1fusion gene with genetic heterogeneity indicates a good prognosis in a chronic myeloid leukemia case.

    Science.gov (United States)

    Zhou, Fen; Jin, Runming; Hu, Yu; Mei, Heng

    2017-01-01

    Chronic myelogenous leukemia (CML) is a pluripotent hematopoietic stem cell disorder caused by the fusion of the BCR and ABL1 genes. Quantitative RT-PCR (qRT-PCR) is a routinely performed screening technique to identify BCR-ABL1 fusion genes, but a limitation of this method is its inability to recognize novel fusions that have not been previously characterized. Next-generation sequencing (NGS) is an effective and sensitive detection method for the determination of novel BCR-ABL1 fusion genes as well as previously characterized ones. The oncoprotein tyrosine kinase BCR-ABL1 is a constitutively active kinase involved in the activation of a number of signaling pathways, and it has been the therapeutic target for tyrosine kinase inhibitors (TKIs) such as imatinib. Reports have presented opposing viewpoints about the effect of the disrupted Src homology 3 (SH3) domain on TKI efficacy. We here report that using NGS we identified a novel BCR-ABL1 fusion gene with breakpoints in the BCR intron 14 and the ABL1 intron 2, leading to partial deletion of its SH3 domain. In the present case, the patient received targeted therapy with the TKI imatinib at 400 mg/day and no adverse reaction was reported. The patient eventually entered remission with decreased proliferation of karyocytes and granulocytes. We also identified mutations in genes, including TP53 , FLT3 , ASXL1 , SETBP1 , CEBPA and CBL, that seemed to have an influence on the outcome of TKI therapy targeting the BCR-ABL1 protein. Together with previously reported results, it is clear that the genetic heterogeneity of CML patients significantly affects the presentation of the disease and its progression and therefore should inform the design of the therapeutic strategy.

  18. 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.

  19. 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

  20. Coexistence of p190 BCR/ABL Transcript and CALR 52-bp Deletion in Chronic Myeloid Leukemia Blast Crisis: A Case Report.

    Science.gov (United States)

    Seghatoleslami, Mohammad; Ketabchi, Neda; Ordo, Alireza; Asl, Javad Mohammadi; Golchin, Neda; Saki, Najmaldin

    2016-01-01

    We introduce a 78-year-old woman presented with thrombocytosis and high blast count who had a history of splenectomy. Her cytogenetic analysis revealed aberrant chromosomal rearrangements in different clonal populations harboring 46XX karyotype with t(9;22) (q34;q11). RT-PCR assay detected the e1a2 BCR-ABL translocation resulting from rearrangement of the minor breakpoint cluster region (m-bcr) in BCR gene. Subsequent evaluation of the disease showed calreticulin (CALR) 52-bp deletion as well as the absence of JAK2 (V617F) heterozygous mutation in granulocyte population of peripheral blood using allele-specific PCR and bi-directional DNA sequencing. To our knowledge, this is the first case of a patient initially diagnosed as p190 BCR-ABL transcript positive CML in blast crisis characterized by a 52-bp deletion in CALR gene.

  1. Coexistence of P190 BCR/ABL transcript and CALR 52-bp deletion in chronic myeloid leukemia blast crisis: a case report

    Directory of Open Access Journals (Sweden)

    najmaldin saki

    2016-01-01

    Full Text Available We present a case of a 78-year-old woman presented with thrombocytosis and high blast count, who had a history of splenectomy. Her cytogenetic analysis revealed aberrant chromosomal rearrangements in different clonal populations harboring 46XX karyotype with t(9;22(q34;q11. RT-PCR assay detected the e1a2 BCR-ABL translocation resulting from a rearrangement of the minor breakpoint cluster region (m-bcr in the BCR gene. Subsequent evaluations of the disease showed calreticulin (CALR 52-bp deletion as well as the absence of JAK2V617F heterozygous mutation in granulocyte population of peripheral blood using allele-specific PCR and bi-directional DNA sequencing. To our knowledge, this is the first case of a patient initially diagnosed as p190 BCR-ABL transcript positive CML in blastic crisis characterized with a 52-bp deletion in CALR gene.

  2. A novel BCR-ABL transcript e2a2 in a chronic myelogenous leukaemia patient with a duplicated Ph-chromosome and monosomy 7.

    Science.gov (United States)

    Leibundgut, E O; Jotterand, M; Rigamonti, V; Parlier, V; Mühlematter, D; Tobler, A; Solenthaler, M

    1999-09-01

    A novel BCR-ABL transcript was detected by multiplex RT-PCR in a patient with Philadelphia chromosome (Ph) positive chronic myelogenous leukaemia (CML) in accelerated phase. Sequencing of the aberrant transcript revealed an in-frame e2a2 fusion that included a 9 basepairs insertion. Cytogenetic analysis showed t(9;22), an additional Ph chromosome and monosomy 7. The clinical course was dismal: therapy was poorly tolerated, and the patient died in blast crisis 10 months after diagnosis. These data support the association of additional Ph and monosomy 7 with poor prognosis and suggest that the novel e2a2 BCR-ABL transcript may be related to an aggressive clinical course.

  3. Frequency and clinical impact of ETV6/RUNX1, AF4-MLL, and BCR/ABL fusion genes on features of acute lymphoblastic leukemia at presentation.

    Science.gov (United States)

    Ajuba, I C; Madu, A J; Okocha, C; Ibegbulam, O G; Okpala, I; Nna, O E

    2016-01-01

    Variations in disease presentation and outcome of leukemia treatment has been associated with the presence of certain mutant genes. Three major translocations (ETV6-RUNX1, BCR-ABL, and AF4-MLL) in acute lymphoblastic leukemia (ALL) have been shown to affect treatment outcome. This study is aimed at assessing the relationship between these translocations and the presence of other indicators of disease severity (white cell count, hemoglobin concentration, platelet count, and hematocrit) in ALL. Forty chemotherapy naïve patients aged between 9 months and 54 years had their marrow samples analyzed for the prevalent mutations. Their clinical and laboratory details on presentation were also obtained. Abnormal genes detected were BCR/ABL1 major transcript in 5 (12.5%), ETV6/RUNX1 in 2 (5.0%), MLL/AF4 none and none of the patients had more than one fusion gene. There was no relationship between the presence of these fusion genes and the clinical and laboratory features of ALL. An association exists between the fusion genes and ethnic origin of the patients (P = 0.005). There is no significant association between the abnormal fusion genes detected and some laboratory features of prognostic importance, which include total white blood cell count (P = 0.416) and FAB subtype (P = 0.576). Presence of fusion the genes BCR/ABL1, ETV6/RUNX1, and MLL/AF4 does not have any impact on the clinical and laboratory features of ALL at presentation.

  4. 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. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    National Research Council Canada - National Science Library

    Reddy, E. P

    2007-01-01

    Because it is now apparent that a significant proportion of patients chronically treated with imatinib develop resistance due to the acquisition of mutations in the kinase domain of BCR-ABL our aim...

  6. BCR-ABL, ETV6-RUNX1 and E2A-PBX1: prevalence of the most common acute lymphoblastic leukemia fusion genes in Mexican patients.

    Science.gov (United States)

    Jiménez-Morales, S; Miranda-Peralta, E; Saldaña-Alvarez, Y; Perez-Vera, P; Paredes-Aguilera, R; Rivera-Luna, R; Velázquez-Cruz, R; Ramírez-Bello, J; Carnevale, A; Orozco, L

    2008-10-01

    This study was conducted to determine the frequency of the most common fusion genes in Mexican pediatric patients with acute lymphoblastic leukemia (ALL). Molecular analysis using RT-PCR was carried out in 53-blood samples: 52 patients with de novo ALL and one with relapsed ALL. The ETV6-RUNX1 fusion was found in 7 cases (13.5%), BCR-ABL fusion was detected in 2 cases (3.8%), and 6 patients (11.5%) expressed the chimeric gene E2A-PBX1. The prevalence of E2A-PBX1 is one of the highest that has been described thus far in childhood ALL. Furthermore, we detected both the BCR-ABL, and E2A-PBX1 fusion in the relapsed patient. With regards to the immunophenotype, ETV6-RUNX1 was expressed in both pre-B and T-cell cases, while the presence of E2A-PBX1 and BCR-ABL was associated with the pre-B ALL phenotype. The prevalence of E2A-PBX1 in Mexican pediatric cases supports the existence of ethnic differences in the frequency of molecular markers of ALL.

  7. BCR-ABL1 tyrosine kinase inhibitors for the treatment of chronic myeloid leukemia.

    Science.gov (United States)

    Cuellar, Sandra; Vozniak, Michael; Rhodes, Jill; Forcello, Nicholas; Olszta, Daniel

    2017-01-01

    The management of chronic myeloid leukemia with BCR-ABL1 tyrosine kinase inhibitors has evolved chronic myeloid leukemia into a chronic, manageable disease. A patient-centered approach is important for the appropriate management of chronic myeloid leukemia and optimization of long-term treatment outcomes. The pharmacist plays a key role in treatment selection, monitoring drug-drug interactions, identification and management of adverse events, and educating patients on adherence. The combination of tyrosine kinase inhibitors with unique safety profiles and individual patients with unique medical histories can make managing treatment difficult. This review will provide up-to-date information regarding tyrosine kinase inhibitor-based treatment of patients with chronic myeloid leukemia. Management strategies for adverse events and considerations for drug-drug interactions will not only vary among patients but also across tyrosine kinase inhibitors. Drug-drug interactions can be mild to severe. In instances where co-administration of concomitant medications cannot be avoided, it is critical to understand how drug levels are impacted and how subsequent dose modifications ensure therapeutic drug levels are maintained. An important component of patient-centered management of chronic myeloid leukemia also includes educating patients on the significance of early and regular monitoring of therapeutic milestones, emphasizing the importance of adhering to treatment in achieving these targets, and appropriately modifying treatment if these clinical goals are not being met. Overall, staying apprised of current research, utilizing the close pharmacist-patient relationship, and having regular interactions with patients, will help achieve successful long-term treatment of chronic myeloid leukemia in the age of BCR-ABL1 tyrosine kinase inhibitors.

  8. 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.

  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. [Early monitoring of BCR-ABL transcript levels and cytogenetic in assessing the prognosis of chronic myeloid leukemia].

    Science.gov (United States)

    Huang, Qin; Zhang, Xiao-yan; Li, Yan; Wang, Xiao-min

    2013-10-15

    To explore the prognostic significance of early monitoring of BCR-ABL transcript levels and cytogenetic evaluations for chronic myeloid leukemia in chronic phase (CML-CP). From July 2007 to May 2012, 56 CML-CP patients received oral imatinib 400 mg/d. The BCR-ABL transcript levels were monitored and cytogenetic examinations performed after 3 and 6 months respectively. The median follow-up time was 48 months. The 3-month BCR-ABL transcript levels ≤ 10% of patients 5-year overall survival (OS) and progression-free survival (PFS) were better than BCR-ABL transcript levels >10% of patients (OS: 100% vs 84.6%, P = 0.011; PFS: 94.6% vs 67.7%, P = 0.045); cytogenetics: Ph(+) ≤ 35 % of patients 5-year OS and PFS better than Ph(+) > 35% of patients (OS: 100% vs 76.2%, P = 0.001; PFS: 95.2% vs 38.1%, P = 0.001); the 6-month BCR-ABL transcripts level ≤ 1% of patients 5-year OS and PFS also better than BCR-ABL transcript levels> 1% of patients (OS: 100% vs 71.4%, P = 0.000; PFS: 95.2% vs 47.6%, P = 0.001); Ph(+) = 0% and Ph(+)> 0% patients, 5-year OS and PFS were significantly different (OS: 100% vs 68.6%, P = 0.000; PFS: 95.3% vs 45.7%, P = 0.000). Early molecular biology and cytogenetics monitoring have some significance in the prognostic assessment of CML-CP. And individualized treatment strategies should be based upon the monitoring results in conjunctions with comprehensive judgments.

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

    Directory of Open Access Journals (Sweden)

    P. V. B. Carvalho

    2003-01-01

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

  12. 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

    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

  13. 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 (R CT ) 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  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. Combined inhibition of β-catenin and Bcr-Abl synergistically targets tyrosine kinase inhibitor-resistant blast crisis chronic myeloid leukemia blasts and progenitors in vitro and in vivo.

    Science.gov (United States)

    Zhou, H; Mak, P Y; Mu, H; Mak, D H; Zeng, Z; Cortes, J; Liu, Q; Andreeff, M; Carter, B Z

    2017-10-01

    Tyrosine kinase inhibitor (TKI) resistance and progression to blast crisis (BC), both related to persistent β-catenin activation, remain formidable challenges for chronic myeloid leukemia (CML). We observed overexpression of β-catenin in BC-CML stem/progenitor cells, particularly in granulocyte-macrophage progenitors, and highest among a novel CD34 + CD38 + CD123 hi Tim-3 hi subset as determined by CyTOF analysis. Co-culture with mesenchymal stromal cells (MSCs) induced the expression of β-catenin and its target CD44 in CML cells. A novel Wnt/β-catenin signaling modulator, C82, and nilotinib synergistically killed KBM5 T315I and TKI-resistant primary BC-CML cells with or without BCR-ABL kinase mutations even under leukemia/MSC co-culture conditions. Silencing of β-catenin by short interfering RNA restored sensitivity of primary BCR-ABL T315I/E255V BC-CML cells to nilotinib. Combining the C82 pro-drug, PRI-724, with nilotinib significantly prolonged the survival of NOD/SCID/IL2Rγ null mice injected with primary BCR-ABL T315I/E255V BC-CML cells. The combined treatment selectively targeted CML progenitors and inhibited CD44, c-Myc, survivin, p-CRKL and p-STAT5 expression. In addition, pretreating primary BC-CML cells with C82, or the combination, but not with nilotinib alone, significantly impaired their engraftment potential in NOD/SCID/IL2Rγ-null-3/GM/SF mice and significantly prolonged survival. Our data suggest potential benefit of concomitant β-catenin and Bcr-Abl inhibition to prevent or overcome Bcr-Abl kinase-dependent or -independent TKI resistance in BC-CML.

  16. bcr-abl oncogene activation in Philadelphia chromosome-positive acute lymphoblastic leukemia

    NARCIS (Netherlands)

    Hermans, A.; Gow, J.; Selleri, L.; von Lindern, M.; Hagemeijer, A.; Wiedemann, L. M.; Grosveld, G.

    1988-01-01

    Tumor-specific alterations in oncogenes are thought to play a central role in the development of cancer. An example is the consistent fusion of the bcr gene to the c-abl oncogene on the Ph chromosome in CML. The Ph chromosome can also be observed in ALL. About 50% of Ph+ ALL cases, in contrast to

  17. SÍNTESES E PROPRIEDADES DE FÁRMACOS INIBIDORES DA TIROSINA QUINASE BCR-ABL, UTILIZADOS NO TRATAMENTO DA LEUCEMIA MIELOIDE CRÔNICA

    Directory of Open Access Journals (Sweden)

    Liviane D. de Azevedo

    Full Text Available The chronic myeloid leukemia (CML is characterized by presence of the Philadelphia chromosome (Ph, originated from the translocation between chromosomes 9 and 22. This chromosome generates an abnormal protein tyrosine kinase which is responsible for tumor cell proliferation. The emergence of tyrosine kinase inhibitors (TKIs has transformed the treatment of CML and imatinib being the first representative of this class. Although treatment with imatinib has reached surprising results, approximately 30% of patients exhibited resistance, especially in later stages of the disease. This fact stimulated the development of novel BCR-ABL enzyme inhibitors drugs classified as tyrosine kinase inhibitors (TKIs of second and third generations. The TKIs have different chemical functions in their structure, and the knowledge of synthetic methods for preparation of these compounds can be a powerful tool for the development of new derivatives. The five approved BCR-ABL Tyrosine Kinase inhibitors (TKI used in Chronic Myeloid Leukemia (CML are reviewed aiming the main synthetic routes, highlighting the advantages and disadvantages associated with them.

  18. 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

    Cancer Research, a biochemist with a research focus on tumor suppressor and cell cycle checkpoint signaling pathways. For more information about...the treatment of cancer. Cell Cycle . 2004;3:779-788. 12. Nimmanapalli R, Fuino L, Stobaugh C, Richon V, Bhalla K. Cotreatment with the histone...deacetylase inhibitors for the treatment of cancer. Cell Cycle . 2004;3:779-788. 26. Guo F, Sigua C, Tao J, et al. Cotreatment with histone deacetylase

  19. Measurement of adherence to BCR-ABL inhibitor therapy in chronic myeloid leukemia: current situation and future challenges.

    Science.gov (United States)

    Noens, Lucien; Hensen, Marja; Kucmin-Bemelmans, Izabela; Lofgren, Christina; Gilloteau, Isabelle; Vrijens, Bernard

    2014-03-01

    BCR-ABL inhibitors for treating chronic myeloid leukemia in chronic phase have transformed a previously incurable malignancy into a manageable condition. However, suboptimal medication adherence has been observed with these agents affecting clinical outcomes and healthcare costs. In order to raise awareness of the problem of adherence, and before developing pragmatic strategies to enhance medication adherence, a deep understanding of the best approaches for measuring adherence in chronic myeloid leukemia patients and identifying non-adherence is required. A systematic literature review on the prevalence, measurement methods, consequences and risk factors for non-adherence to BCR-ABL inhibitors and adherence-enhancing interventions was performed and critically appraised. Of the 19 included articles, 9 were retrospective. Average adherence varied from 19% to almost 100% of the proportion of prescribed drug taken, but it was measured through various different methods and within different study groups. Suboptimal adherence was associated with a negative impact on both clinical and economic outcomes. There is a lack of supportive evidence demonstrating a difference in adherence across BCR-ABL inhibitors and even contradictory results between the 2(nd) generation inhibitors. Drug-related adverse events and forgetfulness were common reasons for intentional and unintentional non-adherence, respectively, but further research is required to identify additional reasons behind non-adherence or patients at risk of non-adherence. Non-adherence in chronic myeloid leukemia patients treated with BCR-ABL inhibitors is common and associated with critical outcomes. However, this review highlights important existing gaps, reveals inconsistent definitions, and a lack of standardized methods for measuring adherence in chronic myeloid leukemia. All require further investigation.

  20. 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.

  1. 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.

  2. 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

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

    Directory of Open Access Journals (Sweden)

    Ganguly Bani

    2007-01-01

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

  4. Screening und Charakterisierung von Peptidliganden für den BCR-ABL mRNA Translokationsbereich

    OpenAIRE

    Bäumler, Jörg

    2006-01-01

    Die reziproke Translokation t(9;22) ist in 95% der chronischen myeloischen Leukämie vorhanden. Bei der Translokation entsteht ein Fusionsprotein BCR-ABL, welches ausreichend für die Entstehung von Leukämien ist. 30% aller akuten lymphatischen Leukämien sind ebenfalls positiv für diese Translokation. Durch die Translokation entsteht am Translokationsbruchpunkt eine einzigartige RNA-Sequenz, welche als Ziel für eine RNA-Liganden Suche dienen kann. Ziel dieser Arbeit war es, Peptidliganden zu fi...

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    Stroncek David F

    2006-10-01

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

  7. Andrographolide downregulates the v-Src and Bcr-Abl oncoproteins and induces Hsp90 cleavage in the ROS-dependent suppression of cancer malignancy.

    Science.gov (United States)

    Liu, Sheng-Hung; Lin, Chao-Hsiung; Liang, Fong-Ping; Chen, Pei-Fen; Kuo, Cheng-Deng; Alam, Mohd Mujahid; Maiti, Barnali; Hung, Shih-Kai; Chi, Chin-Wen; Sun, Chung-Ming; Fu, Shu-Ling

    2014-01-15

    Andrographolide is a diterpenoid compound isolated from Andrographis paniculata that exhibits anticancer activity. We previously reported that andrographolide suppressed v-Src-mediated cellular transformation by promoting the degradation of Src. In the present study, we demonstrated the involvement of Hsp90 in the andrographolide-mediated inhibition of Src oncogenic activity. Using a proteomics approach, a cleavage fragment of Hsp90α was identified in andrographolide-treated cells. The concentration- and time-dependent induction of Hsp90 cleavage that accompanied the reduction in Src was validated in RK3E cells transformed with either v-Src or a human truncated c-Src variant and treated with andrographolide. In cancer cells, the induction of Hsp90 cleavage by andrographolide and its structural derivatives correlated well with decreased Src levels, the suppression of transformation, and the induction of apoptosis. Moreover, the andrographolide-induced Hsp90 cleavage, Src degradation, inhibition of transformation, and induction of apoptosis were abolished by a ROS inhibitor, N-acetyl-cysteine. Notably, Hsp90 cleavage, decreased levels of Bcr-Abl (another known Hsp90 client protein), and the induction of apoptosis were also observed in human K562 leukemia cells treated with andrographolide or its active derivatives. Together, we demonstrated a novel mechanism by which andrographolide suppressed cancer malignancy that involved inhibiting Hsp90 function and reducing the levels of Hsp90 client proteins. Our results broaden the molecular basis of andrographolide-mediated anticancer activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Identification of novel tyrosine kinase inhibitors for drug resistant T315I mutant BCR-ABL: a virtual screening and molecular dynamics simulations study

    Science.gov (United States)

    Banavath, Hemanth Naick; Sharma, Om Prakash; Kumar, Muthuvel Suresh; Baskaran, R.

    2014-11-01

    BCR-ABL tyrosine kinase plays a major role in the pathogenesis of chronic myeloid leukemia (CML) and is a proven target for drug development. Currently available drugs in the market are effective against CML; however, side-effects and drug-resistant mutations in BCR-ABL limit their full potential. Using high throughput virtual screening approach, we have screened several small molecule databases and docked against wild-type and drug resistant T315I mutant BCR-ABL. Drugs that are currently available, such as imatinib and ponatinib, were also docked against BCR-ABL protein to set a cutoff value for our screening. Selected lead compounds were further evaluated for chemical reactivity employing density functional theory approach, all selected ligands shows HLG value > 0.09900 and the binding free energy between protein-ligand complex interactions obtained was rescored using MM-GBSA. The selected compounds showed least ΔG score -71.53 KJ/mol to maximum -126.71 KJ/mol in both wild type and drug resistant T315I mutant BCR-ABL. Following which, the stability of the docking complexes were evaluated by molecular dynamics simulation (MD) using GROMACS4.5.5. Results uncovered seven lead molecules, designated with Drug-Bank and PubChem ids as DB07107, DB06977, ST013616, DB04200, ST007180 ST019342, and DB01172, which shows docking scores higher than imatinib and ponatinib.

  9. A case of acute myeloid leukemia with e6a2 BCR-ABL fusion transcript acquired after progressing from chronic myelomonocytic leukemia

    OpenAIRE

    Jinjuan Yao; Dan Douer; Lu Wang; Maria E. Arcila; Khedoudja Nafa; April Chiu

    2017-01-01

    Philadelphia (Ph) chromosome is a cytogenetic hallmark of chronic myeloid leukemia (CML). Most patients with CML harbor either the e13a2 or e14a2 BCR-ABL fusion product, while a small subset of the cases expresses e1a2 or e19a2 transcripts. We report a patient with chronic myelomonocytic leukemia (CMML), initially Ph chromosome negative at presentation, with rapid disease progression to acute myeloid leukemia (AML) and appearance of Ph chromosome and BCR-ABL e6a2, a very uncommon fusion trans...

  10. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Characteristics and outcome of chronic myeloid leukemia patients with E255K/V BCR-ABL kinase domain mutations.

    Science.gov (United States)

    Naqvi, Kiran; Cortes, Jorge E; Luthra, Raja; O'Brien, Susan; Wierda, William; Borthakur, Gautam; Kadia, Tapan; Garcia-Manero, Guillermo; Ravandi, Farhad; Rios, Mary Beth; Dellasala, Sara; Pierce, Sherry; Jabbour, Elias; Patel, Keyur; Kantarjian, Hagop

    2018-02-20

    Kinase domain (KD) mutations of ABL1 represent the most common resistance mechanism to tyrosine kinase inhibitors (TKI) in CML. Besides T315I, mutations in codon 255 are highly resistant mutations in vitro to all TKI. We aimed to study the incidence, prognosis, and response to treatment in patients with E255K/V. We evaluated 976 patients by sequencing of BCR-ABL1 fusion transcript for ABL1 KD mutations. We identified KD mutations in 381 (39%) patients, including E255K/V in 48 (13% of all mutations). At mutation detection, 14 patients (29%) were in chronic phase (CP), 12 (25%) in accelerated phase (AP), and 22 (46%) in blast phase (BP). 9/14 CP patients responded to treatment (best response complete hematologic response-CHR-4; complete cytogenetic response-CCyR-1; major molecular response-MMR-4); only 4/12 AP patients (CHR 3; MMR 1) and 7/22 BP patients responded (CCyR 2; MMR 2; partial cytogenetic response-PCyR-3). After a median follow-up of 65 months from mutation detection, 36 patients (75%) died: 9/14 (64%) in CP, 9/12 (75%) in AP, and 18/22 (82%) in BP (p = 0.003); median overall survival was 12 months. Patients with E255K/V mutation have a poor prognosis, regardless of the stage of the disease at detection.

  12. Frequency of the minor BCR-ABL (e1;a2 transcript oncogene in a Mexican population with adult acute lymphoblastic leukaemia

    Directory of Open Access Journals (Sweden)

    I. Olarte-Carrillo

    2015-07-01

    Conclusion: Prevalence of BCR-ABL expression by RT-PCR has not previously been reported in Mexico. Our laboratory found a higher prevalence than that reported in Latin-American series, but lower than that reported for the European population.

  13. 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.

  14. 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...

  15. “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.

  16. 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.

  17. Measurement of BCR-ABL1 by RT-qPCR in chronic myeloid leukaemia: findings from an International EQA Programme.

    Science.gov (United States)

    Scott, Stuart; Travis, Debbie; Whitby, Liam; Bainbridge, John; Cross, Nicholas C P; Barnett, David

    2017-05-01

    Sequential measurement of BCR-ABL1 mRNA levels by reverse transcription quantitative polymerase chain reaction (RT-qPCR) is embedded in the management of patients with chronic myeloid leukaemia (CML), and has played an important role in the remarkable improvement in patient outcomes seen in this disease. As a provider of external quality assessment (EQA) in this area, UK NEQAS for Leucocyte Immunophenotyping (UKNEQAS LI) has a unique perspective on the changing face of BCR-ABL1 testing in CML. To assess the impact of technical standardisation and the development of the International Scale (IS) upon the accuracy of BCR-ABL1 testing, we reviewed EQA trial data from 2007 to 2015. Comparison of participant results identified considerable variability at both high and low levels of disease, including therapeutically important decision points; however, results converted to the IS showed less variability compared to unconverted data sets. We also found that different methods of converting to the IS produce consistently different median results within UKNEQAS LI IS data sets. This data suggests that whilst the development of the IS has improved the comparability of results between centres, there is still the need for further improvement in the processes of converting raw results to the IS in order to fully realise the benefits of molecular monitoring of CML. © 2017 John Wiley & Sons Ltd.

  18. Design and analytic validation of BCR-ABL1 quantitative reverse transcription polymerase chain reaction assay for monitoring minimal residual disease.

    Science.gov (United States)

    Jennings, Lawrence J; Smith, Frederick A; Halling, Kevin C; Persons, Diane L; Kamel-Reid, Suzanne

    2012-01-01

    Monitoring minimal residual disease by quantitative reverse transcription polymerase chain reaction has proven clinically useful, but as yet there are no Food and Drug Administration-approved tests. Guidelines have been published that provide important information on validation of such tests; however, no practical examples have previously been published. To provide an example of the design and validation of a quantitative reverse transcription polymerase chain reaction test. To describe the approach used by an individual laboratory for development and validation of a laboratory-developed quantitative reverse transcription polymerase chain reaction test for BCR-ABL1 fusion transcripts. Elements of design and analytic validation of a laboratory-developed quantitative molecular test are discussed using quantitative detection of BCR-ABL1 fusion transcripts as an example. Validation of laboratory-developed quantitative molecular tests requires careful planning and execution to adequately address all required analytic performance parameters. How these are addressed depends on the potential for technical errors and confidence required for a given test result. We demonstrate how one laboratory validated and clinically implemented a quantitative BCR-ABL1 assay that can be used for the management of patients with chronic myelogenous leukemia.

  19. 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.

  20. 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, Genoveva; 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

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

    International Nuclear Information System (INIS)

    Xia, D.Y.; Liu, L.; Hao, M.W.; Liu, Q.; Chen, R.A.; Liang, Y.M.

    2014-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-10-14

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

  3. Adaptive immunity to leukemia is inhibited by cross-reactive induced regulatory T cells

    OpenAIRE

    Manlove, Luke S.; Berquam-Vrieze, Katherine E.; Pauken, Kristen E.; Williams, Richard T.; Jenkins, Marc K.; Farrar, Michael A.

    2015-01-01

    BCR-ABL+ acute lymphoblastic leukemia patients have transient responses to current therapies. However, the fusion of BCR to ABL generates a potential leukemia-specific antigen that could be a target for immunotherapy. We demonstrate that the immune system can limit BCR-ABL+ leukemia progression although ultimately this immune response fails. To address how BCR-ABL+ leukemia escapes immune surveillance, we developed a peptide: MHC-II tetramer that labels endogenous BCR-ABL-specific CD4+ T cell...

  4. 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. © 2016 by the Association of Clinical Scientists, Inc.

  5. 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

    Chronic myeloid leukemia (CML) is genetically characterized by the Philadelphia (Ph) chromosome, formed through a reciprocal translocation between chromosomes 9 and 22 and giving rise to the constitutively active tyrosine kinase P210 BCR/ABL1. Therapeutic strategies aiming for a cure of CML...... 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...

  6. Comparative study of different methodologies to detect the JAK2 V617F mutation in chronic BCR-ABL1 negative myeloproliferative neoplasms

    Directory of Open Access Journals (Sweden)

    Alline Didone

    2016-04-01

    Full Text Available Objectives: A mutation in the JAK2 gene, V617F, has been identified in several BCR-ABL1 negative myeloproliferative neoplasms (MPN: polycythemia vera (PV, essential thrombocythemia (ET, and primary myelofibrosis (PMF. Defining the presence or absence of this mutation is an essential part of clinical diagnostic algorithms and patient management. Here, we aimed to evaluate the performance of three PCR-based assays: Amplification Refractory Mutation System (ARMS, High-Resolution Melting analysis (HRM, and Sanger direct sequencing, and compare their results with those obtained by a PCR restriction fragment polymorphism assay (PCR-RFLP. Design and methods: We used blood samples from 136 patients (PV=20; PMF=20; ET=28, and other MPN suspected cases=68. Results: Comparable results were observed among the four assays in patients with PV, PMF, and MPN suspected cases. In patients with a diagnosis of ET, the JAK2 V617F mutation was detected in 67.8% of them by the PCR-ARMS and PCR-HRM assay and in 64% of them by the conventional Sanger sequence approach. The PCR-ARMS and PCR-HRM assays were 100% concordant. With these tests, only one of the 20 patients with ET and one of the three patients with clinically suspected MPN gave different results compared with those obtained by the PCR-RFLP. Conclusions: Our results have demonstrated that the PCR-ARMS and PCR-HRM assays could detect the JAK2 V617F mutation effectively in MPN patients, but PCR-HRM assays are rapid and the most cost-effective procedures. Keywords: Myeloproliferative, JAK2 V617F, Mutation, Wild type, Screening

  7. In chronic myeloid leukemia patients on second-line tyrosine kinase inhibitor therapy, deep sequencing of BCR-ABL1 at the time of warning may allow sensitive detection of emerging drug-resistant mutants.

    Science.gov (United States)

    Soverini, Simona; De Benedittis, Caterina; Castagnetti, Fausto; Gugliotta, Gabriele; Mancini, Manuela; Bavaro, Luana; Machova Polakova, Katerina; Linhartova, Jana; Iurlo, Alessandra; Russo, Domenico; Pane, Fabrizio; Saglio, Giuseppe; Rosti, Gianantonio; Cavo, Michele; Baccarani, Michele; Martinelli, Giovanni

    2016-08-02

    Imatinib-resistant chronic myeloid leukemia (CML) patients receiving second-line tyrosine kinase inhibitor (TKI) therapy with dasatinib or nilotinib have a higher risk of disease relapse and progression and not infrequently BCR-ABL1 kinase domain (KD) mutations are implicated in therapeutic failure. In this setting, earlier detection of emerging BCR-ABL1 KD mutations would offer greater chances of efficacy for subsequent salvage therapy and limit the biological consequences of full BCR-ABL1 kinase reactivation. Taking advantage of an already set up and validated next-generation deep amplicon sequencing (DS) assay, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse. a total of 125 longitudinal samples from 51 CML patients who had acquired dasatinib- or nilotinib-resistant mutations during second-line therapy were analyzed by DS from the time of failure and mutation detection by conventional sequencing backwards. BCR-ABL1/ABL1%(IS) transcript levels were used to define whether the patient had 'optimal response', 'warning' or 'failure' at the time of first mutation detection by DS. DS was able to backtrack dasatinib- or nilotinib-resistant mutations to the previous sample(s) in 23/51 (45 %) pts. Median mutation burden at the time of first detection by DS was 5.5 % (range, 1.5-17.5 %); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 1-9 months). In 5 cases, the mutations were detectable at baseline. In the remaining cases, response level at the time mutations were first detected by DS could be defined as 'Warning' (according to the 2013 ELN definitions of response to 2nd-line therapy) in 13 cases, as 'Optimal response' in one case, as 'Failure' in 4 cases. No dasatinib- or nilotinib-resistant mutations were detected by DS in 15 randomly selected patients with 'warning' at various timepoints, that later turned into optimal

  8. 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. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  9. Adaptive Immunity to Leukemia Is Inhibited by Cross-Reactive Induced Regulatory T Cells.

    Science.gov (United States)

    Manlove, Luke S; Berquam-Vrieze, Katherine E; Pauken, Kristen E; Williams, Richard T; Jenkins, Marc K; Farrar, Michael A

    2015-10-15

    BCR-ABL(+) acute lymphoblastic leukemia patients have transient responses to current therapies. However, the fusion of BCR to ABL generates a potential leukemia-specific Ag that could be a target for immunotherapy. We demonstrate that the immune system can limit BCR-ABL(+) leukemia progression although ultimately this immune response fails. To address how BCR-ABL(+) leukemia escapes immune surveillance, we developed a peptide: MHC class II tetramer that labels endogenous BCR-ABL-specific CD4(+) T cells. Naive mice harbored a small population of BCR-ABL-specific T cells that proliferated modestly upon immunization. The small number of naive BCR-ABL-specific T cells was due to negative selection in the thymus, which depleted BCR-ABL-specific T cells. Consistent with this observation, we saw that BCR-ABL-specific T cells were cross-reactive with an endogenous peptide derived from ABL. Despite this cross-reactivity, the remaining population of BCR-ABL reactive T cells proliferated upon immunization with the BCR-ABL fusion peptide and adjuvant. In response to BCR-ABL(+) leukemia, BCR-ABL-specific T cells proliferated and converted into regulatory T (Treg) cells, a process that was dependent on cross-reactivity with self-antigen, TGF-β1, and MHC class II Ag presentation by leukemic cells. Treg cells were critical for leukemia progression in C57BL/6 mice, as transient Treg cell ablation led to extended survival of leukemic mice. Thus, BCR-ABL(+) leukemia actively suppresses antileukemia immune responses by converting cross-reactive leukemia-specific T cells into Treg cells. Copyright © 2015 by The American Association of Immunologists, Inc.

  10. New mutations detected by denaturing high performance liquid chromatography during screening of exon 6 bcr-abl mutations in patients with chronic myeloid leukemia treated with tyrosine kinase inhibitors.

    Science.gov (United States)

    Mascarenhas, Cintia C; Cunha, Anderson F; Miranda, Eliana C; Zulli, Roberto; Silveira, Rosana A; Costa, Fernando F; Pagnano, Katia B B; De Souza, Carmino A

    2009-07-01

    Point mutations within the ABL kinase domain are the most frequent mechanism for reactivation of kinase activity of the BCR-ABL gene and have been associated with clinical resistance to tyrosine kinase (TK) inhibitors in patients with CML, conferring a poor prognosis. T315I (Treonine-->Isoleucine) is a mutation in the exon 6 of BCR-ABL gene that makes the protein resistant to kinase inhibitors currently used for treating CML. Denaturing High-performance liquid chromatography (D-HPLC) allows for high throughput mutation screening. In this study, we screened mutations in exon 6 of the BCR-ABL gene in patients presenting failure or sub optimal response according to Leukemia Net criteria and correlated the presence of mutations with clinical outcome. Genomic DNA was extracted from peripheral blood samples from 93 patients with CML (5 intolerant and 88 resistant). The PCR product was analysed by D-HPLC, and the patients samples with abnormal D-HLPC profiles were submitted to automated sequencing, using specific primers. Overall survival (OS) was calculated from the date of mutation analysis, for the whole group and for both groups (mutation versus no mutation). We screened mutations in exon 6 of the BCR-ABL gene in 93 CML TKI - resistant patients. Twenty-three out of 93 samples (25%) showed an abnormal elution profile. Automated sequencing confirmed the presence of a nucleotide change in 19 out of 23 cases: one polymorphism, T315T, seven known point mutations: T315I, F317L, V339L, M351T, E355G and F359V and three novel mutations: C305R, D325D and I360S. OS for the whole group was 80% in a median observation time of 30 months. OS for patients without the mutation was 87% and with the mutation was 56%, in a median observation time of 37 and 10 months, respectively (p < 0.0001, RR = 68). D-HPLC is a practical and sensitive method for routine clinical monitoring for emergence of kinase domain mutations and may be useful for optimising therapy in CML. The screening of

  11. The mTOR inhibitor, everolimus (RAD001), overcomes resistance to imatinib in quiescent Ph-positive acute lymphoblastic leukemia cells

    International Nuclear Information System (INIS)

    Kuwatsuka, Y; Minami, M; Minami, Y; Sugimoto, K; Hayakawa, F; Miyata, Y; Abe, A; Goff, D J; Kiyoi, H; Naoe, T

    2011-01-01

    In Ph-positive (Ph + ) leukemia, the quiescent cell state is one of the reasons for resistance to the BCR-ABL-kinase inhibitor, imatinib. In order to examine the mechanisms of resistance due to quiescence and the effect of the mammalian target of rapamycin inhibitor, everolimus, for such a resistant population, we used Ph + acute lymphoblastic leukemia patient cells serially xenotransplanted into NOD/SCID/IL2rγ null (NOG) mice. Spleen cells from leukemic mice showed a higher percentage of slow-cycling G 0 cells in the CD34 + CD38 − population compared with the CD34 + CD38 + and CD34 − populations. After ex vivo imatinib treatment, more residual cells were observed in the CD34 + CD38 − population than in the other populations. Although slow-cycling G 0 cells were insensitive to imatinib in spite of BCR-ABL and CrkL dephosphorylation, combination treatment with everolimus induced substantial cell death, including that of the CD34 + CD38 − population, with p70-S6 K dephosphorylation and decrease of MCL-1 expression. The leukemic non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse system with the in vivo combination treatment with imatinib and everolimus showed a decrease of tumor burden including CD34 + cells. These results imply that treatment with everolimus can overcome resistance to imatinib in Ph + leukemia due to quiescence

  12. 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.

  13. Radotinib induces high cytotoxicity in c-KIT positive acute myeloid leukemia cells.

    Science.gov (United States)

    Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk

    2017-06-05

    Previously, we reported that radotinib, a BCR-ABL1 tyrosine kinase inhibitor, induced cytotoxicity in acute myeloid leukemia (AML) cells. However, the effects of radotinib in the subpopulation of c-KIT-positive AML cells were unclear. We observed that low-concentration radotinib had more potent cytotoxicity in c-KIT-positive cells than c-KIT-negative cells from AML patients. To address this issue, cell lines with high c-KIT expression, HEL92.1.7, and moderate c-KIT expression, H209, were selected. HEL92.1.7 cells were grouped into intermediate and high c-KIT expression populations. The cytotoxicity of radotinib against the HEL92.1.7 cell population with intermediate c-KIT expression was not different from that of the population with high c-KIT expression. When H209 cells were grouped into c-KIT expression-negative and c-KIT expression-positive populations, radotinib induced cytotoxicity in the c-KIT-positive population, but not the c-KIT-negative population. Thus, radotinib induces cytotoxicity in c-KIT-positive cells, regardless of the c-KIT expression intensity. Therefore, radotinib induces significant cytotoxicity in c-KIT-positive AML cells, suggesting that radotinib is a potential target agent for the treatment of c-KIT-positive malignancies including AML. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Clinical end points for drug treatment trials in BCR-ABL1-negative classic myeloproliferative neoplasms: consensus statements from European LeukemiaNET (ELN) and Internation Working Group-Myeloproliferative Neoplasms Research and Treatment (IWG-MRT).

    Science.gov (United States)

    Barosi, G; Tefferi, A; Besses, C; Birgegard, G; Cervantes, F; Finazzi, G; Gisslinger, H; Griesshammer, M; Harrison, C; Hehlmann, R; Hermouet, S; Kiladjian, J-J; Kröger, N; Mesa, R; Mc Mullin, M F; Pardanani, A; Passamonti, F; Samuelsson, J; Vannucchi, A M; Reiter, A; Silver, R T; Verstovsek, S; Tognoni, G; Barbui, T

    2015-01-01

    The discovery of somatic mutations, primarily JAK2V617F and CALR, in classic BCR-ABL1-negative myeloproliferative neoplasms (MPNs) has generated interest in the development of molecularly targeted therapies, whose accurate assessment requires a standardized framework. A working group, comprised of members from European LeukemiaNet (ELN) and International Working Group for MPN Research and Treatment (IWG-MRT), prepared consensus-based recommendations regarding trial design, patient selection and definition of relevant end points. Accordingly, a response able to capture the long-term effect of the drug should be selected as the end point of phase II trials aimed at developing new drugs for MPNs. A time-to-event, such as overall survival, or progression-free survival or both, as co-primary end points, should measure efficacy in phase III studies. New drugs should be tested for preventing disease progression in myelofibrosis patients with early disease in randomized studies, and a time to event, such as progression-free or event-free survival should be the primary end point. Phase III trials aimed at preventing vascular events in polycythemia vera and essential thrombocythemia should be based on a selection of the target population based on new prognostic factors, including JAK2 mutation. In conclusion, we recommended a format for clinical trials in MPNs that facilitates communication between academic investigators, regulatory agencies and drug companies.

  15. The fundamental prevalence of chronic myeloid leukemia-generating clonogenic cells in the light of the neutrality theory of evolution.

    Science.gov (United States)

    Jankovic, G M; Pavlovic, M; Vukomanovic, D J; Colovic, M D; Lazarevic, V

    2001-01-01

    A variety of normal human tissues have been reported to harbor small cell populations carrying potentially oncogenic gene rearrangements. This backdrop of mutant cells may be present in the majority of healthy individuals and is apparently weakly selected against. This may provide empirical support for the concept of global neutrality, or near-neutrality (very weak selection), of many somatic mutations. Many healthy individuals, as well as patients with chronic myeloid leukemia, manifest the BCR-ABL fusion gene in blood cells. The presumed neutrality of the BCR-ABL rearrangement-carrying pluripotential hematopoietic stem cells and the relative uniformity of the incidence rate of CML worldwide were used to estimate the extent of the background of BCR-ABL-positive stem cells and the numerical size of the human pluripotential hematopoietic stem cell pool. Three different approaches (molecular-epidemiological, statistical, and population genetical) were employed. Each resulted in very similar estimates of the size of the stem cells carrying the BCR-ABL allele fusions (1.4 x 10(4) cells) and the size of the total human stem cell pool (1.6 x 10(9) cells per individual). The implication of these estimates in the context of the hierarchical nature of the stem cell pool is also considered. The presumptive smaller-sized population of CD34(-) stem cells could not be characterized by any of the approaches used as a "founding" population, representing an ultimate source of all hematopoietic progenitors, or as a subset of stem cells comprising a deeper "kinetic" segment of the total (10(9)-sized) stem cell compartment. (c)2001 Elsevier Science.

  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. No influence of BCR-ABL1 transcript types e13a2 and e14a2 on long-term survival: results in 1494 patients with chronic myeloid leukemia treated with imatinib.

    Science.gov (United States)

    Pfirrmann, Markus; Evtimova, Dobromira; Saussele, Susanne; Castagnetti, Fausto; Cervantes, Francisco; Janssen, Jeroen; Hoffmann, Verena S; Gugliotta, Gabriele; Hehlmann, Rüdiger; Hochhaus, Andreas; Hasford, Joerg; Baccarani, Michele

    2017-05-01

    The genomic break on the major breakpoint cluster region of chromosome 22 results in two BCR-ABL1 transcripts of different sizes, e14a2 and e13a2. Favorable survival probabilities of patients with chronic myeloid leukemia (CML) in combination with too small patient samples may yet have obstructed the observation of differences in overall survival of patients according to transcript type. To overcome potential power problems, overall survival (OS) probabilities and probabilities of CML-related death were analyzed in 1494 patients randomized to first-line imatinib treatment. OS probabilities and probabilities of dying of CML were compared using the log-rank or Gray test whichever was appropriate. Both tests were stratified for the EUTOS long-term survival score. Between the groups with a single transcript, neither OS probabilities (stratified log-rank test: p = 0.106) nor probabilities of CML-related death were significantly different (stratified Gray test: p = 0.256). Regarding OS, the Cox hazard ratio (HR) of transcript type e13a2 (n = 565) to type e14a2 (n = 738) was 1.332 (95% CI 0.940-1.887). Considering probabilities of leukemia-related death, the corresponding subdistribution HR resulted in 1.284 (95% CI 0.758-2.176). Outcome did not change if patients with both transcripts (n = 191) were added to the 738 with type e14a2 only. The prognostic association of transcript type and long-term survival outcome was weak and without clinical relevance. However, earlier reported differences in the rate and the depth of molecular response could be relevant for the chance of successfully discontinuing TKI treatment. The effect of transcript type on molecular relapse after discontinuation is unknown, yet.

  18. Assessment of response to imatinib therapy in patients with chronic myeloid leukemia with e13a2 and e14a2 transcripts of BCR/ABL1 gene

    International Nuclear Information System (INIS)

    Dmitrenko, Yi.V.; Fedorenko, V.Yi.; Shlyakhtichenko, T.Yu.; And others

    2015-01-01

    The influence of e13a2 and e14a2 transcripts of BCR/ABL1 gene on the efficiency of imatinib therapy in patients with chronic myeloid leukemia was assessed. We examined 508 patients with the chronic phase of chronic myeloid leukemia without radiation in anamnesis as well as 13 patients with the similar diagnosis and with confirmed presence of radiation exposure due to the Chornobyl Nuclear Power Plant accident. No significant differences in hematologic parameters, rate of additional chromosomal aberrations and variant translocations were observed between patients with e13a2 and e14a2 transcript. The overall survival and progression-free survival rates were not statistically different between two groups with different transcripts. However, the rate of event-free survival was statistically lower for the patients with e13a2 transcript compared to the ones with e14a2 transcript (51 % versus 62.0 %, p = 0.039). The number of primary resistant patients was 40 % regardless of the transcript expressed. A significant prevalence in incidence either of lost complete cytogenetic response or failure of the major molecular response was shown in patients with e13a2 transcript compared to patients with e14a2 transcripts (43.5 % versus 24.8 %, p = 0.015). Imatinib therapy is more effective for CML patients with e14a2 transcript compared to patients with e13a2 transcript expression. The transcript e13a2 can be viewed as a adverse prognostic factor for imatinib therapy of chronic myeloid leukemia

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

    Science.gov (United States)

    2018-03-05

    Acute Biphenotypic Leukemia; Acute Erythroid Leukemia; Acute Lymphoblastic Leukemia in Remission; Acute Megakaryoblastic Leukemia; Acute Myeloid Leukemia Arising From Previous Myelodysplastic Syndrome; Acute Myeloid Leukemia in Remission; Blasts Under 10 Percent of Bone Marrow Nucleated Cells; Blasts Under 5 Percent of Bone Marrow Nucleated Cells; Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Mixed Phenotype Acute Leukemia; Myelodysplastic Syndrome; Myelodysplastic Syndrome With Excess Blasts; Pancytopenia; Refractory Anemia; Secondary Acute Myeloid Leukemia

  20. Clinical Efficacy and Safety of First-Line Dasatinib Therapy and the Relevance of Velocity of BCR-ABL1 Transcript Decline for Achievement of Molecular Responses in Newly Diagnosed Chronic-Phase Chronic Myeloid Leukemia: Report from the Juntendo Yamanashi Cooperative Study Group.

    Science.gov (United States)

    Takaku, Tomoiku; Iriyama, Noriyoshi; Mitsumori, Toru; Sato, Eriko; Gotoh, Akihiko; Kirito, Keita; Noguchi, Masaaki; Koike, Michiaki; Sakamoto, Junichi; Oba, Koji; Komatsu, Norio

    2018-01-01

    The use of tyrosine kinase inhibitors led to an improvement in the prognoses of patients with chronic myeloid leukemia (CML). The aims of this study were to investigate the efficacy and safety of dasatinib in Japanese patients and to explore the factors that affect the achievement of molecular responses. The primary endpoint was a major molecular response (MMR) by 12 months. The halving time for BCR-ABL1 transcripts was calculated using transcript levels. Thirty-two patients with chronic-phase CML (CML-CP) were enrolled and 30 received 100 mg dasatinib once daily. At 24 months of follow-up, 21 (72%) and 24 (83%) patients achieved an MMR by 12 and 24 months, respectively; the rates of a deep molecular response (DMR) by 12 and 24 months were 48 and 59%, respectively. A shorter halving time of BCR-ABL1 transcripts (≤10.6 days) accurately predicted both an MMR and a DMR. The incidence of pleural effusion was 50%. Our study reconfirmed the efficacy and safety of dasatinib treatment in Japanese patients with newly diagnosed CML-CP. In addition, the usefulness of the halving time of BCR-ABL1 transcripts was validated. These data emphasize the significance of an early treatment response in achieving a DMR during dasatinib therapy. © 2017 S. Karger AG, Basel.

  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)

    2018-03-23

    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. Identification of Novel Genes and Candidate Targets in CML Stem Cells

    National Research Council Canada - National Science Library

    Eaves, Connie

    2008-01-01

    .... Though imatinib mesylate(IM) that targets BCR-ABL kinase activity is now widely used, its curative potential as a single agent is not sure, moreover it is unlikely to eliminate the CML stem cells either, which highlights...

  3. Naturally occurring CD4+ CD25+ FOXP3+ T-regulatory cells are increased in chronic myeloid leukemia patients not in complete cytogenetic remission and can be immunosuppressive.

    Science.gov (United States)

    Rojas, Jose M; Wang, Lihui; Owen, Sally; Knight, Katy; Watmough, Sarah J; Clark, Richard E

    2010-12-01

    Clinical presentation of chronic myeloid leukemia (CML) requires not only the deregulated tyrosine kinase BCR-ABL, but also the failure of an immune response against BCR-ABL-expressing cells. T-cell responses against BCR-ABL and other antigens are well-described, but their relevance to the in vivo control of CML is unclear. The suppressive role of naturally occurring T regulatory (T-reg) cells in antitumor immunity is well-established, although little is known about their role in modulating the T-cell response to BCR-ABL. Naturally occurring T-reg cells were characterized and quantified by flow cytometry in 39 CML patients and 10 healthy donors. Their function was studied by observing their effect on responses to purified protein derivative, a recall antigen, and on the response of an autologous T-cell line recognizing BCR-ABL. T-reg cells were CD4(+), CD25(+), FOXP3(+), CD127(low), and CD62L(high). T-reg numbers in patients in complete cytogenetic remission were significantly lower than in patients not in complete cytogenetic remission (p T-reg cell depletion using anti-CD25 selection enhanced proliferative responses to purified protein derivative. Furthermore, the interferon-γ and/or granzyme-B production of effector cells specific for viral peptides or a BCR-ABL HLA-A3-restricted peptide was inhibited when autologous T-reg cells were present. Taken together, these data suggest a role for T-reg cells in limiting immune responses in CML patients and this may include immune responses to BCR-ABL. The increased frequency of T-reg cells in patients with high levels of BCR-ABL transcripts indicates that an immune mechanism may be important in the control of CML. Copyright © 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  5. Oncogenic STAT5 signaling promotes oxidative stress in chronic myeloid leukemia cells by repressing antioxidant defenses.

    Science.gov (United States)

    Bourgeais, Jerome; Ishac, Nicole; Medrzycki, Magdalena; Brachet-Botineau, Marie; Desbourdes, Laura; Gouilleux-Gruart, Valerie; Pecnard, Emmanuel; Rouleux-Bonnin, Florence; Gyan, Emmanuel; Domenech, Jorge; Mazurier, Frederic; Moriggl, Richard; Bunting, Kevin D; Herault, Olivier; Gouilleux, Fabrice

    2017-06-27

    STAT5 transcription factors are frequently activated in hematopoietic neoplasms and are targets of various tyrosine kinase oncogenes. Evidences for a crosstalk between STAT5 and reactive oxygen species (ROS) metabolism have recently emerged but mechanisms involved in STAT5-mediated regulation of ROS still remain elusive. We demonstrate that sustained activation of STAT5 induced by Bcr-Abl in chronic myeloid leukemia (CML) cells promotes ROS production by repressing expression of two antioxidant enzymes, catalase and glutaredoxin-1(Glrx1). Downregulation of catalase and Glrx1 expression was also observed in primary cells from CML patients. Catalase was shown not only to reduce ROS levels but also, to induce quiescence in Bcr-Abl-positive leukemia cells. Furthermore, reduction of STAT5 phosphorylation and upregulation of catalase and Glrx1 were also evidenced in leukemia cells co-cultured with bone marrow stromal cells to mimic a leukemic niche. This caused downregulation of ROS levels and enhancement of leukemic cell quiescence. These data support a role of persistent STAT5 signaling in the regulation of ROS production in myeloid leukemias and highlight the repression of antioxidant defenses as an important regulatory mechanism.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-06-07

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

  7. Targeting of the BLT2 in chronic myeloid leukemia inhibits leukemia stem/progenitor cell function

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Meifang; Ai, Hongmei; Li, Tao [Department of Laboratory Medicine, JingZhou Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), Jingzhou (China); Rajoria, Pasupati; Shahu, Prakash [Department of Clinical Medicine, Medical School of Yangtze University, Jingzhou (China); Li, Xiansong, E-mail: lixiansongjz@hotmail.com [Department of Neurosurgery, JingZhou Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), Jingzhou (China)

    2016-04-15

    Imatinib, a tyrosine kinase inhibitor (TKI) has significantly improved clinical outcome for chronic myeloid leukemia (CML) patients. However, patients develop resistance when the disease progresses to the blast phase (BP) and the mechanisms are not well understood. Here we show that BCR-ABL activates BLT2 in hematopoietic stem/progenitor cells to promote leukemogenesis and this involves the p53 signaling pathway. Compared to normal bone marrow (NBM), the mRNA and protein levels of BLT2 are significantly increased in BP-CML CD34{sup +} stem/progenitor cells. This is correlated with increasing BCR-ABL expression. In contrast, knockdown of BCR-ABL or inhibition of its tyrosine kinase activity decreases Blt2 protein level. BLT2 inhibition induces apoptosis, inhibits proliferation, colony formation and self-renewal capacity of CD34{sup +} cells from TKI-resistant BP-CML patients. Importantly, the inhibitory effects of BCR-ABL TKI on CML stem/progenitor cells are further enhanced upon combination with BLT2 inhibition. We further show that BLT2 activation selectively suppresses p53 but not Wnt or BMP-mediated luciferase activity and transcription. Our results demonstrate that BLT2 is a novel pathway activated by BCR-ABL and critically involved in the resistance of BP-CML CD34{sup +} stem/progenitors to TKIs treatment. Our findings suggest that BLT2 and p53 can serve as therapeutic targets for CML treatment. - Highlights: • BCR-ABL regulates BLT2 expression to promote leukemogenesis. • BLT2 is essential to maintain CML cell function. • Activation of BLT2 suppresses p53 signaling pathway in CML cells. • Inhibition of BLT2 and BCR-ABL synergize in eliminating CML CD34{sup +} stem/progenitors.

  8. Targeting of the BLT2 in chronic myeloid leukemia inhibits leukemia stem/progenitor cell function

    International Nuclear Information System (INIS)

    Xiao, Meifang; Ai, Hongmei; Li, Tao; Rajoria, Pasupati; Shahu, Prakash; Li, Xiansong

    2016-01-01

    Imatinib, a tyrosine kinase inhibitor (TKI) has significantly improved clinical outcome for chronic myeloid leukemia (CML) patients. However, patients develop resistance when the disease progresses to the blast phase (BP) and the mechanisms are not well understood. Here we show that BCR-ABL activates BLT2 in hematopoietic stem/progenitor cells to promote leukemogenesis and this involves the p53 signaling pathway. Compared to normal bone marrow (NBM), the mRNA and protein levels of BLT2 are significantly increased in BP-CML CD34 + stem/progenitor cells. This is correlated with increasing BCR-ABL expression. In contrast, knockdown of BCR-ABL or inhibition of its tyrosine kinase activity decreases Blt2 protein level. BLT2 inhibition induces apoptosis, inhibits proliferation, colony formation and self-renewal capacity of CD34 + cells from TKI-resistant BP-CML patients. Importantly, the inhibitory effects of BCR-ABL TKI on CML stem/progenitor cells are further enhanced upon combination with BLT2 inhibition. We further show that BLT2 activation selectively suppresses p53 but not Wnt or BMP-mediated luciferase activity and transcription. Our results demonstrate that BLT2 is a novel pathway activated by BCR-ABL and critically involved in the resistance of BP-CML CD34 + stem/progenitors to TKIs treatment. Our findings suggest that BLT2 and p53 can serve as therapeutic targets for CML treatment. - Highlights: • BCR-ABL regulates BLT2 expression to promote leukemogenesis. • BLT2 is essential to maintain CML cell function. • Activation of BLT2 suppresses p53 signaling pathway in CML cells. • Inhibition of BLT2 and BCR-ABL synergize in eliminating CML CD34 + stem/progenitors.

  9. Intracellular Retention of ABL Kinase Inhibitors Determines Commitment to Apoptosis in CML Cells

    Science.gov (United States)

    Dziadosz, Marek; Schnöder, Tina; Heidel, Florian; Schemionek, Mirle; Melo, Junia V.; Kindler, Thomas; Müller-Tidow, Carsten; Koschmieder, Steffen; Fischer, Thomas

    2012-01-01

    Clinical development of imatinib in CML established continuous target inhibition as a paradigm for successful tyrosine kinase inhibitor (TKI) therapy. However, recent reports suggested that transient potent target inhibition of BCR-ABL by high-dose TKI (HD-TKI) pulse-exposure is sufficient to irreversibly commit cells to apoptosis. Here, we report a novel mechanism of prolonged intracellular TKI activity upon HD-TKI pulse-exposure (imatinib, dasatinib) in BCR-ABL-positive cells. Comprehensive mechanistic exploration revealed dramatic intracellular accumulation of TKIs which closely correlated with induction of apoptosis. Cells were rescued from apoptosis upon HD-TKI pulse either by repetitive drug wash-out or by overexpression of ABC-family drug transporters. Inhibition of ABCB1 restored sensitivity to HD-TKI pulse-exposure. Thus, our data provide evidence that intracellular drug retention crucially determines biological activity of imatinib and dasatinib. These studies may refine our current thinking on critical requirements of TKI dose and duration of target inhibition for biological activity of TKIs. PMID:22815843

  10. Targeting of heat shock protein 32 (Hsp32)/heme oxygenase-1 (HO-1) in leukemic cells in chronic myeloid leukemia: a novel approach to overcome resistance against imatinib.

    Science.gov (United States)

    Mayerhofer, Matthias; Gleixner, Karoline V; Mayerhofer, Julia; Hoermann, Gregor; Jaeger, Eva; Aichberger, Karl J; Ott, Rene G; Greish, Khaled; Nakamura, Hideaki; Derdak, Sophia; Samorapoompichit, Puchit; Pickl, Winfried F; Sexl, Veronika; Esterbauer, Harald; Schwarzinger, Ilse; Sillaber, Christian; Maeda, Hiroshi; Valent, Peter

    2008-02-15

    Resistance toward imatinib and other BCR/ABL tyrosine kinase inhibitors remains an increasing clinical problem in the treatment of advanced stages of chronic myeloid leukemia (CML). We recently have identified the heat shock protein 32 (Hsp32)/heme oxygenase-1 (HO-1) as a BCR/ABL-dependent survival molecule in CML cells. We here show that silencing Hsp32/HO-1 in CML cells by an siRNA approach results in induction of apoptosis. Moreover, targeting Hsp32/HO-1 by either pegylated zinc protoporphyrine (PEG-ZnPP) or styrene maleic acid-micelle-encapsulated ZnPP (SMA-ZnPP) resulted in growth inhibition of BCR/ABL-transformed cells. The effects of PEG-ZnPP and SMA-ZnPP were demonstrable in Ba/F3 cells carrying various imatinib-resistant mutants of BCR/ABL, including the T315I mutant, which exhibits resistance against all clinically available BCR/ABL tyrosine kinase inhibitors. Growth-inhibitory effects of PEG-ZnPP and SMA-ZnPP also were observed in the CML-derived human cell lines K562 and KU812 as well as in primary leukemic cells obtained from patients with freshly diagnosed CML or imatinib-resistant CML. Finally, Hsp32/HO-1-targeting compounds were found to synergize with either imatinib or nilotinib in producing growth inhibition in imatinib-resistant K562 cells and in Ba/F3 cells harboring the T315I mutant of BCR/ABL. In summary, these data show that HO-1 is a promising novel target in imatinib-resistant CML.

  11. Comparison of imatinib, dasatinib, nilotinib and INNO-406 in imatinib-resistant cell lines.

    Science.gov (United States)

    Deguchi, Yasuyuki; Kimura, Shinya; Ashihara, Eishi; Niwa, Tomoko; Hodohara, Keiko; Fujiyama, Yoshihide; Maekawa, Taira

    2008-06-01

    We compared the growth-inhibitory effects and inhibition profile of the SRC family kinases (SFKs) of imatinib, dasatinib, nilotinib and INNO-406. Dasatinib exhibited the strongest potency against BCR-ABL with little selectivity over SFKs. Nilotinib exhibited a weaker affinity than the other inhibitors, but was highly specific for ABL and may be useful for the treatment of P-glycoprotein overexpressing leukemic cells. INNO-406 had an intermediate affinity for BCR-ABL between that of dasatinib and nilotinib, and inhibited only SFKs LCK and LYN among SFKs. Both nilotinib and INNO-406 were potent inhibitors of the dasatinib-resistant T315A, F317L and F317V BCR-ABL mutations.

  12. Modeling chronic myeloid leukemia in immunodeficient mice reveals expansion of aberrant mast cells and accumulation of pre-B cells

    International Nuclear Information System (INIS)

    Askmyr, M; Ågerstam, H; Lilljebjörn, H; Hansen, N; Karlsson, C; Palffy, S von; Landberg, N; Högberg, C; Lassen, C; Rissler, M; Richter, J; Ehinger, M; Järås, M; Fioretos, T

    2014-01-01

    Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm that, if not treated, will progress into blast crisis (BC) of either myeloid or B lymphoid phenotype. The BCR-ABL1 fusion gene, encoding a constitutively active tyrosine kinase, is thought to be sufficient to cause chronic phase (CP) CML, whereas additional genetic lesions are needed for progression into CML BC. To generate a humanized CML model, we retrovirally expressed BCR-ABL1 in the cord blood CD34 + cells and transplanted these into NOD-SCID (non-obese diabetic/severe-combined immunodeficient) interleukin-2-receptor γ-deficient mice. In primary mice, BCR-ABL1 expression induced an inflammatory-like state in the bone marrow and spleen, and mast cells were the only myeloid lineage specifically expanded by BCR-ABL1. Upon secondary transplantation, the pronounced inflammatory phenotype was lost and mainly human mast cells and macrophages were found in the bone marrow. Moreover, a striking block at the pre-B-cell stage was observed in primary mice, resulting in an accumulation of pre-B cells. A similar block in B-cell differentiation could be confirmed in primary cells from CML patients. Hence, this humanized mouse model of CML reveals previously unexplored features of CP CML and should be useful for further studies to understand the disease pathogenesis of CML

  13. Translocation of BCR to chromosome 9: A new cytogenetic variant detected by FISH in two Ph-negative, BCR-positive patients with chronic myeloid leukemia

    NARCIS (Netherlands)

    A. Hagemeijer (Anne); A. Buijs (Arjan); E.M.E. Smit (Elisabeth); L.A.J. Janssen (Bart); G.J.M. Creemers (Geert-Jan); D. van der Plas (D.); G.C. Grosveld (Gerard)

    1993-01-01

    textabstractLeukemic cells from two patients with Philadelphia-negative chronic myeloid leukemia (CML) were investigated: I) Cytogenetics showed a normal 46.XY karyotype in both cases, 2) molecular studies revealed rearrangement of the M-BCR region and formation of BCR-ABL fusion mRNA with b2a2

  14. The generation of cytotoxic T cell epitopes and their generation for cancer immunotherapy

    NARCIS (Netherlands)

    Kessler, Jan

    2009-01-01

    Cytotoxic T cell epitopes are the targets for a T cell mediated immunotherapy of cancer. The thesis reports on their identification in the tumor associated proteins BCR-ABL and PRAME by the reverse immunology (prediction) strategy. An extended strategy is used, including the analysis of the

  15. SU-G-TeP3-07: On the Development of Mechano-Biological Assessment of Leukemia Cells Using Optical Tweezers

    Energy Technology Data Exchange (ETDEWEB)

    Brost, E; Brooks, J; Piepenburg, J; Watanabe, Y; Hui, S [Therapeutic Radiology and Masonic Cancer Center, University of Minnesota, Minneapolis, MN (United States); Chakraborty, S; Das, T [Max Planck Institute for Intelligent Systems Department of New Materials and Biosystems Department of Mechanical Engineering, Indian Institute of Technology, Kharagpur (India); Green, A [Department of Physics, University of Saint Thomas, Saint Paul, MN (United States)

    2016-06-15

    Purpose: Patients with BCR-ABL (Ph +ve) acute lymphoblastic leukemia are at very high risk of relapse and mortality. In line with the NIH mission to understand the physical and biological processes, we seek to report mechano-biological method to assessment and distinguish treated/untreated leukemia cells. Methods: BCR-ABL leukemia cell populations and silica microspheres were trapped in a 100x magnification optical trapping system (λ=660 nm, 70 mW). Light refracted through the trapped sample was collected in the back focal plane by a quadrant detector to measure the positions of individual cells. The sample was driven at a known frequency and amplitude with a flexure translation stage, and the target’s response was recorded. The measured response was calibrated using the known driving parameters, and information about cell movements due to mechano-biological effects was extracted. Two leukemia cell populations were tested: a control group and a group treated with 2 Gy. Results: The mechano-biological movements of 10 microspheres, control cells, and treated cells were tracked over a ∼30 minute window at 1 minute intervals. The microsphere population did not see significant change in mechano-biological movements over the testing interval and remained constant. The control cell population saw a two-fold rise in activity that peaked around 1200 seconds, then dropped off sharply. The treated cell population saw a two-fold rise in activity that peaked at 400 seconds, and dropped off slowly. Conclusion: The investigated technique allows for direct measurement the movements of a trapped object due to mechano-biological effects such as thermal and extracellular motion. When testing microspheres, the mechano-biological activity remained constant over time due to the lack of biological factors. In both the control and treated cell populations, the mechano-biological activity was increased, possibly due to mitochondrial activation. This extra activity decreased over time

  16. Rac2-MRC-cIII-generated ROS cause genomic instability in chronic myeloid leukemia stem cells and primitive progenitors.

    Science.gov (United States)

    Nieborowska-Skorska, Margaret; Kopinski, Piotr K; Ray, Regina; Hoser, Grazyna; Ngaba, Danielle; Flis, Sylwia; Cramer, Kimberly; Reddy, Mamatha M; Koptyra, Mateusz; Penserga, Tyrone; Glodkowska-Mrowka, Eliza; Bolton, Elisabeth; Holyoake, Tessa L; Eaves, Connie J; Cerny-Reiterer, Sabine; Valent, Peter; Hochhaus, Andreas; Hughes, Timothy P; van der Kuip, Heiko; Sattler, Martin; Wiktor-Jedrzejczak, Wieslaw; Richardson, Christine; Dorrance, Adrienne; Stoklosa, Tomasz; Williams, David A; Skorski, Tomasz

    2012-05-03

    Chronic myeloid leukemia in chronic phase (CML-CP) is induced by BCR-ABL1 oncogenic tyrosine kinase. Tyrosine kinase inhibitors eliminate the bulk of CML-CP cells, but fail to eradicate leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) displaying innate and acquired resistance, respectively. These cells may accumulate genomic instability, leading to disease relapse and/or malignant progression to a fatal blast phase. In the present study, we show that Rac2 GTPase alters mitochondrial membrane potential and electron flow through the mitochondrial respiratory chain complex III (MRC-cIII), thereby generating high levels of reactive oxygen species (ROS) in CML-CP LSCs and primitive LPCs. MRC-cIII-generated ROS promote oxidative DNA damage to trigger genomic instability, resulting in an accumulation of chromosomal aberrations and tyrosine kinase inhibitor-resistant BCR-ABL1 mutants. JAK2(V617F) and FLT3(ITD)-positive polycythemia vera cells and acute myeloid leukemia cells also produce ROS via MRC-cIII. In the present study, inhibition of Rac2 by genetic deletion or a small-molecule inhibitor and down-regulation of mitochondrial ROS by disruption of MRC-cIII, expression of mitochondria-targeted catalase, or addition of ROS-scavenging mitochondria-targeted peptide aptamer reduced genomic instability. We postulate that the Rac2-MRC-cIII pathway triggers ROS-mediated genomic instability in LSCs and primitive LPCs, which could be targeted to prevent the relapse and malignant progression of CML.

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    Grzegorz Helbig

    2014-06-01

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

  19. Combined treatment of 3-hydroxyflavone and imatinib mesylate increases apoptotic cell death of imatinib mesylate-resistant leukemia cells.

    Science.gov (United States)

    Kim, Jung-Hyun; Song, Minjung; Kang, Geun-Ho; Lee, Eung-Ryoung; Choi, Hye-Yeon; Lee, Chung; Kim, Jin-Hoi; Kim, Youngsoo; Koo, Bon-Nyeo; Cho, Ssang-Goo

    2012-09-01

    Imatinib mesylate, a Bcr/Abl tyrosine kinase inhibitor, is widely used in treating chronic myeloid leukemia. However, drug-resistance of leukemia cells becomes an emergent problem. Herein, various flavonoids were screened for applicability in leukemia treatment, and 3-hydroxyflavone (3-HF) was found to be most effective in reducing cancer cell viability. The combination of 3-HF and imatinib mesylate resulted in significant apoptotic cell death in imatinib mesylate-resistant leukemia cells. Combined treatment resulted in apparent activation of caspases and decrease of the oncoprotein phosphor-Bcr/Abl in leukemia cells. Our results suggest that this combined treatment is beneficial in imatinib mesylate-resistant chronic myelogenous leukemia. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Heterogeneity of genomic fusion of BCR and ABL in Philadelphia chromosome-positive acute lymphoblastic leukemia

    International Nuclear Information System (INIS)

    Rubin, C.M.; Carrino, J.J.; Dickler, M.N.; Leibowitz, D.; Smith, S.D.; Westbrook, C.A.

    1988-01-01

    Philadelphia chromosome-positive acute lymphoblastic leukemia occurs in two molecular forms, those with and those without rearrangement of the breakpoint cluster region on chromosome 22. The molecular abnormality in the former group is similar to that found in chronic myelogenous leukemia. To characterize the abnormality in the breakpoint cluster region-unrearranged form, the authors have mapped a 9; 22 translocation from the Philadelphia chromosome-positive acute lymphoblastic leukemia cell line SUP-B13 by using pulsed-field gel electrophoresis and have cloned the DNA at the translocation junctions. They demonstrate a BCR-ABL fusion gene on the Philadelphia chromosome. The exons from ABL are the same. Analysis of leukemic cells from four other patients with breakpoint cluster region-unrearranged Philadelphia chromosome-positive acute lymphoblastic leukemia revealed a rearrangement on chromosome 22 close to the breakpoint in SUP-B13 in only one patient. These data indicate that breakpoints do not cluster tightly in this region but are scattered, possibly in a large intron. Given the large size of BCR and the heterogeneity in breakpoint location, detection of BCR rearrangement by standard Southern blot analysis is difficult. Pulsed-field gel electrophoresis should allow detection at the DNA level in every patient and thus will permit clinical correlation of the breakpoint location with prognosis

  1. A Phase 2 Trial of Ponatinib in Philadelphia Chromosome–Positive Leukemias

    Science.gov (United States)

    Cortes, J.E.; Kim, D.-W.; Pinilla-Ibarz, J.; le Coutre, P.; Paquette, R.; Chuah, C.; Nicolini, F.E.; Apperley, J.F.; Khoury, H.J.; Talpaz, M.; DiPersio, J.; DeAngelo, D.J.; Abruzzese, E.; Rea, D.; Baccarani, M.; Müller, M.C.; Gambacorti-Passerini, C.; Wong, S.; Lustgarten, S.; Rivera, V.M.; Clackson, T.; Turner, C.D.; Haluska, F.G.; Guilhot, F.; Deininger, M.W.; Hochhaus, A.; Hughes, T.; Goldman, J.M.; Shah, N.P.; Kantarjian, H.

    2013-01-01

    BACKGROUND Ponatinib is a potent oral tyrosine kinase inhibitor of unmutated and mutated BCR-ABL, including BCR-ABL with the tyrosine kinase inhibitor–refractory threonine-to-isoleucine mutation at position 315 (T315I). We conducted a phase 2 trial of ponatinib in patients with chronic myeloid leukemia (CML) or Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph-positive ALL). METHODS We enrolled 449 heavily pretreated patients who had CML or Ph-positive ALL with resistance to or unacceptable side effects from dasatinib or nilotinib or who had the BCR-ABL T315I mutation. Ponatinib was administered at an initial dose of 45 mg once daily. The median follow-up was 15 months. RESULTS Among 267 patients with chronic-phase CML, 56% had a major cytogenetic response (51% of patients with resistance to or unacceptable side effects from dasatinib or nilotinib and 70% of patients with the T315I mutation), 46% had a complete cytogenetic response (40% and 66% in the two subgroups, respectively), and 34% had a major molecular response (27% and 56% in the two subgroups, respectively). Responses were observed regardless of the baseline BCR-ABL kinase domain mutation status and were durable; the estimated rate of a sustained major cytogenetic response of at least 12 months was 91%. No single BCR-ABL mutation conferring resistance to ponatinib was detected. Among 83 patients with accelerated-phase CML, 55% had a major hematologic response and 39% had a major cytogenetic response. Among 62 patients with blast-phase CML, 31% had a major hematologic response and 23% had a major cytogenetic response. Among 32 patients with Ph-positive ALL, 41% had a major hematologic response and 47% had a major cytogenetic response. Common adverse events were thrombocytopenia (in 37% of patients), rash (in 34%), dry skin (in 32%), and abdominal pain (in 22%). Serious arterial thrombotic events were observed in 9% of patients; these events were considered to be treatment-related in 3

  2. A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias.

    Science.gov (United States)

    Cortes, J E; Kim, D-W; Pinilla-Ibarz, J; le Coutre, P; Paquette, R; Chuah, C; Nicolini, F E; Apperley, J F; Khoury, H J; Talpaz, M; DiPersio, J; DeAngelo, D J; Abruzzese, E; Rea, D; Baccarani, M; Müller, M C; Gambacorti-Passerini, C; Wong, S; Lustgarten, S; Rivera, V M; Clackson, T; Turner, C D; Haluska, F G; Guilhot, F; Deininger, M W; Hochhaus, A; Hughes, T; Goldman, J M; Shah, N P; Kantarjian, H

    2013-11-07

    Ponatinib is a potent oral tyrosine kinase inhibitor of unmutated and mutated BCR-ABL, including BCR-ABL with the tyrosine kinase inhibitor-refractory threonine-to-isoleucine mutation at position 315 (T315I). We conducted a phase 2 trial of ponatinib in patients with chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL). We enrolled 449 heavily pretreated patients who had CML or Ph-positive ALL with resistance to or unacceptable side effects from dasatinib or nilotinib or who had the BCR-ABL T315I mutation. Ponatinib was administered at an initial dose of 45 mg once daily. The median follow-up was 15 months. Among 267 patients with chronic-phase CML, 56% had a major cytogenetic response (51% of patients with resistance to or unacceptable side effects from dasatinib or nilotinib and 70% of patients with the T315I mutation), 46% had a complete cytogenetic response (40% and 66% in the two subgroups, respectively), and 34% had a major molecular response (27% and 56% in the two subgroups, respectively). Responses were observed regardless of the baseline BCR-ABL kinase domain mutation status and were durable; the estimated rate of a sustained major cytogenetic response of at least 12 months was 91%. No single BCR-ABL mutation conferring resistance to ponatinib was detected. Among 83 patients with accelerated-phase CML, 55% had a major hematologic response and 39% had a major cytogenetic response. Among 62 patients with blast-phase CML, 31% had a major hematologic response and 23% had a major cytogenetic response. Among 32 patients with Ph-positive ALL, 41% had a major hematologic response and 47% had a major cytogenetic response. Common adverse events were thrombocytopenia (in 37% of patients), rash (in 34%), dry skin (in 32%), and abdominal pain (in 22%). Serious arterial thrombotic events were observed in 9% of patients; these events were considered to be treatment-related in 3%. A total of 12% of patients

  3. Prognostic significance of BCR-ABL rearrangement in childhood acute lymphoblastic leukemia

    OpenAIRE

    Styczyński, Jan; Jatczak-Gaca, Agnieszka; Matiakowska, Karolina; Bartoszewska-Kubiak, Alicja; Kołtan, Andrzej; Dębski, Robert; Pogorzała, Monika; Skonieczka, Katarzyna; Morgut-Klimkiewicz, Małgorzata; Soszyńska, Krystyna; Wysocki, Mariusz

    2015-01-01

    B a c k g r o u n d. Acute lymphoblastic leukemia (ALL) is the most frequent pediatric malignancy. Presence of adverse risk factors determines risk group stratification in this disease. O b j e c t i v e. The aim of study was the analysis of results of therapy and role of prognostic risk factors in treatment of childhood ALL in kujawsko-pomorskie region in 1995-2010. P a t i e n t s a n d m e t h o d s. During this period, ALL was diagnosed in 223 patients. With respect to time period and the...

  4. Oncogenic Kinase Bcr-Abl and Its Resistance to Pharmacological Inhibitors

    Czech Academy of Sciences Publication Activity Database

    Kryštof, Vladimír

    2008-01-01

    Roč. 102, č. 9 (2008), s. 795-800 E-ISSN 1213-7103 Institutional research plan: CEZ:AV0Z50380511 Keywords : imatinib * inhibitor * chronic myeloid leukaemia * kinase * cancer Subject RIV: CE - Biochemistry www.chemicke-listy.cz/docs/full/2008_09_795-800.pdf

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

    Directory of Open Access Journals (Sweden)

    Murat Albayrak

    2010-09-01

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

  6. A BCR-ABL Kinase Activity-Independent Signaling Pathway in Chronic Myelogenous Leukemia

    Science.gov (United States)

    2008-02-01

    PA) as a series of tagged image files. All images were then constructed in Adobe Photoshop 6.0 (Adobe, San Jose, CA). Antibodies and Western blot...hereby marked ‘‘ advertisement ’’ in accordance with 18 USC section 1734. © 2007 by The American Society of Hematology 678 BLOOD, 15 JULY 2007 ! VOLUME 110

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

    Directory of Open Access Journals (Sweden)

    Michele Tanturli

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

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

    Directory of Open Access Journals (Sweden)

    Fei Fei

    2012-06-01

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

  9. Gravity and positional homeostasis of the cell

    Science.gov (United States)

    Nace, G. W.

    1983-01-01

    The effect of gravity upon cytoplasmic aggregates of the size present in eggs and upon cells is investigated. An expression is developed to describe the tendency of torque to rotate the egg and reorganize its constituents. This expression provides the net torque resulting from buoyancy and gravity acting upon a dumbbell-shaped cell, with heavy and light masses at either end and floating in a medium. Torques of approximately 2.5 x 10 to the -13th to 0.85 dyne-cm are found to act upon cells ranging from 6.4 microns to 31 mm (chicken egg). It is noted that cells must expend energy to maintain positional homeostasis against gravity, as demonstrated by results from Skylab 3, where tissue cultures used 58 percent more glucose on earth than in space. The implications for developmental biology, physiology, genetics, and evolution are discussed. It is argued that at the cellular and tissue levels the concept of gravity receptors may be unnecessary.

  10. 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

    2017-05-01

    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. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Discovery of a fusion kinase in EOL-1 cells and idiopathic hypereosinophilic syndrome

    OpenAIRE

    Griffin, John H.; Leung, Joey; Bruner, Rebecca J.; Caligiuri, Michael A.; Briesewitz, Roger

    2003-01-01

    Idiopathic hypereosinophilic syndrome (HES) is a myeloproliferative disease of unknown etiology. Recently, it has been reported that imatinib mesylate (Gleevec), an inhibitor of Bcr-Abl kinase useful in the treatment of chronic myeloid leukemia, is also effective in treating HES; however, the molecular target of imatinib in HES is unknown. This report identifies a genetic rearrangement in the eosinophilic cell line EOL-1 that results in the expression of a fusion protein comprising an N...

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

    Directory of Open Access Journals (Sweden)

    Ryosuke Shirasaki

    2012-01-01

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

  13. [Imatinib Combined with VP Low Dose Regiment for Treating Newly Diagnosed Adult Patients with Ph-positive ALL].

    Science.gov (United States)

    Liu, Kui; Guo, Yue-Lu; Yao, Zi-Long; Jin, Xiang-Shu; Zhang, Ran; Han, Xiao-Pin; Gao, Xiao-Ning; Yu, Li; Jing, Yu

    2015-12-01

    To investigate the inductive therapeutic effects of imatinib combined with VP low dose regiment on adult patients with Ph-positive acute lymphoblastic leukemia (Ph(+) ALL). Fourteen newly diagnosed adult patients with Ph(+) ALL were treated with VP regimen, and imatinib (400 mg/d) was added at the 8(th) day. VP regimen would be stopped when neutropenia lasted for 1 week or complicated with infection, fever, etc. Therapeutic effects were assessed by bone marrow morphology and quantitative analysis of BCR/ABL:ABL at the 28(th) - 33(rd) day. Patients could be treated with imatinib combined with chemotherapy for consolidation and maintenance therapy or were treated with allogeneic hematopoietic stem cell transplantation after complete remission. Fourteen cases obtained CR1 after first course of treatment, the median decline of BCR/ABA:ABL was 55.89 (10.25 -180.97) %; during the induction chemotherapy, pulmonary infection occurred in 3 patients, diarrhea in 1 patients, facial edema in 3 patients, however, all these patients were cured after symptomatic treatment, only 1 patient died of relapse after transplantation. In the period of tyrosine kinase inhibitor (TKI), inductive chemotherapy combined with imatinib and low dose VP can obtaine satisfactory CR rate and decrease the toxicity of the traditional drugs. It is suggested that TKI combined with VP regimen chemotherapy can achieve CR1 and make possible for allo-HSCT, from which patients can achieve the long-term survival.

  14. Cytokeratin-positive folliculo-stellate cells in chicken adenohypophysis.

    Science.gov (United States)

    Nishimura, Shotaro; Yamashita, Miyu; Kaneko, Takane; Kawabata, Fuminori; Tabata, Shoji

    2017-11-01

    Folliculo-stellate (FS) cells are non-endocrine cells found in the adenohypophysis and are identified in many animals by the S100 protein marker. Although keratin is another FS marker in several animals, there is no information on localization of keratin in the avian adenohypophysis. In this study, localization of cytokeratin in chicken adenohypophyseal cells was investigated immunohistochemically. Basic cytokeratin (bCK)-positive cells were arranged radially in the cell cords with their cytoplasmic processes reaching the basal lamina. The cell bodies encircled a follicle in the center of the cell cord. Furthermore, the bCK-positive cells were also S100B-positive. Growth hormone, prolactin, adrenocorticotrophic hormone, and luteinizing hormone β-subunit did not co-localize with the bCK-positive cells. In addition, the bCK-positive cells had a laminin-positive area in their cytoplasm. Transmission electron microscopy observed agranular cells equipped with several microvilli that encircled a follicle. These results indicate that bCK-positive cells in the chicken adenohypophysis may be a predominant FS cell population and produce laminin. It is suggested that they function as sustentacular cells to sustain the adjacent endocrine cells and the structure of the cell cords in the chicken adenohypophysis. © 2017 Japanese Society of Animal Science.

  15. Wnt5a enhances the response of CML cells to Imatinib Mesylate through JNK activation and γ-catenin inhibition.

    Science.gov (United States)

    Niu, Chang-Chun; Zhao, Chen; Zhang, Xiao-Li; Pan, Jing; Zhao, Chen; Wu, Wei-Ru; Li, Zhi-Qiang; Liu, Tao; Yang, Zhong; Si, Wei-Ke

    2013-11-01

    Imatinib Mesylate is widely used for the treatment of chronic myelogenous leukaemia (CML), and its effects on CML cells are influenced by several signalling proteins. The research is aimed at determining whether Wnt5a affects the effects of Imatinib Mesylate against BCR-ABL positive CML cells (K562 cells and KU812 cells) and which signalling proteins are involved in. The results showed that Wnt5a augmented the effects of Imatinib Mesylate on inhibiting CML cells proliferation and inducing apoptosis in vitro; Wnt5a enhanced the inhibition effect of Imatinib Mesylate on the growth of K562 cells xenograft tumour in an animal model. Furthermore, Wnt5a inhibited β-catenin and its target gene Survivin, increased the activity of JNK and suppressed γ-catenin expression. When inhibiting the activity of JNK, the influence of Wnt5a on the effects of Imatinib Mesylate was attenuated. Moreover, JNK suppressed β-catenin and its target gene Survivin, and enhanced the effects of Imatinib Mesylate. These results suggest that Wnt5a can enhance the efficacy of Imatinib Mesylate through JNK/β-catenin/Survivin and γ-catenin/β-catenin/Survivin pathways. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Current Concepts in Pediatric Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL

    Directory of Open Access Journals (Sweden)

    Kathrin M. Bernt

    2014-03-01

    Full Text Available The t(9;22(q34;q11 or Philadelphia chromosome that creates a BCR-ABL1 fusion gene encoding for a chimeric BCR-ABL1 protein is present in 3-4% of pediatric acute lymphoblastic leukemia (Ph+ ALL, and about 25% of adult ALL cases. Prior to the advent of tyrosine kinase inhibitors (TKI, Ph+ ALL was associated with a very poor prognosis despite use of intensive chemotherapy and frequently hematopoietic stem cell transplantation (HSCT in first remission. The development of TKIs revolutionized the therapy of Ph+ ALL. Addition of the first generation ABL1 class TKI imatinib to intensive chemotherapy dramatically increased survival for children with Ph+ ALL and established that many patients can be cured without HSCT. In parallel, the mechanistic understanding of Ph+ ALL expanded exponentially through careful mapping of pathways downstream of BCR-ABL1, the discovery of mutations in master regulators of B-cell development such as IKZF1 (Ikaros, PAX5 and EBF, the recognition of the complex clonal architecture of Ph+ ALL, and the delineation of genomic, epigenetic and signaling abnormalities contributing to relapse and resistance. Still, many important basic and clinical questions remain unanswered. Current clinical trials are testing second generation TKIs in patients with newly diagnosed Ph+ ALL. Neither the optimal duration of therapy nor the optimal chemotherapy backbone are currently defined. The role of HSCT in first remission and post-transplant TKI therapy also require further study. In addition, it will be crucial to continue to dig deeper into understanding Ph+ ALL at a mechanistic level, and translate findings into complementary targeted approaches. Expanding targeted therapies holds great promise to decrease toxicity and improve survival in this high risk disease, which provides a paradigm for how targeted therapies can be incorporated into treatment of other high risk leukemias.

  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. Copyright Georg Thieme Verlag KG Stuttgart . New York.

  18. 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.

  19. Analyzing the spatial positioning of nuclei in polynuclear giant cells

    International Nuclear Information System (INIS)

    Stange, Maike; Hintsche, Marius; Sachse, Kirsten; Gerhardt, Matthias; Beta, Carsten; Valleriani, Angelo

    2017-01-01

    How cells establish and maintain a well-defined size is a fundamental question of cell biology. Here we investigated to what extent the microtubule cytoskeleton can set a predefined cell size, independent of an enclosing cell membrane. We used electropulse-induced cell fusion to form giant multinuclear cells of the social amoeba Dictyostelium discoideum . Based on dual-color confocal imaging of cells that expressed fluorescent markers for the cell nucleus and the microtubules, we determined the subcellular distributions of nuclei and centrosomes in the giant cells. Our two- and three-dimensional imaging results showed that the positions of nuclei in giant cells do not fall onto a regular lattice. However, a comparison with model predictions for random positioning showed that the subcellular arrangement of nuclei maintains a low but still detectable degree of ordering. This can be explained by the steric requirements of the microtubule cytoskeleton, as confirmed by the effect of a microtubule degrading drug. (paper)

  20. Analyzing the spatial positioning of nuclei in polynuclear giant cells

    Science.gov (United States)

    Stange, Maike; Hintsche, Marius; Sachse, Kirsten; Gerhardt, Matthias; Valleriani, Angelo; Beta, Carsten

    2017-11-01

    How cells establish and maintain a well-defined size is a fundamental question of cell biology. Here we investigated to what extent the microtubule cytoskeleton can set a predefined cell size, independent of an enclosing cell membrane. We used electropulse-induced cell fusion to form giant multinuclear cells of the social amoeba Dictyostelium discoideum. Based on dual-color confocal imaging of cells that expressed fluorescent markers for the cell nucleus and the microtubules, we determined the subcellular distributions of nuclei and centrosomes in the giant cells. Our two- and three-dimensional imaging results showed that the positions of nuclei in giant cells do not fall onto a regular lattice. However, a comparison with model predictions for random positioning showed that the subcellular arrangement of nuclei maintains a low but still detectable degree of ordering. This can be explained by the steric requirements of the microtubule cytoskeleton, as confirmed by the effect of a microtubule degrading drug.

  1. Stem cell antigen 1-positive mesenchymal cells are the origin of follicular cells during thyroid regeneration.

    Science.gov (United States)

    Okamoto, Minoru; Hayase, Suguru; Miyakoshi, Masaaki; Murata, Tsubasa; Kimura, Shioko

    2013-01-01

    Many tissues are thought to contain adult stem/progenitor cells that are responsible for repair of the tissue where they reside upon damage and/or carcinogenesis, conditions when cellular homeostasis becomes uncontrolled. While the presence of stem/progenitor cells of the thyroid has been suggested, how these cells contribute to thyroid regeneration remains unclear. Here we show the origin of thyroid follicular cells and the process of their maturation to become follicular cells during regeneration. By using β-galactosidase (β-gal) reporter mice in conjunction with partial thyroidectomy as a model for thyroid regeneration, and bromodeoxyuridine (BrdU) long label-retaining cell analysis, we demonstrated that stem cell antigen 1 (Sca1) and BrdU-positive, but β-gal and NKX2-1 negative cells were found in the non-follicular mesenchymal area 7 days after partial thyroidectomy. They temporarily co-expressed cytokeratin 14, and were observed in part of follicles by day 35 post-partial thyroidectomy. Sca1, BrdU, β-gal, and NKX2-1-positive cells were found 120 days post-partial thyroidectomy. These results suggested that Sca1 and BrdU positive cells may participate in the formation of new thyroid follicles after partial thyroidectomy. The process of thyroid follicular cell regeneration was recapitulated in ex vivo thyroid slice collagen gel culture studies. These studies will facilitate research on thyroid stem/progenitor cells and their roles in thyroid diseases, particularly thyroid carcinomas.

  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. Tubulointerstitial Nephritis with IgM-Positive Plasma Cells.

    Science.gov (United States)

    Takahashi, Naoki; Saeki, Takako; Komatsuda, Atsushi; Munemura, Chishio; Fukui, Takeaki; Imai, Naofumi; Homma, Noriyuki; Hatta, Tsuguru; Samejima, Ken-Ichi; Fujimoto, Takashi; Omori, Hiroki; Ito, Yumi; Nishikawa, Yudai; Kobayashi, Mamiko; Morikawa, Yukie; Fukushima, Sachiko; Yokoi, Seiji; Mikami, Daisuke; Kasuno, Kenji; Kimura, Hideki; Nemoto, Tomoyuki; Nakamoto, Yasunari; Sada, Kiyonao; Sugai, Manabu; Naiki, Hironobu; Yoshida, Haruyoshi; Narita, Ichiei; Saito, Yoshihiko; Iwano, Masayuki

    2017-12-01

    Infiltration by IgG-positive plasma cells is a common finding in tubulointerstitial nephritis. Indeed, it has been thought that CD138-positive mature plasma cells secrete mainly IgG, and the occurrence of tubulointerstitial nephritis with CD138-positive plasma cells secreting IgM has rarely been reported. Routine immunofluorescence of fresh frozen sections is considered the gold standard for detection of immune deposits. However, the immunoenzyme method with formalin-fixed, paraffin-embedded sections is superior for detecting IgM- or IgG-positive cells within the renal interstitium, thus histologic variants may often go undetected. We recently discovered a case of tubulointerstitial nephritis showing IgM-positive plasma cell accumulation within the interstitium. To further explore the morphologic and clinical features of such cases, we performed a nationwide search for patients with biopsy-proven tubulointerstitial nephritis and high serum IgM levels. We identified 13 patients with tubulointerstitial nephritis and IgM-positive plasma cell infiltration confirmed with the immunoenzyme method. The clinical findings for these patients included a high prevalence of distal renal tubular acidosis (100%), Fanconi syndrome (92%), and anti-mitochondrial antibodies (82%). The pathologic findings were interstitial nephritis with diffusely distributed CD3-positive T lymphocytes and colocalized IgM-positive plasma cells, as well as tubulitis with CD3-positive T lymphocytes in the proximal tubules and collecting ducts. Additionally, levels of H + -ATPase, H + , K + -ATPase, and the HCO 3 - -Cl - anion exchanger were markedly decreased in the collecting ducts. We propose to designate this group of cases, which have a common histologic and clinical form, as IgM-positive plasma cell-tubulointerstitial nephritis. Copyright © 2017 by the American Society of Nephrology.

  4. Classical cadherins control nucleus and centrosome position and cell polarity.

    Science.gov (United States)

    Dupin, Isabelle; Camand, Emeline; Etienne-Manneville, Sandrine

    2009-06-01

    Control of cell polarity is crucial during tissue morphogenesis and renewal, and depends on spatial cues provided by the extracellular environment. Using micropatterned substrates to impose reproducible cell-cell interactions, we show that in the absence of other polarizing cues, cell-cell contacts are the main regulator of nucleus and centrosome positioning, and intracellular polarized organization. In a variety of cell types, including astrocytes, epithelial cells, and endothelial cells, calcium-dependent cadherin-mediated cell-cell interactions induce nucleus and centrosome off-centering toward cell-cell contacts, and promote orientation of the nucleus-centrosome axis toward free cell edges. Nucleus and centrosome off-centering is controlled by N-cadherin through the regulation of cell interactions with the extracellular matrix, whereas the orientation of the nucleus-centrosome axis is determined by the geometry of N-cadherin-mediated contacts. Our results demonstrate that in addition to the specific function of E-cadherin in regulating baso-apical epithelial polarity, classical cadherins control cell polarization in otherwise nonpolarized cells.

  5. 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

  6. Prevalence and pattern of Lupus erythematosus cell positivity in ...

    African Journals Online (AJOL)

    The prevalence and pattern of lupus erythematosus (LE) cell positivity in diseases in Ile-Ife, Osun state was carried out between January 1999 and June 2004 (5½ years). A total of 96 patients with different diseases were screened for LE cell using standard techniques. Of this number, 63 (65.6%) were females and 33 ...

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

    Science.gov (United States)

    2010-05-01

    autophosphorylation of JAK2 kinase itself (Fig. 7b) as well as the transphosphorylation of GST -Abltide substrate (Fig. 7c). In vivo inhibition of Jak2...presented in International Symposium on Genomic Instability and Cancer at the University of Kashmir, Srinagar, India , 2007.   Conclusions Our...of recombinant kinase was used with 1 lg GST -Sam68 (aa 331– 443; Santa Cruz Biotechnologies sc-4249), dephosphorylated a- Casein (Sigma C8032) or

  8. Use of deferasirox, an iron chelator, to overcome imatinib resistance of chronic myeloid leukemia cells.

    Science.gov (United States)

    Kim, Dae Sik; Na, Yoo Jin; Kang, Myoung Hee; Yoon, Soo-Young; Choi, Chul Won

    2016-03-01

    The treatment of chronic myeloid leukemia (CML) has achieved impressive success since the development of the Bcr-Abl tyrosine kinase inhibitor, imatinib mesylate. Nevertheless, resistance to imatinib has been observed, and a substantial number of patients need alternative treatment strategies. We have evaluated the effects of deferasirox, an orally active iron chelator, and imatinib on K562 and KU812 human CML cell lines. Imatinib-resistant CML cell lines were created by exposing cells to gradually increasing concentrations of imatinib. Co-treatment of cells with deferasirox and imatinib induced a synergistic dose-dependent inhibition of proliferation of both CML cell lines. Cell cycle analysis showed an accumulation of cells in the subG1 phase. Western blot analysis of apoptotic proteins showed that co-treatment with deferasirox and imatinib induced an increased expression of apoptotic proteins. These tendencies were clearly identified in imatinib-resistant CML cell lines. The results also showed that co-treatment with deferasirox and imatinib reduced the expression of BcrAbl, phosphorylated Bcr-Abl, nuclear factor-κB (NF-κB) and β-catenin. We observed synergistic effects of deferasirox and imatinib on both imatinib-resistant and imatinib-sensitive cell lines. These effects were due to induction of apoptosis and cell cycle arrest by down-regulated expression of NF-κB and β-catenin levels. Based on these results, we suggest that a combination treatment of deferasirox and imatinib could be considered as an alternative treatment option for imatinib-resistant CML.

  9. Use of deferasirox, an iron chelator, to overcome imatinib resistance of chronic myeloid leukemia cells

    Science.gov (United States)

    Kim, Dae Sik; Na, Yoo Jin; Kang, Myoung Hee; Yoon, Soo-Young; Choi, Chul Won

    2016-01-01

    Background/Aims: The treatment of chronic myeloid leukemia (CML) has achieved impressive success since the development of the Bcr-Abl tyrosine kinase inhibitor, imatinib mesylate. Nevertheless, resistance to imatinib has been observed, and a substantial number of patients need alternative treatment strategies. Methods: We have evaluated the effects of deferasirox, an orally active iron chelator, and imatinib on K562 and KU812 human CML cell lines. Imatinib-resistant CML cell lines were created by exposing cells to gradually increasing concentrations of imatinib. Results: Co-treatment of cells with deferasirox and imatinib induced a synergistic dose-dependent inhibition of proliferation of both CML cell lines. Cell cycle analysis showed an accumulation of cells in the subG1 phase. Western blot analysis of apoptotic proteins showed that co-treatment with deferasirox and imatinib induced an increased expression of apoptotic proteins. These tendencies were clearly identified in imatinib-resistant CML cell lines. The results also showed that co-treatment with deferasirox and imatinib reduced the expression of BcrAbl, phosphorylated Bcr-Abl, nuclear factor-κB (NF-κB) and β-catenin. Conclusions: We observed synergistic effects of deferasirox and imatinib on both imatinib-resistant and imatinib-sensitive cell lines. These effects were due to induction of apoptosis and cell cycle arrest by down-regulated expression of NF-κB and β-catenin levels. Based on these results, we suggest that a combination treatment of deferasirox and imatinib could be considered as an alternative treatment option for imatinib-resistant CML. PMID:26874514

  10. An activating mutation of GNB1 is associated with resistance to tyrosine kinase inhibitors in ETV6-ABL1-positive leukemia

    Czech Academy of Sciences Publication Activity Database

    Zimmermannova, O.; Doktorova, E.; Stuchlý, J.; Kanderová, V.; Kuzilkova, D.; Strnad, Hynek; Starková, J.; Alberich-Jorda, Meritxell; Falkenburg, J.H.F.; Trka, J.; Petrák, J.; Zuna, J.; Žaliová, M.

    2017-01-01

    Roč. 36, č. 43 (2017), s. 5985-5994 ISSN 0950-9232 R&D Projects: GA ČR(CZ) GBP302/12/G101; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:68378050 Keywords : acute lymphoblastic-leukemia * chronic myeloid-leukemia * bcr-abl * tel-abl * cytogenetic characterization * imatinib-mesylate * fusion proteins * blast crisis * rearrangement * patient Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 7.519, year: 2016

  11. Eμ/miR-125b transgenic mice develop lethal B-cell malignancies.

    Science.gov (United States)

    Enomoto, Y; Kitaura, J; Hatakeyama, K; Watanuki, J; Akasaka, T; Kato, N; Shimanuki, M; Nishimura, K; Takahashi, M; Taniwaki, M; Haferlach, C; Siebert, R; Dyer, M J S; Asou, N; Aburatani, H; Nakakuma, H; Kitamura, T; Sonoki, T

    2011-12-01

    MicroRNA-125b-1 (miR-125b-1) is a target of a chromosomal translocation t(11;14)(q24;q32) recurrently found in human B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This translocation results in overexpression of miR-125b controlled by immunoglobulin heavy chain gene (IGH) regulatory elements. In addition, we found that six out of twenty-one BCP-ALL patients without t(11;14)(q24;q32) showed overexpression of miR-125b. Interestingly, four out of nine patients with BCR/ABL-positive BCP-ALL and one patient with B-cell lymphoid crisis that had progressed from chronic myelogenous leukemia overexpressed miR-125b. To examine the role of the deregulated expression of miR-125b in the development of B-cell tumor in vivo, we generated transgenic mice mimicking the t(11;14)(q24;q32) (Eμ/miR-125b-TG mice). Eμ/miR-125b-TG mice overexpressed miR-125b driven by IGH enhancer and promoter and developed IgM-negative or IgM-positive lethal B-cell malignancies with clonal proliferation. B cells obtained from the Eμ/miR-125b-TG mice were resistant to apoptosis induced by serum starvation. We identified Trp53inp1, a pro-apoptotic gene induced by cell stress, as a novel target gene of miR-125b in hematopoietic cells in vitro and in vivo. Our results provide direct evidence that miR-125b has important roles in the tumorigenesis of precursor B cells.

  12. 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.

  13. Highly parallel identification of essential genes in cancer cells.

    Science.gov (United States)

    Luo, Biao; Cheung, Hiu Wing; Subramanian, Aravind; Sharifnia, Tanaz; Okamoto, Michael; Yang, Xiaoping; Hinkle, Greg; Boehm, Jesse S; Beroukhim, Rameen; Weir, Barbara A; Mermel, Craig; Barbie, David A; Awad, Tarif; Zhou, Xiaochuan; Nguyen, Tuyen; Piqani, Bruno; Li, Cheng; Golub, Todd R; Meyerson, Matthew; Hacohen, Nir; Hahn, William C; Lander, Eric S; Sabatini, David M; Root, David E

    2008-12-23

    More complete knowledge of the molecular mechanisms underlying cancer will improve prevention, diagnosis and treatment. Efforts such as The Cancer Genome Atlas are systematically characterizing the structural basis of cancer, by identifying the genomic mutations associated with each cancer type. A powerful complementary approach is to systematically characterize the functional basis of cancer, by identifying the genes essential for growth and related phenotypes in different cancer cells. Such information would be particularly valuable for identifying potential drug targets. Here, we report the development of an efficient, robust approach to perform genome-scale pooled shRNA screens for both positive and negative selection and its application to systematically identify cell essential genes in 12 cancer cell lines. By integrating these functional data with comprehensive genetic analyses of primary human tumors, we identified known and putative oncogenes such as EGFR, KRAS, MYC, BCR-ABL, MYB, CRKL, and CDK4 that are essential for cancer cell proliferation and also altered in human cancers. We further used this approach to identify genes involved in the response of cancer cells to tumoricidal agents and found 4 genes required for the response of CML cells to imatinib treatment: PTPN1, NF1, SMARCB1, and SMARCE1, and 5 regulators of the response to FAS activation, FAS, FADD, CASP8, ARID1A and CBX1. Broad application of this highly parallel genetic screening strategy will not only facilitate the rapid identification of genes that drive the malignant state and its response to therapeutics but will also enable the discovery of genes that participate in any biological process.

  14. Induction Chemotherapy for p16 Positive Oropharyngeal Squamous Cell Carcinoma

    OpenAIRE

    Saito, Yuki; Ando, Mizuo; Omura, Go; Yasuhara, Kazuo; Yoshida, Masafumi; Takahashi, Wataru; Yamasoba, Tatsuya

    2016-01-01

    Objectives/Hypothesis We aimed to determine the effectiveness of induction chemotherapy for treating p16?positive oropharyngeal cancer in our department. Study Design This was a retrospective case series to assess treatment effectiveness. Methods We administered induction chemotherapy to patients with stage III to IV oropharyngeal p16?positive squamous cell carcinoma between 2008 and 2013. Induction chemotherapy was administered using combinations of docetaxel, cisplatin, and 5?fluorouracil. ...

  15. Distribution of CD163-positive cell and MHC class II-positive cell in the normal equine uveal tract.

    Science.gov (United States)

    Sano, Yuto; Matsuda, Kazuya; Okamoto, Minoru; Takehana, Kazushige; Hirayama, Kazuko; Taniyama, Hiroyuki

    2016-02-01

    Antigen-presenting cells (APCs) in the uveal tract participate in ocular immunity including immune homeostasis and the pathogenesis of uveitis. In horses, although uveitis is the most common ocular disorder, little is known about ocular immunity, such as the distribution of APCs. In this study, we investigated the distribution of CD163-positive and MHC II-positive cells in the normal equine uveal tract using an immunofluorescence technique. Eleven eyes from 10 Thoroughbred horses aged 1 to 24 years old were used. Indirect immunofluorescence was performed using the primary antibodies CD163, MHC class II (MHC II) and CD20. To demonstrate the site of their greatest distribution, positive cells were manually counted in 3 different parts of the uveal tract (ciliary body, iris and choroid), and their average number was assessed by statistical analysis. The distribution of pleomorphic CD163- and MHC II-expressed cells was detected throughout the equine uveal tract, but no CD20-expressed cells were detected. The statistical analysis demonstrated the distribution of CD163- and MHC II-positive cells focusing on the ciliary body. These results demonstrated that the ciliary body is the largest site of their distribution in the normal equine uveal tract, and the ciliary body is considered to play important roles in uveal and/or ocular immune homeostasis. The data provided in this study will help further understanding of equine ocular immunity in the normal state and might be beneficial for understanding of mechanisms of ocular disorders, such as equine uveitis.

  16. 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.

  17. 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

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    for FOXP3 expression in tumour cells and tumour infiltrating Tregs. Labelling of a majority of the neoplastic cells was seen in one case of C-ALCL. Another three cases (one LyP and two C-ALCL) displayed weak labelling of very occasional atypical T-cells. In the remaining 38 cases the atypical lymphoid...... infiltrate was FOXP3 negative. By contrast, all biopsies contained tumour infiltrating FOXP3-positive Tregs. Significant higher numbers were recorded in ALK negative S-ALCL and LyP than in C-ALCL and S-ALCL positive for ALK. In conclusion, it is shown that FOXP3 expression in cutaneous and systemic CD30...

  19. Anaphylaxis related with positively charged white-cell reduction filters.

    Science.gov (United States)

    Topal, Yaşar; Topal, Hatice; Çapanoğlu, Murat; Çetinkaya, Petek Uzay; Kocabas, Can Naci

    2014-04-01

    Allergic reactions related to blood transfusion frequently occur and most of them are mild reactions such as urticaria, erythema, pruritus and flushing. More severe and life threatening allergic reactions such as anaphylactic shock rarely occur. Application of white cell reduction filters during transfusions may prevent alloimmunization, febrile nonhemolytic reactions and transmission of intracellular infectious agents. Despite their beneficial effects, white-cell reduction filters may cause allergic reactions. In this article we present three patients who had anaphylactic reactions during blood transfusion with positively charged leucocyte filters. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. 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.

  1. Inhibition of protein kinase CK2 by CX-5011 counteracts imatinib-resistance preventing rpS6 phosphorylation in chronic myeloid leukaemia cells: new combined therapeutic strategies

    Science.gov (United States)

    Salizzato, Valentina; Borgo, Christian; Cesaro, Luca; Pinna, Lorenzo A.; Donella-Deana, Arianna

    2016-01-01

    Chronic myeloid leukaemia (CML) is a myeloproliferative disorder promoted by the constitutive tyrosine kinase activity of Bcr-Abl oncoprotein. Although treatment with the Bcr-Abl-inhibitor imatinib represents the first-line therapy against CML, almost 20-30% of patients develop chemotherapeutic resistance and require alternative therapy. Here we show that a strong hyper-phosphorylation/activation of ERK1/2, Akt Ser473, and 40S ribosomal protein S6 (rpS6) is detectable in imatinib-resistant KCL22 and K562 CML cells as compared to the -sensitive cell variants. In imatinib-resistant CML cells, high concentration of imatinib is required to strongly inhibit Bcr-Abl, ERK1/2 and Akt Ser473 phosphorylation, but under these conditions the phosphorylation of rpS6, a common downstream effector of MEK/ERK1/2 and PI3K/Akt/mTOR pathways is only slightly reduced. By contrast, down-regulation of the protein kinase CK2 by the inhibitor CX-5011 or by silencing the CK2 subunits does not affect the activation state of MEK/ERK1/2 or PI3K/Akt/mTOR signalling, but causes a drop in rpS6 phosphorylation in parallel with reduced protein synthesis. CK2-inhibition by CX-5011 induces cell death by apoptosis and acts synergistically with imatinib or the MEK-inhibitor U0126 in reducing the viability of imatinib-resistant CML cells. The ternary mixture containing CX-5011, imatinib and U0126 represents the most effective synergistic combination to counteract CML cell viability. These results disclose a novel CK2-mediated mechanism of acquired imatinib-resistance resulting in hyper-phosphorylation of rpS6. We suggest that co-targeting CK2 and MEK protein kinases is a promising strategy to restore responsiveness of resistant CML cells to imatinib. PMID:26919095

  2. CD34-positive stromal cells and alpha-smooth muscle actin-positive stromal cells in the tumor capsule of skin sweat gland neoplasms.

    Science.gov (United States)

    Nakayama, Hirofumi; Enzan, Hideaki; Miyazaki, Eriko; Moriki, Toshiaki; Toi, Makoto; Zhang, Yanhu

    2002-01-01

    To elucidate the roles of CD34-positive stromal cells and alpha-smooth muscle actin-positive stromal cells at the tumor border of skin sweat gland neoplasms, we examined expression of stromal cell markers in the tumor capsule of 19 skin sweat gland neoplasms (16 mixed tumors of the skin and three nodular hidradenomas) using monoclonal antibodies to CD34, CD31, cytokeratin 14 (CK14), alpha-smooth muscle actin (ASMA) and high molecular weight caldesmon (HCD). We regarded CD34-positive, CD31-, CK14-, ASMA- and HCD-negative stromal cells to be CD34-positive stromal cells, and ASMA-positive, HCD-, CK14-, CD34- and CD31-negative stromal cells to be ASMA-positive stromal cells. CD34-positive stromal cells were detected in the tumor capsule of all 19 of the tumors examined. In nine of the 16 mixed tumors (56%) and all of the three nodular hidradenomas, ASMA-positive stromal cells were detected at the immediate inner side of the CD34-positive stromal cell layers. These results indicate that cellular components in the tumor capsules of mixed tumors of the skin and nodular hidradenomas are CD34-positive stromal cells and ASMA-positive stromal cells, and suggest that stromal cells of these two cell types are associated with tumor capsule formation of skin sweat gland neoplasms.

  3. Prognostic impact of IKZF1 deletion in adults with common B-cell acute lymphoblastic leukemia.

    Science.gov (United States)

    Yao, Qiu-Mei; Liu, Kai-Yan; Gale, Robert Peter; Jiang, Bin; Liu, Yan-Rong; Jiang, Qian; Jiang, Hao; Zhang, Xiao-Hui; Zhang, Mei-Jie; Chen, Shan-Shan; Huang, Xiao-Jun; Xu, Lan-Ping; Ruan, Guo-Rui

    2016-04-11

    Interrogate the impact of IKZF1 deletion on therapy-outcomes of adults with common B-cell acute lymphoblastic leukemia. One hundred sixty-five consecutive adults with common B-cell ALL were tested for IKZF1 deletion and for BCR/ABL. Deletions in IKZF1 were detected using multiplex RQ-PCR, multiplex fluorescent PCR, sequence analysis and multiplex ligation-dependent probe amplification (MLPA). BCR/ABL was detected using RQ-PCR. All subjects received chemotherapy and some also received an allotransplant and tyrosine kinase-inhibitors. Multivariate analyses were done to identify associations between IKZF1 deletion and other variables on non-relapse mortality (NRM), cumulative incidence of relapse (CIR), leukemia-free survival (LFS) and survival. Amongst subjects achieving complete remission those with IKZF1 deletion had similar 5-year non-relapse mortality (NRM) (11% [2-20%] vs. 16% [4-28%]; P = 0.736), a higher 5-year cumulative incidence of relapse (CIR) (55% [35-76%] vs. 25% [12-38%]; P = 0.004), and worse 5-year leukemia-free survival (LFS) (33% [16-52%] vs. 59% [42-73%]; P = 0.012) and survival (48% [33-62%] vs. 75% [57-86%]; P = 0.002). In multivariate analyses IKZF1 deletion was associated with an increased relapse (relative risk [RR] =2.7, [1.4-5.2]; P = 0.002), a higher risk of treatment-failure (inverse of LFS; RR = 2.1, [1.2-3.6]; P = 0.007) and a higher risk of death (RR = 2.8, [1.5-5.5]; P = 0.002). The adverse impact of IKZF1 deletion on outcomes was stronger in subjects without vs. with BCR-ABL1 and in subjects receiving chemotherapy-only vs. an allotransplant. IKZF1 deletion was independently-associated with a higher relapse risk and worse LFS and survival in adults with common B-cell ALL after adjusting for other prognostic variables and differences in therapies. These data suggest IKZF1 deletion may be a useful prognostic variable in adults with common B-cell ALL, especially in persons without BCR-ABL1 and those receiving chemotherapy

  4. 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.

  5. Abnormal chromatin clumping in leucocytes of Ph positive Chronic Myeloid Leukemia cases - extending the morphological spectrum

    Directory of Open Access Journals (Sweden)

    Adhya Amit

    2008-10-01

    Full Text Available The syndrome of abnormal chromatin clumping is largely a morphological entity characterized by exaggerated chromatin clumping seen in the neutrophils. According to the recent World Health Organization (WHO classification, it is categorized as a variant of atypical chronic myeloid leukemia (aCML or Ph-negative CML. Most of the cases reported in literature have been negative for the Ph chromosome or the BCR-ABL gene. Till date, Ph positivity has been demonstrated in just one case. We report two more Ph-positive CML cases with abnormal chromatin clumping in neutrophils. To the best of our knowledge, this is only the second time in literature that such cases have been described. These two unusual cases go on to extend the morphological spectrum of granulocytic changes seen in Ph-positive CML.

  6. Increased peroxisome proliferator-activated receptor γ 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

    2017-05-01

    Imatinib is actively transported by organic cation transporter-1 (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. Herein we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1 + cell lines, peroxisome proliferator-activated receptor γ agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor γ 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 γ-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor γ transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; P chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor γ ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor γ 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 γ transcriptional activity. Copyright© Ferrata Storti Foundation.

  7. The first cell-fate decision of mouse preimplantation embryo development: integrating cell position and polarity.

    Science.gov (United States)

    Mihajlović, Aleksandar I; Bruce, Alexander W

    2017-11-01

    During the first cell-fate decision of mouse preimplantation embryo development, a population of outer-residing polar cells is segregated from a second population of inner apolar cells to form two distinct cell lineages: the trophectoderm and the inner cell mass (ICM), respectively. Historically, two models have been proposed to explain how the initial differences between these two cell populations originate and ultimately define them as the two stated early blastocyst stage cell lineages. The 'positional' model proposes that cells acquire distinct fates based on differences in their relative position within the developing embryo, while the 'polarity' model proposes that the differences driving the lineage segregation arise as a consequence of the differential inheritance of factors, which exhibit polarized subcellular localizations, upon asymmetric cell divisions. Although these two models have traditionally been considered separately, a growing body of evidence, collected over recent years, suggests the existence of a large degree of compatibility. Accordingly, the main aim of this review is to summarize the major historical and more contemporarily identified events that define the first cell-fate decision and to place them in the context of both the originally proposed positional and polarity models, thus highlighting their functional complementarity in describing distinct aspects of the developmental programme underpinning the first cell-fate decision in mouse embryogenesis. © 2017 The Authors.

  8. CD163-positive cancer cells are potentially associated with high malignant potential in clear cell renal cell carcinoma.

    Science.gov (United States)

    Ma, Chaoya; Horlad, Hasita; Ohnishi, Koji; Nakagawa, Takenobu; Yamada, Sohsuke; Kitada, Shohei; Motoshima, Takanobu; Kamba, Tomomi; Nakayama, Toshiyuki; Fujimoto, Naohiro; Takeya, Motohiro; Komohara, Yoshihiro

    2018-03-01

    CD163 is preferentially expressed by monocyte/macrophages; however, recent studies using immunohistochemistry (IHC) have reported that some cancer cells also express CD163. In the present IHC study, we investigated CD163 staining of cancer cells and macrophages in clear cell renal cell carcinoma (ccRCC) tissues and determined the relationship between cancer cell CD163 expression and clinical prognosis in patients with ccRCC. IHC for CD163 was performed in ccRCC tissues from 103 patients. CD163-positive cancer cells were detected in 35% of the patients (36/103); however, the positive signals on cancer cells were significantly lower than those on macrophages. CD163-positive cancer cells were preferentially detected in patients with high T classification, and females, and were significantly associated with shortened progression-free survival and a lower overall survival ratio. Notably, a high intensity of CD163-positive macrophage infiltration was detected in the CD163-positive cancer cell-high tumor areas. Although CD163 mRNA was detected in cultured macrophages, no CD163 mRNA was detected in two cultured RCC cell lines. The detailed mechanism by which a positive signal is detected on cancer cells has not been clarified. Detection of the CD163 antigen on cancer cells might be a useful marker for evaluating the clinical course of patients with ccRCC.

  9. Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells.

    Science.gov (United States)

    Gaya, Mauro; Barral, Patricia; Burbage, Marianne; Aggarwal, Shweta; Montaner, Beatriz; Warren Navia, Andrew; Aid, Malika; Tsui, Carlson; Maldonado, Paula; Nair, Usha; Ghneim, Khader; Fallon, Padraic G; Sekaly, Rafick-Pierre; Barouch, Dan H; Shalek, Alex K; Bruckbauer, Andreas; Strid, Jessica; Batista, Facundo D

    2018-01-25

    B cells constitute an essential line of defense from pathogenic infections through the generation of class-switched antibody-secreting cells (ASCs) in germinal centers. Although this process is known to be regulated by follicular helper T (TfH) cells, the mechanism by which B cells initially seed germinal center reactions remains elusive. We found that NKT cells, a population of innate-like T lymphocytes, are critical for the induction of B cell immunity upon viral infection. The positioning of NKT cells at the interfollicular areas of lymph nodes facilitates both their direct priming by resident macrophages and the localized delivery of innate signals to antigen-experienced B cells. Indeed, NKT cells secrete an early wave of IL-4 and constitute up to 70% of the total IL-4-producing cells during the initial stages of infection. Importantly, the requirement of this innate immunity arm appears to be evolutionarily conserved because early NKT and IL-4 gene signatures also positively correlate with the levels of neutralizing antibodies in Zika-virus-infected macaques. In conclusion, our data support a model wherein a pre-TfH wave of IL-4 secreted by interfollicular NKT cells triggers the seeding of germinal center cells and serves as an innate link between viral infection and B cell immunity. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  10. 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.

  11. Planar cell polarity breaks bilateral symmetry by controlling ciliary positioning.

    Science.gov (United States)

    Song, Hai; Hu, Jianxin; Chen, Wen; Elliott, Gene; Andre, Philipp; Gao, Bo; Yang, Yingzi

    2010-07-15

    Defining the three body axes is a central event of vertebrate morphogenesis. Establishment of left-right (L-R) asymmetry in development follows the determination of dorsal-ventral and anterior-posterior (A-P) body axes, although the molecular mechanism underlying precise L-R symmetry breaking in reference to the other two axes is still poorly understood. Here, by removing both Vangl1 and Vangl2, the two mouse homologues of a Drosophila core planar cell polarity (PCP) gene Van Gogh (Vang), we reveal a previously unrecognized function of PCP in the initial breaking of lateral symmetry. The leftward nodal flow across the posterior notochord (PNC) has been identified as the earliest event in the de novo formation of L-R asymmetry. We show that PCP is essential in interpreting the A-P patterning information and linking it to L-R asymmetry. In the absence of Vangl1 and Vangl2, cilia are positioned randomly around the centre of the PNC cells and nodal flow is turbulent, which results in disrupted L-R asymmetry. PCP in mouse, unlike what has been implicated in other vertebrate species, is not required for ciliogenesis, cilium motility, Sonic hedgehog (Shh) signalling or apical docking of basal bodies in ciliated tracheal epithelial cells. Our data suggest that PCP acts earlier than the unidirectional nodal flow during bilateral symmetry breaking in vertebrates and provide insight into the functional mechanism of PCP in organizing the vertebrate tissues in development.

  12. Apoptosis in chronic myeloid leukaemia: normal responses by progenitor cells to growth factor deprivation, X-irradiation and glucocorticoids

    Energy Technology Data Exchange (ETDEWEB)

    Amos, T.A.S.; Lewis, J.L.; Grand, F.H.; Gooding, R.P.; Goldman, J.M.; Gordon, M.Y. [Royal Postgraduate Medical School, London (United Kingdom)

    1995-10-01

    Inhibition of apoptosis (genetically programmed active cell death) by p210 BCR-ABL expression is a mechanism that might contribute to clonal expansion in chronic myeloid leukaemia (CML). Since cell death following exposure to ionizing radiation and many chemotherapeutic agents can occur by the apoptotic pathway, inhibition of apoptosis would be expected to confer a relative resistance to these treatments. Similarly, cells deprived of growth factors in vitro die by apoptosis, and inhibition of apoptosis would therefore be expected to allow cells to survive better in growth factor-deprived conditions. We found that the survival of normal and CML myeloid progenitors was the same after in vitro incubation in deprived conditions and after treatment with X-irradiation or glucocorticoids. We also found that mature cells in colonies produced by CML progenitors (CFU-GM) did not survive better than those produced by normal progenitor cells. Flow cytometric analysis of propidium iodide-stained cells provided a direct indication that the degree of apoptosis may correspond to the degree of deprivation. These results suggest that inhibition of apoptosis may not be the primary mechanism whereby BCR-ABL influences the expansion of the malignant clone in CML. (Author).

  13. Apoptosis in chronic myeloid leukaemia: normal responses by progenitor cells to growth factor deprivation, X-irradiation and glucocorticoids

    International Nuclear Information System (INIS)

    Amos, T.A.S.; Lewis, J.L.; Grand, F.H.; Gooding, R.P.; Goldman, J.M.; Gordon, M.Y.

    1995-01-01

    Inhibition of apoptosis (genetically programmed active cell death) by p210 BCR-ABL expression is a mechanism that might contribute to clonal expansion in chronic myeloid leukaemia (CML). Since cell death following exposure to ionizing radiation and many chemotherapeutic agents can occur by the apoptotic pathway, inhibition of apoptosis would be expected to confer a relative resistance to these treatments. Similarly, cells deprived of growth factors in vitro die by apoptosis, and inhibition of apoptosis would therefore be expected to allow cells to survive better in growth factor-deprived conditions. We found that the survival of normal and CML myeloid progenitors was the same after in vitro incubation in deprived conditions and after treatment with X-irradiation or glucocorticoids. We also found that mature cells in colonies produced by CML progenitors (CFU-GM) did not survive better than those produced by normal progenitor cells. Flow cytometric analysis of propidium iodide-stained cells provided a direct indication that the degree of apoptosis may correspond to the degree of deprivation. These results suggest that inhibition of apoptosis may not be the primary mechanism whereby BCR-ABL influences the expansion of the malignant clone in CML. (Author)

  14. Single-cell analysis of the fate of c-kit-positive bone marrow cells

    Science.gov (United States)

    Czarna, Anna; Sanada, Fumihiro; Matsuda, Alex; Kim, Junghyun; Signore, Sergio; Pereira, João D.; Sorrentino, Andrea; Kannappan, Ramaswamy; Cannatà, Antonio; Hosoda, Toru; Rota, Marcello; Crea, Filippo; Anversa, Piero; Leri, Annarosa

    2017-10-01

    The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.

  15. Spindle Positioning and Cell Division in Caenorhabditis elegans

    OpenAIRE

    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 diversity and maintenance of stem cell populations throughout the development and life of the organism. Two different mechanisms of asymmetric cell division exist. In one case the fate of the daughter ce...

  16. Management of Platelet Transfusion Therapy in Patients With Blood Cancer or Treatment-Induced Thrombocytopenia

    Science.gov (United States)

    2018-02-15

    Acute Biphenotypic Leukemia; Acute Lymphoblastic Leukemia; Acute Myeloid Leukemia; B-Cell Non-Hodgkin Lymphoma; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Hematologic and Lymphocytic Disorder; Hematopoietic Cell Transplantation Recipient; Myelodysplastic Syndrome; Primary Myelofibrosis; Secondary Myelofibrosis; T-Cell Non-Hodgkin Lymphoma; Thrombocytopenia; Venous Thromboembolism

  17. Induction Chemotherapy for p16 Positive Oropharyngeal Squamous Cell Carcinoma.

    Science.gov (United States)

    Saito, Yuki; Ando, Mizuo; Omura, Go; Yasuhara, Kazuo; Yoshida, Masafumi; Takahashi, Wataru; Yamasoba, Tatsuya

    2016-04-01

    We aimed to determine the effectiveness of induction chemotherapy for treating p16-positive oropharyngeal cancer in our department. This was a retrospective case series to assess treatment effectiveness. We administered induction chemotherapy to patients with stage III to IV oropharyngeal p16-positive squamous cell carcinoma between 2008 and 2013. Induction chemotherapy was administered using combinations of docetaxel, cisplatin, and 5-fluorouracil. We measured the survival rates using the Kaplan-Meier method and log-rank test. We reviewed 23 patients (18 men and 5 women; age, 42-79 years). Induction chemotherapy resulted in partial or complete remission (20 patients) and in stable (2 patients) or progressive (1 patient) disease. In partial or complete remission, subsequent radiotherapy was performed in 16 patients, chemoradiotherapy in two, and transoral resection in two. In stable or progressive disease, subsequent open surgery was performed. Overall, one patient died of cervical lymph node metastasis, one died of kidney cancer, and one died of myocardial infarction. Event-free, distant-metastasis-free survival was present for 20 patients. The 3-year disease-specific survival was 95%; the overall survival was 87%. Two patients required gastrostomies during chemoradiotherapy and three required tracheotomies, but these were closed in all patients. The therapeutic response to induction chemotherapy for p16-positive oropharyngeal cancer was good. Partial or complete remission was achieved in almost 90% patients, and control of local and distant metastases was possible when it was followed by radiotherapy alone or with transoral resection of the primary tumor. A multicenter study is required to confirm these findings. 4.

  18. 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

  19. 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.

  20. Sickle cell, habitual dys-positions and fragile dispositions: young people with sickle cell at school

    Science.gov (United States)

    Dyson, Simon M; Atkin, Karl; Culley, Lorraine A; Dyson, Sue E; Evans, Hala

    2011-01-01

    The experiences of young people living with a sickle cell disorder in schools in England are reported through a thematic analysis of forty interviews, using Bourdieu’s notions of field, capital and habitus. Young people with sickle cell are found to be habitually dys-positioned between the demands of the clinic for health maintenance through self-care and the field of the school, with its emphases on routines, consistent attendance and contextual demands for active and passive pupil behaviour. The tactics or dispositions that young people living with sickle cell can then employ, during strategy and struggle at school, are therefore fragile: they work only contingently, transiently or have the unintended consequences of displacing other valued social relations. The dispositions of the young people with sickle cell are framed by other social struggles: innovations in school procedures merely address aspects of sickle cell in isolation and are not consolidated into comprehensive policies; mothers inform, liaise, negotiate and advocate in support of a child with sickle cell but with limited success. Reactions of teachers and peers to sickle cell have the enduring potential to drain the somatic, cultural and social capital of young people living with sickle cell. PMID:21375541

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  2. Stem cell tourism in South Africa: The legal position | Mahomed ...

    African Journals Online (AJOL)

    Stem cell tourism has become a common phenomenon worldwide and is increasingly affecting South Africa, as is evident from recent media reports. We examine the South African legal framework regulating stem cell therapy, focusing first on the effects of unproven stem cell treatments, and provide recommendations that ...

  3. Radiation-induced apoptosis in differentially modulated by PTK inhibitora in K562 cells

    International Nuclear Information System (INIS)

    Lee, Hyung Sik; Moon, Chang Woo; Hur, Won Joo; Jeong, Su Jin; Jeong Min Ho; Lee, Jeong Hyeon; Lim, Young Jin; Park, Heon Joo

    2000-01-01

    The effect of PTK inhibitors (herbimycin A and genistein) on the induction of radiation-induced apoptosis in Ph-positive K562 leukemia cell line was investigated. K562 cells in exponential growth phase were irradiated with a linear accelerator at room temperature. For 6 MV X-ray irradiation and drug treatment, cultures were initiated at 2x10 6 cells/ml. The cells were irradiated with 10Gy. Stock solutions of herbimycin A and genistein were prepared in dimethyl sulphoxide (DMSO). After incubation at 37 .deg. for 0-48 h, the extent of apoptosis was determined using agarose gel electrophoresis and TUNEL assay. The progression of cells through the cell cycle after irradiation and drug treatment was also determined with flow cytometry. Western blot analysis was used to monitor bcl-2, bcl-X-L and bax protein levels. Treatment with 10 Gy X-irradiation did not result in the induction of apoptosis. The HMA alone (500 nM) also failed to induce apoptosis. By contrast, incubation of K562 cells with HMA after irradiation resulted in a substantial induction of nuclear condensation and fragmentation by agarose gel electrophoresis and TUNEL assay. Genistein failed to enhance the ability of X-irradiation to induce DNA fragmentation. Enhancement of apoptosis by HMA was not attributable to downregulation of the bcl-2 or bcl-X-L anti-apoptotic proteins. When the cells were irradiated and maintained with HMA, the percentage of cells in G2/M phase decreased to 30-40% at 48 h. On the other hand, cells exposed to 10 Gy X-irradiation alone or maintained with genistein did not show marked cell cycle redistribution. We have shown that nanomolar concentrations of the PTK inhibitor HMA synergize with X-irradiation in inducing the apoptosis in Ph (+) K562 leukemia cell line. While, genistein, a PTK inhibitor which is not selective for p210 bcr/abl failed to enhance the radiation induced apoptosis in K562 cells. It is unlikely that the ability of HMA to enhance apoptosis in K562 cells is

  4. Molecular pathways of early CD105-positive erythroid cells as compared with CD34-positive common precursor cells by flow cytometric cell-sorting and gene expression profiling

    International Nuclear Information System (INIS)

    Machherndl-Spandl, S; Suessner, S; Danzer, M; Proell, J; Gabriel, C; Lauf, J; Sylie, R; Klein, H-U; Béné, M C; Weltermann, A; Bettelheim, P

    2013-01-01

    Special attention has recently been drawn to the molecular network of different genes that are responsible for the development of erythroid cells. The aim of the present study was to establish in detail the immunophenotype of early erythroid cells and to compare the gene expression profile of freshly isolated early erythroid precursors with that of the CD34-positive (CD34 + ) compartment. Multiparameter flow cytometric analyses of human bone marrow mononuclear cell fractions (n=20) defined three distinct early erythroid stages. The gene expression profile of sorted early erythroid cells was analyzed by Affymetrix array technology. For 4524 genes, a differential regulation was found in CD105-positive erythroid cells as compared with the CD34 + progenitor compartment (2362 upregulated genes). A highly significant difference was observed in the expression level of genes involved in transcription, heme synthesis, iron and mitochondrial metabolism and transforming growth factor-β signaling. A comparison with recently published data showed over 1000 genes that as yet have not been reported to be upregulated in the early erythroid lineage. The gene expression level within distinct pathways could be illustrated directly by applying the Ingenuity software program. The results of gene expression analyses can be seen at the Gene Expression Omnibus repository

  5. 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...... characteristics, CD163lo and CD163hi, together constituting a substantial fraction of DCs. Both subsets were characterized as [lin]- CD4+ ILT3+ HLA-DR+ CD11c+ by flow cytometry, and CD163 mRNA was readily detectable in MACS purified human DCs. CD163 on DCs was upregulated by glucocorticoid, and treatment...

  6. The Role of Backup NHEJ Repair in Creating Genomic Instability in CML

    Science.gov (United States)

    2008-03-01

    reagents and critical reading of the manuscript. 20 Figure Legends 1 BCR-ABL positive CML cell lines show down-regulation of major NHEJ proteins...2006;26:3935-3941. 52. Tebbs RS, Thompson LH, Cleaver JE. Rescue of Xrcc1 knockout mouse embryo lethality by transgene-complementation. DNA Repair (Amst

  7. Long Noncoding RNA PANDA Positively Regulates Proliferation of Osteosarcoma Cells.

    Science.gov (United States)

    Kotake, Yojiro; Goto, Taiki; Naemura, Madoka; Inoue, Yasutoshi; Okamoto, Haruna; Tahara, Keiichiro

    2017-01-01

    A long noncoding RNA, p21-associated ncRNA DNA damage-activated (PANDA), associates with nuclear transcription factor Y subunit alpha (NF-YA) and inhibits its binding to promoters of apoptosis-related genes, thereby repressing apoptosis in normal human fibroblasts. Here, we show that PANDA is involved in regulating proliferation in the U2OS human osteosarcoma cell line. U2OS cells were transfected with siRNAs against PANDA 72 h later and they were subjected to reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR and cell-cycle analysis. PANDA was highly expressed in U2OS cells, and its expression was induced by DNA damage. Silencing PANDA caused arrest at the G 1 phase of the cell cycle, leading to inhibition of cell proliferation. Quantitative RT-PCR showed that silencing PANDA increased mRNA levels of the cyclin-dependent kinase inhibitor p18, which caused G 1 phase arrest. These results suggest that PANDA promotes G 1 -S transition by repressing p18 transcription, and thus promotes U2OS cell proliferation. Copyright© 2017 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  8. ER-α36-Mediated Rapid Estrogen Signaling Positively Regulates ER-Positive Breast Cancer Stem/Progenitor Cells

    Science.gov (United States)

    Deng, Hao; Zhang, Xin-Tian; Wang, Mo-Lin; Zheng, Hong-Yan; Liu, Li-Jiang; Wang, Zhao-Yi

    2014-01-01

    The breast cancer stem cells (BCSC) play important roles in breast cancer occurrence, recurrence and metastasis. However, the role of estrogen signaling, a signaling pathway important in development and progression of breast cancer, in regulation of BCSC has not been well established. Previously, we identified and cloned a variant of estrogen receptor α, ER-α36, with a molecular weight of 36 kDa. ER-α36 lacks both transactivation domains AF-1 and AF-2 of the 66 kDa full-length ER-α (ER-α66) and mediates rapid estrogen signaling to promote proliferation of breast cancer cells. In this study, we aim to investigate the function and the underlying mechanism of ER-α36-mediated rapid estrogen signaling in growth regulation of the ER-positive breast cancer stem/progenitor cells. ER-positive breast cancer cells MCF7 and T47D as well as the variants with different levels of ER-α36 expression were used. The effects of estrogen on BCSC's abilities of growth, self-renewal, differentiation and tumor-seeding were examined using tumorsphere formation, flow cytometry, indirect immunofluorence staining and in vivo xenograft assays. The underlying mechanisms were also studied with Western-blot analysis. We found that 17-β-estradiol (E2β) treatment increased the population of ER-positive breast cancer stem/progenitor cells while failed to do so in the cells with knocked-down levels of ER-α36 expression. Cells with forced expression of recombinant ER-α36, however, responded strongly to E2β treatment by increasing growth in vitro and tumor-seeding efficiency in vivo. The rapid estrogen signaling via the AKT/GSK3β pathway is involved in estrogen-stimulated growth of ER-positive breast cancer stem/progenitor cells. We concluded that ER-α36-mediated rapid estrogen signaling plays an important role in regulation and maintenance of ER-positive breast cancer stem/progenitor cells. PMID:24558373

  9. CD3-positive B cells: a storage-dependent phenomenon.

    Directory of Open Access Journals (Sweden)

    Angela Nagel

    Full Text Available The majority of clinical studies requires extensive management of human specimen including e.g. overnight shipping of blood samples in order to convey the samples in a central laboratory or to simultaneously analyze large numbers of patients. Storage of blood samples for periods of time before in vitro/ex vivo testing is known to influence the antigen expression on the surface of lymphocytes. In this context, the present results show for the first time that the T cell antigen CD3 can be substantially detected on the surface of human B cells after ex vivo storage and that the degree of this phenomenon critically depends on temperature and duration after blood withdrawal. The appearance of CD3 on the B cell surface seems to be a result of contact-dependent antigen exchange between T and B lymphocytes and is not attributed to endogenous production by B cells. Since cellular subsets are often classified by phenotypic analyses, our results indicate that ex vivo cellular classification in peripheral blood might result in misleading interpretations. Therefore, in order to obtain results reflecting the in vivo situation, it is suggested to minimize times of ex vivo blood storage after isolation of PBMC. Moreover, to enable reproducibility of results between different research groups and multicenter studies, we would emphasize the necessity to specify and standardize the storage conditions, which might be the basis of particular findings.

  10. Intracellular position of mitochondria in mesophyll cells differs between C3and C4grasses.

    Science.gov (United States)

    Hatakeyama, Yuto; Ueno, Osamu

    2017-09-01

    In C 3 plants, part of the CO 2 fixed during photosynthesis in chloroplasts is released from mitochondria during photorespiration by decarboxylation of glycine via glycine decarboxylase (GDC), thereby reducing photosynthetic efficiency. The apparent positioning of most mitochondria in the interior (vacuole side of chloroplasts) of mesophyll cells in C 3 grasses would increase the efficiency of refixation of CO 2 released from mitochondria by ribulose 1,5-bisphosphate carboxylase/​oxygenase (Rubisco) in chloroplasts. Therefore, in mesophyll cells of C 4 grasses, which lack both GDC and Rubisco, the mitochondria ought not to be positioned the same way as in C 3 mesophyll cells. To test this hypothesis, we investigated the intracellular position of mitochondria in mesophyll cells of 14 C 4 grasses of different C 4 subtypes and subfamilies (Chloridoideae, Micrairoideae, and Panicoideae) and a C 3 -C 4 intermediate grass, Steinchisma hians, under an electron microscope. In C 4 mesophyll cells, most mitochondria were positioned adjacent to the cell wall, which clearly differs from the positioning in C 3 mesophyll cells. In S. hians mesophyll cells, the positioning was similar to that in C 3 cells. These results suggest that the mitochondrial positioning in C 4 mesophyll cells reflects the absence of both GDC and Rubisco in the mesophyll cells and the high activity of phosphoenolpyruvate carboxylase. In contrast, the relationship between the mitochondrial positioning and enzyme distribution in S. hians is complex, but the positioning may be related to the capture of respiratory CO 2 by Rubisco. Our study provides new possible insight into the physiological role of mitochondrial positioning in photosynthetic cells.

  11. 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 accumulation of FOXP3-positive immune cells in the tumor microenvironment in metastatic renal cell carcinoma is unknown. EXPERIMENTAL DESIGN: Baseline (n = 58) and on-treatment (n = 42) tumor core biopsies were prospectively obtained from patients with clear cell metastatic renal cell carcinoma before...... and during IL-2-based immunotherapy. Immunohistochemical expression of FOXP3 was estimated by stereological counting technique and correlated with other immune cell subsets and overall survival. RESULTS: A significant increase in absolute intratumoral FOXP3-positive immune cells was observed comparing...

  12. Characterization of nonlymphoid cells in rat spleen, with special reference to strongly Ia-positive branched cells in T-cell areas

    International Nuclear Information System (INIS)

    Dijkstra, C.D.

    1982-01-01

    By use of a monoclonal antibody against Ia antigen in an immunoperoxidase method, strongly Ia-positive branched cells are found in the T-cell areas of the splenic white pulp of the rat. In order to further characterize these cells, enzyme histochemical characteristics, phagocytic capacity, and irradiation sensitivity have been studied. Evidence is presented that these strongly Ia-positive branched cells represent interdigitating cells. The influence of whole-body irradiation on interdigitating cells is discussed. Comparison with data from the literature on the in vitro dendritic cell isolated from spleen cell suspensions reveals many similarities between the described interdigitating cell in vivo and the dendritic cell in vitro

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  14. Role of Foxp3-positive regulatory T cells during infection

    Science.gov (United States)

    Belkaid, Yasmine

    2012-01-01

    Surviving an infection requires the generation of an immune response that controls the invading pathogen while limiting collateral damage to self tissues that may result from an exuberant immune response. Various populations of regulatory cells, including Foxp3+ Treg, have been shown to play a central role in the establishment of these controlled immune responses. In this review, I discuss current hypotheses and points of polemic associated with the origin, mode of action and antigen specificity of Foxp3+ Treg during infection. PMID:18395860

  15. 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

  16. Prevalence of t(12;21)[ETV6-RUNX1]-positive cells in healthy neonates

    DEFF Research Database (Denmark)

    Lausten-Thomsen, Ulrik; Madsen, Hans Ole; 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......-RUNX1-positive cells in healthy neonates, mononuclear cells from 1417 umbilical cord blood samples were isolated within 24 hours from birth and subsequently screened for ETV6-RUNX1 transcripts using a highly sensitive real-time reverse transcription polymerase chain reaction assay. In first...... mononuclear cells from the same cord blood samples (mean sorted: 18 × 10(6) cells) revealed no positive findings, which demonstrates that the level and/or frequency of ETV6-RUNX1-positive cells is markedly lower than suggested in previous studies....

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

    Directory of Open Access Journals (Sweden)

    Corrêa Stephany

    2012-03-01

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

  18. Positive Captopril Renography Without Renal Artery Stenosis but a Renal Cell Carcinoma.

    Science.gov (United States)

    Hsieh, Ping-Ju; Kuo, Yen-Shu; Chen, Meng-Lin; Lin, Ching-Ling; Su, Hung-Yi

    2017-11-01

    A positive captopril renography indicates that patient's hypertension is renin dependent, most commonly caused by renal artery stenosis. The authors reported a case of positive captopril renography; however, CT demonstrated that renal arteries were intact, but there was a huge chromophobe renal cell carcinoma. Renin-dependent hypertension was relieved soon after nephrectomy. It is an uncommon cause of positive captopril renography.

  19. Tracking infrared signatures of drugs in cancer cells by Fourier transform microspectroscopy.

    Science.gov (United States)

    Bellisola, Giuseppe; Della Peruta, Marco; Vezzalini, Marzia; Moratti, Elisabetta; Vaccari, Lisa; Birarda, Giovanni; Piccinini, Massimo; Cinque, Gianfelice; Sorio, Claudio

    2010-12-01

    Aimed at developing accurate, reliable and cost-saving analytical techniques for drugs screening we evaluated the potential of Fourier Transform (FT) InfraRed (IR) microspectroscopy (microFTIR) as a quantitative pre-diagnostic approach for the rapid identification of IR signatures of drugs targeting specific molecular pathways causing Chronic Myeloid Leukemia (CML). To obtain reproducible FTIR absorbance spectra at the necessary spatial resolution we optimized sample preparation and acquisition parameters on a single channel Mercury-Cadmium-Telluride (MCT) detector in the spectral interval of frequencies from 4000 to 800 cm(-1). Single K562 cells were illuminated by Synchrotron Radiation (SR) and a number of ~15 K562 cells spread in monolayer were illuminated by a conventional IR source (Globar), respectively. Combining IR spectral data with the results of complementary biochemical investigations carried out in samples by different analytical methods we identified and cross-validated IR signatures of drugs targeting the oncogenic protein BCR/ABL and its associated abnormal tyrosine kinase activity in K562 cell line. Unsupervised pattern recognition performed by Hierarchical Cluster Analysis (HCA) clustered the spectra of single K562 cells in two distinct groups roughly corresponding to living and to apoptotic cells, respectively. The corresponding IR spectral profiles were assumed to represent drug-resistant and drug-sensitive cells. Significant variations with increasing percentages of apoptotic cells were observed after the treatment of K562 cells with drugs that directly or indirectly target BCR/ABL. In conclusion, we suggest that microFTIR associated with multivariate data analysis may be useful to assess drug compounds in ex vivo cancer cell models and possibly peripheral blast cells from CML patients.

  20. Thy-1 (CD90)-Positive Hepatic Progenitor Cells, Hepatoctyes, and Non-parenchymal Liver Cells Isolated from Human Livers.

    Science.gov (United States)

    Weiss, Thomas S; Dayoub, Rania

    2017-01-01

    In response to liver injury, hepatic cells, especially hepatocytes, can rapidly proliferate to repair liver damage. Additionally, it was shown that under certain circumstances liver resident cells with progenitor capabilities are involved in liver cell proliferation and differentiation. These hepatic progenitor cells (HPCs), known as oval cells in rodents, are derived from the canals of Hering, which are located in the periportal region of the liver. Regarding to different cell niches, which were defined for human HPCs, several markers have been used to identify these cells such as CD34, c-kit, OV-6, and Thy-1 (CD90). The latter was shown to be expressed on HPCs in human liver tissue with histological signs of regeneration. In this chapter we describe a detailed method for the isolation of Thy-1 positive cells from human resected liver tissue. Based on a procedure for isolating primary human hepatocytes and non-parenchymal cells (NPCs) we expanded this protocol to additional enzymatic dissociation, filtration, and centrifugation steps. This results in a bile duct cell enriched fraction of NPCs from which Thy-1 (CD90) positive cells were purified by Thy-1 positivity selection using MACS technique. Bipotential progenitor cells from human liver resections can be isolated using Thy-1 and was shown to be a suitable tool for the enrichment of liver resident progenitor cells for xenotransplantation.

  1. 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.

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

    International Nuclear Information System (INIS)

    Choi, Seon Young; Jang, Soo Hwa; Park, Jin; Jeong, Saeromi; Park, Jin Ho; Ock, Kwang Su; Lee, Kangtaek; Yang, Sung Ik; Joo, Sang-Woo; Ryu, Pan Dong; Lee, So Yeong

    2012-01-01

    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. 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.

  4. 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

  5. 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, <1 × 104 cells ml-1). Here we observe ˜6× increase in the elastic (Young’s) modulus of the cell body and ˜3.6× decrease in the pericellular brush length of LD cells compared to HD ALL3 cells. The difference observed in the elastic modulus is much larger than typically reported for pathologically transformed cells. Thus, cell-cell communication must be taken into account when studying biomechanics of cells, in particular, correlating cell phenotype and its biophysical properties.

  6. Distinct transcriptional programs in thymocytes responding to T cell receptor, Notch, and positive selection signals

    OpenAIRE

    Huang, Yina H.; Li, Dongling; Winoto, Astar; Robey, Ellen A.

    2004-01-01

    T cell antigen receptor (TCR) signaling is necessary but not sufficient to promote the positive selection of CD4+CD8+ thymocytes into CD4+ or CD8+ mature T cells. Notch signaling has also been implicated as a potential regulator of both CD4/CD8 T cell development and TCR signaling. However, the relationship between positive selection, TCR signaling, and Notch remains unclear. Here we use DNA microarray analysis to compare gene expression changes in CD4+CD8+ double-positive thymocytes undergoi...

  7. Crizotinib in Combination with Everolimus Synergistically Inhibits Proliferation of ALK-Positive Anaplastic Large Cell Lymphoma.

    Science.gov (United States)

    Xu, Wendan; Kim, Ji-Won; Jung, Woo June; Koh, Youngil; Yoon, Sung-Soo

    2017-06-19

    Anaplastic large cell lymphoma (ALCL) is a rare aggresive non-Hodgkin lymphoma, of which over 50% of cases have an aberrant NPM-ALK fusion protein. Both mTOR inhibitor everolimus and ALK inhibitor crizotinib have shown promising antitumor activity in ALK-positive cancer cell lines. However, their combined effect has not yet been investigated. We evaluated the anti-proliferative effects of everolimus and/or crizotinib in ALK-positive ALCL cell lines, Karpas 299 and SU-DHL-1, and lung adenocarcinoma cell line, NCI-H2228. We found that individually, both everolimus and crizotinib potently inhibited cell growth in a dose-dependent manner in both Karpas 299 and SU-DHL-1 cells. A combination of these agents synergistically inhibited proliferation in the two cell lines. Crizotinib down-regulated aberrant AKT and ERK phosphorylation induced by everolimus. Combination treatment also significantly increased G0/G1 cell-cycle arrest, DNA damage, and apoptosis compared with everolimus or crizotinib alone in ALK-positive ALCL cells. In the Karpas 299 xenograft model, the combination treatment exerted a stronger antitumor effect than monotherapies, without significant change in body weight. The synergistic effect of everolimus and crizotinib was also reproduced in the ALK-positive lung adenocarcinoma cell line NCI-H2228. The combination treatment abrogated PI3K/AKT and mTOR signaling pathways with little effect on the Ras/ERK pathway in NCI-H2228 cells. Crizotinib combined with everolimus synergistically inhibits proliferation of ALK-positive ALCL cells. Our results suggest that this novel combination is worthy of further clinical development in patients with ALK-positive ALCL.

  8. Cell-Fate Specification in Arabidopsis Roots Requires Coordinative Action of Lineage Instruction and Positional Reprogramming.

    Science.gov (United States)

    Yu, Qiaozhi; Li, Pengxue; Liang, Nengsong; Wang, Hong; Xu, Meizhi; Wu, Shuang

    2017-10-01

    Tissue organization and pattern formation within a multicellular organism rely on coordinated cell division and cell-fate determination. In animals, cell fates are mainly determined by a cell lineage-dependent mechanism, whereas in plants, positional information is thought to be the primary determinant of cell fates. However, our understanding of cell-fate regulation in plants mostly relies on the histological and anatomical studies on Arabidopsis ( Arabidopsis thaliana ) roots, which contain a single layer of each cell type in nonvascular tissues. Here, we investigate the dynamic cell-fate acquisition in modified Arabidopsis roots with additional cell layers that are artificially generated by the misexpression of SHORT-ROOT ( SHR ). We found that cell-fate determination in Arabidopsis roots is a dimorphic cascade with lineage inheritance dominant in the early stage of pattern formation. The inherited cell identity can subsequently be removed or modified by positional information. The instruction of cell-fate conversion is not a fast readout during root development. The final identity of a cell type is determined by the synergistic contribution from multiple layers of regulation, including symplastic communication across tissues. Our findings underline the collaborative inputs during cell-fate instruction. © 2017 American Society of Plant Biologists. All Rights Reserved.

  9. Bitter Taste Responses of Gustducin-positive Taste Cells in Mouse Fungiform and Circumvallate Papillae.

    Science.gov (United States)

    Yoshida, Ryusuke; Takai, Shingo; Sanematsu, Keisuke; Margolskee, Robert F; Shigemura, Noriatsu; Ninomiya, Yuzo

    2018-01-15

    Bitter taste serves as an important signal for potentially poisonous compounds in foods to avoid their ingestion. Thousands of compounds are estimated to taste bitter and presumed to activate taste receptor cells expressing bitter taste receptors (Tas2rs) and coupled transduction components including gustducin, phospholipase Cβ2 (PLCβ2) and transient receptor potential channel M5 (TRPM5). Indeed, some gustducin-positive taste cells have been shown to respond to bitter compounds. However, there has been no systematic characterization of their response properties to multiple bitter compounds and the role of transduction molecules in these cells. In this study, we investigated bitter taste responses of gustducin-positive taste cells in situ in mouse fungiform (anterior tongue) and circumvallate (posterior tongue) papillae using transgenic mice expressing green fluorescent protein in gustducin-positive cells. The overall response profile of gustducin-positive taste cells to multiple bitter compounds (quinine, denatonium, cyclohexamide, caffeine, sucrose octaacetate, tetraethylammonium, phenylthiourea, L-phenylalanine, MgSO 4 , and high concentration of saccharin) was not significantly different between fungiform and circumvallate papillae. These bitter-sensitive taste cells were classified into several groups according to their responsiveness to multiple bitter compounds. Bitter responses of gustducin-positive taste cells were significantly suppressed by inhibitors of TRPM5 or PLCβ2. In contrast, several bitter inhibitors did not show any effect on bitter responses of taste cells. These results indicate that bitter-sensitive taste cells display heterogeneous responses and that TRPM5 and PLCβ2 are indispensable for eliciting bitter taste responses of gustducin-positive taste cells. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  10. E2a/Pbx1 induces the rapid proliferation of stem cell factor-dependent murine pro-T cells that cause acute T-lymphoid or myeloid leukemias in mice.

    Science.gov (United States)

    Sykes, David B; Kamps, Mark P

    2004-02-01

    Oncoprotein E2a/Pbx1 is produced by the t(1;19) chromosomal translocation of human pre-B acute lymphoblastic leukemia. E2a/Pbx1 blocks differentiation of primary myeloid progenitors but, paradoxically, induces apoptosis in established pre-B-cell lines, and no transforming function of E2a/Pbx1 has been reported in cultured lymphoid progenitors. Here, we demonstrate that E2a/Pbx1 induces immortal proliferation of stem cell factor (SCF)-dependent pro-T thymocytes by a mechanism dependent upon both its transactivation and DNA-binding functions. E2a-Pbx1 cooperated with cytokines or activated signaling oncoproteins to induce cell division, as inactivation of conditional E2a/Pbx1 in either factor-dependent pro-T cells or pro-T cells made factor independent by expression of Bcr/Abl resulted in pro-T-cell quiescence, while reactivation of E2a/Pbx1 restored cell division. Infusion of E2a/Pbx1 pro-T cells in mice caused T lymphoblastic leukemia and, unexpectedly, acute myeloid leukemia. The acute lymphoblastic leukemia did not evidence further maturation, suggesting that E2a/Pbx1 establishes an early block in pro-T-cell development that cannot be overcome by marrow or thymic microenvironments. In an E2a/Pbx1 pro-T thymocyte clone that induced only pro-T acute lymphoblastic leukemia, coexpression of Bcr/Abl expanded its leukemic phenotype to include acute myeloid leukemia, suggesting that unique functions of cooperating signaling oncoproteins can influence the lymphoid versus myeloid character of E2a/Pbx1 leukemia and may cooperate with E2a/Pbx1 to dictate the pre-B-cell phenotype of human leukemia containing t(1;19).

  11. 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,

  12. Concomitant use of polarization and positive phase contrast microscopy for the study of microbial cells

    Czech Academy of Sciences Publication Activity Database

    Žižka, Zdeněk; Gabriel, Jiří

    2015-01-01

    Roč. 60, č. 6 (2015), s. 545-550 ISSN 0015-5632 Institutional support: RVO:61388971 Keywords : polarization microscopy * microbial cells * positive phase contrast Subject RIV: EE - Microbiology, Virology Impact factor: 1.335, year: 2015

  13. Wnt signaling positively regulates endothelial cell fate specification in the Fli1a-positive progenitor population via Lef1.

    Science.gov (United States)

    Hübner, Kathleen; Grassme, Kathrin S; Rao, Jyoti; Wenke, Nina K; Zimmer, Cordula L; Korte, Laura; Mu Ller, Katja; Sumanas, Saulius; Greber, Boris; Herzog, Wiebke

    2017-10-01

    During vertebrate embryogenesis, vascular endothelial cells (ECs) and primitive erythrocytes become specified within close proximity in the posterior lateral plate mesoderm (LPM) from a common progenitor. However, the signaling cascades regulating the specification into either lineage remain largely elusive. Here, we analyze the contribution of β-catenin dependent Wnt signaling to EC and erythrocyte specification during zebrafish embryogenesis. We generated novel β-catenin dependent Wnt signaling reporters which, by using destabilized fluorophores (Venus-Pest, dGFP), specifically allow us to detect Wnt signaling responses in narrow time windows as well as in spatially restricted domains, defined by Cre recombinase expression (Tg(axin2 BAC :Venus-Pest) mu288 ; Tg(14TCF:loxP-STOP-loxP-dGFP) mu202 ). We therefore can detect β-catenin dependent Wnt signaling activity in a subset of the Fli1a-positive progenitor population. Additionally, we show that mesodermal Wnt3a-mediated signaling via the transcription factor Lef1 positively regulates EC specification (defined by kdrl expression) at the expense of primitive erythrocyte specification (defined by gata1 expression) in zebrafish embryos. Using mesoderm derived from human embryonic stem cells, we identified the same principle of Wnt signaling dependent EC specification in conjunction with auto-upregulation of LEF1. Our data indicate a novel role of β-catenin dependent Wnt signaling in regulating EC specification during vasculogenesis. Copyright © 2017. Published by Elsevier Inc.

  14. Intracellular Position of Centrioles and the Direction of Homeostatic Epithelial Cell Movements in the Mouse Cornea.

    Science.gov (United States)

    Silverman, Erika; Zhao, Jin; Merriam, John C; Nagasaki, Takayuki

    2017-02-01

    Corneal epithelial cells exhibit continuous centripetal movements at a rate of about 30 µm per day, but neither the driving force nor the mechanism that determines the direction of movements is known. To facilitate the investigation of homeostatic cell movement, we examined if the intracellular position of a centriole can be used as a directional marker of epithelial cell movements in the mouse cornea. A direction of cell movements was estimated in fixed specimens from a pattern of underlying subepithelial nerve fibers. Intracellular position of centrioles was determined by gamma-tubulin immunohistology and plotted in a narrow strip along the entire diameter of a cornea from limbus to limbus. When we determined the position of centrioles in the peripheral cornea where cell movements proceed generally along a radial path, about 55% of basal epithelial cells contained a centriole in the front half of a cell. However, in the central cornea where cells exhibit a spiral pattern of movements, centrioles were distributed randomly. These results suggest that centrioles tend to be positioned toward the direction of movement in corneal basal epithelial cells when they are moving centripetally at a steady rate.

  15. Evaluation of Commercial-off-the-Shelf Materials for the Preservation of Gram Positive Vegetative Cells

    Science.gov (United States)

    2017-02-01

    EVALUATION OF COMMERCIAL-OFF-THE-SHELF MATERIALS FOR THE PRESERVATION OF GRAM-POSITIVE VEGETATIVE CELLS ECBC-TR-1435 Daniel Angelini...Commercial-off-the-Shelf Materials for the Preservation of Gram-Positive Vegetative Cells 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM...through direct culture of the agents themselves. Materials have been developed to preserve the viability of pathogens contained within clinical

  16. A pericentric inv(9)(p22q34) of the der(9)t(9;22)(q34;q11.2) is a recurrent secondary anomaly in Ph-positive leukemia.

    Science.gov (United States)

    Pan, Jinlan; Xue, Yongquan; Qiu, Huiying; Chen, Suning; Zhang, Jun; Wu, Yafang; Shen, Juan; Wang, Yong

    2010-12-01

    A pericentric inv(9)(p22q34) of the derivative chromosome 9 that resulted from a standard t(9;22)(q34;q11.2) was identified by R-banding karyotypic analysis and fluorescence in situ hybridization (FISH) assays in 4 (0.18%) of 2,200 Philadelphia chromosome (Ph)-positive leukemia patients, including 3 with chronic myeloid leukemia (CML) in chronic phase and 1 with acute myeloid leukemia (AML) in our hospital since 2004. All four patients had two malignant clones: one with only t(9;22)(q34;q11.2) and another with der(9)t(9;22)(q34;q11.2)inv(9)(p22q34) that resulted in the separation of the ABL1/BCR fusion gene. No metaphases with only inv(9)(p22q34) were seen in any of them. FISH also found a deletion of partial sequence of BCR on der(9)t(9;22)(q34;q11.2)inv(9)(p22q34) in 67.5% of bone marrow cells in the AML patient, but did not detect the deletion of the sequence of ASS/9q34 in these four patients. Reverse transcriptase-polymerase chain reaction revealed a b3a2 type of BCR/ABL1 fusion transcript in all of them, proving their disease to be Ph-positive leukemia. On reviewing the literature, only two solitary Ph-positive leukemia patients have been noticed to have the inv(9)(p22q34) anomaly. These two patients, together with our four documented patients, indicate that inv(9)(p22q34) is a novel, rare, but recurrent secondary chromosomal abnormality for Ph-positive leukemia. Despite receiving hydroxyurea therapy (n = 3 patients), combined chemotherapy (n = 2), even imatinib treatment (n = 1), three patients, including one with AML and two with CML (one of whom progressed into the lymphoblastic blast phase), died with survival times of 28 days, 13 months, and 34 months, respectively. Only one patient with CML remained alive for 5.5 months. Their negative outcome implies that inv(9)(p22q34) has an unfavorable impact on prognosis. Presently, no firm conclusions can be drawn from this study. Because the case number reported here is very small, more patients with this anomaly

  17. 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.

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

    Directory of Open Access Journals (Sweden)

    Hannes C A Drexler

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

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

    DEFF Research Database (Denmark)

    Bonnavion, Rémy; Teinturier, Romain; Jaafar, Rami

    2015-01-01

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

  20. 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 ...

  1. High expression of Mcl-1 in ALK positive and negative anaplastic large cell lymphoma

    NARCIS (Netherlands)

    Rust, R; Harms, G; Blokzijl, T; Boot, M; Diepstra, A; Kluiver, J; Visser, L; Peh, SC; Lim, M; Kamps, WA; Poppema, S; van den Berg, Anke

    Aim: To gain more insight into the genes involved in the aetiology and pathogenesis of anaplastic large cell lymphoma (ALCL). Methods: Serial analysis of gene expression ( SAGE) was undertaken on the CD4+ALK+ ( anaplastic lymphoma kinase positive) ALCL derived cell line Karpas299 and as comparison

  2. 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...

  3. 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.

  4. S100A6 and c-Kit-Positive Spindle Cell Melanoma of the Dorsal Foot

    Directory of Open Access Journals (Sweden)

    Yasutaka Mitamura

    2014-05-01

    Full Text Available Spindle cell melanoma, which is a rare form of melanoma, is clinically and histopathologically difficult to diagnose from a variety of nonmelanocytic spindle cell tumors. We describe a 42-year-old Japanese woman with amelanotic melanoma that comprised spindle cells with positive c-kit and S100A6 staining. The use of c-kit and S100A6 might be useful for improving the diagnosis.

  5. 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.

  6. 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.

  7. Positive Feedback Keeps Duration of Mitosis Temporally Insulated from Upstream Cell-Cycle Events.

    Science.gov (United States)

    Araujo, Ana Rita; Gelens, Lendert; Sheriff, Rahuman S M; Santos, Silvia D M

    2016-10-20

    Cell division is characterized by a sequence of events by which a cell gives rise to two daughter cells. Quantitative measurements of cell-cycle dynamics in single cells showed that despite variability in G1-, S-, and G2 phases, duration of mitosis is short and remarkably constant. Surprisingly, there is no correlation between cell-cycle length and mitotic duration, suggesting that mitosis is temporally insulated from variability in earlier cell-cycle phases. By combining live cell imaging and computational modeling, we showed that positive feedback is the molecular mechanism underlying the temporal insulation of mitosis. Perturbing positive feedback gave rise to a sluggish, variable entry and progression through mitosis and uncoupled duration of mitosis from variability in cell cycle length. We show that positive feedback is important to keep mitosis short, constant, and temporally insulated and anticipate it might be a commonly used regulatory strategy to create modularity in other biological systems. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  8. The Antiproliferative Activity of Kinase Inhibitors in Chronic Myeloid Leukemia Cells Is Mediated by FOXO Transcription Factors

    Science.gov (United States)

    Pellicano, Francesca; Scott, Mary T; Helgason, G Vignir; Hopcroft, Lisa E M; Allan, Elaine K; Aspinall-O'Dea, Mark; Copland, Mhairi; Pierce, Andrew; Huntly, Brian J P; Whetton, Anthony D; Holyoake, Tessa L

    2014-01-01

    Chronic myeloid leukemia (CML) is initiated and maintained by the tyrosine kinase BCR-ABL which activates a number of signal transduction pathways, including PI3K/AKT signaling and consequently inactivates FOXO transcription factors. ABL-specific tyrosine kinase inhibitors (TKIs) induce minimal apoptosis in CML progenitor cells, yet exert potent antiproliferative effects, through as yet poorly understood mechanisms. Here, we demonstrate that in CD34+ CML cells, FOXO1 and 3a are inactivated and relocalized to the cytoplasm by BCR-ABL activity. TKIs caused a decrease in phosphorylation of FOXOs, leading to their relocalization from cytoplasm (inactive) to nucleus (active), where they modulated the expression of key FOXO target genes, such as Cyclin D1, ATM, CDKN1C, and BCL6 and induced G1 arrest. Activation of FOXO1 and 3a and a decreased expression of their target gene Cyclin D1 were also observed after 6 days of in vivo treatment with dasatinib in a CML transgenic mouse model. The over-expression of FOXO3a in CML cells combined with TKIs to reduce proliferation, with similar results seen for inhibitors of PI3K/AKT/mTOR signaling. While stable expression of an active FOXO3a mutant induced a similar level of quiescence to TKIs alone, shRNA-mediated knockdown of FOXO3a drove CML cells into cell cycle and potentiated TKI-induced apoptosis. These data demonstrate that TKI-induced G1 arrest in CML cells is mediated through inhibition of the PI3K/AKT pathway and reactivation of FOXOs. This enhanced understanding of TKI activity and induced progenitor cell quiescence suggests that new therapeutic strategies for CML should focus on manipulation of this signaling network. Stem Cells 2014;32:2324–2337 PMID:24806995

  9. Lineage-Biased Stem Cells Maintain Estrogen-Receptor-Positive and -Negative Mouse Mammary Luminal Lineages

    Directory of Open Access Journals (Sweden)

    Chunhui Wang

    2017-03-01

    Full Text Available Delineating the mammary differentiation hierarchy is important for the study of mammary gland development and tumorigenesis. Mammary luminal cells are considered a major origin of human breast cancers. However, how estrogen-receptor-positive (ER+ and ER− luminal cells are developed and maintained remains poorly understood. The prevailing model suggests that a common stem/progenitor cell generates both cell types. Through genetic lineage tracing in mice, we find that SOX9-expressing cells specifically contribute to the development and maintenance of ER− luminal cells and, to a lesser degree, basal cells. In parallel, PROM1-expressing cells give rise only to ER+ luminal cells. Both SOX9+ and PROM1+ cells specifically sustain their respective lineages even after pregnancy-caused tissue remodeling or serial transplantation, demonstrating characteristic properties of long-term repopulating stem cells. Thus, our data reveal that mouse mammary ER+ and ER− luminal cells are two independent lineages that are maintained by distinct stem cells, providing a revised mammary epithelial cell hierarchy.

  10. 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.

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

    International Nuclear Information System (INIS)

    Zhang, Qiao; Yang, Zhe; Wang, Weiping; Guo, Ting; Jia, Zhuqing; Ma, Kangtao; Zhou, Chunyan

    2014-01-01

    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

  12. 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.

  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. Label-free quantitative proteomics of CD133-positive liver cancer stem cells

    Directory of Open Access Journals (Sweden)

    Tsai Sheng-Ta

    2012-11-01

    Full Text Available Abstract Background CD133-positive liver cancer stem cells, which are characterized by their resistance to conventional chemotherapy and their tumor initiation ability at limited dilutions, have been recognized as a critical target in liver cancer therapeutics. In the current work, we developed a label-free quantitative method to investigate the proteome of CD133-positive liver cancer stem cells for the purpose of identifying unique biomarkers that can be utilized for targeting liver cancer stem cells. Label-free quantitation was performed in combination with ID-based Elution time Alignment by Linear regression Quantitation (IDEAL-Q and MaxQuant. Results Initially, IDEAL-Q analysis revealed that 151 proteins were differentially expressed in the CD133-positive hepatoma cells when compared with CD133-negative cells. We then analyzed these 151 differentially expressed proteins by MaxQuant software and identified 10 significantly up-regulated proteins. The results were further validated by RT-PCR, western blot, flow cytometry or immunofluorescent staining which revealed that prominin-1, annexin A1, annexin A3, transgelin, creatine kinase B, vimentin, and EpCAM were indeed highly expressed in the CD133-positive hepatoma cells. Conclusions These findings confirmed that mass spectrometry-based label-free quantitative proteomics can be used to gain insights into liver cancer stem cells.

  15. Efficient isolation of human CD34 positive hemopoietic progenitor cells by immune panninga.

    Science.gov (United States)

    Holyoake, T L; Alcorn, M J; Richmond, L J; Freshney, M G; Pearson, C; Fitzsimons, E; Steward, W P; Dunlop, D J; Pragnell, I B

    1994-01-01

    In this study we have assessed the use of soybean agglutinin (SBA) and CD34 microcellector devices for the selection of CD34 positive hemopoietic progenitor cells. Burst forming unit-erythroid (BFU-E), colony forming unit-granulocyte/macrophage (CFU-GM) and the recently developed multipotential human colony forming unit-type A (CFU-A) clonogenic assays were used to measure progenitor numbers in the starting mononuclear cell (MNC), the SBA negative, the nonadherent CD34 negative and the adherent CD34 positive fractions during panning. CFU-A progenitors were present at a relatively high incidence in the MNC fraction (220 per 10(5) MNC) and were enriched 15-fold in the adherent CD34 positive fraction. This progenitor incidence and enrichment were similar to those of CFU-GM and BFU-E. The mean recovery for CD34 positive cells was 2.3 x 10(6) cells per marrow aspirate. Analyses by flow cytometry demonstrated that 1-5% of input MNC were CD34 positive, that the purity of the CD34 fraction was approximately 80% and that the calculated recovery for CD34 positive cells was 61%. Recoveries for CFU-GM, BFU-E and CFU-A were between 18 and 40%. CFU-A progenitors were found exclusively in the adherent CD34 positive fraction, whereas a significant proportion of both CFU-GM and BFU-E were present in the nonadherent CD34 negative fraction. We propose that the Applied Immune Sciences (AIS) flasks preferentially bind the cells which express CD34 most strongly and that this is reflected in the finding of primitive CFU-A only in the CD34 positive fraction, with lineage-restricted progenitors found in both CD34 positive and negative fractions. This hypothesis is strengthened by data on long-term bone marrow cultures in which the CD34 positive fraction is better able to maintain output of CFU-GM compared with the CD34 negative fraction. In conclusion, relatively pure populations of CD34 positive cells may be rapidly and efficiently isolated from bone marrow samples with good recovery. The

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

    Directory of Open Access Journals (Sweden)

    Karuza Maria Alves Pereira

    2011-08-01

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

  17. The study of resistant mechanisms and reversal in an imatinib resistant Ph+ acute lymphoblastic leukemia cell line.

    Science.gov (United States)

    Xing, Hongyun; Yang, Xi; Liu, Ting; Lin, Juan; Chen, Xiaoyi; Gong, Yuping

    2012-04-01

    In this study, we established an imatinib resistant Ph+ acute lymphoblastic leukemia (ALL) cell line SUP-B15/RI in vitro and studied the mechanism of imatinib resistance. Our results showed that the BCR-ABL1 fusion gene and the mdr1 gene were 6.1 times and 1.7 times, respectively, as high as that of parental SUP-B15 cell line. We found no mutation in the Abl kinase domain of SUP-B15/RI. Furthermore, the detection of cell signaling pathway of PI3K/AKT/mTOR, RAS/RAF, NF-κB, JNK and STAT showed the up-regulation of phosphorylation of AKT, mTOR, P70S6K, and RAF, ERK, and MEK, down-regulation of PTEN and 4EBP-1, and no change in other cell signaling pathways in SUP-B15/RI. However, dasatinib and nilotinib showed partial resistance. Interestingly, bortezomib had no resistance. Imatinib combination with rapamycin had synergistic effect on overcoming the resistance. Altogether, over-expression of BCR-ABL1 and mdr1 gene were involved in the resistance mechanisms, and up-regulation of the cell signaling pathways of PI3K/AKT/mTOR, RAS/RAF in SUP-B15/RI cell line may be correlated with them. The SUP-B15/RI cell line was also resistant to the second generation tyrosine kinase, dasatinib, and nilotinib, not bortezomib. The combination of imatinib with rapamycin can partially overcome the resistance and blockade of the ubiquitin-proteasome can be also a promising pathway to overcome imatinib resistance. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. 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.

  19. Synergy of photoacoustic and fluorescence flow cytometry of circulating cells with negative and positive contrasts

    Science.gov (United States)

    Nedosekin, Dmitry A.; Sarimollaoglu, Mustafa; Galanzha, Ekaterina I.; Sawant, Rupa; Torchilin, Vladimir P.; Verkhusha, Vladislav V.; Ma, Jie; Frank, Markus H.; Biris, Alexandru S.; Zharov, Vladimir P.

    2012-01-01

    In vivo photoacoustic (PA) and fluorescence flow cytometry were previously applied separately using pulsed and continuous wave lasers respectively, and positive contrast detection mode only. This paper introduces a real-time integration of both techniques with positive and negative contrast modes using only pulsed lasers. Various applications of this new tool are summarized, including detection of liposomes loaded with Alexa-660 dye, red blood cells labeled with Indocyanine Green, B16F10 melanoma cells co-expressing melanin and green fluorescent protein (GFP), C8161-GFP melanoma cells targeted by magnetic nanoparticles, MTLn3 adenocarcinoma cells expressing novel near-infrared iRFP protein, and quantum dot-carbon nanotube conjugates. Negative contrast flow cytometry provided label-free detection of low absorbing or weakly fluorescent cells in blood absorption and autofluorescence background, respectively. The use of pulsed laser for time-resolved discrimination of objects with long fluorescence lifetime (e.g., quantum dots) from shorter autofluorescence background (e.g., blood plasma) is also highlighted in this paper. The supplementary nature of PA and fluorescence detection increased the versatility of the integrated method for simultaneous detection of probes and cells having various absorbing and fluorescent properties, and provided verification of PA data using a more established fluorescence based technique. The principles of integrated photoacoustic and fluorescence flow cytometry using positive contrast for detection of strongly absorbing and fluorescent cells and negative contrast for detection of weakly absorbing and fluorescent cells in blood absorption and autofluorescence background, respectively. PMID:22903924

  20. Olig2/Plp-positive progenitor cells give rise to Bergmann glia in the cerebellum.

    Science.gov (United States)

    Chung, S-H; Guo, F; Jiang, P; Pleasure, D E; Deng, W

    2013-03-14

    NG2 (nerve/glial antigen2)-expressing cells represent the largest population of postnatal progenitors in the central nervous system and have been classified as oligodendroglial progenitor cells, but the fate and function of these cells remain incompletely characterized. Previous studies have focused on characterizing these progenitors in the postnatal and adult subventricular zone and on analyzing the cellular and physiological properties of these cells in white and gray matter regions in the forebrain. In the present study, we examine the types of neural progeny generated by NG2 progenitors in the cerebellum by employing genetic fate mapping techniques using inducible Cre-Lox systems in vivo with two different mouse lines, the Plp-Cre-ER(T2)/Rosa26-EYFP and Olig2-Cre-ER(T2)/Rosa26-EYFP double-transgenic mice. Our data indicate that Olig2/Plp-positive NG2 cells display multipotential properties, primarily give rise to oligodendroglia but, surprisingly, also generate Bergmann glia, which are specialized glial cells in the cerebellum. The NG2+ cells also give rise to astrocytes, but not neurons. In addition, we show that glutamate signaling is involved in distinct NG2+ cell-fate/differentiation pathways and plays a role in the normal development of Bergmann glia. We also show an increase of cerebellar oligodendroglial lineage cells in response to hypoxic-ischemic injury, but the ability of NG2+ cells to give rise to Bergmann glia and astrocytes remains unchanged. Overall, our study reveals a novel Bergmann glia fate of Olig2/Plp-positive NG2 progenitors, demonstrates the differentiation of these progenitors into various functional glial cell types, and provides significant insights into the fate and function of Olig2/Plp-positive progenitor cells in health and disease.

  1. 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.

  2. Anti-inflammatory effects of cell-based therapy with tyrosine hydroxylase-positive catecholaminergic cells in experimental arthritis.

    Science.gov (United States)

    Jenei-Lanzl, Zsuzsa; Capellino, Silvia; Kees, Frieder; Fleck, Martin; Lowin, Torsten; Straub, Rainer H

    2015-02-01

    Studies in rheumatoid arthritis (RA), osteoarthritis (OA) and mice with arthritis demonstrated tyrosine hydroxylase-positive (TH(+)) cells in arthritic synovium and parallel loss of sympathetic nerve fibres. The exact function of TH(+) cells and mode of TH induction are not known. Synovial cells of RA/OA were isolated and cultured under normoxic/hypoxic conditions with/without stimulating enzyme cofactors of TH and inhibitors of TH. We studied TH expression and release of cytokines/catecholamines. In vivo function was tested by cell therapy with TH(+) neuronal precursor cells (TH(+) neuronal cells) in DBA/1 mice with collagen type II-induced arthritis (CIA). Compared with normoxic conditions, hypoxia increased TH protein expression and catecholamine synthesis and decreased release of tumour necrosis factor (TNF) in OA/RA synovial cells. This inhibitory effect on TNF was reversed by TH inhibition with α-methyl-para-tyrosine (αMPT), which was particularly evident under hypoxic conditions. Incubation with specific TH cofactors (tetrahydrobiopterin and Fe(2+)) increased hypoxia-induced inhibition of TNF, which was also reversed by αMPT. To address a possible clinical role of TH(+) cells, murine TH(+) neuronal cells were generated from mesenchymal stem cells. TH(+) neuronal cells exhibited a typical catecholaminergic phenotype. Adoptive transfer of TH(+) neuronal cells markedly reduced CIA in mice, and 6-hydroxydopamine, which depletes TH(+) cells, reversed this effect. The anti-inflammatory effect of TH(+) neuronal cells on experimental arthritis has been presented for the first time. In RA/OA, TH(+) synovial cells have TH-dependent anti-inflammatory capacities, which are augmented under hypoxia. Using generated TH(+) neuronal cells might open new avenues for cell-based therapy. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  3. Parthenolide reduces the frequency of ABCB5-positive cells and clonogenic capacity of melanoma cells from anchorage independent melanospheres

    Science.gov (United States)

    Czyz, Malgorzata; Koprowska, Kamila; Sztiller-Sikorska, Malgorzata

    2013-01-01

    Growing evidence suggests that the cancer stem cell phenotype in melanoma is dynamically regulated. Therefore, effective therapies have to target simultaneously bulk tumor cells and melanoma stem-like cells. The aim of the present study was to investigate the effects of parthenolide on heterogeneous cancer cell populations from anchorage-independent melanospheres. Cells derived from nodular melanoma specimens were grown under serum-free sphere-forming conditions. The effects of parthenolide on cellular viability, immunophenotype and self-renewing capacity were assessed with cells from dissociated melanospheres. Its penetration capacity was evaluated with intact melanospheres. In melanoma cells that survived treatment with parthenolide, a different immunophenotype than that in untreated control was found. The frequency of cells expressing the ABCB5 transporter was markedly reduced. Most importantly, melanoma cells that survived parthenolide treatment lost their self-renewing capacity. Significantly lower influence of drug on cellular viability and frequency of ABCB5-positive cells was observed in intact melanospheres. The potential clinical significance of our findings is based on the ability of parthenolide to affect both bulk and melanoma stem-like cells with clonogenic capacity and high expression of the ABCB5 transporter. Its low penetration capacity, however, may limit its action to easily accessible melanoma cells, either circulating in the blood or those in the vicinity to blood vessels within the tumor. Because of limited penetration capacity of parthenolide, this drug should be further explored as a part of multimodal therapies rather than as a stand-alone therapeutic agent. PMID:23192276

  4. Distinct oxysterol requirements for positioning naïve and activated dendritic cells in the spleen

    Science.gov (United States)

    Lu, Erick; Dang, Eric V.; McDonald, Jeffrey G.; Cyster, Jason G.

    2017-01-01

    Correct positioning of dendritic cells (DCs) is critical for efficient pathogen encounter and antigen presentation. Epstein-Barr virus–induced gene 2 (EBI2) has been identified as a chemoattractant receptor required for naïve CD4+DCIR2+ DC positioning in response to 7α,25-hydroxycholesterol (7α,25-HC). We now provide evidence that a second EBI2 ligand, 7α,27-HC, is involved in splenic DCIR2+ DC positioning and homeostasis. Cyp27a1, the enzyme uniquely required for 7α,27-HC synthesis, is expressed by stromal cells in the region of naïve DC localization. After activation, DCIR2+ DCs move into the T cell zone. We find that EBI2 is rapidly up-regulated in DCIR2+ DCs under certain activation conditions, and positioning at the B-T zone interface depends on EBI2. Under conditions of type I interferon induction, EBI2 ligand levels are elevated, causing activated DCIR2+ DCs to disperse throughout the T zone. Last, we provide evidence that oxysterol metabolism by Batf3-dependent DCs is important for EBI2-dependent positioning of activated DCIR2+DCs. This work indicates that 7α,27-HC functions as a guidance cue in vivo and reveals a multitiered role for EBI2 in DC positioning. Deficiency in this organizing system results in defective CD4+ T cell responses. PMID:28738017

  5. Tyrosine hydroxylase positive nerves and mast cells in the porcine gallbladder

    Directory of Open Access Journals (Sweden)

    I. Stefanov

    2017-03-01

    Full Text Available The aim of this study was to detect the localisation of tyrosine hydroxylase (TH positive nerve fibres (THN and distribution of tyrosine hydroxylase positive mast cells (THMC in the wall of porcine gallbladder. THN were observed as single fibres, nerve fibres forming perivascular plexuses and nerve fibres grouped within the nerve fascicles. In the gallbladder`s fundus, body and neck, the TH+ fibres formed mucosal, muscular and serosal nonganglionated nerve plexuses. Toluidine blue (TB staining was used to confirm that the TH positive cells were mast cells. The number of THMC in the propria of gallbladder`s fundus, body and neck was significantly higher than in the muscular and serosal layers in both genders. The number of mast cells in the musculature was higher than in the serosa. The density and location of the THMC were similar to the TB positive (with gamma meta-chromasia mast cells in both males and females, and statistically significant difference was not established. In conclusion, original data concerning the existence and localisation of catecholaminergic nerves and THMC distribution in the porcine gallbladder’s wall are presented. The results could con-tribute to the body of knowledge of functional communication between autonomic nerves and mast cells in the gallbladder.

  6. A Positive Control for Detection of Functional CD4 T Cells in PBMC: The CPI Pool.

    Science.gov (United States)

    Schiller, Annemarie; Zhang, Ting; Li, Ruliang; Duechting, Andrea; Sundararaman, Srividya; Przybyla, Anna; Kuerten, Stefanie; Lehmann, Paul V

    2017-12-07

    Testing of peripheral blood mononuclear cells (PBMC) for immune monitoring purposes requires verification of their functionality. This is of particular concern when the PBMC have been shipped or stored for prolonged periods of time. While the CEF (Cytomegalo-, Epstein-Barr and Flu-virus) peptide pool has become the gold standard for testing CD8 cell functionality, a positive control for CD4 cells is so far lacking. The latter ideally consists of proteins so as to control for the functionality of the antigen processing and presentation compartments, as well. Aiming to generate a positive control for CD4 cells, we first selected 12 protein antigens from infectious/environmental organisms that are ubiquitous: Varicella, Influenza, Parainfluenza, Mumps, Cytomegalovirus, Streptococcus , Mycoplasma , Lactobacillus , Neisseria , Candida , Rubella, and Measles. Of these antigens, three were found to elicited interferon (IFN)-γ-producing CD4 cells in the majority of human test subjects: inactivated cytomegalo-, parainfluenza-, and influenza virions (CPI). While individually none of these three antigens triggered a recall response in all donors, the pool of the three (the 'CPI pool'), did. One hundred percent of 245 human donors tested were found to be CPI positive, including Caucasians, Asians, and African-Americans. Therefore, the CPI pool appears to be suitable to serve as universal positive control for verifying the functionality of CD4 and of antigen presenting cells.

  7. Detection of circulating tumor cells in cervical cancer using a conditionally replicative adenovirus targeting telomerase-positive cells.

    Science.gov (United States)

    Takakura, Masahiro; Matsumoto, Takeo; Nakamura, Mitsuhiro; Mizumoto, Yasunari; Myojyo, Subaru; Yamazaki, Rena; Iwadare, Jyunpei; Bono, Yukiko; Orisaka, Shunsuke; Obata, Takeshi; Iizuka, Takashi; Kagami, Kyosuke; Nakayama, Kentaro; Hayakawa, Hideki; Sakurai, Fuminori; Mizuguchi, Hiroyuki; Urata, Yasuo; Fujiwara, Toshiyoshi; Kyo, Satoru; Sasagawa, Toshiyuki; Fujiwara, Hiroshi

    2018-01-01

    Circulating tumor cells (CTC) are newly discovered biomarkers of cancers. Although many systems detect CTC, a gold standard has not yet been established. We analyzed CTC in uterine cervical cancer patients using an advanced version of conditionally replicative adenovirus targeting telomerase-positive cells, which was enabled to infect coxsackievirus-adenovirus receptor-negative cells and to reduce false-positive signals in myeloid cells. Blood samples from cervical cancer patients were hemolyzed and infected with the virus and then labeled with fluorescent anti-CD45 and anti-pan cytokeratin antibodies. GFP (+)/CD45 (-) cells were isolated and subjected to whole-genome amplification followed by polymerase chain reaction analysis of human papillomavirus (HPV) DNA. CTC were detected in 6 of 23 patients with cervical cancers (26.0%). Expression of CTC did not correlate with the stage of cancer or other clinicopathological factors. In 5 of the 6 CTC-positive cases, the same subtype of HPV DNA as that of the corresponding primary lesion was detected, indicating that the CTC originated from HPV-infected cancer cells. These CTC were all negative for cytokeratins. The CTC detected by our system were genetically confirmed. CTC derived from uterine cervical cancers had lost epithelial characteristics, indicating that epithelial marker-dependent systems do not have the capacity to detect these cells in cervical cancer patients. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  8. 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.

  9. Membrane tension controls adhesion positioning at the leading edge of cells

    Science.gov (United States)

    Pontes, Bruno; Gole, Laurent; Kosmalska, Anita Joanna; Tam, Zhi Yang; Luo, Weiwei; Kan, Sophie; Viasnoff, Virgile; Roca-Cusachs, Pere; Tucker-Kellogg, Lisa

    2017-01-01

    Cell migration is dependent on adhesion dynamics and actin cytoskeleton remodeling at the leading edge. These events may be physically constrained by the plasma membrane. Here, we show that the mechanical signal produced by an increase in plasma membrane tension triggers the positioning of new rows of adhesions at the leading edge. During protrusion, as membrane tension increases, velocity slows, and the lamellipodium buckles upward in a myosin II–independent manner. The buckling occurs between the front of the lamellipodium, where nascent adhesions are positioned in rows, and the base of the lamellipodium, where a vinculin-dependent clutch couples actin to previously positioned adhesions. As membrane tension decreases, protrusion resumes and buckling disappears, until the next cycle. We propose that the mechanical signal of membrane tension exerts upstream control in mechanotransduction by periodically compressing and relaxing the lamellipodium, leading to the positioning of adhesions at the leading edge of cells. PMID:28687667

  10. Induction of a new alkaline band at a target position in internodal cells of Chara corallina.

    Science.gov (United States)

    Shimmen, Teruo; Yamamoto, Ako

    2002-09-01

    Characean cells develop alternating alkaline and acid bands on their surface upon illumination. However, the mechanism of band formation is not fully understood. In the present study, we succeeded in inducing a new alkaline band at an original acid band in internodal cells of Chara corallina. Chloroplasts in an acid band were locally removed by wounding the cell in the absence of the cell turgor pressure. The chloroplast-removed area was observed as a white belt in a green cylindrical internodal cell. This internodal cell developed a new alkaline band on the surface at the chloroplast-removed area. The narrower the chloroplast-removed area, the less significant the extent of OH(-) extrusion. This is the first success in inducing a new alkaline band at a target position in Characeae.

  11. Invariant NKT 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

    2011-01-01

    Natural killer T (NKT) cells recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell receptor (TCR), but the forces driving TCR conservation have remained uncertain. Here we show that NKT cells recognize 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 are required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is found at a low level in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR, and most important, they extend the range of microbes recognized by this conserved TCR to several clinically important bacteria. PMID:21892173

  12. Human adipose tissue-derived tenomodulin positive subpopulation of stem cells: A promising source of tendon progenitor cells.

    Science.gov (United States)

    Gonçalves, A I; Gershovich, P M; Rodrigues, M T; Reis, R L; Gomes, M E

    2018-03-01

    Cell-based therapies are of particular interest for tendon and ligament regeneration given the low regenerative potential of these tissues. Adipose tissue is an abundant source of stem cells, which may be employed for the healing of tendon lesions. However, human adult multipotent adipose-derived stem cells (hASCs) isolated from the stromal vascular fraction of adipose tissue originate highly heterogeneous cell populations that hinder their use in specific tissue-oriented applications. In this study, distinct subpopulations of hASCs were immunomagnetic separated and their tenogenic differentiation capacity evaluated in the presence of several growth factors (GFs), namely endothelial GF, basic-fibroblast GF, transforming GF-β1 and platelet-derived GF-BB, which are well-known regulators of tendon development, growth and healing. Among the screened hASCs subpopulations, tenomodulin-positive cells were shown to be more promising for tenogenic applications and therefore this subpopulation was further studied, assessing tendon-related markers (scleraxis, tenomodulin, tenascin C and decorin) both at gene and protein level. Additionally, the ability for depositing collagen type I and III forming extracellular matrix structures were weekly assessed up to 28 days. The results obtained indicated that tenomodulin-positive cells exhibit phenotypical features of tendon progenitor cells and can be biochemically induced towards tenogenic lineage, demonstrating that this subset of hASCs can provide a reliable source of progenitor cells for therapies targeting tendon regeneration. Copyright © 2017 John Wiley & Sons, Ltd.

  13. MR Tracking of Transplanted Cells With “Positive Contrast” Using Manganese Oxide Nanoparticles

    Science.gov (United States)

    Gilad, Assaf A.; Walczak, Piotr; McMahon, Michael T.; Na, Hyon Bin; Lee, Jung Hee; An, Kwangjin; Hyeon, Taegwhan; van Zijl, Peter C.M.; Bulte, Jeff W.M.

    2008-01-01

    Rat glioma cells were labeled using electroporation with either manganese oxide (MnO) or superparamagnetic iron oxide (SPIO) nanoparticles. The viability and proliferation of SPIO-labeled cells (1.9 mg Fe/ml) or cells electroporated with a low dose of MnO (100 μg Mn/ml) was not significantly different from unlabeled cells; a higher MnO dose (785 μg Mn/ml) was found to be toxic. The cellular ion content was 0.1−0.3 pg Mn/cell and 4.4 pg Fe/cell, respectively, with cellular relaxivities of 2.5−4.8 s−1 (R1) and 45−84 s−1 (R2) for MnO-labeled cells. Labeled cells (SPIO and low-dose MnO) were each transplanted in contralateral brain hemispheres of rats and imaged in vivo at 9.4T. While SPIO-labeled cells produced a strong “negative contrast” due to the increase in R2, MnO-labeled cells produced “positive contrast” with an increased R1. Simultaneous imaging of both transplants with opposite contrast offers a method for MR “double labeling” of different cell populations. PMID:18581402

  14. Microcirculation within Grooved Substrates regulates Cell Positioning and Cell Docking inside Microfluidic Channels

    Science.gov (United States)

    Manbachi, Amir; Shrivastava, Shamit; Cioffi, Margherita; Chung, Bong Geun; Moretti, Matteo; Demirci, Utkan; Yliperttula, Marjo; Khademhosseini, Ali

    2009-01-01

    Immobilization of cells inside microfluidic devices is a promising approach for enabling studies related to drug screening and cell biology. Despite extensive studies in using grooved substrates for immobilizing cells inside channels, a systematic study of the effects of various parameters that influence cell docking and retention within grooved substrates has not been performed. We demonstrate using computational simulations that the fluid dynamic environment within microgrooves significantly varies with groove width, generating micro-circulation areas in smaller microgrooves. Wall shear stress simulation predicted that shear stresses were in opposite direction in smaller grooves (25 and 50 μm wide) in comparison to those in wider grooves (75 and 100 μm wide). To validate the simulations, cells were seeded within microfluidic devices, where microgrooves of different widths were aligned perpendicularly to the direction of the flow. Experimental results showed that, as predicted, the inversion of the local direction of shear stress within the smaller grooves resulted in alignment of cells on two opposite sides of the grooves under the same flow conditions. Also, the amplitude of shear stress within microgrooved channels significantly influenced cell retainment in the channels. Therefore, our studies suggest that microscale shear stresses greatly influence cellular docking, immobilization, and retention in fluidic systems and should be considered for the design of cell-based microdevices. PMID:18432345

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

    DEFF Research Database (Denmark)

    Fløe, Andreas; Brix, Liselotte; Wejse, Christian

    Background: Understanding the CD8+ response against Mycobacterium tuberculosis (MTB) may be a key to improved TB diagnostics and vaccine development. Aims and Objectives: To detect a CD8+ T-cell response against Mycobacterium tuberculosis (MTB) in active tuberculosis (TB) and latent TB (LTBI...... candidates, from which we constructed MHC multimers (Dextramers). Peripheral blood mononuclear cells (PBMC) from 7 TB-patients, 16 LTBI patients and 8 MTB-exposed, IGRA-negative, healthy subjects (HE), all HLA A*02 positive, were stained with the Dextramers and with anti-CD8 and anti-CD3, and analyzed...... on a flow cytometer. The MTB epitopes were analyzed in 5 pools (3-7 epitopes each). Positive responses included >0.001 % of CD8+, CD3+ cells, supported by inspection of flow cytometry plots. Results: MTB-specific CD8+ T-cells were detected more often in TB patients (57%) than in LTBI patients (41...

  16. Oral squamous cell carcinoma in human immunodeficiency virus positive patients: clinicopathological audit.

    Science.gov (United States)

    Butt, F M A; Chindia, M L; Rana, F

    2012-03-01

    Most human immunodeficiency virus positive patients now have a longer life expectancy, with the advent of highly active antiretroviral therapy. However, they are now at increased risk of developing a malignancy during their lives. To investigate the age at which oral squamous cell carcinoma presents in patients infected with human immunodeficiency virus. Prospective, clinicohistopathological audit of patients infected with human immunodeficiency virus. Of 200 human immunodeficiency virus positive patients, 16 (8 per cent) presented with oral squamous cell carcinoma (nine women and seven men; age range 18-43 years, mean age 31.7 years). The majority of patients (62.5 per cent) had stage III and IV disease (tumour-node-metastasis staging). There was a predilection for poorly differentiated oral squamous cell carcinoma (using Broder's histopathological classification). Oral squamous cell carcinoma associated with human immunodeficiency virus infection appears to present at a relatively young age.

  17. Identification of alpha beta and gamma delta T cell receptor-positive cells

    DEFF Research Database (Denmark)

    Geisler, C; Larsen, J K; Plesner, T

    1988-01-01

    distribution and function of these different T cells. In immunofluorescence studies gamma delta TCR+ cells have been identified as CD3+WT-31- or CD3+CD4-CD8- cells. However, this may not be the optimal procedure because gamma delta TCR+ cells are weakly WT-31+, and some are CD8+. The aim of this study...... was to evaluate a panel of monoclonal antibodies (MoAb) directed against different chains of the TCR-T3 complex for a more precise identification of alpha beta+ and gamma delta TCR+ cells in flow cytometric studies. We found that the MoAb anti-Ti-gamma A and delta-TCS-1, recognizing the TCR-gamma and the TCR...

  18. Similar cisplatin sensitivity of HPV-positive and -negative HNSCC cell lines.

    Science.gov (United States)

    Busch, Chia-Jung; Becker, Benjamin; Kriegs, Malte; Gatzemeier, Fruzsina; Krüger, Katharina; Möckelmann, Nikolaus; Fritz, Gerhard; Petersen, Cordula; Knecht, Rainald; Rothkamm, Kai; Rieckmann, Thorsten

    2016-06-14

    Patients with HPV-positive head and neck squamous cell carcinoma (HNSCC) show better survival rates than those with HPV-negative HNSCC. While an enhanced radiosensitivity of HPV-positive tumors is clearly evident from single modality treatment, cisplatin is never administered as monotherapy and therefore its contribution to the enhanced cure rates of HPV-positive HNSCC is not known. Both cisplatin and radiotherapy can cause severe irreversible side effects and therefore various clinical studies are currently testing deintensified regimes for patients with HPV-positive HNSCC. One strategy is to omit cisplatin-based chemotherapy or replace it by less toxic treatments but the risk assessment of these approaches remains difficult. In this study we have compared the cytotoxic effects of cisplatin in a panel of HPV-positive and -negative HNSCC cell lines alone and when combined with radiation.While cisplatin-treated HPV-positive strains showed a slightly stronger inhibition of proliferation, there was no difference regarding colony formation. Cellular responses to the drug, namely cell cycle distribution, apoptosis and γH2AX-induction did not differ between the two entities but assessment of cisplatin-DNA-adducts suggests differences regarding the mechanisms that determine cisplatin sensitivity. Combining cisplatin with radiation, we generally observed an additive but only in a minority of strains from both entities a clear synergistic effect on colony formation. In summary, HPV-positive and -negative HNSCC cells were equally sensitive to cisplatin. Therefore replacing cisplatin may be feasible but the substituting agent should be of similar efficacy in order not to jeopardize the high cure rates for HPV-positive HNSCC.

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

    Science.gov (United States)

    Mahy, T. X.

    1982-03-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Jhong-Yin Chen

    2013-05-01

    Full Text Available The objective of the current research was not only to provide a fast and automatic positioning platform for single cells, but also improved biomolecular manipulation techniques. In this study, an automatic platform for cell positioning using electroosmotic flow and image processing technology was designed. The platform was developed using a PCI image acquisition interface card for capturing images from a microscope and then transferring them to a computer using human-machine interface software. This software was designed by the Laboratory Virtual Instrument Engineering Workbench, a graphical language for finding cell positions and viewing the driving trace, and the fuzzy logic method for controlling the voltage or time of an electric field. After experiments on real human leukemic cells (U-937, the success of the cell positioning rate achieved by controlling the voltage factor reaches 100% within 5 s. A greater precision is obtained when controlling the time factor, whereby the success rate reaches 100% within 28 s. Advantages in both high speed and high precision are attained if these two voltage and time control methods are combined. The control speed with the combined method is about 5.18 times greater than that achieved by the time method, and the control precision with the combined method is more than five times greater than that achieved by the voltage method.

  1. Targeting Sindbis virus-based vectors to Fc receptor-positive cell types

    International Nuclear Information System (INIS)

    Klimstra, William B.; Williams, Jacqueline C.; Ryman, Kate D.; Heidner, Hans W.

    2005-01-01

    Some viruses display enhanced infection for Fc receptor (FcR)-positive cell types when complexed with virus-specific immunoglobulin (Ig). This process has been termed antibody-dependent enhancement of viral infection (ADE). We reasoned that the mechanism of ADE could be exploited and adapted to target alphavirus-based vectors to FcR-positive cell types. Towards this goal, recombinant Sindbis viruses were constructed that express 1 to 4 immunoglobulin-binding domains of protein L (PpL) as N-terminal extensions of the E2 glycoprotein. PpL is a bacterial protein that binds the variable region of antibody kappa light chains from a range of mammalian species. The recombinant viruses incorporated PpL/E2 fusion proteins into the virion structure and recapitulated the species-specific Ig-binding phenotypes of native PpL. Virions reacted with non-immune serum or purified IgG displayed enhanced binding and ADE for several species-matched FcR-positive murine and human cell lines. ADE required virus expression of a functional PpL Ig-binding domain, and appeared to be FcγR-mediated. Specifically, ADE did not occur with FcγR-negative cells, did not require active complement proteins, and did not occur on FcγR-positive murine cell lines when virions were bound by murine IgG-derived F(ab') 2 fragments

  2. A Notch positive feedback in the intestinal stem cell niche is essential for stem cell self-renewal.

    Science.gov (United States)

    Chen, Kai-Yuan; Srinivasan, Tara; Tung, Kuei-Ling; Belmonte, Julio M; Wang, Lihua; Murthy, Preetish Kadur Lakshminarasimha; Choi, Jiahn; Rakhilin, Nikolai; King, Sarah; Varanko, Anastasia Kristine; Witherspoon, Mavee; Nishimura, Nozomi; Glazier, James A; Lipkin, Steven M; Bu, Pengcheng; Shen, Xiling

    2017-04-28

    The intestinal epithelium is the fastest regenerative tissue in the body, fueled by fast-cycling stem cells. The number and identity of these dividing and migrating stem cells are maintained by a mosaic pattern at the base of the crypt. How the underlying regulatory scheme manages this dynamic stem cell niche is not entirely clear. We stimulated intestinal organoids with Notch ligands and inhibitors and discovered that intestinal stem cells employ a positive feedback mechanism via direct Notch binding to the second intron of the Notch1 gene. Inactivation of the positive feedback by CRISPR/Cas9 mutation of the binding sequence alters the mosaic stem cell niche pattern and hinders regeneration in organoids. Dynamical system analysis and agent-based multiscale stochastic modeling suggest that the positive feedback enhances the robustness of Notch-mediated niche patterning. This study highlights the importance of feedback mechanisms in spatiotemporal control of the stem cell niche. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  3. 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. © 2010 Blackwell Publishing Ltd.

  4. Diagnostic sensitivity of cutoff values of IgG4-positive plasma cell number and IgG4-positive/CD138-positive cell ratio in typical multiple lesions of patients with IgG4-related disease.

    Science.gov (United States)

    Mizushima, Ichiro; Yamada, Kazunori; Harada, Kenichi; Matsui, Shoko; Saeki, Takako; Kondo, Satoru; Takahira, Masayuki; Waseda, Yuko; Hamaguchi, Yasuhito; Fujii, Hiroshi; Yamagishi, Masakazu; Kawano, Mitsuhiro

    2018-03-01

    This study aimed to investigate the diagnostic sensitivity of the cutoff values of IgG4-positive plasma cell (PC) number and IgG4-positive/CD138-positive cell ratio proposed by the International consensus statement (ICS) on the pathology of IgG4-related disease (IgG4-RD) in typical multiple lesions of patients with IgG4-RD. We evaluated IgG4-positive PC number and IgG4-positive/CD138-positive cell ratio in 39 samples from 18 IgG4-RD patients having more than two typical lesions of IgG4-RD. We evaluated 12 submandibular, 12 ophthalmic, six skin, five kidney, two pancreatic, and one bronchus and prostate lesion each in 18 patients with typical clinical, serological, and radiographic features. Concerning IgG4 + PC number per high-power field, most ophthalmic (11/12), kidney (5/5), pancreatic (2/2), and bronchial lesions (1/1) fulfilled the cutoff value of ICS, whereas many of the submandibular (6/12) and skin lesions (0/6) did not. In contrast to the absolute number, all lesions fulfilled the cutoff value of IgG4+/CD138 + cell ratio. In eight cases, only one or two lesions in the same patient fulfilled the cutoff value of ICS, while the others did not. These results suggest that ICS criteria have different sensitivities among the affected organs for the diagnosis of IgG4-RD.

  5. 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

    2016-01-01

    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. PMID:26096934

  6. Compartment-specific tyrosine hydroxylase-positive innervation to AII amacrine cells in the rabbit retina.

    Science.gov (United States)

    Völgyi, B; Debertin, G; Balogh, M; Popovich, E; Kovács-Öller, T

    2014-06-13

    Tyrosine-hydroxylase-positive (TH(+)) amacrine cells release dopamine in a paracrine manner and also form GABA-ergic contact sites with inner retinal neurons. The best known sites are formed by TH(+) fibrous rings and AII amacrine cell somata in stratum 1 of the inner plexiform layer (IPL). An AII amacrine cell is a highly compartmentalized neuron with relatively large soma, a stout dendritic stalk and two sets of processes, one showing lobular appearance and extending horizontally in stratum 1 and a second transversally elongated group of fibers in strata 4 and 5. Although, all of these compartments have been reported as tic sites, it is uncertain if TH(+) amacrine cell inputs are homogeneously distributed or they rather target specific AII cell compartments. In this study we investigated the TH(+)/AII cell system by immunohistochemistry to map the potential synaptic contacts in the rabbit retina. We found numerous intimate contacts between the two amacrine cell populations throughout the IPL. However, TH(+) fibers favored the soma/main stalk region of AII amacrine cells and only contacted lobular appendages and transversal processes sporadically. In addition to the well-studied contacts between AII cell somata and TH(+) rings in stratum 1 we found that the main stalk region in stratum 3 serves as a secondary major target for TH(+) axons. These data thus clearly show that TH(+) contacts to AII amacrine cells are highly compartment specific. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  7. Oncolytic adenoviruses targeted to Human Papilloma Virus-positive head and neck squamous cell carcinomas.

    Science.gov (United States)

    LaRocca, Christopher J; Han, Joohee; Salzwedel, Amanda O; Davydova, Julia; Herzberg, Mark C; Gopalakrishnan, Rajaram; Yamamoto, Masato

    2016-05-01

    In recent years, the incidence of Human Papilloma Virus (HPV)-positive head and neck squamous cell carcinomas (HNSCC) has markedly increased. Our aim was to design a novel therapeutic agent through the use of conditionally replicative adenoviruses (CRAds) that are targeted to the HPV E6 and E7 oncoproteins. Each adenovirus included small deletion(s) in the E1a region of the genome (Δ24 or CB016) intended to allow for selective replication in HPV-positive cells. In vitro assays were performed to analyze the transduction efficiency of the vectors and the cell viability following viral infection. Then, the UPCI SCC090 cell line (HPV-positive) was used to establish subcutaneous tumors in the flanks of nude mice. The tumors were then treated with either one dose of the virus or four doses (injected every fourth day). The transduction analysis with luciferase-expressing viruses demonstrated that the 5/3 fiber modification maximized virus infectivity. In vitro, both viruses (5/3Δ24 and 5/3CB016) demonstrated profound oncolytic effects. The 5/3CB016 virus was more selective for HPV-positive HNSCC cells, whereas the 5/3Δ24 virus killed HNSCC cells regardless of HPV status. In vivo, single injections of both viruses demonstrated anti-tumor effects for only a few days following viral inoculation. However, after four viral injections, there was statistically significant reductions in tumor growth when compared to the control group (p<0.05). CRAds targeted to HPV-positive HNSCCs demonstrated excellent in vitro and in vivo therapeutic effects, and they have the potential to be clinically translated as a novel treatment modality for this emerging disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. 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. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  9. 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...

  10. Isolation and characterization of portal branch ligation-stimulated Hmga2-positive bipotent hepatic progenitor cells

    International Nuclear Information System (INIS)

    Sakai, Hiroshi; Tagawa, Yoh-ichi; Tamai, Miho; Motoyama, Hiroaki; Ogawa, Shinichiro; Soeda, Junpei; Nakata, Takenari; Miyagawa, Shinichi

    2010-01-01

    Research highlights: → Hepatic progenitor cells were isolated from the portal branch-ligated liver of mice. → Portal branch ligation-stimulated hepatic progenitor cells (PBLHCs) express Hmga2. → 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.

  11. Coexistence of chronic myeloid leukemia and diffuse large B-cell lymphoma with antecedent chronic lymphocytic leukemia: a case report and review of the literature.

    Science.gov (United States)

    Abuelgasim, Khadega A; Rehan, Hinna; Alsubaie, Maha; Al Atwi, Nasser; Al Balwi, Mohammed; Alshieban, Saeed; Almughairi, Areej

    2018-03-11

    Chronic lymphocytic leukemia and chronic myeloid leukemia are the most common types of adult leukemia. However, it is rare for the same patient to suffer from both. Richter's transformation to diffuse large B-cell lymphoma is frequently observed in chronic lymphocytic leukemia. Purine analog therapy and the presence of trisomy 12, and CCND1 gene rearrangement have been linked to increased risk of Richter's transformation. The coexistence of chronic myeloid leukemia and diffuse large B-cell lymphoma in the same patient is extremely rare, with only nine reported cases. Here, we describe the first reported case of concurrent chronic myeloid leukemia and diffuse large B-cell lymphoma in a background of chronic lymphocytic leukemia. A 60-year-old Saudi man known to have diabetes, hypertension, and chronic active hepatitis B was diagnosed as having Rai stage II chronic lymphocytic leukemia, with trisomy 12 and rearrangement of the CCND1 gene in December 2012. He required no therapy until January 2016 when he developed significant anemia, thrombocytopenia, and constitutional symptoms. He received six cycles of fludarabine, cyclophosphamide, and rituximab, after which he achieved complete remission. One month later, he presented with progressive leukocytosis (mostly neutrophilia) and splenomegaly. Fluorescence in situ hybridization from bone marrow aspirate was positive for translocation (9;22) and reverse transcription polymerase chain reaction detected BCR-ABL fusion gene consistent with chronic myeloid leukemia. He had no morphologic or immunophenotypic evidence of chronic lymphocytic leukemia at the time. Imatinib, a first-line tyrosine kinase inhibitor, was started. Eight months later, a screening imaging revealed new liver lesions, which were confirmed to be diffuse large B-cell lymphoma. In chronic lymphocytic leukemia, progressive leukocytosis and splenomegaly caused by emerging chronic myeloid leukemia can be easily overlooked. It is unlikely that chronic myeloid

  12. Concomitant use of polarization and positive phase contrast microscopy for the study of microbial cells.

    Science.gov (United States)

    Žižka, Zdeněk; Gabriel, Jiří

    2015-11-01

    Polarization and positive phase contrast microscope were concomitantly used in the study of the internal structure of microbial cells. Positive phase contrast allowed us to view even the fine cell structure with a refractive index approaching that of the surrounding environment, e.g., the cytoplasm, and transferred the invisible phase image to a visible amplitude image. With polarization microscopy, crossed polarizing filters together with compensators and a rotary stage showed the birefringence of different cell structures. Material containing algae was collected in ponds in Sýkořice and Zbečno villages (Křivoklát region). The objects were studied in laboratory microscopes LOMO MIN-8 Sankt Petersburg and Polmi A Carl Zeiss Jena fitted with special optics for positive phase contrast, polarizers, analyzers, compensators, rotary stages, and digital SLR camera Nikon D 70 for image capture. Anisotropic granules were found in the cells of flagellates of the order Euglenales, in green algae of the orders Chlorococcales and Chlorellales, and in desmid algae of the order Desmidiales. The cell walls of filamentous algae of the orders Zygnematales and Ulotrichales were found to exhibit significant birefringence; in addition, relatively small amounts of small granules were found in the cytoplasm. A typical shape-related birefringence of the cylindrical walls and the septa between the cells differed in intensity, which was especially apparent when using a Zeiss compensator RI-c during its successive double setting. In conclusion, the anisotropic granules found in the investigated algae mostly showed strong birefringence and varied in number, size, and location of the cells. Representatives of the order Chlorococcales contained the highest number of granules per cell, and the size of these granules was almost double than that of the other monitored microorganisms. Very strong birefringence was exhibited by cell walls of filamentous algae; it differed in the intensity

  13. CD207+/langerin positive dendritic cells in invasive and in situ cutaneous malignant melanoma

    Directory of Open Access Journals (Sweden)

    Grzegorz Dyduch

    2017-05-01

    Full Text Available Introduction : Dendritic cells are crucial for cutaneous immune response. Their role in melanoma progression is however a matter of controversy. Material and methods : The number of dendritic cells within epidermis and in peri- and intratumoral location was analyzed using CD207 immunostain in 17 cases of in situ and 25 case of invasive melanoma. Results : Average peritumoral CD207+ cells count was 22.88 for all cases, 17.94 for in situ lesions and 26.24 for invasive cases. Average epidermal CD207+ cells count was 164.47 for all cases, 183.00 for in situ lesions and 150.78 – for invasive cases. In case of invasive melanomas, peritumoral CD207+ cells count was positively correlated with Breslow stage (R = 0.59 mitotic activity within the tumor (R = 0.62. Invasive cases with regression showed higher intratumoral and epidermal CD207+ cells count than the ones without (275.00 vs. 95.32 and 173.20 vs. 148.35 but lower peritumoral CD207+ cells count (17.60 vs. 27.26. Invasive cases with ulceration showed higher intratumoral and peritumoral CD207+ cells count than the ones without ulceration (220.08 vs. 55.67 and 44.17 vs. 9.69. Conclusions : CD207+ cells play a role in both progression and regression of melanoma but their exact role needs further studies.

  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. 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.

  16. Reciprocal positive regulation between TRPV6 and NUMB in PTEN-deficient prostate cancer cells

    International Nuclear Information System (INIS)

    Kim, Sung-Young; Hong, Chansik; Wie, Jinhong; Kim, Euiyong; Kim, Byung Joo; Ha, Kotdaji; Cho, Nam-Hyuk; Kim, In-Gyu; Jeon, Ju-Hong; So, Insuk

    2014-01-01

    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. 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.

  18. Placental Kisspeptins Differentially Modulate Vital Parameters of Estrogen Receptor-Positive and -Negative Breast Cancer Cells

    Science.gov (United States)

    Rasoulzadeh, Zahra; Ghods, Roya; Kazemi, Tohid; Mirzadegan, Ebrahim; Ghaffari-Tabrizi-Wizsy, Nassim; Rezania, Simin; Kazemnejad, Somaieh; Arefi, Soheila; Ghasemi, Jamileh; Vafaei, Sedigheh; Mahmoudi, Ahmad-Reza; Zarnani, Amir-Hassan

    2016-01-01

    Kisspeptins (KPs) are major regulators of trophoblast and cancer invasion. Thus far, limited and conflicting data are available on KP-mediated modulation of breast cancer (BC) metastasis; mostly based on synthetic KP-10, the most active fragment of KP. Here, we report for the first time comprehensive functional effects of term placental KPs on proliferation, adhesion, Matrigel invasion, motility, MMP activity and pro-inflammatory cytokine production in MDA-MB-231 (estrogen receptor-negative) and MCF-7 (estrogen receptor-positive). KPs were expressed at high level by term placental syncytiotrophoblasts and released in soluble form. Placental explant conditioned medium containing KPs (CM) significantly reduced proliferation of both cell types compared to CM without (w/o) KP (CM-w/o KP) in a dose- and time-dependent manner. In MDA-MB-231 cells, placental KPs significantly reduced adhesive properties, while increased MMP9 and MMP2 activity and stimulated invasion. Increased invasiveness of MDA-MB-231 cells after CM treatment was inhibited by KP receptor antagonist, P-234. CM significantly reduced motility of MCF-7 cells at all time points (2–30 hr), while it stimulated motility of MDA-MB-231 cells. These effects were reversed by P-234. Co-treatment with selective ER modulators, Tamoxifen and Raloxifene, inhibited the effect of CM on motility of MCF-7 cells. The level of IL-6 in supernatant of MCF-7 cells treated with CM was higher compared to those treated with CM-w/o KP. Both cell types produced more IL-8 after treatment with CM compared to those treated with CM-w/o KP. Taken together, our observations suggest that placental KPs differentially modulate vital parameters of estrogen receptor-positive and -negative BC cells possibly through modulation of pro-inflammatory cytokine production. PMID:27101408

  19. Controlled Positioning of Cells in Biomaterials—Approaches Towards 3D Tissue Printing

    Science.gov (United States)

    Wüst, Silke; Müller, Ralph; Hofmann, Sandra

    2011-01-01

    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. PMID:24956301

  20. 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.

  1. Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus.

    Science.gov (United States)

    Scheler, Björn; Schnepf, Vera; Galgenmüller, Carolina; Ranf, Stefanie; Hückelhoven, Ralph

    2016-05-01

    RHO GTPases are regulators of cell polarity and immunity in eukaryotes. In plants, RHO-like RAC/ROP GTPases are regulators of cell shaping, hormone responses, and responses to microbial pathogens. The barley (Hordeum vulgare L.) RAC/ROP protein RACB is required for full susceptibility to penetration by Blumeria graminis f.sp. hordei (Bgh), the barley powdery mildew fungus. Disease susceptibility factors often control host immune responses. Here we show that RACB does not interfere with early microbe-associated molecular pattern-triggered immune responses such as the oxidative burst or activation of mitogen-activated protein kinases. RACB also supports rather than restricts expression of defence-related genes in barley. Instead, silencing of RACB expression by RNAi leads to defects in cell polarity. In particular, initiation and maintenance of root hair growth and development of stomatal subsidiary cells by asymmetric cell division is affected by silencing expression of RACB. Nucleus migration is a common factor of developmental cell polarity and cell-autonomous interaction with Bgh RACB is required for positioning of the nucleus near the site of attack from Bgh We therefore suggest that Bgh profits from RACB's function in cell polarity rather than from immunity-regulating functions of RACB. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  2. Radotinib Induces Apoptosis of CD11b+ Cells Differentiated from Acute Myeloid Leukemia Cells.

    Directory of Open Access Journals (Sweden)

    Sook-Kyoung Heo

    Full Text Available Radotinib, developed as a BCR/ABL tyrosine kinase inhibitor (TKI, is approved for the second-line treatment of chronic myeloid leukemia (CML in South Korea. However, therapeutic effects of radotinib in acute myeloid leukemia (AML are unknown. In the present study, we demonstrate that radotinib significantly decreases the viability of AML cells in a dose-dependent manner. Kasumi-1 cells were more sensitive to radotinib than NB4, HL60, or THP-1 cell lines. Furthermore, radotinib induced CD11b expression in NB4, THP-1, and Kasumi-1 cells either in presence or absence of all trans-retinoic acid (ATRA. We found that radotinib promoted differentiation and induced CD11b expression in AML cells by downregulating LYN. However, CD11b expression induced by ATRA in HL60 cells was decreased by radotinib through upregulation of LYN. Furthermore, radotinib mainly induced apoptosis of CD11b+ cells in the total population of AML cells. Radotinib also increased apoptosis of CD11b+ HL60 cells when they were differentiated by ATRA/dasatinib treatment. We show that radotinib induced apoptosis via caspase-3 activation and the loss of mitochondrial membrane potential (ΔΨm in CD11b+ cells differentiated from AML cells. Our results suggest that radotinib may be used as a candidate drug in AML or a chemosensitizer for treatment of AML by other therapeutics.

  3. Analysis of pancreas tissue in a child positive for islet cell antibodies.

    Science.gov (United States)

    Oikarinen, M; Tauriainen, S; Honkanen, T; Vuori, K; Karhunen, P; Vasama-Nolvi, C; Oikarinen, S; Verbeke, C; Blair, G E; Rantala, I; Ilonen, J; Simell, O; Knip, M; Hyöty, H

    2008-10-01

    Type 1 diabetes is caused by an immune-mediated process, reflected by the appearance of autoantibodies against pancreatic islets in the peripheral circulation. Detection of multiple autoantibodies predicts the development of diabetes, while positivity for a single autoantibody is a poor prognostic marker. The present study assesses whether positivity for a single autoantibody correlates with pathological changes in the pancreas. We studied post mortem pancreatic tissue of a child who repeatedly tested positive for islet cell antibodies (ICA) in serial measurements. Paraffin sections were stained with antibodies specific for insulin, glucagon, somatostatin, interferon alpha, CD3, CD68, cyclooxygenase-2 (COX-2), beta-2-microglobulin, coxsackie B and adenovirus receptor (CAR), natural killer and dendritic cells. Apoptosis was detected using Fas-specific antibody and TUNEL assay. Enterovirus was searched for using immunohistochemistry and in situ hybridisation, as well as enterovirus-specific RT-PCR from serum samples. The structure of the pancreas did not differ from normal. The number of beta cells was not reduced and no signs of insulitis were observed. Beta-2-microglobulin and CAR were strongly produced in the islets, but not in the exocrine pancreas. Enterovirus protein was detected selectively in the islets by two enterovirus-specific antibodies, but viral RNA was not found. These observations suggest that positivity for ICA alone, even when lasting for more than 1 year, is not associated with inflammatory changes in the islets. However, it is most likely that the pancreatic islets were infected by an enterovirus in this child.

  4. Spontaneous regression in an ulcerated CK7 positive Merkel cell carcinoma

    Directory of Open Access Journals (Sweden)

    Anza Khader

    2015-01-01

    Full Text Available Merkel cell carcinoma is an aggressive and frequently lethal tumor of the elderly, associated with sun exposure and immunosuppression which is less common in the dark-skinned. We report the case of a 40-year-old woman who presented with multiple slowly progressive, mildly itchy ulcerated plaques of size ranging from 2 × 3 cm to 5 × 7 cm on the left knee of 1 year duration. Skin biopsy showed diffuse dermal infiltration by small round cells with molding of cells and lymphocyte infiltration. The cells stained positive for cytokeratin (CK 20, CK7, neuron-specific enolase, and chromogranin. The skin lesions underwent spontaneous regression within 1 month of skin biopsy and have not recurred during the past 2 years. The immune mechanisms triggered by biopsy possibly explain the spontaneous regression.

  5. 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 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.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.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-neurogenic neural function

  6. 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.

  7. Activated WNT signaling in postnatal SOX2-positive dental stem cells can drive odontoma formation.

    Science.gov (United States)

    Xavier, Guilherme M; Patist, Amanda L; Healy, Chris; Pagrut, Ankita; Carreno, Gabriela; Sharpe, Paul T; Martinez-Barbera, Juan Pedro; Thavaraj, Selvam; Cobourne, Martyn T; Andoniadou, Cynthia L

    2015-09-28

    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.

  8. RBX2 maintains final retinal cell position in a DAB1-dependent and -independent fashion.

    Science.gov (United States)

    Fairchild, Corinne L; Hino, Keiko; Han, Jisoo S; Miltner, Adam M; Peinado Allina, Gabriel; Brown, Caileigh E; Burns, Marie E; La Torre, Anna; Simó, Sergi

    2018-02-02

    The laminated structure of the retina is fundamental for the organization of the synaptic circuitry that translates light input into patterns of action potentials. However, the molecular mechanisms underlying cell migration and layering of the retina are poorly understood. Here, we show that RBX2, a core component of the E3 ubiquitin ligase CRL5, is essential for retinal layering and function. RBX2 regulates the final cell position of rod bipolar cells, cone photoreceptors and Muller glia. Our data indicate that sustained RELN/DAB1 signaling, triggered by depletion of RBX2 or SOCS7 - a CRL5 substrate adaptor known to recruit DAB1 - causes rod bipolar cell misposition. Moreover, whereas SOCS7 also controls Muller glia cell lamination, it is not responsible for cone photoreceptor positioning, suggesting that RBX2, most likely through CRL5 activity, controls other signaling pathways required for proper cone localization. Furthermore, RBX2 depletion reduces the number of ribbon synapses and disrupts cone photoreceptor function. Together, these results uncover RBX2 as a crucial molecular regulator of retina morphogenesis and cone photoreceptor function. © 2018. Published by The Company of Biologists Ltd.

  9. Positive intergenic feedback circuitry, involving EBF1 and FOXO1, orchestrates B-cell fate.

    Science.gov (United States)

    Mansson, Robert; Welinder, Eva; Åhsberg, Josefine; Lin, Yin C; Benner, Christopher; Glass, Christopher K; Lucas, Joseph S; Sigvardsson, Mikael; Murre, Cornelis

    2012-12-18

    Recent studies have identified a number of transcriptional regulators, including E2A, early B-cell factor 1 (EBF1), FOXO1, and paired box gene 5 (PAX5), that promote early B-cell development. However, how this ensemble of regulators mechanistically promotes B-cell fate remains poorly understood. Here we demonstrate that B-cell development in FOXO1-deficient mice is arrested in the common lymphoid progenitor (CLP) LY6D(+) cell stage. We demonstrate that this phenotype closely resembles the arrest in B-cell development observed in EBF1-deficient mice. Consistent with these observations, we find that the transcription signatures of FOXO1- and EBF1-deficient LY6D(+) progenitors are strikingly similar, indicating a common set of target genes. Furthermore, we found that depletion of EBF1 expression in LY6D(+) CLPs severely affects FOXO1 mRNA abundance, whereas depletion of FOXO1 activity in LY6D(+) CLPs ablates EBF1 transcript levels. We generated a global regulatory network from EBF1 and FOXO1 genome-wide transcription factor occupancy and transcription signatures derived from EBF1- and FOXO1-deficient CLPs. This analysis reveals that EBF1 and FOXO1 act in a positive feedback circuitry to promote and stabilize specification to the B-cell lineage.

  10. Analysis of Exocyst-Positive Organelle (EXPO)-Mediated Unconventional Protein Secretion (UPS) in Plant Cells.

    Science.gov (United States)

    Ding, Yu; Wang, Juan

    2017-01-01

    Unconventional protein secretion (UPS) together with conventional protein secretion (CPS) is responsible for protein secretion in plants. We have previously identified a novel UPS pathway in plants, which is mediated by exocyst-positive organelle-EXPO. Here, we describe detailed protocols to study UPS in plants by using Arabidopsis protoplasts or transgenic suspension cells, expressing the EXPO marker Exo70E2-XFP, as materials. Via drug and osmotic treatment plus secretion assay, we illustrate several major methods to analyze EXPO-mediated UPS in plant cells, which also supplys mining tools for similar study.

  11. CD44 and SSEA-4 positive cells in an oral cancer cell line HSC-4 possess cancer stem-like cell characteristics.

    Science.gov (United States)

    Noto, Zenko; Yoshida, Toshiko; Okabe, Motonori; Koike, Chika; Fathy, Moustafa; Tsuno, Hiroaki; Tomihara, Kei; Arai, Naoya; Noguchi, Makoto; Nikaido, Toshio

    2013-08-01

    Cancer may be derived from cancer stem-like cells (CSCs), which are tumor-initiating cells that have properties similar to those of stem cells. Identification and isolation of CSCs needs to be improved further. CSCs markers were examined in human oral cancer cell lines by flow cytometry. The stem cell properties of subpopulations expressing different markers were assessed further by in vitro sphere formation assays, expression of stemness genes, drug resistance assays, and the ability to form tumors in nude mice. We demonstrated that CSCs could be isolated by the cell surface markers CD44 and stage-specific embryonic antigen-4 (SSEA-4). CD44+SSEA-4+ cells exhibited cancer stem-like properties, including extensive self-renewal into the bulk of cancer cells. In vivo xenograft experiments indicated that CD44+SSEA-4+ cells exhibit the highest tumorigenic capacity compared with the remaining subpopulations and parental cells. Double-positive cells for CD44 and SSEA-4 exhibited preferential expression of some stemness genes and were more resistant to the anticancer drugs, cisplatin and 5-fluorouracil (5-FU). In addition, cells expressing CD44 and SSEA-4 were detected in all tumor specimens analyzed, while coexpression of CD44 and SSEA-4 was not detectable in normal oral mucosa. Our findings suggest that CD44+SSEA-4+ cells exhibit the characteristics of CSCs in oral squamous cell carcinoma and provide a target for the development of more effective therapies. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Signalome-wide RNAi screen identifies GBA1 as a positive mediator of autophagic cell death

    Science.gov (United States)

    Dasari, Santosh K; Bialik, Shani; Levin-Zaidman, Smadar; Levin-Salomon, Vered; Merrill, Alfred H; Futerman, Anthony H; Kimchi, Adi

    2017-01-01

    Activating alternative cell death pathways, including autophagic cell death, is a promising direction to overcome the apoptosis resistance observed in various cancers. Yet, whether autophagy acts as a death mechanism by over consumption of intracellular components is still controversial and remains undefined at the ultrastructural and the mechanistic levels. Here we identified conditions under which resveratrol-treated A549 lung cancer cells die by a mechanism that fulfills the previous definition of autophagic cell death. The cells displayed a strong and sustained induction of autophagic flux, cell death was prevented by knocking down autophagic genes and death occurred in the absence of apoptotic or necroptotic pathway activation. Detailed ultrastructural characterization revealed additional critical events, including a continuous increase over time in the number of autophagic vacuoles, in particular autolysosomes, occupying most of the cytoplasm at terminal stages. This was followed by loss of organelles, disruption of intracellular membranes including the swelling of perinuclear space and, occasionally, a unique type of nuclear shedding. A signalome-wide shRNA-based viability screen was applied to identify positive mediators of this type of autophagic cell death. One top hit was GBA1, the Gaucher disease-associated gene, which encodes glucocerebrosidase, an enzyme that metabolizes glucosylceramide to ceramide and glucose. Interestingly, glucocerebrosidase expression levels and activity were elevated, concomitantly with increased intracellular ceramide levels, both of which correlated in time with the appearance of the unique death characteristics. Transfection with siGBA1 attenuated the increase in glucocerebrosidase activity and the intracellular ceramide levels. Most importantly, GBA1 knockdown prevented the strong increase in LC3 lipidation, and many of the ultrastructural changes characteristic of this type of autophagic cell death, including a significant

  13. 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. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  14. 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.

  15. Strategy for personalized treatment of human papillomavirus-positive oropharyngeal squamous cell carcinoma

    International Nuclear Information System (INIS)

    Mizumachi, Takatsugu; Hatakeyama, Hiromitsu; Kano, Satoshi; Sakashita, Tomohiro; Suzuki, Seigo; Homma, Akihiro; Oridate, Nobuhiko; Fukuda, Satoshi

    2011-01-01

    We performed a retrospective analysis of the association between tumor HPV status and the demographic and clinicopathological parameters of 83 patients with oropharyngeal squamous cell carcinoma at Hokkaido University Hospital, Japan, between 1998 and 2010. The parameters included age, gender, tumor subsite, Tumor-Node-Metastasis (TNM) stage, and overall survival. HPV status was established by multiplex polymerase chain reaction analysis. Of the 83 oropharyngeal cancers, 22 were positive for HPV-16, two for HPV-18, and one for HPV-35 and HPV-58. Kaplan-Meier survival analysis showed improved overall survival rates in patients with HPV-positive tumors (p=0.0024) compared with HPV-negative tumors. Of the 51 patients who received chemoradiotherapy, HPV-positive patients experienced better overall survival than HPV-negative patients (p=0.0024). HPV status is a significantly favorable prognostic factor in oropharyngeal cancer in Japan. (author)

  16. 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.

  17. AMPK promotes survival of c-Myc-positive melanoma cells by suppressing oxidative stress.

    Science.gov (United States)

    Kfoury, Alain; Armaro, Marzia; Collodet, Caterina; Sordet-Dessimoz, Jessica; Giner, Maria Pilar; Christen, Stefan; Moco, Sofia; Leleu, Marion; de Leval, Laurence; Koch, Ute; Trumpp, Andreas; Sakamoto, Kei; Beermann, Friedrich; Radtke, Freddy

    2018-03-01

    Although c-Myc is essential for melanocyte development, its role in cutaneous melanoma, the most aggressive skin cancer, is only partly understood. Here we used the Nras Q61K INK4a -/- mouse melanoma model to show that c-Myc is essential for tumor initiation, maintenance, and metastasis. c-Myc-expressing melanoma cells were preferentially found at metastatic sites, correlated with increased tumor aggressiveness and high tumor initiation potential. Abrogation of c-Myc caused apoptosis in primary murine and human melanoma cells. Mechanistically, c-Myc-positive melanoma cells activated and became dependent on the metabolic energy sensor AMP-activated protein kinase (AMPK), a metabolic checkpoint kinase that plays an important role in energy and redox homeostasis under stress conditions. AMPK pathway inhibition caused apoptosis of c-Myc-expressing melanoma cells, while AMPK activation protected against cell death of c-Myc-depleted melanoma cells through suppression of oxidative stress. Furthermore, TCGA database analysis of early-stage human melanoma samples revealed an inverse correlation between C-MYC and patient survival, suggesting that C-MYC expression levels could serve as a prognostic marker for early-stage disease. © 2018 The Authors.

  18. Fibroid polyps of intestinal tract are inflammatory-reactive proliferations of CD34-positive perivascular cells.

    Science.gov (United States)

    Wille, P; Borchard, F

    1998-06-01

    Our aim was to determine the histogenesis of fibroid polyps (FP). These polyps are rare inflammatory-reactive, tumour-like lesions of unknown aetiology, arising in the submucosa or mucosa of the gastrointestinal tract. They are mainly due to a proliferation of characteristic spindle cells. Nine FP were investigated by light microscopy and immunohistochemistry with endothelial markers (Factor VIII, CD34, CD31), a neuronal marker (S100), muscular markers (desmin, alpha-smooth muscle actin) and histiocytic markers (PGMI, KP1, MAC387) using the highly sensitive avidin-biotin-peroxidase technique. We demonstrate, for the first time, a consistent positivity of the characteristic spindle cells of FP for CD34. The proposed endothelial, histiocytic or neuronal origin of FP could be completely ruled out. Because of the consistent positivity of the spindle cells of FP for CD34 we suggest an origin of these lesions from primitive perivascular or vascular cells. This origin and a probable relationship to gastrointestinal stromal tumours (GIST) is discussed.

  19. 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.

  20. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Improved accuracy of cell surface shaving proteomics in Staphylococcus aureus using a false-positive control

    DEFF Research Database (Denmark)

    Solis, Nestor; Larsen, Martin Røssel; Cordwell, Stuart J

    2010-01-01

    Proteolytic treatment of intact bacterial cells is an ideal means for identifying surface-exposed peptide epitopes and has potential for the discovery of novel vaccine targets. Cell stability during such treatment, however, may become compromised and result in the release of intracellular proteins...... that complicate the final analysis. Staphylococcus aureus is a major human pathogen, causing community and hospital-acquired infections, and is a serious healthcare concern due to the increasing prevalence of multiple antibiotic resistances amongst clinical isolates. We employed a cell surface "shaving" technique...... to trypsin and three identified in the control. The use of a subtracted false-positive strategy improved enrichment of surface-exposed peptides in the trypsin data set to approximately 80% (124/155 peptides). Predominant surface proteins were those associated with methicillin resistance-surface protein SACOL...

  2. 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

  3. PDGFBB promotes PDGFRα-positive cell migration into artificial bone in vivo.

    Science.gov (United States)

    Yoshida, Shigeyuki; Iwasaki, Ryotaro; Kawana, Hiromasa; Miyauchi, Yoshiteru; Hoshi, Hiroko; Miyamoto, Hiroya; Mori, Tomoaki; Kanagawa, Hiroya; Katsuyama, Eri; Fujie, Atsuhiro; Hao, Wu; Kobayashi, Tami; Sato, Yuiko; Miyamoto, Kana; Morioka, Hideo; Matsumoto, Morio; Chiba, Kazuhiro; Toyama, Yoshiaki; Nakagawa, Taneaki; Miyamoto, Takeshi

    2012-05-18

    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 α (PDGFRα)-positive mesenchymal stem cells/pre-osteoblastic cells into artificial bone in vivo. Growth factors such as transforming growth factor beta (TGFβ) 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. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Measuring relative utilization of aerobic glycolysis in breast cancer cells by positional isotopic discrimination.

    Science.gov (United States)

    Yang, Da-Qing; Freund, Dana M; Harris, Benjamin R E; Wang, Defeng; Cleary, Margot P; Hegeman, Adrian D

    2016-09-01

    The ability of cancer cells to produce lactate through aerobic glycolysis is a hallmark of cancer. In this study, we established a positional isotopic labeling and LC-MS-based method that can specifically measure the conversion of glucose to lactate in glycolysis. We show that the rate of aerobic glycolysis is closely correlated with glucose uptake and lactate production in breast cancer cells. We also found that the production of [3-(13) C]lactate is significantly elevated in metastatic breast cancer cells and in early stage metastatic mammary tumors in mice. Our findings may enable the development of a biomarker for the diagnosis of aggressive breast cancer. © 2016 Federation of European Biochemical Societies.

  5. Parallel arrangements of positive feedback loops limit cell-to-cell variability in differentiation.

    Directory of Open Access Journals (Sweden)

    Anupam Dey

    Full Text Available Cellular differentiations are often regulated by bistable switches resulting from specific arrangements of multiple positive feedback loops (PFL fused to one another. Although bistability generates digital responses at the cellular level, stochasticity in chemical reactions causes population heterogeneity in terms of its differentiated states. We hypothesized that the specific arrangements of PFLs may have evolved to minimize the cellular heterogeneity in differentiation. In order to test this we investigated variability in cellular differentiation controlled either by parallel or serial arrangements of multiple PFLs having similar average properties under extrinsic and intrinsic noises. We find that motifs with PFLs fused in parallel to one another around a central regulator are less susceptible to noise as compared to the motifs with PFLs arranged serially. Our calculations suggest that the increased resistance to noise in parallel motifs originate from the less sensitivity of bifurcation points to the extrinsic noise. Whereas estimation of mean residence times indicate that stable branches of bifurcations are robust to intrinsic noise in parallel motifs as compared to serial motifs. Model conclusions are consistent both in AND- and OR-gate input signal configurations and also with two different modeling strategies. Our investigations provide some insight into recent findings that differentiation of preadipocyte to mature adipocyte is controlled by network of parallel PFLs.

  6. Controlling cell position in complex heterotypic 3D microtissues by tissue fusion.

    Science.gov (United States)

    Rago, Adam P; Dean, Dylan M; Morgan, Jeffrey R

    2009-03-01

    Tissue fusion and cell sorting are processes fundamental to developmental biology with applications in tissue engineering. We have designed a fusion assay to investigate the factors governing the fusion of microtissues and the cell sorting that occurs after fusion. Normal human fibroblast (NHF) spheroids were self-assembled and cultured for 1, 4, or 7 days, then combined in trough shaped recesses. Over a 24-h period the spheroids fused to become a rod shaped microtissue and the kinetics and extent of fusion could be quantified by assessing rod contraction. By varying the amount of spheroid culture time prior to fusion (1-7 days), the rate of fusion, the coherence of the building units (as measured by fusion angle) and the steady state length of the structure could be easily controlled. Longer pre-culture times for the spheroids resulted in slower fusion, less coherence and increased length of rod microtissues. The fusion kinetics and steady length of rods formed by smaller versus larger spheroids ( approximately 100 vs. 300 microm diameter) were indistinguishable, even though smaller spheroids had twice the surface area and greater numbers of contacts between units. Both small and large spheroids were strongly influenced by spheroid pre-culture time. Pre-culture time could also be used to control cell sorting and cell position when combinations of NHFs and H35s, a rat hepatocyte cell line, were fused to form heterotypic microtissues. Control of fusion and cell position are important parameters for creating functional heterotypic microtissues as well as the use of microtissues as building units to create larger tissue structures.

  7. Locomotor response to novelty correlates with the number of midbrain tyrosine hydroxylase positive cells in rats.

    Science.gov (United States)

    Jerzemowska, Grażyna; Plucińska, Karolina; Kulikowski, Michał; Trojniar, Weronika; Wrona, Danuta

    2012-01-04

    The present study investigated whether the higher dopaminergic system activation in rats with high (HRs) rather than low (LRs) locomotor activity in response to novelty depend on the number of cells containing the enzyme tyrosine hydroxylase (TH(+)) and/or differences in the morphology of these cells. One week after the novelty test, brains from male Wistar rats (HRs and LRs) were collected and stained for TH expression (immunohistochemistry) and for morphological analysis (immunofluorescent staining). The morphology and total number of TH(+) cells was analyzed for each A9 (substantia nigra) and A10 (ventral tegmental area) group of the midbrain dopaminergic cells. We found that HRs had a higher total number of TH(+) cells in the whole midbrain dopaminergic region (A9-A10) and in the A9 group only than LRs. In particular midbrain dopaminergic groups of neurons, HR/LR differences were regionally specific: HRs had a higher total number of TH(+) cells in the A9, and in the anterior part of the A10. In contrast, the LRs had a higher number of TH(+) cells in the parabrachial pigmented nucleus (A10) and in the posterior part of the A9. There were no significant differences in the morphology of the midbrain dopamine neurons between HRs and LRs. Moreover, there was a positive correlation between the total number of TH(+) neurons and the locomotor activity score in response to novelty in the whole A9-A10 region and in the particular A9 group only. The results obtained indicate that the higher behavioral activation in resting conditions correlates with the higher number rather than changes in the morphology of the midbrain dopaminergic TH(+) cells. It supports findings on the higher level of dopaminergic system activation in high responders to novelty that depends on the number of midbrain dopaminergic TH(+) neurons. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. S100 protein-positive Langerhans cells in 80 dentigerous cysts

    Directory of Open Access Journals (Sweden)

    Chun-Han Chang

    2017-12-01

    Full Text Available Background/purpose: Langerhans cells (LCs are antigen-presenting cells. This study assessed the LC counts in 80 dentigerous cysts (DCs. Materials and methods: The S100-positive LC numbers in the lining epithelia and subepithelial connective tissues were counted at 80 DC sites without inflammation, 33 DC sites with mild/moderate inflammation, and 9 DC sites with severe inflammation from 80 DC specimens. Results: The mean S100-positive LC counts in the lining epithelia and subepithelial connective tissues increased significantly from no inflammation (0.6 ± 0.6 and 0.7 ± 0.6 cell/high-power field or HPF, respectively through mild/moderate inflammation (8.1 ± 2.0 and 4.5 ± 2.3 cells/HPF, respectively to severe inflammation DC sites (21.0 ± 7.0 and 11.1 ± 6.5 cells/HPF, respectively; P-value 50 μm group (8.6 ± 7.1 and 4.8 ± 4.5 cells/HPF, respectively than in the thinner lining epithelium (≦50 μm group (0.6 ± 0.6 and 0.6 ± 0.6 cells/HPF, respectively; both P-values < 0.001. Conclusion: A significant association of high-grade inflammation and thick lining epithelium with the increased LC number in DCs is found. Very few LCs in the lining epithelia of DCs without inflammation indicate the reduced immunosurveillance ability against DC lining epithelial cells in DC patients. It needs further studies to confirm the role of reduced immunosurveillance in the enlargement of the DC. Keywords: Langerhans cell, dentigerous cyst, inflammation, lining epithelium, immunosurveillance

  9. Hypoxia inducible factor-1 alpha expression is increased in infected positive HPV16 DNA oral squamous cell carcinoma and positively associated with HPV16 E7 oncoprotein

    Directory of Open Access Journals (Sweden)

    Di Fede Olga

    2011-10-01

    Full Text Available Abstract Background There is increasing evidence for the role of High Risk (HR Human PapillomaVirus (HPV in the pathogenesis of Oral Squamous Cell Carcinoma (OSCC. The E6 and E7 oncogenes from HR HPVs are responsible for the deregulation of p53 and pRB proteins involved in cell cycle and apoptotic pathways. In cell lines experiments, the HPV E7 protein seems to be able to enhance Hypoxia Inducible Factor-1 alpha (HIF-1α activity, normally involved in the response to hypoxia and able to enhance angiogenesis. Results We studied tumor specimens from 62 OSCC; a higher prevalence of tumors in TNM stage II and also in pT2 class between OSCC infected positive HPV16 DNA than non-infected ones was observed. HIF-1α positivity was detected throughout the analysed fields, not associated with areas of necrosis and also observed in cells immediately adjacent to blood vessels. A significant increase in mean values of the HIF-1α labeling indexes was observed for pT1-T2, as well for stage I-II, in the infected positive HPV16 DNA tumors than non-infected ones. HIF-1α and HPV16 E7 labeling indexes showed a significantly positive correlation which suggested a positive association between HPV16 E7 and HIF-1α expression. Conclusions In our specimens HIF-1α immunoreactivity hints for an O2-independent regulatory mechanism in infected positive HPV16 DNA tumors, especially for pT1-T2 and stage I-II tumors, suggesting a very early involvement in the development of HPV-induced OSCC. HIF-1α and HPV16 E7 labeling indexes suggest also a positive association between the two proteins in infected positive HPV16 DNA OSCC.

  10. Revised mechanism of d-alanine incorporation into cell wall polymers in Gram-positive bacteria

    Science.gov (United States)

    Reichmann, Nathalie T.; Cassona, Carolina Picarra

    2013-01-01

    Teichoic acids (TAs) are important for growth, biofilm formation, adhesion and virulence of Gram-positive bacterial pathogens. The chemical structures of the TAs vary between bacteria, though they typically consist of zwitterionic polymers that are anchored to either the peptidoglycan layer as in the case of wall teichoic acid (WTA) or the cell membrane and named lipoteichoic acid (LTA). The polymers are modified with d-alanines and a lack of this decoration leads to increased susceptibility to cationic antimicrobial peptides. Four proteins, DltA–D, are essential for the incorporation of d-alanines into cell wall polymers and it has been established that DltA transfers d-alanines in the cytoplasm of the cell onto the carrier protein DltC. However, two conflicting models have been proposed for the remainder of the mechanism. Using a cellular protein localization and membrane topology analysis, we show here that DltC does not traverse the membrane and that DltD is anchored to the outside of the cell. These data are in agreement with the originally proposed model for d-alanine incorporation through a process that has been proposed to proceed via a d-alanine undecaprenyl phosphate membrane intermediate. Furthermore, we found that WTA isolated from a Staphylococcus aureus strain lacking LTA contains only a small amount of d-alanine, indicating that LTA has a role, either direct or indirect, in the efficient d-alanine incorporation into WTA in living cells. PMID:23858088

  11. Concentration of Glial Cell Line-Derived Neurotrophic Factor Positively Correlates with Symptoms in Functional Dyspepsia.

    Science.gov (United States)

    Tanaka, Fumio; Tominaga, Kazunari; Fujikawa, Yoshiko; Nagami, Yasuaki; Kamata, Noriko; Yamagami, Hirokazu; Tanigawa, Tetsuya; Shiba, Masatsugu; Watanabe, Toshio; Fujiwara, Yasuhiro; Arakawa, Tetsuo

    2016-12-01

    In patients with functional dyspepsia (FD), mild duodenal inflammation correlates with increased mucosal permeability. Enteric glial cells can produce glial cell line-derived neurotrophic factor (GDNF) to repair disrupted epithelial barrier function. We examined the role of duodenal GDNF in FD pathophysiology and its association with dyspeptic symptoms. Duodenal biopsies taken from FD patients and control subjects were used for analysis. GDNF protein expression and localization were examined. Cellular infiltration of eosinophils and mast cells was measured. We also examined the intercellular space between the adjacent epithelial cells at the apical junction complex using transmission electron microscopy. In FD patients, expression of GDNF protein was significantly increased compared with controls, 107.3 (95.3-136.7) versus 49.3 (38.0-72.6) pg/mg protein (median (interquartile range), p = 0.006), respectively. GDNF was localized in enteric glial cells, eosinophils, and epithelial cells. The number of eosinophils was significantly greater in FD patients than in controls, 1039 (923-1181) versus 553 (479-598) cells/mm 2 (p = 0.021), respectively. The intercellular space was dilated at the adherent junction in FD patients compared to control patients, 32.4 (29.8-34.8) versus 22.0 (19.9-26.1) nm (p = 0.002), respectively. Intercellular distance positively correlated with the frequency of postprandial fullness and early satiation (p = 0.001, r = 0.837 and p = 0.009, r = 0.693, respectively). Expression of GDNF correlated with epigastric burning (p = 0.041, r = 0.552). Increased expression of duodenal GDNF might be involved in FD pathophysiology and symptom perception.

  12. Prognostic Significance of Progesterone Receptor–Positive Tumor Cells Within Immunohistochemically Defined Luminal A Breast Cancer

    Science.gov (United States)

    Prat, Aleix; Cheang, Maggie Chon U.; Martín, Miguel; Parker, Joel S.; Carrasco, Eva; Caballero, Rosalía; Tyldesley, Scott; Gelmon, Karen; Bernard, Philip S.; Nielsen, Torsten O.; Perou, Charles M.

    2013-01-01

    Purpose Current immunohistochemical (IHC)-based definitions of luminal A and B breast cancers are imperfect when compared with multigene expression-based assays. In this study, we sought to improve the IHC subtyping by examining the pathologic and gene expression characteristics of genomically defined luminal A and B subtypes. Patients and Methods Gene expression and pathologic features were collected from primary tumors across five independent cohorts: British Columbia Cancer Agency (BCCA) tamoxifen-treated only, Grupo Español de Investigación en Cáncer de Mama 9906 trial, BCCA no systemic treatment cohort, PAM50 microarray training data set, and a combined publicly available microarray data set. Optimal cutoffs of percentage of progesterone receptor (PR) –positive tumor cells to predict survival were derived and independently tested. Multivariable Cox models were used to test the prognostic significance. Results Clinicopathologic comparisons among luminal A and B subtypes consistently identified higher rates of PR positivity, human epidermal growth factor receptor 2 (HER2) negativity, and histologic grade 1 in luminal A tumors. Quantitative PR gene and protein expression were also found to be significantly higher in luminal A tumors. An empiric cutoff of more than 20% of PR-positive tumor cells was statistically chosen and proved significant for predicting survival differences within IHC-defined luminal A tumors independently of endocrine therapy administration. Finally, no additional prognostic value within hormonal receptor (HR) –positive/HER2-negative disease was observed with the use of the IHC4 score when intrinsic IHC-based subtypes were used that included the more than 20% PR-positive tumor cells and vice versa. Conclusion Semiquantitative IHC expression of PR adds prognostic value within the current IHC-based luminal A definition by improving the identification of good outcome breast cancers. The new proposed IHC-based definition of luminal A

  13. Expression of Slug in S100β-protein-positive cells of postnatal developing rat anterior pituitary gland.

    Science.gov (United States)

    Horiguchi, Kotaro; Fujiwara, Ken; Tsukada, Takehiro; Yako, Hideji; Tateno, Kozue; Hasegawa, Rumi; Takigami, Shu; Ohsako, Shunji; Yashiro, Takashi; Kato, Takako; Kato, Yukio

    2016-02-01

    Among heterogeneous S100β-protein-positive (S100β-positive) cells, star-like cells with extended cytoplasmic processes, the so-called folliculo-stellate cells, envelop hormone-producing cells or interconnect homophilically in the anterior pituitary. S100β-positive cells are known, from immunohistochemistry, to emerge from postnatal day (P) 10 and to proliferate and migrate in the parenchyma of the anterior pituitary with growth. Recent establishment of S100β-GFP transgenic rats expressing specifically green fluorescent protein (GFP) under the control of the S100β-promoter has allowed us to observe living S100β-positive cells. In the present study, we first confirmed that living S100β-positive cells in tissue cultures of S100β-GFP rat pituitary at P5 were present prior to P10 by means of confocal laser microscopy and that they proliferated and extended their cytoplasmic processes. Second, we examined the expression of the Snail-family zinc-finger transcription factors, Snail and Slug, to investigate the mechanism behind the morphological changes and the proliferation of S100β-positive cells. Interestingly, we detected Slug expression in S100β-positive cells and its increase together with development in the anterior pituitary. To analyze downstream of SLUG in S100β-positive cells, we utilized specific small interfering RNA for Slug mRNAs and observed that the expression of matrix metalloprotease (Mmp) 9, Mmp14 and chemokine Cxcl12 was down-regulated and that morphological changes and proliferation were decreased. Thus, our findings suggest that S100β-positive cells express Slug and that its expression is important for subsequent migration and proliferation.

  14. Transcriptomics and methylomics of CD4-positive T cells in arsenic-exposed women.

    Science.gov (United States)

    Engström, Karin; Wojdacz, Tomasz K; Marabita, Francesco; Ewels, Philip; Käller, Max; Vezzi, Francesco; Prezza, Nicola; Gruselius, Joel; Vahter, Marie; Broberg, Karin

    2017-05-01

    Arsenic, a carcinogen with immunotoxic effects, is a common contaminant of drinking water and certain food worldwide. We hypothesized that chronic arsenic exposure alters gene expression, potentially by altering DNA methylation of genes encoding central components of the immune system. We therefore analyzed the transcriptomes (by RNA sequencing) and methylomes (by target-enrichment next-generation sequencing) of primary CD4-positive T cells from matched groups of four women each in the Argentinean Andes, with fivefold differences in urinary arsenic concentrations (median concentrations of urinary arsenic in the lower- and high-arsenic groups: 65 and 276 μg/l, respectively). Arsenic exposure was associated with genome-wide alterations of gene expression; principal component analysis indicated that the exposure explained 53% of the variance in gene expression among the top variable genes and 19% of 28,351 genes were differentially expressed (false discovery rate arsenic group. Arsenic exposure was associated with genome-wide DNA methylation; the high-arsenic group had 3% points higher genome-wide full methylation (>80% methylation) than the lower-arsenic group. Differentially methylated regions that were hyper-methylated in the high-arsenic group showed enrichment for immune-related gene ontologies that constitute the basic functions of CD4-positive T cells, such as isotype switching and lymphocyte activation and differentiation. In conclusion, chronic arsenic exposure from drinking water was related to changes in the transcriptome and methylome of CD4-positive T cells, both genome wide and in specific genes, supporting the hypothesis that arsenic causes immunotoxicity by interfering with gene expression and regulation.

  15. Rhabdomyolysis in a Sickle Cell Trait Positive Active Duty Male Soldier.

    Science.gov (United States)

    Saxena, Pulkit; Chavarria, Christopher; Thurlow, John

    2016-01-01

    Exertional rhabdomyolysis is a complication of sickle cell trait (SCT) likely first reported in the military population over 40 years ago. Although commonly a benign condition, numerous studies and case reports have identified SCT positive patients to be at increased risk for rhabdomyolysis, compartment syndrome and sudden cardiac death. We report a recent case of an SCT positive African American active duty male Soldier who suffered exertional rhabdomyolysis following an Army Physical Fitness Test. His course was complicated by acute renal failure requiring hemodialysis, and he eventually recovered renal function. The diagnosis was significantly delayed despite a typical clinical presentation and available SCT screening results. The case highlights the importance of the recognition of SCT as a risk factor for severe rhabdomyolysis, and suggests more must be done for an effective SCT screening program for the active duty military population.

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

    Directory of Open Access Journals (Sweden)

    Sara V. Bates

    2013-10-01

    Full Text Available Hemorrhagic pustules with a “blueberry muffin” appearance accompanied by respiratory failure in a neonate present a challenging differential diagnosis that includes infections and neoplasms. We present a case of multiorgan, multisite Langerhans cell histiocytosis (LCH, positive for the oncogenic BRAF V600E mutation, in a preterm neonate. Infants with LCH pose a diagnostic challenge due to their heterogeneous presentations. This case is unusual in that the newborn presented with severe multiorgan involvement. Due to the rare incidence, wide spectrum of clinical manifestations, and high mortality rate, clinicians must maintain a high index of suspicion for LCH.

  17. Positioning of the NOR-bearing chromosomes in relation to nucleoli in daughter cells after mitosis

    Czech Academy of Sciences Publication Activity Database

    Kalmárová, Markéta; Smirnov, Evgeny; Kováčik, Lubomír; Popov, Alexey; Raška, Ivan

    2008-01-01

    Roč. 57, č. 3 (2008), s. 421-425 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA304/06/1691 Grant - others:GA ČR(CZ) GA304/06/1662; GA MŠk(CZ) LC535; Wellcome Trust(XE) 075834/04/Z Program:LC Institutional research plan: CEZ:AV0Z50110509 Keywords : chromosome positioning * NORs * daughter cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.653, year: 2008

  18. False positive indium-111 white blood cell scan in a closed clavicle fracture

    International Nuclear Information System (INIS)

    Friedman, R.J.; Gordon, L.

    1988-01-01

    Aggressive treatment of the multiply injured patient often requires early fixation of many fractures, some of which may be open. Often, patients develop postoperative fevers requiring a thorough workup to rule out infection. Recently, indium-111 white blood cell (WBC) imaging has become a valuable adjunct in the diagnosis of acute infection. The patient described had a simple, closed clavicle fracture with markedly increased activity on an indium-111 WBC scan obtained for fever workup. This subsequently proved to be a normal, healing, noninfected fracture by other diagnostic techniques. Noninfected, simple closed fractures should be added to the list of causes for a false-positive indium-111 WBC scan

  19. First-line treatment of advanced ALK-positive non-small-cell lung cancer

    Directory of Open Access Journals (Sweden)

    Gandhi S

    2015-09-01

    Full Text Available Shipra Gandhi,1 Hongbin Chen,2 Yujie Zhao,2 Grace K Dy2 1Department of Internal Medicine, State University of New York, 2Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY, USA Abstract: Non-small-cell lung cancer (NSCLC is one of the leading causes of cancer deaths, both within the US and worldwide. There have been major treatment advances in NSCLC over the past decade with the discovery of molecular drivers of NSCLC, which has ushered in an era of personalized medicine. There are several actionable genetic aberrations in NSCLC, such as epidermal growth factor receptor and anaplastic lymphoma kinase (ALK. In 3%–7% of NSCLC, a chromosomal inversion event in chromosome 2 leads to fusion of a portion of the ALK gene with the echinoderm microtubule–associated protein-like 4 (EML4 gene. The constitutive activation of the ALK fusion oncogene renders it vulnerable to therapeutic intervention. This review focuses on the first-line treatment of advanced ALK-positive NSCLC using ALK inhibitors. Crizotinib was the first agent proven to be efficacious as first-line treatment for ALK-positive NSCLC. However, acquired resistance inevitably develops. The central nervous system is a sanctuary site that represents a common site for disease progression as well. Hence, more potent, selective next-generation ALK inhibitors that are able to cross the blood–brain barrier have been developed for treatment against crizotinib-resistant ALK-positive NSCLC and are also currently being evaluated for first-line therapy as well. In this review, we provide summary of the clinical experience with these drugs in the treatment of ALK-positive NSCLC. Keywords: non-small-cell lung cancer, ALK, first line, crizotinib, pemetrexed

  20. Human immunodeficiency virus-positive secondary syphilis mimicking cutaneous T-cell lymphoma.

    Science.gov (United States)

    Yamashita, Michiko; Fujii, Yoshiyuki; Ozaki, Keiji; Urano, Yoshio; Iwasa, Masami; Nakamura, Shingen; Fujii, Shiro; Abe, Masahiro; Sato, Yasuharu; Yoshino, Tadashi

    2015-10-08

    Malignant syphilis or lues maligna is a severe form of secondary syphilis that was commonly reported in the pre-antibiotic era, and has now reemerged with the advent of the human immunodeficiency virus (HIV) epidemic. However, the characteristic histopathological findings of malignant syphilis remain controversial. The aim of this case report was to clarify the clinical and histopathological findings of HIV-positive malignant secondary syphilis. A Japanese man in his forties complained of fever, skin lesions, headache, and myalgia without lymphadenopathy during the previous 4 weeks. The skin lesions manifested as erythematous, nonhealing, ulcerated papules scattered on his trunk, extremities, palm, and face. Although the skin lesions were suspected to be cutaneous T-cell lymphomas on histological analyses, they lacked T-cell receptor Jγ rearrangement; moreover, immunohistochemical analyses confirmed the presence of spirochetes. The patient was administered antibiotics and anti-retroviral therapy, which dramatically improved the symptoms. On the basis of these observations of the skin lesions, we finally diagnosed the patient with HIV-associated secondary syphilis that mimicked cutaneous T-cell lymphoma. The patient's systemic CD4+ lymphocyte count was very low, and the infiltrate was almost exclusively composed of CD8+ atypical lymphocytes; therefore, the condition was easily misdiagnosed as cutaneous lymphoma. Although the abundance of plasma cells is a good indicator of malignant syphilis on skin histological analyses, in some cases, the plasma cell count may be very low. Therefore, a diagnosis of malignant secondary syphilis should be considered before making a diagnosis of primary cutaneous peripheral T-cell lymphoma or lymphoma associated with HIV infection.

  1. The Transcription Factors EBF1 and EBF2 Are Positive Regulators of Myelination in Schwann Cells.

    Science.gov (United States)

    Moruzzo, Diego; Nobbio, Lucilla; Sterlini, Bruno; Consalez, G Giacomo; Benfenati, Fabio; Schenone, Angelo; Corradi, Anna

    2017-12-01

    Myelin formation by Schwann cells is tightly controlled by multiple pathways and regulatory molecules. The Ebf2 gene, belonging to the Ebf family of transcription factors regulating cell development and differentiation, is expressed in Schwann cells, and Ebf2 knockout mice show peripheral nerve defects. We also found that Ebf1 is expressed in Schwann cells. To investigate Ebf function in myelination, we silenced Ebf genes in myelinating dorsal root ganglia cultures. Combined downregulation of Ebf genes leads to a severe impairment of myelin formation that is completely rescued by their specific overexpression, suggesting that the expression level of Ebf genes strongly influences axon myelination. In addition, by profiling Ebf target genes, we found several transcripts belonging to pathways actively involved in peripheral myelination, including Gliomedin, a gene with a role in the formation of the nodes of Ranvier and recently implicated in the pathogenesis of the nodo-paranodopathies. Our results suggest that Ebf genes act as positive regulators of myelination and directly regulate the promoter of Gliomedin.

  2. A positive feedback pathway of estrogen biosynthesis in breast cancer cells is contained by resveratrol

    International Nuclear Information System (INIS)

    Wang Yun; Ye Lan; Leung, Lai K.

    2008-01-01

    Cytochrome P450 (CYP) 19 enzyme or aromatase catalyses the rate-determining step of estrogen synthesis. The transcriptional control of CYP19 gene is highly specific in different cell types, for instance, Promoter I.3/II is commonly used for regulation in breast cancer cells. Recently, a positive feedback pathway for estrogen synthesis has been identified in ERα expressing SK-BR-3 cells. CYP19 mRNA abundance and activity are increased in this pathway and the promoter usage is switched from Promoter I.3/II to I.1 through a non-genomic process. In the present study, effect of the phytocompound resveratrol on this Promoter I.1-controlled expression of aromatase was investigated. Results indicated that resveratrol reduced the estradiol-induced mRNA abundance in SK-BR-3 cells expressing ERα. Luciferase reporter gene assays revealed that resveratrol could also repress the transcriptional control dictated by Promoter I.1. Since the ERE-driven luciferase activity was not repressed by resveratrol, the nuclear events of estrogen were unlikely to be suppressed by resveratrol. Instead the phytochemical reduced the amount of ERK activated by estradiol, which could be the pathway responsible for Promoter I.1 transactivation and the induced CYP19 expression. The present study illustrated that resveratrol impeded the non-genomic induction of estrogen on CYP19

  3. Regulation of the Hippocampal Network by VGLUT3-Positive CCK- GABAergic Basket Cells

    Directory of Open Access Journals (Sweden)

    Caroline Fasano

    2017-05-01

    Full Text Available Hippocampal interneurons release the inhibitory transmitter GABA to regulate excitation, rhythm generation and synaptic plasticity. A subpopulation of GABAergic basket cells co-expresses the GABA/glycine vesicular transporters (VIAAT and the atypical type III vesicular glutamate transporter (VGLUT3; therefore, these cells have the ability to signal with both GABA and glutamate. GABAergic transmission by basket cells has been extensively characterized but nothing is known about the functional implications of VGLUT3-dependent glutamate released by these cells. Here, using VGLUT3-null mice we observed that the loss of VGLUT3 results in a metaplastic shift in synaptic plasticity at Shaeffer’s collaterals – CA1 synapses and an altered theta oscillation. These changes were paralleled by the loss of a VGLUT3-dependent inhibition of GABAergic current in CA1 pyramidal layer. Therefore presynaptic type III metabotropic could be activated by glutamate released from VGLUT3-positive interneurons. This putative presynaptic heterologous feedback mechanism inhibits local GABAergic tone and regulates the hippocampal neuronal network.

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

    Science.gov (United States)

    Scimeca, Manuel; Bonanno, Elena; Piccirilli, Eleonora; Baldi, Jacopo; Mauriello, Alessandro; Orlandi, Augusto; Tancredi, Virginia; Gasbarra, Elena; Tarantino, Umberto

    2015-01-01

    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. PMID:26101529

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Role of neuropeptide Y (NPY) in the differentiation of Trpm-5-positive olfactory microvillar cells.

    Science.gov (United States)

    Doyle, Kharen L; Cunha, Carla; Hort, Yvonne; Tasan, Ramon; Sperk, Günther; Shine, John; Herzog, Herbert

    2018-04-01

    The mouse olfactory neuroepithelium (ON) is comprised of anatomically distinct populations of cells in separate regions; apical (sustentacular and microvillar), neuronal (olfactory sensory neurons) and basal (horizontal and globose basal cells). The existence of microvillar cells (MVCs) is well documented but their nature and function remains unclear. An important transcription factor for the differentiation of MVCs is Skn-1a, with loss of function of Skn-1a in mice resulting in a complete loss of Trpm-5 expressing MVCs, while olfactory sensory neuron differentiation is normal. Our previous research has shown that neuropeptide Y (NPY) is expressed in MVCs and is important in the neuroproliferation of olfactory precursors. This study showed that following X-ray irradiation of the snout of wildtype mice, which decreases the proliferation of basal precursor cells, the numbers of Trpm-5-positive MVCs is increased at 2 and 5 weeks post-irradiation compared to controls. Skn-1a expression in the ON following X-ray irradiation also increases at 2 weeks post-irradiation in a regionally specific manner matching the expression pattern of Trpm-5-positive MVCs. In parallel, NPYCre knock-in mice were used to examine the expression of Skn-1a following activation of NPY unilaterally in the ON (unilateral nasal irrigation of AAV-NPY-FLEX). These experiments demonstrated that Skn-1a is only expressed when NPY is activated in MVCs. Therefore the expression of NPY is necessary for the transcription factor-mediated differentiation of olfactory MVCs. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Localization of preferential sites of rearrangement within the BCR gene in Philadelphia chromosome-positive acute lymphoblastic leukemia

    International Nuclear Information System (INIS)

    Denny, C.T.; Shah, N.P.; Ogden, S.; Willman, C.; McConnell, T.; Crist, W.; Carroll, A.; Witte, O.N.

    1989-01-01

    The Philadelphia chromosome associated with acute lymphoblastic leukemia (ALL) has been linked to a hybrid BCR/ABL protein product that differs from that found in chronic myelogenous leukemia. This implies that the molecular structures of the two chromosomal translocations also differ. Localization of translocation breakpoints in Philadelphia chromosome-positive ALL has been impeded due to the only partial characterization of the BCR locus. The authors have isolated the entire 130-kilobase BCR genomic locus from a human cosmid library. They have demonstrated that these breakpoints are all located at the 3' end of the intron around an unusual restriction fragment length polymorphism caused by deletion of a 1-kilobase fragment containing Alu family reiterated sequences. This clustering is unexpected in light of previous theories of rearrangement in Philadelphia chromosome-positive chronic myelogenous leukemia that would have predicted a random dispersion of breakpoints in the first intron in Philadelphia chromosome-positive ALL. The proximity of the translocation breakpoints to this constitutive deletion may indicate shared mechanisms of rearrangement or that such polymorphisms mark areas of the genome prone to recombination

  8. 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; Isa, Adiba; Jeffery, 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...

  9. Detection of (Leu-7)-positive cells with NK activity in human gingival tissues from patients with periodontitis

    International Nuclear Information System (INIS)

    Komiyama, K.; Hirsch, H.Z.; Mestecky, J.; Moro, I.

    1986-01-01

    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 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

  10. 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.

  11. 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.

  12. Ascorbic Acid Kills Epstein-Barr Virus (EBV) Positive Burkitt Lymphoma Cells and EBV Transformed B-Cells in Vitro, but not in Vivo

    Science.gov (United States)

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

    2014-01-01

    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 ascorbic acid-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. PMID:23067008

  13. 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.

  14. The human trigeminal ganglion: c-kit positive neurons and interstitial cells.

    Science.gov (United States)

    Rusu, M C; Pop, F; Hostiuc, S; Dermengiu, D; Lală, A I; Ion, D A; Mănoiu, V S; Mirancea, N

    2011-10-20

    The presence of c-kit positive neurons in sensory ganglia has been verified in various species but not in humans. Our aim has been to identify whether human primary trigeminal neurons label with c-kit/CD117 and thus, whether data gathered in animal studies can be extrapolated to humans. We also intended to establish whether, and which non-neuronal cells also label with c-kit in the trigeminal ganglion. Human adult trigeminal ganglia from eight cadavers were processed for immunohistochemistry on paraffin embedded samples using monoclonal antibodies for CD117/c-kit, and three additional trigeminal ganglia were used for transmission electron microscopy (TEM). To evaluate which neuronal type (A or B) was labeled with c-kit, we evaluated the same neurons on adjacent sections labeled with antibodies for neurofilaments (NF). c-kit has labeled trigeminal neurons (TNs), mast cells and interstitial cells (ICs) within the trigeminal ganglion. c-kit+TNs were NF-and thus were strongly presumed to be nociceptive, as such neurons are known to be NF-poor. c-kit+ICs with long and moniliform processes intermingled with the satellite glial cells (SGCs) of the neuronal envelopes. TEM evaluations confirmed this mixed composition of the neuronal envelopes and demonstrated that the perineuronal ICs are in fact interstitial Cajal-like cells (ICLCs) and/or telocytes. c-kit+TNs were objectified in humans and strongly presumed to be nociceptive. TNs envelopes mostly consist of SGCs, but are also combined with ICLCs/telocytes. Copyright © 2011 Elsevier GmbH. All rights reserved.

  15. Degenerative disc disease of herniated intervertebral discs is associated with extracellular matrix remodeling, vimentin-positive cells and cell death.

    Science.gov (United States)

    Loreto, Carla; Musumeci, Giuseppe; Castorina, Alessandro; Loreto, Corrado; Martinez, Giuseppa

    2011-03-01

    annulus fibrosus and nucleus pulposus cells. In conclusion, as demonstrated by the vimentin-positive cells, the injured IVD has endogenous resources that can stem the DDD damage, including substitution of damaged elastic fibers by oxytalan fibers. In addition, induction of apoptosis suggests an increased cell turnover in response to repair needs. Copyright © 2011 Elsevier GmbH. All rights reserved.

  16. PIK3CA, HRAS and PTEN in human papillomavirus positive oropharyngeal squamous cell carcinoma

    International Nuclear Information System (INIS)

    Chiosea, Simion I; Nikiforova, Marina N; Grandis, Jennifer R; Lui, Vivian W Y; Diergaarde, Brenda; Maxwell, Jessica H; Ferris, Robert L; Kim, Seungwon W; Luvison, Alyssa; Miller, Megan

    2013-01-01

    Recent genomic evidence suggests frequent phosphatidylinositide 3-kinase (PI3K) pathway activation in human papillomavirus (HPV) positive oropharyngeal squamous cell carcinoma. Mutations/amplification of the gene encoding p110α catalytic subunit of phosphoinositide 3-kinase (PIK3CA), loss of phosphatase and tensin homolog (PTEN) and HRAS mutations are known to activate PI3K pathway. PIK3CA mutations were identified by Sanger sequencing in 23 of 75 (31%) HPV-positive oropharyngeal carcinomas, including exon 9 (p.E545K [n = 10] and p.E542K [n = 5]) or exon 20 (p.H1047Y, n = 2) mutations. Five rare and one novel (p.R537Q) PIK3CA mutations were identified. HRAS mutation (p.Q61L) was detected in 1 of 62 tested cases. PIK3CA amplification by fluorescence in situ hybridization (FISH) was identified in 4 cases (4/21, 20%), while PTEN loss was seen in 7 (7/21, 33%) cases (chromosome 10 monosomy [n = 4], homozygous deletion [n = 3]). Overall, genetic alterations that likely lead to PI3K pathway activation were identified in 34 of 75 cases (45%) and did not correlate with disease specific survival. These findings offer a molecular rationale for therapeutic targeting of PI3K pathway in patients with HPV-positive oropharyngeal carcinoma

  17. 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.

  18. Significance of CD133 positive cells in four novel HPV-16 positive cervical cancer-derived cell lines and biopsies of invasive cervical cancer.

    Science.gov (United States)

    Javed, Shifa; Sharma, Bal Krishan; Sood, Swati; Sharma, Sanjeev; Bagga, Rashmi; Bhattacharyya, Shalmoli; Rayat, Charan Singh; Dhaliwal, Lakhbir; Srinivasan, Radhika

    2018-04-02

    Cervical cancer is a major cause of cancer-related mortality in women in the developing world. Cancer Stem cells (CSC) have been implicated in treatment resistance and metastases development; hence understanding their significance is important. Primary culture from tissue biopsies of invasive cervical cancer and serial passaging was performed for establishing cell lines. Variable Number Tandem Repeat (VNTR) assay was performed for comparison of cell lines with their parental tissue. Tumorsphere and Aldefluor assays enabled isolation of cancer stem cells (CSC); immunofluorescence and flow cytometry were performed for their surface phenotypic expression in cell lines and in 28 tissue samples. Quantitative real-time PCR for stemness and epithelial-mesenchymal transition (EMT) markers, MTT cytotoxicity assay, cell cycle analysis and cell kinetic studies were performed. Four low-passage novel cell lines designated RSBS-9, - 14 and - 23 from squamous cell carcinoma and RSBS-43 from adenocarcinoma of the uterine cervix were established. All were HPV16+. VNTR assay confirmed their uniqueness and derivation from respective parental tissue. CSC isolated from these cell lines showed CD133 + phenotype. In tissue samples of untreated invasive cervical cancer, CD133 + CSCs ranged from 1.3-23% of the total population which increased 2.8-fold in radiation-resistant cases. Comparison of CD133 + with CD133 - bulk population cells revealed increased tumorsphere formation and upregulation of stemness and epithelial-mesenchymal transition (EMT) markers with no significant difference in cisplatin sensitivity. Low-passage cell lines developed would serve as models for studying tumor biology. Cancer Stem Cells in cervical cancer display CD133 + phenotype and are increased in relapsed cases and hence should be targeted for achieving remission.

  19. 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.

  20. Microchannel-connected SU-8 honeycombs by single-step projection photolithography for positioning cells on silicon oxide nanopillar arrays

    International Nuclear Information System (INIS)

    Larramendy, Florian; Paul, Oliver; Blatche, Marie Charline; Mazenq, Laurent; Laborde, Adrian; Temple-Boyer, Pierre

    2015-01-01

    We report on the fabrication, functionalization and testing of SU-8 microstructures for cell culture and positioning over large areas. The microstructure consists of a honeycomb arrangement of cell containers interconnected by microchannels and centered on nanopillar arrays designed for promoting cell positioning. The containers have been dimensioned to trap single cells and, with a height of 50 µm, prevent cells from escaping. The structures are fabricated using a single ultraviolet photolithography exposure with focus depth in the lower part of the SU-8 resist. With optimized process parameters, microchannels of various aspect ratios are thus produced. The cell containers and microchannels serve for the organization of axonal growth between neurons. The roughly 2 µm-high and 500 nm-wide nanopillars are made of silicon oxide structured by deep reactive ion etching. In future work, beyond their cell positioning purpose, the nanopillars could be functionalized as sensors. The proof of concept of the novel microstructure for organized cell culture is given by the successful growth of interconnected PC12 cells. Promoted by the honeycomb geometry, a dense network of interconnections between the cells has formed and the intended intimate contact of cells with the nanopillar arrays was observed by scanning electron microscopy. This proves the potential of these new devices as tools for the controlled cell growth in an interconnected container system with well-defined 3D geometry. (paper)

  1. Cytotoxic T Cells in PD-L1-Positive Malignant Pleural Mesotheliomas Are Counterbalanced by Distinct Immunosuppressive Factors.

    Science.gov (United States)

    Awad, Mark M; Jones, Robert E; Liu, Hongye; Lizotte, Patrick H; Ivanova, Elena V; Kulkarni, Meghana; Herter-Sprie, Grit S; Liao, Xiaoyun; Santos, Abigail A; Bittinger, Mark A; Keogh, Lauren; Koyama, Shohei; Almonte, Christina; English, Jessie M; Barlow, Julianne; Richards, William G; Barbie, David A; Bass, Adam J; Rodig, Scott J; Hodi, F Stephen; Wucherpfennig, Kai W; Jänne, Pasi A; Sholl, Lynette M; Hammerman, Peter S; Wong, Kwok-Kin; Bueno, Raphael

    2016-12-01

    PD-L1 immunohistochemical staining does not always predict whether a cancer will respond to treatment with PD-1 inhibitors. We sought to characterize immune cell infiltrates and the expression of T-cell inhibitor markers in PD-L1-positive and PD-L1-negative malignant pleural mesothelioma samples. We developed a method for immune cell phenotyping using flow cytometry on solid tumors that have been dissociated into single-cell suspensions and applied this technique to analyze 43 resected malignant pleural mesothelioma specimens. Compared with PD-L1-negative tumors, PD-L1-positive tumors had significantly more infiltrating CD45 + immune cells, a significantly higher proportion of infiltrating CD3 + T cells, and a significantly higher percentage of CD3 + cells displaying the activated HLA-DR + /CD38 + phenotype. PD-L1-positive tumors also had a significantly higher proportion of proliferating CD8 + T cells, a higher fraction of FOXP3 + /CD4 + Tregs, and increased expression of PD-1 and TIM-3 on CD4 + and CD8 + T cells. Double-positive PD-1 + /TIM-3 + CD8 + T cells were more commonly found on PD-L1-positive tumors. Compared with epithelioid tumors, sarcomatoid and biphasic mesothelioma samples were significantly more likely to be PD-L1 positive and showed more infiltration with CD3 + T cells and PD-1 + /TIM-3 + CD8 + T cells. Immunologic phenotypes in mesothelioma differ based on PD-L1 status and histologic subtype. Successful incorporation of comprehensive immune profiling by flow cytometry into prospective clinical trials could refine our ability to predict which patients will respond to specific immune checkpoint blockade strategies. Cancer Immunol Res; 4(12); 1038-48. ©2016 AACR. ©2016 American Association for Cancer Research.

  2. The inhibition of PARP but not EGFR results in the radiosensitization of HPV/p16-positive HNSCC cell lines

    International Nuclear Information System (INIS)

    Güster, Julian David; Weissleder, Stephanie Valerie; Busch, Chia-Jung; Kriegs, Malte; Petersen, Cordula; Knecht, Rainald; Dikomey, Ekkehard; Rieckmann, Thorsten

    2014-01-01

    Background and purpose: HPV-negative and HPV-positive HNSCC comprise distinct tumor entities with different biological characteristics. Specific regimens for the comparably well curable HPV-positive entity that reduce side effects without compromising outcome have yet to be established. Therefore, we tested here whether the inhibition of EGFR or PARP may be used to specifically enhance the radiosensitivity of HPV-positive HNSCC cells. Materials and methods: Experiments were performed with five HPV/p16-positive HNSCC cell lines. Inhibitors used were cetuximab, olaparib and PF-00477736. The respective inhibition of EGFR, PARP and Chk1 was evaluated by Western blot, immunofluorescence analysis and assessment of cell cycle distribution. Cell survival was assessed by colony formation assay. Results: Inhibition of EGFR by cetuximab failed to radiosensitize any of the HPV-positive HNSCC cell lines tested. In contrast, PARP-inhibition resulted in a substantial radiosensitization of all strains, with the sensitization being further enhanced by the additional inhibition of Chk1. Conclusions: PARP-inhibition effectively radiosensitizes HPV-positive HNSCC cells and may therefore represent a viable alternative to chemotherapy possibly even allowing for a reduction in radiation dose. For the latter, PARP-inhibition may be combined with the inhibition of Chk1. In contrast, the inhibition of EGFR cannot be expected to radiosensitize HPV-positive HNSCC through the modulation of cellular radiosensitivity

  3. Mitotic Spindle Positioning in the EMS Cell of Caenorhabditis elegans Requires LET-99 and LIN-5/NuMA.

    Science.gov (United States)

    Liro, Małgorzata J; Rose, Lesilee S

    2016-11-01

    Asymmetric divisions produce daughter cells with different fates, and thus are critical for animal development. During asymmetric divisions, the mitotic spindle must be positioned on a polarized axis to ensure the differential segregation of cell fate determinants into the daughter cells. In many cell types, a cortically localized complex consisting of Gα, GPR-1/2, and LIN-5 (Gαi/Pins/Mud, Gαi/LGN/NuMA) mediates the recruitment of dynactin/dynein, which exerts pulling forces on astral microtubules to physically position the spindle. The conserved PAR polarity proteins are known to regulate both cytoplasmic asymmetry and spindle positioning in many cases. However, spindle positioning also occurs in response to cell signaling cues that appear to be PAR-independent. In the four-cell Caenorhabditis elegans embryo, Wnt and Mes-1/Src-1 signaling pathways act partially redundantly to align the spindle on the anterior/posterior axis of the endomesodermal (EMS) precursor cell. It is unclear how those extrinsic signals individually contribute to spindle positioning and whether either pathway acts via conserved spindle positioning regulators. Here, we genetically test the involvement of Gα, LIN-5, and their negative regulator LET-99, in transducing EMS spindle positioning polarity cues. We also examined whether the C. elegans ortholog of another spindle positioning regulator, DLG-1, is required. We show that LET-99 acts in the Mes-1/Src-1 pathway for spindle positioning. LIN-5 is also required for EMS spindle positioning, possibly through a Gα- and DLG-1-independent mechanism. Copyright © 2016 by the Genetics Society of America.

  4. Cytomorphological features of ALK-positive lung adenocarcinomas: psammoma bodies and signet ring cells.

    Science.gov (United States)

    Pareja, Fresia; Crapanzano, John P; Mansukhani, Mahesh M; Bulman, William A; Saqi, Anjali

    2015-03-01

    Correlation between histology and genotype has been described in lung adenocarcinomas. For example, studies have demonstrated that adenocarcinomas with an anaplastic lymphoma kinase (ALK) gene rearrangement may have mucinous features. The objective of the current study was to determine whether a similar association can be identified in cytological specimens. A retrospective search for ALK-rearranged cytopathology (CP) and surgical pathology (SP) lung carcinomas was conducted. Additional ALK-negative (-) lung adenocarcinomas served as controls. For CP and SP cases, the clinical data (i.e., age, sex, and smoking history), architecture, nuclear features, presence of mucin-containing cells (including signet ring cells), and any additional salient characteristics were evaluated. The search yielded 20 ALK-positive (+) adenocarcinomas. Compared with patients with ALK(-) lung adenocarcinomas (33 patients; 12 with epidermal growth factor receptor [EGFR]-mutation, 11 with Kristen rat sarcoma [KRAS]-mutation, and 10 wild-type adenocarcinomas), patients with ALK(+) adenocarcinoma presented at a younger age; and there was no correlation noted with sex or smoking status. The most common histological pattern in SP was papillary/micropapillary. Mucinous features were associated with ALK rearrangement in SP specimens. Signet ring cells and psammoma bodies were evident in and significantly associated with ALK(+) SP and CP specimens. However, psammoma bodies were observed in rare adenocarcinomas with an EGFR mutation. Both the ALK(+) and ALK(-) groups had mostly high nuclear grade. Salient features, including signet ring cells and psammoma bodies, were found to be significantly associated with ALK(+) lung adenocarcinomas and are identifiable on CP specimens. Recognizing these may be especially helpful in the molecular triage of scant CP samples. © 2014 American Cancer Society.

  5. 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.

  6. Acacetin and Chrysin, Two Polyphenolic Compounds, Alleviate Telomeric Position Effect in Human Cells

    Directory of Open Access Journals (Sweden)

    Amina Boussouar

    2013-01-01

    Full Text Available We took advantage of the ability of human telomeres to silence neighboring genes (telomere position effect or TPE to design a high-throughput screening assay for drugs altering telomeres. We identified, for the first time, that two dietary flavones, acacetin and chrysin, are able to specifically alleviate TPE in human cells. We further investigated their influence on telomere integrity and showed that both drugs drastically deprotect telomeres against DNA damage response. However, telomere deprotection triggered by shelterin dysfunction does not affect TPE, indicating that acacetin and chrysin target several functions of telomeres. These results show that TPE-based screening assays represent valuable methods to discover new compounds targeting telomeres.

  7. Energy-positive wastewater treatment and desalination in an integrated microbial desalination cell (MDC)-microbial electrolysis cell (MEC)

    Science.gov (United States)

    Li, Yan; Styczynski, Jordyn; Huang, Yuankai; Xu, Zhiheng; McCutcheon, Jeffrey; Li, Baikun

    2017-07-01

    Simultaneous removal of nitrogen in municipal wastewater, metal in industrial wastewater and saline in seawater was achieved in an integrated microbial desalination cell-microbial electrolysis cell (MDC-MEC) system. Batch tests showed that more than 95.1% of nitrogen was oxidized by nitrification in the cathode of MDC and reduced by heterotrophic denitrification in the anode of MDC within 48 h, leading to the total nitrogen removal rate of 4.07 mg L-1 h-1. Combining of nitrogen removal and desalination in MDC effectively solved the problem of pH fluctuation in anode and cathode, and led to 63.7% of desalination. Power generation of MDC (293.7 mW m-2) was 2.9 times higher than the one without salt solution. The electric power of MDC was harvested by a capacitor circuit to supply metal reduction in a MEC, and 99.5% of lead (II) was removed within 48 h. A kinetic MDC model was developed to elucidate the correlation of voltage output and desalination efficiency. Ratio of wastewater and sea water was calculated for MDC optimal operation. Energy balance of nutrient removal, metal removal and desalination in the MDC-MEC system was positive (0.0267 kW h m-3), demonstrating the promise of utilizing low power output of MDCs.

  8. 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.

  9. Alloimmunization due to red cell antibodies in Rhesus positive Omani Pregnant Women: Maternal and Perinatal outcome

    Directory of Open Access Journals (Sweden)

    Tamima Al-Dughaishi

    2015-01-01

    Full Text Available Objective: This study is aimed to determine the prevalence of alloimmunization due to antibodies to red blood cell (RBC antigens (other than rhesus [Rh] antigen and report the maternal, perinatal, and neonatal outcomes. Materials and Methods: A retrospective review of medical records of all patients with minor RBCs antibodies alloimmunization who were followed and delivered at Sultan Qaboos University Hospital, Oman from June 2011 to June 2013. Maternal characteristics, antibody type, antibody titer in addition to perinatal and neonatal outcomes were reviewed. Results: There were 1160 patients with Rh positive status in the study. The most common ABO blood group was O, followed by A, B, and AB. We found 33 out of 1160 Rh positive women alloimmunized with minor RBCs antibodies that gave a prevalence of minor RBCs alloimmunization of 2.7%. The most frequent antibody was anti-E 38%, followed by anti-c 17% and anti-kell 17%. 6 of these 33 patients were identified to have significant antibody titer, and two cases showed evidence of fetal anemia. Only one case required an intrauterine blood transfusion. The most common neonatal complication was jaundice in 53%, followed by respiratory distress syndrome in 28%. Two cases complicated by neonatal anemia required a postnatal blood transfusion. Conclusion: Alloimmunization with anti-E, anti-c, and anti-kell were the most common antibodies among the study group. Minor RBCs alloimmunization was an important cause of neonatal morbidity.

  10. 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.

  11. Association between red blood cell indices and CD4 count in HIV-positive reproductive women

    Science.gov (United States)

    Lumbanraja, S. N.; Siregar, D. I. S.

    2018-03-01

    Red blood cell indices, hemoglobin, and hematocrit reflect rapidity of HIV disease progression. This study aims to determine red blood cell indices and CD4 count in HIV-positive reproductive women. This study was a cross sectional study conducted at AIDS outpatient clinic at Haji Adam Malik General Hospital, Medan Indonesia. All seropositive reproductive women within antiretroviral therapy consented for blood count and CD4 examination. Data were collected and analyzed with SPSS 19. In subjects with CD4≤350 mm3, mean hemoglobin was 10.95 ± 2.01, hematocrit was 31.83 ± 5.04%, MCV was 84.17 ± 11.41, MCH was 25.98 ± 2.65, and MCHC was 32.18 ± 2.17. Mean hemoglobin, hematocrit, and MCH value was significantly lower in subjects with CD4 ≤350 mm3 (p=0.014; p=0.001; p=0.01; respectively). Lower Hb, Ht, and MCH associated with thelower CD4 count.

  12. A double mechanism for the mesenchymal stem cells' positive effect on pancreatic islets.

    Directory of Open Access Journals (Sweden)

    Arianna Scuteri

    Full Text Available The clinical usability of pancreatic islet transplantation for the treatment of type I diabetes, despite some encouraging results, is currently hampered by the short lifespan of the transplanted tissue. In vivo studies have demonstrated that co-transplantation of Mesenchymal Stem Cells (MSCs with transplanted pancreatic islets is more effective with respect to pancreatic islets alone in ensuring glycemia control in diabetic rats, but the molecular mechanisms of this action are still unclear. The aim of this study was to elucidate the molecular mechanisms of the positive effect of MSCs on pancreatic islet functionality by setting up direct, indirect and mixed co-cultures. MSCs were both able to prolong the survival of pancreatic islets, and to directly differentiate into an "insulin-releasing" phenotype. Two distinct mechanisms mediated these effects: i the survival increase was observed in pancreatic islets indirectly co-cultured with MSCs, probably mediated by the trophic factors released by MSCs; ii MSCs in direct contact with pancreatic islets started to express Pdx1, a pivotal gene of insulin production, and then differentiated into insulin releasing cells. These results demonstrate that MSCs may be useful for potentiating pancreatic islets' functionality and feasibility.

  13. Tropoelastin bridge region positions the cell-interactive C terminus and contributes to elastic fiber assembly.

    Science.gov (United States)

    Yeo, Giselle C; Baldock, Clair; Tuukkanen, Anne; Roessle, Manfred; Dyksterhuis, Leanne B; Wise, Steven G; Matthews, Jacqueline; Mithieux, Suzanne M; Weiss, Anthony S

    2012-02-21

    The tropoelastin monomer undergoes stages of association by coacervation, deposition onto microfibrils, and cross-linking to form elastic fibers. Tropoelastin consists of an elastic N-terminal coil region and a cell-interactive C-terminal foot region linked together by a highly exposed bridge region. The bridge region is conveniently positioned to modulate elastic fiber assembly through association by coacervation and its proximity to dominant cross-linking domains. Tropoelastin constructs that either modify or remove the entire bridge and downstream regions were assessed for elastogenesis. These constructs focused on a single alanine substitution (R515A) and a truncation (M155n) at the highly conserved arginine 515 site that borders the bridge. Each form displayed less efficient coacervation, impaired hydrogel formation, and decreased dermal fibroblast attachment compared to wild-type tropoelastin. The R515A mutant protein additionally showed reduced elastic fiber formation upon addition to human retinal pigmented epithelium cells and dermal fibroblasts. The small-angle X-ray scattering nanostructure of the R515A mutant protein revealed greater conformational flexibility around the bridge and C-terminal regions. This increased flexibility of the R515A mutant suggests that the tropoelastin R515 residue stabilizes the structure of the bridge region, which is critical for elastic fiber assembly.

  14. Positive-unlabeled learning for the prediction of conformational B-cell epitopes

    Science.gov (United States)

    2015-01-01

    Background The incomplete ground truth of training data of B-cell epitopes is a demanding issue in computational epitope prediction. The challenge is that only a small fraction of the surface residues of an antigen are confirmed as antigenic residues (positive training data); the remaining residues are unlabeled. As some of these uncertain residues can possibly be grouped to form novel but currently unknown epitopes, it is misguided to unanimously classify all the unlabeled residues as negative training data following the traditional supervised learning scheme. Results We propose a positive-unlabeled learning algorithm to address this problem. The key idea is to distinguish between epitope-likely residues and reliable negative residues in unlabeled data. The method has two steps: (1) identify reliable negative residues using a weighted SVM with a high recall; and (2) construct a classification model on the positive residues and the reliable negative residues. Complex-based 10-fold cross-validation was conducted to show that this method outperforms those commonly used predictors DiscoTope 2.0, ElliPro and SEPPA 2.0 in every aspect. We conducted four case studies, in which the approach was tested on antigens of West Nile virus, dihydrofolate reductase, beta-lactamase, and two Ebola antigens whose epitopes are currently unknown. All the results were assessed on a newly-established data set of antigen structures not bound by antibodies, instead of on antibody-bound antigen structures. These bound structures may contain unfair binding information such as bound-state B-factors and protrusion index which could exaggerate the epitope prediction performance. Source codes are available on request. PMID:26681157

  15. The MYB23 gene provides a positive feedback loop for cell fate specification in the Arabidopsis root epidermis.

    Science.gov (United States)

    Kang, Yeon Hee; Kirik, Victor; Hulskamp, Martin; Nam, Kyoung Hee; Hagely, Katherine; Lee, Myeong Min; Schiefelbein, John

    2009-04-01

    The specification of cell fates during development requires precise regulatory mechanisms to ensure robust cell type patterns. Theoretical models of pattern formation suggest that a combination of negative and positive feedback mechanisms are necessary for efficient specification of distinct fates in a field of differentiating cells. Here, we examine the role of the R2R3-MYB transcription factor gene, AtMYB23 (MYB23), in the establishment of the root epidermal cell type pattern in Arabidopsis thaliana. MYB23 is closely related to, and is positively regulated by, the WEREWOLF (WER) MYB gene during root epidermis development. Furthermore, MYB23 is able to substitute for the function of WER and to induce its own expression when controlled by WER regulatory sequences. We also show that the MYB23 protein binds to its own promoter, suggesting a MYB23 positive feedback loop. The localization of MYB23 transcripts and MYB23-green fluorescent protein (GFP) fusion protein, as well as the effect of a chimeric MYB23-SRDX repressor construct, links MYB23 function to the developing non-hair cell type. Using mutational analyses, we find that MYB23 is necessary for precise establishment of the root epidermal pattern, particularly under conditions that compromise the cell specification process. These results suggest that MYB23 participates in a positive feedback loop to reinforce cell fate decisions and ensure robust establishment of the cell type pattern in the Arabidopsis root epidermis.

  16. The role of NFκB in spheroid formation of human breast cancer cells cultured on the Random Positioning Machine

    DEFF Research Database (Denmark)

    Kopp, Sascha; Sahana, Jayashree; Islam, Tawhidul

    2018-01-01

    Human MCF-7 breast cancer cells were exposed to a Random Positioning Machine (RPM). After 24 hours (h) the cells grew either adherently within a monolayer (AD) or within multicellular spheroids (MCS). AD and MCS populations were separately harvested, their cellular differences were determined per...

  17. Niemann-Pick disease, type B with TRAP-positive storage cells and secondary sea blue histiocytosis

    Directory of Open Access Journals (Sweden)

    R. Saxena

    2009-09-01

    Full Text Available We present 2 cases of Niemann Pick disease, type B with secondary sea-blue histiocytosis. Strikingly, in both cases the Pick cells were positive for tartrate resistant acid phosphatase, a finding hitherto described only in Gaucher cells. This report highlights the importance of this finding as a potential cytochemical diagnostic pitfall in the diagnosis of Niemann Pick disease.

  18. Palmitate-induced ER stress increases trastuzumab sensitivity in HER2/neu-positive breast cancer cells

    International Nuclear Information System (INIS)

    Baumann, Jan; Wong, Jason; Sun, Yan; Conklin, Douglas S.

    2016-01-01

    HER2/neu-positive breast cancer cells have recently been shown to use a unique Warburg-like metabolism for survival and aggressive behavior. These cells exhibit increased fatty acid synthesis and storage compared to normal breast cells or other tumor cells. Disruption of this synthetic process results in apoptosis. Since the addition of physiological doses of exogenous palmitate induces cell death in HER2/neu-positive breast cancer cells, the pathway is likely operating at its limits in these cells. We have studied the response of HER2/neu-positive breast cancer cells to physiological concentrations of exogenous palmitate to identify lipotoxicity-associated consequences of this physiology. Since epidemiological data show that a diet rich in saturated fatty acids is negatively associated with the development of HER2/neu-positive cancer, this cellular physiology may be relevant to the etiology and treatment of the disease. We sought to identify signaling pathways that are regulated by physiological concentrations of exogenous palmitate specifically in HER2/neu-positive breast cancer cells and gain insights into the molecular mechanism and its relevance to disease prevention and treatment. Transcriptional profiling was performed to assess programs that are regulated in HER2-normal MCF7 and HER2/neu-positive SKBR3 breast cancer cells in response to exogenous palmitate. Computational analyses were used to define and predict functional relationships and identify networks that are differentially regulated in the two cell lines. These predictions were tested using reporter assays, fluorescence-based high content microscopy, flow cytometry and immunoblotting. Physiological effects were confirmed in HER2/neu-positive BT474 and HCC1569 breast cancer cell lines. Exogenous palmitate induces functionally distinct transcriptional programs in HER2/neu-positive breast cancer cells. In the lipogenic HER2/neu-positive SKBR3 cell line, palmitate induces a G2 phase cell cycle delay and

  19. Palmitate-induced ER stress increases trastuzumab sensitivity in HER2/neu-positive breast cancer cells.

    Science.gov (United States)

    Baumann, Jan; Wong, Jason; Sun, Yan; Conklin, Douglas S

    2016-07-27

    HER2/neu-positive breast cancer cells have recently been shown to use a unique Warburg-like metabolism for survival and aggressive behavior. These cells exhibit increased fatty acid synthesis and storage compared to normal breast cells or other tumor cells. Disruption of this synthetic process results in apoptosis. Since the addition of physiological doses of exogenous palmitate induces cell death in HER2/neu-positive breast cancer cells, the pathway is likely operating at its limits in these cells. We have studied the response of HER2/neu-positive breast cancer cells to physiological concentrations of exogenous palmitate to identify lipotoxicity-associated consequences of this physiology. Since epidemiological data show that a diet rich in saturated fatty acids is negatively associated with the development of HER2/neu-positive cancer, this cellular physiology may be relevant to the etiology and treatment of the disease. We sought to identify signaling pathways that are regulated by physiological concentrations of exogenous palmitate specifically in HER2/neu-positive breast cancer cells and gain insights into the molecular mechanism and its relevance to disease prevention and treatment. Transcriptional profiling was performed to assess programs that are regulated in HER2-normal MCF7 and HER2/neu-positive SKBR3 breast cancer cells in response to exogenous palmitate. Computational analyses were used to define and predict functional relationships and identify networks that are differentially regulated in the two cell lines. These predictions were tested using reporter assays, fluorescence-based high content microscopy, flow cytometry and immunoblotting. Physiological effects were confirmed in HER2/neu-positive BT474 and HCC1569 breast cancer cell lines. Exogenous palmitate induces functionally distinct transcriptional programs in HER2/neu-positive breast cancer cells. In the lipogenic HER2/neu-positive SKBR3 cell line, palmitate induces a G2 phase cell cycle delay and

  20. Erythropoietin receptor expression and its relationship with trastuzumab response and resistance in HER2-positive breast cancer cells.

    Science.gov (United States)

    Zhang, Chi; Duan, Xuening; Xu, Ling; Ye, Jingming; Zhao, Jianxin; Liu, Yinhua

    2012-12-01

    Resistance to trastuzumab is a major issue in the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Several potential resistance mechanisms have been investigated, but the results are controversial and no conclusion has been reached. Erythropoietin receptor (EPOR) may function in cell growth, and expressed in various cancer cells. Because the downstream signaling pathways for EPOR and HER2 partially overlapped, we hypothesized that EPOR may play a role in the inhibition effect of trastuzumab and resistance to trastuzumab. Here, we detected the expression of EPOR mRNA and protein in HER2-positive breast cancer cell lines and tissues. EPOR expressed in SKBR3, MDA-MB-453, and UACC-812 cell lines, but not in BT474. Of the 55 HER2-positive cancer tissues, EPOR was positive in 42 samples and highly expressed (H-score ≥ 25) in 24 by immunohistochemistry. The difference between EPOR expression and Ki67 index was significant (P = 0.033), and EPOR expression also positively correlated with higher pathological stage (Spearman correlation coefficient = 0.359; P = 0.007). Exogenous EPO antagonized trastuzumab-induced inhibition of cell proliferation in HER2/EPOR dual-positive breast cancer cells. We then exposed SKBR3 cells to trastuzumab for 4 months to obtain trastuzumab-resistant SKBR3 cell line, which demonstrated higher phosphorylated EPOR level, higher EPO expression and more extracellular secretion than non-resistant parental SKBR3 cells. Downregulation EPOR expression using short hairpin RNA resensitized trastuzumab-resistant cells to this drug, and SKBR3 cells with EPOR downregulation demonstrated attenuated trastuzumab resistance after the same resistance induction. EPOR downregulation plus trastuzumab produced a synergetic action in the inhibition of cell proliferation and invasion in SKBR3 and MDA-MB-453 cell lines. Therefore, EPOR expression may be involved in tumor progression and proliferation in HER2-positive breast cancer

  1. Diethylstilbestrol alters positive and negative selection of T cells in the thymus and modulates T-cell repertoire in the periphery

    International Nuclear Information System (INIS)

    Brown, Nicole; Nagarkatti, Mitzi; Nagarkatti, Prakash S.

    2006-01-01

    Prenatal exposure to diethylstilbestrol (DES) is known to cause altered immune functions and increased susceptibility to autoimmune disease in humans. In the current study, we investigated the effects of DES on T-cell differentiation in the thymus using the HY-TCR transgenic (Tg) mouse model in which the female mice exhibit positive selection of T cells bearing the Tg TCR, while the male mice show negative selection of such T cells. In female HY-TCR-Tg mice, exposure to DES showed more pronounced decrease in thymic cellularity when compared to male mice. Additionally, female mice also showed a significant decrease in the proportion of double-positive (DP) T cells in the thymus and HY-TCR-specific CD8 + T cells in the periphery. Male mice exhibiting negative selection also showed decreased thymic cellularity following DES exposure. Moreover, the male mice showed increased proportion of double-negative (DN) T cells in the thymus and decreased proportion of CD8 + T cells. The density of expression of HY-TCR on CD8 + cells was increased following DES exposure in both females and males. Finally, the proliferative response of thymocytes to mitogens and peripheral lymph node T cells to male H-Y antigen was significantly altered in female and male mice following DES treatment. Taken together, these data suggest that DES alters T-cell differentiation in the thymus by interfering with positive and negative selection processes, which in turn modulates the T-cell repertoire in the periphery

  2. Tracheal ulcer due to Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly.

    Science.gov (United States)

    Ito, Takeo; Fujisaki, Hideaki; Nishio, Suehiro; Hiroshige, Shigeo; Miyazaki, Eishi; Kadota, Jun-ichi

    2014-03-01

    A 74-year-old man was referred to our hospital because of a tracheal stenosis circumscribed with soft tissue density and a left pulmonary nodule. Open biopsy of a right submandibular lymph node revealed diffuse large B-cell lymphoma, and the malignant cells were positive for Epstein-Barr virus gene products. Bronchofiberscopy revealed a tracheal necrotizing ulcer. After chemotherapy, the tracheal ulcer resolved. To our knowledge, this is the first report of a case of Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly with a tracheal ulcer. © 2013 Published by The Japanese Respiratory Society on behalf of The Japanese Respiratory Society.

  3. Human Aortic Endothelial Cell Labeling with Positive Contrast Gadolinium Oxide Nanoparticles for Cellular Magnetic Resonance Imaging at 7 Tesla

    Directory of Open Access Journals (Sweden)

    Yasir Loai

    2012-03-01

    Full Text Available Positive T1 contrast using gadolinium (Gd contrast agents can potentially improve detection of labeled cells on magnetic resonance imaging (MRI. Recently, gadolinium oxide (Gd2O3 nanoparticles have shown promise as a sensitive T1 agent for cell labeling at clinical field strengths compared to conventional Gd chelates. The objective of this study was to investigate Gado CELLTrack, a commercially available Gd2O3 nanoparticle, for cell labeling and MRI at 7 T. Relaxivity measurements yielded r1 = 4.7 s−1 mM−1 and r2/r1 = 6.2. Human aortic endothelial cells were labeled with Gd2O3 at various concentrations and underwent MRI from 1 to 7 days postlabeling. The magnetic resonance relaxation times T1 and T2 of labeled cell pellets were measured. Cellular contrast agent uptake was quantified by inductively coupled plasma–atomic emission spectroscopy, which showed very high uptake compared to conventional Gd compounds. MRI demonstrated significant positive T1 contrast and stable labeling on cells. Enhancement was optimal at low Gd concentrations, attained in the 0.02 to 0.1 mM incubation concentration range (corresponding cell uptake was 7.26 to 34.1 pg Gd/cell. Cell viability and proliferation were unaffected at the concentrations tested and up to at least 3 days postlabeling. Gd2O3 is a promising sensitive and stable positive contrast agent for cellular MRI at 7 T.

  4. Spontaneous Remission of Epstein-Barr Virus-Positive Diffuse Large B-Cell Lymphoma of the Elderly

    Directory of Open Access Journals (Sweden)

    T. Mizuno

    2013-05-01

    Full Text Available A 94-year-old female patient presented with anorexia and left axillar lymphadenopathy on admission. Her past history was angina pectoris at 83 years of age and total gastrectomy due to gastric cancer at 87 years. The family history revealed that her son had had a malignant lymphoma, the histopathological diagnosis of which was diffuse large B-cell lymphoma. A physical examination showed both cervical, axillar, and inguinal lymphadenopathy without tenderness. She had elevated lactate dehydrogenase, ferritin, and soluble interleukin-2 receptor (sIL-2R. Whole-body computed tomography confirmed the cervical, axillary, and inguinal lymphadenopathy. Gallium-68 imaging revealed positive accumulation in these superficial lymph nodes. A right inguinal lymph node biopsy showed features of Epstein-Barr virus-associated lymphoproliferative disorder. Immunohistological studies on this lymph node biopsy showed CD20-positive large cells, CD3-positive small cells, and CD30-partly-positive large cells. In situ hybridization showed Epstein-Barr virus-positive, LMP-partly-positive, and EBNA2-negative cells. She refused chemotherapy as her son had died from hematemesis during chemotherapy. She received intravenous hyperalimentation for 1 month after admission. No palpable lymph nodes were identified by physical examination or computed tomography 3 months after admission, and regression of lactate dehydrogenase, ferritin, and sIL-2R was observed. She recovered from anorexia and was discharged. She died from pneumonia 10 months later after initial symptoms of anorexia. The autopsy showed no superficial lymphadenopathy.

  5. CD146- and CD105-positive phenotypes of retinal ganglion cells. Are these in situ proofs of neuronal regeneration?

    Science.gov (United States)

    Vrapciu, A D; Rusu, M C; Voinea, L M; Corbu, C G

    2014-10-01

    The in vivo identity of stem cells is not yet clear. Numerous studies involve the perivascular niches as providers of stem cells during regenerative processes. CD146, in humans, as well as gicerin, at chicken, play roles in neuronal development and neurites extension. CD146 is a marker of stemness but also a pericytary marker. Stem cells in vascular niches can differentiate in neural cells. By applying CD146 and CD105 antibodies on human retinas from glaucomatous eyes, CD146-positive retinal ganglion cells (RGCs) were found, some being placed in perivascular positions; ongoing processes of neurites extension were related to these neurons. On other hand, RGCs were positively labeled by CD105 antibodies. These results support the hypothesis that in glaucoma eyes the CD146-positive RGCs result from regenerative processes driven by stem cells in the retinal perivascular niches. Further experiments are needed to evaluate whether CR146-positive neurons indicate also a physiological process of maintenance of retina. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Number of serotonin positive cells and acute cellular rejection in the early period after small bowel transplantation in pigs.

    Science.gov (United States)

    Honsova, Eva; Lodererova, Alena; Balaz, Peter; Oliverius, Martin

    2010-03-01

    Small bowel transplantations (SBT) are being increasingly performed to treat patients with irreversible intestinal failure or short-bowel syndrome. Histologic evaluation of small bowel allograft biopsies is important for the diagnosis of acute cellular rejection (ACR). Serotonin (5-hydroxytriptamin) is a biogenous amine of which up to 95% is present in the enterochromaffin cells of the gastrointestinal tract. The aim of our study was to analyze rejection and number of serotonin positive cells in the intestinal graft biopsy samples early after SBT in pigs. 24 pigs were used and divided into 4 groups. Group A, autologous SBT (n = 3) as a control group; group B, allogeneic SBT with tacrolimus monotherapy (n = 7); group C, allogeneic SBT immunosuppressed with tacrolimus and sirolimus (n = 8); and group D, without immunosuppression (n = 6). Observation period was 30 days. Mucosal biopsies were obtained on days 0, 3, 5, 7, 10, 14, 20, 28 after transplantation. ACR was classified according to standardized grading schema on a scale of indeterminate, mild, moderate, and severe. Serotonin positive cells were quantified as the number of positive cells in 20 high power fields. There were no significant differences in the number of serotonin positive cells and different grades of ACR. In our experiment the number of serotonin positive cells was not a sensitive marker of ACR in the early period after small bowel transplantation.

  7. Acid sphingomyelinase activity as an indicator of the cell stress in HPV-positive and HPV-negative head and neck squamous cell carcinoma.

    Science.gov (United States)

    Gerle, Mirko; Medina, Tuula Peñate; Gülses, Aydin; Chu, Hanwen; Naujokat, Hendrik; Wiltfang, Jörg; Açil, Yahya

    2018-03-21

    Human papillomavirus (HPV) infection, especially HPV-16 and HPV-18, has been increasingly associated with head and neck squamous cell carcinoma. The treatment of HPV-positive squamous cell carcinoma has a better response to both radiotherapy and chemotherapy and presents a better prognosis for the patient. Defining the underlying mechanism of the difference might help in developing future treatment options and could be an important factor in personal therapy planning. Endogenously secreted acid sphingomyelinase (ASMase) levels in the cellular stress caused by irradiation and cisplatin were investigated. MTT assay was performed to evaluate the viability of the treated cells. Keratinocytes were used to evaluate the effects of radiation on normal tissues. Irradiation caused a dose-dependent increase in ASMase activity in both SCC9 HPV-negative, and UDSCC2 HPV-positive cells. ASMase activity in UDSCC2 cells was significantly higher than that in SCC9 cells. UDSCC cells were more sensitive to cisplatin treatment than SCC cells, and the dose-response in the activity was observed in long-time treatments when high doses of cisplatin were used. The results of the current study have clearly showed that HPV positivity should be considered as one of the determinative factors which should be considered when tumor treatments are planned. However, further studies are needed to determine the differences in cellular responses and pathways among HPV-negative and HPV-positive cells.

  8. The extent of B-cell activation and dysfunction preceding lymphoma development in HIV-positive people

    DEFF Research Database (Denmark)

    Shepherd, L; Borges, Á H; Harvey, R

    2018-01-01

    OBJECTIVES: B-cell dysfunction and activation are thought to contribute to lymphoma development in HIV-positive people; however, the mechanisms are not well understood. We investigated levels of several markers of B-cell dysfunction [free light chain (FLC)-κ, FLC-λ, immunoglobulin G (IgG), IgA, Ig......M and IgD] prior to lymphoma diagnosis in HIV-positive people. METHODS: A nested matched case-control study was carried out within the EuroSIDA cohort, including 73 HIV-positive people with lymphoma and 143 HIV-positive lymphoma-free controls. Markers of B-cell dysfunction were measured in prospectively...

  9. A subset of high Gleason grade prostate carcinomas contain a large burden of prostate cancer syndecan-1 positive stromal cells.

    Science.gov (United States)

    Sharpe, Benjamin; Alghezi, Dhafer A; Cattermole, Claire; Beresford, Mark; Bowen, Rebecca; Mitchard, John; Chalmers, Andrew D

    2017-05-01

    There is a pressing need to identify prognostic and predictive biomarkers for prostate cancer to aid treatment decisions in both early and advanced disease settings. Syndecan-1, a heparan sulfate proteoglycan, has been previously identified as a potential prognostic biomarker by multiple studies at the tissue and serum level. However, other studies have questioned its utility. Anti-Syndecan-1 immunohistochemistry was carried out on 157 prostate tissue samples (including cancerous, adjacent normal tissue, and non-diseased prostate) from three independent cohorts of patients. A population of Syndecan-1 positive stromal cells was identified and the number and morphological parameters of these cells quantified. The identity of the Syndecan-1-positive stromal cells was assessed by multiplex immunofluorescence using a range of common cell lineage markers. Finally, the burden of Syndecan-1 positive stromal cells was tested for association with clinical parameters. We identified a previously unreported cell type which is marked by Syndecan-1 expression and is found in the stroma of prostate tumors and adjacent normal tissue but not in non-diseased prostate. We call these cells Prostate Cancer Syndecan-1 Positive (PCSP) cells. Immunofluorescence analysis revealed that the PCSP cell population did not co-stain with markers of common prostate epithelial, stromal, or immune cell populations. However, morphological analysis revealed that PCSP cells are often elongated and displayed prominent lamellipodia, suggesting they are an unidentified migratory cell population. Analysis of clinical parameters showed that PCSP cells were found with a frequency of 20-35% of all tumors evaluated, but were not present in non-diseased normal tissue. Interestingly, a subset of primary Gleason 5 prostate tumors had a high burden of PCSP cells. The current study identifies PCSP cells as a novel, potentially migratory cell type, which is marked by Syndecan-1 expression and is found in the stroma

  10. The Importance of Positive Immunomagnetic Cell Separation Prior to Autologous Hematopoetic Stem Cell Transplantation for Advanced Stage Lymphomas

    Directory of Open Access Journals (Sweden)

    Benedek István

    2016-12-01

    Full Text Available We present the method of immunomagnetic stem cell separation with the ISOLEX 300i device (Isolex® 300i Magnetic Cell Selection System, Nextell Therapeutics Inc. Irvine California 21618 USA and the results obtained using this method in patients admitted to the Hematology and Bone Marrow Transplantation Clinic of Tîrgu Mureş, Romania. Cell selection has a great importance in separating stem cells from tumor cells, therefore contributing to the success of autologous stem cell transplantation.

  11. Nestin-positive mesenchymal stem cells favour the astroglial lineage in neural progenitors and stem cells by releasing active BMP4

    Directory of Open Access Journals (Sweden)

    Leprince Pierre

    2004-09-01

    Full Text Available Abstract Background Spontaneous repair is limited after CNS injury or degeneration because neurogenesis and axonal regrowth rarely occur in the adult brain. As a result, cell transplantation has raised much interest as potential treatment for patients with CNS lesions. Several types of cells have been considered as candidates for such cell transplantation and replacement therapies. Foetal brain tissue has already been shown to have significant effects in patients with Parkinson's disease. Clinical use of the foetal brain tissue is, however, limited by ethical and technical problems as it requires high numbers of grafted foetal cells and immunosuppression. Alternatively, several reports suggested that mesenchymal stem cells, isolated from adult bone marrow, are multipotent cells and could be used in autograft approach for replacement therapies. Results In this study, we addressed the question of the possible influence of mesenchymal stem cells on neural stem cell fate. We have previously reported that adult rat mesenchymal stem cells are able to express nestin in defined culture conditions (in the absence of serum and after 25 cell population doublings and we report here that nestin-positive (but not nestin-negative mesenchymal stem cells are able to favour the astroglial lineage in neural progenitors and stem cells cultivated from embryonic striatum. The increase of the number of GFAP-positive cells is associated with a significant decrease of the number of Tuj1- and O4-positive cells. Using quantitative RT-PCR, we demonstrate that mesenchymal stem cells express LIF, CNTF, BMP2 and BMP4 mRNAs, four cytokines known to play a role in astroglial fate decision. In this model, BMP4 is responsible for the astroglial stimulation and oligodendroglial inhibition, as 1 this cytokine is present in a biologically-active form only in nestin-positive mesenchymal stem cells conditioned medium and 2 anti-BMP4 antibodies inhibit the nestin-positive mesenchymal

  12. Characterization of a GM7 glioblastoma cell line showing CD133 positivity and both cytoplasmic and nuclear localization of nestin.

    Science.gov (United States)

    Loja, Tomas; Chlapek, Petr; Kuglik, Petr; Pesakova, Martina; Oltova, Alexandra; Cejpek, Pavel; Veselska, Renata

    2009-01-01

    A newly established GM7 cell line was derived from the tumor tissue of a 65-year-old man surgically treated for a relapse of glioblastoma multiforme that occurred 10 months after first surgery following radiotherapy. GM7 cells exhibit spindle or glia-like morphology, and multinucleated giant cells are also present in the culture. The cells proliferate rapidly (PDT is about 18 h) and tend to grow in multilayer without contact inhibition. Using G-banding and SKY, the GM7 cell line was identified as near-triploid with a large number of structural and numerical abnormalities. Repeated karyotyping during long-term cultivation confirmed a chromosome number of 70+/-3 chromosomes per cell. Special attention was paid to the immunocytochemical analysis of protein markers in this cell line; GM7 cells showed strong positivity for CD133, vimentin, nestin, NF-160 and S-100 protein and weak positivity for GFAP and NSE, but were negative for synaptophysin. The most important features of the GM7 cell line are its stable phenotype CD133+/nestin+, which are accepted as stem cell markers in neural stem/progenitor cells, and especially unusual intracellular localization of the IF protein nestin, which was detected and repeatedly confirmed both in the cytoplasm and cell nucleus. For this reason, the new GM7 glioblastoma cell line represents an important model suitable not only for further studies on glioblastoma biology and cancer stem cells, but particularly for the detailed investigation of the role of nestin in transformed cells.

  13. P-glycoprotein is expressed and causes resistance to chemotherapy in EBV-positive T-cell lymphoproliferative diseases

    International Nuclear Information System (INIS)

    Yoshimori, Mayumi; Takada, Honami; Imadome, Ken-Ichi; Kurata, Morito; Yamamoto, Kouhei; Koyama, Takatoshi; Shimizu, Norio; Fujiwara, Shigeyoshi; Miura, Osamu; Arai, Ayako

    2015-01-01

    Epstein–Barr virus-positive T-cell lymphoproliferative diseases (EBV-T-LPDs) are rare lymphomas with poor prognosis. Although chemotherapeutic strategies such as CHOP have been often selected, they have exhibited only limited efficacy. To clarify the mechanism of chemoresistance, we examined P-glycoprotein (P-gp) expression. P-gp acts as an energy-dependent efflux pump that excretes drugs from the cytoplasm, resulting in low-intracellular drug concentrations and poor sensitivity to chemotherapy. We examined P-gp expression in EBV-positive cells by immunohistochemistry staining in three patients of EBV-T-LPDs and the expression was detected in all patients. We also examined mdr1 mRNA expression by reverse-transcriptase polymerase-chain reaction (RT-PCR) in EBV-positive tumor cells from these patients and additional three patients. The expression was detected in all examined patients. In five EBV-T-LPDs patients, P-gp function was detected by Rhodamine-123 efflux assay in these cells. The efflux was inhibited by treatment with a P-gp inhibitor, cyclosporine A (CsA). We also examined and detected P-gp expression in EBV-positive T-cell lines SNT8 and SNT16 established from EBV-T-LPDs patients, by RT-PCR and western blotting. The function was also detected by Rhodamine-123 efflux in these cell lines. Inhibition and knock down of P-gp by CsA and siRNA, respectively, enhanced etoposide- and doxorubicin-induced cell death in the EBV-positive T-cell lines. Finally, we infected the T-cell line MOLT4 with EBV, and found that mdr1 mRNA expression and Rhodamine 123 efflux were upregulated after infection. These results indicated that enhanced P-gp expression contributed to the chemoresistance of EBV-T-LPDs

  14. WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning.

    Science.gov (United States)

    Lee, M M; Schiefelbein, J

    1999-11-24

    The formation of the root epidermis of Arabidopsis provides a simple and elegant model for the analysis of cell patterning. A novel gene, WEREWOLF (WER), is described here that is required for position-dependent patterning of the epidermal cell types. The WER gene encodes a MYB-type protein and is preferentially expressed within cells destined to adopt the non-hair fate. Furthermore, WER is shown to regulate the position-dependent expression of the GLABRA2 homeobox gene, to interact with a bHLH protein, and to act in opposition to the CAPRICE MYB. These results suggest a simple model to explain the specification of the two root epidermal cell types, and they provide insight into the molecular mechanisms used to control cell patterning.

  15. Relevance of pituitary aromatase and estradiol on the maintenance of the population of prolactin-positive cells in male mice.

    Science.gov (United States)

    García-Barrado, María José; Blanco, Enrique J; Catalano-Iniesta, Leonardo; Sanchez-Robledo, Virginia; Iglesias-Osma, María Carmen; Carretero-Hernández, Marta; Rodríguez-Cobos, Javier; Burks, Deborah Jane; Carretero, José

    2016-07-01

    In previous studies we demonstrated the expression of aromatase in pituitary cells. This expression is gender related, and is also associated with the presence of prolactinomas. To ascertain the relevance of aromatase in modulating the populations of prolactin-positive pituitary cells an immunocytochemical and morphometric study of prolactin-positive pituitary cells was carried out using the pituitary glands of adult male and female aromatase-knockout (ArKO) mice. Additionally has been determined if pituitary aromatase is involved in a gender-linked differentiated regulation of the prolactin-producing pituitary cells. Compared to wild-type mice, the knockout animals of both genders showed a significant decrease (pprolactin cells, as well as in the percentages of the prolactin-positive cells and the proliferating prolactin cells. Our results suggest that estradiol is responsible for the maintenance of the population of prolactin cell in males and, so as not to disturb the endocrine reproductive environment, estradiol is synthesized inside the pituitary by circulating testosterone via means of aromatase P450, which acts in paracrine way. This new role for pituitary aromatase may well explain the previous findings establishing that the pituitary expression of aromatase is higher in males than in females, and the association between the development of prolactinomas and the increased expression of aromatase in tumours. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. The differential role of HTRA1 in HPV-positive and HPV-negative cervical cell line proliferation

    International Nuclear Information System (INIS)

    Stuqui, Bruna; Conceição, André Luis Giacometti; Termini, Lara; Sichero, Laura; Villa, Luisa Lina; Rahal, Paula; Calmon, Marília de Freitas

    2016-01-01

    High-risk human papillomaviruses (HPVs) are strongly associated with the development of some malignancies. The E6 and E7 viral oncoproteins are the primary proteins responsible for cell homeostasis alteration and immortalization. Furthermore, the E6 protein from high-risk HPVs can interact with the PDZ (PSD-90/Dlg/ZO-1) domains of cellular proteins, triggering cell transformation. One protein that is associated with pathological conditions and has a PDZ domain is the protease HTRA1 (high temperature requirement 1). This protein is poorly expressed in some cancers, suggesting a tumor suppressor role. The aim of this study was to evaluate the effect of HTRA1 overexpression in HPV16-positive (CasKi) and HPV-negative (C33) cervical cell lines. The cells were transfected with a vector containing the HTRA1 ORF or an empty vector. HTRA1 overexpression was confirmed by qRT-PCR. The cells were subjected to cell proliferation, colony formation, apoptosis and cell cycle assays. C33 cells expressing HTRA1 grew significantly fewer colonies and showed less proliferation than cells without HTRA1 expression. In contrast, in the CasKi cells overexpressing HTRA1, there was an increase in the cell growth rate and in the colonies density compared to cells expressing low levels of HTRA1. An apoptosis assay showed that HTRA1 does not interfere with the apoptosis rate in these cells. A cell cycle immunofluorescence assay revealed more CasKi cells overexpressing HTRA1 in the S phase and more C33 HTRA1-transfected cells in the G0/G1 phase, suggesting that HTRA1 plays different roles in the cell cycle progression of these cells. HTRA1 overexpression prevents cell proliferation in the HPV-negative cell line and increases cell proliferation in the HPV-positive cell line. Although the E6/HTRA1 interaction has already been described in the literature, more studies are required to confirm whether the present functional findings are a result of this interaction

  17. High Level of Tregs Is a Positive Prognostic Marker in Patients with HPV-Positive Oral and Oropharyngeal Squamous Cell Carcinomas

    Directory of Open Access Journals (Sweden)

    E. Lukesova

    2014-01-01

    Full Text Available Background. Human papillomaviruses (HPVs have been proved as one of the etiological factors of oropharyngeal squamous cell carcinoma (OPSCC. Patients with tumors of viral etiology have a lower recurrence rate and better prognosis. OPSCC is linked to an alteration in the immune system. Only a limited number of studies have correlated both the immunological parameters and HPV status with patient prognosis. The aim of this study was to determine whether HPV infection and the immunological status influence patient prognosis individually or in concurrence. Material and Methods. Sixty patients with oral and oropharyngeal carcinomas were enrolled. They were divided into HPV-positive and HPV-negative groups based on the expression of HPV 16 E6 mRNA. Basic lymphocyte subpopulations were determined in the peripheral blood by means of flow cytometry. Results. Significantly better disease-specific survival (DSS was observed in patients with HPV-positive tumors. Nodal status, tumor grade, recurrence, and CD8+/Tregs ratio were identified as factors influencing DSS. A higher level of Tregs and a lower ratio of CD8/Tregs influenced overall survival (OS independently of HPV status and age. Patients with HPV-positive tumors and high levels of Tregs survived significantly better than patients from the other groups. Conclusion. Better survival is associated with HPV positivity and elevated Tregs levels. Our data suggest that HPV infection and Tregs do not influence patient prognosis in concurrence.

  18. Preferential antitumor effect of the Src inhibitor dasatinib associated with a decreased proportion of aldehyde dehydrogenase 1-positive cells in breast cancer cells of the basal B subtype

    Directory of Open Access Journals (Sweden)

    Watanabe Mika

    2010-10-01

    Full Text Available Abstract Background Recent studies have suggested that the Src inhibitor dasatinib preferentially inhibits the growth of breast cancer cells of the basal-like subtype. To clarify this finding and further investigate combined antitumor effects of dasatinib with cytotoxic agents, a panel of breast cancer cell lines of various subtypes was treated with dasatinib and/or chemotherapeutic agents. Methods Seven human breast cancer cell lines were treated with dasatinib and/or seven chemotherapeutic agents. Effects of the treatments on c-Src activation, cell growth, cell cycle, apoptosis and the proportion of aldehyde dehydrogenase (ALDH 1-positive cells were examined. Results The 50%-growth inhibitory concentrations (IC50s of dasatinib were much lower in two basal B cell lines than those in the other cell lines. The IC50s of chemotherapeutic agents were not substantially different among the cell lines. Dasatinib enhanced antitumor activity of etoposide in the basal B cell lines. Dasatinib induced a G1-S blockade with a slight apoptosis, and a combined treatment of dasatinib with etoposide also induced a G1-S blockade in the basal B cell lines. Dasatinib decreased the expression levels of phosphorylated Src in all cell lines. Interestingly, dasatinib significantly decreased the proportion of ALDH1-positive cells in the basal B cell lines but not in the other cell lines. Conclusions The present study indicates that dasatinib preferentially inhibits the growth of breast cancer cells of the basal B subtype associated with a significant loss of putative cancer stem cell population. A combined use of dasatinib with etoposide additively inhibits their growth. Further studies targeting breast cancers of the basal B subtype using dasatinib with cytotoxic agents are warranted.

  19. Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming

    NARCIS (Netherlands)

    Kwakkenbos, Mark J.; Diehl, Sean A.; Yasuda, Etsuko; Bakker, Arjen Q.; van Geelen, Caroline M. M.; Lukens, Michaël; van Bleek, Grada M.; Widjojoatmodjo, Myra N.; Bogers, Willy M. J. M.; Mei, Henrik; Radbruch, Andreas; Scheeren, Ferenc A.; Spits, Hergen; Beaumont, Tim

    The B cell lymphoma-6 (Bcl-6) and Bcl-xL proteins are expressed in germinal center B cells and enable them to endure the proliferative and mutagenic environment of the germinal center. By introducing these genes into peripheral blood memory B cells and culturing these cells with two factors produced

  20. Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming

    NARCIS (Netherlands)

    Kwakkenbos, Mark J.; Diehl, Sean A.; Yasuda, Etsuko; Bakker, Arjen Q.; van Geelen, Caroline M. M.; Lukens, Michaël V.; van Bleek, Grada M.; Widjojoatmodjo, Myra N.; Bogers, Willy M. J. M.; Mei, Henrik; Radbruch, Andreas; Scheeren, Ferenc A.; Spits, Hergen; Beaumont, Tim

    2010-01-01

    The B cell lymphoma-6 (Bcl-6) and Bcl-xL proteins are expressed in germinal center B cells and enable them to endure the proliferative and mutagenic environment of the germinal center. By introducing these genes into peripheral blood memory B cells and culturing these cells with two factors produced

  1. Model-based analysis of anion-exchanger positioning in direct methanol fuel cell systems

    Science.gov (United States)

    Kraus, Maik; Schröder, Daniel; Krewer, Ulrike

    2014-09-01

    In this work we present a model based study to investigate the presence of anion exchangers in direct methanol fuel